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Item Open Access A Comparative Study of Habitat Complexity, Neuroanatomy, and Cognitive Behavior in Anolis Lizards(2012) Powell, Brian JamesChanging environmental conditions may present substantial challenges to organisms experiencing them. In animals, the fastest way to respond to these changes is often by altering behavior. This ability, called behavioral flexibility, varies among species and can be studied on several levels. First, the extent of behavioral flexibility exhibited by a species can be determined by observation of that species' behavior, either in nature or in experimental settings. Second, because the central nervous system is the substrate determining behavior, neuroanatomy can be studied as the proximate cause of behavioral flexibility. Finally, the ultimate causation can be examined by studying ecological factors that favor the evolution of behavioral flexibility. In this dissertation, I investigate behavioral flexibility across all three levels by examining the relationship between habitat structure, the size of different structures within the brain and total brain size, and behavioral flexibility in six closely-related species of Puerto Rican Anolis lizards. Anolis lizards provide an excellent taxon for this study as certain species, including those used here, are classified as belonging to different ecomorphs and are morphologically and behaviorally specialized to distinct structural habitat types.
In order to determine the presence of behavioral flexibility in Anolis, I first presented Anolis evermanni with a series of tasks requiring motor learning and a single instance of reversal learning. Anolis evermanni demonstrated high levels of behavioral flexibility in both tasks.
To address the pattern of brain evolution in the Anolis brain, I used a histological approach to measure the volume of the whole brain, telencephalon, dorsal cortex, dorsomedial cortex, medial cortex, dorsal ventricular ridge, cerebellum, and medulla in six closely-related species of Puerto Rican Anolis lizards belonging to three ecomorphs. These data were analyzed to determine the relative contribution of concerted and mosaic brain evolution to Anolis brain evolution. The cerebellum showed a trend toward mosaic evolution while the remaining brain structures matched the predictions of concerted brain evolution.
I then examined the relationship between the complexity of structural habitat occupied by each species and brain size in order to determine if complex habitats are associated with relatively large brains. I measured brain volume using histological methods and directly measured habitat complexity in all six species. Using Principal Component Analysis, I condensed the measures of habitat structure to a single variable and corrected it for the scale of each lizard species' movement, calling the resulting measurement relevant habitat complexity. I tested the relationship between relative volume of the telencephalon, dorsal cortex, dorsomedial cortex, and whole brain against both relative habitat complexity and ecomorph classification. There was no relationship between the relative volume of any brain structure examined and either relevant habitat complexity or ecomorph. However, relevant habitat complexities for each species did not completely match their ecomorph classifications.
Finally, I tested the levels of behavioral flexibility of three species of Anolis, A. evermanni, A. pulchellus, and A. cristatellus, belonging to three distinct ecomorphs, by presenting them with tasks requiring motor and reversal learning. Anolis evermanni performed well in both tasks, while A. pulchellus required more trials to learn the motor task. Only a single Anolis cristatellus was able to perform either task. Anolis evermanni displayed lower levels of neophobia than the other species, which may be related to its superior performance.
In combination, this research suggests that Anolis of different ecomorphs display different levels of behavioral flexibility. At the proximate level, this difference in behavioral flexibility cannot be explained by changes in the relative size of the total brain or brain structures associated with cognitive abilities in other taxa. At the ultimate level, the size of the brain and several constituent structures cannot be predicted by habitat complexity. However, behavioral flexibility in certain tasks may be favored by utilization of complex habitats. Flexibility in different tasks is not correlated, rendering broad comparisons to a habitat complexity problematic.
Item Open Access A Molecular Phylogenetic Study of Historical Biogeography and the Evolution of Self-Incompatibility RNases in Indian Ocean Coffea (Rubiaceae)(2010) Nowak, Michael DennisA fundamental goal in the diverse field of evolutionary biology is reconstructing the historical processes that facilitated lineage diversification and the current geographic distribution of species diversity. Oceanic islands provide a view of evolutionary processes that may otherwise be obscured by the complex biogeographic histories of continental systems, and have thus provided evolutionary biology with some of its most lasting and significant theories. The Indian Ocean island of Madagascar is home to an extraordinarily diverse and endemic biota, and reconstructing the historical processes responsible for this diversity has consumed countless academic careers. While the flowering plant genus Coffea is but one lineage contributing to Madagascar's staggering floral diversity, it is representative of the common evolutionary theme of adaptive radiation and local endemism on the island. In this dissertation, I employ the genus Coffea as a model for understanding historical biogeographic processes in the Indian Ocean using methods of molecular phylogenetics and population genetics. In the molecular phylogenetic study of Coffea presented in chapter 2, I show that Madagascan Coffea diversity is likely the product of at least two independent colonization events from Africa, a result that contradicts current hypotheses for the single origin of this group.
Species of Coffea are known to exhibit self-incompatibly, which can have a dramatic affect on the geographic distribution of plant genetic diversity. In chapter 3, I identify the genetic mechanism of self-incompatibility in Coffea as homologous to the canonical eudicot S-RNase system. Baker's Rule suggests that self-incompatible lineages are very unlikely to colonize oceanic islands, and in chapter 4, I test this hypothesis by characterizing the strength of self-incompatibility and comparing S-RNase polymorphism in Coffea populations endemic to isolated Indian Ocean islands (Grande Comore and Mauritius) with that of Madagascan/African species. My findings suggest that while island populations show little evidence for genetic bottleneck in S-RNase allelic diversity, Mauritian endemic Coffea may have evolved a type of "leaky" self-incompatibility allowing self-fertilization at some unknown rate. Through the application of traditional phylogenetic methods and novel data from the self-incompatibly locus, my dissertation contributes a wealth of new information regarding the evolutionary and biogeographic history of Coffea in the Indian Ocean.
Item Open Access A Next-Generation Approach to Systematics in the Classic Reticulate Polypodium vulgare Species Complex (Polypodiaceae)(2014) Sigel, Erin MackeyThe Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.
Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.
Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.
Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.
This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.
Item Open Access A Phylogenetic, Ecological, and Functional Characterization of Non-Photoautotrophic Bacteria in the Lichen Microbiome(2011) Hodkinson, Brendan P.Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, Cladonia grayi, and one from a cyanolichen, Peltigera praetextata). Non-photobiont bacterial genes for nitrogen fixation were not detected in the Cladonia thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.
Item Open Access A Preliminary Study of Threonine Deaminase Duplication in Solanaceae(2013) Huang, JieOne of the most important questions in evolutionary biology is how new genes and new functions arise and evolve. Among the theories addressing this question, gene duplication is one of the most popular. Previous study has shown that two threonine deaminase (TD) gene copies exist in Solanum lycopersicum, and these two copies have very different functions and low sequence similarities. The primary objective of this study was to widen our understanding of this gene duplication and the subsequent evolutionary processes affecting the duplicate copies by first collecting additional TD sequences from related species, building a gene tree, and inferring the point of gene duplication. The evolutionary processes acting on this gene were then analyzed using the program PAML. Results indicate that 1) The TD duplication probably occurred in before the split of the Solanoideae from the Nicotianoidea; and 2) there is strong evidence for positive selection on one of the TD copies after gene duplication, while for the other TD copy, only weak evidence for positive selection was found; and 3) adaptive improvement of the copy with new function probably spanned a period of at least 25 million years.
Item Open Access A Survey of Fungal Community Composition along a Gradient of Recovery on the Mine Sites in the Carolinas(2019-05-06) Miao, RuolinIn the era of Anthropocene, an increasing part of the terrestrial environments is losing their ecosystem services and function, negatively affecting both human economics and the ecological system. Phytoremediation, the use of plants to reverse degradation and to restore ecological function, has been a promising approach. However, the symbiotic soil microbiota that influence the effectiveness of this method is not fully understood. I sampled the soil and roots of Pinus spp. (pines) at four sites along a gradient of vegetation recovery on the Superfund Site Brewer Gold Mine (SC), the Henry Knob Mine (SC), and Russell Gold Mine. The acidity, nutrient profile and heavy-metal contamination of collected soil is determined. DNA is extracted from the soil and root samples with PowerSoil DNA Isolation Kit, followed by preparation of multiplex PCR samples of the ITS region. Sequence reads generated through Illumina Miseq is processed through QIIME pipeline and taxonomy assigned through UNITE database. The results show a pattern of succession in fungal communities along a recovery gradient. While the mycorrhizal fungi on the least recovered site are dominated by Rhizopogon sp. and Pisolithus sp., sites with more recovered vegetation reveal a more diverse array of symbiotic fungi, including Amanita sp. and Russula sp.. These diverse fungi, although came later in the succession pattern, likely brings more diverse benefits to help their hosts cope with the stressful environment. This “bioprospecting” method could be applied to extract and amplify symbiotic fungi to facilitate revegetation efforts.Item Open Access A Systems Level Analysis of the Transcription Factor FoxN2/3 and FGF Signal Transduction in Sea Urchin Larval Skeleton Development and Body Axis Formation(2011) Rho, Ho KyungSpecification and differentiation of a cell is accomplished by changing its gene expression profiles. These processes require temporally and spatially regulated transcription factors (TFs), to induce the genes that are necessary to a specific cell type. In each cell a set of TFs interact with each other or activate their targets; as development progresses, transcription factors receive regulatory inputs from other TFs and a complex gene regulatory network (GRN) is generated. Adding complexity, each TF can be regulated not only at the transcriptional level, but also by translational, and post-translational mechanisms. Thus, understanding a developmental process requires understanding the interactions between TFs, signaling molecules and target genes which establish the GRN.
In this thesis, two genes, FoxN2/3, a TF and FGFR1, a component of the FGF signaling pathway are investigated. FoxN2/3 and FGFR1 have different mechanisms that function in sea urchin development; FoxN2/3 regulates gene expression and FGFR1 changes phosphorylation of target proteins. However, their ultimate goals are the same: changing the state of an earlier GRN into the next GRN state.
First, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN. Expression of foxN2/3 begins in the descendants of micromeres at the early blastula stage; and then is lost from PMCs at the mesenchyme blastula stage. foxN2/3 expression then shifts to the secondary mesenchyme cells (SMCs) and later to the endoderm. Here we show that, Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in the descendants of micromeres. The later endomesoderm expression is independent of the earlier expression of FoxN2/3 in micromeres and independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of larval skeleton. A number of proteins are necessary for skeletogenesis, and early expression of at least several of these is dependent on FoxN2/3. Furthermore, knockdown (KD) of FoxN2/3 inhibits normal PMC ingression. PMCs lacking FoxN2/3 protein are unable to join the skeletogenic syncytium and they fail to repress the transfating of SMCs into the skeletogenic lineage. Thus, FoxN2/3 must be present for the PMC GRN to control normal ingression, expression of skeletal matrix genes, prevention of transfating, and control fusion of the PMC syncytium.
Second, we show that the FGF-FGFR1 signaling is required for the oral-aboral axis formation in the sea urchin embryos. Without FGFR1, nodal is induced in all of the cells at the early blastula stage and this ectopic expression of nodal requires active p38 MAP kinase. The loss of oral restriction of nodal expression results in the abnormal organization of PMCs and the larval skeleton; it also induces ectopic expression of oral-specific genes and represses aboral-specific genes. The abnormal oral-aboral axis formation also affected fgf and vegf expression patterns; normally these factors are expressed in two restricted areas of the ectoderm between the oral and the aboral side, but when FGFR1 is knocked down, Nodal expands, and in response the expression of the FGF and VEGF ligands expands, and this in turn affects the abnormal organization of larval skeleton.
Item Open Access A Systems-Level Analysis of an Epithelial to Mesenchymal Transition(2012) Saunders, Lindsay RoseEmbryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive in vitro research has identified many EMT regulators. However, most in vitro studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.
In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype in vivo from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion.
A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression.
The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.
Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (∑Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of ∑Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by ∑Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg.
Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of ∑Erg targets.
Item Open Access An Analysis of the Correlation between Cortisol Levels and Anxious Behavior of Captive Aye-Ayes (Daubentonia madagscariensis) at the Duke Lemur Center(2016-05-06) Audra, BassI sought to determine if there existed a correlation between anxious behavior and cortisol levels for captive Aye-Ayes. I measured stress-related behavior by using an ethnographic methodology and focused on five specific behavior patterns: pacing, self-grooming, vigilance, human interaction, and vocalizations. I conducted 10 hours of observations on eight Aye-Aye individuals for a total of 80 hours. These observations were split between direct observations in an Aye-Aye’s enclosure and indirect observations by videotaping. Saliva samples were collected from each individual using chewed-on swabs; swabs were centrifuged and frozen for later extraction of cortisol concentrations. There was a general increase in anxious behavior when the Aye-Ayes were being observed directly; pacing showed the greatest difference. There was also a slight increase in cortisol concentrations when comparing weeks with direct and indirect observations. This study demonstrated that there is a potential relationship between anxious behavior and cortisol levels in Aye-Ayes that could be better understood with more research. Future studies should conduct more observation hours that are equally split between male and female Aye-Ayes. In addition, saliva samples should be consistently collected immediately after each observation. Finally, cortisol levels from saliva samples should be bolstered with other collection methods, specifically serum and fecal.Item Open Access An Avian Basal-Ganglia Forebrain Circuit Modulates the Reversal of Externally Reinforced Changes to Adult Zebra Finch Song(2017-05-20) Blazing, RobinSongbirds learn their songs through a trial and error process that shows remarkable similarities to human language learning, making them an ideal model for studying the neural substrates of vocal learning. Although adult zebra finch song is generally highly stable, a recent white noise aversive reinforcement learning paradigm has made it possible to shift the pitch of targeted song syllables. When aversive reinforcement is stopped, syllable pitch recovers to its stable baseline value over the course of several days. This recovery provides evidence that zebra finches are intrinsically motivated to match song performance to a previously memorized target version of the song. In this study, I tested the hypothesis that the lateral magnocellular nucleus of the anterior nidopallium (LMAN), a cortico-basal ganglia outflow nucleus implicated in both juvenile and externally reinforced adult learning, is necessary for intrinsically motivated pitch recovery. I drove down the fundamental frequency of targeted song syllables using white noise aversive reinforcement. I then performed bilateral electrolytic lesions of LMAN to determine whether normal pitch recovery would take place without LMAN activity. All three birds lesioned demonstrated significantly reduced recovery rates, providing convincing preliminary evidence that LMAN is implicated in song recovery. However, these results were not conclusive due to small sample size and the lack of histological data to verify lesion efficacy. Further characterization of the role of LMAN in pitch recovery could provide a valuable context for explaining phenomena associated with human language re-learning, such as how stroke victims might have difficulty recovering speech, or how adults are able access and easily re-learn elements of languages to which they were exposed during early childhood.Item Open Access An Experimental and Quantitative Analysis of E. coli Stress Response: Metabolic and Antibiotic Stressors(2014) Jalli, Inderpreet SinghA series of experiments and mathematical models explore the response of the bacteria E. coli to stressors. Experimentally, the effect of L-homocysteine, a non-protein amino acid, is explored, and via math models, the effect of trimethoprim, a common antibiotic, is also explored. Previous work on L-homocysteine labels it a stressor, and this assertion is refined via the presented work. A mathematical model that improves on a previous work published by Kwon et al. (2008) explores the response of E. coli to various supplementations of amino acids when exposed to trimethoprim. New methods of developing antibiotics and therapeutic drug treatments are also explored.
Item Open Access Analysis of crinkled Function in Drosophila melanogaster Hair and Bristle Morphogenesis(2012) Singh, VinayMutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia.
In Drosophila melanogaster mutations in MyoVIIa encoded by crinkled (ck) cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in Drosophila melanogaster to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.
The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.
We further demonstrate that over-expression of Quail (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of Alpha-actinin-GFP, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of Alpha-actinin-GFP in a ck/MyoVIIa null or near null background shows that Alpha-actinin-GFP cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing Alpha-actinin-GFP in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that Alpha-actinin-GFP over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that Twinfilin, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability.
In addition, we use yeast two-hybrid strategies to identify Slam as a protein that directly binds to ck/MyoVIIa. We show that Slam, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of Slam and loss-of-function mutations in Slam phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of Slam and RhoGEF2 association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via RhoGEF2.
Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.
Item Open Access Application of Phylogenetic Analysis in Cancer Evolution(2018) Ding, YuantongCancer is a major threat to human health and results in 1 in 6 deaths globally. Despite an extraordinary amount of effort and money spent, eradication or control of advanced disease has not yet been achieved. Understanding cancer from an evolutionary point of view may provide new insight to more effective control and treatment of the disease. Cancer as a disease of dynamic, stochastic somatic genomic evolution was first described by Nowell in 1976, and since then researchers have identified clonal expansions and genetic heterogeneity within many different types of neoplasms. The advancement in sequencing technology, especially single-cell sequencing, has open up new frontier by bringing the study of genomes to the cellular level. Phylogenetic analysis, which is a powerful tool inferring evolutionary relationships among various biological species or other entities based upon similarities and differences in their physical or genetic characteristics, has recently been applied to cancer studies and start to show promises in deciphering cancer evolution. However, new challenges have also arisen in experimental design, methodology and interpretation regarding to phylogeny of cancer cells. The overarching theme of this dissertation is to bring phylogenetic analysis to the context of cancer evolution. By using in silico simulations, I show the advantages and disadvantages of different sampling designs for phylogenetic analysis. Although bulk sequencing can hardly recover the topology of phylogenetic trees, I then developed a new method to infer sub-clone spatial distribution utilizing phased haplotypes from bulk sequencing. And lastly, I demonstrate the usage of phylogenetic analysis in breast cancer with multi-regional bulk sequencing and lung cancer with single cell sequencing.
Item Open Access Applications of Mathematical Modelling to Infectious Disease Dynamics in Developing Countries.(2013) Castorena, Christopher RobertMathematical modeling has proven to be an essential tool for the development of
control strategies and in distinguishing driving factors in disease dynamics. A key
determinant of a given model's potential to aid in such measures is the availability
of data to parameterize and verify the model. For developing countries in particular,
data is often sparse and difficult to collect. It is therefore important to understand
the types of data that are necessary for a modeling project to be successful. In this
thesis I analyze the value of particular types of data for a set of infections. The first
project analyzes the importance of considering age-specific mixing patterns in vaccine
preventable infections in which disease severity varies with age. The second project
uses a simulated data set to explore the plausibility of recovering the parameters of an
epidemiological model from a time series data set of monthly dengue haemorrhagic
fever reports.
Item Open Access Assessing Anti-B7-H3 Antibody and gp70 Cancer Vaccine Therapy for TNBC(2022-04-18) Sheu, LaurenImmunotherapy has emerged as a promising approach for addressing TNBC, an aggressive breast cancer subtype for which few targeted therapies exist. TNBC immunotherapy, however, is dominated by PD-1 immune checkpoint blockade (ICB), which only benefits a small minority of patients. To improve upon these initial efforts, we sought to target B7-H3 with TNBC immunotherapy, as this marker is expressed in a vast majority of TNBCs. In searching for an immunotherapy strategy, we decided to develop and assess a B7-H3-targeting antibody with cancer vaccine combination, as this regimen has recently shown resounding therapeutic benefits for another breast cancer type in the clinic (NCT00524277). To this end, we adapted NCT00524277’s treatment components for murine studies, creating M-m276, a B7-H3-targeting antibody, and a gp70-targeting cancer vaccine; both B7-H3 and gp70 are human/mouse TNBC-specific biomarkers. M-m276 and gp70 vaccine were administered in a B7-H3+ murine in vivo model of TNBC (i.e., Balb/C mice with lung-seeded 4T1). Significant survival extension was observed in mice treated with the combination therapy, relative to the monotherapies alone. Given these results, we next sought to better understand the mechanism of this combination therapy. Upon finding that M-m276 mediates antibody-dependent cellular phagocytosis (ADCP), we hypothesized that M-m276 augments the efficacy of cancer vaccines by increasing tumor antigen presentation to cytotoxic T lymphocytes (CTLs) through either cross-presentation or trogocytosis, two ADCP-linked processes. Although M-m276 was found to have no impact on cross-presentation, we found that M-m276 significantly increases the trogocytosis of tumor membrane by antigen-presenting cells (APCs) in vitro, enabling these APCs to present greater amounts of tumor antigen to CTLs. Altogether, our results support B7-H3-targeting antibody with cancer vaccine as a TNBC treatment strategy and propose a potential mechanism for how these therapy components interact.Item Open Access B-cyclin/CDK Regulation of Mitotic Spindle Assembly through Phosphorylation of Kinesin-5 Motors in the Budding Yeast, Saccharomyces cerevisiae(2012) Chee, Mark Kuan LengAlthough it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast, Saccharomyces cerevisiae, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in S. cerevisiae that may also be regulated by CDK phosphorylation.
In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in S. cerevisiae that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2 episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2 and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.
Item Open Access Balance Between Plant Growth and Defense: Transcriptional and Translational Control of Plant Immune System(2012) Wang, WeiThe activation and maintenance of plant immune responses require a significant amount of energy because they are accompanied by massive transcriptional reprogramming. Spurious activation of plant defense machinery can lead to autoimmune diseases and growth inhibition. So it is important for plants to tightly regulate the immune system to ensure the balance between growth and defense. However, neither the molecular mechanisms nor the design principles of how plants reach this balance are understood.
In this dissertation work, I showed how transcriptional and translational control of plant immune system can help avoid the constant immune surveillance and elicit a proper level of defense responses when necessary. These fine tunings of the immune system ensure the balance between growth and defense.
My research on the transcriptional regulation of plant defense responses led to the surprising discovery that even without pathogen, plant can 'anticipate' potential infection according to a circadian schedule under conditions that favor the initiation of infection. Functional analysis of 22 novel immune components unveiled their transient expression at dawn, when the infection is most likely to happen. This pulse expression pattern was shown to be regulated by the central circadian oscillator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) since these 22 genes are no longer induced in the cca1 mutant. Moreover, the temporal control of the transcription level of these 22 immune genes by CCA1 also fine tunes their expression pattern according to the perceptions of different pathogenic signals. At the basal defense level, the expression of these genes can be transiently induced upon perceptions of critical infection stages of the pathogen. When an elevated level of defense response is needed, the high expression levels of these genes are maintained to confer a stronger immunity against pathogen. Since this stronger form of defense may also cause the suicidal death of the plant cells, the interplay between the circadian clock and defense allows a better decision on the proper level of the immunity to minimize the sacrificial death. The circadian clock is also known to regulate the growth-related cellular functions extensively. So the circadian clock can help to balance the energy used in growth and defense through transcriptional regulation on both sides.
Besides the integrated control by the circadian clock, the translational control on a key transcription factor involved in the growth-to-defense transition can also maintain the balance between growth and defense.TBF1 is a major transcription factor that can initiate the growth-to-defense transition through transcriptional repression of growth-associated cellular functions and induction of defense-related machinery. Bioinformatics studies identified 2 upstream open reading frames (uORFs) encoding multiple phenylalanine at 5' of the translation initiation codon of TBF1. Under normal conditions, these 2 uORFs can repress the translation of TBF1 to prevent accidental activation. However, pathogen infection may cause rapid and transient depletion of phenylalanine, a well-known precursor for cell wall components and the SAR signal SA. This depletion signal can be reflected by the increase of uncharged tRNAPhe, which subsequently leads to the phosphorylation of eIF2á and the release of uORFs' repression on TBF1. These findings provided the molecular details of how uORF-based translational control can couple transcriptional reprogramming with metabolic status to coordinately trigger the growth-to-defense transition.
In summary, my dissertation work has identified previously unrecognized regulatory mechanisms by which plant immune responses are balanced with growth. These new findings will further investigations into these novel interfaces between plants and pathogens. Future studies will definitely further improve our understandings of the plant-microbe interactions.
Item Open Access Balancing the good and the bad: Assessing the positive and negative effects of alien species on native plant demography(2022) Loomis, AlexanderAlien species are considered one of the primary threats to native plant populations and their control is often prominent among proposed management actions. While negative alien effects are well documented, there are also many ways that alien species can have positive effects on native plant populations that may actually contribute to their persistence. Moreover, the effect of alien species on native plants can change in magnitude and direction over varying abiotic conditions. The success of native plant populations is determined by a mix of ecological and genetic factors. Alien (and native) species and abiotic conditions could also drive selection of plant traits. In order to understand the drivers of native plant population success in the face of changing climate and increasing prevalence of alien species, it is vital to understand the relationship between genotype, phenotype, and fitness of native plants. In chapter one, I quantified the effects of neighboring alien and native plants on all demographic rates in a population of the Hawaiian endemic plant Schiedea globosa, performing biannual censuses for 4 years to encompass relatively harsh and as well as benign seasons and years. The effects of alien neighbors were mixed but most often positive across many demographic rates in both harsh and more benign abiotic conditions, suggesting that alien neighbors benefit S. globosa plants through multiple mechanisms, such as nurse plant effects and associational resistance. The effects of heterospecific native neighbors were less often positive, indicating fundamentally different effects of native and alien neighbors on the demography of the focal native. These mixed effects highlight the need to consider potential benefits of alien species in the management of threatened native plants and that those benefits may be altered by changing abiotic conditions. In chapter two, I constructed population models for multiple Schiedea species across populations and years, using demographic rate regressions driven by the effects of alien and native neighbors, integrating the mixed effects of alien and native species on demographic rates of populations to project the net effect on population growth of native populations. The effects of alien and heterospecific native plant neighbors were mixed but most often positive across many demographic rates in both harsh and more benign abiotic conditions, suggesting that alien and native neighbors benefit native plants through multiple mechanisms, such as nurse plant effects and associational resistance. The effect of alien and heterospecific native neighbors on population growth was generally positive-- the mixed, but largely positive, net effects of alien and native neighbors on population growth highlight the need to consider potential benefits of alien, as well as native, species in the management of at-risk native plant populations, and that those benefits may be altered by changing abiotic conditions, as indicated by differing effects across (and within) years and populations. In chapter 3, I used paternal half-sibship pairs to measure the heritability of morphological traits under field conditions of the critically endangered Schiedea adamantis, which were found to be heritable in prior studies in greenhouse conditions, in reintroduced populations. I also performed a selection analysis, regressing fitness components against traits of outplants that I hypothesized might influence response to climate and alien and native neighbors to study the relationship between genotype, phenotype, and fitness of plants in restoration outplantings and assessing potential for evolutionary rescue. I found no significant heritability of any of the morphological traits. I did find evidence of selection, as leaf shape, area, and whole plant morphology had significant effects on fitness components (growth, survival, and reproduction), and significant interaction effects showing traits influenced fitness components differently at different levels of shade. Together, these results suggest that while variation in traits benefit individual plants in differing field conditions, these outplantings may not have the ability to respond to selection through evolution.
Item Open Access Behavioral and Geophysical Factors Influencing Success in Long Distance Navigation(2023) Granger, JesseMany animals can sense the earth’s magnetic field and use it to perform incredible feats of navigation; however, studying this phenomenon in the lab is difficult because behavioral responses to magnetic cues can be highly variable. My Ph.D. research attempts to fill this knowledge gap in the following ways: we first explore potential sources for this variability, including both natural and artificial sources of noise. We then examine the ways in which these natural sources of noise could be used to study magnetoreception in animals that are not feasible to study in the laboratory. Finally, we propose a possible solution for how navigating animals may overcome noise to still accomplish highly accurate migrations. Chapter 1 contains the relevant background and introduction. In Chapter 2, we conduct a synthetic review of natural and anthropogenic sources of radio frequency electromagnetic noise (RF) and its effects on magnetoreception. Anthropogenic RF has been shown to disrupt magnetic orientation behavior in some animals. Two sources of natural RF might also have the potential to disturb magnetic orientation behavior under some conditions: solar RF and atmospheric RF. In this review, we outline the frequency ranges and electric/magnetic field magnitudes of RF that have been shown to disturb magnetoreceptive behavior in laboratory studies and compare these to the ranges of solar and atmospheric RF. Frequencies shown to be disruptive in laboratory studies range from 0.1 to 10 MHz, with magnetic magnitudes as low as 1 nT reported to have effects. Based on these values, it appears unlikely that solar RF alone routinely disrupts magnetic orientation. In contrast, atmospheric RF does sometimes exceed the levels known to disrupt magnetic orientation in laboratory studies. We provide a reference for when and where atmospheric RF can be expected to reach these levels, as well as a guide for quantifying RF measurements.
In Chapter 3, we explore how these natural sources of noise may allow us to study magnetoreception in animals that are not feasible to study in the laboratory. Although it is difficult to perform behavioral experiments on baleen whales, it may be possible to use live stranding data (strandings that indicate the whale may have made a navigational error, rather than those having died at sea and washed ashore) as a tool to investigate the cues they use while navigating. Here we show that there is a 2.1-fold increase in the likelihood of a live gray whale (Eschrichtius robustus) stranding (n=186) on days with a high sunspot count than on low sunspot days (p<0.0001). Increased sunspot count is strongly correlated with solar storms – sudden releases of high-energy particles from the sun which have the potential to disrupt magnetic orientation behavior when they interact with earth’s magnetosphere. We further explore this relationship by examining portions of earth’s electromagnetic spectrum that are affected by solar storms and found a 3.7-fold increase in the likelihood of a live stranding on days with high solar radio flux (RF) as measured from earth (p<0.0001). One hypothesized mechanism for magnetoreception, the radical-pair theory, predicts that magnetoreception can be disrupted by RF radiation, and RF noise has been shown to disrupt magnetic orientation in certain species. To our knowledge, this is the first evidence that provides support for a specific magnetoreception mechanism in whales.
Finally, in Chapter 4, we propose a mechanism for how magnetoreceptive animals may overcome noise to perform incredibly accurate migrations. Many animals use the geomagnetic field to migrate long distances with high accuracy; however, research has shown that individual responses to magnetic cues in the laboratory can be highly variable. Thus, it has been hypothesized that magnetoreception alone is insufficient for accurate migrations and animals must either switch to a more accurate sensory cue or integrate their magnetic sense over time. Here we suggest that magnetoreceptive migrators could also use collective navigation strategies. Using agent-based models, we compare agents utilizing collective navigation to both the use of a secondary sensory system and time-integration. In our models, by using collective navigation alone, over 70% of the group is still able to successfully reach their goal even as their ability to navigate becomes extremely noisy. To reach the same success rates, in our models, a secondary sensory system must provide perfect navigation for over 73% of the migratory route, and time integration must integrate over 50 time-steps, indicating that magnetoreceptive animals could benefit from using collective navigation. Finally, we explore the impact of population loss on animals relying on collective navigation. We show that as population density decreases, a greater proportion of individuals fail to reach their destination and, in our models, a 50% population reduction resulted in up to a 37% decrease in the proportion of individuals completing their migration. We additionally show that this process is compounding, eventually resulting in complete population collapse.
Item Open Access Behavioral Measures and Ecological Correlates of Vision in Poeciliid Fishes(2022) Solie, SarahUnderstanding how animals see the world and how visual systems have evolved to meet the needs of particular animals are major goals of visual ecology research. The Poeciliidae are a diverse family of Neotropical freshwater fishes and are excellent models for visual ecology research given longstanding interest in visual signaling in this group. However, despite extensive research investigating the form and function of visual signals in the poeciliids, there remains a surprising paucity of research regarding poeciliid visual system function and evolution. To address this gap, my dissertation research sought to investigate: (1) how Trinidadian guppies (Poecilia reticulata) perceive visual stimuli that vary in spatial detail and contrast, (2) correlates of eye size and eye investment across P. reticulata populations that experience different threats from predation and, (3) visual signaling correlates of eye size across the family Poeciliidae.
The first chapter of this dissertation introduces the questions and the study system. In Chapter 2, I examine the ability of Trinidadian guppies (Poecilia reticulata) to perceive visual stimuli that vary in spatial frequency and contrast. Male P. reticulata bear complex body patterning made up of patches that vary in color, contrast, and size, and these visual signals that are known to be important in mate choice. However, the extent to which conspecifics are able to resolve the details of these patterns has historically been overlooked. I used an optomotor assay to measure the behavioral responses of eight individual P. reticulata (N = 4 males; 4 females) to rotating achromatic stimuli. Unsurprisingly, I found that P. reticulata are better able to perceive stimuli as they increase in contrast and decrease in spatial frequency. Moreover, I found that female P. reticulata may outperform males on optomotor tasks.
In Chapter 3, I investigate how predation environment contributes to eye size variation in P. reticulata. Eye size is an important predictor of visual abilities, and it varies widely across taxa. Moreover, eye size is known to be correlated with numerous ecological factors including habitat complexity, light availability, and predation risk. However, less is known about how differences in ecological parameters across populations influence variation in eye size within species. I measured the eye diameter and standard length of 45 females and 307 males from 21 populations of known geographic origin and predation environment. I found that eye diameter was correlated with predation environment after controlling for standard length, with fish from low-predation environments having eyes that are 5.5% - 7.9% larger in diameter than those from high-predation environments. I also found that sexual dimorphism in eye diameter appears to be driven by sexual dimorphism in standard length, as there was no significant effect of sex on eye diameter after accounting for standard length.
Finally, in Chapter 4 I examine variation in eye size across the Poeciliidae. The poeciliids are a diverse family of freshwater fishes to which Poecilia reticulata belongs, and the group exhibits substantial variation in the distribution and types of visual signals used in mate choice. I measured eye size and eye investment for 66 species of poeciliids and took a phylogenetic approach to test whether variation in eye morphology was correlated with aspects of visual signaling. I found that the presence of sexually selected visual signals was associated with greater eye investment and, in particular, that sexual dichromatism was associated with an approximately 6% increase in eye diameter investment compared to species without sexual dichromatism.