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Analyses of human oral microbiomes reveal substantial amounts of information about health, diet, and diseases of individuals and their communities within the archaeological record. In order to examine bacterial genomes from the past, specific archaeological samples that contain remnants of the microbial communities in question must be utilized. Recent developments in high-throughput, next-generation DNA sequencing have enabled the characterization of entire oral microbiomes and genomes from the remains of the bacteria trapped in calcified dental plaque. This project analyzed samples of ancient human dental calculus from North and Central America, which were examined for the changes within the oral microbiome in relation to the adoption of agriculture. Additionally, the conditional presence of pathogens associated with an increasingly agricultural and carbohydrate-rich diet was examined. The overarching goal was to examine and determine the level of microbial shifts within the past oral microbiomes of North and Central America, and by virtue of using the associated archaeological reports and analyses, place the data into the proper context. Three distinct sets of dental calculus were used for this Dissertation; The first is from Indigenous samples (N=56), spanning from the Archaic to the Mississippian, recovered from excavations in the Guntersville Basin in Northern Alabama. The second set is from a Late-Terminal Classic Maya city center and satellite village in the Upper Belize Valley (N=11). The final sample set comes from an archaeologically recovered early 20th century Cemetery near Jackson Mississippi (N=12). After individual analyses, they are all examined along a temporal axis to examine the effect of agriculture on the human oral microbiome. The findings from this study have shown that oral microbiomes of the Americas were affected by the introduction of agriculture, but remained biologically diverse. Because various subsistence strategies can shape and affect the oral microbiome, the composition is seen to change over time. Our understanding of the evolution of oral microbiomes throughout human history is more complex than previously thought; there is no global trend for the oral microbiome, but is highly location dependent.

The complementarity of ancient DNA lends itself to integration with paleopathological inquiries of disease, particularly in scenarios where there is limited or conflicting historical, skeletal, and archaeological information in a given spatio-temporal context. This thesis expands on molecular approaches applied to the detection of “invisible” pathogens associated with non-catastrophic morbidity and mortality that are embedded in a unique biosocial context of the disease experience. Presented in ‘sandwich-thesis’ format, I explore the historical narrative surrounding malaria in Imperial period Italy (1st-4th c. A.D.) using a molecular approach that is integrated with an ecosocial framework, as well as addressing the methodological challenges of identifying pathogens in contexts without a priori knowledge or incongruous evidentiary sources. My research presents the first partial mitochondrial genome for P. falciparum recovered from two adults (prioritized from a subset of 58 individuals) in disparate ecological and social localities in Imperial period Italy. This provides a timestamp for this ancient protozoan parasite, with an emphasis on a multi-faceted approach to frame the human-parasite-vector-environment interactions in the studied localities. Additionally, I successfully applied an in-solution hybridization capture technique designed to detect over 1,000 human pathogens in archaeological samples, both of known and unknown pathogen constituents. This technique qualitatively and quantitatively assesses the likelihood of low abundance pathogenic targets that are present, in order to prioritize candidates to further pursue with downstream analyses, as well as beginning to explore the synergistic landscape of pathogen-pathogen interactions. In combination, the research outlined in this thesis emphasizes the molecular and biosocial experience of disease as interconnected elements in dynamic epidemiological environments of the past. / Dissertation / Doctor of Philosophy (PhD)

Denisova Cave is located in the Altai mountains of Russia. Excavations from this cave have yielded two large hominin molars and three hominin phalanxes from the Pleistocene. One of the phalanxes (Denisova 3) had extraordinary DNA preservation allowing the sequencing of high quality nuclear and mitochondrial DNA (mtDNA) genomes and has been shown to belong to a young girl from hereto unknown sister group of Neandertals, called Denisovans. The mtDNA of Denisova 3 surprisingly split from the mtDNA ancestor of modern humans and Neandertals twice as long ago as the split of modern humans and Neandertals. The mtDNA of one of the molars (Denisova 4) was also sequenced and differs at only two positions from the mtDNA of Denisova 3. A second phalanx (Altai 1) also yielded a high quality genome, and was a Neandertal. While Neandertals show an admixture signal of 1-4% into present-day non-Africans, Denisovans show an admixture of up to 5% in present-day Oceanians, and to a much lesser extent East Asians. This thesis encompasses two studies. In the first study, we sequenced the complete mtDNA genome of the additional molar (Denisova 8), as well as a few megabases of nuclear DNA from Denisova 4 and Denisova 8. While the mtDNA of Denisova 8 is clearly of the Denisova type, its branch to the most recent common ancestor of Denisovans is half as long as the branch leading to Denisova 3 or Denisova 4, indicating that Denisova 8 lived many millenia before the other two. Both Denisova 4 and 8 fall together with Denisova 3 based on nuclear DNA, bringing the number of known Denisovans from one to three. In the second study, we sequenced an almost complete mtDNA and a few megabases of nuclear DNA from the third hominin phalanx from Denisova Cave, Altai 2. Both the mtDNA and the nuclear DNA show Altai 2 to be a Neandertal. The mtDNA also showed the presence of substantial Pleistocene spotted hyena contamination. Low levels of spotted hyena contamination were also found in Altai 1, Denisova 3 and Denisova 4. Partial mtDNA genomes of the contaminating spotted hyenas from these four hominins were compared to mtDNA genomes of other extant and extinct spotted hyenas. We show that the spotted hyenas that contaminated the two Denisovans come from a population of spotted hyenas found in Pleistocene Europe as well as present-day Africa, while the spotted hyenas that contaminated Altai 2, and possibly Altai 1, come from a population of spotted hyenas found in Pleisticene eastern Russia and northern China. This indicates that Denisova Cave was a meeting point of eastern and western hominins as well as eastern and western spotted hyena populations.

Botryococcus braunii is a microscopic, colonial green alga that may be found in fresh and brackish waters throughout the globe. B. braunii is unique in that it constitutively synthesises and secretes copious amounts of various long-chain (C23-C40) hydrocarbons, generically termed “botryococcenes”. Botryococcanes, the hydrogenated forms of botryococcenes, comprise 1% of the fossil hydrocarbons found in petroleum deposits and in oil-shales. Microfossils identified as Botryococcus by optical and scanning electron microscopy are also abundant in these strata, but the actual identity and precise relationship between these microfossils and extant Botryococcus species is not known. In this investigation, the relationship between living Botryococcus algae and microfossils identified as Botryococcus using traditional palaeontological analysis and light-microscopy was investigated by analysis of ancient DNA (aDNA). The material used was identified in sediments from Boswell Lake (British Columbia, Canada), a Holocene lake that had remained undisturbed since the glacial retreat. New flow-cytometry methods were developed to rapidly purify enough of the relevant microfossils, from which aDNA was extracted and sequenced. Pollen grains were purified using the same flow-cytometry method and from the same horizons as the Botryococcus microfossils and used to age the sedimentary horizons by 14C radiocarbon dating. Samples of the purified microfossils were imaged by scanning electron microscopy for comparison with published images of fossils identified as Botryococcus from kerogens. In addition, metaDNA from the relevant horizons was extracted and sequenced by NGS, and a chemical analysis for botryococcene derivatives performed using two-dimensional gas chromatography (2D-GC). The genomic analyses show that the sub-fossils identified in Boswell Lake are likely to be representatives of B. braunii, race B. The geochemical analysis identified hydrocarbons that migrate as botryococcenes on 2D-GC in the strata whence the sub-fossils were purified. The SEM images indicate that the microfossils purified from Boswell Lake have similar morphologies to those found in kerogens. Taken together, these data strongly support the proposition that petroleum and kerogen deposits are unusually rich in B. braunii and that these algae have a lineage potentially dating 500 million years. The metagenomic analysis enabled similar conclusions to be reached regarding the presence of B. braunii within the sediment, without the need for targeted microfossil purification. While this analysis was less precise due to the under-representation of algal genomes in the public sequence databases, the metagenomics approach employed was particularly well suited to the temporal analysis of prokaryotic microcosms within Lake Boswell, the succession of which could be associated with periods of climatic variation. The analytical methods described herein are generally applicable to understanding microbial systems over geological periods, and may be used to generate important insights into the cause and effect relationships between microbial populations and environmental perturbation.

In paleopathology, the assessment of disease occurs through macroscopic observation, which is dependent on the preservation of the sample and the experience of the observer. Many disease events do not leave any visible signatures and therefore go undetected. The relatively new field of paleomicrobiology incorporates molecular techniques where microbial DNA, if present, is amplified from an archaeological sample. The identification of genetic material from micro-organisms, including bacteria and viruses, can confirm a diagnosis that was originally based on visible osteological or mummified tissue changes. Even more promising is the capability of molecular technology to detect microbial DNA evidence of disease processes that were not visibly evident.<p> Based on phylogenetic analyses of modern isolates, scientists have concluded that micro-organisms such as <i>Mycobacterium tuberculosis</i> and <i>Helicobacter pylori</i> have been associated with humans for thousands of years. <i>M. tuberculosis</i> is the causative agent of the disease tuberculosis, and <i>H. pylori</i> is known for its role in gastritis and peptic ulcers. Both are pathogenic bacteria that still impact the health of modern populations. Through the analysis of microbial DNA from these two bacteria in skeletal and mummified tissue, data can be accumulated regarding the spatial and temporal impact of these infections. Interestingly, due to the lengthy association between these bacteria and humans, phylogenetic studies on modern strains have shown that strain characterizations of both <i>M. tuberculosis</i> and <i>H. pylori</i> bacteria reveal connections with past human migrations.<p> In 1999, human remains were discovered eroding out of a glacier in northern British Columbia, Canada on the traditional territory of the Champagne and Aishihik First Nations. The Aboriginal elders named the site Kwäday Dän Tsìnchi, which means long ago person found. Radiocarbon testing of bone collagen and artifacts from the site suggested a time-frame of approximately AD 1670 to 1850, which is either pre-European contact or early post-contact for that area. I analyzed the tissues of the ancient individual specifically for genetic evidence of <i>M. tuberculosis</i> and <i>H. pylori</i> to identify partial health status and determine if a connection could be made to strains associated with European populations to clarify whether the site was pre or post-European contact.<p> Through polymerase chain reaction (PCR) testing of the individuals tissues with primers specific for the IS<i>6100</i> insertion sequence, <i>TbD1</i>, and <i>Rv3479</i>, <i>katG</i> and <i>gyrB</i> genes, I identified evidence of a possible latent tuberculosis infection. Genetic characterization of the <i>katG</i> gene associated with the ancient <i>M. tuberculosis</i> strain revealed a potential connection with European strains. Amplification and sequencing of the <i>gyrB</i> gene fragment indicated the presence of two alleles that may have been the result of a selective pressure.<p> PCR testing of the individuals stomach tissue with specific primers for regions with the <i>vacA</i> gene resulted in a positive identification of <i>H. pylori</i> DNA. Genetic characterization of this virulence-associated gene indicated that the strain contained a <i>vacA</i> signal (s) region s2 allele. This allele is more commonly identified in Western strains that do not cause disease, which suggests that the individual had no gastric symptoms and that European strains were present in northwestern Canada at that time. The vacA middle (m) region contained a hybrid m2a/m1d sequence. Modern hybrids are rare but they have been identified in Asian strains. Studies have shown that the m2a allele is more common in Western strains. A phylogenetic analysis identified that the m1d region clusters with previously published novel strains associated with Aboriginal individuals that are closely related to Asian strains. This indicates a past connection between the ancient individual and his ancestors who arrived in the New World from Asia thousands of years ago.

In paleopathology, the assessment of disease occurs through macroscopic observation, which is dependent on the preservation of the sample and the experience of the observer. Many disease events do not leave any visible signatures and therefore go undetected. The relatively new field of paleomicrobiology incorporates molecular techniques where microbial DNA, if present, is amplified from an archaeological sample. The identification of genetic material from micro-organisms, including bacteria and viruses, can confirm a diagnosis that was originally based on visible osteological or mummified tissue changes. Even more promising is the capability of molecular technology to detect microbial DNA evidence of disease processes that were not visibly evident.<p> Based on phylogenetic analyses of modern isolates, scientists have concluded that micro-organisms such as <i>Mycobacterium tuberculosis</i> and <i>Helicobacter pylori</i> have been associated with humans for thousands of years. <i>M. tuberculosis</i> is the causative agent of the disease tuberculosis, and <i>H. pylori</i> is known for its role in gastritis and peptic ulcers. Both are pathogenic bacteria that still impact the health of modern populations. Through the analysis of microbial DNA from these two bacteria in skeletal and mummified tissue, data can be accumulated regarding the spatial and temporal impact of these infections. Interestingly, due to the lengthy association between these bacteria and humans, phylogenetic studies on modern strains have shown that strain characterizations of both <i>M. tuberculosis</i> and <i>H. pylori</i> bacteria reveal connections with past human migrations.<p> In 1999, human remains were discovered eroding out of a glacier in northern British Columbia, Canada on the traditional territory of the Champagne and Aishihik First Nations. The Aboriginal elders named the site Kwäday Dän Tsìnchi, which means long ago person found. Radiocarbon testing of bone collagen and artifacts from the site suggested a time-frame of approximately AD 1670 to 1850, which is either pre-European contact or early post-contact for that area. I analyzed the tissues of the ancient individual specifically for genetic evidence of <i>M. tuberculosis</i> and <i>H. pylori</i> to identify partial health status and determine if a connection could be made to strains associated with European populations to clarify whether the site was pre or post-European contact.<p> Through polymerase chain reaction (PCR) testing of the individuals tissues with primers specific for the IS<i>6100</i> insertion sequence, <i>TbD1</i>, and <i>Rv3479</i>, <i>katG</i> and <i>gyrB</i> genes, I identified evidence of a possible latent tuberculosis infection. Genetic characterization of the <i>katG</i> gene associated with the ancient <i>M. tuberculosis</i> strain revealed a potential connection with European strains. Amplification and sequencing of the <i>gyrB</i> gene fragment indicated the presence of two alleles that may have been the result of a selective pressure.<p> PCR testing of the individuals stomach tissue with specific primers for regions with the <i>vacA</i> gene resulted in a positive identification of <i>H. pylori</i> DNA. Genetic characterization of this virulence-associated gene indicated that the strain contained a <i>vacA</i> signal (s) region s2 allele. This allele is more commonly identified in Western strains that do not cause disease, which suggests that the individual had no gastric symptoms and that European strains were present in northwestern Canada at that time. The vacA middle (m) region contained a hybrid m2a/m1d sequence. Modern hybrids are rare but they have been identified in Asian strains. Studies have shown that the m2a allele is more common in Western strains. A phylogenetic analysis identified that the m1d region clusters with previously published novel strains associated with Aboriginal individuals that are closely related to Asian strains. This indicates a past connection between the ancient individual and his ancestors who arrived in the New World from Asia thousands of years ago.

abstract: Leprosy and tuberculosis are age-old diseases that have tormented mankind and left behind a legacy of fear, mutilation, and social stigmatization. Today, leprosy is considered a Neglected Tropical Disease due to its high prevalence in developing countries, while tuberculosis is highly endemic in developing countries and rapidly re-emerging in several developed countries. In order to eradicate these diseases effectively, it is necessary to understand how they first originated in humans and whether they are prevalent in nonhuman hosts which can serve as a source of zoonotic transmission. This dissertation uses a phylogenomics approach to elucidate the evolutionary histories of the pathogens that cause leprosy and tuberculosis, Mycobacterium leprae and the M. tuberculosis complex, respectively, through three related studies. In the first study, genomes of M. leprae strains that infect nonhuman primates were sequenced and compared to human M. leprae strains to determine their genetic relationships. This study assesses whether nonhuman primates serve as a reservoir for M. leprae and whether there is potential for transmission of M. leprae between humans and nonhuman primates. In the second study, the genome of M. lepraemurium (which causes leprosy in mice, rats, and cats) was sequenced to clarify its genetic relationship to M. leprae and other mycobacterial species. This study is the first to sequence the M. lepraemurium genome and also describes genes that may be important for virulence in this pathogen. In the third study, an ancient DNA approach was used to recover M. tuberculosis genomes from human skeletal remains from the North American archaeological record. This study informs us about the types of M. tuberculosis strains present in post-contact era North America. Overall, this dissertation informs us about the evolutionary histories of these pathogens and their prevalence in nonhuman hosts, which is not only important in an anthropological context but also has significant implications for disease eradication and wildlife conservation. / Dissertation/Thesis / Appendix_A / Appendix_B / Appendix_F / Doctoral Dissertation Evolutionary Biology 2017

Humans are dependent on the animals they raise and breed for food and secondary products. Archaeological and genetic investigations can provide critical insights into the history and development of these breeds and help understand human activities in the past. Furthermore, many well-adapted breeds are endangered and archaeological and genetic data can help inform future breed conservation choices. Utilising ancient DNA data could potentially permit detailed diachronic analyses of the development of animal breeds. Ancient DNA analyses have typically focussed on large-scale biogeographic patterns in time and space, such as the spread of domesticates or the movements of peoples. Few studies have attempted fine-scale diachronic analysis within single animal populations or breeds. This is largely due to restricted sample availability and the limited phylogenetic resolution provided by the mitochondrial genome, the most commonly used ancient DNA marker. In this thesis, I demonstrate that fine-scale diachronic analyses within single animal populations and breeds over short time scales are feasible. First, in order to address the limitations of sample size, I assessed three sample screening methods’ abilities (maximum mitochondrial DNA amplicon length, NanoDrop® spectrometry and collagen preservation) to select samples in which DNA was preserved and analysed the utility of parchment as a novel source of ancient and historic DNA. None of the screening methods accurately predicted DNA preservation, but collagen preservation was able to weed out extremely poorly preserved samples from further analysis. All but one of the tested parchments produced multiple sequences matching several different species. Parchment therefore was not appropriate for fine-scale diachronic analyses. Next, I assessed whether analysing the nuclear genome could permit fine-resolution diachronic genetic studies. Since single nucleotide polymorphisms are ideal candidate nuclear markers for diachronic DNA analyses, I assessed the accuracy of the nuclear SNP-typing methodology, SNaPshot™, by genotyping three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. The SNaPshot™ protocol was found to provide accurate genotypic information in all cases. Finally, as a proof of method, I compared the diachronic information provided by the mitochondrial and nuclear genomes in Icelandic and Thoroughbred horses. Specifically, in the Icelandic horse, I analysed the mitochondrial D-loop and three coat colour genes in modern and historic populations. In the Icelandic horse, I found statistically significant evidence for genetic change in the mitochondrial genome over the last 150 years. I found no evidence for change in coat colour allele frequencies. Conversely, in the biased and small historic Thoroughbred dataset, the mitochondrial genome was insufficient to provide population-level information, but I was able to show that allele frequencies in the nuclear MSTN gene, a gene previously shown to influence racing performance, have changed significantly in the past century.

Longtemps utilisés en palynologie pour l’étude des paléo-environnements, les coprolithes, excréments fossilisés, sont également d’importantes sources d’information pour des espèces disparues, producteurs ou proies. Grâce à de nombreux échantillons archéologiques provenant d’une dizaine de grottes, comme la Grotte Chauvet-Pont d’Arc, deux espèces ont pu être étudiées : le loup, Canis lupus, et l’hyène des cavernes, Crocuta crocuta spelaea.À partir d’un coprolithe de canidé, daté à 34 500 ans, un génome mitochondrial complet de Canis lupus a pu être reconstitué. Les analyses phylogénétiques ont montré que ce spécimen se situe en dehors de la diversité des chiens et loups actuels. Puis, les analyses menées sur des gènes nucléaires ont montré que le spécimen de Chauvet ne présente pas de traces évidentes de domestication. L’étude du coprolithe met en évidence un régime carné, avec un bol alimentaire comportant des traces d’ADN d’ours des cavernes, Ursus spelaeus.Dans un second temps, grâce à l’étude de nombreux coprolithes d’hyène des cavernes, une alimentation variée composée d’animaux de grande comme de petite taille a été mise en évidence pour ce carnivore. L’analyse plus fine des séquences d’ADN contenues dans l’un des échantillons a permis de reconstituer un génome mitochondrial complet pour une espèce aujourd’hui éteinte : le bison des forêts, Bison schoetensacki. En parallèle, grâce à l’étude d’un ossement de bison des steppes, Bison priscus, un génome mitochondrial complet a été obtenu pour cette espèce éteinte. L’ajout de ces deux nouvelles séquences mitochondriales à la phylogénie des bovidés a permis d’apporter des éclaircissements à cette dernière. / Coprolites have long been used in palynology for paleoenvironments reconstruction. They also are an important source of information on the DNA of the producing species and its diet. Using numerous archeological samples from several caves, including the Chauvet-Pont d’Arc cave, we studied coprolites for two species: the wolf, Canis lupus and the cave hyena, Crocuta crocuta spelaea.Using a canid coprolite from the Chauvet cave, dated back to 34 500 years, we obtained a complete mitochondrial genome sequence. Phylogenetic analyses highlight a maternal lineage that positions outside the diversity of extant dogs and wolfs. Then, analyzes conducted on the nuclear genes showed that the Chauvet canis lupus specimen does not display obvious indication of domestication. Analysing the coprolite for other species to indicate the diet of this specimen, we detected cave bear (Ursus spelaeus) DNA sequences.Second, using many cave hyena coprolites, a flexible diet consisting of large as well as small animals was demonstrated for this extinct carnivore. Focusing the analysis on a coprolite samples that contained large amounts of bovine DNA, we obtained for the first time a complete mitochondrial genome sequence for the extinct European forest bison, Bison schoetensacki. In parallel, a bone sample for the extinct steppe bison provided the first complete mitochondrial genome sequence for Bison priscus. These two genome sequences shed new lights on the phylogeny of Bovinae.