Dissertations / Theses on the topic 'Evolution (Biology)'

The bacterial origin of mitochondria from an ancient endosymbiosis is now widely accepted and the mitochondrial ancestor is generally believed to belong to the bacterial subdivision α-proteobacteria. The high fraction of mitochondrial proteins encoded in the nucleus has commonly been explained with a massive transfer of genes from the genome of the ancestral mitochondrion.

The aim of this work was to get a better understanding of the mitochondrial origin and evolution by comparative genomics and phylogenetic analyses on mitochondria and α-proteobacteria. To this end, we sequenced the genomes of the intracellular parasites Bartonella henselae and Bartonella quintana, the causative agents of cat-scratch disease and trench fever, and compared them with other α-proteobacteria as well as mitochondrial eukaryotes.

Our results suggest that the adaptation to an intracellular life-style is coupled to an increased rate of genome degradation and a reduced ability to accommodate environmental changes. Reconstruction of the α-proteobacterial ancestor and phylogenetic analyses of the mitochondrial proteome in yeast revealed that only a small fraction of the proteins used for mitochondrial functions could be traced to the α-proteobacteria. Furthermore, a substantial fraction of the mitochondrial proteins was of eukaryotic origin and while most of the genes of the α-proteobacterial ancestor have been lost, many of those that have been transferred to the nuclear genome seem to encode non-mitochondrial proteins.

Symbiosis is a widely common phenomenon in nature and has undoubtedly contributed to the evolution of all organisms on earth. Symbiotic associations can be of varying character, such as parasitic or mutualistic, but all imply a close relationship. To study the evolution of genomes of insect endosymbionts, we have sequenced the genomes of the mutualist Buchnera aphidicola from the aphid Schizaphis graminum (Sg) and the reproductive manipulator Wolbachia pipientis strain wRi from Drosophila simulans that show strikingly different evolutionary patterns.

The comparison between the genome of B.aphidicola (Sg) and the genome of B.aphidicola from the aphid Acyrthosiphon pisum (Ap), that are believed to have diverged 50 million years ago, revealed a perfect gene order conservation and loss of only 14 genes in either of the lineages. In contrast, the rate of nucleotide turnover is very fast probably due to relaxed selection and loss of DNA repair genes. The genomic stasis observed in Buchnera was attributed to the loss of repeats and of the gene recA.

In striking contrast to the genomes of B.aphidicola, a vast amount of repeats were found in the genome sequence of W.pipientis strain wMel. The comparison between the genomes of W.pipientis strain wRi and W.pipientis strain wMel shows that a lot of rearrangements have occurred since their divergence. The massive amount of repeats might stem from relaxed selection pressure but possibly also from selection to create variability via recombination.

Comparisons between pairs of genomes from closely related bacteria showed that the stability of gene order and content is connected to an intracellular lifestyle and indicated that homologous recombination between repeats is an important mechanisms for causing intrachromosomal rearrangements. Our studies show that the lifestyle of a bacterium to a great extent shapes the evolution of their genetic material and future capabilities to adapt to new environments.

Individuals of a given population share more behavioural traits with each other than with members of other populations. For example, in humans, traditions are specific to regions or countries. These cultural relationships can tell us about the history of the populations, their origin and the amount of exchange between them. In birds, regional dialects have been described in many species. However, the mechanisms with which dialects form in populations is not fully understood because it is difficult to analyse experimentally. Translocated populations, with their known histories, offer an opportunity to study these mechanisms. From the study of bird vocalisations we can make inferences regarding population structure and relationships as well as their history, individual behavioural state, neuronal and physiological mechanisms or development of neuronal learning. Too achieve this, cross-disciplinary approaches are necessary, combining field work, bioacoustic methods, statistical tools such as machine learning, ecological knowledge and phylogenetic methods. Here, I will describe computational methods for the treatment and classification of bird vocalisations and will use them to depict the relationships between bird populations. First, I discretise the data in order to define the cultural traits. Then phylogenetic tree-building methods are used. Two approaches are possible, first to map these traits onto known phylogenies and, second, to directly build the phylogeny of these traits. I describe the application of these methods to test several hypothesis on bird songs evolution related to both their history and the mechanisms with which they evolve. Evidence for the presence of dialects in the Puget Sound white-crowned sparrow (Zonotrichia leucophrys pugetensis) is provided on the basis of the syllable content of the songs. The absence of vocal sexual dimorphism is reported in the Australasian gannet (or takapu, Morus serrator), a member of the Sulidae family for which extensive sexual dimorphism has been reported in other species. Subsequently, convergence between the begging calls of several cuckoo species and their respective hosts is suggested by various bioacoustic methods. In addition, the male calls of the hihi (or stitchbird, Notiomystis cincta) is analysed in an island population. The corresponding pattern of variation suggests a post-dispersal acquisition of calls via learning which is in agreement with the most related species in the revised phylogeny of the hihi. Finally, the mechanisms of song evolution are depicted in translocated populations of tieke (or saddleback, Philesturnus carunculatus rufusater), resulting in the development of island dialects.

Species evolution can often be adequately described with a phylogenetic tree. Interestingly, this is the case also for the evolution of homologous genes; a gene in an ancestral species may – through gene duplication, gene loss, lateral gene transfer (LGT), and speciation events – give rise to a gene family distributed across contemporaneous species. However, molecular sequence evolution and genetic recombination make the history – the gene tree – non-trivial to reconstruct from present-day sequences. This history is of biological interest, e.g., for inferring potential functional equivalences of extant gene pairs. In this thesis, we present biologically sound probabilistic models for gene family evolution guided by species evolution – effectively yielding a gene-species tree reconciliation. Using Bayesian Markov-chain Monte Carlo (MCMC) inference techniques, we show that by taking advantage of the information provided by the species tree, our methods achieve more reliable gene tree estimates than traditional species tree-uninformed approaches. Specifically, we describe a comprehensive model that accounts for gene duplication, gene loss, a relaxed molecular clock, and sequence evolution, and we show that the method performs admirably on synthetic and biological data. Further-more, we present two expansions of the inference procedure, enabling it to pro-vide (i) refined gene tree estimates with timed duplications, and (ii) probabilistic orthology estimates – i.e., that the origin of a pair of extant genes is a speciation. Finally, we present a substantial development of the model to account also for LGT. A sophisticated algorithmic framework of dynamic programming and numerical methods for differential equations is used to resolve the computational hurdles that LGT brings about. We apply the method on two bacterial datasets where LGT is believed to be prominent, in order to estimate genome-wide LGT and duplication rates. We further show that traditional methods – in which gene trees are reconstructed and reconciled with the species tree in separate stages – are prone to yield inferior gene tree estimates that will overestimate the number of LGT events.
Arters evolution kan i många fall beskrivas med ett träd, vilket redan Darwins anteckningsböcker från HMS Beagle vittnar om. Detta gäller också homologa gener; en gen i en ancestral art kan – genom genduplikationer, genförluster, lateral gentransfer (LGT) och artbildningar – ge upphov till en genfamilj spridd över samtida arter. Att från sekvenser från nu levande arter rekonstruera genfamiljens framväxt – genträdet – är icke-trivialt på grund av genetisk rekombination och sekvensevolution. Genträdet är emellertid av biologiskt intresse, i synnerhet för att det möjliggör antaganden om funktionellt släktskap mellan nutida genpar. Denna avhandling behandlar biologiskt välgrundade sannolikhetsmodeller för genfamiljsevolution. Dessa modeller tar hjälp av artevolutionens starka inverkan på genfamiljens historia, och ger väsentligen upphov till en förlikning av genträd och artträd. Genom Bayesiansk inferens baserad på Markov-chain Monte Carlo (MCMC) visar vi att våra metoder presterar bättre genträdsskattningar än traditionella ansatser som inte tar artträdet i beaktning. Mer specifikt beskriver vi en modell som omfattar genduplikationer, genförluster, en relaxerad molekylär klocka, samt sekvensevolution, och visar att metoden ger högkvalitativa skattningar på både syntetiska och biologiska data. Vidare presenterar vi två utvidgningar av detta ramverk som möjliggör (i) genträdsskattningar med tidpunkter för duplikationer, samt (ii) probabilistiska ortologiskattningar – d.v.s. att två nutida gener härstammar från en artbildning. Slutligen presenterar vi en modell som inkluderar LGT utöver ovan nämnda mekanismer. De beräkningsmässiga svårigheter som LGT ger upphov till löses med ett intrikat ramverk av dynamisk programmering och numeriska metoder för differentialekvationer. Vi tillämpar metoden för att skatta LGT- och duplikationsraten hos två bakteriella dataset där LGT förmodas ha spelat en central roll. Vi visar också att traditionella metoder – där genträd skattas och förlikas med artträdet i separata steg – tenderar att ge sämre genträdsskattningar, och därmed överskatta antalet LGT-händelser.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

The architecture of a plant is intimately tied to its fitness. Knowledge of the processes and patterns of growth form evolution can therefore contribute to a richer understanding of plant evolution. The genus Adenia (Passifloraceae) of ca. 100 species is an Old World lineage in which growth form radiated. I constructed a molecular phylogeny of the group, analyzed the stem and tuber anatomy of over half the species, and investigated patterns of growth form evolution in a phylogenetic context. I also described four new species and a new combination. Predictions based on evolutionary developmental models of growth form evolution were tested in Adenia, and one of them, the homeotic switch hypothesis, was tested throughout the eudicots. The switch hypothesis claims that the storage tissue of tubers and stems results from a common developmental origin. Phylogenetic analyses revealed that growth form transitions were frequent, and anatomical studies revealed traits that are associated with each growth form; moreover, traits are shared between tubers and succulent stems as predicted by the switch hypothesis. As expected, tuberous plants and succulents are also closely related across the eudicots. The switch hypothesis is substantiated in Adenia and the eudicots as a whole.

The evolution of a genome is shaped by spatial interactions at multiple scales. At the angstrom level, structural constraints on both RNA molecules and proteins contribute to the evolution of a gene sequence. Such optimized genes are weaved together in a particular order, out of a near-infinite number of combinations, to result in a genome. The fate of a genome is intricately linked to the evolutionary fate of its host organism; in turn, the fate of an organism is governed by where it resides in space. In this dissertation, I investigate how structure shapes the evolution of a gene, genome content, and pathogen populations residing in a diseased human lung. Chapter 1 provides a brief historical overview of population genetics in structured environments. I motivate why it is important to determine the migration rate of new alleles. Chapter 2 investigates how pathogens evolve within the structure of the cystic fibrosis lung. I find that migration rate and mutation rate are on similar timescales. Selection, rather than spatial isolation, maintains diversity within a pathogen population. Chapter 3 presents a new method to probe how codon choice is optimized throughout a gene. I find that codon choice is dictated by preference for particular RNA secondary structures, rather than intrinsic properties of a codon. Chapter 4 describes an ongoing study of how rapidly P. aeruginosa populations evolve in short-term infections. Preliminary results show that gene duplication events can sweep through a population in just 11 days. Chapter 5 introduces ideas for future directions. I pose questions regarding how pathogens evolve molecular mimicry that can trigger autoimmune disease in the human host, and how cancer-inducing inflammation might be detected from mutational signatures in the microbiome.
Systems Biology

Includes abstract.
Includes bibliographical references (leaves 138-161).
This dissertation describes three major sets of experiments, all of which involved the construction and use of various reciprocal chimaeric MSV constructs. First, chimaeric viruses were used in genetic complementation-type experiments to investigate the biological significance of interactions between the two virion-sense open reading frames (ORFs) of MSV, their products, and the rest of the genome. Six chimaeric MSV constructs were made by reciprocally exchanging the ORFs encoding movement protein (MP) and coat protein (CP) individually, and in pairs, between MSV-Kom and MSV-Set, which share just 78% overall nucleotide identity. Analysis of symptomatology and infection efficiency of chimaeras and wild-type parental viruses revealed evidence of functionally relevant specific interactions between MSV MP and CP.

Onychophorans are closely allied to the arthropods and possess a body organisation more similar to Middle Cambrian fossils than to recent arthropods. This means that onychophorans in some respects can be regarded as a model for the last common ancestor to both the Arthropoda and the Onychophora. This thesis mainly deals with the morphology of the head region of the Onychophora, but developmental investigations of the expression of a key regulatory gene, engrailed, are also carried out. The innervation of the head was found to differ from that reported in earlier investigations. The nerves that support the mouth were found to originate from three different regions of the brain. That innervation pattern suggests that present day onychophorans with a ventrally placed mouth, have evolved from an ancestor with a terminal mouth. Furthermore it was confirmed that the onychophoran structure with the unfortunately chosen term labrum is not homologous to the structure in arthropods that bears this name. Instead it is a muscular outgrowth from the pharynx. The embryological investigations gave further support for an ancestral and terminal mouth. The two most anterior oral lips are first located on the dorso-frontal side of the head, and later migrate to their final position at the ventral side. This phenomenon also explains their somewhat unexpected innervation from the dorsum. It was also established that the eye originates at a position posterior to the antenna. This is reversed compared to the condition in arthropods, were the eye is innervated from the protocerebrum and the first antenna from the dutocerebrum, and implies that the eye and antenna are not serially homologous between the two groups. A structure in the onychophoran head that has gained little attention is the hypocerebral organ, also termed infracerebral organ. It has been suggested as a corpora allata analog by earlier workers. Its ultrastructure was investigated, and great similarities to the corpora allata of the stick insect Carausius morosus were found. However, the lack of innervation of the hypocerebral organ of Onychophora poses a problem since the corpora allata of insects is controlled by nerves. Instead, cellular strands were found that connected the hypocerebral organ with the brain, and it is possible that these strands act as an alternative communication. The expression pattern of the segment polarity gene engrailed was found to be different from that reported in an earlier account of onychophorans. Engrailed was expressed in a subset of developing neurons in the brain anlage and in the ectoderm and mesoderm of the limb buds. The engrailed positive cells in the brain anlage were located in the area were the first commissure will form. This indicates that engrailed might have a function in axon guidance, as has been reported in other organisms. Later embryonic stages showed expression in the neuropile of the brain. There were no indications of this gene acting in determination of segment polarity. This suggests that there may be at least two copies of engrailed in onychophorans.

A major challenge in biology is to identify how different species arose and acquired distinct phenotypic traits. High-throughput sequencing is transforming our understanding of biology by allowing us to study genomes and cellular processes at genome-wide levels. Only a decade subsequent to the publication of the first human genome draft, genome assemblies of hundreds of organisms have been produced. Yet, genome analysis remains challenging and advances have lagged far behind our sequencing abilities and other technological advances. The next generation of comparative genomicists must therefore understand, invent and apply a wide number of computational tools in order to study biology in the most efficient manner and in order to pose the most interesting questions. This thesis spans areas covering evolutionary genomics, gene regulation, and computational methods development. A major aim was to understand how genetic variation contributes to variation in phenotypic traits. This was approached using a large variety of evolutionary and comparative genomics tools. In particular, high-throughput sequencing datasets were analysed to study single-cell transcriptomics, gene duplications, gene architecture evolution, and alternative splicing. Additionally, in cases where off-the-shelf analysis tools were inexistent, novel pipelines and programs were designed and implemented to solve algorithmic problems such as scaffolding genome assemblies and short-read mapping onto small exons.

Includes bibliographical references (leaves 101-122).
Aquatic species,notably fish, can reveal details of drainage evolution, especially where their evolutionary relationships can be reconstructed using phylogeographic methods. In this study, the mitochondrial DNA sequence diversity of the characiform genus, Hydrocynus, which is widespread across tropical Africa, is reconstructed using a phylogenetic framework and divergences are dated using the cytochrome b (cyt) region.

The diversity of many groups of organisms is related to the evolution of features that contribute to rapid radiations. This project reconstructed the phylogeny of carabid beetles in the subfamily Harpalinae, a speciose group of terrestrial predators. The phylogenetic inference focused on the sister group relationships, the monophyly of the subfamily and the tribal relationships within harpalines. Molecular sequence data, primarily from 28S ribosomal DNA and the wingless gene, were collected from more than 200 carabid beetles. Parsimony, minimum evolution distance, maximum likelihood, and Bayesian phylogenetic analysis methods were used to reconstruct the phylogeny of harpalines. Brachinine bombardier beetles and austral psydrines were found to be closely related to the harpaline clade. Within harpalines, zuphiites formed a clear clade as did pseudomorphines + graphipterines + orthogoniines. However the lebiomorph assemblage and the tribe Lebiini were not monophyletic. With the use of harpaline phylogenetic hypotheses, the evolution of the arboreal lifestyle was elucidated within the subfamily, including the rate and number of origins and losses of arboreality. Correlated evolution of several morphological characters and habitat was explored. Significant correlation of adhesive subtarsal setae and bilobed fourth tarsomeres on carabid legs were found with arboreality and may be arboreal adaptations, while long legs and long elytra are probably not associated with arboreality. The relationship of other morphological characters with arboreality is not clear. Harpalines may have been part of a rapid radiation of species diversity, where many lineages invaded new ecological niches and evolved novel morphological features to become adapted to their environment.

In this study, we investigate the causes and consequences of directional neutral evolution. We believe this is an evolutionary force that has not received the attention its importance to DNA and protein evolution warrants. Traditional sequence analyses rely upon models that assume homogeneity of nucleotide and amino acid compositions across different lineages, but our examinations of modern sequences show that this assumption is often violated. Our findings imply that periods of directional changes to the nucleotide or amino acid compositions of biological sequences have occurred in the past. Moreover, we demonstrate that these directional changes are very common and can be quite extreme. We show that modifications to the genetic code---a phenomenon that is common among mitochondrial genomes---can cause directional changes to the amino acid composition of proteins. Codon reassignments shift the relative number of codons assigned to each amino acid, and when coupled with neutral evolution over a period of time these changes will lead to a corresponding shift in the relative amino acid usages within the protein. More common causes of directional changes to the amino acid composition of proteins are mutation biases in the DNA. In particular, we show that biases to the relative proportions of A+T and G+C are capable of bringing about amino acid composition changes to the proteins the DNA encodes, with AT-rich DNA favouring the encoding of the FYMINK amino acids, and GC-rich DNA favouring the encoding of GARP amino acids. We show that this effect is both pervasive across all kingdoms of life, and that it also affects every protein within the genome---whether highly or loosely conserved. Finally, we show that directional neutral evolution in the DNA can operate in the presence of purifying selection in transmembrane domains. Since selection does not preclude neutral evolution if there are multiple favourable mutations possible, DNA mutation bias is able to influence the direction of evolution of the transmembrane domains without interfering with their fitness. We extend this same notion to include the possibility of mutation biases biasing the outcome of positive selection, as well.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Although the treatment of cancer is a major focus of biomedical research, many cancers are extremely hard to treat. Tumors likely resist treatment because each tumor is heterogeneous, and can evolve. Although tumor evolution has long been appreciated, it remains incompletely understood. In this thesis, I will explore two questions related to cancer heterogeneity and evolution: how evolution can affect plastic phenotypes, and the role of purifying selection in cancer evolution. Different cell states or phenotypes have been observed within tumors, and they are associated with treatment resistance and metastasis. The observation that these phenotypes are plastic leads to a conundrum: how can selection act on such an unstable phenotype? We determined that plasticity, in the form of cell state bias, varies widely across clones in a tumor. These different biases are heritable, with each cell faithfully passing its differentiation bias to its daughters. Simulations revealed that this makes plasticity an evolvable phenotype--- in a changing environment, an optimal state bias will be selected. The second question explored in this thesis is the role of purifying selection in cancer evolution. It is widely thought that tumor evolution is dominated by positive selection. We posited that, as in the evolution of species, purifying selection would prevent the fixation of deleterious mutations in tumors. Through computational analysis of tumor genomes, we determined that purifying selection acts to remove deleterious mutations. Genes under purifying selection must be important to tumors in vivo, as only mutations in these genes would be problematic. Consistent with this prediction, most genes under purifying selection in tumors were essential in cancer cell lines. To find genes essential to tumors but not generally cell-essential, we developed a method to find genes under increased purifying selection in one tumor type over others. This revealed a number of pathways under selection in melanomas, but not other tumor types, such as DNA damage pathways. By seeking genes important to tumors, but not generally essential, our analysis revealed potential therapeutic targets. Purifying selection offers an unprecedented view into which genes are essential to tumors in vivo, a finding predominantly inaccessible through experimentation.
by Robert Austin Mathis.
Ph. D.

The sheer complexity of evolutionary systems biology requires us to develop more sophisticated tools for analysis, as well as more probing and biologically relevant representations of the data. My research has focused on three aspects of evolutionary systems biology. I ask whether a gene’s position in the human metabolic network affects the degree to which natural selection prunes variation in that gene. Using a novel orthology inference tool that uses both sequence similarity and gene synteny, I inferred orthologous groups of genes for the full genomes of 8 mammals. With these orthologs, I estimated the selective constraint (the ratio of non-synonymous to synonymous nucleotide substitutions) on 1190 (or 80.2%) of the genes in the metabolic network using a maximum likelihood model of codon evolution and compared this value to the betweenness centrality of each enzyme (a measure of that enzyme’s relative global position in the network). Second, I have focused on the evolution of metabolic systems in the presence of gene and genome duplication. I show that increases in a particular gene’s copy number are correlated with limiting metabolic flux in the reaction associated with that gene. Finally, I have investigated the proliferative cell programs present in 6 different cancers (breast, colorectal, gastrointestinal, lung, oral squamous and prostate cancers). I found an overabundance of genes that share expression between cancer and embryonic tissue and that these genes form modular units within regulatory, proteininteraction, and metabolic networks. This despite the fact that these genes, as well as the proteins they encode and reactions they catalyze show little overlap among cancers, suggesting parallel independent reversion to an embryonic pattern of gene expression.

Mammals have three members of the small taste receptor gene family responsible for the perception of sweet and savory tastes: two genes (T1R2 and T1R3) comprise the canonical sweet receptor, and a third gene, T1R1, acts with T1R3 to make the savory receptor. Here, in a joint effort with a team of international collaborators, we show that even though birds are missing the taste receptor gene (T1R2) required by other vertebrates to perceive carbohydrates and sweeteners, hummingbirds still detect sugars—but in a novel way. This project spanned multiple fields and field sites, integrating taste tests on wild birds, behavioral analysis of captive animals, bioinformatics, receptor cloning, and cell-based functional assays. The first published avian genome, that of the chicken, revealed a surprising lack of T1R2. Chickens are sweet-insensitive: however, many nectar-feeding birds appear highly attuned to sugars like sucrose, fructose and glucose. Our initial field experiments with a panel of artificial sweeteners as well as high-speed filming and choice tests on captive birds indicated a rapid response to sugars rather than a post-ingestive metabolic sensing of caloric value. As the response appeared sensory, we pursued a candidate gene approach to search for possible taste receptors, and cloned T1R taste receptors from chickens, hummingbirds, and swifts. By analyzing genomes from an additional 10 birds and an alligator, we documented widespread absence of T1R2 and identified signatures of positive selection in the remaining hummingbird T1Rs. Together with Dr. Yasuka Toda at the University of Tokyo, we were able to test the function of these receptors in cell culture. We used a cell-based luminescence assay to measure functional responses. As expected, chicken and swift receptors responded to amino acids, but, surprisingly, the umami receptor in hummingbirds had acquired a new function and was now sensitive to carbohydrates as well. Chimeric studies of receptors containing hummingbird and chicken sequence identified 19 mutations involved in this functional change: since divergence from swifts, the umami receptor underwent extensive re-modeling. Further behavioral tests with wild hummingbirds revealed that most agonists from the cell-based assay were appetitive, while artificial sweeteners which did not activate the receptors were not preferred—a concordance between in vivo and in vitro studies that indicates that this re-purposed receptor guides hummingbird taste behavior. Diet shifts have profound physiological effects and evolutionary ramifications: the radiation of hummingbirds is likely due, at least in part, to their ability to colonize an empty niche. However, much remains to be learned about the roles of taste in changes in diet, and the causes and effects of shifts in diet and perception are often unclear. For instance, birds appear to have lost T1R2 early in their evolutionary history. As they are the descendants of carnivorous dinosaurs, birds may have experienced relaxed selection on the sweet receptor similar to that seen in mammalian carnivores; alternatively, the loss could be due to the overall genome-wide reduction seen in birds. In Chapter 3, we begin to investigate causes and consequences of the changes in hummingbird taste receptors, and present new behavioral studies regarding the ability to detect amino acids. Together, these findings raise questions about the evolution of sensory systems and of neural circuits underlying perception: studying taste receptors in a comparative context yields insight into basic aspects of the evolutionary process as well as molecular mechanisms underlying behavior.
Biology, Organismic and Evolutionary

One of the large lineages of parasitic wasps, the Cynipoidea, exhibits three distinctly different life modes. Slightly more than half of the about 3000 species are parasitoids in insect larvae, whereas the remaining species are associated with plants, either as gall inducers or as inquilines (guests feeding on plant tissue in galls). The main focus of this thesis has been to identify morphological changes associated with the shifts between life modes. Particular attention was paid to structures believed to be important in gall initiation. Comparative anatomical studies of the egg, larva and venom apparatus were performed, including representatives of parasitoids, gall inducers and inquilines. Examination of gross morphology and ultrastructure revealed that the eggs of the gall inducers are larger and surrounded by a thicker shell than the parasitoid eggs. These differences may be related to the fact that the gall inducer egg contains sufficient egg yolk for the embryo during the entire egg period, whereas the parasitoid egg often absorbs nutrients through the eggshell. Furthermore, the gall inducer egg is probably more exposed to desiccation and therefore a thicker and more resistant eggshell is crucial. Comparing the terminal-instar larvae of about 30 species of parasitoids, gall inducers and inquilines, extensive morphological variation was found, particularly in the head and mouthpart features. The variation was summarized in 33 morphological and one life-history character and parsimony analyses were performed. The resulting phylogenetic estimates were largely in accordance with previous analyses of adult morphology and molecular data. The larval data point to a single origin of the inquilines, in agreement with adult morphology but in conflict with molecular data. The venom apparatus was found to be quite uniform in structure among a sample of 25 species of cynipoid species. It consists of a very short venom duct, a reservoir and a single unbranched venom gland. With few exceptions, the venom apparatus is conspicuously larger relative to the female metasoma in the gall inhabiting species than in the parasitoids. We found little evidence of anatomical structures that could facilitate chemical communication between the gall-inducer embryo and the surrounding plant tissue through the thick eggshell. On the other hand, the enormous venom glands and reservoirs, which are apparently not used for defence, suggest that the adult female plays a significant role in gall induction by injecting secretions into the host plant when laying eggs.

Auxin is a phytohormone that has long been known to control many aspects of plant growth and development. The YUCCA (YUC) gene family is a large group of genes that catalyze auxin biosynthesis and have been shown to be critical for vegetative growth and inflorescence development in grasses. There is genetic redundancy present with Arabidopsis YUCs, but in Zea mays (maize), a single gene knockout of ZmSPI1 causes a severe inflorescence phenotype. Since Oryza sativa (rice), another grass species, does not show an inflorescence phenotype when OsYUC1/SPI1 is knocked down, SPI1 appears to have undergone an evolutionary shift in function within the grass family. This study shows that SPI1 expression in PACMAD (Panicoideae, Arundinoideae, Chlorodoideae, Micrairoideae, Aristidoideae, and Danthoniodeae subfamilies) clade grasses Sorghum bicolor and Setaria italica occurs at sites of inflorescence branching and is consistent with maize, but in BEP (Bambusoideae, Ehrhartoideae, and Pooideae subfamilies) clade grasses rice and Brachypodium distachyon SPI1 shifts from localized expression to more generalized expression and potentially becomes weaker. Artificial microRNA (amiRNA) knockdowns of SPI1 expression in Brachypodium did not show a phenotype when expression was reduced to 28.01% (+/- 6.39%) of wild type. In rice and Brachypodium, other YUC genes were shown to be expressed in the inflorescence by quantitative RT-PCR (qPCR), suggesting YUC proteins are more redundant in BEP grasses such as B. distachyon and O. sativa, than in maize and potentially its relatives.

The Tachinidae is a taxonomically and ecologically diverse clade of parasitoids for which evolutionary and ecological relationships with hosts are largely unknown. Here, I employed a multidisciplinary approach to evaluate the determinants of patterns of host use in the Tachinidae. First, I examined spatio-temporal variation in the tachinid-dominated parasitoid assemblage of one lepidopteran species Grammia geneura . The parasitoid assemblage and parasitism rates varied dramatically among and within sampling sites, seasons, and years. I show that this variability may be a function of habitat-specific parasitism and indirect interactions between this host and other Macrolepidoptera through shared tachinid parasitoids. I then experimentally examined the host selection process in the tachinid Exorista mella. Host movement was an important elicitor of attack behavior. Flies also responded to odors associated with food plants of their host. Experienced flies attacked hosts more readily than did inexperienced flies. Based on these results, I proposed a host selection scenario for this tachinid species. E. mella also teamed to associate colors with hosts and avoided deterrent models that they had experienced. However, I failed to find evidence for odor learning. Learning of host-associated cues by E. mella may allow this parasitoid to take advantage of abundant host populations and maintain host-searching efficiency in an unpredictable environment. To examine how host-associated characteristics evolved in the Tachinidae, I reconstructed the evolutionary relationships within the subfamily Exoristinae using molecular data. Phylogenetic analyses generally supported recent classifications. Analyses of host-related characters indicated that tachinids show great evolutionary lability in behavior, morphology, and host range. Finally, I sampled host species to assess the determinants of tachinid community structure and host range. Several host characteristics were found to affect tachinid species richness. These patterns may be due to the opportunistic use of abundant hosts by polyphagous tachinids, enemy-free space provided by well-defended hosts, and the process of host location. Patterns of tachinid host use varied significantly with sample size, host diet breadth, host gregariousness, plant form, and host morphology. Taken together, these studies indicate high levels of plasticity in tachinid-host associations. This may be responsible for their ecological and evolutionary success.

Genetic information is encoded by the linear sequence of the DNA double helix, while epigenetic information is overlayed as the packaging of DNA and associated proteins into the chromatin structure. Variations in chromatin structure play a vital role in establishing and maintaining patterns of gene expression during differentiation and development of higher eukaryotes, and disruption of this epigenetic gene regulation can lead to cancer. Mammals display an epigenetic phenomenon known as genomic imprinting, which provides an ideal model system for the study of epigenetics. Genes subject to genomic imprinting are differentially expressed within a single cell depending on the parental origin of the chromosome. Imprinting of the maternally expressed H19 gene and the adjacent paternally expressed Igf2 gene is regulated by the chromatin insulator protein CTCF. The studies presented in this thesis aim to investigate the functional mechanisms of CTCF-dependent gene regulation at the H19/Igf2 locus and at numerous other target sites throughout the genome. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. We have developed novel microarray based methods to identify and characterize numerous new CTCF target sites throughout the mouse genome. We have shown that CTCF acts as part of the RNA polymerase II complex. We have identified the post-translational modification by addition of ADP-ribose polymers to CTCF, and demonstrated that this modification regulates its insulating ability. The results of these studies of CTCF-dependent epigenetic gene regulation are discussed in light of the evolution of genomic imprinting and chromatin insulators, and a novel role for poly ADP-ribosylation of CTCF in the progression of cancer is proposed.

The relative timing and sequence of events during embryonic development plays an important role in adult shape and thus in evolution. The sequence in which bones form in the developing embryo should therefore contain a component capable of revealing evolutionary history, however processes relating to ossification sequence and the sequences themselves are poorly known and rarely discussed. In this thesis, I describe the embryonic skeletal development of Meleagris gallopavo, Sterna hirundo, Somateria mollissima, Anas platyrhynchos, Cairina moschata, Dromaius novaehollandiae, Rhea americana and Struthio camelus for the first time in the scientific literature, focusing on ossification. All species exhibited intraspecific variation in ossification sequence, but the level of polymorphism present was generally quite low. Specimens collected from the wild did not show more variability in ossification sequence than those incubated under constant conditions in the lab. Dermal bones did not always ossify before endochondral bones, nor did neural-crest derived elements always form before elements derived from the paraxial mesoderm. All of this suggests that the factors controlling ossification sequence are complex, and more than one variable may play a role. In order to examine sequences in a more explicit phylogenetic context, I converted them into a form that is easily analyzed (event-pairs) and used these as characters for phylogenetic reconstruction. While this technique is plagued with problems involving logical and biological non-independence, it is an efficient tool for surveying conservation and divergence in ossification sequences at different levels of phylogenetic relatedness. I also reconstructed shifts on an accepted topology. The analysis indicates that ossification sequences are influenced by relative evolutionary reduction or expansion in element size. Reduced elements ossify late in sequence, and also temporally later, as measured by stage. Enlarged elements
Le temps de formation et la séquence d'événements du développement embryonnaire jouent un rôle important dans la forme adulte et dans l'évolution. La séquence selon laquelle les os se forment dans l'embryon devrait donc contenir des informations capables de révéler l'histoire évolutionnaire. Cependant, les facteurs qui influencent la séquence d'ossification et les séquences elles-mêmes sont mal compris et rarement étudiés. Dans cette thèse, je décris le développement squelettique embryonnaire chez Meleagris gallopavo, Sterna hirundo, Somateria mollissima, Anas platyrhynchos, Cairina moschata, Dromaius novaehollandiae, Rhea americana et Struthio camelus pour la première fois dans la littérature scientifique, en me concentrant sur l'ossification. Une variabilité intraspécifique entre les séquences d'ossification a été observée chez toutes espèces, mais le niveau de polymorphisme était généralement bas. Les spécimens d'espèces sauvages n'ont pas montré plus de variabilité dans la séquence d'ossification que ceux incubés dans les conditions constantes du laboratoire. Les os membraneux n'ossifient pas toujours avant les os de cartilage, et les os dérivés de la crête neurale ne se forment pas toujours avant les éléments dérivés du mésoderme paraxial. Ceci suggère que les facteurs qui contrôlent la séquence d'ossification sont complexes et que plus qu'une facteur peuvent y jouer un rôle. Afin d'examiner les séquences dans un contexte phylogénetique, je les ai convertis en une forme facile à analyser (‘paires d'événements') et ai utilisé les caractères de séquence d'ossification pour la reconstruction phylogénetique. Bien que cette technique présente des problèmes liés à un manque d'indépendence logique et biologique, c'est un outil efficace pour examiner la conservation et la divergence des séquences d'ossification à différents niveaux de rélation phylogénetique. J'ai aussi reconstruit les changements$

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006.
Title from PDF t.p. (viewed Nov. 18, 2008). Source: Dissertation Abstracts International, Volume: 67-12, Section: B, page: 6862. Adviser: Michael Lynch.

Cancer research has made tremendous progress in understanding the basic biology of tumors. One of the key insights that has informed work in this area is the recognition that a tumor is an evolutionary system, in which individual cells undergo a process of rapid mutation and selection leading to a progression in phenotypes and, typically, aggressiveness of the tumor. Tumor phylogenetics is a strategy for interpreting the evolution of tumors using computer algorithms for phylogenetics, i.e., the inference of evolutionary trees. The approach takes advantage of a large body of phylogenetic theory and algorithms, developed primarily for inferring evolution among species, to interpret complex tumor data sets as evidence for evolutionary processes. The result is a tumor phylogeny, or phylogenetic tree, a reconstruction of the sequences of mutations that cells within a tumor or class of tumors accumulate over the course of their progression. The goals of finding such trees are to better interpret heterogeneity within and among tumors, identify and classify tumor subtypes with possible underlying mechanisms of action, learn markers of progression for key steps in tumor evolution, and enable predictive modeling of likely tumor progression steps that may ultimately assist in diagnosis and treatment. In this dissertation, we discuss a computational framework for reconstructing phylogenies from genome-scale tumor array and sequencing data. We first present a novel phylogenetic pipeline for building tumor phylogenies from whole-genome copy number variation data. The steps included computational unmixing for resolving heterogeneity in genomic data from tumors, a statistical method for progression marker discovery, a statistical method for data discretization, application of character-based phylogeny reconstruction, and analyses of the resulting trees to draw biological significance. We then describe HMM-CNA, an improved model for discovering progression markers from cohorts of patient tumor copy number data that are especially relevant for phylogeny reconstruction via a custom multi-sample Hidden Markov model (HMM). We next present a novel strategy for phylogeny building from single cell sequencing data by inferring features that can accurately capture the composition of the individual genome sequences and distinguish among stages of tumor progression. We demonstrate these contributions on both simulated and human breast tumor biopsy and cell line data assuming a maximum parsimony model of evolution. Finally, we discuss future directions for building a more realistic model of tumor evolution by integrating patterns in genome structural changes with the functional elements they encode. We close with a discussion of recent research, current trends, and challenges and opportunities facing the field.

All organisms must interact with and adapt to their surrounding environment. There are myriad ways in which species accomplish this; ultimately resulting in the vast diversity of life on earth today. Changes in the environment can have profound impacts on an organisms' ability to compete and utilize their surroundings. Plants are particularly impacted by local environmental differences because of the fact that they are immobile. This environmental variation exists at both large and small spatial scales. For example, on larger scales, forces such as fire and grazers can remove dominant plant competitors. on smaller scales, variation in resource availability (e.g. light, nutrients, water) may benefit more phenotypically plastic species. to better understand how changes in the environment, on both large and small spatial scales, I established a two part study using milkweed (Asclepias spp.) as a model system. in the first chapter, I ask how fire, large grazers, and nutrients have affected milkweed abundance over relatively long time and large spatial scales. Here I found that most milkweed species increase in abundance with burning alone but expressed species-specific responses to other treatment combinations. This indicates that milkweed species have likely experienced unique fluctuations in abundance as fire and large herbivores moved across the landscape. The second aspect of this research focuses in on a single year and relatively small spatial scales. Here, using common milkweed (A. syriaca), I ask how environmental variation shapes spatial structuring of phenotypes within fine-scale physical distance and how genotypes impact phenotypes. I found that environment, not genotype, had a relatively larger role on fine-scale phenotypic variation. Combined, these results have implications for understanding the role of large and small scale environmental variations in plant phenotypes and plant abundance.

Orientador: Cesar Martins
Abstract: One of the biggest challenges in chromosome biology is to understand the occurrence and complex genetics of extra, non-essential karyotype elements, commonly known as supernumerary B chromosomes (Bs). Bs are present in diverse species of eukaryotes and their molecular characterization remains elusive for years. A distinguished feature that makes them different from the normal chromosomes (called A chromosomes) is their way of inheritance in irregular fashion. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. The non-Mendelian inheritance and unpairing/non-recombining abilities make the B chromosomes immensely interesting for genomics studies, thus arising different questions about their genetic composition, survival, maintenance and role inside the cell. This study aims to uncover these phenomena in different species. Here, we sequenced the genomes of three model organisms including fish species Astyanax mexicanus and Astyanax correntinus, and grasshopper Abracris flavolineata with (B+) and without Bs (B-) to identify the B-localized sequences, called B chromosome blocks (“B-blocks”). We established approaches for this analysis that comprised of steps such as comparative genomics analysis and annotation of B chromosomal genes and DNA repeat types. The next generation sequencing (NGS) analyses identified thousands of genes fragments as well as... (Complete abstract click electronic access below)
Doutor

Small GTP binding proteins function as molecular switches which cycles between GTP-bound ON and GDP-bound OFF states, and regulate a wide variety of cellular processes as biological timers. The first characterized member of the small GTPase family, the mutated oncogene p21 src, later known as Harvey-Ras, was identified in the early 1980s (Shih, T. Y. et al. 1980). In the following years small Ras-lik GTPases were found in several organisms and it was soon discovered that they took part in processes, such as signal transduction, gene expression, cytoskeleton reorganisation, microtubule organisation, and vesicular and nuclear transport. The first Rho (Ras homology) gene was cloned in 1985 from the sea slug Aplysia (Madaule, P. et al. 1985) and because of their homology to Ras it was first suspected that they could act as oncogenes. Later studies have shown that even though they participate in processes such as cell migration and motility they are not mutated in cancers.

The first indications that Rho was a signaling protein regulating the actin cytoskeleton, came from experiments where activated forms of human RhoA was microinjected into 3T3 cells (Paterson, H. F. et al. 1990). Another Rho-like GTPase Rac1 (named after Ras-related C3 botulinum toxin substrate) was later shown to regulate actin cytoskeletal dynamics as well, suggesting that Rho-family members cooperate in controlling these processes (Ridley, A. J. et al. 1992). The Rac GTPase was also implicated in regulating the phagocytic NADPH oxidase, which produce superoxide for killing phagocytized microorganisms (Abo, A. et al. 1991). Thus, it soon became clear that Rac/Rho and the related GTPase Cdc42 (cell division cycle 42) had central functions in many important cellular processes.

There are at least three types of regulators for Rho-like proteins. The GDP/GTP exchange factors (GEFs) which stimulates conversion from the GDPbound form to the GTP-bound form. GDP dissociation inhibitors (GDIs) decrease the nucleotide dissociation from the GTPase and retrieve them from membranes to the cytosol. GTPase activating proteins (GAPs) stimulates the intrinsic GTPase activity and GTP hydrolysis. In addition there are probably regulators that dissociate GDI from the GTPase leaving it open for activation by the RhoGEFs.

Ras and Rho-family proteins participate in a coordinated regulation of cellular processes such as cell motility, cell growth and division. The Ral GTPase is closely related to Ras and recent studies have shown that this GTPase is involved in crosstalk between both Ras and Rho proteins (Feig, L. A. et al. 1996; Oshiro, T. et al. 2002). Ral proteins are not found in plants and they appear to be restricted to animalia and probably yeast. During a screen for small GTPases in Drosophila melanogaster I discovered in 1993 several new members of the Ras-family, such as Drosophila Ral (DRal), Ric1 and Rap2. The functions of Ral GTPases in Drosophila have until recently been poorly known, but in paper 2 we present some of the new findings.

Rho-like GTPases have been identified in several eukaryotic organisms such as, yeast (Bender, A. et al. 1989), Dictyostelium discoideum (Bush, J. et al. 1993), plants (Yang, Z. et al. 1993), Entamoeba histolytica (Lohia, A. et al. 1993) and Trypanosoma cruzi (Nepomuceno-Silva, J. L. et al. 2001). In our first publication, (Winge, P. et al. 1997), we describe the cloning of cDNAs from RAC-like GTPases in Arabidopsis thaliana and show mRNA expressions pattern for five of the genes. The five genes analyzed were expressed in most plant tissues with the exception of AtRAC2 (named Arac2 in the paper), which has an expression restricted to vascular tissues. We also discuss the evolution and development of RAC genes in plants. The third publication, (Winge, P. et al. 2000), describe the genetic structure and the genomic sequence of 11 RAC genes from Arabidopsis thaliana. As most genomic sequences of the AtRACs we analyzed came from the Landsberg erecta ecotype and the Arabidopsis thaliana genome was sequenced from the Columbia ecotype, it was possible to compare the sequences and identify new polymorphisms. The genomic location of the AtRAC genes plus the revelation of large genomic duplications provided additional information regarding the evolution of the gene family in plants. A summary and discussion of these new findings are presented together with a general study of small Ras-like GTPases and their evolution in cellular organisms. This study suggests that the small GTPases in eukaryots evolved from two bacterial ancestors, a Rab-like and a MglA/Arp-like (Arf-like) protein. The MglA proteins (after the mgl locus in Myxococcus xanthus) are required for gliding motility, which is a type of movement that take place without help of flagella.

The second publication describes experiments done with the Drosophila melanogaster DRal gene and its effects on cell shape and development. Ectopic expression of dominant negative forms of DRal reveals developmental defects in eye facets and hairs, while constitutive activated forms affects dorsal closure, leaving embryos with an open dorsal phenotype. Results presented in this publication suggest that DRal act through the Jun N-terminal kinase (JNK) pathway to regulate dorsal closure, but recent findings may point to additional explanations as well. The results also indicate a close association between processes regulated by Rac/Rho and Ral proteins in Drosophila.

Of the four most diverse insect orders, the Lepidoptera contain remarkably few predatory and/or parasitic taxa, and while species with carnivorous life histories have evolved independently numerous times in moths and butterflies, this has rarely led to diversification. As a rule, aphytophagous taxa seem prone to extinction. In this dissertation, I explore the ecological and evolutionary consequences of entomophagy in the butterfly family Lycaenidae using several approaches: natural history observation, phylogenetics, population genetics and stable isotope chemistry. A striking exception to the lack of radiation and persistence in aphytophagous lineages is the lycaenid subfamily Miletinae, which with 13 genera and 190 species is among the largest and most diverse groups of aphytophagous Lepidoptera. Most miletines eat Hemiptera, although some consume ant brood or are fed by trophallaxis from their host ant. I inferred the higher-level phylogeny of this group using data from one mitochondrial and six nuclear genes sampled from representatives of all genera and nearly half the described species. Biogeographic analyses indicate that Miletinae likely diverged from an African ancestor near the start of the Eocene, and four lineages dispersed between Africa and Asia. Phylogenetic constraint in prey selection is apparent at two levels: related miletine species are more likely to feed on related Hemiptera and are also more likely to associate with closely related ants species, either directly by eating the ants, or indirectly by eating hemipteran prey attended by those ants. I then examined the influence of diet on the population structure of lycaenid butterflies, and more specifically, I investigated whether particular feeding habits are correlated with traits that might make species vulnerable to extinction. To do this, I compared the phylogeography and population genetics of two endemic lycaenid species of roughly similar age from southern Africa: Chrysoritis chrysaor, whose caterpillars are strictly herbivorous, and Thestor protumnus, whose cuckoo-like caterpillars survive by soliciting regurgitations from their host ants. I sampled both species from populations throughout their entire known ranges, and found that in contrast to C. chrysaor, T. protumnus has exceedingly small effective population sizes and individuals disperse poorly. With its aphytophagous life history, T. protumnus exhibits a high degree of host dependence and specialization. Although these results are correlative and based on only a single comparison, it seems likely that small population sizes and extreme ecological specialization make populations of T. protumnus more susceptible to disturbance and prone to extinction. Having focused in detail on the population biology of just one species, I then analysed the evolution of Thestor as a whole. This genus is exceptional because all of its 27 described species are thought to be entomophagous, and all are thought to be predators or parasites of a single species of ant, Anoplolepis custodiens. Using representatives sampled from all known species and populations of Thestor as well as 15 outgroup species, I inferred the phylogeny of the genus in two ways: first by using characters from mitochondrial and nuclear genes, and second by analyzing genome-wide SNPs generated for each species using double digest RADseq. I also sequenced the ants associated with each of these taxa using ddRADseq. This investigtion showed that all 24 of the species in the Western Cape utilize Anoplolepis custodiens, while T. protumnusand T. dryburghi (the two species that are found in the north-western part of South Africa) use a closely related, but different species of Anoplolepis, and T. basutus (the species found in the eastern part of South Africa) utilizes yet a third species. Thus factors driving diversity in the genus Thestor may have initially involved ant associations and/or geographic isolation, but other forces are likely to be responsible for generating and maintaining the more recent diversity in the group. Flight time may have separated the “black” and “yellow” groups of Thestor: the black group fly predominantly in the summer months, while the yellow group fly predominantly in the spring. And while species spread across the genus fly in the spring and summer months, only members of the yellow group fly during the winter and fall months. Despite these broad scale differences, species in the genus Thestor show little evidence of niche partitioning, especially those in the Western Cape, and represent an extreme example of the coexistence of 24 species apparently utilizing a single food resource. While working on the previous three projects, I was surprised by the number of species of South African Lycaenidae with incomplete life histories despite decades of work by avid lepidopterists in the region. For example, in the genus Thestor, although all 27 species are assumed to be aphytophagous, partial life histories have been described for only four species. In part the paucity of data is due to the difficult terrain occupied by these butterflies, and the fact that those whose caterpillars associate with ants often spend significant portions of their lives hidden in ant nests in crevices of rock that are intractable for excavation and observation. To deepen our understanding of South African lycaenid life histories, I used nitrogen and carbon stable isotopic methods to survey a large number of species and their potential food sources. With these methods, I confirmed some known or suspected life histories and showed that in any one area, a species can have a highly variable diet. I also discovered that some of the nitrogen stable isotope values are much higher than expected for land animals, implying longer than average food chains and/or extreme environmental conditions. Together, these studies shed light on how carnivorous life histories affect the evolution of lycaenid butterflies, and help to explain why entomophagous lineages appear to be an evolutionary “dead end” in contrast to their herbivorous counterparts.
Biology, Organismic and Evolutionary

We develop a new class of computationally feasible stochastic models for statistical analysis of genetic sequence evolution and inference of properties of the underlying substitution processes in the context of maximum likelihood framework. Existing models for evolution of protein coding sequences allow site to site variation in non-synonymous substitution rates, but assume that the rate of synonymous substitutions is constant for all sites. New models provide a rigorous statistical framework for testing the hypothesis of synonymous rate constancy, and enable a host of data exploration and analysis tools. For several indicative data sets, the constancy assumption is shown to be violated, and some possible explanations are given. We also present an algorithm for improving efficiency of maximum likelihood evaluations, and discuss HyPhy--a user friendly and publicly distributed software implementation of our methods.

The vertebrate head and face, one of the defining features of vertebrates, is an evolutionary novelty that enabled the major radiation of Vertebrata. The emergence of the multipotent neural crest cells, often mentioned as the “fourth germ layer”, which produces most of the bone, cartilage and dentine of the vertebrate head, was the key to this innovation. Unlike the appendicular and axial skeletons in the rest of the body, which have relatively uncomplicated anatomy and are generated from cells of mesodermal origin, most of the cranial bones have complex structures generated by a unique mechanism termed intramembranous ossification and originate from the cranial neural crest cells. The unique and innovative nature of the neural crest and cranial bone is also reflected in the morphogenic processes that create diversity in craniofacial shape throughout the course of vertebrate evolution, exemplified in the bird lineage. In this thesis, I seek to expand our knowledge of the development and evolution of craniofacial elements, by developing a 3-D platform to generate cranial bone in vitro and investigating the evolution of beak and tooth loss in birds. In the first chapter, I introduce the first system for generating intramembranous bone with mouse cranial neural crest cells in 3-D hydrogel culture. The flexibility of the platform will allow it to be utilized for generating 3-D cranial bones from neural crest cells using various sources, including birds and human patients with craniofacial disorders. In the second chapter, the process of beak evolution and the latent developmental potential to generate teeth in birds are analyzed using an intriguing chick mutant, Talpid2. The geometric morphometric analysis of, and the gene expression patterns observed in, the Talpid2 mutant reveal the atavistic nature of the mutant phenotypes, including ancestral beak shape and the possible resurrection of teeth development. Preliminary efforts to manipulate the gene responsible for the phenotypes are also addressed for future studies. Together, the results of these studies provide useful tools and knowledge to further advance our understanding in the craniofacial development in amniotes.
Biology, Molecular and Cellular

The Andean mountain chains of South America are home to exceptionally high levels of biodiversity, including one-sixth of all plant species. Among them, the 550 species in the Neotropical bellflower clade (Campanulaceae: Lobelioideae) represent one of the single largest endemic radiations. In this dissertation, I perform a series of studies, ranging from alpha taxonomy to phylogenetic comparative biology, to understand this group’s evolution. In Chapter 1, I resolve phylogenetic relationships of the Neotropical bellflowers from five plastid DNA regions using maximum likelihood and Bayesian inference. This phylogeny greatly improves the resolution within this group and highlights the need for taxonomic revisions. My results demonstrate that the majority of species of Centropogon, Burmeistera, and Siphocampylus together form a primarily mainland Neotropical clade, collectively termed the centropogonids. Within the centropogonids, I identify high support for the monophyly of Burmeistera and the polyphyly of Centropogon and mainland Siphocampylus. Caribbean Siphocampylus, however, group with other Caribbean lobelioid species. Ancestral character state reconstruction identifies a dynamic pattern of fruit evolution within the centropogonids, which underscores the difficulty of diagnosing broad taxonomic groups on the basis of fruit type. Finally, we identify that the centropogonids form a Pan-Andean radiation with broad habitat diversity, which sets the stage for a subsequent chapter in my thesis (Chapter 4). In Chapters 2 and 3, I describe four species of centropogonids as new to science. In Chapter 2, two species of Burmeistera from the Cordillera de Talamanca are described: B. serratifolia Lagom. & D. Santam., endemic to Panama, and B. monroi D. Santam. & Lagom., from both Panama and Costa Rica. These species are included in a dichotomous key to all Central American species of Burmeistera. In Chapter 3, I describe two species of Siphocampylus from the Central Andes of Peru and Bolivia: S. antonellii Lagom. & D. Santam., endemic to high elevation grasslands of Calca, Peru, and S. siberiensis Lagom. & D. Santam., endemic to cloud forests of Cochabamba, Bolivia. Both species are robust shrubs that produce tubular pink flowers that are likely pollinated by hummingbirds. In Chapter 4, I use the Neotropical bellflowers as a model system to understand the triggers of rapid diversification in the Andes. I explore the interaction of abiotic and biotic factors by applying a series of diversification models that incorporate orogeny, climate, and biological trait evolution to a time-calibrated phylogeny of the group. Here, I demonstrate that speciation rates rose with increasing Andean elevation, while extinction decreased during global cooling. Of these, climate appears to have played the more important role, which I attribute to preadaptation to cool climates. Biotic traits that facilitate plant-animal mutualisms, specifically fruit type and pollination syndrome, additionally enhanced diversification rates. These results suggest that, while the Andes acted as a species pump for this lineage, a synergistic interaction of abiotic and biotic factors underlies its rapid diversification, which culminated in one of the fastest plant radiations documented to date: the centropogonids. This study underscores the complex interplay of ecological and historical determinants in generating the world’s biodiversity. Finally, in Chapter 5, I use comparative methods to explore shifts between vertebrate pollination syndromes in the centropogonid clade. Such shifts, which are often associated with convergent floral phenotypes, are invoked as key factors underlying floral diversification in angiosperms. Using phylogenetic principal components analyses, I show that, despite morphological similarities between these pollination syndromes, centropogonid flowers adapted to pollination by bats and by hummingbirds fall into separate regions of morphospace. I further document the repeated evolution of pollination syndromes: the clade is ancestrally hummingbird pollinated, and bat pollination has evolved independently fourteen times, with nine reversions to hummingbird pollination. An obligate relationship with sicklebill hummingbirds, characterized by extremely curved bills, evolved only once. While the evolution of floral morphology has been dynamic in this clade, there is no difference in diversification rate in hummingbird and bat pollinated linages. These results suggest that specialized pollination syndromes are not evolutionary dead-ends. Instead, shifts between vertebrate pollinators may have greatly facilitated the maintenance of an ancestrally high diversification rate among centropogonid species.
Biology, Organismic and Evolutionary

Reproduction constitutes the principle determinant of organismal fitness and, therefore, a central theme in biology is devoted to understanding variation in the mode of reproduction and its effects within and among species. Different breeding systems lead to varying levels of inbreeding, outcrossing, and sex ratios---with concomitant effects on patterns of genetic variation, effective population size, and adaptation by natural selection. Theoretical studies have proposed many mechanisms to explain the diversity of breeding systems observed in nature, several models of which invoke deleterious effects of mutation as a dominant force in patterning breeding system evolution. These notions motivated the present investigations into (1) the factors contributing to outcrossing rates and sex ratio in Caenorhabditis elegans and their population genetic effects, and (2) the general importance of deleterious mutation in the evolution of breeding system, emphasizing the Caenorhabditis clade. Nematodes of the genus Caenorhabditis provide a convenient system for studying these issues because species vary in breeding system, self-fertile C. elegans hermaphrodites can be genetically transformed into females to create dioecious populations, and complete genomic sequences of two species allow genomic analysis. With this context, I constructed a quantitative model describing sex ratio and outcrossing as a function of male copulatory ability and sex-chromosome non-disjunction (as a consequence of the XO sex-determination mechanism). The sex ratio and amount of outcrossing in C. elegans were then predicted from laboratory experiments and nucleotide polymorphism data, by applying population genetic theory of selection at linked sites when partial selfing is taken into account. These analyses suggest that outcrossing occurs on the order of one percent. Subsequent computational and multigeneration experimental evolution studies of the potential influence of deleterious mutations on breeding system evolution demonstrated that (1) selfing species do not exhibit lower rates of deleterious mutation than outcrossing species, (2) a higher deleterious mutation rate does not prevent the invasion of selfing alleles into a population, and (3) adaptive modulation of sex-chromosome non-disjunction is unlikely to underlie the persistence of males in androdioecious species. These results indicate that mutational theories are unlikely to fully explain the evolution of breeding system and sex.

In this dissertation I employ a comparative gene expression approach to address the evolution of butterfly wing pattern formation at several levels, with emphasis on early pattern determination and pigment gene regulation during late development. Expression analysis of the receptor molecule Notch suggested previously unknown roles for Notch signaling in butterfly wing patterning. Notch upregulation was found to precede the activation of the transcription factor Distal-less during early eyespot color pattern determination. A phylogenetic comparison of expression time series from multiple moth and butterfly species suggested that changes in a Notch/Distal-less temporal pattern formation process were associated with the gain and loss of both eyespot and midline color patterns during wing pattern evolution. Additionally, Notch expression was found to occur in a grid-like pattern in the butterfly wing epithelium shortly after pupation. This observation, together with previous expression and simulation studies, support a Notch-mediated lateral inhibition model of wing scale organization. Tryptophan-derived ommochrome pigments are a derived feature of nymphalid butterfly wings. I found that multiple genes in the ommochrome biosynthetic pathway were expressed in the wings of selected nymphalid butterflies. Additionally, transcriptional regulation of genes encoding the ommochrome synthesis enzymes vermilion and cinnabar was found to be temporally and spatially associated with the polymorphism and development of forewing band patterns in the mimetic butterfly Heliconius erato. These findings provide evidence that changes in ommochrome gene regulation underlie the evolution and development of major nymphalid wing pattern elements.

Natural selection gives rise to biodiversity by purging the less-fit among variants that are too similar (a principle known as character displacement), but to predict how fit or different an organism needs to be to survive is hard. In the simplest theoretical case, the probability whether one lineage versus another survives depends only on their relative fitness and random fluctuations. In more complex scenarios, this probability may depend on the fitness of all the other lineages in the population, mutations that these and other lineages acquire before the outcome of competition is decided, and additional ecological interactions. These complexities evolve readily in laboratory microbial populations, suggesting that they are the norm in Nature, and have been extensively studied theoretically. This thesis provides one of the few empirical examples in which the evolution and mechanism of some of these complexities have been characterized and modeled sufficiently to make basic predictions, such as whether a mutation will fix or go extinct, which competing lineages may or may not coexist, and how do these processes relate? This work was carried out in an established system for experimental evolution, populations of asexual budding yeast (S. cerevisiae) in microtiter plates. Chapter 2 demonstrates an experimental design and modeling approach to infer the distribution of fitness effects of beneficial mutations from the population-dynamics of genetic markers. The inferred distribution accurately predicts fixation probabilities of lineages and adaptation rates of populations. Chapter 3 describes a new example of spontaneously-evolved coexistence between types competing for the same resources, including the physical mechanism, genetic basis and a mathematical model of the coexistence. The conclusion provides additional analyses to connect the findings from these two chapters and discusses their implications for microbial evolution more generally and directions for future work.
Biophysics

Ett generellt problem i dagens svenska gymnasieskola är att elever ofta använder vardagliga förklaringar istället för vetenskapliga när de ska förklara naturvetenskapliga teorier och begrepp. När elever ska förklara orsaken till att biologisk evolution sker, uppkommer olika alternativa idéer, idéer som inte är förenliga med vetenskapliga förklaringar. Denna studie beskriver vilka alternativa idéer som elever ger när de skriftligt besvarar en uppgift innehållande ett evolutionsbiologiskt fenomen. De förklaringar eleverna ger på uppgiften diskuteras och utvecklas i intervjuer, för att öka kunskapen och förståelsen för vilka uppfattningar gymnasieelever har om biologisk evolution. Den vanligaste alternativa idén som framkommer när den skriftliga uppgiften löses är att evolution drivs för att ett behov finns(giraffen behöver utvecklas för att klara sig). Andra förekommande alternativa idéer är inlärning (giraffen sträcker på sig och därför blir halsen längre) och anpassning (giraffen anpassar sig till en ny omgivning). Fem begrepp är centrala för att förstå helheten i evolutionsteorin; variation, överlevnad, ärftlighet, reproduktion och anpassning. Variation visar sig vara ett nyckelbegrepp för elevens förmåga att frångå en alternativ idé om evolution och uppnå en högre grad av vetenskaplighet i sin förklaring.

In this thesis, I used the filamentous ascomycete genus Neurospora as a model to test theoretical predictions on the evolutionary consequences of different reproductive strategies and reproductive gene evolution. The genus Neurospora contains taxa representing a diversity of reproductive strategies, and here I constructed a phylogeny by which I was able to show that several independent transitions in reproductive mode have occurred in the evolutionary history of the genus. This feature makes Neurospora a suitable model for the evolution of reproductive modes. Molecular evolutionary analysis of housekeeping genes revealed an accelerated protein evolution in the highly inbreeding homothallic taxa, in accordance with theory predictions of lower efficiency of selection in asexual and highly inbreeding taxa. Furthermore, self-sterile (heterothallic) taxa capable of asexual propagation was found to be associated with a three-fold higher neutral substitution rate, indicative of a higher mutation accumulation due to elevated number of cell divisions per unit time in these taxa. Further, I have shown a general pattern of rapid evolution of genes involved in reproduction in Neurospora, thus extending the pattern of general high divergence of reproductive genes previously well known in animals, to fungi. Two rapidly evolving reproductive genes: the pheromone receptor genes pre-1 and pre-2 involved in mate recognition were studied in detail. For the gene pre-1 the rapid divergence was found to be driven by positive selection in both heterothallic and homothallic taxa. The rapid divergence of the pheromone receptor gene pre-2 cannot be explained by positive selection and for this gene a subtle differences in evolutionary constraints between heterothallic and homothallic taxa were found. The general similarity in evolutionary constraints of pre-genes in taxa of both mating-systems indicates that these genes serve other functions beside mate recognition.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.
Title as it appears in MIT commencement exercises program, June 6, 2014: The evolution of mRNA splicing in mammalian tissues Cataloged from PDF version of thesis.
Includes bibliographical references (pages 167-172).
In this thesis, I describe investigations into the evolution of splicing in mammals. I first investigate a small class of alternative splicing events, tandem splice sites, and show how they are used to introduce and remove coding sequence in a species-specific manner. I then describe the generation and analysis of a large RNA-seq dataset from 9 matched tissues in 5 species, with the aim to investigate the evolution of splicing in mammals. I first investigate the evolution of exons that predate the most ancient divergence of species studied, finding that their splicing is frequently poorly conserved. For a subset of these exons, I identify unique regulatory properties and provide evidence linking alternative splicing to phosphorylation potential of proteins. I then consider sources of novel exons, in these species. I use these and other published data to identify one way in which splicing of novel exons impacts the biology of the cell. I also present evidence implicating genomic indels in exon creation and splicing variation.
by Jason Jay Merkin.
Ph. D.

Forty-four actin genes from five angiosperm species, whose evolutionary relationships are well characterized, were PCR-cloned and sequenced. Phylogenetic analysis of 34 of these actin genes, along with those previously published, indicate that plant actin genes are monophyletic and underwent a rapid radiation early in land plant evolution. Six sets of putative orthologues have been identified and their sequences were used to calculate rates of evolution. The synonomous rate of substitution $(5.44\times10\sp{-9}$/site/year) is similar to that of other nuclear protein-encoding genes but the non-synonomous rate $(0.13\times10\sp{-9}$/site/year) is 4-10 times higher than that of vertebrate actin genes. Relative-rate tests do not support a faster rate in plants than in vertebrates. Evidence is also provided that some members of the actin multigene family in maize are undergoing gene conversion. Finally, we show that some plant actin genes have undergone intron loss probably as a consequence of a gene conversion event between the genomic copy and the reverse transcript.

Gene loss is the most important event in the process of genome reduction that appears associated with bacterial endosymbionts of insects. These small genomes were derived features evolved from ancestral prokaryotes with larger genome sizes, consequence of a massive process of genome reduction due to drastic changes in the ecological conditions and evolutionary pressures acting on these prokaryotic lineages during their ecological transition to host-dependent lifestyle. In the present thesis, the process of genome reduction is studied from different perspectives. In the first chapter, genome rearrangements have been studied in a set of 31 complete γ-proteobacterial genomes that includes five genomes of bacterial endosymbionts of insects. This is carried out by comparing the order of a subset of 244 single-copy orthologous genes presents in all the genomes and calculating the number of inversions and breakpoints between each genome pair. This reveals that inversions were the main rearrangement event in γ-proteobacteria evolution, with a progressive increase in the number of rearrangements with increased evolutionary distance. However, significant heterogeneity in different γ-proteobacterial lineages was also detected, with a significant acceleration in the rates of genome rearrangements in bacterial endosymbionts of insects at initial stages of the association. In the second chapter, the structure and functional capabilities of Sodalis glossinidius has been studied. S. glossinidius is the secondary endosymbiont of tsetse flies, and it´s at very initial stages of genome reduction process. It´s genome is experiencing a massive process of gene inactivation, with 972 pseudogenes (inactivated genes) that were described but not annotated in the original annotation of the genome. In this chapter, a complete functional re-annotation of this genome was carried out, that includes the characterization of 1501 pseudogenes though analysis of S. glossinidius intergenic regions. A massive presence of CDSs related with mobile genetic elements and surface proteins were detected, being also the functional classes most affected by pseudogenization. The reconstruction of the metabolic map of S. glossinidius revealed a functional profile very similar to that of free-living enterics, with inactivation of L-arginine biosynthesis pathway, whereas the comparison with Wigglesworthia glossinidia (tsetse primary endosymbiont) reveals possible cases of metabolic complementation between both tsetse endosymbionts at thiamine, coenzyme A and tetrahydrofolate biosynthesis level. Finally, in the third chapter of the thesis, the complete reductive evolution process associated with S. glossinidius was studied from a systems biology perspective through the reconstruction of their genome-scale metabolic networks at different stages of this process and the prediction of their internal reaction fluxes under different external conditions through Flux Balance Analysis. This revealed the decisive role of the pseudogenization of genes involved in L-arginine and glycogen biosynthesis and specially the pseudogenization of the key anaplerotic enzyme phosphoenolpyruvate carboxylase in the ecological transition to a host-dependent lifestyle experienced by S. glossinidius. A progressive decrease in network robustness to gene deletion events and to changes in particular reaction fluxes were detected. Finally, reductive evolution simulations over the functional network of S. glossinidius under different external conditions revealed a higher plasticity in minimal networks evolved in a nutrient-rich environment, and allow defining different sets of essential and disposable genes based on their presence or absence in minimal metabolic networks. These essential genes had more optimized patterns of codon usage and more restricted patterns of sequence evolution than disposable genes that could be lost without affecting the functionality of the network. However, lineage-specific estimates of dN and dS in S. glossinidius and Escherichia coli revealed that common features of ancient bacterial endosymbionts like acceleration in the rates of sequence evolution and the loss of adaptative codon usage were starting to affect S. glossinidius evolution.
En esta tesis doctoral, el proceso de reducción genómica característico de bacterias endosimbiontes de insectos ha sido estudiado utilizando diferentes aproximaciones computacionales basadas en la genómica comparada y la biología de sistemas. Por un lado, las dinámicas de reordenaciones genómicas han sido estudiadas en un subconjunto de 31 genomas completos de γ-proteobacterias que incluyen 5 genomas completos de endosimbiontes bacterianos de insectos, revelando una aceleración significativa de las tasas de reordenaciones en estos genomas en etapas iniciales del proceso de reducción. Posteriormente, el genoma de Sodalis glossinidius, el endosimbionte secundario de la mosca tsétsé, fue re-anotado con el objetivo de evaluar el impacto de los procesos de inactivación génica y proliferación de elementos genéticos móviles en etapas tempranas del proceso de reducción, asi como su impacto sobre las capacidades funcionales de la bacteria en el contexto ecológico de su coexistencia con el endosimbionte primario ancestral Wigglesworthia glossinidia. Finalmente, el proceso completo de reducción genómica en S. glossinidius ha sido estudiado a través de la reconstrucción de su red metabólica a diferentes etapas de este proceso y su análisis funcional mediante Análisis de Balance de Flujos, evaluando la robustez de las redes frente a sucesos de deleción asi como las dinámicas evolutivas de genes esenciales y no esenciales en base a su presencia en redes mínimas evolucionadas a partir de la red funcional. Este análisis permitió identificar sucesos de inactivación génica con efectos drásticos sobre las capacidades funcionales del sistema como los genes implicados en la biosíntesis de arginina y glicógeno, y especialmente la inactivación de la enzima fosfoenolpiruvato carboxilasa, asi como una disminución progresiva de la robustez de las redes frente a diferentes sucesos mutacionales asociada al proceso de pérdida génica. Finalmente, simulaciones de evolución reductiva sobre la red funcional bajo diferentes condiciones de entorno ha permitido definir conjuntos de genes esenciales y delecionables en base a su presencia o ausencia en las redes mínimas producto de las simulaciones, revelando una mayor conservación a nivel de secuencia y un uso de codones más optimizado en genes esenciales frente a genes cuya pérdida no afecta a la funcionalidad del sistema.