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Browsing by Author "Mueller, Rachel, advisor"

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    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.
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    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 member
    Salamanders 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.
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    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 member
    Hybridization 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.
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    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.
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    The effects of genome expansion on transposable element diversity in salamanders
    (Colorado State University. Libraries, 2021) Haley, Ava, author; Mueller, Rachel, advisor; Sloan, Daniel, committee member; Stenglein, Mark, committee member
    Transposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using ecological models for species distribution that consider richness and evenness of communities. It is currently not well studied how genome expansion impacts the diversity of transposable elements. However, there are a few models that predict TE diversity decreasing as genomes expand due to varying mechanisms such as selection against ectopic recombination and competition between TEs and silencing machinery. Salamanders are known to have some of the largest vertebrate genomes. Salamanders of the genus Plethodon in particular have very large genomes consisting of high levels of TEs, with sizes ranging from 30 to 70 Gigabases (Gb). Here, I use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade and one in the western clade: Plethodon glutinosus (41.4 Gb), P. cinereus (30.5 Gb), P. idahoensis (71.7 Gb), and P. vehiculum (50.5 Gb). I classified the TEs in these datasets using RepeatMasker and DnaPipeTE and found ~51 superfamilies which accounted for 27-32% of the genomes. For each genome I calculated the Simpson's and Shannon's diversity indices to quantify diversity, taking into account both TE richness and evenness. In all cases, the values for Simpson's index were within 0.75 and 0.79, and for Shannon's index all species were within 1.88 and 1.99. We conclude that once genomes reach large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to observations made by previous studies done on smaller genomes.