Browsing by Author "Sloan, Dan, committee member"
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Item Open Access Causes and consequences of plant climate adaptation(Colorado State University. Libraries, 2019) Monroe, John Grey, author; McKay, John, advisor; Ghalambor, Cameron, committee member; Hufbauer, Ruth, committee member; Sloan, Dan, committee member; Des Marais, Dave, committee memberClimatic conditions such as temperature and drought can sources of strong selection on natural populations. In plants, whose sessile nature forces them to adapt to local climate conditions, extensive evidence of local adaptation has been observed. However, the consequences of this adaptation on ecosystem processes such as carbon cycling remain poorly understood. Additionally, the molecular basis of adaptation is often unresolved and the specific climatic factors that drive adaptive evolution unclear. Addressing these knowledge gaps has become increasingly urgent as climate change threatens to rapidly alter selection regimes. Fortunately, conceptual and technical advances provide new opportunities to characterize and integrate environments, phenotypes, and genes, and thus advance our understanding of the causes and consequences of climate adaptation. In Chapter 2 of this dissertation, I consider the consequences of climate adaptation through the lens of ecoevolutionary dynamics. Integrating environments and phenotypes by considering ecosystem impacts of adaptive evolution, I review empirical evidence that contemporary climate adaptation could significantly alter the carbon cycle. In Chapter 3, I investigate the molecular basis of adaptation to winter temperatures in the model plant Arabidopsis thaliana by integrating genes and environments through the framework of landscape and population genetics. Specifically, I address the hypothesis that loss-of-function in a family of transcription factors contributes to adaptation to warmer climates. In Chapter 4, I develop methods combining whole genome sequence data, long term remote sensing, and reverse genetics to study drought as an agent of selection on flowering time and identify loss-of-function variants contributing to this evolution in Arabidopsis thaliana. Together, this work has inspired my interest in combining conceptual, computational, experimental innovations into an integrated research program to understand climate adaptation.Item Open Access Ectopic expression of R2R3-MYB transcription factors to control suberin biosynthesis(Colorado State University. Libraries, 2022) Berning, Nick, author; Medford, June, advisor; Peebles, Christie, committee member; Sloan, Dan, committee memberMinimizing the deleterious effects of abiotic stresses on cultivated plants is critical to maximizing crop yield. Suberin is a glycerol based polyester found in the endodermis, seed coat, cork cells, and areas of wounding in the epidermis. Recently, suberin biosynthesis has been shown to be at least partially regulated by a set of R2R3-MYB transcription factors. The ability to control suberin biosynthesis in specific plant tissues could be a valuable biotechnological tool in designing plants which can withstand higher degrees of abiotic stress. In this thesis, I detail a genetic screen of four different R2R3-MYB transcription factor's ability to induce ectopic suberin formation in the root epidermis of Arabidopsis thaliana. Subsequently, I characterize the transcription factor with the greatest ability to induce ectopic suberin biosynthesis, MYB92. MYB92, when expressed in the root epidermis, consistently forms a suberin barrier within that tissue. Plants expressing MYB92 in the root epidermis may be stunted and chlorotic under typical growth conditions, however, they outperform wild-type Col-0 plants under salt stress. More characterization of ectopic, suberin barrier's ability to confer salt tolerance could be performed in order to understand how epidermal suberin might perform in crop plants.Item Open Access Expanding on expansion: genome gigantism and its effects on DNA methylation, RNA splicing and organellar scaling(Colorado State University. Libraries, 2023) Adams, Alexander Nichols, author; Mueller, Rachel, advisor; Hanson, Jeffrey, committee member; Hoke, Kim, committee member; Sloan, Dan, committee memberAcross the tree of life, the correlated traits of genome size and cell size both vary by orders of magnitude, with the increase in genome size being largely attributable to an increase in transposable elements (TEs) throughout the genome. This accumulation of TEs affects many facets of the cell including DNA regulation, organellar scaling, and RNA transcription. This dissertation will explore all 3 of these facets through the lens of genome gigantism and address how these facets differ in large cells in comparison to cells that are more typical in size. The first chapter will discuss methylation of cytosines at genomic CpG dinucleotide sites that silence TEs. TE abundance drives differences in genome size, but TE silencing variation across genomes of different sizes remains largely unexplored. Salamanders include most of the largest C-values — 9 to 120 Gb. We measured CpG methylation levels in salamanders with genomes ranging from 2N = ~58 Gb to 4N = ~116 Gb. We compared these levels to results from endo- and ectothermic vertebrates with more typical genomes. Salamander methylation levels are ~90%, higher than all endotherms. However, salamander methylation does not differ from the other ectotherms, despite a ~100-fold difference in nuclear DNA content. Because methylation affects the nucleotide compositional landscape through 5-methylcytosine deamination to thymine, we quantified salamander CpG dinucleotide levels and compared them to other vertebrates. Salamanders have comparable CpG levels to other ectotherms, and ectotherm levels are higher than endotherms. These data show no shift in global methylation at the base of salamanders, despite a dramatic increase in TE load and genome size. This result is reconcilable with previous studies by considering endothermy and ectothermy, which may be more important drivers of methylation in vertebrates than genome size. The next chapter will look at how an increase in cell size affects organellar structure and abundance. Depending on their shape, organelles can scale in larger cells by increasing volume, length, or number. Scaling may also reflect demands placed on organelles by increased cell size. The 8,653 species of amphibians exhibit diverse cell sizes, providing a powerful system to investigate organellar scaling. Using transmission electron microscopy and stereology, we analyzed three frog and salamander species whose enterocyte cell volumes range from 228 to 10,593 μm3. We show that the nucleus increases in radius (i.e. spherical volume) while the mitochondria increase in total network length; the endoplasmic reticulum and Golgi apparatus, with their complex shapes, are intermediate. Notably, all four organelles increase in volume proportionate to cell volume. This pattern suggests that protein concentrations are the same across amphibian species that differ 50-fold in cell size, and that organellar building blocks are incorporated into more or larger organelles following the same "rules" across cell sizes, despite variation in metabolic and transport demands. This conclusion contradicts results from experimental cell size increases, which produce severe proteome dilution. We hypothesize that salamanders have evolved the biosynthetic capacity to maintain a functional proteome despite a huge cell volume. Finally, the last chapter will be discussing differences in intronic splicing, an important step that pre-mRNA transcripts undergo during processing in the nucleus to become mature mRNAs. Although long thought to occur exclusively in a single step, some introns are now also known to be removed in multiple steps through a process called recursive splicing. This non-canonical form of splicing is hypothesized to aid with intron splicing fidelity, particularly in longer introns. Using West African lungfish (Protopterus annectens; genome size ~40Gb) as a model, we use total RNA-seq data to test the hypothesis that gigantic genomes, which have relatively long introns, have increased levels of recursive splicing compared to genomes of more typical size. Our results reveal levels of recursive splicing at conserved sites similar to those seen in humans, suggesting that genome-wide intronic expansion accompanying evolutionary increase in genome size is not associated with the evolution of high levels of recursive splicing. However, in addition to these results, we also observed patterns of decreasing RNA-seq read depths across entire intron lengths and note that both canonical co-transcriptional splicing and stochastic recursive splicing using many random splice sites could produce this pattern. Thus, we infer canonical co-transcriptional splicing and/or stochastic recursive splicing — but not widespread recursive splicing at conserved sites — manage the removal of long introns.Item Open Access Extrinsic incubation temperature impacts on Zika virus evolution and vector competence during systemic Aedes infection(Colorado State University. Libraries, 2020) Murrieta, Reyes David A., author; Ebel, Gregory, advisor; Olson, Kenneth, committee member; Stenglein, Mark, committee member; Sloan, Dan, committee memberArthropod-borne viruses (arboviruses) are distinctive in that they are required to constantly replicate in different hosts and in a wide range of temperatures for their perpetuation in nature. Vertebrate hosts tend to maintain temperatures of approximately 37°C - 40°C, but arthropods hosts are poikilotherms and subject to ambient temperatures which can have a daily temperature fluctuation of > 10°C. Invertebrate host genus, species, and strain in combination with arbovirus strain and preparation methods are known to have large impacts on vector competence and vectorial capacity. Seemingly small differences in host geographic isolation, virus strain, and preparation methods can have significant impacts on vector competence studies. The role of temperature on the ability of an arthropod vector to acquire, maintain, and transmit a pathogen has been investigated for numerous arboviruses. Changing the extrinsic incubation temperature between distinct constant temperatures has been shown to alter arbovirus vector competence, extrinsic incubation period, and mosquito survival, in which moderate temperatures of 28°C-32°C are optimal and temperatures higher and lower have deleterious effects. The mean and range of daily temperature fluctuations (diurnal temperature) have likewise been shown to influence arbovirus perpetuation and vector competence, in which large daily temperature fluctuations negatively affect mosquito development, survival, and vector competence. However, little is known as to how temperature alters arbovirus genetic diversity during systemic mosquito infection or how differences in arbovirus hosts and viral strains impact arbovirus genetic diversity in relationship to temperature. Therefore in the study completed in chapter two, we characterized the impact that constant temperatures of 25°C, 28°C, 32°C, and 35°C, and the diurnal fluctuation from 25°C to 35°C during extrinsic incubation periods have on the Puerto Rican isolate of Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti (Poza Rica) and Aedes albopictus (Florida) mosquitoes. To characterize the impact that temperature has on ZIKV population diversity in different host species and viral isolates, in the study completed in chapter three, we used a Tapachula, Mexico Aedes aegypti line and a Chiapas, Mexico ZIKV isolate to assess ZIKV population dynamics during 20°C, 24°C, 28°C, 32°C, 34°C, and 36°C constant extrinsic incubation temperatures. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species, while transmission peaked at 10 days post-infection for Aedes aegypti and 14 days post-infection in Aedes albopictus. The diurnal temperature group is not predicted by the constant temperature distribution. Instead, when using the mean daily temperature of the diurnal group as a predicter, its VC lies between the moderate (28°C and 32°C) and extreme (25°C and 35°C) temperature group VCs. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the ZIKV selective environment during infection. During mosquito infection, constant temperatures more often elicited positive selection whereas diurnal temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology within mosquitoes and has distinct effects on the selective environment within mosquitoes. Additionally, the selective pressures induced by temperature are consistent across host species and viral strain and have similar impacts on shaping the viral population structure. However, input viral populations are still a driving factor of diversity and expansion during systemic mosquito infection. While our findings and those of others suggest that vector competence is impacted unimodally regardless of temperature, this is only applicable for constant temperatures. Future work assessing daily temperature fluctuation range and mean are needed to have a clear understanding of the role extrinsic incubation temperature plays on vector competence.Item Open Access Genetic drift and mutational hazard in the evolution of salamander genomic gigantism(Colorado State University. Libraries, 2016) Mohlhenrich, Erik, author; Mueller, Rachel, advisor; Sloan, Dan, committee member; Black, William, committee memberSalamanders have the largest nuclear genome sizes among tetrapods and, with the exception of lungfishes, among vertebrates as a whole. Lynch and Conery (2003) have proposed the mutational hazard hypothesis to explain variation in genome size and complexity. Under this hypothesis, non-coding DNA imposes a selective cost by increasing the target for degenerative mutations, i.e. the mutational hazard. Expansion of non-coding DNA, and thus genome size, is expected to be driven by increased levels of genetic drift and/or decreased mutation rates; the former determines the efficiency with which excess non-coding DNA can be selected against, while the latter determines the level of mutational hazard. Here, we test the hypothesis that salamanders have experienced stronger long-term, persistent genetic drift than frogs, a clade with more typically sized vertebrate genomes. To test this hypothesis, we compared dN/dS and Kr/Kc values between these clades. Our results reject this hypothesis; we find that salamanders have not experienced stronger genetic drift than frogs. Additionally, we find evidence consistent with a lower nucleotide substitution rate in salamanders. This result, along with previous work showing lower rates of small deletions and ectopic recombination in salamanders, suggests that a lower mutational hazard may contribute to genome expansion in this clade. Taken together, these results further underscore the importance of studying large genomes and indicate that salamanders provide an important model system for the study of how non-drift processes (i.e. mutation, natural selection) shape the evolution of genome size.Item Open Access HopBA1, a pathogen virulence factor, reveals tissue-specific immune responses within the Pseudomonas syringae pv. tomato–Nicotiana benthamiana pathosystem(Colorado State University. Libraries, 2024) Todd, Tyler Scott, author; Nishimura, Marc, advisor; Sloan, Dan, committee member; Roberts, Robyn, committee memberPlant pathogens represent a major threat to food security as they dramatically reduce crop yield, impact the expression of desirable traits, and reduce post-harvest longevity. To infect host plants, bacteria like Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) utilize a type III secretion system to deliver virulence proteins (also known as effectors) into the host cytoplasm to suppress immunity. In response, plants have evolved intracellular immune receptors that perceive immunosuppression and reactivate immunity. Thus, a given pathogen effector can both suppress and activate immunity depending on the host genome. Dissecting the molecular mechanisms of plant pathogen effectors helps inform our understanding of both disease and resistance. The present work reveals an uncharacterized role for Pst DC3000 as an aggressive vascular pathogen, causing systemic infection in the model plant Nicotiana benthamiana (Nb). Further, it establishes that the bacterial effector HopBA1 inhibits movement through the vascular system, despite increasing pathogen persistence within the primary infection site in leaves. Simultaneously, HopBA1 was found to induce irreversible upward vertical bending (i.e., hyponasty) in the petioles of infiltrated leaves, a novel phenotype for a bacterial effector. LC-MS/MS and RNA-Seq revealed phytohormone alteration (notably, a reduction in auxin and jasmonic acid-related metabolites) and transcriptional reprogramming of both developmental and defense genes. HopBA1-dependent growth restriction was suppressed in Nb eds1 (ENHANCED DISEASE SUSCEPTIBILITY 1) mutant plants, which still undergo HopBA1-induced hyponasty. Together, these results suggest that (1) HopBA1 triggers tissue-specific immune responses and (2) hyponasty is due to HopBA1's virulence activity, rather than host immune activation. Thus, HopBA1 in combination with the model pathogen Pst DC3000 becomes an important tool to dissect the poorly understood area of vascular-specific immunity. Vascular pathogens are particularly devastating and difficult to manage in crop species owing to the pathogen's internal location and systemic route of infection, making this research useful in crop improvement.Item Open Access Influence of management practices on virulence factors, antimicrobial resistance genes and heavy metal resistance genes in broiler chicken production(Colorado State University. Libraries, 2023) Woyda, Reed Richard, author; Abdo, Zaid, advisor; Oladeinde, Adelumola, committee member; Daniels, Josh, committee member; Sloan, Dan, committee member; Stenglein, Mark, committee memberThe main bacterial species associated with food-borne illness in humans are Escherichia coli, Salmonella species and Campylobacter species. The ability of a bacterial strain to survive the food-production pipeline and to mount an infection and cause disease in humans is dependent on an array of genetic factors. The presence of specific virulence factors will influence the severity of disease while antimicrobial resistance genes affect the choice and efficacy of treatment. Management practices in poultry production aim at reducing the incidence of poultry and human bacterial pathogens and, in general, at maintaining a healthy flock and a healthy global population. However, the influence of management practices, in a post-antibiotic era, on pathogenic bacterial species, and in particular the selective pressures imposed on genetic factors such as antimicrobial and metal resistance and virulence factors, are understudied. In Chapter 2, we provide a robust bacterial genomic analysis pipeline which is used for subsequent analysis in the following chapters. Chapter 3 provides an understanding of the current antimicrobial resistance and virulence factors present in chicken production and human clinical settings. This work found these host sources harbored different antimicrobial resistance genes and virulence factors that can classify them into phylogroups and host origin. In Chapter 4, through characterization of Campylobacter species isolated from broiler litter, we determined the reused litter environment selected for Campylobacter species lacking virulence factors aiding in colonization of chicken and human hosts. In Chapter 5, we determined the practice of adding copper sulfate to drinking water, commonly used for growth promotion or sanitization, may have selected for, and provided a reservoir for, Salmonella strains harboring plasmid-borne copper resistance genes. Overall, this work provides a computational pipeline for the high-throughput analysis of bacterial genomes and provides insights into selective pressures imposed on pathogenic bacterial species by modern-day management practices.Item Open Access Mating system transitions impact population structure and biodiversity in Solanum habrochaites(Colorado State University. Libraries, 2018) Miller, Chris, author; Bedinger, Patricia, advisor; Ward, Sarah, committee member; Richards, Chris, committee member; Sloan, Dan, committee memberAlthough many plant species have evolved means of preventing self-fertilization, self-compatibility (SC), the ability to set self-seed using pollen and ovules of the same plant is exceedingly common. In the wild tomatoes (Solanum section Lycopersicon) and other Solanaceous species, plants have evolved a genetic mechanism for preventing self-fertilization. Although many species are entirely either SC or self-incompatible (SI, unable to self-fertilize), one species of wild tomato, Solanum habrochaites, is notable for having separate SI and SC populations. In Chapter 1 of this thesis, I introduce mating system and self-incompatibility in Solanum habrochaites. Briefly, Solanaceous plants exhibit a specific type of gametophytic self-incompatibility controlled by cytotoxic, stylar-expressed S-RNases (and other factors) and pollen-expressed male resistance factors. At the northern species margin in southern and central Ecuador, S. habrochaites has undergone as least one SI → SC mating system transition. The loss of SI in this region coincides with a unique geographical feature, the Amotape Huancabamba Depression. In Chapter 2, I explore the loss of SI at the northern species margin using population genetics and reproductive biology. By analyzing the population structure of these populations in combination with controlled crosses, protein expression, and S-RNase allele screening, I identified at least four SC groups resulting from independent transitions from SI→SC. I also identified a fifth SC group of populations which likely arose due to the interbreeding of two separately derived SC populations. Stylar S-RNase protein expression can also be detected in this region, suggesting previously inactivated S-RNase genes in the parental groups may have become reactivated upon hybridization. In Chapter 3, I present the analysis of reproductive characters and morphology of newly collected populations of S. habrochaites. In one SC group, I find evidence of the "selfing syndrome," a phenomenon in which SC populations are predicted to possess small flowers and unexserted stigmas compared to their SI counterparts. This syndrome was not detected in the other SC populations, however. In Chapter 4, I describe two new "selfing" S-RNase alleles (hab-7, and hab-8) in different SC groups using degenerate primers and RNA-seq. hab-7 likely could encode a functional S-RNase protein, but it is likely unable to function in the SI response due to very low gene expression. The other allele, hab-8, detected in a different SC group, cannot express a functional S-RNase protein due to a single nucleotide substitution that produces a premature stop codon. Finally, in Chapter 5, I summarize my conclusions which support multiple SI→SC mating system transitions at the northern margin of S. habrochaites and review evidence that two distinct SC populations have interbred. I also suggest that some S-RNase alleles can be reversibly silenced and reactivated.Item Open Access Natural cases of salamander hybridization suggest a consistent relationship between genetic distance and reproductive isolation across tetrapods(Colorado State University. Libraries, 2019) Melander, Scott, author; Mueller, Rachel, advisor; Sloan, Dan, committee member; Ebel, Greg, committee memberHybridization between populations along the path to complete reproductive isolation can provide snapshots of speciation in action. Here, we present the first comprehensive list of natural salamander hybrids and estimate genetic distances between the parental hybridizing species using a mitochondrial and nuclear gene (MT-CYB and RAG1). Salamanders are outliers among tetrapod vertebrates in having low metabolic rates and highly variable sex chromosomes. Both of these features might be expected to impact speciation; mismatches between the mitochondrial and nuclear genomes that encode the proteins for oxidative metabolism, as well as mismatches in heteromorphic sex chromosomes, can lead to reproductive isolation. We compared the genetic distances between hybridizing parental species across four main tetrapod clades that differ in metabolic rates and sex chromosome diversity: salamanders, lizards, mammals, and birds. Our results reveal no significant differences, suggesting that variation in these traits across vertebrates does not translate into predictable patterns of genetic divergence and incompatible loci in hybrids.Item Open Access Phenotype to genotype and back in emerging and established crop species(Colorado State University. Libraries, 2023) Woods, Patrick O'Neal David, author; McKay, John, advisor; Hufbauer, Ruth, committee member; Funk, Chris, committee member; Sloan, Dan, committee memberUnderstanding the relationship between the phenotype and genotype is a fundamental goal of genetics. Through the years, two primary approaches have been developed for studying the phenotype-genotype relationship: forward genetic and reverse genetics. Forward genetics enables the potential discovery of numerous candidate genes controlling a phenotype while reverse genetics allows for the mechanistic validation of a single gene's role in controlling a phenotype. Applying these two approaches to crops enables the discovery of genetic targets that can be used for crop improvement through breeding. In this dissertation, I focused on understanding the phenotype-genotype relationship in both the emerging crop Cannabis sativa and the established crop Maize. In Chapter 1, I used both a forward a reverse genetics approach to identify and validate candidate genes controlling agriculturally important traits (agronomic and biochemical) in Cannabis sativa. In Chapter 2, I used a reverse population genetics approach to identify the genetics underlying local adaptation in feral and domesticated populations of Cannabis sativa. In Chapter 3, I used a forward genetics approach to identify candidate genes controlling variation in root system architecture in Maize. Collectively, this work demonstrates how modern genomic techniques can be applied to both new and old crop systems to identify genetic targets for use in crop innovation through breeding.Item Embargo SARS-CoV-2 evolution and within-host variation in nonhuman animals(Colorado State University. Libraries, 2024) Bashor, Laura, author; VandeWoude, Sue, advisor; Stenglein, Mark, committee member; Bosco-Lauth, Angela, committee member; Sloan, Dan, committee member; Gagne, Roderick B., committee memberThe COVID-19 pandemic originated following spillover of SARS-CoV-2 from non-human animals into humans. Despite concentrated efforts before and after the pandemic, current research is constrained by the impracticality of witnessing initial host shift events and transmission dynamics that shape infectious disease emergence. SARS-CoV-2 transmission from humans to a range of domestic and wild species has been well documented; furthermore, spillback into humans from white-tailed deer, mink, hamsters, domestic cats, and lions has also been reported. SARS-CoV-2, like other RNA viruses, has the ability to adapt rapidly following host shifts. These cross-species transmission events can accelerate novel variant emergence through selection for genetic variation that improves virus fitness in a novel host environment. To evaluate the possibility that cross-species transmission accelerates SARS-CoV-2 evolution and variant emergence, we employed next-generation sequencing of viral genomes recovered from experimentally and naturally infected animals to characterize within-host virus populations. We demonstrated the use of experimental exposure studies as a controlled system to test hypotheses surrounding SARS-CoV-2 adaptation in cats (Felis catus), dogs (Canis lupus familiaris), hamsters (Mesocricetus auratus), ferrets (Mustela putorius furo), deer mice (Peromyscus maniculatus), bushy-tailed woodrats (Neotoma cinerea), Brazilian free-tailed bats (Tadarida brasiliensis), striped skunks (Mephitis mephitis), red foxes (Vulpes vulpes) and mule deer (Odocoileus hemionus). We also evaluated publicly available sequencing data from infected felids, and investigated within-host dynamics in natural infections of Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) in a zoo environment. Our initial work investigated SARS-CoV-2 evolution across three passages in Vero cells and experimentally infected cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1). We observed the rapid selection and fixation of five SARS-CoV-2 mutations in Vero cells, followed by their reversion in dogs, cats and hamsters 1-3 days post-infection. We noted 14 emergent variants across the SARS-CoV-2 genome, including increased variation in the SARS-CoV-2 spike protein. Emergent variants included mutations not detected in the original virus stocks used for inoculation, and several defining mutations of variant lineages of concern in humans. Finally, we noted increased signs of adaptation in dogs, which did not shed infectious virus, including six nonsynonymous mutations in the SARS-CoV-2 open-reading frames (ORFs) encoding proteins for virus replication. In particular, this work underscored the potential for accelerated viral evolution in cell culture systems used commonly in virological research. This work has been published and represents Chapter 2 of this dissertation. Our next study built upon this work by investigating SARS-CoV-2 evolution in three experimental cohorts of domestic cats (n=23) infected through direct inoculation and cat-to-cat contact transmission. We observed high numbers of within-host variants in SARS-CoV-2 genomes recovered from cats compared to what is documented in humans, over half of which were nonsynonymous changes. The number of variants detected was positively correlated with the experimental dose of virus inoculum, and fewer variants were observed in contact cats. Similar to the previous study, mutations occurring at the same positions as defining VOC mutations, and signatures of positive selection in the viral spike (S) gene were observed. Our concurrent analysis of publicly available SARS-CoV-2 sequences showed no evidence for independent evolutionary trajectories associated with natural infections of domestic cats or other felids, and confirmed susceptibility of felids to the breadth of variants circulating in human populations. This work has also been published and represents Chapter 3 of this dissertation. We subsequently investigated SARS-CoV-2 evolution in longitudinal samples collected from Amur tigers (n=2), African lions (n=11), and spotted hyenas (n=4) infected during an outbreak at the Denver Zoo. Longitudinal nasal swabs were collected from infected individuals over an approximately three-month sampling period. We determined that the outbreak was caused by a single introduction of the Delta sublineage AY.20, which was a rare variant circulating in human populations at the time. We inferred a transmission chain from tigers to lions to hyenas, which was consistent with the appearance of clinical signs in infected animals. We observed expansion and diversification of within-host virus populations, and signatures of both purifying and positive selection. The strongest signs of positive selection were evident in the viral nucleocapsid (N) gene, and in viruses recovered from hyenas. Four candidate species-specific adaptive mutations, two of which are in the N gene, were identified in lions and hyenas (N A254V) and hyenas alone (ORF1ab E1724D, S T274I, and N P326). This work is presented in Chapter 4 of this dissertation. In Chapter 5, we evaluated a large dataset of peridomestic wildlife species experimentally infected with two SARS-CoV-2 variants, WA01 and Delta. Study species included deer mice (n=3), bushy-tailed woodrats (n=3), Brazilian free-tailed bats (n=4), striped skunks (n=5), red foxes (n=9), and mule deer (n=6). Distinct dynamics were observed in within-host virus populations recovered from WA01- and Delta- infected animals. This included increased within-host variation, relative effective population size, and genomic signatures of positive selection in WA01 animals. In contrast to our first study in domestic dogs, Brazilian free-tailed bats, which also did not shed infectious virus, did not show increased signs of adaptation. We also observed a potential host barrier to infection in skunks and one fox, followed by the emergence of potential de novo mutations. Six novel mutations were also detected in contact-exposed mule deer. Our findings suggest that mule deer populations, similar to what has been documented in closely related white-tailed deer, should be investigated for accelerated SARS-CoV-2 evolution. Collectively, our work reveals the unique dynamics of SARS-CoV-2 evolution and transmission in both naturally- and experimentally- infected felids. We observed rapid viral adaptation both in vitro and in vivo, highlighting advantages and limitations of experimental animal infections for studies of viral evolution. In each study, we used publicly available data to contextualize our experimental data and identify broader patterns. Furthermore, we identified specific SARS-CoV-2 mutations and genomic regions under selective pressures across a range of animal species, setting the groundwork for future mechanistic studies. Our findings underscore the importance of a One Health approach to understanding SARS-CoV-2 evolution, and the need for surveillance in animal populations.Item Open Access The evolution of plasticity in the transcriptome of the Trinidadian guppy(Colorado State University. Libraries, 2023) Whedbee, Miles, author; Hoke, Kim, advisor; Sloan, Dan, committee member; Montgomery, Taiowa, committee member; Ben-Hur, Asa, committee memberPhenotypic plasticity is a ubiquitous feature of all living systems, and there is much interest in how plasticity influences long term evolutionary trajectories. One of the major complications with modeling evolutionary trajectories is that plasticity itself is known to evolve. The evolution of plasticity has mainly been focused on at the level of the whole organism, and it is unclear if plasticity at all levels of biological organization evolve. Models that assume no generational change in plasticity may be overly simplistic; a more nuanced approach could incorporate the evolution of plasticity into the modeling. A first step towards this end is to determine what levels of biological organization plasticity evolves, and then to determine if there are predictable patterns of evolved plasticity. RNA is an intermediate to DNA and protein, that can undergo changes in response to environmental conditions, thereby modifying the genetic information passed on to non-coding RNAs, functional RNAs, and proteins. Responses to environment include both changes in abundance of RNAs, as well as changes to the composition of the molecules. This dissertation focuses on the evolution of plasticity within the transcriptome of Poecilia reticulata (Trinidadian guppy). One of the major known regulators of transcript abundance are small RNAs (sRNAs). Micro RNAs (miRNAs), are a specific type of sRNA that bind transcripts, typically leading to translational silencing. We investigated two forms of plasticity, an abundance measure of plasticity (miRNA differential expression), and a compositional measure of plasticity (A-to-I RNA editing). A-to-I RNA editing is the chemical nucleotide change from adenosine to inosine, catalyzed by the enzyme ADAR. We first produced a set of miRNAs in guppies, and confirmed the presence of key biogenesis pathway components, i.e. argonaute proteins in the genome. Tissue-specific miRNA expression patterns were identified for three tissues in Poecilia reticulata (Trinidadian guppy), brain, ovary and testis. We found most discovered miRNAs were located in intergenic regions of the genome. Some miRNAs matched known miRBase sequences, while others were considered novel guppy miRNAs. We observed miRNAs expressed from tandem clusters and analyzed piRNA distribution in ovary samples. This study provides important insights into guppy small RNA expression, laying the groundwork for future investigations into their regulatory roles. The 3rd chapter of this dissertation revealed many miRNAs with differential expression (DE), including population main effects, rearing condition, and their interactions. Population DE miRNAs showed a wide range of expression levels. Rearing condition main effects were (slightly) less common. We identified miRNAs with evolved expression plasticity, distributed across four categories: reversed, evolved plastic, assimilated, and accommodated. Both populations showed similar numbers of miRNAs exhibiting plasticity. In the final chapter of this dissertation we characterized the "editome" of guppies. The majority of the edits were consistent with A-to-I editing, with a smaller proportion of C-to-U edits. The intragenic edits were distributed among a number of genes. However, there were no significant differences in editing between populations, rearing conditions, or their interaction. This dissertation revealed significant miRNA expression differences and provided insights into A-to-I editing patterns in guppies.Item Open Access Time series analysis of limber pine (Pinus flexilis) health in the U.S. Rocky Mountains in response to white pine blister rust (Cronartium ribicola) and bark beetles(Colorado State University. Libraries, 2018) Leddy, K. A., author; Stewart, Jane E., advisor; Abdo, Zaid, committee member; Sloan, Dan, committee member; Schoettle, Anna, committee member; Liber, Howard, committee memberFrom 2004-2007, 106 permanent limber pine monitoring plots were established and measured throughout the U.S. Rocky Mountains (MT, WY, CO) to characterize health trends in response to white pine blister rust (WPBR) and bark beetles (including mountain pine beetle, "MPB", and Ips spp., "Ips") over time. These plots were subsequently measured in 2011-2013 and again in 2016-17 to form a time series analysis of limber pine health. Data were gathered on 8,206 monumented trees (4,176 limber pine) and included measurements on various stand, ground cover, and landscape characteristics over the three time intervals. The overall percentage of live trees infected with WPBR was 29.4% in 2004-07 and 25.7% in 2016-17, with incidence decreasing in parts of Wyoming (Pole Mountain, Laramie Peak), increasing in southern Colorado (Sangre de Cristo Mountains), and stable in other subregions. However, of limber pines that were healthy during the first measurement, 22.2% were declining/dying and 21.1% had died by the end of the study period due to WPBR and/or bark beetle damages. Due to this, it is likely that new WPBR infections are occurring as the large number of live, infected trees dying during the survey may have masked newly infected trees in incidence calculations. In heavily WPBR-infected areas such as Pole Mountain, Wyoming, 65% of live trees were infected (in 2004-07), and of trees that began the study as healthy, 23% were declining or dying and 38% had died by the end of the study period (2016-17). Additionally, WPBR severity increased significantly from the beginning of the study with 4 previously uninfected sites gaining WPBR infections, 29 sites advancing to 'moderately infected' and 5 sites becoming 'heavily infected'. The overall average number of cankers per tree (3.5) was stable, but the number of infected limber pine with a canker in the lower 1/3 of the stem (18%) increased significantly (+4.2%, P = 0.001). When examining all limber pine in the study, 8%, 3% and 3% were killed by MPB/Ips., WPBR, and combined effects of these agents, respectively. Of the 887 live, but declining or dying limber pine, 52% had WPBR infections and 38% had damage from twig beetles (Pityophthorus spp., Pityogenes spp.) in 2016-17. Though all sites had ≥ 20% limber pine composition, 34% of sites had no limber pine regeneration and 7% had no regeneration of any tree species over the entirety of the study period. The results of this time series indicate that limber pine populations in the U.S. Rocky Mountains are declining due to effects from WPBR and MPB/Ips. Long-term surveys capture the effects of these damage agents on native tree populations and provide critical guidance for future management and restoration of these ecologically valuable species. Limber pine is at risk due to the various biotic and abiotic agents threatening their health. Thus, future directions involve restorative management practices for highly impacted areas where limber pine is a climax species and proactive management for healthy limber stands to promote resilience to likely damage agents. In highly impacted areas (WPBR incidence, mortality, or bark beetle damage on >50% of trees and low limber pine density and regeneration), where limber pine co-exists with other tree species, it may be favorable to allow the natural succession of other tree species to become dominant. However in xeric, harsh sites where limber pine is a climax species, these highly impacted areas are at-risk for losing all tree cover and should be considered for protective and restorative planting strategies. As natural resistance to WPBR occurs on the landscape, genetic screening and protection of mature limber pine carrying either complete or partial resistance to the pathogen should be pursued to preserve this genetic diversity. A priority should be to protect resistant against bark beetles and fire using established management practices. Additionally, seed-sourcing from resistant trees can allow for resistant progeny to be out-planted into high priority areas, thus buffering stands at risk for high WPBR mortality. Moreover management plans that promote diversification of age and diameter classes within stands can provide resilience against pest and pathogen attacks, as bark beetles vary in diameter preference and WPBR infections tend to cause higher mortality in smaller diameter trees. Lastly in healthy limber pine stands, proactive management of pest impacts to promote stand resilience is recommended as in Schoettle & Sniezko (2007) in order to preserve these healthy populations.