The main objective of this thesis was the elucidation of the host-pathogen interaction between Pinus patula and Fusarium circinatum. This was accomplished by studying differential gene expression at the molecular level. Therefore, the first chapter reports the use of PCR-based methods in gene discovery and transcriptome analysis. The use of these techniques in the identification of novel transcripts in host-pathogen interactions addressed. These examples illustrate the differences and strong features of each technique. Chitinases are linked to defence responses in plants. In chapter tw0, the induction of chitinases in P. patula was assessed at both the protein and genetic level. Western blot analysis and enzyme activity assays indicate that chitinase enzyme is not detected a part of the defence response by P. patula after infection by F. circinatum. This was further confirmed by the lack of significant induction of two Pinus chitinase genes, LP6 and PSCHI4, as determined by RT-PCR analysis. Partial DNA sequence homologues for the LP6 and PSCH14 genes were determined and compared with a variety of plant chitinases. The low levels of detectable chitinase induction in P. patula might explain the high levels of susceptibility to the pitch canker fungus observed in seedlings of this tree. Pinus patula, the most widely planted species in South Africa, is highly susceptible to infection by F. circinatum. In chapter three, suppression subtractive hybridisation was used to elucidate the changes taking place at the molecular level early on in this interaction. Most of the identified transcripts shared homology to both biotic and abiotic stress in plants. The induction of one fragment, displaying homology to phytocyanin proteins, as followed through RT-PCR. Induction levels for this fragment differed significantly between less and more susceptible plants. Although most of the sequences isolated in this study can be Iiked to stress, most have not been linked with specific plant-pathogen interactions. This raises questions in regard to the function of these genes in host-pathogen interactions. Further research identify the function of these sequences in the defence response will be needed. These sequences can also be tested against a family of Pinus trees to ascertain if they will be useful in marker assisted selection. A molecular analysis of culture degeneration and pathogenicity of F. circinatum was attempted in chapter four. In this chapter, the differential induction of transcripts in F. circinatum was determined against several other Fusarium spp. Several of he identified fragments shared homology with stress related proteins. One transcript shared homology to a polyketide synthase, FUM5, that could be linked to fumonisin production in other Fusarium spp. ELISA detected no fumonisin production, although the FUM5 transcripts were detected. The identification of all the transcripts could provide a basis for more intensive gene discovery studies in F. circinatum and other Fusarium spp. The induction of these sequences in different isolates needs to be studied to prove their function in F. circinatum. This study also complements several other studies that studied the morphological characteristics of culture degeneration. Resistance gene analogues have been reported from a diverge set of plant species. In chapter five, degenerate PCR amplification was used to isolate TI-NBS-LRR like resistance gene analogues from a range of Pinus species. These sequences w re further characterised through comparative analysis with previously reported Pinus resistance gene analogues. Through motif analysis, several of the known conserved motifs found in NBS domains were identified and conservation with other plant NBS motifs is indicated. The P-Ioop and GLPL motifs displayed a high level of conservation on amino acid level with other plant NBS motifs. However, slight differences in several of the conserved regions were observed when the Pinus analogues were compared with Arabidopsis thaliana. The identification of differences between angiosperm and gymnosperm NBS sequences indicates that design of new degenerate probes and primers for the isolation of more ancient NBS sequences is needed. Further, phylogenetic and structural analyses of these sequences will also aid in understanding the relationship between angiosperm and gymnosperm NBS sequences. The knowledge gained from such a study will highlight the similarities between angiosperm and gymnosperm defence responses. This study represents the first detailed report on RGA in Pinus. / Thesis (PhD)--University of Pretoria, 2006. / Genetics / Unrestricted

Orientador: Clarisse Palma da Silva / Resumo: A filogeografia surgiu como uma ponte entre várias disciplinas evolutivas, como a genética populacional, filogenia e biogeografia, estabelecendo ligações entre os estudos micro e macro evolutivos. A filogeografia tem sido cada vez mais utilizada para estudar a evolução de regiões com altos índices de diversidade, como a região Neotrópical. Os Neotrópicos apresentam uma grande variedade de biomas, incluindo a Floresta Atlântica, a segunda maior floresta tropical da América do Sul, contendo mais de 60% de todas as espécies terrestres do planeta. Um número crescente de estudos filogeográficos com espécies da Floresta Atlântica nos ajudado a entender os processos evolutivos que gradualmente formaram essa grande biodiversidade. Realizamos análises filogeográficas, avaliando padrões de estrutura e diversidade genética, tempo de divergência das linhagens e a demografia histórica do complexo de espécies Pitcairnia flammea (Bromeliaceae), grupo adaptado a inselbergs neotropicais naturalmente fragmentados, baseados em conjuntos de dados de microssatélites nucleares e sequências plastidiais. Nossos resultados mostraram baixa a moderada diversidade genética nuclear dentro de populações de P. flammea, e alta estrutura genética populacional, com poucos haplótipos de DNA plastidial compartilhados entre populações, indicando fluxo gênico limitado e baixa conectividade entre afloramentos rochosos. Não encontramos nenhuma estrutura filogeográfica clara, além de duas linhagens evolutivas que di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Phylogeography has emerged as a bridge between several evolutionary disciplines, such as population genetics, phylogeny and biogeography, establishing the link between micro and macroevolutionary studies. Phylogeography has been increasingly used to study the evolution of regions with high diversity indexes, such as the Neotropic region. The Neotropics presents a wide variety of biomes, including the Atlantic Rainforest, the second largest tropical rainforest in South America containing more than 60% of all terrestrial species on the planet. An increasing number of phylogeographic studies in Atlantic Rainforest species have helped us to understand the evolutionary process that gradually formed such great biodiversity. We performed phylogeographic analyses, assessing genetic structure patterns, timing of lineage divergence and historical demography of Pitcairnia flammea species complex (Bromeliaceae), which are adapted to naturally fragmented Neotropical inselbergs, based on nuclear microsatellites and plastid sequence data sets. Our results showed low to moderate nuclear genetic diversity within P. flammea populations, and high population genetic structure, with few plastid DNA haplotype shared among populations, indicating limited gene flow and low connectivity among rock outcrops. We found no clear phylogeographic structure, besides two evolutionary lineage which diverged approximately 2 Mya., suggesting an important role of early Pleistocene climatic changes in the diversi... (Complete abstract click electronic access below) / Mestre

No description available.

O veado-mão-curta (Mazama nana) é uma espécie de cervídeo que ocupa a região Sul do Brasil, norte da Argentina e leste do Paraguai, tendo sido severamente afetada pela redução drástica das áreas florestadas. Trata-se, também, da espécie de cervídeo neotropical menos estudada pela ciência. Frente a essa situação, o presente projeto propôs-se a entender como essa espécie se distribui em sua área de ocorrência, propôs áreas prioritárias para sua conservação e estimou a densidade de duas populações. Dada a raridade e a alta elusividade da espécie, propôs-se o uso de metodologias indiretas para se atingir o objetivo proposto. Assim, foram usadas metodologias baseadas na coleta de amostras fecais, extração do DNA e posterior análises molecular e genética. Foram coletadas amostras fecais com o auxílio de um cão farejador em unidades de conservação distribuídas ao longo do Sul do Brasil. Após a identificação da espécie por meio da amplificação de um fragmento do citocromo B e corte com enzimas de restrição (PCR/RFLP) e com os dados de localização das amostras, foram feitas modelagens de distribuição com o software Maxent. Foram escolhidas duas unidades de conservação, onde foram realizadas coletas de amostras fecais, baseadas em faixas, para possibilitar a estimativa da densidade de animais nestas populações. Estabeleceu-se a distribuição geográfica potencial de M. nana no Brasil para os Estados do Paraná, Santa Catarina, norte e centro do Rio Grande do Sul, extremo sul de São Paulo e Mato Grosso do Sul. Porção leste do Paraguai e, na Argentina para a província de Missiones. A densidade da espécie para a região norte do Parque Nacional do Iguaçu foi de 1,9 ind/km2 e para o Parque Estadual Vila Rica do Espírito Santo foi de 5,5 ind/km2. A população potencial da espécie foi de 152.991 indivíduos, sendo 15.524 indivíduos a população dentro das áreas protegidas. Sugere-se a manutenção do estado de conservação da espécie como Vulnerável, tanto na lista Brasileira como na lista Internacional de fauna ameaçada de extinção. / The Brazilian dwarf brocket (Mazama nana) is a deer species that occupies the forests of southern Brazil, north of Argentina and east of Paraguay. It has been greatly affected by the drastic reduction of forested areas. It is also the less studied neotropical deer. Considering this situation, this project aimed to shed light on the species distribution along its range, to indicate conservation priority areas and estimate the density of two populations. Given the rarity and high elusiveness of the species, it is proposed the use of indirect methods to achieve this goal. Fecal samples collection based methodologies were used, followed by DNA extraction and subsequent molecular and genetic analysis. Fecal samples were tracked and collected in protected areas spread over south Brazil, with the help of a scat detection dog. After species identification by PCR/RFLP and samples spatialization, species distribution modeling was carried out using Maxent software suit. Two protected areas were chosen for a faecal sampling based on transects, in order to estimate the population density. Potential geographical distribution of M. nana in Brazil was stablished at states of Paraná, Santa Catarina, northern and center Rio Grande do Sul, extreme South of São Paulo and Mato Grosso do Sul. Also at Eastern Paraguay and Missiones province in Argentina. Species density at the northern area of Iguaçu National Park was 1.9 ind/km2 and at the State Park of Vila Rica do Espírito Santo was 5.5 ind/km2. The species potential population was 152,991 individuals, including 15,524 individuals inside protected areas. It is suggested to maintain species conservation status as vulnerable on the Brazilian and on the International red list of threatened species.

The population genetics of species occupying formerly glaciated regions are not only impacted by glacial retreat but also agricultural land use that is typical of such regions. Areas which have experienced glaciation often display a lowered amount of genetic variability and minimal population structure, and these effects become more predominant with increasing distance from a potential refugial population. Meanwhile, agricultural land use over the recent past has also been demonstrated to disrupt population structure distribution through disturbance regimes. The purpose of this study was to assess potential post-glacial and agricultural effects on populations of creek chub (Semotilus atromaculatus) in two agricultural watersheds that differ in the glacial history. The Saint Joseph River (SJR) watershed, Indiana and Michigan, USA was entirely glaciated during the last glacial maxima, while the Little Miami River (LMR) watershed in Ohio, USA, is situated on the boundary of the glacier. The degree of agricultural land use also varies between and within the two watersheds. Using eight microsatellite loci, 312 individuals were genotyped from 13 sites in SJR and 2,318 individuals from 29 sites in LMR. Measures of genetic differentiation showed that there was strong differentiation between watersheds. Analyses within watersheds recovered additional but weaker differentiation that was mostly associated with the geography of sub-watersheds and isolation by distance. Proximity to the glacial boundary appeared to play a minimal role in genetic differentiation and genetic variation. Differentiation among localities was not directly associated with the glacial boundary within LMR, and localities in this watershed had lower allelic richness and heterozygosity than those in the fully glaciated SJR. After accounting for the positive correlation of stream distance in LMR using partial Mantel test, both glacial history and agricultural land use were positively correlated with genetic differentiation. However, these predictor variables were also strongly correlated with one another which prevented disentangling the two potential effects. Within SJR, no 10 relationship of genetic differentiation with agricultural land use was recovered. My study shows that there is not a simple relationship between glacial history, contemporary land use, and genetic differentiation in creek chub. Rather, it appears that the patterns of genetic variation observed may be more closely linked to the dispersal behavior of creek chub within and among watersheds, and the history of effective population size within watersheds.

O gênero Mazama é composto por cinco espécies no Brasil. São animais de visualização dificultada por causa de comportamentos evasivos, o que torna as capturas e os estudos comportamentais quase impossíveis. Assim, o uso de metodologias não invasivas para estudos ecológicos e genéticos destas espécies se torna necessário. A análise do DNA fecal está dentro das técnicas mais promissoras para esse fim. Este estudo objetivou genotipar amostras fecais, de uma população de veado-mateiro (Mazama americana) para a obtenção de informações genéticas e ecológicas. Para tanto, foram coletadas, com auxílio de um cão farejador, georreferenciadas e estocadas em etanol absoluto, 52 amostras fecais de cervídeos. A coleta realizou-se em um fragmento (21o20S 47o17W) de 600 ha de floresta estacional semidecidual. Dessas amostras coletadas, 31% (n=16) foram classificadas como frescas e 69% (n=36) como não frescas. O DNA foi extraído em torno de 30 dias após a coleta, usando o kit comercial QIAamp® DNA Stool Mini Kit, seguindo o protocolo do fabricante. Das 52 amostras, 45 foram identificadas por PCR/RFLP como pertencentes a M. americana e as demais apresentaram problemas de amplificação e digestão, permanecendo sem identificação. Amplificaram-se por PCR cinco locos microssatélites, e o sucesso de amplificação, visualizado em gel de agarose, variou com o tamanho dos locos e com a classe das amostras. O sucesso de amplificação foi de 65% das amostras da categoria fresca e 35% das amostras da categoria não fresca. Encontrou-se uma correlação negativa (R= -0,82) entre o tamanho dos fragmentos dos locos de microssatélites e o sucesso de amplificação. Foi possível identificar o sexo do animal em 43,7% das amostras fecais, pela amplificação do gene da amelogenina. Os locos microssatélites amplificados foram analisados em sequenciador automático. Os eletroferogramas gerados pelo seqüenciador impossibilitaram a genotipagem da maioria dos locos e amostras, tornando inviável qualquer análise genética e ecológica com confiabilidade. Fica evidente a dificuldade de se trabalhar com a metodologia do DNA fecal para a identificação individual e sexagem de amostras obtidas de Cervídeos florestais em vida livre. Algumas melhorias metodológicas (coleta de amostras fecais frescas, seleção de iniciadores para locos menores e quantificação do DNA extraído por PCR em tempo real) são sugeridas para o aumento nos índices de sucesso na genotipgem em estudos futuros. / Mazama genus is composed by five species in Brazil. All of them are difficult to observe due to their evasive behaviors, what makes the captures and behavioral studies almost impossible. Thus, the use of non invasive methodologies is necessary to study the ecology and genetics of these species. The fecal DNA analysis is one of the most promising techniques for this purpose. This study aimed to genotype a Mazama americana population faecal samples for obtaining genetics and ecological information. For this, 52 deer faecal samples were collected in a 600ha seasonal semideciduos forest fragment (21o20S 47o17W), with the help of a detection dog, stored in ethanol and georeferenced. Of these samples 31% (n=16) was classified as fresh and 69% (n=36) as not fresh. About thirty days after the collection the DNA was extracted using the QIAamp® DNA Stool Mini Kit following the manufacturers instructions. From the 52 samples collected and extracted, 45 were identified by PCR/RFLP as M. americana and the others showed amplification and digestion problems, remaining without identification. Five microsatellite loci were amplified by PCR and the amplification success, visualized in agarose gel, varied with the loco size and age class. The amplifications success occurred in 65% of the fresh samples and in 35% of the non-fresh samples and a negative correlation (R= -0.82) was found between amplification success and loci sizes. It was possible to identify the animal sex in 43% of the samples by the amelogenin gene. The microsatellite loci amplifications were analyzed in an automatic sequencer. The majority of the samples and loci were impossible to genotype because of the quality of the elestroferograms, what made impossible any reliable genetic and ecological analysis. It is evident the difficulty to work with the faecal DNA methodology using field collected forests deer samples for individual and sexual identifications. Some methodological improvements (collect fresh samples, select primers for shorter loci and quantify the extracted DNA by real time PCR) are suggested to increase the genotyping success indexes in future studies

O percevejo-marrom-da-soja, Euschistus heros (F.) (Hemiptera: Pentatomidae), é uma praga chave da soja na América do Sul. Recentemente, foi descoberto a presença de duas linhagens alopátricas de E. heros no Brasil. Uma linhagem de ocorrência predominante na região Norte/Nordeste (N) e outra predominante na região Sul (S) e uma zona sobreposição entre as linhagens no Cerrado. O encontro de dois pools gênicos pode representar aumento ou diminuição de vigor das populações mediado pelo grau de isolamento reprodutivo. O encontro das linhagens no Cerrado é uma hipótese para as explosões populacionais de E. heros em cultivos de soja e constante ocorrência em cultivos de algodão relatados nos últimos anos. Questões sobre o potencial adaptativo dessas linhagens são de grande importância para entendimento da dinâmica e dos surtos populacionais de E. heros. Assim, nossos objetivos foram: 1) verificar a presença de barreiras reprodutivas entre as duas linhagens alopátricas de E. heros que promova uma prole híbrida de baixo fitness; e 2) avaliar o efeito da temperatura e fotoperíodo no tempo de desenvolvimento e viabilidade de fases imaturas e na expressão da dormência nas linhagens alopátricas de E. heros. Cruzamentos recíprocos foram realizados com intuito de avaliar o desenvolvimento da prole híbrida quando comparada com a prole coespecífica. Os parâmetros biológicos e de tabela de vida evidenciaram que o cruzamento entre as duas linhagens não representou diminuição de fitness da prole híbrida, portanto, não existe incompatibilidade pós-zigótica entre as linhagens de E. heros. Adicionalmente, ovos das linhagens (S e N) foram incubados em câmaras climáticas sob diferentes regimes de luz (10: 14, 11,5: 12,5 e 14: 10 (L: E)); e temperaturas (14,18, 21 e 25°C). Ficou evidente que existe uma relação entre temperatura e fotoperíodo no tempo de desenvolvimento e viabilidade de E. heros. A temperatura de 18°C e fotoperíodo de 10: 14 (L:E) aumentam o tempo necessário para completar o ciclo e reduzem a viabilidade. Temperatura de 25°C e fotoperíodo de 14: 10 (L:E) diminuem tempo de desenvolvimento e aumentam a viabilidade nas fases imaturas das duas linhagens. A expressão do fenótipo da dormência foi positivamente afetada por temperaturas menores e fotoperíodos mais curtos, especialmente para a linhagem Sul. A linhagem Norte não expressou características fenotípicas de dormência mesmo sob condições de temperaturas baixas e dias curtos. As diferenças encontradas possivelmente estão relacionadas ao local de origem das duas linhagens. Assim, o encontro de dois pools gênicos onde não há barreira reprodutiva aumenta a diversidade genética nas populações e permite a combinação de alelos que podem aumentar o vigor das populações e a capacidade de adaptação a ambientes novos e perturbados, como o ambiente agrícola. Os resultados demonstram que as duas linhagens possuem respostas similares aos fatores de estresses abióticos nas fases imaturas e distintas na expressão do fenótipo da dormência. As respostas deste trabalho oferecem informações úteis para melhor entender a dinâmica populacional contemporânea e para implementação de estratégias de manejo mais eficientes e sustentáveis de E. heros no cenário agrícola nacional. / The brown soybean stink bug, Euschistus heros (F.) (Hemiptera: Pentatomidae), is a key soybean pest in South America. It was recently discovered the presence of two allopatric lineages of E. heros in Brazil. A lineage predominantly occurring in the Northern/Northeast region (N) and another predominate in the Southern region (S) and an overlapping hybridization zone in the Cerrado region. The meeting of two gene pools may represent an increase or decrease in the vigor of populations in the absence of reproductive isolation. The reunion of the lineages in the Cerrado is a hypothesis for the expressive increase of the species in soybean cultivations and expansion of problems in the cotton crop. Questions about the adaptive potential of these lineages are of great importance for understanding the dynamics and population outbreaks of E. heros. The present study aims to: 1) verify the presence of reproductive barriers between the two lineages by means of a low fitness hybrid offspring; 2) to evaluate the effect of temperature and photoperiod on the development time and viability of immature phases and expression of dormancy in the allopatric lineages of E. heros. Reciprocal crosses were performed with the purpose of evaluating the development of the hybrid offspring when compared to the co-specific offspring. The biological and life table parameters showed that the crossbreeding between the two lineages did not represent a decrease in the fitness of the hybrid offspring. Therefore, there is no post-zygotic incompatibility among the lineages of E. heros. Additionally, eggs from both lineages (S and N) were incubated in climatic chambers under different light regimes (10: 14, 11,5: 12,5 and 14: 10 (L: E) and temperatures (14, 18, 21 and 25°C). It was evident that there is a relationship between temperature and photoperiod in the development time and viability of E. heros. The temperature of 18°C and photoperiod of 10: 14 (L: E) increase the time required to complete the cycle and reduce viability. Temperature of 25°C and photoperiod of 14: 10 (L: E) decrease development time and increase viability in the immature phases of the two lineages. The expression of the dormancy phenotype was positively affected by shorter temperatures and shorter photoperiods, especially for the Southern lineage. The Northern lineage did not express phenotypic characteristics of dormancy even under low temperature and short-day conditions. The differences found are possibly related to the geographical origin of the two lineages. Thus, the meeting of two gene pools where there is no reproductive barrier increases the genetic diversity in populations and, allows the combination of alleles that can increase the vigor of populations and the ability to adapt to new and disturbed environments, such as the agricultural environment. The results demonstrate that the two strains have similar responses to the abiotic stress factors in the immature phases and distinct in the expression of the dormancy phenotype. The answers of this work offer useful information to better understand contemporary population dynamics and to implement more efficient and sustainable management strategies of E. heros in the national agricultural scenario.

The McMurdo Dry Valleys comprise some 4,800km2 of ice-free terrain in east Antarctica and this constitutes the coldest and most arid desert on Earth. The ecosystem of the Dry Valleys is characterized by microbial processes since environmental extremes severely limit higher plant and animal life. A major international collaborative research effort co-ordinated by the International Center for Terrestrial Antarctic Research (ICTAR), identified long-term study sites representative of maritime and inland Dry Valleys environments. The maritime site, Miers Valley, has been the subject of intensive multi-disciplinary study in recent years, of which the work in this thesis is a part. Previous studies have identified soil microbial communities and their putative functional roles, but lithic communities have not been previously appreciated. This thesis reports aspects on the biodiversity and ecology of lithic microbial communities in Miers Valley. A survey of terrain revealed extensive weathered granite, but no porous sandstone or limestone rocks more commonly associated with cryptoendolithic communities (those colonizing pore spaces within rock substrates). Granite was extensively colonized (30-100% of available substrate) by chasmoendolithic microorganisms (colonizing cracks and fissures in weathered rock). Visual examination of colonized rocks revealed a distinct zone of biomass 2-5mm below the rock surface, and this was overlain by a weathered and friable matrix of rock. Microscopy revealed a community dominated by diverse cyanobacterial morphotypes, plus other unidentifiable microbes of varied morphology. A quantitative approach to broad-scale community fingerprinting was adopted, utilizing terminal restriction fragment length polymorphism (TRFLP) and sequence based identifications of restriction fragments. The multi-domain approach encompassed Archaea, Bacteria and Eukarya. The results revealed relatively low species richness (0.6-1.8) for each domain with community richness estimates also relatively low (<3). Nonetheless very clear and statistically supported patterns in the occurrence of phylotypes within chasmolithic communities were related to aspect (which strongly affects temperature and moisture availability in Dry Valleys locations). The bacterial assemblages formed two groups (cold-dry south facing slopes and valley floor moraine). The eukaryal assemblages also formed two groups although here the moraine samples grouped with the warmer wetter north facing slope and the cold-dry south facing slope assemblages formed a separate group. The archaeal assemblages displayed no difference within different valley terrain. Extensive sequence based interrogation of community structure using clone libraries revealed a community dominated by cyanobacteria, Actinobacteria, Deinococci and putative lichens. These phyla are all known for their extreme tolerance to desiccation and occurrence in arid landscapes. Phylogenetic analysis revealed that these abundant taxa shared close affiliation with those from other Antarctic refuge niches such as hypoliths and cryptoendoliths. The cyanobacteria were mainly Oscillatoriales, but other genera such as Chroococcidiopsis and Nostoc commonly recovered in hot desert lithic communities were generally absent. The eukaryal community was dominated by chlorophyte algae, whilst the archaeal phylotypes were a diverse collection spanning both euryachaeal and crenarchaeal lineages. Overall the data revealed the chasmoendolithic community in Miers Valley was widespread and with relatively restricted diversity. The selection pressures related to topology of the valley have resulted in different community structure within the valley. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

O veado-mão-curta (Mazama nana) é uma espécie de cervídeo que ocupa a região Sul do Brasil, norte da Argentina e leste do Paraguai, tendo sido severamente afetada pela redução drástica das áreas florestadas. Trata-se, também, da espécie de cervídeo neotropical menos estudada pela ciência. Frente a essa situação, o presente projeto propôs-se a entender como essa espécie se distribui em sua área de ocorrência, propôs áreas prioritárias para sua conservação e estimou a densidade de duas populações. Dada a raridade e a alta elusividade da espécie, propôs-se o uso de metodologias indiretas para se atingir o objetivo proposto. Assim, foram usadas metodologias baseadas na coleta de amostras fecais, extração do DNA e posterior análises molecular e genética. Foram coletadas amostras fecais com o auxílio de um cão farejador em unidades de conservação distribuídas ao longo do Sul do Brasil. Após a identificação da espécie por meio da amplificação de um fragmento do citocromo B e corte com enzimas de restrição (PCR/RFLP) e com os dados de localização das amostras, foram feitas modelagens de distribuição com o software Maxent. Foram escolhidas duas unidades de conservação, onde foram realizadas coletas de amostras fecais, baseadas em faixas, para possibilitar a estimativa da densidade de animais nestas populações. Estabeleceu-se a distribuição geográfica potencial de M. nana no Brasil para os Estados do Paraná, Santa Catarina, norte e centro do Rio Grande do Sul, extremo sul de São Paulo e Mato Grosso do Sul. Porção leste do Paraguai e, na Argentina para a província de Missiones. A densidade da espécie para a região norte do Parque Nacional do Iguaçu foi de 1,9 ind/km2 e para o Parque Estadual Vila Rica do Espírito Santo foi de 5,5 ind/km2. A população potencial da espécie foi de 152.991 indivíduos, sendo 15.524 indivíduos a população dentro das áreas protegidas. Sugere-se a manutenção do estado de conservação da espécie como Vulnerável, tanto na lista Brasileira como na lista Internacional de fauna ameaçada de extinção. / The Brazilian dwarf brocket (Mazama nana) is a deer species that occupies the forests of southern Brazil, north of Argentina and east of Paraguay. It has been greatly affected by the drastic reduction of forested areas. It is also the less studied neotropical deer. Considering this situation, this project aimed to shed light on the species distribution along its range, to indicate conservation priority areas and estimate the density of two populations. Given the rarity and high elusiveness of the species, it is proposed the use of indirect methods to achieve this goal. Fecal samples collection based methodologies were used, followed by DNA extraction and subsequent molecular and genetic analysis. Fecal samples were tracked and collected in protected areas spread over south Brazil, with the help of a scat detection dog. After species identification by PCR/RFLP and samples spatialization, species distribution modeling was carried out using Maxent software suit. Two protected areas were chosen for a faecal sampling based on transects, in order to estimate the population density. Potential geographical distribution of M. nana in Brazil was stablished at states of Paraná, Santa Catarina, northern and center Rio Grande do Sul, extreme South of São Paulo and Mato Grosso do Sul. Also at Eastern Paraguay and Missiones province in Argentina. Species density at the northern area of Iguaçu National Park was 1.9 ind/km2 and at the State Park of Vila Rica do Espírito Santo was 5.5 ind/km2. The species potential population was 152,991 individuals, including 15,524 individuals inside protected areas. It is suggested to maintain species conservation status as vulnerable on the Brazilian and on the International red list of threatened species.

O gênero Mazama é composto por cinco espécies no Brasil. São animais de visualização dificultada por causa de comportamentos evasivos, o que torna as capturas e os estudos comportamentais quase impossíveis. Assim, o uso de metodologias não invasivas para estudos ecológicos e genéticos destas espécies se torna necessário. A análise do DNA fecal está dentro das técnicas mais promissoras para esse fim. Este estudo objetivou genotipar amostras fecais, de uma população de veado-mateiro (Mazama americana) para a obtenção de informações genéticas e ecológicas. Para tanto, foram coletadas, com auxílio de um cão farejador, georreferenciadas e estocadas em etanol absoluto, 52 amostras fecais de cervídeos. A coleta realizou-se em um fragmento (21o20S 47o17W) de 600 ha de floresta estacional semidecidual. Dessas amostras coletadas, 31% (n=16) foram classificadas como frescas e 69% (n=36) como não frescas. O DNA foi extraído em torno de 30 dias após a coleta, usando o kit comercial QIAamp® DNA Stool Mini Kit, seguindo o protocolo do fabricante. Das 52 amostras, 45 foram identificadas por PCR/RFLP como pertencentes a M. americana e as demais apresentaram problemas de amplificação e digestão, permanecendo sem identificação. Amplificaram-se por PCR cinco locos microssatélites, e o sucesso de amplificação, visualizado em gel de agarose, variou com o tamanho dos locos e com a classe das amostras. O sucesso de amplificação foi de 65% das amostras da categoria fresca e 35% das amostras da categoria não fresca. Encontrou-se uma correlação negativa (R= -0,82) entre o tamanho dos fragmentos dos locos de microssatélites e o sucesso de amplificação. Foi possível identificar o sexo do animal em 43,7% das amostras fecais, pela amplificação do gene da amelogenina. Os locos microssatélites amplificados foram analisados em sequenciador automático. Os eletroferogramas gerados pelo seqüenciador impossibilitaram a genotipagem da maioria dos locos e amostras, tornando inviável qualquer análise genética e ecológica com confiabilidade. Fica evidente a dificuldade de se trabalhar com a metodologia do DNA fecal para a identificação individual e sexagem de amostras obtidas de Cervídeos florestais em vida livre. Algumas melhorias metodológicas (coleta de amostras fecais frescas, seleção de iniciadores para locos menores e quantificação do DNA extraído por PCR em tempo real) são sugeridas para o aumento nos índices de sucesso na genotipgem em estudos futuros. / Mazama genus is composed by five species in Brazil. All of them are difficult to observe due to their evasive behaviors, what makes the captures and behavioral studies almost impossible. Thus, the use of non invasive methodologies is necessary to study the ecology and genetics of these species. The fecal DNA analysis is one of the most promising techniques for this purpose. This study aimed to genotype a Mazama americana population faecal samples for obtaining genetics and ecological information. For this, 52 deer faecal samples were collected in a 600ha seasonal semideciduos forest fragment (21o20S 47o17W), with the help of a detection dog, stored in ethanol and georeferenced. Of these samples 31% (n=16) was classified as fresh and 69% (n=36) as not fresh. About thirty days after the collection the DNA was extracted using the QIAamp® DNA Stool Mini Kit following the manufacturers instructions. From the 52 samples collected and extracted, 45 were identified by PCR/RFLP as M. americana and the others showed amplification and digestion problems, remaining without identification. Five microsatellite loci were amplified by PCR and the amplification success, visualized in agarose gel, varied with the loco size and age class. The amplifications success occurred in 65% of the fresh samples and in 35% of the non-fresh samples and a negative correlation (R= -0.82) was found between amplification success and loci sizes. It was possible to identify the animal sex in 43% of the samples by the amelogenin gene. The microsatellite loci amplifications were analyzed in an automatic sequencer. The majority of the samples and loci were impossible to genotype because of the quality of the elestroferograms, what made impossible any reliable genetic and ecological analysis. It is evident the difficulty to work with the faecal DNA methodology using field collected forests deer samples for individual and sexual identifications. Some methodological improvements (collect fresh samples, select primers for shorter loci and quantify the extracted DNA by real time PCR) are suggested to increase the genotyping success indexes in future studies