RNA sequencing (RNA-seq) technologies facilitate the characterisation of genes and transcripts in different cell types as well as their expression analysis across various conditions. Due to its ability to provide in-depth insights into transcription and post-transcription mechanisms, RNA-seq has been extensively used in functional genetics and transcriptomics, system biology and developmental biology in animals, plants, diseases, etc. The aim of this project is to use mathematical and computational models to integrate big genomic and transcriptomic data from high-throughput technologies in plant biology and develop new methods to identify which genes or transcripts have significant expression variation across experimental conditions of interest, then to interpret the regulatory causalities of these expression changes by distinguishing the effects from the transcription and alternative splicing. We performed a high resolution ultra-deep RNA-seq time-course experiment to study Arabidopsis in response to cold treatment where plants were grown at 20^oC and then the temperature was reduced to 4^oC. We have developed a high quality <i>Arabidopsis thaliana</i> Reference Transcript Dataset (AtRTD2) transcriptome for accurate transcript and gene quantification. This high quality time-series dataset was used as the benchmark for novel method development and downstream expression analysis. The main outcomes of this project include three parts. i) A pipeline for differential expression (DE) and differential alternative splicing (DAS) analysis at both gene and transcript levels. Firstly, we implemented data pre-processing to reduce the noise/low expression, batch effects and technical biases of read counts. Then we used the limma-voom pipeline to compare the expression at corresponding time-points of 4^oC to the time-points of 20^oC. We identified 8,949 genes with altered expression of which 2,442 showed significant DAS and 1,647 were only regulated by AS. Compared with current publications, 3,039 of these genes were novel cold-responsive genes. In addition, we identified 4,008 differential transcript usage (DTU) transcripts of which the expression changes were significantly different to their cognate DAS genes. ii) A TSIS R package for time-series transcript isoform switch (IS) analysis was developed. IS refers to the time-points when a pair of transcript isoforms from the same gene reverse their relative expression abundances. By using a five metric scheme to evaluate robustly the qualities of each switch point, we identified 892 significant ISs between the high abundance transcripts in the DAS genes and about 57% of these switches occurred very rapidly between 0-6h following transfer to 4^oC. iii) A RLowPC R package for co-expression network construction was generated. The RLowPC method uses a two-step approach to select the high-confidence edges first by reducing the search space by only picking the top ranked genes from an initial partial correlation analysis, and then computes the partial correlations in the confined search space by only removing the linear dependencies from the shared neighbours, largely ignoring the genes showing lower association. In future work, we will construct dynamic transcriptional and AS regulatory networks to interpret the causalities of DE and DAS. We will study the coupling and de-coupling of expression rhythmicity to the Arabidopsis circadian clock in response to cold. We will develop new methods to improve the statistical power of expression comparative analysis, such as by taking into account the missing values of expression and by distinguishing the technical and biological variabilities.

<p>Fungi display diverse growth forms. Some grow as unicellular yeasts, some grow as multicellular hyphae, while others switch between these two growth forms, i.e., the dimorphic fungi. Dimorphism is found in many pathogenic fungi, and it is thought to be a strategy to maximize their fitness during different stages of life cycles. The corn smut fungus <i>Ustilago maydis</i> serves as a renowned model organism for studying fungal dimorphism and its role in pathogenesis. However, knowledge only from the model species may not be expanded to other species unless multispecies studies have been demonstrated. In this dissertation, I performed comparative analyses to examine if knowledge from <i>U. maydis</i> is translational to other dimorphic fungi. First, a physiological study was conducted to find what can serve as a common signal for dimorphic transition of several Ustilaginomycotina species. I found that the lipid serves as a potential common cue for yeast-to-hyphal transition in most dimorphic species, while alternate types of energy-source carbohydrate do not affect fungal dimorphism. In addition, pectin and high temperature can also trigger filamentous growth in some Ustilaginomycotina species. Second, I performed comparative transcriptomics to determine if a mechanism for yeast-to-hyphal dimorphic transition is conserved across multiple dimorphic species. Three species of Ustilaginomycotina (<i>U. maydis</i>, <i>Tilletiopsis washingtonensis </i>and <i>Meira miltonrushii</i>) plus one species from Ascomycota (<i>Ophiostoma novo-ulmi</i>) were included in the analyses. I found that the similarity of transcriptomic alteration is not dependent on phylogenetic relatedness. Genes in amino acid transport and metabolism, energy production and conversion and cytoskeleton are commonly altered during the dimorphic transition of all studied species. Moreover, I discovered several core genes which can play a conserved role in transducing signals for the dimorphic transition. Finally, I performed comparative analyses of 190 fungal genomes to determine genomic properties that are associated with types of fungal growth form. I found that small genome size is a characteristic for yeast-like fungi. Few indicator genes, such as genes encoding proteins in the NADPH oxidase complex and cytoskeletons, which are predominantly lost in yeast-like fungi in both Ascomycota and Basidiomycota. However, many other genes are associated with types of growth form in a lineage-specific manner. Findings from this dissertation will serve as fundamentals for future research in fungal cell biology, especially in fungal dimorphism. Additionally, results from this study suggest cautions when extrapolating results from model species onto non-model species.</p>

Coregonines, including cisco (Coregonus artedi), kiyi (Coregonus kiyi), and bloater (Coregonus hoyi), are a focus for prey fish conservation and restoration efforts throughout the Laurentian Great Lakes. However, fundamental questions about coregonine ecology and genetics remain. For example, we know little about how the early life stages of coregonines respond to environmental change at either the genotypic or phenotypic level. We also have limited knowledge about how to identify different species at the larval stage and the genetic relationships among species, which makes the different species difficult to study at the larval stage. To increase the probability for success in restoration efforts, current and future research need to integrate traditional and novel approaches to better understand what leads to current and future coregonine successes. We used DNA and RNA omics tools, genomics and transcriptomics to boost our comprehension of current coregonine populations and to help understand how C. artedi may respond to environmental change. During the winter of 2017, we conducted a pilot experiment to evaluate how C. artedi eggs may respond to increased light exposure resulting from current and expected reductions in annual ice and snow cover due to global warming. We used transcriptomics to assess differences in gene expression between a continuous light and continuous dark treatment. Our results indicate that light is an environmental factor that could lead to earlier hatch dates, smaller yolk sacs, changes in mortality and differential gene expression in metabolic related and other functionally important genes. In 2018, we sampled larval coregonines in the Apostle Islands of Lake Superior each week from hatch in May until late July. We used genomic sequencing to genetically identify 197 larvae to species: C. artedi, C. hoyi, and C. kiyi. The larval demographic characteristics of each species was assessed and revealed that length ranges, growth rates, yolk sac condition, and effective population size varied among species. Larvae of all three species were found throughout the entirety of the Apostle Islands and the genetic diversity within each species appears high. The results from our pilot experiment and field observations help advance our understanding of the important early life stages of coregonines and how changes in light exposure or growth rates could affect their success or failure in a changing climate.

Listeria monocytogenes est un pathogène d'origine alimentaire à multiples facettes caractérisé par sa capacité d'adaptation dans des conditions défavorables et par sa prolifération dans une vaste gamme d'environnements, du sol aux cellules hôtes des mammifères. L'hétérogénéité génétique de L. monocytogenes se reflète dans sa structure clonale diversifiée, ce qui corrèle, dans une certaine mesure, avec des traits phénotypiques tels que la virulence ou la résistance au stress. La thèse portait sur deux phénotypes les plus éminents, la formation d'un biofilm et le potentiel de virulence, sous différents angles et à l'aide des technologies les plus récentes. Tout au long des études, des grands panels d'isolats ont été utilisés pour représenter la diversité intraspécifique. Stimulants défavorables tels que le choc froid et la privation d'éléments nutritifs induits par l'étape d'adhésion bactérienne. L'ajout de NaCl aux cultures de croissance a stimulé la production de biofilm et, de manière surprenante, il a considérablement intensifié la maturation du biofilm de cellules privées de nutriments. Un degré élevé de variation de la productivité relative du biofilm a été observé parmi les sérotypes, les génotypes, de même que les isolats selon les conditions de culture. Cependant, un certain génotype (complexe clonal 26) a révélé de manière caractéristique une production de biofilm plus élevée à froid (10°C), suggérant une association du génotype avec le phénotype du biofilm. Pan-GWAS a identifié un certain nombre de gènes parmi lesquels ceux impliqués dans des fonctions telles que la ‘transformation/compétence’, les ‘gènes liés aux phages’ et le ‘métabolisme du phosphate’ devront faire l'objet d'études plus approfondies sur leur rôle dans la formation du biofilm. L'analyse du séquençage de l'ARN a révélé une grande hétérogénéité intraspécifique dans les profils de transcriptome basal qui mettaient en évidence le rôle du réseau de régulation, y compris certains facteurs transcriptionnels avec des rôles clés dans la virulence tels que σB, PrfA, et CodY. La plasticité transcriptomique entre les lignées I et II ainsi que les génotypes hyper et hypovirulents ont confirmé les caractéristiques évolutives et épidémiologiques de L. monocytogenes. De plus, la voie métabolique centrale a été impliquée dans l'infection dans le système modèle de Galleria mellonella. En conclusion, la thèse a exploré la diversité intraspécifique de L. monocytogenes et a donné lieu à de nombreux résultats phénotypiques, génomiques et transcriptomiques. Grâce à l'approche intégrative des omiques en listeriologie, le présent travail contribuera à dévoiler la physiologie et la pathogenèse de la bactérie. / Conditions and proliferation in a wide range of environments from soil to mammalian host cells. The genetic heterogeneity in L. monocytogenes is reflected on its diversified clonal structure which correlates, to some extent, with phenotypic traits such as virulence or stress resistance. The thesis investigated two most prominent phenotypes, biofilm formation and virulence potential, from various perspectives using state-of-the art technologies. Throughout the studies, large panels of isolates were used to represent the intraspecific diversity. Unfavourable stimuli such as cold shock and nutrient deprivation induced bacterial adhesion step. Addition of NaCl to growth cultures stimulated biofilm production and, surprisingly, it significantly intensified biofilm maturation of nutrient-deprived cells. High degree of variation in relative biofilm productivity was observed among serotypes, genotypes, as well as isolates across culture conditions, however, certain genotype (clonal complex 26) revealed distinctively higher biofilm production under cold temperature (10°C) suggesting an association of genotype with biofilm phenotype. Pan-GWAS identified a number of genes among which those implicated in functions such as ‘transformation/competence’, ‘phage-related genes’, and ‘metabolism of phosphate’ will need further investigations for their roles in biofilm formation. RNA sequencing analysis revealed high intraspecific heterogeneity in basal transcriptome profiles that featured the role of regulatory network including certain transcriptional factors with key roles in virulence such as σB, PrfA, and CodY. The transcriptomic plasticity between lineage I and II as well as hyper- and hypovirulent genotypes supported the evolutionary and epidemiological characteristics of L. monocytogenes. Moreover, the central metabolic pathway was implicated in the infection in Galleria mellonella model system. Conclusively, the thesis explored intraspecific diversity in L. monocytogenes and resulted in ample phenotypic, genomic, and transcriptomic findings. With the integrative omics approach in listeriology, the present work will contribute to unveiling the physiology and pathogenesis of the bacterium.

<p>During photosynthesis sunlight is absorbed by photosynthetic pigments and converted into organic compounds, such as carbohydrates. Photosynthesis needs to be highly regulated, since both too much and too little light are harmful to plant. If too little light is absorbed, a plant cannot store enough energy, which will have effects on growth and fitness of the plant. With too much light absorbed, a dangerous side reaction of photosynthesis, the production of reactive oxygen species can happen. These reactive oxygen species can damage the proteins in the chloroplast and the lipids of the chloroplast.</p><p>To avoid the production of reactive oxygen species, plants have evolved many mechanisms, which act on different time-scales and different levels of organization. As a first measure, when the absorbed light is exceeding the capacity for its utilization, is to switch the light-harvesting antenna from efficient light harvesting to energy dissipation. This process is called feedback de-excitation (FDE). The protein PsbS is essential for this process as well as a functioning xanthophylls cycle with the enzyme violaxanthin de-epoxidase (VDE).</p><p>I have investigated the effects of plants with changes in their ability to dissipate excess excitation energy in the model plants species Arabidopsis thaliana. Three genotypes with either increased or decreased capacity for FDE were used during my experiments. The first genotype over-expresses the PsbS gene, having approximately two-fold increased amounts of PsbS and FDE. The second is a PsbS deletion mutant with no PsbS protein and no FDE. The third genotype cannot perform the conversion of violaxanthin to zeaxanthin, because the enzyme VDE is missing. This mutant has some FDE left. </p><p><i>Arabidopsis thaliana</i> is an annual plant, which flowers only once in its lifetime. Therefore, when counting the seeds produced an estimation of fitness can be made from the amount of seeds produced. This was done during my experiments and shown that FDE is a trait and that plants with increased FDE have a higher fitness and vice versa. </p><p>This was also the case for a collection of plants lacking a single protein from the light harvesting antenna. All of these genotypes had a fitness reduction, proving that their function is not redundant. </p><p>In an attempt to explain why the fitness is reduced in plants with altered FDE, photosynthetic measurements, as well as a determination of the transcriptome and the metabolome was performed. Plants lacking FDE had higher levels of photoinhibition, leading both to lower rates of photosynthesis and to higher repair cost. This could in part explain the reduction in fitness. These plants also had major changes in their transcriptome and their metabolome. Primary metabolism was most effected, for example carbohydrate and amino acid metabolism. But there were also changes in secondary metabolism such as an up regulation of the biosynthesis of anthocyanins.</p>

Die Mykorrhiza (griechisch: mýkēs für „Pilz”; rhiza für „Wurzel”) stellt eine Symbiose zwischen Pilzen und einem Großteil der Landpflanzen dar. Der Pilz verbessert durch die Symbiose die Versorgung der Pflanze mit Nährstoffen, während die Pflanze den Pilz mit Kohlenhydraten versorgt. Die arbuskuläre Mykorrhiza (AM) stellt dabei einen beson-dere Form der Mykorrhiza dar. Der AM-Pilz bildet dabei während der Symbiose die namensgebenden Arbuskeln innerhalb der Wurzelzellen als Ort des primären Nährstoff- austausches aus. Die AM-Symbiose (AMS) ist der Forschungsschwerpunkt dieser Arbeit. Als Modellorganismen wurden Medicago truncatula und Glomus intraradices verwendet. Es wurden Transkriptionsanalysen durchgeführt um u.a. AMS regulierte Transkriptions- faktoren (TFs) zu identifizieren. Die Aktivität der Promotoren von drei der so identifizier-ten AMS-regulierten TFs (MtOFTN, MtNTS, MtDES) wurde mit Hilfe eine Reportergens visualisiert. Der Bereich der größten Promotoraktivität waren in einem Fall nur die ar- buskelhaltigen Zellen (MtOFTN). Im zweiten Fall war der Promotor auch aktiv in nicht arbuskelhaltigen Zellen, jedoch am stärksten aktiv in den arbuskelhaltigen Zellen (MtNTS). Ein weiterer Promotor war in arbuskelhaltigen Zellen und den diesen benach-barten Zellen gleich aktiv (MtDES). Zusätzlich wurden weitere Gene als AMS-reguliert identifiziert und es wurde für drei dieser Gene (MtPPK, MtAmT, MtMDRL) ebenfalls eine Promotor::Reporter-Aktivitäts- studie durchgeführt. Die Promotoren der Kinase (MtPPK) und des Ammoniumtrans-porters (MtAmt) waren dabei ausschließlich in arbuskelhaltigen Zellen aktiv, während die Aktivität des ABC-Transporters (MtMDRL) keinem bestimmten Zelltyp zuzuordnen war. Für zwei weitere identifizierte Gene, ein Kupfertransporter (MtCoT) und ein Zucker- bzw. Inositoltransporter (MtSuT), wurden RNA-Interferenz (RNAi)-Untersuchungen durchgeführt. Dabei stellte sich in beiden Fällen heraus, dass, sobald ein RNAi-Effekt in den transformierten Wurzeln vorlag, diese in einem deutlich geringerem Ausmaß wie in der Wurzelkontrolle von G. intraradices kolonisiert worden sind. Im Falle von MtCoT könnte das aus dem selben Grund geschehen, wie im Falle von MtPt4. Welche Rolle MtSuT genau in der Ausbildung der AMS spielt und welche Rolle Inositol in der Aus- bildung der AMS spielt müsste durch weitere Untersuchungen am Protein untersucht werden. Weitere Untersuchen an den in dieser Arbeit als spezifisch für arbuskelhaltige Zellen gezeigten Genen MtAmT, MtPPK und MtOFTN könnten ebenfalls aufschlussreich für das weitere Verständnis der AMS sein. Dies trifft auch auf die TFs MtNTS und MtDES zu, die zwar nicht ausschließlich arbuskelspezifisch transkribiert werden, aber auch eine Rolle in der Regulation der AMS innerhalb von M. truncatula Wurzeln zu spielen scheinen. / The mycorrhiza (Greek: mýkēs for "mushroom"; rhiza for "root") is a symbiosis between fungi and the vast majority of land plants. The fungus improves the nutrient supply of the plant, while the plant provides the fungus with carbohydrates. The arbuscular my-corrhiza (AM) represents a special type of mycorrhiza. The AM forms during the sym-biosis eponymous arbuscules within the root cells as the supposed site of the major nu-trient exchange. The AM symbiosis (AMS) is the research focus of this work. Medicago truncatula and Glomus intraradices were used as model organisms. During the project several transcription analysis were performed to identify AMS re-gulated transcription factors (TFs). The activity of the promoters of three of the identified AMS regulated TFs (MtOFTN, MtNTS, MtDES) were visualised using a reporter gene. Cells with promoter activity were in one case the arbuscle containing cells (MtOFTN). In the another case, the promoter was also weakly active in non arbuscle containing cells, however the major site of activity were the arbuscle containing cells (MtNTS). Another promoter was active in arbuscle containing and adjacent cells (MtDES). In addition, other genes were identified as AMS regulated and for three of these genes (MtPPK, MtAmT, MtMDRL) a promoter::reporter activity study was conducted, too. The promoters of the kinase (MtPPK) and the ammonium transporter (MtAmT) were active exclusively in arbuscle containing cells, whereas the activity of the ABC-transporter (MtMDRL) could not be assigned to a specific cell type. For two other identified genes (a copper transporter (MtCoT) and a sugar/ inositol transporter (MtSuT)) RNA-interference (RNAi) studies were carried out. The studies revealed in both cases that, once an RNAi effect was present in the transformed roots, the roots were colonised by G. intraradices in a much lesser extent as in the vector-control. In the case of MtCoT it maybe has the same basic principle as in the case of the phosphate transporter MtPt4. Which role MtSuT and inositol plays during the fo-rmation of the AMS has to be reviewed. Further examinations on the genes MtAmT, MtPPK and MtOFTN could also be reveal-ing for the understanding of the AMS, as their promotors, as shown in this thesis, are exclusively active in arbuscle containing cells The same can be said for the TFs MtNFTS and MtDES. They are not exclusively transcripted in arbuscle containing cells, but nevertheless seem to play a role in the formation of the AMS within M. truncatula roots.

Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients).The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease.

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.

<p>The <italic>Polypodium vulgare<italic> complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, <italic>P. vulgare<italic> has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the <italic>P. vulgare<italic> complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the <italic>Polypodium vulgare<italic> complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns. </p><p>Chapter I presents a diploids-only phylogeny of the <italic>P. vulgare<italic> complex and related species to test previous hypotheses concerning relationships within <italic>Polypodium<italic> sensu stricto. Analyses of sequence data from four plastid loci (<italic>atpA<italic>, <italic>rbcL<italic>, <italic>matK<italic>, and <italic>trnG-trnR<italic>) recovered a monophyletic <italic>P. vulgare<italic> complex comprising four well-supported clades. The <italic>P. vulgare<italic> complex is resolved as sister to the Neotropical <italic>P. plesiosorum<italic> group and these, in turn, are sister to the Asian endemic <italic>Pleurosoriopsis makinoi<italic>. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the <italic>P. vulgare<italic> complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the <italic>P. vulgare<italic> complex. </p><p>Chapter II addresses reported discrepancies regarding the occurrence of <italic>Polypodium calirhiza<italic> in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent <italic>Pteridophytes of Mexico<italic>. Originally treated as a tetraploid cytotype of <italic>P. californicum<italic>, <italic>P. calirhiza<italic> now is hypothesized to have arisen through hybridization between <italic>P. glycyrrhiza<italic> and <italic>P. californicum<italic>. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from <italic>P. californicum<italic>. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes <italic>P. calirhiza<italic> from <italic>P. californicum<italic> and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where <italic>P. californicum<italic> is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and <italic>P. calirhiza<italic> grows at high elevations in central and southern Mexico. The occurrence of <italic>P. calirhiza<italic> in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.</p><p>Chapter III examines a case of reciprocal allopolyploid origins in the fern <italic>Polypodium hesperium<italic> and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in <italic>Polypodium hesperium<italic>, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of <italic>Polypodium hesperium<italic> are allopatric, with populations north and south of 42&#730; N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of <italic>P. hesperium<italic>. Several features of <italic>Polypodium hesperium<italic> make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages. </p><p>This dissertation concludes by demonstrating the utility of the allotetraploid <italic>Polypodium hesperium<italic> for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for <italic>Polypodium<italic>, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of <italic>P. hesperium<italic>. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from <italic>P. amorphum<italic> are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in <italic>P. hesperium<italic> supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.</p> / Dissertation