All eukaryotes contain at least three homologous RNA polymerases (RNAPI, RNAPII, and RNAPIII), which are directed towards their target genes by interactions with RNAP-specific general transcription factors (GTFs). Although the RNAPs transcribe a subset of nuclear genes, it is unknown if they have evolved altered functionality. An in silico approach to identify putative differences in the three RNAPs was used to identify whether any functional differences likely exist by identifying amino acids that have experienced shifts in evolutionary rates and by identifying length differences between the cleft loops of the RNAPs. The difference in the levels of concerted evolution experienced by the genes transcribed by RNAPs was then exploited to test the molecular coevolution hypothesis of Dover and Flavell (1984. Cell . 38:622-623.). According to the molecular coevolution hypothesis, concertedly evolving DNA increases the evolutionary rate of any interacting proteins. We thus compared the evolutionary rates of the three RNAPs and their GTFs, since their target genes undergo different levels of concerted evolution. Finally, the origins of the 5 subunits that are specific to RNAPIII were examined, since no homologous relationships have been identified thus far for any of these subunits. Several sites that have experienced shifts in substitution rates in the ancestral RNAPs were found in all three enzymes and these sites were clustered near the active sites in all cases. Several cleft loops with different lengths (in amino acids) between the three RNAPs were also identified. The validity of the molecular coevolution hypothesis was largely confirmed via the demonstration that most subunits of RNAPI evolve faster than those of RNAPIII and most subunits of RNAPIII evolve faster than RNAPII. This is consistent with the molecular coevolution hypothesis because RNAPI experiences higher levels of concerted evolution than the target genes of RNAPIII. The evolutionary rates of the GTFs of RNAPI and RNAPIII were also higher than those of RNAPII. Finally, four of the five RNAPIII-specific subunits were identified as homologs of RNAPII GTFs, indicating that several of the GTF paralogs existed in ancestral RNAPIII and were subsequently recruited to the enzyme before the diversification of eukaryotes.

Antibiotic resistance is generally associated with a cost in terms of reduced competitive fitness in the absence of antibiotics. Despite this 'cost of resistance', the cessation of antibiotic treatment does not result in significant reductions in the prevalence of resistance. The maintenance of resistance, in spite of the costs, has been attributed to the rarity of reversion mutations, relative to compensatory mutations at other loci in the genome. However, the large size of bacteria populations, and the potential for migration, suggest that reversion mutations should occasionally be introduced to resistant populations. In this thesis, I show that additional mechanisms can prevent fixation of reversion mutations even if they do occur. Using an experimental evolution approach, with rifampicin resistance in Pseudomonas aeruginosa as a model system, I measured the costs of resistance in several environments and followed the adaptive dynamics of resistant populations where a sensitive lineage had invaded by migration. The results suggest that several additional mechanisms contribute to the maintenance of antibiotic resistance. Most rifampicin resistance mutations are not unconditionally costly in all environments, suggesting that migration between environments could maintain a resistant reservoir population. In environments where resistance is initially costly, the fixation of a revertant is not guaranteed, even if introduced through migration. Revertant fixation was impeded or prevented by clonal interference from adaptation in the resistant strain. Revertants that did successfully replace the resistant strain were forced to adapt to do so. Contrary to assumptions in the existing literature, fitness in the resistant strains was not recovered by general compensatory mutations, but instead by adaptive mutations specific to the environment. The data challenge several assumptions about the maintenance of antibiotic resistance: that resistance mutations are always costly, that the rarity of back mutations prevents the reversion of resistance, and that resistant strains recover fitness by compensatory mutations.

<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> 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>

<p>Centromeres are the chromosomal loci where microtubule spindles bind, via the kinetochore, during mitosis and meiosis. Paradoxically the centromere, as a functional unit, is essential to guarantee faithful chromosome segregation, whereas its underlying DNA sequences and associated kinetochore proteins are fast evolving. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. In spite of their importance, very little is known about the degree to which centromeric tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from species using publicly available genomic sequence and our own data. We found that despite an overall lack of sequence conservation, centromeric tandem repeats from diverse species showed similar modes of evolution. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. In addition, we performed a survey of fungi genomes for the presence of high-copy tandem repeats, but found little evidence to suggest that high-copy centromeric repeats are a common feature feature in fungi, with the possible exception of the <i>Zygomycota</i>. phylum. Finally, in most species the kinetochore assembles at a single locus, but in some cases the kinetochore forms along the entire length of the chromosomes forming holocentric chromosomes. Following a literature review we estimate that holocentricity is very common and has evolved at least thirteen times.

<p> In this work, I describe my doctoral work studying the regulation of transcription with both computational and experimental methods on the natural genetic variation in a population. This works integrates an investigation of the consequences of polymorphisms at three stages of gene regulation in the developing fly embryo: the diversity at <i>cis</i>-regulatory modules, the integration of transcription factor binding into changes in chromatin state and the effects of these inputs on the final phenotype of embryonic gene expression.</p>