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Ancient pathogenomics is the field that studies past pathogens by recovering ancient DNA from archaeological remains. This discipline has been highly reliant on phylogenetic analyses, which provide information on: how past strains are related to their modern relatives; past diversity of the pathogen in question; and their geographical dispersal during past epidemics. However, in order to understand what genomic changes contributed to the dispersal, adaptation, and virulence evolution of pathogens, as well as to understand their past ecology, one needs to employ other analytical tools. In this thesis, I have employed and adapted the concepts of gene content, de novo assembly, and pangenomics to study the differences in virulence and functional potential of ancient bacterial strains. I have applied these approaches to Yersinia pestis, the bacterium responsible for plague, with a focus on its early genomic evolution in Eurasia between 5,000 to 3,000 years ago. I also demonstrate these concepts by applying these workflows to the first recovered ancient Streptococcus mutans genome from South Africa. This pathobiont is strongly associated with caries formation, one of the top ten health burdens affecting present day human populations. Overall, this thesis showcases the importance of expanding the ancient pathogenomics toolkit to include functional analytical approaches to study past pathogen genomes. Only in the light of archaeological contexts, will we gain insight into the emergence, ecology and long-term evolution of ancient pathogens. Introduction ... p. 4 Health and disease: connecting the past to the present ... p. 4 How can we learn from past disease? texts, mummies, bones and their tales ... p. 5 - Ancient dna ... p. 6 - Plague ... p. 9 -- Yersinia pestis ... p. 10 -- History of plague and contributions of ancient dna ... p. 12 - Caries ... p. 15 -- Evidence of caries in the archaeological record ... p. 16 -- Streptococcus mutans: a commensal with disease potential ... p. 17 - Comparative ancient pathogenomics ... p. 19 Aim ... p. 23 Manuscripts overview and author contribution ... p. 24 Manuscript a ... p. 30 Manuscript b ... p. 58 Manuscript c ... p. 89 Manuscript d ... p. 105 Manuscript e ... p. 122 Discussion ... p. 156 - Where to look for ancient pathogen dna? ... p. 156 - What have we learned from phylogenies? ... p. 160 - New insights from known genes and deletions ... p. 162 - Moving on from a single reference genome: de novo assembly and pangenomics ... p. 164 - What is knowledge without context: future outlooks for the field of ancient pathogenomics ... p. 167 Conclusion ... p. 171

Archaeogenetics is the research field of studying the genetic information contained in ancient DNA (aDNA) to gain insight into the past. Analysis of human aDNA from archaeological material has allowed archaeogeneticists to observe changes in the genetic composition of populations in an area through time. By using aDNA in this manner, a higher degree of resolution can be gained into the timing of past genetic transitions, compared to the resolution that is available when inferring the past from modern genomic data alone. In this thesis, I focus on the movement of genes, via migration of people and/or admixture, and the information that this movement can provide about human history. I introduce the differences between the inheritance mechanisms of uniparental (mitochondrial DNA and the Y-chromosome) and autosomal markers; the forces of evolution in population genetics; some methods commonly used in the analysis of human aDNA in the manuscripts included in this thesis; prior (archaeo-)genetics research regarding the population history of West Eurasia and the Americas -as context for my own research in these geographic areas-, and discuss the information gained by my own work about the population history of the areas studied, the limitations of archaeogenetic inferences, and the importance of combining archaeogenetic results with those from other disciplines when studying human history. 1. Introduction 1.1 Introduction to human population genetics 1.1.1 Inheritance patterns in humans 1.1.2 The forces of evolution 1.2 Analytical methods used in ancient DNA 1.2.1 Analysis of common variants in human population history 1.2.1 Using rare variation to study human history 1.3 The major genetic transitions in Western Eurasia from the Mesolithic to the Bronze Age. 1.4 The population history of the Americas 2. Aim of the thesis Manuscript A Manuscript B Manuscript C 3. Overview of manuscripts and author’s contribution 3.1 Manuscript A 3.2 Manuscript B 3.3 Manuscript C 4. Manuscript A 5. Manuscript B 6. Manuscript C 7. Discussion 7.1 Insights gained by the work presented 7.2 Limitations to the archaeogenetic approach 7.3 How many individuals is enough? 7.4 Thoughts about the future of thinking about the past 8. References

The revolution of sequencing technology has brought an exponential increase in the production of genomic data. This thesis tackles global and continental questions on human demographic history from two directions using genetic data. Manuscript A provides a novel analytical method for estimating migration rate and effective population size utilizing highcoverage whole genome sequences, while manuscript B and C reveal the history of population movement and interactions by directly analyzing genome-wide data from ancient individuals. 1. Introduction 1.1 Learning population demographic history from genomic data 1.1.1 Whole genome sequences help on reconstructing population history 1.1.2 Applications of the sequential Markov coalescent for demographic inference 1.1.3 A brief overview on the human demographic history 1.2 Genetic perspectives on the (past) population structure in Africa 1.2.1 Present-day genetic, linguistic and subsistence variations in Africa 1.2.2 Ancient DNA sheds lights on the past population movements in Africa 1.3 A brief introduction into Eurasia's Eastern Steppe 2. Aim of the thesis 3. Overview of Manuscripts and author’s Contribution 3.1 Manuscript A 3.2 Manuscript B 3.3 Manuscript C 4. Manuscript A 5. Manuscript B 6. Manuscript C 7. Discussion 7.1 MSMC-IM and outlook 7.2 aDNA - challenges and future 8. References

I applied state-of-the-art ancient DNA (aDNA) approaches to recover highly degraded DNA molecules from post-glacial hunter-gatherers and early farmers from the Mediterranean to investigate the demographic processes that underlie archaeological transitions after the Ice Age and during the expansion of farming practises. I placed a particular emphasis on examining the genomic evidence for crossings of the Mediterranean Sea, as was proposed by archaeologists based on similarities in material culture. Each of the manuscripts contributes crucial genomic reference points for Mediterranean areas where human occupation locally persisted during the Ice Age, or with some of the earliest indications for farming. In Manuscript A, I report the oldest ancient genome-wide data for Africa to date retrieved from nine ~15,000 calBP microlithic Iberomaurusian hunter-gatherers from the Mediterranean coast in Morocco (Van de Loosdrecht et al. 2018. Science). In Manuscript B, I report a genomic transect of ten individuals from the Iberian Peninsula from various sites with Upper Palaeolithic to Middle Neolithic occupations (~13,000-6,000 calBP) (Villalba-Mouco et al. 2019. Curr. Biol). In Manuscript C, I explore the multi-disciplinary potential of archaeogenetics, and jointly report genomic and isotopic data for dietary inference for a transect of 19 Mesolithic foragers and Early Neolithic farmers from Sicily (~10,700-7,200 calBP) (Van de Loosdrecht et al. 2020. BioRxiv). In the discussion I provide archaeogenetic perspectives on topics long been debated by scholars of Mediterranean prehistory: 1) the origin of the Iberomaurusian industry in Northwest Africa; 2) the admixture dynamics of European foragers after the Ice Age; 3) the genomic evidence for direct population interactions across the Mediterranean Sea; 4) the demographic processes underlying the transition from foraging to farming in the Mediterranean; and 5) the southern Mediterranean Neolithic expansion route. 1. Introduction 1.1. The ancient DNA revolution: current state-of-the-art 1.2. The ~10,000-year trajectory from foraging to farming in the Mediterranean region 1.2.1. Northwestern Africa during and after the last Ice Age 1.2.2. Europe during and after the last Ice Age 1.2.3. The Neolithic Revolution: sedentary farming communities expand into the Mediterranean 2. Aims and objectives 3. Overview of manuscripts 3.1. Manuscript A 3.2. Manuscript B 3.3. Manuscript C 4. Author contributions 5. Manuscript A 6. Manuscript B 7. Manuscript C 8. Discussion 8.1. Archaeogenetic perspectives on the repopulation of northern Africa and Europe after the Last Glacial Maximum 8.1.1. Northwestern Africa 8.1.2. Europe 8.2. Is there genomic evidence that prehistoric Mediterranean peoples were in direct contact across the Mediterranean Sea? 8.3. Limitations of ancestry inferences using ancient DNA 8.4. Conclusion 8.5. Outlook

Ancient DNA has become an indispensable resource of fundamental research. Especially the combination of DNA capture methods and Next Generation Sequencing (NGS) has allowed to exploit the full potential of ancient DNA. In this dissertation I present three studies which involve capture of ancient DNA to answer different scientific questions. In my first study I bioinformatically reconstruct the mitochondrial genome of the extinct giant deer Megaloceros giganteus after applying mitochondrial DNA capture and sequencing. Using the giant deer's reconstructed mitochondrial genome I then apply phylogenetic analyses that allow to resolve the giant deer's placement within the cervid family tree and reaffirm the fallow deer being its closest extant relative. Mitochondrial DNA capture also provides the basis for the next study, in which I investigate the effects of X-rays on ancient DNA. Different radiation settings are explored including those commonly used in computed tomography (CT) of anthropological and palaeontological specimens. The results allow to define 200 Gray (Gy) as the maximum absorbed X-ray dose which can still be regarded harmless to ancient DNA molecules. No X-radiation induced effects can be observed below this threshold. In my third study I develop and apply capture of 488 human immunity genes in order to compare the immunogenetic makeup of Late Medieval plague (Yersinia pestis) victims and their modern day successors from Ellwangen, Southern Germany. Applying bioinformatic tools allows me to investigate kinship within both populations, determine genetic continuity between past and present, and reconstruct the Human Leukocyte Antigen (HLA) allelic profiles of both populations. While the majority of HLA alleles do not differ in their frequencies, HLA-B51:01 and HLA-DRB113 show significant frequency differences between both populations, potentially indicative of selective pressure through Yersinia pestis. 1 ancient DNA research - History and Methods 1.1 A Short History of aDNA Research 1.2 aDNA Retrieval from Old Bone 1.3 DNA Libraries 1.4 Next Generation Sequencing 2 Applications of aDNA Capture 2.1 Current Methods for Target DNA Enrichment 2.2 Study 1: Capture of Giant Deer Mitochondrial DNA 2.2.1 Archaeological Material 2.2.2 Extraction of Giant Deer DNA and Library Preparation 2.2.3 Bait Preparation and Capture 2.2.4 Sequence Processing and Assembly 2.2.5 Multiple Sequence Alignment 2.2.6 Phylogenetic Analyses Publication: Mitochondrial Genomes of Giant Deers Suggest their Late Survival in Central Europe 2.3 Study 2: Effect of X-rays on ancient DNA 2.3.1 Background and Previous Work 2.3.2 Investigating X-ray effects on aDNA after Mitochondrial DNA Capture Publication: Effect of X-ray 'irradfotion on anC'ient DNA 'in s'Ub-fossil bones - G'Uidel'ines for safe X-ray imaging 2.4 Study 3: Immunity Capture 2.4.1 Background: Plague, HLA and other Immunity Genes 2.4.2 Initial Array Design for Immunity Capture 2.4.3 Improved Array Design 2.4.4 In-Solution Capture 2.4.5 Evaluation of the In-Solution Capture Efficiency 2.4.6 Application of the Immunity Capture to 16th Cent. Plague Victims 2.4.7 HLA Typing and Allele Frequency Comparisons 2.4.8 CCR5-6 32 Mutation in Plague Victims and Contemporary Ell- wangen 3 Discussion 3.1 Giant Deer and Megafauna 3.2 X-rays and aDNA 3.3 In-Solution Immunity Capture 3.4 Stability of the CCR5-6 32 Mutation 3.5 Frequency Shifts in particular HLA alleles 4 Conclusion Appendix and Supplementary Material

Objekt des Monats der UB Wien aus der Zoologischen Sammlung (Juli 2013) Text und Foto: Maximilian Petrasko

Die Entstehung, die Entwicklung und der Einfluß einer Daphnia-Dauereibank auf eine Daphnia-Population im Belauer See (Schleswig-Hostein) wurde mit molekularbiologischen Methoden (Mikrosatelliten; SSCP) untersucht. Eine Besonderheit des Belauer Sees ist das stratifizierte Sediment, so daß paläolimnologische Studien anhand älterer Dauereier (ca. 200 Jahre) durchgeführt werden konnten. Die Bedeutung von Daphnia-Dauereiern im Sediment wurde anhand zweier Hypothesen diskutiert. Hypothese 1 postuliert, daß die Dauereier im Sediment ein Spiegel vergangener Populationen sind, Hypothese 2 nimmt an, daß die aus den Dauereiern schlüpfenden Daphnien die Evolutionsrate der Population verlangsamen. Untersuchungen der Produktion, der Selektion und des Schlüpfens der Dauereier zeigten, daß beide Hypothesen nicht für den Belauer See zutreffend sind. Bei den drei im Belauer See koexistierenden Taxa ist eine zeitliche Veränderung in der genetischen Zusammensetzung in der Population zu erkennen. Die Ergebnisse zeigen, daß ein Einfluß von der Dauereibank ausgeht Inhaltsverzeichnis 1 Einleitung ___________________________________________________1 2 Material und Methoden _______________________________________7 2.1 Der Untersuchungsorganismus Daphnia ______________________________________ 7 2.2 Probenahmen ____________________________________________________________ 8 2.2.1 Das Untersuchungsgewässer__________________________________________ 8 2.2.2 Probenahmen von Daphnien aus dem Pelagial ____________________________ 9 2.2.3 Probenahmen von Dauereiern aus dem Sediment_________________________ 10 2.2.3.1 Probenahme von rezenten Dauereiern _____________________________ 10 2.2.3.2 Probenahme von alten Dauereiern ________________________________ 12 2.2.4 Probenahmen von Dauereiern aus dem Spülsaum ________________________ 13 2.3 Populationsdynamische Parameter__________________________________________ 13 2.3.1 Abundanzen von Dauereiern im Sediment ______________________________ 13 2.3.1.1 Abundanzen von rezenten Dauereiern _____________________________ 13 2.3.1.2 Abundanzen von älteren Dauereiern ______________________________ 15 2.3.2 Schwimmfähigkeit von Ephippien ____________________________________ 15 2.3.3 Schlüpffähigkeit von Dauereiern _____________________________________ 15 2.4 Genetische Analyse _______________________________________________________ 17 2.4.1 Probenvorbereitung und DNA-Isolierung_______________________________ 17 2.4.2 Charakterisierung der genetischen Struktur von Daphnien__________________ 20 2.4.2.1 Amplifikation von DNA-Sequenzen ______________________________ 20 2.4.2.2 Mikrosatelliten-Analyse ________________________________________ 23 2.4.2.3 Analyse mitochondrieller rDNA _________________________________ 27 2.4.2.4 Aufreinigung der mitochondriellen PCR-Produkte und Sequenzierung ___ 31 2.4.2.5 Allozym-Analyse _____________________________________________ 32 2.5 Datenanalyse ____________________________________________________________ 34 2.5.1 Genetische Variation_______________________________________________ 34 2.5.2 Hardy-Weinberg-Gleichgewicht (HWE) _______________________________ 35 2.5.3 Test auf Genkopplung (Linkage Equilibrium) ___________________________ 36 2.5.4 Simpson Index____________________________________________________ 37 2.5.5 Exakter Test zur Populationsdifferenzierung ____________________________ 38 2.5.6 Genetische Distanz und Konstruktion genetischer Bäume __________________ 38 2.5.7 Wright’s F-Statistik________________________________________________ 40 2.5.8 Multivariate Statistik_______________________________________________ 40 2.5.8.1 Prinzipielle-Komponenten-Analyse (PCA) _________________________ 41 2.5.8.2 Multidimensionales-Skalieren (MDS) _____________________________ 41 3 Ergebnisse _________________________________________________42 3.1 Auswahl und Etablierung der genetischen Methoden___________________________ 42 3.1.1 Methode zur Bestimmung der Haplotypen ______________________________ 42 3.1.1.1 SSCP-Analyse mit fluoreszenzmarkierten PCR-Produkten_____________ 43 3.1.1.2 SSCP-Analyse mit radioaktiven Isotopen __________________________ 46 3.1.1.3 SSCP-Analyse nach einer Silberfärbung ___________________________ 47 3.1.2 Mikrosatelliten-Analyse zur Bestimmung der klonalen Struktur _____________ 48 3.1.2.1 Auswahl von Mikrosatelliten-Primern _____________________________ 48 3.1.2.2 Optimierung der Mikrosatelliten-Analyse __________________________ 54 3.1.2.3 Einfluß des Probenumfangs auf die Genauigkeit der genetischen Analyse _ 55 3.1.2.4 Vergleich von Allozym- und Mikrosatelliten-Analyse ________________ 56 Inhaltsverzeichnis ii 3.2 Saisonale Entwicklung der genetischen Struktur von Daphnia im Belauer See ______ 60 3.2.1 Unterteilung der saisonalen Entwicklung anhand der abiotischen Parameter im Belauer See ___________________________________________________ 60 3.2.2 Saisonale Entwicklung der genetischen Variation der Daphnien im Belauer See 61 3.2.3 Saisonale Entwicklung der Mikrosatelliten-Allelfrequenzen ________________ 65 3.2.4 Saisonale Entwicklung der Populationsstruktur __________________________ 68 3.3 Die Populationsstruktur von Daphnien während der Phase der sexuellen Reproduktion____________________________________________________________ 72 3.3.1 Populationsstruktur von D. galeata im Mai ’99 __________________________ 72 3.3.1.1 Anteil der treibenden Ephippien__________________________________ 72 3.3.1.2 Genetische Variation bei D. galeata_______________________________ 73 3.3.1.3 Einfluß der sexuellen Reproduktion auf die Heterozygosität____________ 74 3.3.1.4 Klonale Zusammensetzung in der D. galeata-Population ______________ 75 3.3.1.5 Genetische Differenzierung innerhalb der Population _________________ 76 3.3.2 Populationsstruktur von D. hyalina im Herbst ’99 ________________________ 77 3.3.2.1 Genetische Variation bei D. hyalina_______________________________ 77 3.3.2.2 Einfluß der sexuellen Reproduktion auf die Heterozygosität____________ 78 3.3.2.3 Klonale Zusammensetzung in der D. hyalina-Population ______________ 80 3.3.2.4 Genetische Differenzierung innerhalb der Population _________________ 81 3.3.2.5 Entwicklung der Populationsstruktur im Herbst _____________________ 83 3.4 Dauereier im Sediment und im Spülsaum ____________________________________ 87 3.4.1 Abundanzen von Dauereiern im Sediment ______________________________ 87 3.4.2 Gleichmäßigkeit der Verteilung der Dauereier im Sediment ________________ 88 3.4.3 Schlüpferfolg von Daphnia-Dauereiern ________________________________ 90 3.4.3.1 Schlüpferfolg von Daphnia-Dauereiern aus dem Sediment ohne SedimentÜberschichtung_______________________________________________ 90 3.4.3.2 Schlüpferfolg von Daphnia-Dauereiern aus dem Sediment mit SedimentÜberschichtung_______________________________________________ 91 3.4.3.3 Schlüpferfolg von Daphnia-Dauereiern aus dem Spülsaum ____________ 93 3.4.4 Vergleich der Dauereier im Sediment und der geschlüpften Daphnien ________ 94 3.4.4.1 Taxonomische Zusammensetzung der Dauereier im Sediment und der geschlüpften Daphnien_________________________________________ 94 3.4.4.2 Genetische Variation der Dauereier im Sediment und der geschlüpften Daphnien ___________________________________________________ 95 3.4.4.3 Klonale Zusammensetzung der Dauereier im Sediment und der geschlüpften Daphnien_________________________________________ 96 3.5 Paläolimnologische Ergebnisse _____________________________________________ 99 3.5.1 Abundanzen von älteren Dauereiern___________________________________ 99 3.5.2 Genetische Variation von älteren Dauereiern im Sediment ________________ 100 3.5.3 Vergleich der Allelfrequenzen von älteren und jüngeren Dauereiern_________ 101 4 Diskussion_________________________________________________103 4.1 Methodendiskussion _____________________________________________________ 103 4.1.1 Bestimmung der maternalen Linien __________________________________ 103 4.1.2 Bestimmung der klonalen Struktur ___________________________________ 106 4.1.3 Einfluß des Probenumfangs auf die Genauigkeit der genetischen Analyse ____ 109 4.1.4 Vergleich von Allozym- und Mikrosatelliten-Analyse____________________ 110 4.1.5 Hybride im Belauer See ___________________________________________ 111 4.1.6 Bestimmung der klonalen Struktur bei älteren Dauereiern_________________ 113 Inhaltsverzeichnis iii 4.2 Populationsstruktur der Daphnien im Belauer See____________________________ 114 4.2.1 Bedeutung der Dauereier im Sediment ________________________________ 115 4.2.1.1 Bildung der Dauereier in der Daphnien-Population__________________ 116 4.2.1.2 Bedeutung der maternalen Linien für die Populationsstruktur von D. cucullata ________________________________________________ 118 4.2.1.3 Bedeutung von treibenden Dauereiern für die Dauereibank ___________ 119 4.2.1.4 Schlüpferfolg und Abundanzen _________________________________ 120 4.2.1.5 Selektion beim Schlüpfen______________________________________ 123 4.2.1.6 Bedeutung der Dauereier im Sediment - Synthese___________________ 124 4.2.2 Einfluß der Daphnia-Dauereier auf die genetische Struktur der Population im Pelagial______________________________________________________ 127 4.2.3 Auswirkung der sexuellen Reproduktion auf das HWE ___________________ 129 4.2.4 Bedeutung der Dauereier im Sediment als paläolimnologische Datenbank ____ 130 4.3 Schlußbetrachtung ______________________________________________________ 132 5 Zusammenfassung __________________________________________134 6 Literaturverzeichnis ________________________________________137 7 Anhang _____________________________________________________I