Theses and Dissertations

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Open Access
Uncovering mechanisms driving variation in mutation rates in organellar and nuclear genomes
(Colorado State University. Libraries, 2024) Waneka, Gus, author; Sloan, Daniel B., advisor; Lockridge Mueller, Rachel, committee member; Argueso, Juan Lucas, committee member; Stenglein, Mark, committee member
Mutations are changes to DNA sequences which drive evolution by supplying raw genetic variation for natural selection to act upon. At the same time, mutations tend to have negative fitness consequences and are the source of genetic diseases. Such costs and benefits of mutation create opposing forces of selection on mutation rate modifiers, which are alleles (typically in DNA repair genes) responsible for increases or decreases to mutation rates. Essentially all eukaryotes possess at least two genomes: the nuclear genome (nucDNA) and the endosymbiotically derived mitochondrial genome (mtDNA). Photosynthetic plants and algae additionally possess endosymbiotically derived plastid genomes (cpDNAs). Together, the mtDNA and cpDNA are referred to as organellar genomes. Chapter 1 of this dissertation provides a framework for understanding how DNA repair machinery and mutation rates have evolved in complex eukaryotic cells. Chapters 2 and 3 focus on specific repair pathways active in organellar genomes. Finally, Chapter 4 shifts focus to understand how environmental perturbations in expression level impact mutation in plant nuclear genomes. Repair of organellar genomes is conducted by nuclear-encoded genes that are translated in the cytosol and targeted to the organelles. In terms of evolutionary history, organellar repair machinery is a mosaic network of bacterial-like repair genes, which came into the cell with the organelles, and nuclear repair genes that have been co-opted for organellar function. In some cases, repair proteins are targeted to both the nucDNA and mtDNA (and/or cpDNA in plants) to perform similar functions. This is the case as for many base excision repair (BER) proteins, which identify and remove of chemically damaged bases. In contrast, organellar repair arsenals are thought to lack canonical mismatch-repair (MMR) and nucleotide excision repair (NER), which are both important repair pathways in nuclear genomes. Instead, the diverse eukaryotic lineages have adopted unique strategies for organellar genome maintenance. As a result, there is a tremendous diversity in mtDNA mutation rates, which show over a 4000-fold variation across eukaryotes. Interestingly, much of this variation is driven by the extremely low point mutation rates plant mtDNAs. In addition, plant organellar genomes are more recombinationally active and plant mtDNAs are structurally unstable compared to the mtDNAs of other eukaryotes. Chapter 2 of this dissertation explores the mechanistic basis of low point mutation rates and recombination-mediated repair in plant organellar genomes. We performed high-fidelity Duplex Sequencing on a panel of Arabidopsis thaliana lines lacking specific organellar genome repair genes. We report large point mutation increases in mutant lines lacking MSH1, a mutS homolog that has been proposed to induce double-stranded breaks at the site of DNA mismatches, effectively shuttling such lesions into homologous recombination (HR) pathways that play important roles in plant organellar genome replication and repair. We see smaller point mutation increases in other mutant lines lacking RADA, RECA1 and RECA3. In addition, we generated long-read Oxford Nanopore sequencing to characterize repeat-mediated recombination in several of the mutants in the panel. Our findings provide valuable insights into the mechanisms driving the fascinating patterns of organellar genome evolution in land plants. The aforementioned lack of NER in organellar genomes is surprising given the importance of NER as a 'catchall' for repair of a variety of bulky DNA lesions in nuclear genomes. Chapter 3 focuses in on the fate of photodamage (an important type of bulky DNA damage) in organellar DNA. To do so, we leverage publicly available XR-seq datasets, which were generated to quantify and map active NER excision products in nuclear genomes following UV exposure. The taxonomic scope of chapter is expanded from plants to also include fungi (the brewer's yeast Saccharomyces cerevisiae) and animals (the model fruit fly Drosophila melanogaster). We find that mtDNA-derived XR-seq reads in A. thaliana and S. cerevisiae have distinct and repeatable patterns in terms of length and internal positioning of pyrimidine dimers (known targets of photodamage formation). These data mirror established patterns of NER-derived reads originating from the nuclear genomes, raising the exciting possibility that NER-like repair pathways may exist in for repair of photodamage in organellar genomes. The focus of chapter 4 shifts to understanding how environmental changes impact mutation in plant nuclear genomes. The textbook view of mutation and adaptation is that mutations occur randomly with respect to their environment-specific fitness consequences. However, this view of random mutation is challenged as evidence increasingly establishes a correlation between increased expression and decreased mutation via the coordination of transcription and DNA repair machinery at the molecular level. As a result of this correlation, intragenomic mutation rates likely vary with changing environments given that expression levels are environmentally labile. Therefore, certain genes may be predisposed to higher or lower mutation rates depending on the environment, though the magnitude and importance of this effect remains largely untested. A technical challenge in addressing these questions is that large scale plant mutation datasets are time and resource intensive to generate. A recent plant study relied on low frequency somatic calls from Illumina based shotgun libraries to generate a large number of mutations, but others report that most of these inferred mutations are sequencing errors. To overcome these challenges, we took a novel approach to measuring somatic mutations by using Duplex Sequencing to quantify mutations in targeted regions of the A. thaliana nucDNA. We identified a set of differentially expressed genes in plants grown at different temperatures, which we then targeted for mutation detection using hybrid capture. In addition to wild type (WT) lines, we also studied mutant lines deficient in BER and MMR to test if either of these pathways are responsible for the correlation between expression and mutation in plants. We found large point mutation increases in the MMR mutants compared to WT plants, which displayed surprisingly few mutations at either temperature. Though the small number of WT mutations precluded a meaningful comparison of expression and mutation in a WT background, this result if nonetheless valuable for establishing that the true frequency of somatic mutations in plants is indeed very low suggesting that previous estimates likely conflated Illumina based sequencing artifacts with mutations. Mutation rates vary by over three orders of magnitude across the tree of life. Much of this variation is captured in mitochondrial mutation rates. The chapters of this dissertation provide valuable insights into the molecular processes that drive mutation rate variation in eukaryotic genomes. Such mechanistic understandings are critical for advancing the broader field of mutation rate evolution.
Open Access
The role of CpNifS in selenium and sulfur plant metabolism: implications for phytoremediation and photosynthesis
(Colorado State University. Libraries, 2008) Van Hoewyk, Doug, author; Pilon, Marinus, advisor; Pilon-Smits, Elizabeth, advisor
NifS-like proteins are a conserved group of proteins that can cleave the sulfur-containing amino acid cysteine in alanine and elemental sulfur (S), and selenocysteine alanine and selenium (Se). In yeast and bacteria, NifS-like proteins are essential for survival because they provide the S for iron(Fe)-S clusters, a prosthetic group that is inserted into various FeS proteins that have a role in electron transfer. Furthermore, NifS-proteins are an essential part of Se metabolism in organisms that require this trace element. The goal of this research was to characterize the function of a chloroplastic NifS-like protein in Arabidopsis thaliana, designated AtCpNifS. As described in this dissertation, overexpression of CpNifS increases plant tolerance to selenate and accumulation of Se. Increased levels of CpNifS prevents toxic incorporation of selenocysteine into proteins, and thus enhances Se tolerance. This may benefit phytoremediation-the use of plants to naturally clean polluted soils and groundwater. In an effort to further the field of phytoremediation, a transcriptome experiment was performed in order to identify other genes and pathways that are involved in responding to Se stress. However, as divulged, plants likely do not require Se for essential metabolism, and the true function of CpNifS is more likely in the maturation of FeS clusters. The knockdown of CpNifS proteins in Arabidopsis using an inducible RNAi approach revealed that chloroplast function and structure became impaired, and that levels of all tested FeS proteins decreased. Consequently, the rate of photosynthetic electron transport, which is dependent on FeS proteins, diminished, and plants became chlorotic and eventually died. Therefore, CpNifS is required for FeS proteins, and is essential for proper photosynthesis and plant growth.
Open Access
Colorado cytospora canker complex on Populus tremuloides Michx.
(Colorado State University. Libraries, 2009) Kepley, Jeff B., author; Reeves, F. Brent, advisor
Cytospora canker is a serious fungal disease affecting aspen in natural and commercial forests as well as urban sites. In Colorado the causal organism responsible for this canker disease is typically reported to be Cytospora chrysosperma (Pers.) Fr. However, a thorough understanding of the species of Cytospora attacking aspen in Colorado is lacking. Fungal identification has been based upon morphological characteristics of fruiting/vegetative structures despite the plasticity known to occur in such diagnostic features. Examinations of cankers on aspen stems in Colorado revealed a morphologically distinct Cytospora-like fungus that frequently co-occurs with C. chrysosperma. This fungus is a new species and is closely associated with and superficially resembles C. chrysosperma. Based on these findings Cytospora canker on aspen in Colorado is a complex of fungi, contrary to what is typically reported in the literature. Isoenzyme analysis was employed as an initial step to determine the genetic/biochemical differences that occur among and between C. chrysosperma and the new non-C. chrysosperma isolates. Of the twelve enzyme systems initially screened only three, viz., alpha esterase, amylase, and glucose-6-phosphate dehydrogenase, provided good resolution for all isolates. Following cluster analysis, two major clades well-delineated the two taxa. Phylogenetic analyses of ITS1-5.8S-ITS2 rDNA and EF-1α sequences produced phylogenetic trees in which non- C. chrysosperma isolates formed a monophyletic clade (with strong bootstrap support and high posterior probability) within a Cytospora spp. phylogeny. Based on these results the non-C. chrysosperma isolates from aspen in Colorado are considered a new Cytospora species. External morphological features of the ascostromata and conidiomata (natural specimens) as well as histological sections of the new Cytospora sp. reveal conceptacles and conceptacle-like tissues which gives fruit bodies a unique target-like appearance. Cultures are darkly pigmented and display robust (large diameter) bead-like hyphae; many hyphal tips from young cultures lyse. Pycnidia produced in vitro do not enclose a multi-lobed locular structure; rather they have indentations/pockets with conidiophores lining these invaginations as well as pycnidial surfaces. In addition to the Cytospora anamorph a Phialocephala-like synanamorph is produced by some isolates. Descriptions of the new Cytospora species and C. chrysosperma, occurring on aspen in Colorado, are provided.
Open Access
Pathogen persistence in wildlife populations: case studies of plague in prairie dogs and rabies in bats
(Colorado State University. Libraries, 2009) George, Dylan, author; Webb, Colleen, advisor
Disease ecology focuses, in part, on how pathogens persist within host wildlife populations For my dissertation my colleagues and I investigated pathogen persistence mechanisms in two host-pathogen systems: Yersinia pestis (plague) in prairie dogs and rabies virus in bats. Plague, caused by the bacterium Yersinia pestis, recently spread into the range of black-tailed prairie dogs (Cynomys ludovicianus) in North America, and has caused drastic and rapid reduction in local prairie dog populations which have generated a metapopulation dynamic for prairie dogs. We developed a stochastic patch occupancy model to determine if prairie dog populations could persist long-term given the effects of plague. Our model demonstrates that metapopulation dynamics can allow prairie dog persistence. Town extinction in this system is caused by plague. Thus, town extinction and plague colonization are two sides of the same coin, which allows to us to interpret plague dynamics implicit within the prairie dog metapopulation. Long-term metapopulation dynamics indicate plague persists within the system and does not require the involvement of additional reservoir hosts (i.e., other resistant rodent species). Bats are a natural reservoir for rabies, and an increasing number of emerging zoonotic viruses. Little is known about mechanisms that generate unique seasonal patterns and allow enzootic pathogen persistence in bat populations. We propose that life history characteristics unique to many bat species coupled with viral adaptations allow for rabies persistence. First, we developed a statistical model to investigate seasonal patterns of rabies cases in bats. Second, we used data from a five-year study of rabies in big brown bats (Eptesicus fuscus) to parameterize a dynamic disease model that elucidates pathogen persistence mechanisms. We show rabies persists in two distinct ways, (1) through effects on bat population viability, and (2) through effects on viral persistence within a viable bat population. Mortality rates vary across seasons, and low rates during hibernation allow long-term bat population viability. Within a viable bat population, viral persistence occurs because of a lengthy incubation period, enhanced by the metabolic effects of host torpor. The mechanisms we identify may be operating in a similar manner for other bat-borne diseases.
Open Access
Regulation of copper transport into and within Arabidopsis thaliana chloroplasts: a focus on copper transport proteins
(Colorado State University. Libraries, 2007) Gogolin, Kathryn Amy, author; Pilon, Marinus, advisor
Copper is an essential micronutrient that is required for the biological processes of photosynthesis and respiration. Nutrients, such as copper, must travel long distances through several organs and across many membranes before they are incorporated into target enzymes. Plastocyanin is a small, copper containing protein that is located within the thylakoid lumen and is vital for photosynthetic activity in higher plants. In addition chloroplasts contain a second target for copper, the superoxide dismutase enzyme CSD2. Although copper is essential it can also be toxic to the cell, therefore there is tight regulation of ion transport. The objective of the research conducted here is to develop a better understanding of copper homeostasis in plant cells. By focusing on the proteins that are involved in the transport of copper new insight can be gained on the delivery pathways of this metal. In this dissertation, I further characterize P-type ATPase of Arabidopsis 1 (PAA1) and P-type ATPase of Arabidopsis 2 (PAA2). An Arabidopsis Copper Chaperone for Cu,Zn Superoxide dimustase (CCS) is identified as a functional homolog of the yeast copper chaperone for Cu,Zn superoxide dimustase (Ccs1/Lys7). I study the effects of altered CCS expression on copper homeostasis in a plant system and I determine that the Heavy Metal Associated 1 transporter functions to transport a metal other than Cu(I) across the chloroplast envelope which affects photosynthetic activity. Finally, I completed a comprehensive analysis of copper transport protein-protein interactions in Arabidopsis studied by the yeast two-hybrid system. With the data gathered here, I propose several new models for copper homeostasis in Arabidopsis. I suggest that there is regulation of Fe Superoxide Dismutase (FeSOD), CCS, and CSD2 in the chloroplast which is controlled by metal cofactor availability, specifically copper. By utilizing the yeast two-hybrid technique, I have identified two new possible delivery pathways for copper. I believe that CCS can deliver copper to Heavy Metal Associated 5 to aid in cell detoxification or possible long distance transport of the ion. Additionally, I propose that copper is transported directly from PAA1 to PAA2 in the chloroplast for delivery to plastocyanin.
Open Access
New roles for calcium channel beta subunits in early zebrafish development
(Colorado State University. Libraries, 2008) Ebert, Alicia Marie, author; Garrity, Deborah, advisor
Voltage-gated calcium channels are present on pre-synaptic terminals and at neuromuscular junctions in the adult. In embryos, the channel is primarily expressed in the developing heart. The auxiliary β subunit is responsible for trafficking the pore-forming α subunit to the membrane, and regulating the calcium channel kinetics. In non-canonical roles, the β subunit regulates gene silencing, vesicle docking, and calcium release from pancreatic cells. We report here the cloning and expression of two zebrafish β2 genes and two β4 genes. Morpholino inhibition of the β4 subunit slowed or blocked the morphogenetic movements of gastrulation, causing the blastoderm to retract and the embryos to assume dorsalized phenotypes. The nuclei of the extra-embryonic yolk syncytial layer (YSL) contained extra centrosomes, which led to formation of abnormal mitotic spindle. Microtubule arrays in the yolk were disrupted or absent. In 48 hpf embryos, the axis of the embryo was expanded mediolaterally and shorter anteroposteriorly. Gastrulation defects were present as early as shield formation. These data combined support the hypothesis for a novel role of the β4 subunit in early zebrafish development.
Open Access
Regulation of copper homeostasis in plants: a focus on chloroplastic superoxide dismutases and copper delivery mechanisms
(Colorado State University. Libraries, 2009) Cohu, Christopher Michael, author; Pilon, Marinus, advisor
Copper (Cu) is an essential micronutrient for higher plant growth and is found in proteins that are important in photosynthesis and respiration. As a cofactor, this trace element is associated with many proteins including plastocyanin, Cu/Zn superoxide dismutase (Cu/ZnSOD), and mitochondrial cytochrome- c oxidase. Due to its redox-active role, Cu is essential for plant life, yet Cu is also dangerous as a free cellular ion and even toxic if in excess. Therefore, delivery and sequestration of Cu must be tightly regulated. The research of this dissertation indicates that sensory mechanisms and signaling pathways exist to coordinate Cu transport and target protein expression based on Cu status. For Arabidopsis and crop species, chloroplastic Cu/ZnSOD is down-regulated during limited Cu availability while at the same time FeSOD is up-regulated. During Cu-limited growth, when Cu/ZnSOD is down-regulated, plastocyanin levels do not change. We suggest that this reduction in Cu/ZnSOD allows for preferential Cu delivery to plastocyanin, which is essential for photosynthesis, while also maintaining chloroplast SOD activity. Cu delivery to Cu/ZnSOD is accomplished by the Cu Chaperone for SOD (CCS). When a CCS loss of function mutant was grown on Cu supplemented soil Cu/ZnSOD and FeSOD activity was not detected. Chloroplast did not exhibit an observable phenotype or photosynthetic deficiencies, even after high light stress treatments. Recent studies have shown that Cu/ZnSODs in the cytosol and chloroplast, along with other Cu proteins, are regulated by Cu via microRNA directed cleavage of Cu protein mRNA. It has also been determined that during Cu-limited growth the SPL7 transcription factor plays a central role in activating Cu-microRNAs and possibly Cu transporters. The research of this dissertation indicates that CCS is also regulated by Cu, mediated by microRNA398, which was not previously predicted by bioinformatic algorithms. Furthermore, data is presented to suggest that SPL7 likely regulates the promoter of FeSOD by activating transcription during limited Cu availability.
Open Access
Population ecology of black-footed ferrets (Mustela nigripes) in relation to sylvatic plague
(Colorado State University. Libraries, 2023) Livieri, Travis M., author; Angeloni, Lisa, advisor; Antolin, Michael, committee member; Biggins, Dean, committee member; Crooks, Kevin, committee member
Infectious diseases can have significant impacts, both direct and indirect, on the conservation of endangered species. A full understanding of these impacts is hindered by the difficulty of teasing apart disease effects from other factors that led to endangerment, the scarcity of population data from before and after disease detection, and the inherent challenge of studying rare species, which are often difficult to detect. Ideally, a disease and population monitoring strategy will detect outbreaks so effective management and mitigation strategies can be implemented. Disease mitigation strategies, such as vaccination or removal of infected individuals, can be effective but costly to implement and rigorous evaluations of such efforts are rare. Here we present a case study and evaluation of a multi-faceted effort to manage multiple impacts of sylvatic plague (plague hereafter), an invasive disease, in a reintroduced population of endangered black-footed ferrets (Mustela nigripes) and their prey, black-tailed prairie dogs (Cynomys ludovicianus), in Conata Basin/Badlands National Park, South Dakota. Since reintroduction in 1994-1999, this is the largest free-ranging ferret population. Chapter One provides a broad introduction to black-footed ferret natural history, ecology, and conservation efforts. We briefly described the life history of black-footed ferrets, their reliance upon prairie dogs (Cynomys spp.) as prey and habitat engineers, and the conflicts between prairie dogs and agricultural interests that motivated human efforts to eradicate prairie dogs and inadvertently drove ferrets towards extinction. Ensuing captive breeding and reintroduction efforts averted extinction of the species, but plague, caused by Yersinia pestis bacteria, led to high mortality in both black-footed ferrets and prairie dogs, was a second factor in ferret decline, and continues to threaten reintroduced populations. Plague management, through flea vector control and vaccination, is a high priority for the black-footed ferret recovery program, along with maintaining genetic diversity and securing habitat. We concluded that black-footed ferret recovery to date has been partially successful, but challenges remain, and plague represents the largest biological threat. In Chapter Two, we evaluated the efforts to manage plague for black-footed ferrets and prairie dogs at Conata Basin/Badlands National Park. We effectively monitored plague using carnivore serology, prairie dog testing, and visual surveys to detect the invasion of plague and inform our mitigation efforts. Both prairie dog colonies and black-footed ferret populations declined precipitously with the plague epizootic. We applied deltamethrin dust into prairie dog burrows to kill fleas and vaccinated black-footed ferrets against plague during annual monitoring efforts. Our results suggested that dusting was effective in maintaining prairie dog colonies compared to non-dusted colonies and significantly increasing survival of black-footed ferrets. Additionally, our vaccination of black-footed ferrets added incremental gains in ferret survival. These combined efforts of plague surveillance, dusting prairie dog burrows, and vaccinating black-footed ferrets likely prevented extirpation of this population. In Chapter Three, we used stable isotope analysis to understand the effects of plague on the proportion of prairie dogs in black-footed ferret diets. Previous studies on black-footed ferrets found up to one-third of ferret diet is comprised of non-prairie dog rodents. Plague causes high mortality in prairie dogs and other small mammals found on prairie dog colonies, potentially increasing variability in prey available for black-footed ferrets. We sampled black-footed ferrets and two prey items, prairie dogs and deer mice (Peromyscus sonoriensis), before and during a plague epizootic and used stable isotope analysis to estimate the diet proportions in relation to plague and dusting. We found that prior to plague black-footed ferret diets in Conata Basin/Badlands National Park were similar to previous studies, but during a plague epizootic ferrets shifted their diet almost completely to prairie dogs. Dusting prairie dog burrows prior to the invasion of plague had a similar effect in shifting black-footed ferret diets. We concluded that despite observed foraging plasticity, black-footed ferrets can be considered prairie dog colony specialists, and any diet effects following deltamethrin dust treatment are likely less severe than the impacts of plague on unprotected ferret populations.
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 member
Across 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.
Open Access
Advancing conservation genomics of migratory species toward a full annual cycle approach
(Colorado State University. Libraries, 2023) DeSaix, Matthew G., author; Ruegg, Kristen C., advisor; Funk, W. Chris, committee member; Koons, David N., committee member; Marra, Peter M., committee member
Global biodiversity loss is one of the foremost concerns of conservation efforts in the 21st century. The maintenance of genetic diversity within species is a critical factor in a species' persistence and adaptive potential in the face of changing environmental conditions. Migratory species make up more than 12% of the global vertebrate biodiversity and pose distinct challenges to conservation efforts due to inhabiting different geographical regions at different times of the year. The field of conservation genomics provides a valuable toolkit to addressing and understanding global biodiversity loss but requires additional methodological developments to better address the conservation challenges posed by migratory species. In my dissertation, I demonstrate advancements in conservation genomics aimed toward better understanding migratory species. In my first study, I addressed the question of ecological and genomic vulnerability to climate change in the Brown-capped Rosy-Finch (Leucosticte australis), an elevational migratory songbird of conservation concern. Second, I addressed a methodological gap in population genomics and developed statistical genetics models for using genotype likelihood data from low-coverage whole genome sequencing data to implement population assignment. In my last study, I demonstrate the utility of low-coverage whole genome sequencing for population assignment with detailing migratory connectivity in the American Redstart (Setophaga ruticilla). Altogether, my doctoral research demonstrates how genomic tools can help unravel the complexities of migratory species conservation. Furthermore, the species-specific results are tied to knowledge gaps identified by wildlife managers and provide valuable information tied to conservation and management applications.
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 member
Phenotypic 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.
Open Access
Slow and noisy: developmental time and gene expression kinetics in big cells
(Colorado State University. Libraries, 2023) Taylor, Alexandra, author; Mueller, Rachel, advisor; Prasad, Ashok, advisor; Hoke, Kim, committee member; Krapf, Diego, committee member
Evolutionary increases in genome size, cell volume, and nuclear volume have been observed across the tree of life, with positive correlations documented between all three traits. It is well documented that developmental tempo slows as genomes, nuclei, and cells increase in size, yet the driving mechanisms are poorly understood. Meanwhile, the dramatic increases in cell volume seen across the tree of life pose interesting questions about a potential relationship between cell volume and stochastic noise at the single cell level, but this remains an underexplored area of research. To bridge these knowledge gaps, we use a mix of deterministic and stochastic, as well as species-specific and more general, models of the somitogenesis clock. In doing so, we explore the impact of changing intra-cellular gene expression kinetics induced by increasing genome size, nuclear volume, and cell volume on developmental tempo and gene expression noise. Results suggest that longer transcriptional and nuclear export times act to slow cell and developmental processes down as genome size and cell volume increase, and that "search processes" undergone by gene products within a cell become noisier with increasing volume. Analyses of stochastic model simulations and existing empirical data bring into question whether or not cell-autonomous oscillations can truly exist in the absence of cell-to-cell signaling.
Open Access
Enrichment as a conservation tool to enhance behavior, morphology, gene expression, and survival in Arkansas darters
(Colorado State University. Libraries, 2023) Kopack, Christopher J., author; Angeloni, Lisa, advisor; Fetherman, Eric, advisor; Ghalambor, Cameron, committee member; Kanno, Yoichiro, committee member
Conservation practitioners often rely on captive breeding programs to supplement wild populations at risk of extinction. While population augmentation has been successful for some taxa, the use of hatchery fish to supplement wild populations can be severely impacted by predation. Elevated predation on hatchery fish may arise because hatchery environments often differ starkly from wild environments, constraining the ability of hatchery fish to phenotypically match the environments in which they are targeted for release. Phenotypic mismatch caused by differences between hatchery and wild environments can limit efforts to conserve fish species at risk of extinction when hatchery-reared fish are used to augment wild populations. Phenotypes adapted to or induced by hatchery environments are thought to be maladapted for life in the wild. Thus, enriching the hatchery environment (abiotically and biotically) to make it more similar to the wild may induce phenotypes, including behavior, morphology, and gene expression profiles, that are better suited to the environments fish will experience after release. Chapter One explores how hatchery-reared fish respond to novel predators and whether those responses can be enhanced to improve survival. Identifying the presence of innate predator recognition and the capacity for learning to recognize predators can inform conservation management practices. We assessed antipredator behavior (time spent moving and distance from a predator) and the efficacy of predator training for three populations of a species of conservation concern, the Arkansas darter (Etheostoma cragini), which is vulnerable to predation by esocid predators like the introduced northern pike (Esox lucius). Arkansas darters demonstrated an innate ability to recognize and respond to a novel esocid predator. Their behavior also changed in response to predator cues (training), though the direction of response to cues was opposite our prediction. Populations differed in their response to the predator treatment, highlighting the potential value of managing populations separately. Our results suggest that antipredator behavior is innate and that exposure to predator cues does affect behavior. This study demonstrates the importance of evaluating enrichment practices and incorporating behavioral observations into conservation programs to guide population-specific management decisions. In Chapter Two, we used a factorial approach to assess whether abiotic enrichment and biotic enrichment (predator recognition training) increase survival of Arkansas darters during encounters with a novel predator. We also assessed the effects of abiotic enrichment on the expression of behavioral and morphological phenotypes across three populations. Morphology and behavior differed among populations and between abiotic treatments, and populations responded differently to the abiotic treatments. Furthermore, we found that in combination with predator training, abiotic enrichment increased the probability of surviving a first encounter with a predator. We therefore recommend conservation practitioners incorporate abiotic enrichment and predator recognition training in the hatchery, as any increase in survival is expected to benefit efforts to conserve this species. In Chapter Three, we took a molecular approach (TagSeq) to elucidate how abiotic enrichment and biotic enrichment impacts the whole-brain gene expression of Arkansas darters, comparing the effects in two hatchery populations to a wild reference population. Although, we found no effect of biotic enrichment on gene expression, we did find that abiotic enrichment has the potential to reduce phenotypic mismatch between hatchery and wild fish, indicating that enrichment may aid current conservation efforts. Overall, these studies suggest a potential role for enrichment in the conservation of imperiled fish, and they highlight the value of a phenotypic approach to managing populations.
Open Access
Genetic background and experience affect courtship behavior in male Trinidadian guppies (Poecilia reticulata)
(Colorado State University. Libraries, 2023) Phipps, Nathan M., author; Hoke, Kim, advisor; Angeloni, Lisa, committee member; Kanno, Yoichiro, committee member
An animal's behavior may be shaped by its genetics and life experience, but the extent to which each of these factors contributes to determining behavioral phenotypes is an outstanding question in biology. Mating behaviors are of particular interest due to their importance in determining fitness. We sought to investigate the genetic architecture of mating behaviors and their plasticity in response to mating experience. Trinidadian guppies (Poecilia reticulata) occur in streams with either high or low predation rates. This genetic background has shaped the evolution of many behavioral phenotypes, including those involved in male courtship strategy. We observed male guppies from high predation, low predation, and intercross populations in their first encounter with a female, then later repeated the encounter to observe how experience affects mating behaviors. We recorded occurrences of three behaviors – sigmoids, forced copulation attempts, and gonopodial swings – to determine how they are affected by sexual experience and genetic background. We found that the frequencies of sigmoids and gonopodial swings vary depending on genetic background and experience. Our findings support existing literature demonstrating that mating behaviors respond plastically to experience. We also found that intercross guppies matched the gonopodial swing and sigmoid frequency phenotypes of the QH genetic line, suggesting that these behaviors may be controlled by loci that are dominant in the high-predation population.
Open Access
Evolutionary increase in genome size drives changes in cell biology and organ structure
(Colorado State University. Libraries, 2022) Itgen, Michael Walter, author; Mueller, Rachel Lockridge, advisor; Sloan, Daniel B., committee member; Hoke, Kim L., committee member; Zhou, Wen, committee member
The evolution of large genome size has been associated with patterns of phenotypic change in cell and organismal biology. The most fundamental of these is between genome size and cell size, which share a strong positive and deterministic relationship. As a result, increases in cell size alter the structure and function of the cell. Genome and cell size, together, are hypothesized to produce emergent consequences on development and physiology at the cellular and organismal level. My dissertation aims to better understand these patterns and identify potential mechanisms underlying these phenotypic changes. I test for the effects of genome and cell size on cell function, cellular physiology, and organ morphology by leveraging the natural variation in genome size found in salamanders (Genus: Plethodon). First, I show that transcriptomic data supports the predictions that large genome and cell size has functional consequences on cell biology. I also reject the hypothesis that large cell size is functionally linked to lower metabolic rate at the cellular level, but I provide transcriptomic evidence that cell size alters the metabolic state of cells. Finally, I show that genome and cell size drives morphological change in organ-specific ways in the heart and liver. I conclude that large cell size does not lower metabolic rate in salamanders. As an alternative, I propose that the evolution of low metabolic rate lifts the constraint of cell size, thus permitting the evolution of genome gigantism.
Open Access
Selenium accumulation in plants and implications for human health: a survey of molecular, biochemical, and ecological cues
(Colorado State University. Libraries, 2022) Lima, Leonardo Warzea, author; Pilon-Smits, Elizabeth, advisor; Schiavon, Michela, committee member; Pilon, Marinus, committee member; Antunes, Mauricio, committee member; Paschke, Mark, committee member
To view the abstract, please see the full text of the document.
Open Access
Paralogy or reality? Exploring gene assembly errors in a target enrichment dataset
(Colorado State University. Libraries, 2022) Rosén, Austin, author; Simmons, Mark P., advisor; Ackerfield, Jennifer, committee member; Richards, Christopher, committee member; Stewart, Jane, committee member
De novo gene assembly of short read data is inherently difficult – similar to the process of assembling a jigsaw puzzle. I describe three errors that occurred with the assembly of target enrichment data in the genus Cirsium (Asteraceae): inconsistent contig selection, artificial recombination, and inconsistent intron determination leading to over-alignment of non-homologous nucleotides. These errors occurred in 39% of loci in the dataset and were often a by-product of undetected paralogs: assembled loci that likely contained paralogous or homoeologous sequences but did not trigger default paralog warnings by the assembly program, HybPiper. Default HybPiper thresholds for identifying paralogy during the assembly process were insufficient to filter such loci. A custom target file was created in which putative paralogs were separated into independent loci. The custom target file was successful in reducing, but not eliminating, assembly errors in the dataset. A final iteration of quality control was performed to create a dataset largely free of assembly errors. However, phylogenetic inferences applied to this final cleansed dataset were unable to resolve the taxonomic relationships between the sampled specimens. Rather, these results affirm that Cirsium is a taxonomically problematic genus and may require population-level genetic data or integrative taxonomy approaches to delimit species boundaries.
Open Access
Phytoremediation with hemp (Cannabis sativa L.): a look at hemp's potential for environmental cleanup and economic recovery
(Colorado State University. Libraries, 2022) Abernathy, Susan M., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Qian, Yaling, committee member
The aim of this thesis study was to test hemp's (Cannabis sativa L.) potential for phytoremediation (environmental clean-up). I tested hemp for tolerance and accumulation of four inorganic pollutants, to evaluate its remediating performance. Hemp has many properties that would make it a likely candidate for phytoremediation however, due to recent regulations, research of this versatile plant has been limited. Phytoremediation is a process of cleaning polluted sites using plants. In this clean-up method, plants may stabilize the pollutant in situ, or take-up the pollutant into the plant tissue. In the latter, there are a few different fates for the pollutant that include degradation, metabolization, sequestration, and/or volatilization. Phytoremediation is a clean process that reestablishes an onsite ecosystem and is a competitive alternative to more conventional scrape-and-remove methods. Hemp is a hardy, fast growing species that produces high biomass. Hemp has deep roots that can be used to reach pollutants deep in the ground. These properties make hemp a potential choice for phytoremediation. Contaminated sites create harsh growing conditions that require hardy plant properties in order for a species to survive. An added benefit to using hemp for remediation is the many economic uses of hemp biomass. Each part of the hemp plant can be used to make goods such as clothing, building material, cosmetics, lotions, animal bedding, fragrances, and medicinal products that have therapeutic qualities. In addition, hemp seeds are nutritious and can be added to the diet. In chapter one of this thesis, phytoremediation is reviewed to explain the remediation process. This review includes explaining the different phytotechnologies that are employed by plants which depend on the plant used and the type of pollutant encountered. Chapter one also reviews hemp, its history, biology, and the properties that make it a viable choice for phytoremediation. Chapter two of this thesis is an experimental chapter presenting data for testing hemp seedlings with four different oxyanions: arsenate (As), molybdate (Mo), vanadate (V), and tungstate (W). The parameters considered were biomass, chlorophyll content, chlorophyll fluorescence, pollutant accumulation levels, and pollutant fate. Brassica juncea (Indian mustard) was used as a reference phytoremediation species. The findings of this thesis study present promising results for hemp as a potential remediator. Arsenic was found to accumulate in the root at levels up to 2700 mg kg-1 DW. Tungsten also accumulated in the root at levels up to 3100 mg kg-1 DW. In both tests, hemp performed well, judged from photosynthetic measurements and relative chlorophyll content, but reduced biomass started at treatments with 3 and 24 mg As L-1 in the shoot and root respectively, and 40 and 80 mg W L-1 in the shoot and root, respectively. Molybdenum accumulated in the shoot at levels up to 4900 mg kg-1 DW and in the root at levels up to 2600 mg kg-1 DW. Biomass reduction of Mo started at treatment with 40 mg Mo L-1 for both shoot and root, while photosynthetic measurements and relative chlorophyll content remained unchanged. Lastly, V accumulated in the root at levels up to 2100 mg V kg-1 DW. Interestingly, hormesis (stimulated growth) was observed in hemp supplied with V: biomass increased at all tested levels. From this study, it was concluded that hemp may have potential for phytoremediation in cleaning contaminated sites with the four elements tested. Hemp performed competitively with the popular phytoremediation species, Indian mustard (Brassica juncea L.) in all levels tested for Mo, V, and W. Hemp's economic recovery with clean post-harvest biomass may offset phytoremediation costs giving this species a unique advantage over other popular phytoremediation choices.
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 member
Minimizing 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.
Open Access
Three guanylyl cyclases in the green shore crab, carcinus maenas: cDNA cloning and effects of molt stage on expression in molting gland (Y-organ)
(Colorado State University. Libraries, 2010) Gunderson, Jennifer L., author; Mykles, Donald L., advisor; Reddy, Anireddy N., committee member; Tjalkens, Ronald B., committee member
YO ecdysteroidogensis is suppressed by molt-inhibiting hormone (MIH) and the pleiotropic neuropeptide crustacean hyperglycemic hormone (CHH) with these neuropeptides being produced in the X-organ of the eyestalk (ES) ganglia. CHH signaling is mediated by a membrane receptor guanylyl cyclase (GC), while MIH signaling may involve a soluble NO-sensitive GC. Here we report the cloning of cDNA sequences encompassing the catalytic domain of the~ subunit of a soluble NO-sensitive GC ( Cm-GC-I ß), membrane receptor GC ( Cm-GC-II), and soluble NO-insensitive GC (Cm-GC-III) from the European green crab, Carcinus maenas, using RT-PCR and RACE. Adult C. maenas occur as two color morphs that differ in growth traits; the "green" morphs molt more frequently than the "red" morphs. These data suggest that YOs in red morphs are less active than the YOs in green morphs, and that the red morphs are refractory to molt induction by eyestalk ablation (ESA). We tested the effects of ESA on the expression of NO synthase ( Cm-NOS), Cm-GC-I ß, Cm-GC-II, and Cm-GC-III in the YOs from green and red morphs using quantitative PCR. Elongation factor-2 (Cm- EF2) served as a constitutively expressed control. ESA caused a small, transient increase in hemolymph ecdysteroid titers in both morphs, with the increase occurring at 3 days post-ESA in green morphs and at 14 days post-ESA in red morphs. ESA had no significant effect on the expression of Cm-EF2, Cm-NOS, Cm-GC-II, and Cm-GC-III, which were not correlated with hemolymph ecdysteroid titers. ESA caused a significant decrease in Cm-GC-I ß expression in red morphs, but had no significant effect in green morphs. Expression was also quantified in green morphs undergoing spontaneous natural molting. There was no significant difference in the expression of Cm-EF2, Cm-NOS, Cm- GC-II, and Cm-GC-II in YOs from intermolt, premolt, and postmolt animals. By contrast, Cm-GC-III mRNA was about 2-fold greater in YOs from postmolt animals than in intermolt and premolt animals. The results show that both color morphs are resistant to ESA and that molting has little effect on NOS and GC expression.