This thesis examines Escherichia coli pyruvate kinase type 1 (PK1), a regulatory enzyme core to energy metabolism. Specifically, this thesis characterises a series of mutations in PK1 that were found when populations of E. coli were evolved in a glucose-limited environment for 20,000 generations. The gene pykF, which codes for the PK1 enzyme, was found to have developed nonsynonymous mutations in all replicate populations. Although the mutations at the nucleotide level were not the same (i.e. not parallel), it is not clear whether parallel adaptation exists at the protein structure/function level. This study aimed to address this question by investigating the kinetic and biophysical properties of the wild-type and seven mutant enzymes. The recombinant wild-type PK1 enzyme used in this study was found to have steady state kinetics consistent with those previously reported. Unlike the rabbit kidney PK enzyme, E. coli PK1 was shown to have a very tight tetrameric structure (picomolar range), which was not affected by the enzyme’s substrates (PEP and ADP), or the allosteric effector (FBP), as judged by analytical ultracentrifugation with fluorescence detection. The mutated residues were highly conserved, and found to fall loosely into three groups: those at the active site (P70T, P70Q and D127N); those at the subunit interface (I264F, A301T and A301S); and at the allosteric binding site (G381A). The seven mutated PK1 enzymes were obtained by mutagenesis followed by protein purification. Steady state kinetic analysis showed that the mutated enzymes displayed a variety of functional changes, suggesting that the populations had not evolved in a parallel manner at the enzyme structure/function level. Mutations within the active site (P70T, P70Q and D127N) all showed a decrease in catalytic potency. P70 is located at the hinge connecting the A and B domains, which forms the active site. PK1-P70Q showed strong cooperative binding to PEP, similar to the wild-type enzyme, in the absence of FBP, whereas PK1-P70T had little cooperativity, suggesting changes in the active site. PK1-D127N showed severely attenuated activity, suggesting, for the first time, that this residue is essential for catalysis. Mutations at the subunit interface (I264F, A301T and A301S) all showed altered allosteric regulation, suggesting that this interface is important in the FBP allosteric response. PK1-I264F, which had lower activity, but greater affinity for PEP, displayed a decreased α-helix content (as judged by CD), indicating that a subunit interface helix that includes this residue had altered. Despite still having a similar response to FBP, PK1-G381A showed an increased affinity for PEP, which, together with an increased α-helix content, suggests that this mutation had changed the structure of the FBP binding domain. None of the mutated enzymes showed altered quaternary structure. Although the populations evolved parallel changes with respect to cell physiology, fitness, and gene expression, this study suggests, for the first time, that the populations have not evolved in a parallel way with respect to protein structure and function.

This PhD dissertation is focused on short tandemly repeated nucleotide patterns which occur extremely often across DNA sequences, called microsatellites. The main characteristic of microsatellites, and probably the reason why they are so abundant across genomes, is the extremely high frequency of specific replication errors occurring within their sequences, which usually cause addition or deletion of one or more complete tandem repeat units. Due to these errors, frequent fluctuations in the number of repetitive units can be observed among cellular and organismal generations. The molecular mechanisms as well as the consequences of these microsatellite mutations, both, on a generational as well as on an evolutionary scale, have sparked debate and controversy among the scientific community. Furthermore, the bioinformatic approaches used to study microsatellites and the ways microsatellites are referred to in the general literature are often not rigurous, leading to misinterpretations and inconsistencies among studies. As an introduction to this complex topic, in Chapter I I present a review of the knowledge accumulated on microsatellites during the past two decades. A major part of this chapter has been published in the Encyclopedia of Life Sciences in a Chapter about microsatellite evolution (see Publication 1 in Appendix II). The ongoing controversy about the rates and patterns of microsatellite mutation was evident to me since before starting this PhD thesis. However, the subtler problems inherent to the computational analyses of microsatellites within genomes only became apparent when retrieving information on microsatellite distribution and abundance for the design of comparative genomic analyses. There are numerous publications analyzing the microsatellite content of genomes but, in most cases, the results presented can neither be reliably compared nor reproduced, mainly due to the lack of details on the microsatellite search process (particularly the program’s algorithm and the search parameters used) and because the results are expressed in terms that are relative to the search process (i.e. measures based on the absolute number of microsatellites). Therefore, in Chapter II I present a critical review of all available software tools designed to scan DNA sequences for microsatellites. My aim in undertaking this review was to assess the comparability of search results among microsatellite programs, and to identify the programs most suitable for the generation of microsatellite datasets for a thorough and reproducible comparative analysis of microsatellite content among genomic sequences. Using sequence data where the number and types of microsatellites were empirical know I compared the ability of 19 programs to accurately identify and report microsatellites. I then chose the two programs which, based on the algorithm and its parameters as well as the output informativity, offered the information most suitable for biological interpretation, while also reflecting as close as possible the microsatellite content of the test files. From the analysis of microsatellite search results generated by the various programs available, it became apparent that the program’s search parameters, which are specified by the user in order to define the microsatellite characteristics to the program, influence dramatically the resulting datasets. This is especially true for programs suited to allow imperfections within tandem repeats, because imperfect repetitions can not be defined accurately as is the case for perfect ones, and because several different algorithms have been proposed to address this problem. The detection of approximate microsatellites is, however, essential for the study of microsatellite evolution and for comparative analyses based on microsatellites. It is now well accepted that small deviations from perfect tandem repeat structure are common within microsatellites and larger repeats, and a number of different algorithms have been developed to confront the challenge of finding and registering microsatellites with all expectable kinds of imperfection. However, biologists have still to apply these tools to their full potential. In biological analyses single tandem repeat hits are consistently interpreted as isolated and independent repeats. This interpretation also depends on the search strategy used to report the microsatellites in DNA sequences and, therefore, I was particularly interested in the capacity of repeat finding programs to report imperfect microsatellites allowing interpretations that are useful in a biological sense. After analzying a series of tandem repeat finding programs I optimized my microsatellite searches to yield the best possible datasets for assessing and comparing the degree of imperfection of microsatellites among different genomes (Chapter III) During the program comparisons performed in Chapter II, I show that the most critical search parameter influencing microsatellite search results is the minimum length threshold. Biologically speaking, there is no consensus with respect to the minimum length, beyond which a short tandem repeat is expected to become prone to microsatellite-like mutations. Usually, a single absolute value of ~12 nucleotides is assigned irrespective of motif length.. In other cases thresholds are assigned in terms of number of repeat units (i.e. 3 to 5 repeats or more), which are better applied individually for each motif. The variation in these thresholds is considerable and not always justifiable. In addition, any current minimum length measures are likely naïve because it is clear that different microsatellite motifs undergo replication slippage at different length thresholds. Therefore, in Chapter III, I apply two probabilistic models to predict the minimum length at which microsatellites of varying motif types become overrepresented in different genomes based on the individual oligonucleotide frequency data of these genomes. Finally, after a range of optimizations and critical analyses, I performed a preliminary analysis of microsatellite abundance among 24 high quality complete eukaryotic genomes, including also 8 prokaryotic and 5 archaeal genomes for contrast. The availability of the methodologies and the microsatellite datasets generated in this project will allow informed formulation of questions for more specific genome research, either about microsatellites, or about other genomic features microsatellites could influence. These datasets are what I would have needed at the beginning of my PhD to support my experimental design, and are essential for the adequate data interpretation of microsatellite data in the context of the major evolutionary units; chromosomes and genomes.

<p> This research examines the evolution, phylogenetic distribution, and functional explanations for a peculiar and often overlooked character seen in birds, herein called tracheal and esophageal displacement. Of special interest to this study is examining whether the trait was present in non-avian theropod dinosaurs. This study found that essentially all birds are characterized by a laterally displaced trachea and/or esophagus. The displacement may occur gradually along the neck, or it may happen immediately upon exiting the oropharynx. Displacement of these organs is the result of a heavily modified neck wherein muscles that create mobility restrictions in lizards, alligators, and mammals (e.g., <i>m. episternocleidomastoideus, m. omohyoideus,</i> and <i> m. sternohyoideus</i>) no longer substantially restrict positions in birds. Rather, these muscles are modified, which may assist with making tracheal movements. </p><p> An exceptionally well-preserved fossil theropod, <i>Scipionyx samniticus </i>, proved to be paramount. Its <i>in situ</i> tracheal and esophageal positions and detailed preservation (showing the hallmarks of displacement including rotation, obliquity, a strong angle, and a dorsal position in a caudad region of the neck) demonstrate that at least some theropods were characterized by tracheal and esophageal displacement. Ultimately, the presence of the trait correlates with a highly flexible neck, allowing slack and permitting for the organs to save length as they avoid the long curves of the S-shaped neck.</p>

No description available.

<p> Flower shape, color and size are extensively studied to both identify and classify different angiosperm taxa. The availability of well-supported molecular phylogenies produced using complex models of sequence evolution, coupled with an understanding of the genes that regulate morphological form in model organisms, and new methods to infer gene expression patterns in diverse species now allow us to understand the genetic basis of morphological differences among closely related species. Studies in Plantaginaceae, Gesneriaceae, Fabaceae and Brassicaceae show the importance of <i>CYCLOIDEA (CYC), RADIALIS (RAD)</i> and <i>DIVARICATA (DIV)</i> in regulating flower shape, but also show divergence in gene function within flowering plants. Previous studies in the zygomorphic model species <i>Antirrhinum majus </i> (snapdragon) have shown that <i>AmCYC</i> is expressed in the adaxial (dorsal) petals of flowers where it activates <i>AmRAD </i>. This expression of <i>AmRAD</i> within adaxial petals represses <i>AmDIV</i> expression causing <i>AmDIV</i> to be restricted to abaxial (ventral) and lateral petals. Like <i>Antirrhinum </i>, traditional <i>Diplacus</i> flowers have distinct dorsal, ventral and lateral petal identities. However, within the clade actinomorphic flowers have evolved independently on two occasions: once in <i>D. pictus </i> and once in <i>D. mohaviensis</i>. mRNA reveal <i> DIV</i> expression to be conserved between <i>D. pictus</i> and snapdragon, whereas <i>CYC</i> and <i>RAD</i> expression, and presumably function, differ between the two species. <i>DpCYC</i> is expressed in a narrow portion on the upper lip of abaxial petals, whereas <i> DpRAD</i> is expressed within both lateral and abaxial petals. <i> D. pictus</i> flowers are characterized by a novel upturned abaxial petal which may be linked to localized <i>CYC</i> expression along the upper surface of the structure. This study sheds new light on the mechanisms regulating flower shape in an endemic Californian monkey flower and shows the importance of testing hypotheses from model species such as <i>Arabidopsis </i> and snapdragon in non-model taxa such as <i>D. pictus</i> to undercover the true variety of mechanisms driving morphological evolution.</p>

<p> Key to understanding the transition that vertebrates made from water to land is determining the developmental and genomic bases for the changes. New bioinformatic tools provide an opportunity to automate the discovery, broaden the number of, and provide an evidence-based ranking for potential candidate genes. I sought to explore this potential for the fin/limb transition, using the substantial genetic and phenotypic data available in model organism databases. Model organism data was used to hypothesize candidate genes for the fin/limb transition. In addition, 131 fin/limb candidate genes from the literature were extracted and used as a basis for comparison with candidates from the model organism databases. Additionally, seven genes specific to limb and 24 genes specific to fin were identified as future fin/limb transition candidates.</p>

<p> Lipid-producing microalgae are a feedstock for commercial products such as nutritional supplements, aquatic animal feed, and biofuels. Unlike most algal phyla, the diatoms (Bacillariophyta) characteristically produce storage lipids throughout their entire lifecycle. In this study, lipids were extracted via chloroform-methanol and quantified as percent dry weight, &mgr;g/mL, and pg/100 &mgr;m³ and then analyzed for a phylogenetic signal by comparing the variability between lineages to the variability within lineages for each metric. These ten taxa were then paired with data gathered from the literature and examined for a phylogenetic signal using previously described methods. In the first analysis, there was greater variability between than within lineages during stationary growth when using percent dry weight as a metric. In the second analysis, a statistically significant phylogenetic signal was detected for nutrient-deplete growth experiments when examining the genus-level phylogeny (P = 0.013).</p>

This project represents an initial examination of the evolutionary history of the genus Pilobolus, and provides a starting point for developing a method to accurately classify members of this genus using molecular genetics. The data analysis presented in this paper suggests a potential evolutionary path in which P. umbonatus and P. sphaerosporus diverged (along a common path) from an original generic ancestor early, but independently, from the evolutionary course taken by P. kleinii. Both Maximum Likelihood and parsimony analyses concur in the branch patterns. Additionally, formation of species-specific clades between samples of P. sphaerosporus, P. kleinii and P. umbonatus suggests that there may be markers present in the ITS sequence that can be used for the development of a molecular test for the classification of species within the genus Pilobolus.Ball State UniversityMuncie, IN 47306 / Department of Biology

<p> Historical biogeography has a diversity of methods for inferring ancestral geographic ranges on phylogenies, but many of the methods have conflicting assumptions, and there is no common statistical framework by which to judge which models are preferable. Probabilistic modeling of geographic range evolution, pioneered by Ree and Smith (2008, <i>Systematic Biology</i>) in their program LAGRANGE, could provide such a framework, but this potential has not been implemented until now. </p><p> I have created an R package, "BioGeoBEARS," described in chapter 1 of the dissertation, that implements in a likelihood framework several commonly used models, such as the LAGRANGE Dispersal-Extinction-Cladogenesis (DEC) model and the Dispersal-Vicariance Analysis (DIVA, Ronquist 1997, <i> Systematic Biology</i>) model. Standard DEC is a model with two free parameters specifying the rate of "dispersal" (range expansion) and "extinction" (range contraction). However, while dispersal and extinction rates are free parameters, the cladogenesis model is fixed, such that the geographic range of the ancestral lineage is inherited by the two daughter lineages through a variety of scenarios fixed to have equal probability. This fixed nature of the cladogenesis model means that it has been indiscriminately applied in all DEC analyses, and has not been subjected to any inference or formal model testing. </p><p> BioGeoBEARS also adds a number of features not previously available in most historical biogeography software, such as distance-based dispersal, a model of imperfect detection, and the ability to include fossils either as ancestors or tips on a time-calibrated tree. </p><p> Several important conclusions may be drawn from this research. First, formal model selection procedures can be applied in phylogenetic inferences of historical biogeography, and the relative importance of different processes can be measured. These techniques have great potential for strengthening quantitative inference in historical biogeography. No longer are biogeographers forced to simply assume, consciously or not, that some processes (such as vicariance or dispersal) are important and others are not; instead, this can be inferred from the data. Second, founder-event speciation appears to be a crucial explanatory process in most clades, the only exception being some intracontinental taxa showing a large degree of sympatry across widespread ranges. This is not the same thing as claiming that founder-event speciation is the <i>only</i> important process; founder event speciation as the only important process is inferred in only one case (<i>Microlophus</i> lava lizards from the Galapagos). The importance of founder-event speciation will not be surprising to most island biogeographers. However, the results are important nonetheless, as there are still some vocal advocates of vicariance-dominated approaches to biogeography, such as Heads (2012, <i>Molecular Panbiogeography of the Tropics</i>), who allows vicariance and range-expansion to play a role in his historical inferences, but explicitly excludes founder-event speciation <i> a priori.</i> The commonly-used LAGRANGE DEC and DIVA programs actually make assumptions very similar to those of Heads, even though many users of these programs likely consider themselves dispersalists or pluralists. Finally, the inclusion of fossils and imperfect detection within the same likelihood and model-choice framework clears the path for integrating paleobiogeography and neontological biogeography, strengthening inference in both. </p><p> Model choice is now standard practice in phylogenetic analysis of DNA sequences: a program such as ModelTest is used to compare models such as Jukes-Cantor, HKY, GTR+I+G, and to select the best model before inferring phylogenies or ancestral states. It is clear that the same should now happen in phylogenetic biogeography. BioGeoBEARS enables this procedure. Perhaps more importantly, however, is the potential for users to create and test new models. Probabilistic modeling of geographic range evolution on phylogenies is still in its infancy, and undoubtedly there are better models out there, waiting to be discovered. It is also undoubtedly true that different clades and different regions will favor different processes, and that further improvements will be had by linking the evolution of organismal traits (e.g., loss of flight) with the evolution of geographic range, within a common inference framework. In a world of rapid climate change and habitat loss, biogeographical methods must maximize both flexibility and statistical rigor if they are to play a role. This research takes several steps in that direction. </p><p> BioGeoBEARS is open-source and is freely available at the Comprehensive R Archive Network (http://cran.r-project.org/web/packages/BioGeoBEARS/index.html). A step-by-step tutorial, using the <i>Psychotria</i> dataset, is available at PhyloWiki (http://phylo.wikidot.com/biogeobears). </p><p> (Abstract shortened by UMI.)</p>

This thesis uses a range of different size spectra to compare contrasting benthic habitats in the aquatic realm. Temporal and spatial variation in benthic size spectra were investigated across a full salinity gradient (i.e. from freshwater, through estuarine to marine) in the River Yealm, south Devon, in order to gauge the influence of large differences in taxonomy and evolutionary history. Abundance and biomass size spectra showed a similar pattern among sites in all seasons but winter, suggesting that the size structure of benthic communities may be similar in sites with very different community compositions. A subsequent study comparing size spectra across salinity by employing artificial substrata suggested that substratum type also had little effect on the size structure of these benthic communities. A technique was developed for obtaining microbial size distributions for benthic communities and showed that microbial size structures were also similar between the marine and freshwater sites within the Yealm system. A final study demonstrated that the shape of size spectra was clearly affected by metal contamination. Size spectra across a salinity gradient -(i.e. from freshwater to lower estuary) in the highly contaminated Fal system were very different to those in the uncontaminated Yealm, due mostly to the low macrofaunal abundance in the former. This thesis is the first to assess patterns in benthic size spectra across a full salinity range in the same system. It is hoped that it will provide a base line for further studies in this exciting research area in macroecology and that biomass spectra might also prove useful as metrics for biomonitoring.