<p> Adaptive Laboratory Evolution (ALE) methodologies are used for studying microbial adaptive mutations that optimize host metabolism. The Systems Biology Research Group (SBRG) at the University of California, San Diego, has implemented high-throughput ALE experiment automation that enables the group to expand their experimental evolutions to scales previously infeasible with manual workflows. The data generated by the high-throughput automation now requires a post-processing, content management and analysis framework that can operate on the same scale. We developed a software system which solves the SBRG's specific ALE big data to knowledge challenges. The software system is comprised of a post-processing protocol for quality control, a software framework and database for data consolidation and a web platform named ALE Analytics for report generation and automated key mutation analysis. The automated key mutation analysis is evaluated against published ALE experiment key mutation results from the SBRG and maintains an average recall of 89.6% and an average precision of 71.2%. The consolidation of all ALE experiments into a unified resource has enabled the development of web applications that compare key mutations across multiple experiments. These features find the genomic regions <i> rph, hns/tdk, rpoB, rpoC</i> and <i>pykF</i> mutated in more than one ALE experiment published by the SBRG. We reason that leveraging this software system relieves the bottleneck in ALE experiment analysis and generates new data mining opportunities for research in understanding system-level mechanisms that govern adaptive evolution.</p>

<p><i>Pediomelum esculentum</i> (commonly prairie turnip) is a perennial legume of the Great Plains, consisting of a deep taproot and large edible tuber, and has served as a nutritious staple in Native American diets. The tuber is capable of storing up to 20 percent protein by weight. <i> P. esculentum</i> is a legume, but not a prominent nodule former; instead, it grows in nitrogen-limited soils and produces large amounts of protein. This suggests the involvement of biological nitrogen fixation. We have investigated the presence of diazotrophic endophytes in <i>P. esculentum</i>. Bacteria were isolated from wild plants on nitrogen free media, identified with their partial 16S rRNA gene sequences, and screened for the presence of the nitrogen fixation gene <i>nifH</i>. Select isolates were applied as a co-inoculum to seedlings grown under gnotobiotic conditions in a growth chamber with no nitrogen source. Seedlings in both the inoculated and uninoculated group developed nodules and showed no signs of nitrogen stress. Bacteria isolated from the nodules and tubers of both groups were closely related to the same <i>Bacillus</i> bacterium isolated from seeds germinated under sterile conditions, according to partial <i>16S rRNA </i> sequences. Bright field and fluorescence imaging revealed bacteria present in the intercellular space of four-week-old tubers and in the sterile germinated seeds. Sectioning and imaging of the nodules show a central nodule vasculature and infected cells extending inwards to the main root vasculature. Nitrogen fixation in the plants was indirectly confirmed by acetylene reduction. Our results suggest <i>P. esculentum</i> has formed a unique symbiosis with a nitrogen fixing <i>Bacillus</i> bacterium that transmits vertically in the seeds and induces nodules. </p>

<p> The deep sea is the Earth&rsquo;s largest ecosystem and harbors a unique and largely endemic fauna. Although most research has focused on the ecological mechanisms that allow coexistence, recent studies have begun to investigate how this remarkable fauna evolved.. My work quantifies geographic patterns of genetic variation and investigates potential mechanisms that shape evolution in the deep ocean. </p><p> Bathymetric genetic divergence is common in the deep sea with population structure typically decreasing with depth. The evolutionary mechanisms that underlie these patterns are poorly understood. Geographic patterns of genetic variation indicated that the protobranch bivalve <i>Neilonella salicensis </i> was composed of two distinct lineages separated bathymetrically. Genetic diversity was greater in the lower-bathyal clade of <i>N. salicensis </i> than the upper to mid-bathyal clade. In a co-occurring mid-bathyal protobranch <i>Malletia johnsoni,</i> population differentiation was greater among samples than the confamilial lower-bathyal <i>Clencharia abyssorum,</i> though, genetic diversity was similar. These patterns suggest general trends do not always hold and fine scale patterns of gene flow need to be thoroughly investigated. </p><p> Little is known about the ecological or evolutionary mechanisms that might promote divergence or maintain population structure. Oxygen minimum zones (OMZs), which cover enormous regions of the deep ocean, might hamper gene flow by precluding larval dispersal. To test this, genetic patterns of the wood-boring bivalve <i>Xylophaga washington</i> were quantified across the northeastern Pacific OMZ. Results indicate two clades were apparent, one throughout the OMZ and one within and below it, possibly segregated by a historically stronger OMZ or other environmental factors that vary with depth. A similarly uninvestigated evolutionary factor with potentially large impacts is selection on mitochondrial DNA. Positive selection is apparent in the mitochondrial DNA of shallow water and deep-sea crabs, shrimp, and fishes, possibly related to any of the myriad factors that differ between the two habitats. </p><p> The deep sea is biogeochemically important and is highly impacted by climate change and anthropogenic factors. Genetic patterns in this habitat are very complex. This work suggests gene flow is inhibited at many scales, both across bathymetric gradients and within small bathymetric ranges.</p><p>

Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In addition, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (Slice Excision, SE). For making excisions precisely we first carried out a cell fate analysis for elbow forming tissue in the developing limb and carefully determined the tissue to be removed. Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, first we made a gene expression analysis for the elbow joint because a specific gene expression analysis for the elbow joint was not available in the literature. Among the genes analyzed, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60 hours after the surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE, whereas Col-9 is down-regulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. Various growth factors have been shown to trigger or enhance the regenerative response in different models and organs. The regeneration response we observe in WE is present in 50% of the embryos. In order to test the effect of growth factors on this response, we implanted window excised limbs with BMP-2, Noggin, or BSA beads as control. BMP-2 inhibits the joint regeneration, while Noggin does not improve regeneration of the joint tissue. On the other hand, Noggin treatment resulted in elongation of the cartilage elements from the amputated surface This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans / acase@tulane.edu

<p> The formation and maintenance of romantic pair bonds is a well-represented topic in human evolutionary sciences. This extensive body of work, drawn mostly from the field of evolutionary psychology, has proposed mechanisms for attracting a mate (e.g., resource display, physical cues), attaining a mate (e.g., intrasexual competition), and keeping a mate (e.g., competitor derogation, emotional manipulation). However, this evolutionary model of human pair bonding has not fully addressed relationship termination. If we accept that we have an evolved suite of behaviors that encourage and facilitate pair bonding, then we must also look to breakups and ask whether evolution has played a role in shaping &ldquo;heartbreak&rdquo;&mdash;the post-relationship grief (PRG) which many individuals endure.</p><p> The evolutionary model of human mating predicts divergent mating &ldquo;agendas&rdquo; for men and women. The first step in our research program was to conduct a modest pilot study to address how and when PRG differs between men and women. This pilot study is included as Chapter One for convenience. Having concluded that many of the existing suppositions about breakups were not supported by our initial inquiry, we set out to expand and revise the current model so that it can be used to make accurate predications regarding a more complex suite of variables (e.g., life history, sexuality). Chapter Two explains the logic and implications of this expansion via the example of a specific breakup scenario: the loss of a woman&rsquo;s partner to a romantic rival.</p><p> After presenting the possible evolutionary cause and adaptive benefits of PRG, we next tested both new and existing hypotheses as they relate to biological sex differences (Chapter Three) and life history variation (Chapter Four) in PRG. This quantitative foundation for ongoing qualitative study concludes with an overview of PRG in a population that is sorely underrepresented in evolutionary literature&mdash;individuals whose sexual orientation is not exclusively heterosexual.</p>

<p> It is often presumed that evolutionary reduction is tantamount to deconstruction, or even destruction, because relaxed selective forces have been insufficient to maintain the organ in its original state. However, studies on reduction are often limited by a lack of diversity, both of related species exhibiting reduction and of the reduced form itself. There have also been very few studies on the reduction of compound eyes, despite the fact that their near ubiquity among arthropods alone makes them perhaps the most common type of eye. Bat flies (Streblidae and Nycteribiidae) are a group of dipterans that exhibit variable degrees of compound eye reduction, and therefore provide the opportunity to study reduction of this organ in a phylogenetic context. The first chapter of this work reports on behavioral experiments demonstrating that the eyes of one bat fly species, <i>Trichobius frequens</i>, are functional, and that they neither exhibit phototaxis typical of other dipteran species, nor move toward a light source. The second chapter uses molecular phylogenetics to identify a correlation between eye and wing morphology. The results also suggest that secondary to their eye reduction, bat flies (at least in the case of New World specie, including <i>Trichobius spp.</i>) have secondarily experienced a shift in the structure of their facets that is convergent with other insects whose eyes have been selected for increased sensitivity. In the final chapter, histological and optical analyses of <i>T. frequens </i> eyes are used to reveal significant structural changes to the microstructure of its ommatidia that increase sensitivity at the expense of acuity. Many of these changes are also convergent with similar adaptations that have been demonstrated to increase sensitivity in organisms that function in reduced light environments. The results of these analyses suggest that reduction in <i> T. frequens</i> eyes may have been part of an active remodeling process resulting from a shift in the relative importance of sensitivity and acuity. As this is a process of reduction not generally considered, the findings here turn our attention to alternative hypotheses that should be considered when studying evolutionary reduction of any organ.</p>

<p> Microbial biofilms on leaf litter can assimilate nutrients, thus altering the elemental composition (i.e. stoichiometric composition) of detritus. Aquatic macroinvertebrates consuming stoichiometrically-altered detritus may increase, decrease, or not change growth rates depending upon their body stoichiometry. Invertebrates with high body phosphorus (P) can allocate more P to rRNA, increasing protein production and growth. Thus, if diet nutrient limitation is alleviated, high P invertebrates are expected to increase growth rates. Conversely, low P invertebrates might not respond or decrease growth rates, due to the metabolic costs of eliminating excess nutrients. Diptera from two families were used in growth studies and fed either maple or oak leaves with different levels of carbon:phosphorus (C:P) to determine if diet P influenced growth rates and body stoichiometry of these high P organisms. </p><p> <i>Tipula abdominalis</i> from the family Tipulidae, were hypothesized to increase growth and consumption rates, and decrease body C:P as diet C:P decreased. It was also hypothesized that the proportion of consumed material composed of a particular element used in growth would increase (GGE), and that the diet ratio at which neither C or P is limiting, and at which maximum growth would occur, would decrease as diet C:P decreased (TERC:P). It was observed that <i>T. abdominalis</i> growth and consumption varied based on leaf type and elemental composition. <i>Tipula abdominalis</i> fed oak had high growth (5.4% day-1) and consumption (1.98mg mg tipulid-1 day-1) compared to maple diets, while individuals fed maple increased growth (2.6 to 5.0% day-1) and consumption (1.1 to 2.1mg mg tipulid-1 day-1) across treatments as maple C:P decreased. <i>Tipula abdominalis</i> were overall homeostatic in body stoichiometry. Contrary to expectations, GGEs decreased as diet C:P decreased, maintaining stoichiometric homeostasis. Also, current TER calculations were not representative of observed growth rates. Thus, results indicate that leaf stoichiometry and leaf type interact to influence growth rates. </p><p> Three genera of Chironomidae (<i>Chironomus, Polypedilum, Micropsectra </i>) were fed a gradient of diet C:P to determine if there are differences in taxon-specific growth responses to changes in diet stoichiometry. It was hypothesized that genera with higher body P would increase growth more than genera with lower body P, and that body P in high P genera would increase as they consumed more dietary P. The three genera had different growth responses to decreased diet C:P that did not appear to be influenced by organism body stoichiometry or diet stoichiometry. <i>Chironomus</i> fed maple and oak and <i>Micropsectra</i> fed maple had similar growth rates (17.5% day^-1), while having significantly different body C:P, 141 and 249 for <i>Chironomus</i> fed oak and maple, respectively, and 359 for <i>Micropsectra</i> fed maple. Despite similarities in diet C:P ranges (1000 to 8000), <i>Chironomus</i> and <i> Polypedilum</i> had different growth responses: <i>Chironomus</i> had high growth, while <i>Polypedilum</i> fed maple did not grow and lost mass (- 4.8% day^-1), and Polypedilum fed oak exhibited relatively low growth rates (5.9% day^-1). Leaf type and genus identity appear to be important factors in determining growth response. However, most Dipterans in this study either increased growth or had no response to dietary P enrichment. Understanding how organism genus identity interacts with leaf type and leaf stoichiometry to influence growth rates will allow more accurate predictions of community changes in nutrient enriched systems. </p>

<p> Many organisms live in temporally or spatially heterogeneous environments. One adaptive response to environmental heterogeneity is phenotypic plasticity, or the ability of an organism to change its phenotype in response to environmental variation. Plasticity is particularly important for plants, which cannot easily escape environmental stresses (e.g., competition, herbivory, or drought), and therefore is common in many morphological and physiological traits that allow plants to cope with stresses. Despite numerous examples of the adaptive value of plasticity in plant traits, the reaction norms of many plant traits do not evolve to their predicted optima: there is often considerable genetic variation in trait plasticities, and some populations display sub-optimal responses to environmental stresses. This mismatch may reflect a balance between the fitness benefits (adaptive value) of plasticity and evolutionary constraints, such as strong genetic correlations with other traits, a lack of genetic variation in the reaction norm, and fitness costs of plasticity. </p><p> This dissertation focuses on the evolution of plasticity in plant defense responses to insect herbivores. Plant induced defenses are well-studied ecologically, but little is known about potential constraints on their evolution or the effects of these constraints on insect herbivores. Using the andromonecious herb <i>Solanum carolinense</i>, I examined the adaptive value of plasticity and tested for several evolutionary constraints on plant induced defenses and tolerance. I found damage-induced plasticity in several defense traits, and herbivores significantly reduced plant fitness in the field. However, neither tolerance nor plasticity in induced defenses was adaptive, and most traits were selected against (i.e., costly) in both environments with and without insect herbivores. </p><p> I uncovered several genetic constraints on plasticity in the plant defense response, including a) a lack of genetic variation in some resistance traits, b) strong genetic correlations between constitutive resistance and inducibility, and c) genetic correlations among the plasticities of different defense traits. Combined, these genetic constraints structured variation in the plant-mediated interaction between the specialist folivores <i>Manduca sexta</i> and <i>Leptinotarsa juncta</i>; this is the first study to demonstrate that genetic trade-offs within a plant species can affect interactions at higher trophic levels. </p><p> Although there were no fitness costs of plasticity in induced chemical/mechanical defenses, there were fitness costs of tolerance, and I was able to detect selection acting directly on trait plasticities. Selection on plasticities was generally positive or stabilizing, indicating fitness benefits of canalization of two defense traits I measured. Fitness costs of tolerance were present both in environments with and without herbivores, meaning that tolerance of herbivory was never adaptive. My dissertation is the first to simultaneously test for both the adaptive value and a breadth of evolutionary constraints (including costs of plasticity) on the plant defense response. </p><p> Combined, my results indicate that selection should reduce both the plasticity and mean expression of <i>S. carolinense</i> defenses; however, correlated selection on plastic traits or a lack of genetic variation in plasticity may prevent this from occurring. Most importantly, these results suggest that two basic predictions of evolutionary theory - that phenotypic plasticity should be both adaptive and costly - do not always hold true for plant defense traits. This underscores the importance of measuring selection on any plastic defense trait assumed to be adaptive, and investigating other potential evolutionary constraints on defensive plasticity besides fitness costs. My study shows that genetic correlations may also be important, but correlations among defense trait plasticities have been rarely investigated and most studies do not account for the presence of these correlations when examining selection on individual defense traits.</p>

<p> The formation of mineralized tissue was critical to the evolution and diversification of metazoans and remains functionally significant in most animal lineages. Of special importance is the protein found occluded within the mineral matrix, which facilitates the process of biomineralization and modulates the final mineral structure. These skeletal matrix proteins have well been described in several species, including the sea urchin <i> Stronglyocentrotus purpuratus,</i> an important model organism. Biomineralization research is limited in other echinoderm classes. This research encompasses the first description of mineral matrix proteins in a member of the echinoderm class Ophiuroidea. This work describes the skeletal matrix proteins of the brittle star <i>Ophiocoma wendtii</i> using bioinformatic and proteomic techniques. General characteristics of matrix protein are described and a number of candidate biomineralization related genes have been identified, cloned, and sequenced. The unique evolutionary and biochemical properties of brittle star skeletal matrix proteins are also described.</p>

<p>This project explores species boundaries, coevolution, biodiversity, parasite life cycles, and ecology using the cestode (tapeworm) fauna parasitizing the spiny dogfish, <i>Squalus acanthias</i>, and several of its close relatives. One of the aims was to verify the species identity of all the shark specimens from which cestodes were collected. Chapter 1 details use of the elasmobranch "barcoding" gene, NADH2, to verify host identifications, as well as to raise doubt about the wisdom of recognizing the Black Sea population of <i>S. acanthias</i> as a distinct subspecies. Chapter 2 examines diversity in the monotypic cestode genus, <i>Trilocularia</i>, throughout the widespread anti-tropical distribution of <i>S. acanthias</i> (i.e., the North and South Pacific, the North Atlantic, and the Black Sea), and in its congeners. An integrative approach, including morphological (i.e., light microscopy, histology, and scanning electron microscopy) and molecular methods (i.e., 28S, ITS1, 16S genes), was employed. Results reveal a large amount of undiscovered diversity in this genus and suggest that species of <i> Trilocularia</i> may be undergoing speciation more rapidly than their hosts. Chapter 3 describes one of the new species discovered, <i>Trilocularia eberti</i> n. sp. from <i>S.</i> cf. <i>mitsukurii</i>, and provides a prototype for future descriptions of species in this genus. Chapter 4 investigates microthrix variation in <i>Trilocularia</i> from the stomach and the spiral intestine of <i>S. acanthias</i> off Rhode Island. Results suggest that variation seen within a host individual is likely due to developmental changes rather than species differences. Chapter 5 aims to further the understanding of cestode infections in a marine environment through space and time by examining seasonal infection parameters in the cestode community of <i>S. acanthias</i> from Rhode Island across three years. While some general trends may be maintained across disparate localities, spatial variation is likely due to differences in accessibility to intermediate hosts and host diet across sites. The knowledge gained from understanding cestode infections in the vast ocean environment allows us to speculate about the factors driving fluctuations in parasite infections in elasmobranchs. </p>