In order to gain a greater understanding of the genetic makeup of the Maltese population, mitochondrial DNA HVR1 and HVR2, and Y-chromosomal and autosomal STRs were amplified in a representative sample of the Maltese population. The results showed that the Maltese have close genetic ties with Sicily and mainland Italy both from a matrilineal and a patrilineal perspective, whilst no conclusive evidence was found for a Phoenician link between the Maltese and the Lebanese population. In order to try and gain an insight into the Maltese population throughout time, a study was conducted on three Maltese archaeological burial places dating from the Neolithic to the Roman period. The study extracted and amplified ancient DNA sequences from these three sites and compared the resulting mtDNA sequences with the modern Maltese population. The results showed that aDNA survives in the Maltese archaeological record, and that some haplotypes found during the Roman period in Malta are also found in the modern day population, whilst other haplotypes present in the archaeological samples are not visible in the modern Maltese population.

One of the biggest innovations in human prehistory was the advent of food production, consisting of the ability to grow crops and domesticate animals for consumption. This wide-scale transition from hunting and gathering to food production led to more permanent settlements, and set in motion major societal changes. In western Eurasia, this revolution spread from the Near East into Europe, Africa and diverse regions of Asia. Agriculture was brought into Europe by the descendants of early Anatolian farmers starting approximately 8,000 years ago. But little was known of the people who developed agriculture in the Fertile Crescent: where they all closely related to the early Anatolian farmers, or were there multiple ethnic groups who developed agriculture in parallel? In the first data chapter, I use the first genome from a Neolithic woman from Ganj Dareh, in the Zagros Mountains (Iran), a site with evidence of early goat domestication 10,000 years ago. I showed that Western Iran wan inhabited by populations mostly similar to Hunter- gatherer populations from the Caucasus, but remarkably, very distinct from the Anatolian farmers who spread the Neolithic package into Europe. While a degree of cultural diffusion between Anatolia, Mesopotamia and the Zagros highlands likely happened, genetic dissimilarity supports a model in which Neolithic societies of that area were distinct. The second chapter deals with how Africa was affected by population movements, originating in the Near East, during the Neolithic times. Characterising genetic diversity in Africa is a crucial step for analyses reconstructing human evolution. Using Mota, an ancient genome from a male from the Ethiopian highlands, I showed a backflow into Africa by populations closely related to the Anatolian Neolithic farmers. The third chapter deals with some common problems and themes in the analysis of ancient DNA, such as merging capture datasets with diverse number of ascertained SNPs, combining capture and shotgun data in the same analysis, and the effect of UDG treatment in ancient samples. I describe the most common problems and their effect in summary statistics, and propose a guide on how to work with ancient DNA to avoid data compatibility problems.

Tuberculosis (TB) has killed millions of people throughout history and still isone of the leading causes of death. Since the early 1990s, ancient DNA(aDNA) research has made considerable contributions to the study of thisinfectious disease in the past. While early studies used polymerase chainreactions (PCRs) solely to identify the TB-causing organisms, namely theMycobacterium tuberculosis complex (MTBC), later approaches extended thefocus to assign the actual disease-causing species or strains of the MTBCbut were either directed at single or few individuals or only provided few data. This research project has screened a large set of European skeletaland dental samples from individuals of the 1st–19th centuries AD for IS6110,an insertion sequence believed to be specific to the MTBC, and has identifieda number of individuals that may indeed have suffered from TB. Reports ofIS6110-like elements in other mycobacteria, however, challenge thesuitability of IS6110 for detecting MTBC. Two sequences similar but notidentical to IS6110 were revealed from several of the samples analysed,supporting the proposal that IS6110 should not serve as the sole target foridentifying MTBC from archaeological material. It cannot be establishedwhere these sequences derive from, but application of a MycobacteriumspecificPCR and targeting of genomic regions of the MTBC that containsingle nucleotide polymorphism (SNPs) indicate that at least some of thesamples contain a range of unknown, most likely environmental, bacterialand/or mycobacterial species. Yet, screening for IS6110 together with thedetection of large sequence polymorphisms (LSPs) and SNPs in othergenomic regions has identified eight individuals to unambiguously containMycobacterium tuberculosis aDNA. Apart from one individual which wasrecovered from Northern France, these skeletons derived from Britisharchaeological excavation sites. The SNP and LSP results enabled theallocation of infecting MTBC strains into various classification systemsreported in clinical literature and revealed that M. tuberculosis strains variedthroughout different time periods, thereby mainly confirming evolutionarypathways suggested in previous studies. Additionally, it was found thatdistinct strains co-existed temporally, and maybe even spatially, in Britainand that at least one individual harboured two different MTBC strains,suggesting a mixed infection. Application of next generation sequencingenabled one of the 19th century strains from Britain to be characterised ineven more detail, revealing closest similarity to a M. tuberculosis strainisolated at the beginning of the 20th century in North America.

Xenarthra form the least diversified major clade of placental mammals, being comprised of 31 described species of sloth, armadillo, and anteater. The past decade has seen a growth in the amount of xenarthran genetic data available, including the recent publication of a phylogenetic framework based on mitochondrial genomes, but more is required to aid in conservation assessments and to elucidate the evolutionary history of this unique order. We aimed to expand upon this by generating a framework based on nuclear genes. Using molecular baits, we enriched nuclear DNA from all extant and a selection of extinct ancient Xenarthrans for 74 phenotypically relevant genes. We aim to build phylogenetic trees based on each successfully enriched loci. Comparing trees against the previous mitochondrial framework will provide a measure of robustness. By seeing how estimated divergence times and rates vary across nuclear genes, we were able to examine how particular xenarthran phenotypes were selected upon in different points of their evolutionary history. While the sequencing of certain genes and species encountered mixed success rates, we have a solid framework for further study and can confirm that nuclear and mitochondrial information yields very similar evolutionary histories. / Thesis / Master of Science (MSc) / Xenarthrans - sloths, armadillos, and anteaters - have a long and complicated evolutionary history. In recent years a growth of new genetic information has made it easier to answer questions about their relation to each other and to other species. By examining many new gene sequences across all living Xenarthra, plus some extinct species, we aim to bolster our understanding of these relationships and the importance of particular traits.

The order Sirenia is one of only two mammalian groups to have completely forgone terrestrial life. While extant sirenians are confined to the tropical waters, the recently extinct Steller’s sea cow (Hydrodamalis gigas) evolved to exploit the frigid waters of the North Pacific Ocean. As limits on O2 availability during submergence and decreased tissue temperature impose severe constraints on oxygen delivery, the oxygen binding (globin) proteins of sirenians are expected to have evolved under strong selection pressures. My comparative molecular analyses indicate that selection pressures on two globin genes (HBA and HBZ-T1) increased in transitional sirenians. As these genes encode the α-chains of all Hb isoforms throughout sirenian development, the resulting functional consequences to adult sirenian Hbs were tested using recombinant Hbs of Steller’s sea cow, the dugong (Dugong dugon), their last common ancestor, and the Florida manatee (Trichechis manatus latirostris). These tests reveal that high affinity Hbs—exceeding those of other mammals examined to date—arose early in sirenian evolution, presumably to maximize O2 extraction from the lungs and limit premature O2 offloading during submergence. Moreover, I demonstrate that the Hb–O2 affinity of the extinct sub-Arctic Steller’s sea cow is less affected by temperature than other sirenians, safeguarding O2 delivery to cool peripheral tissues. However, while this phenotype has primarily been attributed to the binding of additional allosteric effectors to the Hb moiety, Steller’s sea cow Hb binds relatively few of these ligands. Instead, my results suggest the thermodynamic properties of discrete allosteric effector sites are altered by epistatic interactions, a phenomenon that appears to be a critical component to cold adaptation in mammalian Hbs. As the HBA and HBZ-T1 loci also encode sirenian prenatal Hbs, the functional properties these proteins were tested to reveal their O2 affinity increased in parallel to maternal Hb. Notably, Steller’s sea cow HbF has the highest reported O2 affinity of any mammalian Hb tested to date. As the HBA gene encodes the -subunit of both the prenatally expressed HbF and adult expressed HbA proteins, the molecular remodeling of this locus may have concurrently increased the affinity of each protein. / February 2017

The Aztec empire emerged in AD 1428 as a result of the triple alliance among the city-states of Tenochtitlan, Texcoco, and Tlacopan. Although it is well documented that the Aztecs conquered numerous polities in the Basin of Mexico over the next 100 years, the demographic consequences of this expansion remain unclear. At the influential Otomi city-state of Xaltocan, for example, colonial documents suggest that the Aztec conquest led to a replacement of the original Otomi population, whereas archaeological finds suggest that a significant portion of the original population may have remained at the city under Aztec rule. To help resolve questions about Xaltocan’s population history during this period, I extracted ancient DNA from 21 individuals that can be divided into two temporal subpopulations (roughly predating and postdating the hypothesized replacement event). I determined mitochondrial DNA haplogroups through RFLP analyses and constructed haplotypes based on 372 bp of HVR1 sequence. Statistical analyses show significant differences between the mitochondrial composition of the two subpopulations. Altogether, the results of this study support the hypothesis that matrilines at Xaltocan underwent a significant replacement event following the Aztec conquest, and they suggest that the Aztec expansion may have had a substantial genetic impact on certain Mesoamerican populations. / text

Ancient DNA (aDNA) was isolated from the bones of 290 individuals and four species of extinct New Zealand moa. All sampled bones had been recovered from a small geographic area (~10 km radius) near Waikari in North Canterbury. A total of 217 specimens were 14C-AMS dated, providing a temporal framework for the genetic analyses and an unprecedented opportunity to study extinct megafauna at the population level. Taxon and sex were determined for each individual, using aDNA technology. This revealed a large excess of females (overall ♂:♀ = 1:5.1), and significant compositional differences for the moa assemblages between fossil sites. Balanced sex ratios were observed among juvenile moa, suggesting that a gender-bias developed as the birds matured, probably as a result of higher male mortality. Female territoriality and ecological niche-separation are discussed in this context. Mitochondrial DNA (mtDNA), amplified using a quantitative PCR procedure, provided a measure of DNA preservation in each radiocarbon-dated fossil. This assessment showed that DNA degrades over thousands of years according to an exponential decay model, and the average molecular half-life for the here targeted DNA fragment was estimated to be 521 years. By using high-throughput sequencing, six polymorphic moa microsatellite markers were identified and characterised. These are the first microsatellite primers developed exclusively for extinct taxa. A high-resolution genetic study of the four sympatric moa populations was carried out, combining information from mtDNA, microsatellites, sex-identification, and radiocarbon age. Genetic diversity, past demography, kinship, and other aspects of moa biology were analysed. The populations showed a remarkable extent of genetic stability throughout the 3000-4000 years preceding their extinction, suggesting that they were large and viable before suddenly disappearing. The results represent significant advances in aDNA research and thanks to the high resolution in microsatellite markers, moa have here been studied, almost as if they were still alive.

It is acknowledged that some microbes have interrupted distributions, yet these distributions have rarely been correlated with environmental variables. The wild biogeography of the fermenting yeasts Saccharomyces cerevisiae and Saccharomyces paradoxus are explored in this study, considering multiple environmental variables as potential effectors of each species' geographical distributions. I demonstrate that summer temperatures predict maximum species distribution limits for both S. paradoxus and S. cerevisiae on oak bark, and that S. paradoxus is more likely to be isolated from larger, older trees. Modelling these data predicts a generally denser southern European population of S. paradoxus, with S. cerevisiae being scarce on oak bark throughout Europe. It was not possible to recover ancient Saccharomyces DNA sequences from samples of sub-fossilized oaks, from Greco-Roman and North African amphora residues, or from North African 6th-14th Century pottery residues, which may be a consequence of the low concentration of these species in comparable modern environmental samples. Even from air dried breads and recent wines, Saccharomyces aDNA was not recovered as part of this study, although ancient DNA sequences from plants and other yeasts were identified in other samples via the same methods. Any future recovery of ancient Saccharomyces sequences may therefore be challenging. Novel plant sequences possibly belonging to the Musaceae family and Pinus genus were identified from 6th-14th century AD North African pottery; as well as a Vicia-like DNA sequence from a 13th-12th century BC North African amphora.

The Quaternary (the approximately the last 2.6 million years) of North America is a tremendously exciting time period to study with respect to ecology. It saw periods of immense climatic turbidity - the expansion and retreat of continental ice-sheets and large swings in temperature, resulting in the wide scale restructuring of terrestrial ecosystem. It also saw widespread migrations of many species and out of Eurasia, mostly notably of modern humans. Ancient DNA offers powerful tools to examine the relationships and responses of megafunal species to these events, but has largely focused on cold-adapted species, and within radiocarbon-time (i.e. the last 50 thousand years). In this thesis I work to expand our understanding of the genetic landscape of Pleistocene megafuna in three ways. First, I describe the analysis of coprolites from Bechan Cave, Utah and characterize the mammoth inhabitants in the broader context of North American mammoths. Second, I characterize the diversity of American mastodons across the continent and through time, showing that their range likely repeatedly expanded and contracted in response to Pleistocene glaciations. Lastly, I begin to fill in some of the gaps in the American mastodon dataset from chapter 3, and begin to address some of the taxonomic and biogeographic questions about American and Pacific mastodons in Idaho. Understanding how North American megafauna responded to these climatic and anthropogenic stresses may help to explain why so many species went extinct at the end of the last glaciation, and how species may respond to present day warming. However, it is important to include taxa from warmer locales and environments to ensure our models and hypotheses are comprehensive. / Thesis / Doctor of Science (PhD) / Pleistocene North America was a time period of immense climatic turbidity, with temperature swings greater than 15°C in response to the expansion and contraction of continental ice-sheets. Despite these massive swings in temperature, many species managed to thrive on the continent and adapt to glacial-associated ecosystem restructuring. Ancient DNA from Pleistocene megafauna can serve as a very useful tool to answer many questions about the distribution of megafaunal species, and how they may have responded to these climatic events. However, most studies have largely focused on species adapted to cold environments and from the last fifty thousand years. In this thesis I extend our knowledge of the genetic landscape of Pleistocene proboscideans, characterizing the mammoth inhabitants of Bechan Cave, Utah, and producing the first look at American mastodon diversity through space and time. This work increases our representation of warm-adapted specimens and characterizes the effects of glacial cycles on megafauna populations.

Palaeogenetic research on human pathogenic and microbiomic bacteria has been largely restricted to bloodborne pathogens from skeletal tissue and, due to short lengths of degraded ancient DNA, small-scale single loci studies. My thesis has expanded the breadth and depth of palaeomicrobial knowledge via the study of novel specimen types with next-generation technologies. Presented in sandwich thesis format, I discuss genome-scale studies of three previously-unstudied historical pathogens: 19th century Vibrio cholerae (cholera) from an alcohol-preserved intestine from Philadelphia, and medieval Staphylococcus saprophyticus (urinary tract infections) and Gardnerella vaginalis (bacterial vaginosis) from calcified urogenital infections of a Trojan woman. Cholera persists as a dangerous modern disease that was also responsible for severe historic epidemics. My research confirms that 19th century pandemics were caused by an O1 classical strain that may have possessed genomic features that contributed increased virulence. S. saprophyticus and G. vaginalis are opportunistic pathogens of the urogenital microbiome, especially in reproductive-age females. Using very high endogenous DNA content of the calcified infections, I have reconstructed one of the most complete ancient bacterial genomes for S. saprophyticus and coding genome for G. vaginalis. Both ancient pathogens possess most of the virulence and urogenital adaptive genes of modern strains, indicating similar ecological roles for these species in past female health. Finally, I successfully use LLMDA microarray technology (never before utilized for ancient DNA research) to detect ancient pathogens. LLMDA provides an inexpensive and informative alternative to high-throughput sequencing for assessing the metagenomic content of ancient samples. Together, my findings provide a framework emphasizing the need to broadly study past microbiomes in conjunction with specific pathogens. Using molecular data, this work supports anthropological views of infectious disease ecology related to the first epidemiological transition and historical narratives. Taken together with the recent literature on ancient pathogen genomes, my findings indicate that palaeogenome sequences may not necessarily reveal any specific signatures of greater virulence, and interpretations of past diseases must necessarily take into account additional host, environmental, and cultural factors. / Thesis / Doctor of Philosophy (PhD)