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<p>The neocortex expanded spectacularly during human origins. That expansion is thought to form the foundation for our cognitive faculties underlying abstract reasoning and socialization. The human neocortex differs from that of other great apes in several notable regards including altered cell cycle, prolonged corticogenesis, and massively increased size. However, despite decades of effort, little progress has been made in uncovering the genetic contributions that underlie these differences that distinguish our species from closely related primate, such as chimpanzees. A subset of highly conserved non-coding regions that show rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers, but none have linked an expression difference to a organismal traits, such as brain sizes. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) near the Wnt receptor FRIZZLED-8 (FZD8). Using a variety of approaches, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving significantly strong expression. We show that HARE5 likely regulates FZD8 and that expression differences influence cell cycle kinetics, cortical layers, and brain size. At present, this would provide the first evidence of a human-chimpanzee genetic difference influencing the evolution of brain size.</p> / Dissertation

Left/right (L/R) asymmetries, differences in morphology between the otherwise mirrored left- and right-hand sides of the body, are found in animals across the Bilateria. For many years it was thought that the mechanisms for establishment of these asymmetries had evolved separately in the three superphyla that constitute the Bilateria, but the discovery in 2009 that the TGF-beta ligand Nodal shares a conserved role in the Deuterostomia and Lophotrochozoa has re-ignited debate and interest in this field. In this thesis, work examining the establishment and maintenance of L/R asymmetries in the lophotrochozoan superphylum is presented, aimed at uncovering the wider conservation of these pathways across the Bilateria. Illumina sequencing and a range of de novo assembly techniques were used to derive genomic and transcriptomic data respectively for two primary model organisms, the limpet Patella vulgata and the serpulid annelid Pomatoceros lamarckii. Additionally, collaborative work lead to the derivation of transcriptomes for two other mollusc species and the genome of the monogont rotifer Brachionus plicatilis. A range of analysis was performed on these novel resources and is detailed here, with particular reference to the transcription factor cassettes contained in these datasets. These sequence resources formed the basis for examination of the breaking of initial symmetry in these model organisms. Known read-outs of correct establishment of L/R asymmetry, the expression of genes Nodal and Pitx on the right of the body, were codified in the course of normal development in P. vulgata. Pharmacological inhibitors of genes implicated in the establishment of L/R asymmetry, particularly ATPase ion channels, were then applied to embryos. After development, markers of normal development were assayed for signs of bilateral inversion. Although radialised phenotypes were observed, it is unclear whether these are specifically the result of L/R asymmetry defects. The localisation of ATPase mRNA and serotonin, often posited as a small molecule potential morphogen, were also assayed, although no conclusions could be drawn as to a role in the establishment of L/R asymmetry for these molecules, counter to some evidence from vertebrates. Once symmetry is broken, the TGF-beta pathway is responsible for the communication, specification and maintenance of tissue identity across the L/R axis. The novel sequence resources described in this thesis provided a comprehensive window into this signalling cassette, and detailed here is a treatment of the TGF-beta pathway within the Lophotrochozoa. Ligand diversity has increased markedly in some clades, while signal transduction and regulatory steps are relatively unchanged. This work has increased our knowledge of lophotrochozoan biology and particularly the mechanisms underpinning the establishment of asymmetry in this under-researched clade, however, much remains to be discovered about the ultimate origin of asymmetry itself.

<p>For primates, and other arboreal mammals, adopting suspensory locomotion represents one of the strategies an animal can use to prevent toppling off a thin support during arboreal movement and foraging. While numerous studies have reported the incidence of suspensory locomotion in a broad phylogenetic sample of mammals, little research has explored what mechanical transitions must occur in order for an animal to successfully adopt suspensory locomotion. Additionally, many primate species are capable of adopting a highly specialized form of suspensory locomotion referred to as arm-swinging, but few scenarios have been posited to explain how arm-swinging initially evolved. This study takes a comparative experimental approach to explore the mechanics of below branch quadrupedal locomotion in primates and other mammals to determine whether above and below branch quadrupedal locomotion represent neuromuscular mirrors of each other, and whether the patterns below branch quadrupedal locomotion are similar across taxa. Also, this study explores whether the nature of the flexible coupling between the forelimb and hindlimb observed in primates is a uniquely primate feature, and investigates the possibility that this mechanism could be responsible for the evolution of arm-swinging. </p><p> To address these research goals, kinetic, kinematic, and spatiotemporal gait variables were collected from five species of primate (Cebus capucinus, Daubentonia madagascariensis, Lemur catta, Propithecus coquereli, and Varecia variegata) walking quadrupedally above and below branches. Data from these primate species were compared to data collected from three species of non-primate mammals (Choloepus didactylus, Pteropus vampyrus, and Desmodus rotundus) and to three species of arm-swinging primate (Hylobates moloch, Ateles fusciceps, and Pygathrix nemaeus) to determine how varying forms of suspensory locomotion relate to each other and across taxa. </p><p> From the data collected in this study it is evident the specialized gait characteristics present during above branch quadrupedal locomotion in primates are not observed when walking below branches. Instead, gait mechanics closely replicate the characteristic walking patterns of non-primate mammals, with the exception that primates demonstrate an altered limb loading pattern during below branch quadrupedal locomotion, in which the forelimb becomes the primary propulsive and weight-bearing limb; a pattern similar to what is observed during arm-swinging. It is likely that below branch quadrupedal locomotion represents a “mechanical release” from the challenges of moving on top of thin arboreal supports. Additionally, it is possible, that arm-swinging could have evolved from an anatomically-generalized arboreal primate that began to forage and locomote below branches. During these suspensory bouts, weight would have been shifted away from the hindlimbs towards forelimbs, and as the frequency of these boats increased the reliance of the forelimb as the sole form of weight support would have also increased. This form of functional decoupling may have released the hindlimbs from their weight-bearing role during suspensory locomotion, and eventually arm-swinging would have replaced below branch quadrupedal locomotion as the primary mode of suspensory locomotion observed in some primate species. This study provides the first experimental evidence supporting the hypothetical link between below branch quadrupedal locomotion and arm-swinging in primates.</p> / Dissertation

<p>The evolution of reproductive strategies involves a complex calculus of costs and benefits to both parents and offspring. Many marine animals produce embryos packaged in tough egg capsules or gelatinous egg masses attached to benthic surfaces. While these egg structures can protect against environmental stresses, the packaging is energetically costly for parents to produce. In this series of studies, I examined a variety of ecological factors affecting the evolution of benthic development as a life history strategy. I used marine gastropods as my model system because they are incredibly diverse and abundant worldwide, and they exhibit a variety of reproductive and developmental strategies. </p><p>The first study examines predation on benthic egg masses. I investigated: 1) behavioral mechanisms of predation when embryos are targeted (rather than the whole egg mass); 2) the specific role of gelatinous matrix in predation. I hypothesized that gelatinous matrix does not facilitate predation. One study system was the sea slug Olea hansineensis, an obligate egg mass predator, feeding on the sea slug Haminoea vesicula. Olea fed intensely and efficiently on individual Haminoea embryos inside egg masses but showed no response to live embryos removed from gel, suggesting that gelatinous matrix enables predation. This may be due to mechanical support of the feeding predator by the matrix. However, Haminoea egg masses outnumber Olea by two orders of magnitude in the field, and each egg mass can contain many tens of thousands of embryos, so predation pressure on individuals is likely not strong. The second system involved the snail Nassarius vibex, a non-obligate egg mass predator, feeding on the polychaete worm Clymenella mucosa. Gel neither inhibits nor promotes embryo predation for Nassarius, but because it cannot target individual embryos inside an egg mass, its feeding is slow and inefficient, and feeding rates in the field are quite low. However, snails that compete with Nassarius for scavenged food have not been seen to eat egg masses in the field, leaving Nassarius free to exploit the resource. Overall, egg mass predation in these two systems likely benefits the predators much more than it negatively affects the prey. Thus, selection for environmentally protective aspects of egg mass production may be much stronger than selection for defense against predation. </p><p>In the second study, I examined desiccation resistance in intertidal egg masses made by Haminoea vesicula, which preferentially attaches its flat, ribbon-shaped egg masses to submerged substrata. Egg masses occasionally detach and become stranded on exposed sand at low tide. Unlike adults, the encased embryos cannot avoid desiccation by selectively moving about the habitat, and the egg mass shape has high surface-area-to-volume ratio that should make it prone to drying out. Thus, I hypothesized that the embryos would not survive stranding. I tested this by deploying individual egg masses of two age classes on exposed sand bars for the duration of low tide. After rehydration, embryos midway through development showed higher rates of survival than newly-laid embryos, though for both stages survival rates over 25% were frequently observed. Laboratory desiccation trials showed that >75% survival is possible in an egg mass that has lost 65% of its water weight, and some survival (<25%) was observed even after 83% water weight lost. Although many surviving embryos in both experiments showed damage, these data demonstrate that egg mass stranding is not necessarily fatal to embryos. They may be able to survive a far greater range of conditions than they normally encounter, compensating for their lack of ability to move. Also, desiccation tolerance of embryos may reduce pressure on parents to find optimal laying substrata. </p><p>The third study takes a big-picture approach to investigating the evolution of different developmental strategies in cone snails, the largest genus of marine invertebrates. Cone snail species hatch out of their capsules as either swimming larvae or non-dispersing forms, and their developmental mode has direct consequences for biogeographic patterns. Variability in life history strategies among taxa may be influenced by biological, environmental, or phylogenetic factors, or a combination of these. While most prior research has examined these factors singularly, my aim was to investigate the effects of a host of intrinsic, extrinsic, and historical factors on two fundamental aspects of life history: egg size and egg number. I used phylogenetic generalized least-squares regression models to examine relationships between these two egg traits and a variety of hypothesized intrinsic and extrinsic variables. Adult shell morphology and spatial variability in productivity and salinity across a species geographic range had the strongest effects on egg diameter and number of eggs per capsule. Phylogeny had no significant influence. Developmental mode in Conus appears to be influenced mostly by species-level adaptations and niche specificity rather than phylogenetic conservatism. Patterns of egg size and egg number appear to reflect energetic tradeoffs with body size and specific morphologies as well as adaptations to variable environments. Overall, this series of studies highlights the importance of organism-scale biotic and abiotic interactions in evolutionary patterns.</p> / Dissertation

<p>This thesis concerns the use of protein structure to improve phylogenetic inference. There has been growing interest in phylogenetics as the number of available DNA and protein sequences continues to grow rapidly and demand from other scientific fields increases. It is now well understood that phylogenies should be inferred jointly with alignment through use of stochastic evolutionary models. It has not been possible, however, to incorporate protein structure in this framework. Protein structure is more strongly conserved than sequence over long distances, so an important source of information, particularly for alignment, has been left out of analyses.</p><p>I present a stochastic process model for the joint evolution of protein primary and tertiary structure, suitable for use in alignment and estimation of phylogeny. Indels arise from a classic Links model and mutations follow a standard substitution matrix, while backbone atoms diffuse in three-dimensional space according to an Ornstein-Uhlenbeck process. The model allows for simultaneous estimation of evolutionary distances, indel rates, structural drift rates, and alignments, while fully accounting for uncertainty. The inclusion of structural information enables pairwise evolutionary distance estimation on time scales not previously attainable with sequence evolution models. Ideally inference should not be performed in a pairwise fashion between proteins, but in a fully Bayesian setting simultaneously estimating the phylogenetic tree, alignment, and model parameters. I extend the initial pairwise model to this framework and explore model variants which improve agreement between sequence and structure information. The model also allows for estimation of heterogeneous rates of structural evolution throughout the tree, identifying groups of proteins structurally evolving at different speeds. In order to explore the posterior over topologies by Markov chain Monte Carlo sampling, I also introduce novel topology + alignment proposals which greatly improve mixing of the underlying Markov chain. I show that the inclusion of structural information reduces both alignment and topology uncertainty. The software is available as plugin to the package StatAlign. </p><p>Finally, I also examine limits on statistical inference of phylogeny through sequence information models. These limits arise due to the `cutoff phenomenon,' a term from probability which describes processes which remain far from their equilibrium distribution for some period of time before swiftly transitioning to stationarity. Evolutionary sequence models all exhibit a cutoff; I show how to find the cutoff for specific models and sequences and relate the cutoff explicitly to increased uncertainty in inference of evolutionary distances. I give theoretical results for symmetric models, and demonstrate with simulations that these results apply to more realistic and widespread models as well. This analysis also highlights several drawbacks to common default priors for phylogenetic analysis, I and suggest a more useful class of priors.</p> / Dissertation

<p>Sex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.</p><p>Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.</p> / Dissertation

<p>How organisms adapt and survive in continuously fluctuating environments is a central question of evolutionary biology. Additionally, organisms have to deal with the inherent stochasticity in all cellular processes. The purpose of this thesis is to gain insights into how organisms can use epigenetics and the stochasticity of gene expression to deal with a fluctuating environment. To accomplish this, two cases at different temporal and structural scales were explored: (1) the early transcriptional response to an environmental stimulus in single cells, and (2) the evolutionary dynamics of a population adapting to a recurring fluctuating environment. Mathematical models of stochastic gene expression, population dynamics, and evolution were developed to explore these systems.</p><p>First, the information available in sparse single cell measurements was analyzed to better characterize the intrinsic stochasticity of gene expression regulation. A mathematical and statistical model was developed to characterize the kinetics of a single cell, single gene behavior in response to a single environmental stimulus. Bayesian inference approach was used to deduce the contribution of multiple gene promoter states on the experimentally measured cell-to-cell variability. The developed algorithm robustly estimated the kinetic parameters describing the early gene expression dynamics in response a stimulus in single neurons, even when the experimental samples were small and sparse. Additionally, this algorithm allowed testing and comparing different biological hypotheses, and can potentially be applied to a variety of systems.</p><p>Second, the evolutionary adaptation dynamics of epigenetic switches in a recurrent fluctuating environment were studied by observing the evolution of gene regulatory circuit in a population under multiple environmental cycles. The evolutionary advantage of using epigenetics to exploit the natural noise in gene expression was tested by competing this strategy against the classical genetic adaptation through mutations in a variety of evolutionary conditions. A trade-off between minimizing the adaptation time after each environmental transition and increasing the robustness of the phenotype during the constant environment between transitions was observed. Surviving lineages evolved bistable, epigenetic switching to adapt quickly in fast fluctuating environments, whereas genetic adaptation with high robustness was favored in slowly fluctuating environments.</p> / Dissertation

Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.

Doctor of Philosophy / Division of Biology / Theodore J. Morgan / Thermal stress impacts animals around the globe and understanding how organisms adapt to changes in temperature is of particular interest under current climate change predictions. My research focuses on the evolutionary genetics involved in cold tolerance and plasticity of cold tolerance using both artificially selected and naturally segregating populations, while tying the genes of interest to their physiological components. First I address cross-tolerance of stress traits following artificial selection to a non-lethal cold tolerance metric, chill-coma recovery. Using these artificial selection populations, we found that stress traits such as desiccation tolerance, starvation tolerance, acclimation, and chronic and acute cold tolerance do not correlate with level of cold tolerance as defined by chill-coma recovery time. We next assessed lifetime fitness of these different cold tolerance lines and found that only at low temperatures did fitness differ among cold tolerance levels. We then analyzed gene expression differences between resistant and susceptible populations at three time points to understand where selection pressures are hypothesized to act on genomic variation. Our gene expression analyses found many differences between resistant and susceptible lines, primarily manifesting themselves in the recovery period following cold exposure. We next utilized a community resource, the Drosophila melanogaster reference panel, to identify naturally segregating variation in genes associated with cold acclimation and fitness. We specifically asked if long- and short-term acclimation ability had overlapping genetic regions and if plasticity values from constant rearing environments were associated with demographic parameters in fluctuating environments. We found that long- and short-term acclimation are under unique genetic control and functionally tested several genes for acclimation ability. We also found that acclimation ability in constant environments and fitness in fluctuating environments do not correlate, but that genotypes are constrained in their fitness abilities between a warm and cool environment. Our analyses describe several novel genes associated with cold tolerance selection and long- and short-term acclimation expanding our knowledge of the complex relationship between demographic components and survivorship as well as a unique investigation of the change in gene expression during cold exposure.