Assessing the evolutionary patterns of plastid genome reduction in a group of non-photosynthetic parasitic Angiosperms (Orobanchaceae)

Der Plastid, der als Schlüsselfunktion der autotrophen Lebensweise die Photosynthese ausführt, besitzt ein semi-autonomes genetisches System mit eigenem Genom (Plastom), welches für Proteine des Photosyntheseapparates sowie wenige Enzyme anderer metabolischer Prozesse und Untereinheiten grundlegender genetischer Prozesse kodiert. Aufgrund des Überganges zu einer unterschiedlich stark ausgeprägten heterotrophen Lebensweise weisen parasitische Pflanze enorm modifizierte Plastome auf. Diese sind durch eine extreme funktionelle und strukturelle Reduktion sowie stark erhöhte DNS-Substitutionsraten charakterisiert. Gegenstand dieser Dissertation ist es, evolutive Trends der Plastomreduktion bei verminderten Selektionsdrücken zu rekonstruieren. Zu diesem Zweck wurden die Plastome verschiedener zur Photosynthese fähiger und unfähiger Vertreter der Sommerwurzgewächse (Orobanchaceae) vollständig sequenziert und mittels moderner Methoden der vergleichenden Genomanalyse hinsichtlich folgender Aspekte analysiert: (i) strukturelle Änderungen, (ii) potentielle Funktionalität von Photosynthese-assoziierten Plastidengenen, (iii) Pseudogenisierung und Gendeletion, und (iv) Evolution und Auswirkung erhöhter plastidärer DNS-Substitutionsraten. Darüber hinaus behandelt ein Kapitel methodologische Aspekte der Sequenzierung von Plastidengenomen mittels Pyrosequenzierung gesamt-genomischer DNS-Extrakte. Die Analyse plastidärer DNS nah-verwandter Orobanchaceae erlaubt es erstmals, komplexe Muster der Genomreduktion in parasitischen Pflanzen aufzudecken. Unter anderem kann gezeigt werden, dass bereits der Übergang zu einer heterotrophen Lebensweise für strukturelle Änderungen des Plastoms ausschlaggebend ist und zu einem Verlust der Funktionalität bestimmter Gene und einer Erhöhung der Substitutionsraten führt. Innerhalb der Orobanchaceae schreitet die Plastomreduktion mit zunehmender Heterotrophie mit linienspezifischer Geschwindigkeit voran. Das Ausmaß von Pseudogenisierung und Deletion nicht-essentieller Genomabschnitte wird dabei maßgeblich durch die Distanz zu essentiellen genischen Elementen und von der plastidären Operonstruktur beeinflusst. Darüber hinaus weisen die zusammengetragenen Ergebnisse darauf hin, dass parasitische Pflanzen die Funktion einzelner Photosynthese-assoziierter Proteinkomplexe möglicherweise aufrechterhalten und eine erhöhte Rate an intrazellulärem DNS-Transfer aufweisen. Veränderungen der DNS-Substitutionsmuster bei zur Photosynthese fähigen heterotrophen Orobanchaceae implizieren eine Korrelation des Übertritts zur parasitischen Lebensweise mit der Verminderung der Selektion bestimmter plastidärer Genen. Im Vergleich zu photosynthetisch aktiven Pflanzen, weisen Vollparasiten differenzierte Muster bezüglich DNS-Substitutionen auf, einschließlich linienspezifischer Ratenerhöhung und –reduktion. In der vorliegenden Arbeit wird anhand der Analyse von simulierten und experimentell generierten 454-Datensätzen erstmals gezeigt, dass die erfolgreiche Plastomrekonstruktion signifikant von der verwendeten Sequenzdatenmenge bestimmt wird. Darüber hinaus wird eine Methode zur a priori Schätzung der optimalen Datenmenge unter Verwendung weniger Parameter erarbeitet.The prime function of the plastid organelle is to carry out photosynthesis thereby providing autotrophy to the plant kingdom. Plastids retain a semi-autonomous genetic system including a genome (plastome) encoding subunits for photosynthesis-related and unrelated processes as well as proteins for basic functions of the genetic apparatus. Due to the transition from an autotrophic to a semi- or holo-heterotrophic lifestyle, parasitic plants show major plastomic reconfigurations with extreme reductions of plastome size and coding capacity as well as extraordinarily elevated nucleotide substitution rates. Using the broomrape family (Orobanchaceae) as a model group, this dissertation thesis reconstructs molecular evolutionary patterns of reductive plastome evolution of the plastid chromosome under relaxed evolutionary constraints. Employing comparative-evolutionary analyses of completely sequenced plastid genomes from several hemi- and holoparasitic members of Orobanchaceae this work examines aspects concerning the (i) co-linearity and structural rearrangements of plastomes, (ii) potential functionality of genes involved in photosynthesis, (iii) pseudogenization and gene loss, and (iv) accelerated substitution rates in plastid genomes. In addition, one chapter evaluates methodological aspects of plastid genome sequencing employing whole-genome shotgun pyrosequencing. This work reveals that genetic and genomic changes concerning plastome structure, nucleotide substitution rates and selectional constraints occur in a complex and highly lineage specific manner, and it provides novel insights into factors influencing reductive evolution of plastome. Increasing host-dependency notably seeds excessive non-functionalization of plastid genes due to pseudogenization or deletion, and severely relaxes the structural maintenance of the plastid chromosome. Pseudogenization and segmental deletions of newly dispensable regions depend significantly on the operon-structure of the plastid chromosomes as well as on the distribution of essential genes in Orobanchaceae. There is evidence for maintained or alternative function of a photosynthesis-related complex as well as for putatively increased rates of intracellular gene transfer in parasitic plants. Analyses of nucleotide substitutions reveal significantly elevated rates in both housekeeping and photosynthesis genes already in photosynthetic heterotrophs indicating that relaxation of selective constraints relates to the transition to a parasitic lifestyle. Compared to hemiparasites and autotrophs, distinctive trends of rate and selectional changes exist among holoparasite lineages including both local accelerations and rate reductions. Above that, this thesis shows for the first time that the successful reconstruction of plastid chromosomes from whole-genome shotgun pyrosequencing strongly depends on the size of the assembled read pool. Using the results of simulated and empirical 454 datasets in combination with a resampling scheme for automated quality assessment, a method for a parameter-less a priori assessment of the optimal read pool size is established that should ease assembly efforts

Von der Malaria zum Meeresleuchten - Evolution von Organellen und der Wirtszelle in Alveolata (Apicomplexa, Dinoflagellata, Ciliata)

The highly diverse group of alveolates comprises photoautotrophic, heterotrophic and even parasitic species including the malaria pathogen Plasmodium falciparum and the causative agent of toxoplasmosis Toxoplasma gondii. The basis of this conspicuous diversity is their evolutionary history including a complex puzzle of successive endosymbioses. Once, symbionts became permanent residents in their hosts and the derived organelles are represented by mitochondria and plastids. Mitochondria arose from a primary endosymbiosis event with an alpha-proteobacterium, whereas primary plastids developed from an uptaken cyanobacterium. Complex plastids originated by secondary or even tertiary endosymbiosis by the engulfment and reduction of a eukaryotic alga. According to their original bacterial ancestry, the cell organelles harbor nucleus-independent genomes. Over time, most of these genes were transferred to the nucleus by endosymbiotic gene transfer (EGT) and particular presequences mediate the re-import of the corresponding proteins into the organelle. All other organelles of the eukaryotic cell such as peroxisomes were established in a non-endosymbiotic context. This thesis addresses the evolution of plastids and mitochondria in Alveolata, which are characterized by a strong reduction in their organelle genomes. Based on the establishment of transcriptomes and draft genomes from three alveolate key organisms (i.e. Chromera velia, Vitrella brassicaformis and Perkinsus olseni) comparative genome analyses were accomplished. Hence, in the photoautotrophic alga C. velia the smallest mitochondrial genome, consisting of only one single protein-coding coxI gene, could be identifed. Furthermore, high-throughput genome sequencing of the oyster parasite P. olseni documented the complete loss of its plastid genome, but also supported the maintenance of this cryptic organelle by the expression of nuclear-encoded proteins with characteristic bipartite targeting signals for plastid-specific metabolic pathways. Finally, this study showed the common peroxisomal ancestry of the three alveolate phyla ciliates, dinoflagellates and Apicomplexa. A set of diagnostic markers provided an unequivocally proof for the presence of peroxisomes in T. gondii, and it moreover suggested the alga V. brassicaformis as a promising reference organism to study apicomplexan peroxisome biology.Die hoch diverse Gruppe der Alveolata umfasst photoautotrophe, heterotrophe und parasitäre Arten, darunter den Malaria-Erreger Plasmodium falciparum sowie den Erreger der Toxoplasmose Toxoplasma gondii. Die Grundlage dieser hohen Diversität bildet ihre Entstehung aus einem komplexen Puzzle aus aufeinanderfolgenden Endosymbiosen. Die Symbionten wurden einst zu Dauergästen in ihren Wirtszellen und werden heute durch Mitochondrien und Chloroplasten repräsentiert. Mitochondrien entstanden aus einer primären Endosymbiose mit einem alpha-Proteobakterium, wohingegen primäre Plastiden sich aus einem aufgenommenen Cyanobakterium entwickelten. Komplexe Plastiden entstanden über sekundäre oder auch tertiäre Endosymbiosen durch die Aufnahme und Reduzierung eukaryotischer Algen. Gemäß ihrem bakteriellen Ursprung beherbergen diese Organellen eigene, vom Nukleus unabhängige, Genome. Im Laufe der Zeit wurden die meisten Gene über den sogenannten endosymbiontischen Gentransfer (EGT) in den Zellnukleus des Wirts übertragen. Spezielle Präsequenzen vermitteln dabei den Reimport der entsprechenden Proteine in das Organell. Weitere Organellen der eukaryotischen Zelle, wie z.B. Peroxisomen, wurden in einem nicht-endosymbiontischen Kontext etabliert. Diese Dissertation befasst sich mit der Evolution von Plastiden und Mitochondrien in den Alveolata, die stark durch die Reduktion ihrer Organellengenome geprägt sind. Basierend auf der Etablierung von Transkriptomen und Genomen der drei Schlüsselorganismen, Chromera velia, Vitrella brassicaformis und Perkinsus olseni, wurden vergleichende Genomanalysen durchgeführt. Hierbei konnte in der photoautotrophen Alge C. velia das kleinste mitochondriale Genom, bestehend aus nur einem einzigen Protein-kodierenden Gen (coxI), identifiziert werden. Das Genom des Austernparasiten P. olseni dokumentiert den vollständigen Verlust seines Plastid-Genoms, wohingegen der Erhalt eines kryptischen Organells durch die Expression von nukleär kodierten Proteinen plastidärer Stoffwechsel mit charakteristischen zweiteiligen Signalsequenzen unterstützt wird. Abschließend zeigt diese Arbeit den gemeinsamen Ursprung der Peroxisomen in den Alveolata. Ausgewählte diagnostische Marker beweisen eindeutig die Präsenz dieses Organells in T. gondii und unterstützen ebenfalls die Verwendung der Alge V. brassicaformis als vielversprechenden Referenzorganismus im Verständnis der Biologie der Peroxisomen innerhalb der Apicompexa

Genomics and spatial surveillance of Chagas disease and American visceral leishmaniasis

The Trypanosomatidae are a family of parasitic protozoa that infect various animals and plants. Several species within the Trypanosoma and Leishmania genera also pose a major threat to human health. Among these are Trypanosoma cruzi and Leishmania infantum, aetiological agents of the highly debilitating and often deadly vector-borne zoonoses Chagas disease and American visceral leishmaniasis. Current treatment options are far from safe, only partially effective and rarely available in the impoverished regions of Latin America where these ‘neglected tropical diseases’ prevail. Wider-reaching, sustainable protection against T. cruzi and L. infantum might best be achieved by intercepting key routes of zoonotic transmission, but this prophylactic approach requires a better understanding of how these parasites disperse and evolve at various spatiotemporal scales. This dissertation addresses key questions around trypanosomatid parasite biology and spatial epidemiology based on high-resolution, geo-referenced DNA sequence datasets constructed from disease foci throughout Latin America: Which forms of genetic exchange occur in T. cruzi, and are exchange events frequent enough to significantly alter the distribution of important epidemiological traits? How do demographic histories, for example, the recent invasive expansion of L. infantum into the Americas, impact parasite population structure, and do structural changes pose a threat to public health? Can environmental variables predict parasite dispersal patterns at the landscape scale? Following the first chapter’s review of population genetic and genomic approaches in the study of trypanosomatid diseases in Latin America, Chapter 2 describes how reproductive polymorphism segregates T. cruzi populations in southern Ecuador. The study is the first to clearly demonstrate meiotic sex in this species, for decades thought to exchange genetic material only very rarely, and only by non-Mendelian means. T. cruzi subpopulations from the Ecuadorian study site exhibit all major hallmarks of sexual reproduction, including genome-wide Hardy-Weinberg allele frequencies, rapid decay of linkage disequilibrium with map distance and genealogies that fluctuate among chromosomes. The presence of sex promotes the transfer and transformation of genotypes underlying important epidemiological traits, posing great challenges to disease surveillance and the development of diagnostics and drugs. Chapter 3 demonstrates that mating events are also pivotal to L. infantum population structure in Brazil, where introduction bottlenecks have led to striking genetic discontinuities between sympatric strains. Genetic hybridization occurs genome-wide, including at a recently identified ‘miltefosine sensitivity locus’ that appears to be deleted from the majority of Brazilian L. infantum genomes. The study combines an array of genomic and phenotypic analyses to determine whether rapid population expansion or strong purifying selection has driven this prominent > 12 kb deletion to high abundance across Brazil. Results expose deletion size differences that covary with phylogenetic structure and suggest that deletion-carrying strains do not form a private monophyletic clade. These observations are inconsistent with the hypothesis that the deletion genotype rose to high prevalence simply as the result of a founder effect. Enzymatic assays show that loss of ecto-3’-nucleotidase gene function within the deleted locus is coupled to increased ecto-ATPase activity, raising the possibility that alternative metabolic strategies enhance L. infantum fitness in its introduced range. The study also uses demographic simulation modelling to determine whether L. infantum populations in the Americas have expanded from just one or multiple introduction events. Comparison of observed vs. simulated summary statistics using random forests suggests a single introduction from the Old World, but better spatial sampling coverage is required to rule out other demographic scenarios in a pattern-process modelling approach. Further sampling is also necessary to substantiate signs of convergent selection introduced above. Chapter 4 therefore develops a ‘genome-wide locus sequence typing’ (GLST) tool to summarize parasite genetic polymorphism at a fraction of genomic sequencing cost. Applied directly to the infection source (e.g., vector or host tissue), the method also avoids bias from cell purification and culturing steps typically involved prior to sequencing of trypanosomatid and other obligate parasite genomes. GLST scans genomic pilot data for hundreds of polymorphic sequence fragments whose thermodynamic properties permit simultaneous PCR amplification in a single reaction tube. For proof of principle, GLST is applied to metagenomic DNA extracts from various Chagas disease vector species collected in Colombia, Venezuela, and Ecuador. Epimastigote DNA from several T. cruzi reference clones is also analyzed. The method distinguishes 387 single-nucleotide polymorphisms (SNPs) in T. cruzi sub-lineage TcI and an additional 393 SNPs in non-TcI clones. Genetic distances calculated from these SNPs correlate with geographic distances among samples but also distinguish parasites from triatomines collected at common collection sites. The method thereby appears suitable for agent-based spatio-genetic (simulation) analyses left wanted by Chapter 3 – and further formulated in Chapter 5. The potential to survey parasite genetic diversity abundantly across landscapes compels deeper, more systematic exploration of how environmental variables influence the spread of disease. As environmental context is only marginally considered in the population genetic analyses of Chapters 2 – 4, Chapter 5 proposes a new, spatially explicit modelling framework to predict vector-borne parasite gene flow through heterogeneous environment. In this framework, remotely sensed environmental raster values are re-coded and merged into a composite ‘resistance surface’ that summarizes hypothesized effects of landscape features on parasite transmission among vectors and hosts. Parasite population genetic differentiation is then simulated on this surface and fitted to observed diversity patterns in order to evaluate original hypotheses on how environmental variables modulate parasite gene flow. The chapter thereby makes a maiden step from standard population genetic to ‘landscape genomic’ approaches in understanding the ecology and evolution of vector-borne disease. In summary, this dissertation first demonstrates the power of population genetics and genomics to understand fundamental biological properties of important protist parasites, then identifies areas where analytical tools are missing and creates new technical and conceptual frameworks to help fill these gaps. The general discussion (Chapter 6) also outlines several follow-up projects on the key finding of meiotic genetic signatures in T. cruzi. Exploiting recently developed T. cruzi genome-editing systems for the detection of meiotic gene expression and heterozygosis will help understand why and in which life cycle stage some parasite populations use sex and others do not. Long-read sequencing of parental and recombinant genomes will help understand the extent to which sex is diversifying T. cruzi phenotypes, especially virulence and drug resistance properties conferred by surface molecules with repetitive genetic bases intractable to short-read analysis. Chapter 6 also provides follow-up plans for all other research chapters. Emphasis is placed on advancing the complementarity, transferability and public health benefit of the many different methods and concepts employed in this work

The role of visual adaptation in cichlid fish speciation

D. Shane Wright (1) , Ole Seehausen (2), Ton G.G. Groothuis (1), Martine E. Maan (1) (1) University of Groningen; GELIFES; EGDB(2) Department of Fish Ecology & Evolution, EAWAG Centre for Ecology, Evolution and Biogeochemistry, Kastanienbaum AND Institute of Ecology and Evolution, Aquatic Ecology, University of Bern.In less than 15,000 years, Lake Victoria cichlid fishes have radiated into as many as 500 different species. Ecological and sexual sel ection are thought to contribute to this ongoing speciation process, but genetic differentiation remains low. However, recent work in visual pigment genes, opsins, has shown more diversity. Unlike neighboring Lakes Malawi and Tanganyika, Lake Victoria is highly turbid, resulting in a long wavelength shift in the light spectrum with increasing depth, providing an environmental gradient for exploring divergent coevolution in sensory systems and colour signals via sensory drive. Pundamilia pundamila and Pundamilia nyererei are two sympatric species found at rocky islands across southern portions of Lake Victoria, differing in male colouration and the depth they reside. Previous work has shown species differentiation in colour discrimination, corresponding to divergent female preferences for conspecific male colouration. A mechanistic link between colour vision and preference would provide a rapid route to reproductive isolation between divergently adapting populations. This link is tested by experimental manip ulation of colour vision - raising both species and their hybrids under light conditions mimicking shallow and deep habitats. We quantify the expression of retinal opsins and test behaviours important for speciation: mate choice, habitat preference, and fo raging performance

Graph-based modeling and evolutionary analysis of microbial metabolism

Microbial organisms are responsible for most of the metabolic innovations on Earth. Understanding microbial metabolism helps shed the light on questions that are central to biology, biomedicine, energy and the environment. Graph-based modeling is a powerful tool that has been used extensively for elucidating the organising principles of microbial metabolism and the underlying evolutionary forces that act upon it. Nevertheless, various graph-theoretic representations and techniques have been applied to metabolic networks, rendering the modeling aspect ad hoc and highlighting the conflicting conclusions based on the different representations. The contribution of this dissertation is two-fold. In the first half, I revisit the modeling aspect of metabolic networks, and present novel techniques for their representation and analysis. In particular, I explore the limitations of standard graphs representations, and the utility of the more appropriate model---hypergraphs---for capturing metabolic network properties. Further, I address the task of metabolic pathway inference and the necessity to account for chemical symmetries and alternative tracings in this crucial task. In the second part of the dissertation, I focus on two evolutionary questions. First, I investigate the evolutionary underpinnings of the formation of communities in metabolic networks---a phenomenon that has been reported in the literature and implicated in an organism's adaptation to its environment. I find that the metabolome size better explains the observed community structures. Second, I correlate evolution at the genome level with emergent properties at the metabolic network level. In particular, I quantify the various evolutionary events (e.g., gene duplication, loss, transfer, fusion, and fission) in a group of proteobacteria, and analyze their role in shaping the metabolic networks and determining the organismal fitness. As metabolism gains an increasingly prominent role in biomedical, energy, and environmental research, understanding how to model this process and how it came about during evolution become more crucial. My dissertation provides important insights in both directions