Biodiversity is a complex concept encapsulating the variation that occurs within and among levels of biological organization. It is positively linked to ecosystem persistence, adaptability, and function. Biodiversity loss, driven by global change and human activities, is one of the most prominent threats to ecosystems. Characterizing the variation of and processes driving biodiversity is a critical step in understanding the causes, consequences, and magnitude of biodiversity loss. However, characterizing biodiversity comprehensively requires understanding multiple dimensions, or types, of diversity, such as genetic, taxonomic, phylogenetic, and life history diversity, that encompass both ecological and evolutionary processes varying across space and time. In this dissertation, I investigate spatial and temporal variation in frog and toad (order Anura) biodiversity to understand the effects of how diversity is measured on biodiversity characterization and the underlying processes driving biodiversity. In my first chapter, I examined the spatial and temporal variation of genetic diversity and other population genetic metrics to understand the effects of multi-year sampling on population genetic inference in an anuran metapopulation (Arizona treefrog, Hyla (Dryophytes) wrightorum). I found that a single sample year captures global, but not local, population genetic dynamics, as there is considerable temporal variation in genetic metrics within individual populations. In my second chapter, I developed a tool to improve the characterization of anuran life history diversity using species traits. Traits are the measurable attributes of species, and a suite of species traits is used to distinguish ecological strategies found among species. I collated trait data from 411 primary and secondary sources for 106 anuran species found in the United States to develop an anuran traits database for use in conservation, management, and research. In my third chapter, I investigated spatial variation within and among taxonomic, phylogenetic, and life history anuran diversity in the United States and examined the abiotic relationships behind observed patterns. To do this, I developed species distribution models at a 1 km2 resolution for the majority of the native US anurans. I identified relationships among diversity metrics for improved, comprehensive biodiversity characterization and potential ecological and evolutionary processes underlying biodiversity. Spatial variation in multidimensional relationships highlights regional needs for multiple metrics of diversity to comprehensively characterize biodiversity. This spatial variation is driven by temperature, elevation, and water availability, likely related to the biological limits for anurans. Collectively, these chapters highlight the considerable variation that exists within and among species of a broad and diverse biological. Furthermore, these chapters call attention to the importance of measuring multiple biodiversity dimensions for effective conservation in a rapidly changing world. / Doctor of Philosophy / Biodiversity loss, from global change and human activities, is one of the biggest threats to the Earth's ecosystems. Biodiversity is the similarities and differences in organismal characteristics, such as their genetics, evolutionary history, and ecology. Biodiversity is often linked to how well an ecosystem will persist and adapt to global change. To understand the causes and consequences of biodiversity loss, it is important to first measure diversity and what shapes it. However, there are many types of diversity and ways to measure them, such as the number of species in a system, or species richness, the span of evolutionary lineages in a system, or phylogenetic diversity, the number of roles species fill in a system, or life history diversity, and the genetic relationships within individuals and populations, or genetic diversity. In this dissertation, I investigate how our understanding of biodiversity is affected by the ways it can be measured and the relationship among those metrics. To do this, I studied diversity in three ways for frogs and toads of the United States. First, I investigated whether sampling across multiple years is necessary to adequately characterize genetic diversity in a frog species (Arizona treefrog: Hyla (Dryophytes) wrightorum) with populations that fluctuate in size over time. Here, I found that multiple sample years are necessary to capture the genetic variation within individual populations over time, but unnecessary to capture the average genetic variation among all populations over space and time. Second, I developed a tool to improve our ability to measure anuran life history diversity using traits. Traits are measurable attributes of species, and multiple species traits can be used to define the role of a species in an ecosystem. I collected trait data from 411 sources for 106 frog and toad species found in the United States to develop a traits database for use in conservation, management, and research. Third, I investigated the similarities in multiple diversity metrics across the United States using species richness, phylogenetic diversity, and life history diversity. Species richness is highest in the eastern US, phylogenetic diversity is highest in the western US, and life history diversity is clustered around eastern US mountains. These regional relationships among metrics coincided with regional water availability, temperature, and elevation. These results collectively call attention to changes over space and time in frog and toad diversity and how the relationships within and among diversity types relate to our understanding of frog and toad biodiversity. Considering how, where, and when to measure biodiversity can lead to more effect biodiversity conservation in a rapidly changing world.

The elusive jaguar (Panthera onca) is extremely difficult to study due to its wide-ranging behavior, crepuscular activity peaks and its occurrence in low population densities in often dense forest habitats. Jaguars are also a species of concern, but our ability to provide for their survival is hampered by our inability to obtain reliable information on the status of their wild populations. This study combines innovative noninvasive research techniques such as scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of Neotropical felids coexisting across fragmented forest habitats in Belize, Central America. Specifically, we analyzed multi-locus data in jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) collected from 1053 scat samples across their range in the country. First, we optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis), and assessed their utility for cross-species amplification. Additionally, we tested their reliability for species and individual identification using fecal DNA as he primary DNA source. All microsatellite loci examined successfully cross-amplified in the three target species, and were polymorphic. Second, to maximize PCR amplification success and genotyping accuracy rates, and to minimize genotyping error rates for fecal DNA samples, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution/DET buffer, 95% EtOH) suitable for long-term preservation at remote tropical sites. Additionally, we tested fecal DNA samples collected from four different scat locations (top, side, bottom, inside). DET buffer was the superior fecal DNA preservation method and collecting fecal DNA from side and top locations of the scat resulted in the highest PCR success rates. For the main genetic study, we assessed the genetic conservation status of all three target species across the country of Belize. We examined levels of genetic diversity within different sites, (2) defined potential genetic clusters/populations, (3) and examined levels of gene flow and population structure for all three target species on a countrywide scale. Furthermore, we compared genetic diversity and gene flow levels among the three target species. Wild felids in Belize showed moderate levels of heterozygosity (HE = 0.60 - 0.70) with jaguars having the lowest genetic diversity with average expected heterozygosities of HE = 0.60 ± 0.05 and allelic richness (AR) of 4.94 ± 0.44 followed by pumas with HE = 0.65 ± 0.06 and AR of 7.52 ± 0.86 and ocelots with HE = 0.70 ± 0.05 and AR of 3.89 ± 0.23. We observed low to moderate levels of differentiation (FST = 0.00 - 0.15) and weak population structure using spatial Bayesian clustering techniques for all three target species. Although levels of genetic diversity and gene flow across the country are still fairly high, we did detect evidence of fragmentation indicating the risk of further habitat loss and fragmentation for wild felids. Felids were simultaneously monitored across all study sites by remote sensing camera traps, which allows for a comparison of density estimates obtained from two different noninvasive survey approaches. Furthermore, analytical methods for density estimation are advancing rapidly, making it difficult to choose the optimal technique. Thus, we compared a variety of density estimators including the conventional approach of estimating abundance ( ) in programs CAPTURE and MARK and dividing abundance by the effective trapping area (ETA), the recently developed spatially explicit capture-recapture (SECR) models, both the likelihood-based approach (ML-SECR) in program DENSITY and the Bayesian approach (B-SECR) in program SPACECAP, and finally the genetic-based mark-recapture one sampling occasion estimator in program CAPWIRE. Although different survey methods using various density estimators produced similar density estimates, confidence levels and coefficients of variation varied, with SECR methods resulting in the least precise estimates. Detection probabilities were generally higher for noninvasive genetic sampling than for camera trapping. Both techniques were shown to be reliable and highly efficient survey methods for density estimation of low-density Neotropical felids living in challenging environments such as the tropics. While less precise, SECR CMR models are probably a more realistic reflection of our uncertainty. They hold great promise for density estimation studies for wide-ranging and territorial carnivore species, especially if precision can be improved through study design or analysis advancements in the future. In conclusion, our results demonstrated that noninvasive sampling techniques such as molecular scatology and remote camera trapping are efficient research approaches to study multiple Neotropical felids in a multifaceted way and on a countrywide scale. We believe that the techniques and analyses developed in this study are widely applicable and relevant to the conservation and management of other elusive and difficult to study wild felids worldwide. / Ph. D.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第25153号 / 理博第5060号 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 平田 聡, 教授 三谷 曜子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Wälder bedecken 31 % der Landflächen weltweit. Aufgrund ihrer hohen Anzahl an endemischen Arten und ihrem hohen Artenreichtum gehören tropische Regenwälder zu den Biodiversitätshotspots der Welt. Die Ausbreitung von landwirtschaftlich genutzten Flächen führte zu einer verstärkten Degradierung und Waldverlust in Indonesien, die zum heutigen Zeitpunkt global am höchsten ist. Hauptsächliche Ursachen für die Entwaldung des tropischen Regenwaldes in Indonesien sind Holznutzung, Rohstoffabbau und die Produktion von Kautschuk (Hevea brasiliensis) und Palmöl (Elaeis guineensis), daraus folgt eine jährliche Umwandlungsrate von 20 000 km2 von natürlichem Regenwald in genutzte Flächen. Die weltweiten Konsequenzen der Entwaldung können nur geschätzt werden. Lokale Folgen sind Habitatverlust, Fragmentierung und Degradierung von verbleibenden Wäldern. In den verbleibenden Waldfragmenten kommt es zu einer Reduzierung der Artendiversität und einer Veränderung der Artenkombination. Untersuchungen von einzelnen Arten über die Folgen von Habitatfragmentierung auf die genetische Diversität von Pflanzen, zeigen unterschiedliche Ergebnisse, die von den artspezifischen Lebenszyklusstrategien abhängen. Im Allgemeinen ist ein Verlust von genetischen Ressourcen durch genetische Drift und reduzierten Genfluss zu erwarten. Dies entsteht durch die verminderte Austauschkonnektivität der verbleibenden Waldareale und die reduzierte effektive Populationsgröße der dort vorkommenden Arten. Dies kann zu einer Veränderung der genetischen Populationsstruktur der fragmentierten Arten führten, was eine Erhöhung der Wahrscheinlichkeit des Aussterbens der Art zur Folge hat. Der Effekt von Habitat-Fragmentierung auf die genetische Struktur wurde bisher nur für einzelne Pflanzenarten und nicht für Pflanzengemeinschaften untersucht. Weiterhin wurden keine Studien über die Folgen von Landnutzungsveränderungen auf die genetische Diversität von Pflanzen durchgeführt. Das Ziel der vorliegenden Studie war die genetische Diversität von dominanten Pflanzenarten in vier verschiedener Systeme mit unterschiedlicher landwirtschaftlicher Intensität in Sumatra, Indonesien, zu untersuchen. Anonyme AFLP Marker wurden genutzt, um die genetische Diversität von zehn dominanten Pflanzenarten, mit jeweils zehn Individuen, in den folgenden vier Landnutzungssystemen abzuschätzen: altgewachsener tropischer Tieflandregenwald, Kautschuk-Dschungel, Kautschukplantage und Palmölplantage. Die vier Systeme mit jeweils vier Replikaten, wurden in zwei Regionen untersucht, dies ergab eine Gesamtprobenanzahl von 3200. Durch unterschiedliche Artenkompositionen, die durch unterschiedliche Eigenschaften charakterisiert sind, wurde ein Abfall von genetischer Diversität von Wald zu Kautschuk-Dschungel zu Kautschukplantage zu Palmölplantage erwartet. Bei den Analysen wurden zwei Ansätze verwendet, bei dem Ersten wurde jeder Plot als eine Pflanzengemeinschaft betrachtet und bei dem Zweiten einzelne, häufig dominierende, Arten analysiert. Für die Gemeinschaftsanalyse wurden wiederum zwei Ansätze durchgeführt: Erstens der Fragmentpool-Ansatz, bei dem alle AFLP Fragmente der dominanten Arten in einem Fragment-Pool kombiniert wurden und deren genetische Differenzierung berechnet wurden. Zweitens der Artenansatz, bei dem die genetische Diversität pro Art im jeweiligen Plot berechnet wurde. Um die Landnutzungssystem auf genetische Unterschiede zu testen wurde ein „Mixed effect model“ für beide Ansätze der Gemeinschaftsanalyse benutzt. Außerdem wurde die genetische Diversität mit der Diversität von Pflanzenarten, Mykorrhizaarten und Prokaryotenarten korreliert, um die Reaktionsähnlichkeit der Parameter auf Landnutzungsveränderungen abzuschätzen. Die häufig dominanten Arten wurden hinsichtlich ihrer Populationsstruktur und der Populationsdifferenzierung innerhalb und zwischen den Landnutzungssystemen untersucht. Weiterhin wurden Arten nach ihrer Lebensform gruppiert und auf signifikante Unterschiede getestet. Ergebnisse der Gemeinschaftsanalyse mit dem Fragmentpool-Ansatz und dem Artenansatz zeigten keine direkte Korrelation zwischen genetischer Diversität dominanter Pflanzen und dem Landnutzungssystem. Aber aufgrund der Landnutzungsveränderung gibt es unterschiedliche Artenkompositionen im jeweiligen System, die mit ihren unterschiedlichen Eigenschaften, unterschiedliche Diversitäts- und Differenzierungsmuster aufweisen. Die Landnutzungssystem konnten in zwei Gruppen eingeteilt werden, die Baumdominierten Systeme mit hoher genetischer Diversität und die zwei Plantagensysteme mit niedriger genetischer Diversität. Die Analysen basierend auf den einzelnen häufigen Arten zeigen eine hohe Variabilität in der Artenreaktion auf die Landnutzungsveränderungen. Waldarten weisen unterschiedliche Verlustgrade von genetischer Diversität auf. Plantagen werden hauptsächlich von invasiven, kolonisierenden Arten dominiert, die an Störungen adaptiert sind. Daher zeigten die Plantagenplots im Mittel höhere genetische Diversitätslevel als erwartet.

The cacao swollen shoot disease of Theobroma cacao L. (cacao) is caused by Cacao swollen shoot virus (CSSV; genus, Badnavirus, family, Caulimoviridae). The virus is endemic to West Africa, where it poses a serious threat to cocoa production. Despite efforts to control CSSV spread by replacement of infected trees with tolerant cultivars and mealybug vector management, the disease is widespread in West Africa. In Trinidad, leaf mosaic and vein-banding symptoms have been observed in cacao plants in the field since the 1940s, and recently at the International Cocoa Genebank (ICGT), a custodian of cacao germplasm resources. The strains A and B of the suspect Cacao Trinidad virus (CTV) caused the symptoms, and were thought to be related to CSSV, however, viral causality was not demonstrated, until now. To develop molecular detection methods for CSSV in infected plants, polymerase chain reaction (PCR) amplification of eight regions of the CSSV genome was implemented. The PCR results showed variable amplification frequencies of 19 - 42% at each region, for 124 isolates collected in Cote d'Ivoire and Ghana. Pairwise nucleotide (nt) analyses of the eight regions showed 66-99% shared identities, indicating that CSSV isolates exhibit extensive variability with respect to primer design. The results provided preliminary evidence for the existence of a CSSV complex consisting of four divergent species. The full length genome of 14 CSSV isolates from cacao determined using the Illumina HiSeq platform showed 70-99% shared nt identities. The pairwise nt identities placed CSSV sequences into a group of four distinct species, one of which represented a previously undescribed species. Moreover, the full-length genomes grouped phylogenetically with other badnaviruses and revealed two CSSV subclades with three types of genome arrangements; four, five or six open reading frames (ORFs). Predicted functional protein domains were conserved on each ORF. Two distinct, full-length genome sequences were determined using the Illumina HiSeq platform, from DNA isolated from cacao leaves exhibiting distinct symptoms in Trinidad. The sequences were validated by PCR-amplification and sequencing of overlapping viral genome fragments. Pairwise nt analysis indicated that each genome shared 52-62% nt identities with CSSV and other badnaviruses, suggesting that the two are distinct species. Phylogenetic analysis indicated that the two sequences are not strains of the same virus, as supposed, but they represent two previously undescribed species in the genus, Badnavirus, and they have been named Cacao mild mosaic virus (CaMMV) and Cacao yellow-vein-banding virus (CYVBV). Despite sharing the same host and causing similar symptoms in cacao, CSSV, CaMMV, and CYVBV are phylogenetically-distinct species. The discovery of a CSSV species complex and the identification of three new cacao-infecting badnavirus species will support the development of molecular detection tools using the partial and complete genome sequences determined in this study. The ability to develop validated molecular tools for the detection of CSSV and related viruses, CaMMV and CYVBV, in cacao will aid quarantine efforts and safe movement of germplasm from the ICGT in Trinidad to cacao-growing countries, worldwide. Also, molecular diagnostics tools are expected to be useful in efforts underway to develop CSSV-resistant planting material for countries in West Africa, which are currently experiencing continued or new disease outbreaks.

The goals of captive breeding programs for endangered species include preserving genetic diversity and avoiding inbreeding. Typically this is accomplished by minimizing population mean kinship; however, this approach becomes less effective when errors in the pedigree exist and may result in inbreeding depression, or reduced survival. Here, both pedigree- and DNA-based methods were used to assess inbreeding depression in the critically endangered Attwater’s prairie-chicken (Tympanuchus cupido attwateri). Less variation in the pedigree-based inbreeding coefficients and parental relatedness values were observed compared to DNA-based measures suggesting that errors exist in the pedigree. Further, chicks identified with high parental DNA-based relatedness exhibited decreased survival at both 14- and 50-days post-hatch. A similar pattern was observed in later life stages (> 50 days post-hatch) with birds released to the wild; however, the pattern varied depending on the time post-release. While DNA-based inbreeding coefficient was positively correlated with mortality to one month post-release, an opposite pattern was observed at nine months suggesting purging of deleterious alleles. I also investigated whether immunocompetence, or the ability to produce a normal immune response, was correlated with survival; however, no significant correlation was observed suggesting that inbreeding was a more important factor influencing survival. Pairing individuals for breeding by minimizing DNA-based parental relatedness values resulted in a significant increase in chick survival. This study highlights the importance of using DNA-based methods to avoid inbreeding depression when errors exist in the pedigree.

The Red Sea region is one of the important places that allow us to uncover traces of the evolution of anatomically modern humans. Besides the questions related to its expansion out of Africa, this region is also important in terms of the mutual influence between populations of Africa and Arabia that after a long period of isolation and genetic differentiation related to climate change in the Pleistocene and subsequent development of seaways and land routes in the Holocene began to contact with each other more frequently. Number of genetic analyzes has been done but some issues concerning on the later development still remain inadequately answered, mainly because of insufficient material. This work is focused on the analysis of 200 mtDNA sequences of four Sudanese populations - two populations of nomadic herdsmen Rashaida and Beja living in the close neighborhood around the city of Kassala and speaking different languages and two populations of the Nile Valley with settled way of life. Analysis of the intrapopulation level revealed much higher diversity of the sedentary populations (in this work the sedentary populations are represented by the Nubians and Arabs). Interpopulation variability and genetic distances within other 46 populations of the Red Sea showed that although Rashaida and Beja people...

La diversité génétique est à l’origine de l'évolution et de l'adaptation des pop. et des espèces. Dans les agrosystèmes, la div. gén. intra-population est d'une importance majeure : d'une part, elle peut fournir un effet tampon contre les variations climatiquesinterannuelles et les stress biotiques, et d'autre part cette div. peut permettre l’adaptation locale des pop., du fait de leur évolution sous l’effet des pressions sélectives spécifiques aux conditions locales de la région, particulièrement dans le cas d’uneintrod. dans un nouvel environnement. En raison de son importance socio-économique et de son aire de culture étendue, le blé a été choisi comme espèce modèle dans cette étude, en se focalisant sur l’étude de la précocité de de floraison, un caractère adaptatif majeur qui permet au blé de croître sur une large gamme de conditions écologiques et climatiques. Ce projet de thèse a pour objet l’analyse de l'impact de la diversité int.-pop. sur la réponse adaptative à court terme de pop. soumises à des conditions agro-climatiques contrastées, ce par l'étude des variations génétiques, épigénétiques et phénotypiques.L'absence de marqueurs épig. disponible pendant la thèse a conduit à développer 2 études complémentaires. Dans une 1ère partie, sept var. pays. (pop. conservées à la ferme) et une variété moderne ont été distribuées et cultivées pendant trois ans dans sept fermes localisées dans trois pays d'Europe, puis étudiées pour leur réponse aux différentes conditions agro-climatiques, sous l’angle de leurs variations phénotypiques et génotypiques. Dans une 2nde partie, l'effet de la vernalisation sur le profil de méth. de l'ADN du gène VRN-A1 a été étudié, constituant une 1ère étape vers le développement de marqueurs épig. Les résultats de la 1ère partie de l'étude ont révélé que l'histoire de la conservation des var. pays. a fortement influencé leur div. gén. et leur structure génétique fine. Les var. pays. conservées ex situ montrent une faible div. gén., avec une struct. génétique simple. Les var. pays. et les mélanges conservés in situ révèlent une div. gén. plus élevée, avec une struct. génétique complexe. Une différenc. spatio-temporelle génétique et phénotypique a été observée, en relation avec le niveau de diversité initial et avec la complexité de structure des var. pays. Les variétés traditionnelles se différencient plus nettement que les var. modernes, ce qui plaide en faveur de utilisation dans des systèmes d'agriculture biologique et à bas intrants. De façon intéressante, une différenc. phénotypique significative a été observée pour les var. qui présentaient une div. gén. initiale très faible, ce qui suggère que d’autres facteurs, par exemple épig., pourraient intervenir dans les adaptations misesen évidence. La 2nde partie a permis de mettre en évidence un profil de méth. intéressant de l’ADN de VRN-A1 : sur plantes non-vernalisées, ce gène présente des niveaux élevés de méth. dans la partie centrale du gène, mais pas en début et fin de gène. De plus, une partie du 1er intron montre une augmentation significative du niveau de méth. de l'ADN suite au traitement au froid. Ce changement de méth. est positivement associé au niveau d'expression du gène. Si la compréhension du rôle de cette méth. sur la régulation de VRN-A1 nécessite des analyses complémentaires, cette étude a permis de caractériser les modifications de méth. de VRN-A1 en réponse au froid et constitue une 1ère étape vers l’identification de possibles epiallèles dans nos pop. et fournit une base à la construction de marqueurs permettant de suivre la variabilité épig. dans différentes pop. En conclusion, cette étude apporte des connaissances utiles pour une meilleure compréhension de l’origine et l'évolution de la div. gén. présente dans les var. pays. Elles permettront de dvper des méthodes de conservation et desélection à la ferme, en tenant compte de l'importance de la div. int.-pop., afin de rép. aux contraintes posées par l'agriculture bio. / Biodiversity provides the raw material for evolution and adaptation of populations and species. In agricultural biodiversity, the within-population genetic diversity is of major importance. On one hand, it can provide a buffering effect against the year-to-year variation of climate or biotic pressures and on the other hand diversity serves as a resource for the population to respond to selective pressures due to specific local conditions, thus allowing for local adaptation, particularly in the case where a population is introduced into a new location. Due to its wide geographic distribution indicating a high adaptiveotential and its socio-economic importance, wheat was chosen as model crop in this study. Flowering time is a major adaptive trait which has allows wheat to grow over a wide range of ecological and climatic conditions. This PhD study was designed to gain insights about the influence of within population diversity on the short term response of populations to contrasting agro-climatic conditions by studying the genetic, epigenetic and phenotypic variation. But due to the lack of prior existence of epigenetic markers, this thesis study is divided of two parts: In the first part, European wheat populations coming from a set of seven farmer and one modern varieties, each of which was grown on seven farms (distributed across Europe) for three years, were used to study their short term response to contrasting agro-climatic conditions in Europe by analysing their phenotypic and genotypic variations. For the second part the effect of vernalization on the DNA methylation profile of theVRN-A1 gene in winter wheat was studied as a first step towards the development for the epigenetic marker in this gene.The results from the first part of the study revealed that conservation history of these farmer varieties strongly influenced the genetic diversity and fine genetic structure. Ex situ conserved farmer varieties showed low genetic diversity and simpler structure whereas in situ conserved farmer varieties and mixtures revealed higher level of genetic diversity and complex genetic structure. Genetic and phenotypic spatio-temporal differentiation depending upon the level of diversity and structural complexity of the farmer variety was observed. The traditional varieties tend to become more differentiated than the modern variety arguing in favour of use of these diverse traditional (farmer) varieties in organic and low input agriculture systems. Interestingly, a significant phenotypic differentiation for varieties with very low genetic diversity has also been observed in this study, which gives indication of a possible role of epigenetic variation in the process of evolution.From the second part of the study (effect of vernalization on the DNA methylation profile of the VRN-A1 gene), it was found that in addition to the detection of gene body methylation across the VRN-A1 gene, we identified a region within intron 1 that shows significant increase in DNA methylation in response to vernalization treatment that is positively correlated with the gene expression. Although the role of this shift in gene regulation is still unclear due to time limitations in the thesis and the small number of genotypes analysed, this study will provide a good material towards future identification of new epialleles and the development of epigenetic markers to study the epigenetic variability of these populations.This study at large provides useful knowledge on the understanding of farmers' varieties evolutionary response to be used in the development of different breeding and conservation approaches for organic agriculture, taking into consideration of the importance of within population diversity, to satisfactorily address the problems of organic agriculture.

Orientador: Jeffrey Frederico Lui / Banca: Humberto Tonhati / Banca: Selma de Fátima Grossi / Resumo: A ocorrência de híbridos entre javalis e suínos, tanto na natureza como em cativeiro, é bastante comum. Assim, tem-se detectado polimorfismo em javalis, variando o número de cromossomos de 36 a 38. Nesse sentido, objetivou-se, com este trabalho, caracterizar geneticamente javalis (Sus scrofa scrofa) puros e híbridos criados no Brasil, por meio de loci de microssatélites (STRs) do suíno doméstico (Sus scrofa domestica). Para efeito de classificação, os animais foram agrupados, segundo análise de pedigree e número diplóide (2n), em 5 grupos genéticos: grupo I, constituído de 59 suínos domésticos; grupo II, formado por 46 javalis puros de origem; grupo III, constituído de 3 híbridos, com 2n=36, provenientes de acasalamentos entre híbridos e retrocruzamentos; grupo IV, representando 30 híbridos com suíno doméstico de ploidia igual a 37 cromossomos; e grupo V, constituídos de 10 híbridos também com o doméstico, porém com 2n=38, conhecidos popularmente como Javaporcos, devido à similaridade cariotípica e fenotípica com o suíno doméstico. O DNA genômico foi extraído e, posteriormente, amplificou-se, pela técnica de PCR, os fragmentos desses microssatélites - IGF1, ACTG2, TNFB -, os quais foram desenvolvidos para a subespécie Sus scrofa domestica. As condições de amplificação foram padronizadas para as amostras de javali realizadas em um termociclador, com as temperaturas de anelamento variando para cada primer. Ao final das amplificações, os produtos dos microssatélites foram colocados em um seqüenciador capilar modelo ABI 3100 Avant (Applied Biosystems). A partir dos resultados obtidos no presente trabalho, concluiu-se que os microssatélites IGF1, ACTG2 e TNFB, usados em suínos, são eficiente na amplificação heteróloga e podem ser aplicados em javali. Os javalis puros se diferenciam geneticamente dos suínos e dos híbridos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The occurrence of crossbred animals between wild boar and pigs, both in nature and in captivity, is quite common. Thus, polymorphism has been detected among wild boar, varying chromosomes from 36 to 38. Considering this, the objective of the present work was to perform a genetic characterization of wild boar and crossbred boars raised in Brazil, through. the microsatellites loci (STRs) of the domestic pig (Sus scrofa domestica). For classification purposes, the animals were grouped according to pedigree analysis and diploid number (2n) into 5 genetic groups: group I, composed of 59 domestic pigs with 2n = 38; group II, composed of 46 wild boar with 2n = 36 imported from France in 1997; group III, composed of 3 crossbred animals with 2n = 36 from the crossing between crossbred and backcrossing animals; group IV, composed of 30 crossbred animals with domestic pig with ploidy equal to 37 chromosomes and group V, composed of 10 crossbred animals also with domestic pig, but with 2n = 38, popularly known as "Boarpigs" due to their karyotypic and phenotypic similarity with the domestic pig. The genomic DNA was extracted and, after that, the fragments of these microsatellites - IGF1, ACTG2, TNFB - were amplified through the PCR technique, which were developed for the Sus scrofa domestica species. The amplification conditions were standardized for wild boar samples and performed in a thermocycler with the annealing temperatures varying for each primer. At the end of amplifications, the products of microsatellites were placed in a genetic analyzer model ABI 3100 Avant (Applied Biosystems). Considering the results of this research, the microsatellites IGF1, ACTG2 and TNFB used for pigs, were considered to be efficient on the heterologous amplifications and can also be applied on wild boar. The wild boar differs genetically from pigs and crossbreds... (Complete abstract, click electronic access below) / Mestre

Brettanomyces bruxellensis est une levure particulièrement redoutée des vinificateurs pour ses capacités d’altération organoleptique des vins. Elle est également associée à de nombreux produits fermentés et présente une importante diversité génétique en lien avec son origine écologique. L’analyse des profils microsatellites d’une collection importante d’individus (1318) d’origines géographiques variées montre une diversité génétique importante parmi les isolats de vin. Elle met notamment en évidence la coexistence d’individus diploïdes et triploïdes dans différentes régions du monde ainsi qu’à l’échelle d’un chai et d’un vin. La présence de certains génotypes dans plusieurs régions à travers le monde suggère la dispersion de cette espèce et une adaptation importante au milieu difficile qu’est le vin.La relation entre diversité génétique, matrice d’origine et traits physiologiques a été explorée. La nature des sucres utilisables pour supporter la croissance ainsi que les capacités de production de phénols volatils sont peu variables entre les souches étudiées, indépendamment de leur niveau de ploïdie ou de leur origine écologique. Néanmoins, les profils de croissance et de production de phénols volatils (vitesses et rendements) varient et traduisent des différences dans l’adaptation des souches au milieu et aux conditions d’oxygénation. Nos données suggèrent notamment une adaptation plus importante des souches triploïdes aux conditions physico-chimiques du vin. D’un point de vue pratique, l’influence de certains facteurs physico-chimiques, tels que les sucres et la température, sur le développement de B. bruxellensis dans les vins a été étudiée. Dans les vins rouges, la composition en sucres résiduels ne peut pas être considérée comme un outil de diagnostic du risque « Brett ». Néanmoins, les variations importantes de température observées dans les chais, jusqu’alors sous-estimées, pourraient expliquer en partie les phénomènes d’altération de vins rouges fréquemment observés au cours du premier été d’élevage en barrique. / The yeast species Brettanomyces bruxellensis is the most dreaded wine spoilage microorganism because of its repercussions on wine organoleptic wine alteration. It is also present in numerous fermented beverages and its high genetic diversity is partly associated with its ecological origin. Microsatellite analysis of a large collection of isolates (1318) from various geographical origins shows the species’ high genetic diversity, namely among wine strains. Notably, it highlights the coexistence of diploid and triploid individuals worldwide as well as at the region, winery and wine level. Isolation of some of the genotypes in several wine regions in the world suggests this species’ dispersion as well as the putative adaptation of these individuals to the harsh wine environment.The relationship between genetic diversity, matrix type, and physiological traits was further explored. The type of consumable sugars in relation to growth and phenol volatile production capacities of the studied strains, are independent from the ploidy level or ecological origin of the latter. Nevertheless, growth and phenol volatile production profiles (rates and yields) vary, highlighting differences in strains’ growth capacity in different media and aeration conditions. In particular, our data suggests an important adaptation of triploid strains to wine-type environment. From a practical point of view, influence of physicochemical parameters (such as sugars and temperature) on B. bruxellensis’ development in wine has been investigated. In red wine, residual sugar profiles don’t seem to be a relevant tool to estimate the risk associated with “Brett” spoilage. However, the important temperature variations occurring in wine cellars could be a possible explanation for contamination frequency during the first summer of barrel-ageing.