Recovering high-quality DNA from thermally altered human remains poses a significant challenge for research and law enforcement agencies due to high levels of DNA degradation resulting from exposure to extremely high temperatures (e.g., fire). The current standard practice for the DNA identification of badly burned skeletal remains is to extract DNA from dense cortical bone collected from recovered skeletal elements. Some of the problems associated with this method are that it requires specialized equipment and training, is highly invasive (involving the physical destruction of sample material), time-consuming, and does not reliably guarantee the successful identification of the remains in question. At low-medium levels of thermal exposure, charred tissue is often adhered to these skeletal remains and typically discarded. In cases where burned/charred tissue is recoverable, it has the potential to be a more efficient alternative to the sampling of cortical bone. However, little has been done to test the viability of thermally altered soft tissue in terms of DNA identification to date. Burned/charred tissue was collected from skeletal samples provided by the University of Tennessee Forensic Anthropology Center, as a part of a controlled burn from donor individuals, for downstream laboratory processing and DNA analysis as part of the Stone Lab (Arizona State University, School of Human Evolution and Social Change). DNA from this charred tissue was extracted using the Qiagen DNeasy Blood and Tissue Kit, and resulting yields were quantified via fluorometry using the Qubit Fluorometer 2.0 and Agilent TapeStation 4200 High-Sensitivity D5000 assay. It was found that between the temperatures of ~200-300 ℃ (burn category 2) and ~300-350 ℃ (burn category 3), tissue was the most efficient extraction type, especially from tissue taken from the surface of the ilium and the rib. As for bone, both the Dabney and the Loreille protocol performed similarly, so choice in extraction type comes down to personal preference, type of equipment on hand, and training. Although, for samples with low input material, the Dabney protocol is optimal.

‘Describing at Large Their True and Lively Figure, their several Names, Conditions, Kinds, Virtues (both Natural and Fanciful), Countries of their Species, their Love and Hatred to Humankind, and the wonderful work of Natural Selection in their Evolution, Preservation, and Destruction.

Interwoven with curious variety of Creative Narrations out of Academic Literatures, Scholars, Artists, Scientists, and Poets. Illustrated with diverse Graphics and Emblems both pleasant and profitable for Students of all Faculties and Professions.’

Studying human genetic variation opens the possibility of understanding the details of population migrations, how humans develop and function, and why they get sick. To fully understand these things, genetic variation must be comprehensively characterized across globally diverse human populations and evolutionary knowledge can be used to inform studies of disease. In my dissertation I use computational methods to study human genetic variation. Each of my dissertation chapters focuses on a unique topic in the field of human evolutionary genetics. In the first chapter, I present PopInf, a computational pipeline to visualize principal components analysis output and assign ancestry to samples with unknown genetic ancestry, given a reference population panel of known origins. This pipeline facilitates visualization and identification of genetic ancestry across samples, so that this ancestry can be accounted for in studies of health and disease risk. In the next chapter, I investigate factors that shape patterns of genetic variation within and among four small-scale pastoral populations in northern Kenya. I find that geography predominantly shapes patterns of genetic variation in northern Kenyan human populations. In the next chapter, I investigate the extent to which Neanderthal introgression impacts liver cancer etiology. I find a pattern of overall enrichment of somatic mutations on Neanderthal introgressed haplotypes. Finally, through simulations, I investigate the effects of standard autosomal versus sex chromosome complement-informed alignment, variant calling and variant filtering strategies on variants called on the human sex chromosomes. I show that aligning to a reference genome informed on the sex chromosome complement of samples improves variant calling on the sex chromosome compared to aligning to a default reference, and variant calling is improved in males when calling the sex chromosomes haploid rather than diploid and when using haploid-based thresholds for filtering variants on the sex chromosomes. I provide recommendations for alignment, variant calling and filtering on the sex chromosomes based on these findings.

Tuberculosis (TB) is a deadly disease that infects millions of people annually. TB has a global distribution and remains a significant cause of mortality, despite decades of eradication campaigns and antibiotic development. TB is caused by genetically similar pathogens in the Mycobacterium tuberculosis complex (MTBC), and human infections are generally caused by human-associated strains, although humans can contract animal-associated strains. Skeletal evidence of TB on archaeological human skeletal remains and evolutionary dating of MTBC genomes reveal that TB has afflicted humans for approximately 6,000 years. Previous research has shown that MTBC pathogens were introduced into the Americas through a zoonotic transmission from seals and sea lions along the coasts of South America by at least 1000 CE. Characterizing the introduction and enigmatic intercontinental spread of a successful zoonotic transmission over hundreds of years provides valuable insight into the potential of zoonotic MTBC infections. Through the recovery and phylogenomic analysis of the first ancient MTBC genomes (n = 2) from pre-contact North America, I establish that there were multiple contemporaneous MTBC lineages circulating in human populations in the Americas. The high genomic diversity and deep divergence of strains from Mesoamerica suggest that TB was endemic in the region. To reveal the impact of TB within a Mesoamerican city, I examined human skeletons (n = 137) for evidence of disease from sacrificial and natural mortality burial contexts within Tlatelolco, a ceremonial precinct and interregional marketplace at the heart of the Aztec Empire (1300-1521 CE). I found that TB disproportionately affected sacrificial victims, who also exhibited evidence of food insecurity and resource inequality. These results mirror the socioeconomic patterns of TB distribution today. Further, I sampled broadly from sacrificial victims with skeletal evidence of TB not only for biomolecular confirmation of MTBC but also to uncover associations between skeletal TB manifestation and ability to recover ancient MTBC DNA. I identify 10 additional cases of MTBC at Tlatelolco and link ancient MTBC DNA recovery to TB skeletal lesion characteristics and age-at-death of the infected individual. Overall, this body of work combines paleogenomic and paleopathological data to highlight the impact of ancient TB zoonoses.

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.

Although the Caribbean has been continuously inhabited for the last 7,000 years, European contact in the last 500 years dramatically reshaped the cultural and genetic makeup of island populations. Several recent studies have explored the genetic diversity of Caribbean Latinos and have characterized Native American variation present within their genomes. However, the difficulty of obtaining ancient DNA from pre-contact populations and the underrepresentation of non-Latino Caribbean islanders in current research have prevented a complete understanding of genetic variation over time and space in the Caribbean basin. This dissertation uses two approaches to characterize the role of migration and admixture in the demographic history of Caribbean islanders. First, autosomal variants were genotyped in a sample of 55 Afro-Caribbeans from five islands in the Lesser Antilles: Grenada, St. Kitts, St. Lucia, Trinidad, and St. Vincent. These data were used to characterize genetic structure, ancestry and signatures of selection in these populations. The results demonstrate a complex pattern of admixture since European contact, including a strong signature of sex-biased mating and inputs from at least five continental populations to the autosomal ancestry of Afro-Caribbean peoples. Second, ancient mitochondrial and nuclear DNA were obtained from 60 skeletal remains, dated between A.D. 500–1300, from three archaeological sites in Puerto Rico: Paso del Indio, Punta Candelero and Tibes. The ancient data were used to reassesses existing models for the peopling of Puerto Rico and the Caribbean and to examine the extent of genetic continuity between ancient and modern populations. Project findings support a largely South American origin for Ceramic Age Caribbean populations and identify some genetic continuity between pre and post contact islanders. The above study was aided by development and testing of extraction methods optimized for recovery of ancient DNA from tropical contexts. Overall, project findings characterize how ancient indigenous groups, European colonial regimes, the African Slave Trade and modern labor movements have shaped the genomic diversity of Caribbean islanders. In addition to its anthropological and historical importance, such knowledge is also essential for informing the identification of medically relevant genetic variation in these populations.

Studies of ancient pathogens are moving beyond simple confirmatory analysis of diseased bone; bioarchaeologists and ancient geneticists are posing nuanced questions and utilizing novel methods capable of confronting the debates surrounding pathogen origins and evolution, and the relationships between humans and disease in the past. This dissertation examines two ancient human diseases through molecular and bioarchaeological lines of evidence, relying on techniques in paleogenetics and phylogenetics to detect, isolate, sequence and analyze ancient and modern pathogen DNA within an evolutionary framework. Specifically this research addresses outstanding issues regarding a) the evolution, origin and phylogenetic placement of the pathogen causing skeletal tuberculosis in New World prior to European contact, and b) the phylogeny and origins of the parasite causing the human leishmaniasis disease complex. An additional chapter presents a review of the major technological and theoretical advances in ancient pathogen genomics to frame the contributions of this work within a rapidly developing field. This overview emphasizes that understanding the evolution of human disease is critical to contextualizing relationships between humans and pathogens, and the epidemiological shifts observed both in the past and in the present era of (re)emerging infectious diseases. These questions continue to be at the forefront of not only pathogen research, but also

bioarchaeological and paleopathological scholarship.

Skeletal diseases related to reduced bone strength, like osteoporosis, vary in frequency and severity among human populations due in part to underlying genetic differentiation. With >600 disease-associated mutations (DAMs), COL1a1, which encodes the primary subunit of type I collagen, the main structural protein in bone, is most commonly associated with this phenotypic variation. Although numerous studies have explored genotype-phenotype relationships with COL1a1, surprisingly, no study has undertaken an evolutionary approach to determine how changes in constraint over time can be modeled to help predict bone-related disease factors. Here, molecular population and comparative species genetic analyses were conducted to characterize the evolutionary history of COL1a1. First, nucleotide and protein sequences of COL1a1 in 14 taxa representing ~450 million years of vertebrate evolution were used to investigate constraint across gene regions. Protein residues of historically high conservation are significantly correlated with disease severity today, providing a highly accurate model for disease prediction, yet interestingly, intron composition also exhibits high conservation suggesting strong historical purifying selection. Second, a human population genetic analysis of 192 COL1a1 nucleotide sequences representing 10 ethnically and geographically diverse samples was conducted. This random sample of the population shows surprisingly high numbers of amino acid polymorphisms (albeit rare in frequency), suggesting that not all protein variants today are highly deleterious. Further, an unusual haplotype structure was identified across populations, but which is only associated with noncoding variation in the 5' region of COL1a1 where gene expression alteration is most likely. Finally, a population genetic analysis of 40 chimpanzee COL1a1 sequences shows no amino acid polymorphism, yet does reveal an unusual haplotype structure with significantly extended linkage disequilibrium >30 kilobases away, as well as a surprisingly common exon duplication that is generally highly deleterious in humans. Altogether, these analyses indicate a history of temporally and spatially varying purifying selection on not only coding, but noncoding COL1a1 regions that is also reflected in population differentiation. In contrast to clinical studies, this approach reveals potentially functional variation, which in future analyses could explain the observed bone strength variation not only seen within humans, but other closely related primates.