Allelic bias when performing in-solution enrichment of ancient human DNA.

In-solution hybridisation enrichment of genetic variation is a valuable methodology in human paleogenomics. It allows enrichment of endogenous DNA by targeting genetic markers that are comparable between sequencing libraries. Many studies have used the 1240k reagent-which enriches 1,237,207 genome-wide SNPs-since 2015, though access was restricted. In 2021, Twist Biosciences and Daicel Arbor Biosciences independently released commercial kits that enabled all researchers to perform enrichments for the same 1240 k SNPs. We used the Daicel Arbor Biosciences Prime Plus kit to enrich 132 ancient samples from three continents. We identified a systematic assay bias that increases genetic similarity between enriched samples and that cannot be explained by batch effects. We present the impact of the bias on population genetics inferences (e.g. Principal Components Analysis, ƒ-statistics) and genetic relatedness (READ). We compare the Prime Plus bias to that previously reported of the legacy 1240k enrichment assay. In ƒ-statistics, we find that all Prime-Plus-generated data exhibit artefactual excess shared drift, such that within-continent relationships cannot be correctly determined. The bias is more subtle in READ, though interpretation of the results can still be misleading in specific contexts. We expect the bias may affect analyses we have not yet tested. Our observations support previously reported concerns for the integration of different data types in paleogenomics. We also caution that technological solutions to generate 1240k data necessitate a thorough validation process before their adoption in the paleogenomic community.

Insights into the genetic histories and lifeways of Machu Picchu's occupants.

Machu Picchu originally functioned as a palace within the estate of the Inca emperor Pachacuti between ~1420 and 1532 CE. Before this study, little was known about the people who lived and died there, where they came from or how they were related to the inhabitants of the Inca capital of Cusco. We generated genome-wide data for 34 individuals buried at Machu Picchu who are believed to have been retainers or attendants assigned to serve the Inca royal family, as well as 34 individuals from Cusco for comparative purposes. When the ancient DNA results are contextualized using historical and archaeological data, we conclude that the retainer population at Machu Picchu was highly heterogeneous with individuals exhibiting genetic ancestries associated with groups from throughout the Inca Empire and Amazonia. The results suggest a diverse retainer community at Machu Picchu in which people of different genetic backgrounds lived, reproduced, and were interred together.

Ethics of DNA research on human remains: five globally applicable guidelines.

We are a group of archaeologists, anthropologists, curators and geneticists representing diverse global communities and 31 countries. All of us met in a virtual workshop dedicated to ethics in ancient DNA research held in November 2020. There was widespread agreement that globally applicable ethical guidelines are needed, but that recent recommendations grounded in discussion about research on human remains from North America are not always generalizable worldwide. Here we propose the following globally applicable guidelines, taking into consideration diverse contexts. These hold that: (1) researchers must ensure that all regulations were followed in the places where they work and from which the human remains derived; (2) researchers must prepare a detailed plan prior to beginning any study; (3) researchers must minimize damage to human remains; (4) researchers must ensure that data are made available following publication to allow critical re-examination of scientific findings; and (5) researchers must engage with other stakeholders from the beginning of a study and ensure respect and sensitivity to stakeholder perspectives. We commit to adhering to these guidelines and expect they will promote a high ethical standard in DNA research on human remains going forward.

Ancient mitochondrial genomes from the Argentinian Pampas inform the early peopling of the Southern Cone of South America.

The Southern Cone of South America (SCSA) is a key region for investigations about the peopling of the Americas. However, little is known about the eastern sector, the Argentinian Pampas. We analyzed 18 mitochondrial genomes-7 of which are novel-from human skeletal remains from 3 Early to Late Holocene archaeological sites. The Pampas present a distinctive genetic makeup compared to other Middle to Late Holocene pre-Columbian SCSA populations. We also report the earliest individuals carrying SCSA-specific mitochondrial haplogroups D1j and D1g from Early and Middle Holocene, respectively. Using these deep calibration time points in Bayesian phylogenetic reconstructions, we suggest that the first settlers of the Pampas were part of a single and rapid dispersal ∼15,600 years ago. Finally, we propose that present-day genetic differences between the Pampas and the rest of the SCSA are due to founder effects, genetic drift, and a partial population replacement ∼9,000 years ago.

A strontium isoscape for the Conchucos region of highland Peru and its application to Andean archaeology.

Strontium isotope (87Sr/86Sr) analysis of human skeletal remains is an important method in archaeology to examine past human mobility and landscape use. 87Sr/86Sr signatures of a given location are largely determined by the underlying bedrock, and these geology specific isotope signatures are incorporated into skeletal tissue through food and water, often permitting the differentiation of local and non-local individuals in past human populations. This study presents the results of a systematic survey of modern flora and fauna (n = 100) from 14 locations to map the bioavailable 87Sr/86Sr signatures of the Conchucos region, an area where the extent of geologic variability was previously unknown. We illustrate the necessity to examine the variation in 87Sr/86Sr values of the different geological formations available to human land use to document the range of possible local 87Sr/86Sr values. Within the Conchucos region we found significant variation in environmental 87Sr/86Sr values (0.7078-0.7214). The resulting isoscape represents the largest regionally specific bioavailable 87Sr/86Sr map (3,840 km2) to date for the Andes, and will serve as a baseline for future archaeological studies of human mobility in this part of the Peruvian highlands.

A Multidisciplinary Review of the Inka Imperial Resettlement Policy and Implications for Future Investigations.

The rulers of the Inka empire conquered approximately 2 million km2 of the South American Andes in just under 100 years from 1438-1533 CE. Inside the empire, the elite conducted a systematic resettlement of the many Indigenous peoples in the Andes that had been rapidly colonised. The nature of this resettlement phenomenon is recorded within the Spanish colonial ethnohistorical record. Here we have broadly characterised the resettlement policy, despite the often incomplete and conflicting details in the descriptions. We then review research from multiple disciplines that investigate the empirical reality of the Inka resettlement policy, including stable isotope analysis, intentional cranial deformation morphology, ceramic artefact chemical analyses and genetics. Further, we discuss the benefits and limitations of each discipline for investigating the resettlement policy and emphasise their collective value in an interdisciplinary characterisation of the resettlement policy.

Comparing Genetic Variation among Latin American Immigrants: Implications for Forensic Casework in the Arizona- and Texas-Mexico Borderlands.

The humanitarian crisis on the US-Mexico border is a long-standing and evolving crisis in which nearly 8,000 deaths have been reported in the last two decades. These deaths are largely distributed across the Arizona-Mexico and Texas-Mexico border regions, where demographic trends for immigrants attempting to cross into the United States have shifted dramatically. The demographic change and volume of immigrants seeking shelter in the United States present new challenges for the forensic practitioners entrusted with the identification of individuals who lose their lives during the final segment of their journey. Within this border context, this study investigated how genetic variation inferred from forensically significant microsatellites can provide valuable information on regions of origin for unidentified remains at the group level. To explore how to mobilize these genetic data to inform identification strategies, the authors conducted a comparative genetic analysis of identified and unidentified immigrant cases from the Arizona- and Texas-Mexico contexts, as well as 27 other Latin American groups. Allele frequencies were utilized to calculate FST, and relationships were visually depicted in a multidimensional scaling plot. A Spearman correlation coefficient analysis assessed the strength and significance of population relationships, and an agglomerative clustering analysis assessed population clusters. Results indicate that Arizona-Mexico immigrants have the strongest relationship (>80%) with groups from El Salvador, Guatemala, Mexico, and an indigenous group from southern Mexico. Texas-Mexico immigrants have the strongest relationships (>80%) with groups from Belize, Colombia, Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua. These findings agree with, and are discussed in comparison with, previously reported demographic trends, population genetics research, and population history analyses. The authors emphasize the utility and necessity of coupling genetic variation research with a nuanced anthropological perspective for identification processes in the US-Mexico border context.

Increased rate of close-kin unions in the central Andes in the half millennium before European contact.

Analyzing ancient DNA of the central Andes, Ringbauer and colleagues identify a markedly elevated rate of unions of closely related parents after ca. 1000 CE. This change of mating preferences sheds new light on a unique system of social organization based on ancestry ("ayllu") whereby within-group unions were preferred to facilitate sharing of resources.

Integration of ancient DNA with transdisciplinary dataset finds strong support for Inca resettlement in the south Peruvian coast.

Ancient DNA (aDNA) analysis provides a powerful means of investigating human migration, social organization, and a plethora of other crucial questions about humanity's past. Recently, specialists have suggested that the ideal research design involving aDNA would include multiple independent lines of evidence. In this paper, we adopt a transdisciplinary approach integrating aDNA with archaeological, biogeochemical, and historical data to investigate six individuals found in two cemeteries that date to the Late Horizon (1400 to 1532 CE) and Colonial (1532 to 1825 CE) periods in the Chincha Valley of southern Peru. Genomic analyses indicate that these individuals are genetically most similar to ancient and present-day populations from the north Peruvian coast located several hundred kilometers away. These genomic data are consistent with 16th century written records as well as ceramic, textile, and isotopic data. These results provide some of the strongest evidence yet of state-sponsored resettlement in the pre-Colonial Andes. This study highlights the power of transdisciplinary research designs when using aDNA data and sets a methodological standard for investigating ancient mobility in complex societies.

A Paleogenomic Reconstruction of the Deep Population History of the Andes.

There are many unanswered questions about the population history of the Central and South Central Andes, particularly regarding the impact of large-scale societies, such as the Moche, Wari, Tiwanaku, and Inca. We assembled genome-wide data on 89 individuals dating from ∼9,000-500 years ago (BP), with a particular focus on the period of the rise and fall of state societies. Today's genetic structure began to develop by 5,800 BP, followed by bi-directional gene flow between the North and South Highlands, and between the Highlands and Coast. We detect minimal admixture among neighboring groups between ∼2,000-500 BP, although we do detect cosmopolitanism (people of diverse ancestries living side-by-side) in the heartlands of the Tiwanaku and Inca polities. We also highlight cases of long-range mobility connecting the Andes to Argentina and the Northwest Andes to the Amazon Basin. VIDEO ABSTRACT.

"The dead shall be raised": Multidisciplinary analysis of human skeletons reveals complexity in 19th century immigrant socioeconomic history and identity in New Haven, Connecticut.

In July 2011, renovations to Yale-New Haven Hospital inadvertently exposed the cemetery of Christ Church, New Haven, Connecticut's first Catholic cemetery. While this cemetery was active between 1833 and 1851, both the church and its cemetery disappeared from public records, making the discovery serendipitous. Four relatively well-preserved adult skeletons were recovered with few artifacts. All four individuals show indicators of manual labor, health and disease stressors, and dental health issues. Two show indicators of trauma, with the possibility of judicial hanging in one individual. Musculoskeletal markings are consistent with physical stress, and two individuals have arthritic indicators of repetitive movement/specialized activities. Radiographic analyses show osteopenia, healed trauma, and other pathologies in several individuals. Dental calculus analysis did not identify any tuberculosis indicators, despite osteological markers. Isotopic analyses of teeth indicate that all four were likely recent immigrants to the Northeastern United States. Nuclear and mitochondrial DNA were recovered from three individuals, and these analyses identified ancestry, hair/eye color, and relatedness. Genetic and isotopic results upended our initial ancestry assessment based on burial context alone. These individuals provide biocultural evidence of New Haven's Industrial Revolution and the plasticity of ethnic and religious identity in the immigrant experience. Their recovery and the multifaceted analyses described here illuminate a previously undescribed part of the city's rich history. The collective expertise of biological, geochemical, archaeological, and historical researchers interprets socioeconomic and cultural identity better than any one could alone. Our combined efforts changed our initial assumptions of a poor urban Catholic cemetery's membership, and provide a template for future discoveries and analyses.

The Current Genomic Landscape of Western South America: Andes, Amazonia, and Pacific Coast.

Studies of Native South American genetic diversity have helped to shed light on the peopling and differentiation of the continent, but available data are sparse for the major ecogeographic domains. These include the Pacific Coast, a potential early migration route; the Andes, home to the most expansive complex societies and to one of the most widely spoken indigenous language families of the continent (Quechua); and Amazonia, with its understudied population structure and rich cultural diversity. Here, we explore the genetic structure of 176 individuals from these three domains, genotyped with the Affymetrix Human Origins array. We infer multiple sources of ancestry within the Native American ancestry component; one with clear predominance on the Coast and in the Andes, and at least two distinct substrates in neighboring Amazonia, including a previously undetected ancestry characteristic of northern Ecuador and Colombia. Amazonian populations are also involved in recent gene-flow with each other and across ecogeographic domains, which does not accord with the traditional view of small, isolated groups. Long-distance genetic connections between speakers of the same language family suggest that indigenous languages here were spread not by cultural contact alone. Finally, Native American populations admixed with post-Columbian European and African sources at different times, with few cases of prolonged isolation. With our results we emphasize the importance of including understudied regions of the continent in high-resolution genetic studies, and we illustrate the potential of SNP chip arrays for informative regional-scale analysis.

A mass sacrifice of children and camelids at the Huanchaquito-Las Llamas site, Moche Valley, Peru.

Here we report the results of excavation and interdisciplinary study of the largest child and camelid sacrifice known from the New World. Stratigraphy, associated artifacts, and radiocarbon dating indicate that it was a single mass killing of more than 140 children and over 200 camelids directed by the Chimú state, c. AD 1450. Preliminary DNA analysis indicates that both boys and girls were chosen for sacrifice. Variability in forms of cranial modification (head shaping) and stable isotope analysis of carbon and nitrogen suggest that the children were a heterogeneous sample drawn from multiple regions and ethnic groups throughout the Chimú state. The Huanchaquito-Las Llamas mass sacrifice opens a new window on a previously unknown sacrificial ritual from fifteenth century northern coastal Peru. While the motivation for such a massive sacrifice is a subject for further research, there is archaeological evidence that it was associated with a climatic event (heavy rainfall and flooding) that could have impacted the economic, political and ideological stability of one of the most powerful states in the New World during the fifteenth century A.D.

Current evidence allows multiple models for the peopling of the Americas.

Some recent academic and popular literature implies that the problem of the colonization of the Americas has been largely resolved in favor of one specific model: a Pacific coastal migration, dependent on high marine productivity, from the Bering Strait to South America, thousands of years before Clovis, the earliest widespread cultural manifestation south of the glacial ice. Speculations on maritime adaptations and typological links (stemmed points) across thousands of kilometers have also been advanced. A review of the current genetic, archeological, and paleoecological evidence indicates that ancestral Native American population expansion occurred after 16,000 years ago, consistent with the archeological record, particularly with the earliest securely dated sites after ~15,000 years ago. These data are largely consistent with either an inland (ice-free corridor) or Pacific coastal routes (or both), but neither can be rejected at present. Systematic archeological and paleoecological investigations, informed by geomorphology, are required to test each hypothesis.

Genetic Ancestry of Rapanui before and after European Contact.

The origins and lifeways of the inhabitants of Rapa Nui (Easter Island), a remote island in the southeast Pacific Ocean, have been debated for generations. Archaeological evidence substantiates the widely accepted view that the island was first settled by people of Polynesian origin, as late as 1200 CE [1-4]. What remains controversial, however, is the nature of events in the island's population history prior to the first historic contact with Europeans in 1722 CE. Purported contact between Rapa Nui and South America is particularly contentious, and recent studies have reported genetic evidence for Native American admixture in present-day indigenous inhabitants of Rapa Nui [5-8]. Statistical modeling has suggested that this genetic contribution might have occurred prior to European contact [6]. Here we directly test the hypothesis that the Native American admixture of the current Rapa Nui population predates the arrival of Europeans with a paleogenomic analysis of five individual samples excavated from Ahu Nau Nau, Anakena, dating to pre- and post-European contact, respectively. Complete mitochondrial genomes and low-coverage autosomal genomes show that the analyzed individuals fall within the genetic diversity of present-day and ancient Polynesians, and we can reject the hypothesis that any of these individuals had substantial Native American ancestry. Our data thus suggest that the Native American ancestry in contemporary Easter Islanders was not present on the island prior to European contact and may thus be due to events in more recent history.

Mitochondrial DNA haplogroup variation of contemporary mixed South Americans reveals prehistoric displacements linked to archaeologically-derived culture history.

OBJECTIVE: The purpose of this study was to examine South American population structure and prehistoric population displacements prior to the Spanish conquest, utilizing mitochondrial DNA haplogroups of extant mixed populations from Mexico, Costa Rica, Venezuela, Colombia, Ecuador, Peru, Bolivia, Brazil, Argentina, and Chile. METHOD: Relative frequencies of four pan-American haplogroups, obtained from published databases, were analyzed to evaluate patterns of variations, population structure and possible prehistoric migration pathways. RESULTS: Patterns of mtDNA variation verify biogeographic drift processes and possible migratory pathways. CONCLUSIONS: We propose an updated model of South American colonization that is fully compatible with previous studies based on autosomal, mtDNA, and Y chromosome variation and with archaeologically-derived culture history.

Ancient DNA Analysis Suggests Negligible Impact of the Wari Empire Expansion in Peru's Central Coast during the Middle Horizon.

The analysis of ancient human DNA from South America allows the exploration of pre-Columbian population history through time and to directly test hypotheses about cultural and demographic evolution. The Middle Horizon (650-1100 AD) represents a major transitional period in the Central Andes, which is associated with the development and expansion of ancient Andean empires such as Wari and Tiwanaku. These empires facilitated a series of interregional interactions and socio-political changes, which likely played an important role in shaping the region's demographic and cultural profiles. We analyzed individuals from three successive pre-Columbian cultures present at the Huaca Pucllana archaeological site in Lima, Peru: Lima (Early Intermediate Period, 500-700 AD), Wari (Middle Horizon, 800-1000 AD) and Ychsma (Late Intermediate Period, 1000-1450 AD). We sequenced 34 complete mitochondrial genomes to investigate the potential genetic impact of the Wari Empire in the Central Coast of Peru. The results indicate that genetic diversity shifted only slightly through time, ruling out a complete population discontinuity or replacement driven by the Wari imperialist hegemony, at least in the region around present-day Lima. However, we caution that the very subtle genetic contribution of Wari imperialism at the particular Huaca Pucllana archaeological site might not be representative for the entire Wari territory in the Peruvian Central Coast.

Ancient mitochondrial DNA provides high-resolution time scale of the peopling of the Americas.

The exact timing, route, and process of the initial peopling of the Americas remains uncertain despite much research. Archaeological evidence indicates the presence of humans as far as southern Chile by 14.6 thousand years ago (ka), shortly after the Pleistocene ice sheets blocking access from eastern Beringia began to retreat. Genetic estimates of the timing and route of entry have been constrained by the lack of suitable calibration points and low genetic diversity of Native Americans. We sequenced 92 whole mitochondrial genomes from pre-Columbian South American skeletons dating from 8.6 to 0.5 ka, allowing a detailed, temporally calibrated reconstruction of the peopling of the Americas in a Bayesian coalescent analysis. The data suggest that a small population entered the Americas via a coastal route around 16.0 ka, following previous isolation in eastern Beringia for ~2.4 to 9 thousand years after separation from eastern Siberian populations. Following a rapid movement throughout the Americas, limited gene flow in South America resulted in a marked phylogeographic structure of populations, which persisted through time. All of the ancient mitochondrial lineages detected in this study were absent from modern data sets, suggesting a high extinction rate. To investigate this further, we applied a novel principal components multiple logistic regression test to Bayesian serial coalescent simulations. The analysis supported a scenario in which European colonization caused a substantial loss of pre-Columbian lineages.

Ancient DNA reveals selection acting on genes associated with hypoxia response in pre-Columbian Peruvian Highlanders in the last 8500 years.

Archaeological evidence shows that humans began living in the high altitude Andes approximately 12,000 years ago. Andean highlanders are known to have developed the most complex societies of pre-Columbian South America despite challenges to their health and reproductive success resulting from chronic exposure to hypoxia. While the physiological adaptations to this environmental stressor are well studied in contemporary Andean highlanders, the molecular evolutionary processes associated with such adaptations remain unclear. We aim to better understand how humans managed to demographically establish in this harsh environment by addressing a central question: did exposure to hypoxia drive adaptation via natural selection within Andean populations or did an existing phenotype--characterized by reduced susceptibility to hypoxic stress--enable human settlement of the Andes? We genotyped three variable loci within the NOS3 and EGLN1 genes previously associated with adaptation to high altitude in 150 ancient human DNA samples from Peruvian high altitude and coastal low altitude sites in a time frame between ~8500-560 BP. We compare the data of 109 successful samples to forward simulations of genetic drift with natural selection and find that selection, rather than drift, explains the gradual frequency changes observed in the highland populations for two of the three SNPs.

A Re-Appraisal of the Early Andean Human Remains from Lauricocha in Peru.

The discovery of human remains from the Lauricocha cave in the Central Andean highlands in the 1960's provided the first direct evidence for human presence in the high altitude Andes. The skeletons found at this site were ascribed to the Early to Middle Holocene and represented the oldest known population of Western South America, and thus were used in several studies addressing the early population history of the continent. However, later excavations at Lauricocha led to doubts regarding the antiquity of the site. Here, we provide new dating, craniometric, and genetic evidence for this iconic site. We obtained new radiocarbon dates, generated complete mitochondrial genomes and nuclear SNP data from five individuals, and re-analyzed the human remains of Lauricocha to revise the initial morphological and craniometric analysis conducted in the 1960's. We show that Lauricocha was indeed occupied in the Early to Middle Holocene but the temporal spread of dates we obtained from the human remains show that they do not qualify as a single contemporaneous population. However, the genetic results from five of the individuals fall within the spectrum of genetic diversity observed in pre-Columbian and modern Native Central American populations.