Konstanz, Univ., Diss., 2005.

Techn. University, Diss., 2003--Berlin.

Universiẗat, Diss., 2004--Tübingen.

The biosphere harbors a large quantity and diversity of microbial organisms that can thrive in all environments. Estimates of the total number of microbial species reach up to 1012, of which less than 15,000 have been characterized to date. It has been challenging to delineate phenotypically, evolutionary and ecologically meaningful lineages such as for example, species, subspecies and strains. Even within recognized species, gene content can vary considerably between sublineages (for example strains), a problem that can be addressed by analyzing pangenomes, defined as the non-redundant set of genes within a phylogenetic clade, as evolutionary units. Species considered to be ecologically and evolutionary coherent units, however to date it is still not fully understood what are primary habitats and ecological niches of many prokaryotic species and how environmental preferences drive their genomic diversity. Majority of comparative genomics studies focused on a single prokaryotic species in context of clinical relevance and ecology. With accumulation of sequencing data due to genomics and metagenomics, it is now possible to investigate trends across many species, which will facilitate understanding of pangenome evolution, species and subspecies delineation. The major aims of this thesis were 1) to annotate habitat preferences of prokaryotic species and strains; 2) investigate to what extent these environmental preferences drive genomic diversity of prokaryotes and to what extent phylogenetic constraints limit this diversification; 3) explore natural nucleotide identity thresholds to delineate species in bacteria in metagenomics gene catalogs; 4) explore species delineation for applications in subspecies and strain delineation in metagenomics. The first part of the thesis describes methods to infer environmental preferences of microbial species. This data is a prerequisite for the analyses performed in the second part of the thesis which explores how the structure of bacterial pangenomes is predetermined by past evolutionary history and how is it linked to environmental preferences of the species. The main finding in this subchapter that habitat preferences explained up to 49% of the variance for pangenome structure, compared to 18% by phylogenetic inertia. In general, this trend indicates that phylogenetic inertia does not limit evolution of pangenome size and diversity, but that convergent evolution may overcome phylogenetic constraints. In this project we show that core genome size is associated with higher environmental ubiquity of species. It is likely this is due to the fact that species need to have more versatile genomes and most necessary genes need to be present in majority of genomes of that species to be highly prevalent. Taken together these findings may be useful for future predictive analyses of ecological niches in newly discovered species. The third part of the thesis explores data-driven, operational species boundaries. I show that homologous genes from the same species from different genomes tend to share at least 95% of nucleotide identity, while different species within the same genus have lower nucleotide identity. This is in line with other studies showing that genome-wide natural species boundary might be in range of 90-95% of nucleotide identity. Finally, the fourth part of the thesis discusses how challenges in species delineation are relevant for the identification of meaningful within-species groups, followed by a discussion on how advancements in species delineation can be applied for classification of within-species genomic diversity in the age of metagenomics. / Die Biosphäre beherbergt eine große Zahl verschiedener Mikroorganismen, die fast alle bekannten Lebensräume besiedeln können. Die Gesamtzahl mikrobieller Spezies liegt Schätzungen zu Folge bei bis zu 1012, von denen jedoch bis heute erst 15.000 beschrieben worden sind. Die Beschreibung von phänotypisch, evolutionsbiologisch und ökologisch kohärenten Spezies, Sub-Spezies oder Stämmen stellt Forscher vor konzeptionelle Herausforderungen. Selbst innerhalb anerkannter Spezies kann die Kombination einzelner Gene oft stark variieren. Diese Beobachtung ist die Grundlage der Analyse von Pan-Genomen. also der Konstellation originärer Gene innerhalb einer Abstammunsglinie, als evolutionsbiologische Einheiten. Spezies entsprechen prinzipiell ökologisch und evolutionär kohärenten Einheiten, jedoch sind die primären Habitate und ökologischen Nischen vieler prokaryotischer Spezies bis heute nur unzureichend beschrieben, insbesondere mit Blick auf den Einfluss ökologischer Präferenzen auf die Evolution von Genomen. Die Mehrheit vergleichender genomischer Studien untersucht einzelne prokaryotische Spezies mit Bezug auf deren klinische oder ökologische Relevanz. Aufgrund der wachsenden Verfügbarkeit genomischer Daten ist es nun jedoch möglich, vergleichende Studien über Speziesgrenzen hinweg durchzuführen, um allgemeine Prinzipien der Evolution von Pan-Genomen, Spezies und Sub-Spezies zu untersuchen. Die wesentlichen Ziele der vorliegenden Arbeit waren 1) die Annotation von Habitatpräferenzen prokaryotischer Spezies und Stämme; 2) die Quantifizierung des Einflusses von Umwelt und Evolutionsgeschichte (Phylogenie) auf die genomische Diversität von Prokaryoten; 3) die Bestimmung natürlicher Schwellenwerte der Genomsequenzähnlichkeit zwischen Spezies, auch anhand von Genkatalogen; 4) die Untersuchung der Abgrenzung zwischen Spezies, Sub-Spezies und Stämmen mithilfe metagenomischer Daten. Im ersten Teil der Arbeit werden Methoden zur Bestimmung ökologischer Präferenzen mikrobieller Spezies beschrieben. Die so gewonnenen Daten dienen in der Folge als Grundlage für die Quantifizierung von Umwelt- und evolutionsgeschichtlichen Einflüssen auf die Struktur und Evolution bakterieller Pan-Genome im zweiten Teil der Arbeit. Ein zentrales Ergebnis dieser Untersuchung war, dass bis zu 49% der strukturellen Varianz in Pan-Genomen durch Habitatpräferenzen erklärt werden kann, im Gegensatz zu lediglich 18% durch phylogenetische Trägheitseffekte. Dies zeigt, dass die Größe und Diversität von Pan-Genomen nicht phylogenetisch limitiert ist, insbesondere in Fällen von konvergenter Evolution. Große Kern-Genome sind ferner mit einer weiten ökologischen Verbreitung von Spezies assoziiert; eine mögliche Erklärung ist, dass weit verbreitete Spezies vielseitigere Genome mit mehr notwendigen Genen besitzen, die ein Überleben in vielfältigen Umgebungen ermöglichen. Die vorgelegte Arbeit kann weiterhin einen Beitrag zur Vorhersage ökologischer Profile neu beschriebener Spezies leisten. Im dritten Teil der Arbeit werden datenbezogene, operationelle Definition von Spezies-Grenzen untersucht. Es konnte gezeigt werden, dass Gene verschiedener Genome innerhalb derselben Spezies normalerweise mindestens 95% Ähnlichkeit der Nukleotidsequenz aufweisen, während die Ähnlichkeit zwischen Spezies desselben Genus geringer ausfällt. Dieser Wert liegt im Rahmen früherer Schätzungen. Der vierte Teil der Arbeit beschreibt abschließend die Herausforderungen bei der Bestimmung von evolutionären Linien innerhalb von Spezies und diskutiert anschließend, wie konzeptionelle Entwicklungen in dieser Frage für die Klassifizierung und Quantifizierung von Diversität anhand metagenomischer Daten genutzt werden kann.

Bdellovibrionaceae sind außergewöhnliche Bakterien, da sie als die kleinsten bekannten räuberischen Organismen gelten. Seit ihrer Entdeckung in Bodenproben im Jahr 1962 durch Stolp und Starr konnte eine weite Verbreitung in der Natur nachgewiesen werden. Besonderes Interesse erlangten Bdellovibrionaceae durch ihre Fähigkeit bakterielle Erreger wie Escherichia coli, Salmonella und Yersinia zu attackieren. Der bestuntersuchte Vertreter der Familie Bdellovibrionaceae ist Bdellovibrio bacteriovorus. Er durchläuft einen komplexen Lebenszyklus, der nur partiell verstanden ist. In der Attack-Phase werden durch einen noch nicht aufgeklärten Mechanismus potentielle Beutebakterien erkannt und der interzelluläre Kontakt hergestellt. Nachdem eine Pore in der Zellwand der ausschließlich Gram-negativen Beutebakterien erzeugt wurde, dringt B. bacteriovorus in den periplasmatischen Raum ein. Nach etwa 30 Minuten ist der Invasionsprozess abgeschlossen und man kann die Ausbildung sphärischer Bdelloblasten beobachten. Im Beutebakterium verdaut B. bacteriovorus makromolekulare Bestandteile des Wirtes und wächst zu einem langen spiralförmigen Stäbchen aus. Es setzt schließlich die Zellteilung ein bei der 5 bis 30 Tochterzellen in Abhängigkeit zur Größe des Beutebakteriums freiwerden. Mit der Reproduktion ist der parasitäre Lebenszyklus nach etwa 3 Stunden beendet. Die komplexe Regulation der Expression von Proteinen konnte für die einzelnen Wachstumsphasen durch ein- sowie zweidimensionale Gelelektrophorese nachgewiesen werden. In der Vergangenheit haben verschiedene Studien die Komplexität der Wechselwirkung mit den Beutebakterien belegt. Hierbei wurde, neben dem offensichtlichen Abbau makromolekularer Bestandteile der Beutebakterien, ein Recycling und Einbau von Membranbestandteilen wie Lipopolysacchariden (LPS) und Outer Membrane Proteine (OmP) in das Membransystem von B. bacteriovorus diskutiert. Die biologische Interpretation dieses Phänomens ist eine erhöhte Effizienz in der Reproduktion von B. bacteriovorus wenn es Bestandteile des Wirtsbakteriums wiederverwertet im Gegensatz zur Eigensynthese. Trotz der morphologischen Ähnlichkeit und des ungewöhnlichen Lebensstils wurde festgestellt, dass die Familie der Bdellovibrionaceae phylogenetisch sehr heterogen ist. Es werden zur Zeit zwei Gattungen unterschieden Bdellovibrio und Bacteriovorax. Aufgrund von 16S rRNA Untersuchungen konnten auch innerhalb der Gattungen eine Vielzahl von phylogenetisch distinkten Arten nachgewiesen werden. In der Literatur wird eine regulierende Wirkung auf pathogene Gram-negative Bakterien für aquatische Systeme durch Bdellovibrionaceae beschrieben. Weiterhin konnten sie bei verschiedenen Nutztieren im Verdauungstrakt mikrobiologisch nachgewiesen werden. Ein positiver Einfluss auf die Gesundheit der Tiere wurde bei Vorkommen von B. bacteriovorus festgestellt. Für eine Charakterisierung von Umweltisolaten werden in der vorliegenden Arbeit genetische Methoden vorgestellt. Hierfür wurden Hybridisierungsmethoden und PCR-methoden entwickelt, die es ermöglichen aus der Umwelt isolierte Bdellovibrionaceae phylogenetisch einzuordnen. Es konnte gezeigt werden, das sowohl B. bacteriovorus als auch Bacteriovorax stolpii im Verdauungstrakt von Nutztieren vorkommen. Es ist weiterhin gelungen eine PCR-methode für Kotproben zu entwickeln, die einen direkten Nachweis ermöglicht ohne mikrobiologische Anzucht. B. bacteriovorus ist ein Gram-negatives Bakterium, allein dieser Sachverhalt spiegelt die Schwierigkeit wider, wenn der enzymattische Abbau von Zellwandbestandteilen der Beutebakterien Ziel der Untersuchung ist, da auch die Beutebakterien Gram-negativ sind. Es ergibt sich aufgrund eines ähnlichen Zellwandaufbaus ein immenses analytisches Problem die makromolekularen Bestandteile von Wirtsbakterium und Jäger zu trennen. Um dem Lebensstil angepasst Modifikationen von B. bacteriovorus zu untersuchen, wurde in dieser Arbeit LPS, charakteristischer Bestandteil der Äußeren Membran, von Wildtypstamm B. bacteriovorus HD100 und seiner wirtsunabhängigen Mutante HI100 isoliert und das Lipid A strukturell aufgeklärt. Die Isolation gelang durch die Ausnutzung unterschiedlicher Fällungseigenschaften des LPS von B. bacteriovorus HD100 gegenüber des E. coli K-12 LPS aus dem Extraktionsmittel. Weiterhin konnte nachgewiesen werden, das B. bacteriovorus S-Form LPS besitzt. Die außergewöhnliche Struktur des Lipid A von B. bacteriovorus wurde im Detail mit massenspektrometrischen Methoden, ein- und zweidimensionalen NMR Methoden sowie mikrochemischer Methoden in Kombination mit der GC/MS charakterisiert. Der Fettsäureanker besteht aus a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. Dies stellt eine neuartige Struktur dar, da an allen bisher bekannten Lipid A´s sich Brönstedtsäuren am hydrophilen Fettsäureanker befinden, die in physiologischer Lösung durch Protonenabgabe negative Ladungen tragen. Im Lipid A von B. bacteriovorus sind diese Substituenten durch den Neutralzucker Mannose ersetzt. Weiterhin wurden als Fettsäuren nur 3-Hydroxyfettsäuren nachgewiesen, wobei sich auf die 6 gebunden Fettsäuren etwa 1,5 Doppelbindungen verteilen. Dieses Lipid A zeigt eine wesentlich reduzierte endotoxische Aktivität im Vergleich zu E. coli Lipid A und weist als biophysikalische Besonderheit eine erhöhte Fluidität über einen weiten Temperaturbereich auf. Die vorgestellte 16S rRNA Analyse und die Strukturanalyse des Lipid A von B. bacteriovorus belegen seine besondere Stellung in der Welt der Bakterien. / Bdellovibrionaceae are extraordinary bacteria known as the smallest predatory organism so far. Since their discovery in soil samples by Stolp and Starr 1963 they have been detected in a wide range of other natural habitats. Bdellovibrionaceae became the focus of attention concerning their ability to attack pathogens like Escherichia coli, Salmonella and Yersinia. For Bdellovibrio bacteriovorus the most detailed studies are available. The up to now only partially understood lifecycle consists of several complex phases. In the attack phase B. bacteriovorus is motile possessing flagella and the preys are recognized by an unknown mechanism. After attachment on the cell wall within 15 to 30 minutes a pore is formed which is used as entrance to the periplasmatic space of the Gram-negative prey bacteria. The completion of the invasion process can be observed by the change of the prey s shape to spherical bdelloblasts. Inside of the prey B. bacteriovorus degrades macromolecular compounds and transforms into a long spirally shaped rod. At the end of the lifecycle the rod divides yielding 5 up to 30 daughter cells depending on the size of the prey bacteria. This reproduction phase is completed within 3 hours. The complex regulation of expression of a certain number of proteins was observed by one and two dimensional gelelectrophoresis. The complexity of the interaction between predator and prey were examined in several studies. Besides the obvious degradation of macromolecular compounds of the prey, reutilising of lipopolysaccharides (LPS) and Outer Membrane Proteins (OmP) into the membrane system of B. bacteriovorus was discussed. The biological interpretation of such behaviour was that it is more efficient for reproduction to recycle components of the prey than to perform de novo synthesis. Unexpectedly, despite the unique predatory lifecycle and common morphological features, Bdellovibrionaceae show a great phylogenetical diversity based on 16S rRNA analyses. Bdellovibrionaceae are divided into the three species Bdellovibrio bacteriovorus, Bacteriovorax stolpii, Bacteriovorax starrii and some strains yet to be assigned. In two studies Bdellovibrionaceae were found as part of regulation processes for decreasing the number of pathogenic Gram-negative bacteria in aquatic systems. Furthermore, B. bacteriovorus were found in the intestinal tract of several domestic animals showing a positive influence on the state of health. For the phylogenetic characterization of environmental isolates techniques were developed based on hybridisation methods and the PCR. In this work we detected B. bacteriovorus and B. stolpii strains in the gut of animals. Furthermore, a PCR method for direct detection of Bdellovibrionaceae in fecal samples was developed. B. bacteriovorus are Gram-negative bacteria. This fact complicates the study of the degradation of the prey s cell wall as it possesses the architecture of Gram-negative bacteria also. Furthermore, the search for important modifications of the cell wall of B. bacteriovorus concerning the predatory lifestyle becomes an analytical problem. In this study we isolated LPS of the wild type strain B. bacteriovorus HD100 and its host independent mutant strain HI100. For the isolation of pure B. bacteriovorus HD100 S-form LPS we took advantage of different precipitation properties in the extraction solvent of E. coli K-12 LPS and the predator s LPS. The structure of the lipid A was examined in detail by mass spectrometric methods, one- and two-dimensional NMR and chemical analytical techniques. The novel structure consists of backbone built of a-D-ManpII-(1(R)4)-ß-D-GlcpN3NII-(1(R)6)-ß-D-GlcpN3NI-(1(R)1)-a-D-ManpI. This is the first known natural lipid A without negatively charged substituents in physiological solution. The lipid A of B. bacteriovorus carries the neutral sugar mannose instead of Brönstedt acids. Furthermore, the lipid A exclusively consists of 3-hydroxy fatty acids with approximately 1.5 double bounds distributed on six bounded fatty acids. This lipid A shows a significant decreased endotoxic activity in comparison to E. coli lipid A and revealed increased fluidity over broad temperature range as further remarkable biophysical property. The 16S rRNA analysis and the structural analysis of the lipid A of B. bacteriovorus document the unique position in the world of bacteria.

Genealogy reconstruction is widely used in biology when relationships among entities are studied. Phylogenies, or evolutionary trees, show the differences between species. They are of profound importance because they help to obtain better understandings of evolutionary processes. Pedigrees, or family trees, on the other hand visualize the relatedness between individuals in a population. The reconstruction of pedigrees and the inference of parentage in general is now a cornerstone in molecular ecology. Applications include the direct infer- ence of gene flow, estimation of the effective population size and parameters describing the population’s mating behaviour such as rates of inbreeding. In the first part of this thesis, we construct genealogies of various types of cancer. Histopatho- logical classification of human tumors relies in part on the degree of differentiation of the tumor sample. To date, there is no objective systematic method to categorize tumor subtypes by maturation. We introduce a novel algorithm to rank tumor subtypes according to the dis- similarity of their gene expression from that of stem cells and fully differentiated tissue, and thereby construct a phylogenetic tree of cancer. We validate our methodology with expression data of leukemia and liposarcoma subtypes and then apply it to a broader group of sarcomas and of breast cancer subtypes. This ranking of tumor subtypes resulting from the application of our methodology allows the identification of genes correlated with differentiation and may help to identify novel therapeutic targets. Our algorithm represents the first phylogeny-based tool to analyze the differentiation status of human tumors. In contrast to asexually reproducing cancer cell populations, pedigrees of sexually reproduc- ing populations cannot be represented by phylogenetic trees. Pedigrees are directed acyclic graphs (DAGs) and therefore resemble more phylogenetic networks where reticulate events are indicated by vertices with two incoming arcs. We present a software package for pedigree reconstruction in natural populations using co-dominant genomic markers such as microsatel- lites and single nucleotide polymorphism (SNPs) in the second part of the thesis. If available, the algorithm makes use of prior information such as known relationships (sub-pedigrees) or the age and sex of individuals. Statistical confidence is estimated by Markov chain Monte Carlo (MCMC) sampling. The accuracy of the algorithm is demonstrated for simulated data as well as an empirical data set with known pedigree. The parentage inference is robust even in the presence of genotyping errors. We further demonstrate the accuracy of the algorithm on simulated clonal populations. We show that the joint estimation of parameters of inter- est such as the rate of self-fertilization or clonality is possible with high accuracy even with marker panels of moderate power. Classical methods can only assign a very limited number of statistically significant parentages in this case and would therefore fail. The method is implemented in a fast and easy to use open source software that scales to large datasets with many thousand individuals.

No description available.

The presented study comprises a complete morphological and phylotaxonomic revision of basal Sphenacodontia, designated as the paraphyletic ‘haptodontines’. Ianthodon from the Kasimovian is known from newly identified elements, including most of the skull and particular postcrania. This species is determined as the best model for the initial morphology of the Sphenacomorpha (Edaphosauridae and Sphenacodontia). Remarkably older sphenacodontian remains from the Moscovian indicate a derived, though fragmentarily known form, possibly basal Sphenacodontoidea. The genus Haptodus is conclusively revised, including the revalidation of the type species H. baylei from the Artinskian. Haptodus grandis is renamed as Hypselohaptodus, gen. nov. “Haptodus” garnettensis is not monophyletic with Haptodus, moreover the material assigned to it yielded a greater diversity. Thus, its renaming includes Eohaptodus garnettensis, gen. nov., Tenuacaptor reiszi, gen. et spec. nov., and Kenomagnathus scotti, gen. et spec. nov. Along with Ianthodon and the basal edaphosaurid Ianthasaurus, these taxa from a single assemblage are differentiated by dentition and skull proportions, providing a case study of annidation. Since Ianthodon can be excluded from Sphenacomorpha, the larger, stem-based taxon Haptodontiformes is introduced. More derived ‘haptodontines’ apparently form another radiation, named as Pantherapsida. This new taxon includes Cutleria, Tetraceratops, Hypselohaptodus, the Palaeohatteriidae (Pantelosaurus and Palaeohatteria), and the Sphenacodontoidea. The ‘pelycosaur’-therapsid transition is affirmed as a long-term development. An integrative evolutionary hypothesis of basal sphenacodontians is provided, within which the ghost lineage of Early Permian therapsids can be explained by biome shift.

Genealogy reconstruction is widely used in biology when relationships among entities are studied. Phylogenies, or evolutionary trees, show the differences between species. They are of profound importance because they help to obtain better understandings of evolutionary processes. Pedigrees, or family trees, on the other hand visualize the relatedness between individuals in a population. The reconstruction of pedigrees and the inference of parentage in general is now a cornerstone in molecular ecology. Applications include the direct infer- ence of gene flow, estimation of the effective population size and parameters describing the population’s mating behaviour such as rates of inbreeding. In the first part of this thesis, we construct genealogies of various types of cancer. Histopatho- logical classification of human tumors relies in part on the degree of differentiation of the tumor sample. To date, there is no objective systematic method to categorize tumor subtypes by maturation. We introduce a novel algorithm to rank tumor subtypes according to the dis- similarity of their gene expression from that of stem cells and fully differentiated tissue, and thereby construct a phylogenetic tree of cancer. We validate our methodology with expression data of leukemia and liposarcoma subtypes and then apply it to a broader group of sarcomas and of breast cancer subtypes. This ranking of tumor subtypes resulting from the application of our methodology allows the identification of genes correlated with differentiation and may help to identify novel therapeutic targets. Our algorithm represents the first phylogeny-based tool to analyze the differentiation status of human tumors. In contrast to asexually reproducing cancer cell populations, pedigrees of sexually reproduc- ing populations cannot be represented by phylogenetic trees. Pedigrees are directed acyclic graphs (DAGs) and therefore resemble more phylogenetic networks where reticulate events are indicated by vertices with two incoming arcs. We present a software package for pedigree reconstruction in natural populations using co-dominant genomic markers such as microsatel- lites and single nucleotide polymorphism (SNPs) in the second part of the thesis. If available, the algorithm makes use of prior information such as known relationships (sub-pedigrees) or the age and sex of individuals. Statistical confidence is estimated by Markov chain Monte Carlo (MCMC) sampling. The accuracy of the algorithm is demonstrated for simulated data as well as an empirical data set with known pedigree. The parentage inference is robust even in the presence of genotyping errors. We further demonstrate the accuracy of the algorithm on simulated clonal populations. We show that the joint estimation of parameters of inter- est such as the rate of self-fertilization or clonality is possible with high accuracy even with marker panels of moderate power. Classical methods can only assign a very limited number of statistically significant parentages in this case and would therefore fail. The method is implemented in a fast and easy to use open source software that scales to large datasets with many thousand individuals.:Abstract v Acknowledgments vii 1 Introduction 1 2 Cancer Phylogenies 7 2.1 Introduction..................................... 7 2.2 Background..................................... 9 2.2.1 PhylogeneticTrees............................. 9 2.2.2 Microarrays................................. 10 2.3 Methods....................................... 11 2.3.1 Datasetcompilation ............................ 11 2.3.2 Statistical Methods and Analysis..................... 13 2.3.3 Comparison of our methodology to other methods . . . . . . . . . . . 15 2.4 Results........................................ 16 2.4.1 Phylogenetic tree reconstruction method. . . . . . . . . . . . . . . . . 16 2.4.2 Comparison of tree reconstruction methods to other algorithms . . . . 28 2.4.3 Systematic analysis of methods and parameters . . . . . . . . . . . . . 30 2.5 Discussion...................................... 32 3 Wild Pedigrees 35 3.1 Introduction..................................... 35 3.2 The molecular ecologist’s tools of the trade ................... 36 3.2.1 3.2.2 3.2.3 3.2.1 Sibship inference and parental reconstruction . . . . . . . . . . . . . . 37 3.2.2 Parentage and paternity inference .................... 39 3.2.3 Multigenerational pedigree reconstruction . . . . . . . . . . . . . . . . 40 3.3 Background..................................... 40 3.3.1 Pedigrees .................................. 40 3.3.2 Genotypes.................................. 41 3.3.3 Mendelian segregation probability .................... 41 3.3.4 LOD Scores................................. 43 3.3.5 Genotyping Errors ............................. 43 3.3.6 IBD coefficients............................... 45 3.3.7 Bayesian MCMC.............................. 46 3.4 Methods....................................... 47 3.4.1 Likelihood Model.............................. 47 3.4.2 Efficient Likelihood Calculation...................... 49 3.4.3 Maximum Likelihood Pedigree ...................... 51 3.4.4 Full siblings................................. 52 3.4.5 Algorithm.................................. 53 3.4.6 Missing Values ............................... 56 3.4.7 Allelefrequencies.............................. 58 3.4.8 Rates of Self-fertilization.......................... 60 3.4.9 Rates of Clonality ............................. 60 3.5 Results........................................ 61 3.5.1 Real Microsatellite Data.......................... 61 3.5.2 Simulated Human Population....................... 62 3.5.3 SimulatedClonalPlantPopulation.................... 64 3.6 Discussion...................................... 71 4 Conclusions 77 A FRANz 79 A.1 Availability ..................................... 79 A.2 Input files...................................... 79 A.2.1 Maininputfile ............................... 79 A.2.2 Knownrelationships ............................ 80 A.2.3 Allele frequencies.............................. 81 A.2.4 Sampling locations............................. 82 A.3 Output files..................................... 83 A.4 Web 2.0 Interface.................................. 86 List of Figures 87 List of Tables 88 List Abbreviations 90 Bibliography 92 Curriculum Vitae I

Female mating rates vary widely, even among closely related species, but the reasons for this variation are not fully understood. Across Drosophila species, female mating frequencies are positively associated with sperm length. This association may be due in part to sperm limitation, with longer-spermed species transferring fewer sperm, or to cryptic female choice. However, a previously overlooked factor is sperm metabolic rate, which may correlate with sperm length. If faster-metabolizing sperm accumulate agerelated cellular damage more quickly, then females should remate sooner to obtain fresh sperm. Alternatively, frequent female mating may select for increased sperm competitiveness via increased metabolism. Here, we measure sperm metabolism across 13 Drosophila species and compare these measures to published data on female mating rate and on sperm length. Using fluorescent lifetime imaging microscopy, we quantify NAD(P)H metabolism ex vivo, in intact organs. Phylogenetically controlled regression reveals that sperm metabolic rate is positively associated with sperm length and with female mating frequency. Path analysis shows sperm length driving sperm metabolism and sperm metabolism either driving or being driven by female mating rate. While the causal directionality of these relationships remains to be fully resolved, and the effect of sperm metabolism on sperm aging and/or sperm competitiveness remains to be established, our results demonstrate the importance of sperm metabolism in sexual selection.