Genomic analyses correspond with deep persistence of peoples of Blackfoot Confederacy from glacial times.

Mutually beneficial partnerships between genomics researchers and North American Indigenous Nations are rare yet becoming more common. Here, we present one such partnership that provides insight into the peopling of the Americas and furnishes another line of evidence that can be used to further treaty and Indigenous rights. We show that the genomics of sampled individuals from the Blackfoot Confederacy belong to a previously undescribed ancient lineage that diverged from other genomic lineages in the Americas in Late Pleistocene times. Using multiple complementary forms of knowledge, we provide a scenario for Blackfoot population history that fits with oral tradition and provides a plausible model for the evolutionary process of the peopling of the Americas.

Pharmacogenetic Variation in Neanderthals and Denisovans and Implications for Human Health and Response to Medications.

Modern humans carry both Neanderthal and Denisovan (archaic) genome elements that are part of the human gene pool and affect the life and health of living individuals. The impact of archaic DNA may be particularly evident in pharmacogenes-genes responsible for the processing of exogenous substances such as food, pollutants, and medications-as these can relate to changing environmental effects, and beneficial variants may have been retained as modern humans encountered new environments. However, the health implications and contribution of archaic ancestry in pharmacogenes of modern humans remain understudied. Here, we explore 11 key cytochrome P450 genes (CYP450) involved in 75% of all drug metabolizing reactions in three Neanderthal and one Denisovan individuals and examine archaic introgression in modern human populations. We infer the metabolizing efficiency of these 11 CYP450 genes in archaic individuals and find important predicted phenotypic differences relative to modern human variants. We identify several single nucleotide variants shared between archaic and modern humans in each gene, including some potentially function-altering mutations in archaic CYP450 genes, which may result in altered metabolism in living people carrying these variants. We also identified several variants in the archaic CYP450 genes that are novel and unique to archaic humans as well as one gene, CYP2B6, that shows evidence for a gene duplication found only in Neanderthals and modern Africans. Finally, we highlight CYP2A6, CYP2C9, and CYP2J2, genes which show evidence for archaic introgression into modern humans and posit evolutionary hypotheses that explain their allele frequencies in modern populations.

Association between gene methylation and experiences of historical trauma in Alaska Native peoples.

BACKGROUND: Historical trauma experienced by Indigenous peoples of North America is correlated with health disparities and is hypothesized to be associated with DNA methylation. Massive group traumas such as genocide, loss of land and foodways, and forced conversion to Western lifeways may be embodied and affect individuals, families, communities, cultures, and health. This study approaches research with Alaska Native people using a community-engaged approach designed to create mutually-beneficial partnerships, including intentional relationship development, capacity building, and sample and data care. METHODS: A total of 117 Alaska Native individuals from two regions of Alaska joined the research study. Participants completed surveys on cultural identification, historical trauma (historical loss scale and historical loss associated symptoms scale), and general wellbeing. Participants provided a blood sample which was used to assess DNA methylation with the Illumina Infinium MethylationEPIC array. RESULTS: We report an association between historical loss associated symptoms and DNA methylation at five CpG sites, evidencing the embodiment of historical trauma. We further report an association between cultural identification and general wellbeing, complementing evidence from oral narratives and additional studies that multiple aspects of cultural connection may buffer the effects of and/or aid in the healing process from historical trauma. CONCLUSION: A community-engaged approach emphasizes balanced partnerships between communities and researchers. Here, this approach helps better understand embodiment of historical trauma in Alaska Native peoples. This analysis reveals links between the historical trauma response and DNA methylation. Indigenous communities have been stigmatized for public health issues instead caused by systemic inequalities, social disparities, and discrimination, and we argue that the social determinants of health model in Alaska Native peoples must include the vast impact of historical trauma and ongoing colonial violence.

A paleogenome from a Holocene individual supports genetic continuity in Southeast Alaska.

Many specifics of the population histories of the Indigenous peoples of North America remain contentious owing to a dearth of physical evidence. Only few ancient human genomes have been recovered from the Pacific Northwest Coast, a region increasingly supported as a coastal migration route for the initial peopling of the Americas. Here, we report paleogenomic data from the remains of a ∼3,000-year-old female individual from Southeast Alaska, named Tatóok yík yées sháawat (TYYS). Our results demonstrate at least 3,000 years of matrilineal genetic continuity in Southeast Alaska, and that TYYS is most closely related to ancient and present-day northern Pacific Northwest Coast Indigenous Americans. We find no evidence of Paleo-Inuit (represented by Saqqaq) ancestry in present-day or ancient Pacific Northwest peoples. Instead, our analyses suggest the Saqqaq genome harbors Northern Native American ancestry. This study sheds further light on the human population history of the northern Pacific Northwest Coast.

Community partnerships are fundamental to ethical ancient DNA research.

The ethics of the scientific study of Ancestors has long been debated by archaeologists, bioanthropologists, and, more recently, ancient DNA (aDNA) researchers. This article responds to the article "Ethics of DNA research on human remains: five globally applicable guidelines" published in 2021 in Nature by a large group of aDNA researchers and collaborators. We argue that these guidelines do not sufficiently consider the interests of community stakeholders, including descendant communities and communities with potential, but yet unestablished, ties to Ancestors. We focus on three main areas of concern with the guidelines. First is the false separation of "scientific" and "community" concerns and the consistent privileging of researcher perspectives over those of community members. Second, the commitment of the guidelines' authors to open data ignores the principles and practice of Indigenous Data Sovereignty. Further, the authors argue that involving community members in decisions about publication and data sharing is unethical. We argue that excluding community perspectives on "ethical" grounds is convenient for researchers, but it is not, in fact, ethical. Third, we stress the risks of not consulting communities that have established or potential ties to Ancestors, using two recent examples from the literature. Ancient DNA researchers cannot focus on the lowest common denominator of research practice, the bare minimum that is legally necessary. Instead, they should be leading multidisciplinary efforts to create processes to ensure communities from all regions of the globe are identified and engaged in research that affects them. This will often present challenges, but we see these challenges as part of the research, rather than a distraction from the scientific endeavor. If a research team does not have the capacity to meaningfully engage communities, questions must be asked about the value and benefit of their research.

Biological samples taken from Native American Ancestors are human remains under NAGPRA.

In the United States, the Native American Graves Protection and Repatriation Act (NAGPRA) provides a specific framework for the disposition of Native American Ancestral remains within its purview. However, samples such as a bone fragment, tooth, or other biological tissue taken from the remains of these Ancestors have been treated by institutions and researchers as independent of the individual from whom they were removed and used in destructive research such as paleogenomic and other archaeometric analyses without consultation, consent, and collaboration from Native American communities; are not cared for in keeping with the current best practices for Indigenous Ancestors; and are not likely to be repatriated to their communities. Here, we demonstrate that any biological samples removed from Ancestors who are covered under NAGPRA must also be handled according to the stipulations defined for "human remains" within the legislation. As such, we are not proposing a change to existing legislation, but rather best practices, specific to the context of the United States and NAGPRA, relating to the use of and care for biological samples taken from Native American Ancestors.

Numt Parser: Automated identification and removal of nuclear mitochondrial pseudogenes (numts) for accurate mitochondrial genome reconstruction in Panthera.

Nuclear mitochondrial pseudogenes (numts) may hinder the reconstruction of mtDNA genomes and affect the reliability of mtDNA datasets for phylogenetic and population genetic comparisons. Here, we present the program Numt Parser, which allows for the identification of DNA sequences that likely originate from numt pseudogene DNA. Sequencing reads are classified as originating from either numt or true cytoplasmic mitochondrial (cymt) DNA by direct comparison against cymt and numt reference sequences. Classified reads can then be parsed into cymt or numt datasets. We tested this program using whole genome shotgun-sequenced data from 2 ancient Cape lions (Panthera leo), because mtDNA is often the marker of choice for ancient DNA studies and the genus Panthera is known to have numt pseudogenes. Numt Parser decreased sequence disagreements that were likely due to numt pseudogene contamination and equalized read coverage across the mitogenome by removing reads that likely originated from numts. We compared the efficacy of Numt Parser to 2 other bioinformatic approaches that can be used to account for numt contamination. We found that Numt Parser outperformed approaches that rely only on read alignment or Basic Local Alignment Search Tool (BLAST) properties, and was effective at identifying sequences that likely originated from numts while having minimal impacts on the recovery of cymt reads. Numt Parser therefore improves the reconstruction of true mitogenomes, allowing for more accurate and robust biological inferences.

Ancient and modern genomics of the Ohlone Indigenous population of California.

SignificanceCalifornia supports a high cultural and linguistic diversity of Indigenous peoples. In a partnership of researchers with the Muwekma Ohlone tribe, we studied genomes of eight present-day tribal members and 12 ancient individuals from two archaeological sites in the San Francisco Bay Area, spanning ∼2,000 y. We find that compared to genomes of Indigenous individuals from throughout the Americas, the 12 ancient individuals are most genetically similar to ancient individuals from Southern California, and that despite spanning a large time period, they share distinctive ancestry. This ancestry is also shared with present-day tribal members, providing evidence of genetic continuity between past and present Indigenous individuals in the region, in contrast to some popular reconstructions based on archaeological and linguistic information.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.

Social convergence of gut microbiomes in vampire bats.

The 'social microbiome' can fundamentally shape the costs and benefits of group-living, but understanding social transmission of microbes in free-living animals is challenging due to confounding effects of kinship and shared environments (e.g. highly associated individuals often share the same spaces, food and water). Here, we report evidence for convergence towards a social microbiome among introduced common vampire bats, Desmodus rotundus, a highly social species in which adults feed only on blood, and engage in both mouth-to-body allogrooming and mouth-to-mouth regurgitated food sharing. Shotgun sequencing of samples from six zoos in the USA, 15 wild-caught bats from a colony in Belize and 31 bats from three colonies in Panama showed that faecal microbiomes were more similar within colonies than between colonies. To assess microbial transmission, we created an experimentally merged group of the Panama bats from the three distant sites by housing these bats together for four months. In this merged colony, we found evidence that dyadic gut microbiome similarity increased with both clustering and oral contact, leading to microbiome convergence among introduced bats. Our findings demonstrate that social interactions shape microbiome similarity even when controlling for past social history, kinship, environment and diet.

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, "subfossil" koala lemur Megaladapis edwardsi.

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal "subfossil" remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Integrative analysis of DNA, macroscopic remains and stable isotopes of dog coprolites to reconstruct community diet.

Paleofeces or coprolites are often used to reconstruct diet at archaeological sites, usually using macroscopic analyses or targeted DNA amplification and sequencing. Here we present an integrative analysis of dog coprolites, combining macroscopic analyses, stable isotope measurements, and DNA shotgun sequencing to examine diet and health status. Dog coprolites used in this study were recovered from the Janey B. Goode and East Saint Louis archaeological sites, both of which are located in the American Bottom, an extensive Mississippi River floodplain in Southwestern Illinois. Based on the context of recovery, coprolites are assigned to the Late Woodland and Terminal Late Woodland periods (ca. 600-1050 AD). Given the scarcity of human remains from this time period, these dog coprolites can be useful as a proxy for understanding human diet during the Late Woodland period. We find that the Late Woodland dogs consumed a variety of fish as well as bird and plant taxa, possibly including maize, and also harbored intestinal parasites and pathogenic bacteria. By sequencing the fecal microbiome of the coprolites, we find some similarities to modern dog microbiomes, as well as specific taxa that can be used to discriminate between modern and ancient microbiomes, excluding soil contaminants. As dogs are often used as a surrogate to assess human diet, humans living with these dogs likely had a similar diet and were affected by similar parasites. These analyses, when integrated, show a more comprehensive view of ancient dog and human diet and health in the region during the initial expansion of maize agriculture than any individual method could alone.

Dog domestication and the dual dispersal of people and dogs into the Americas.

Advances in the isolation and sequencing of ancient DNA have begun to reveal the population histories of both people and dogs. Over the last 10,000 y, the genetic signatures of ancient dog remains have been linked with known human dispersals in regions such as the Arctic and the remote Pacific. It is suspected, however, that this relationship has a much deeper antiquity, and that the tandem movement of people and dogs may have begun soon after the domestication of the dog from a gray wolf ancestor in the late Pleistocene. Here, by comparing population genetic results of humans and dogs from Siberia, Beringia, and North America, we show that there is a close correlation in the movement and divergences of their respective lineages. This evidence places constraints on when and where dog domestication took place. Most significantly, it suggests that dogs were domesticated in Siberia by ∼23,000 y ago, possibly while both people and wolves were isolated during the harsh climate of the Last Glacial Maximum. Dogs then accompanied the first people into the Americas and traveled with them as humans rapidly dispersed into the continent beginning ∼15,000 y ago.

Sourcing Elephant Ivory from a Sixteenth-Century Portuguese Shipwreck.

The oldest known shipwreck in southern Africa was found in Namibia in 2008.1-4 Forty tons of cargo, including gold and silver coins, helped identify the ship as the Bom Jesus, a Portuguese nau (trading vessel) lost in 1533 while headed to India.4-6 The cargo included >100 elephant tusks,7 which we examined using paleogenomic and stable isotope analyses. Nuclear DNA identified the ivory source as African forest (Loxodonta cyclotis) rather than savanna (Loxodonta africana) elephants. Mitochondrial sequences traced them to West and not Central Africa and from ≥17 herds with distinct haplotypes. Four of the haplotypes are known from modern populations; others were potentially lost to subsequent hunting of elephants for ivory. Stable isotope analyses (δ13C and δ15N) indicated that the elephants were not from deep rainforests but from savanna and mixed habitats. Such habitats surround the Guinean forest block of West Africa8 and accord with the locations of major historic Portuguese trading ports.9,10 West African forest elephants currently range into savanna habitats;11-13 our findings suggest that this was not consequent to regional decimation of savanna elephants for their ivory in the 19th and 20th centuries. During the time of the Bom Jesus, ivory was a central driver in the formation of maritime trading systems connecting Europe, Africa, and Asia. Our integration of paleogenomic, archeological, and historical methods to analyze the Bom Jesus ivory provides a framework for examining vast collections of archaeological ivories around the world, in shipwrecks and other contexts.

Population genetics of wild Macaca fascicularis with low-coverage shotgun sequencing of museum specimens.

OBJECTIVES: Long-tailed macaques (Macaca fascicularis) are widely distributed throughout the mainland and islands of Southeast Asia, making them a useful model for understanding the complex biogeographical history resulting from drastic changes in sea levels throughout the Pleistocene. Past studies based on mitochondrial genomes (mitogenomes) of long-tailed macaque museum specimens have traced their colonization patterns throughout the archipelago, but mitogenomes trace only the maternal history. Here, our objectives were to trace phylogeographic patterns of long-tailed macaques using low-coverage nuclear DNA (nDNA) data from museum specimens. METHODS: We performed population genetic analyses and phylogenetic reconstruction on nuclear single nucleotide polymorphisms (SNPs) from shotgun sequencing of 75 long-tailed macaque museum specimens from localities throughout Southeast Asia. RESULTS: We show that shotgun sequencing of museum specimens yields sufficient genome coverage (average ~1.7%) for reconstructing population relationships using SNP data. Contrary to expectations of divergent results between nuclear and mitochondrial genomes for a female philopatric species, phylogeographical patterns based on nuclear SNPs proved to be closely similar to those found using mitogenomes. In particular, population genetic analyses and phylogenetic reconstruction from the nDNA identify two major clades within M. fascicularis: Clade A includes all individuals from the mainland along with individuals from northern Sumatra, while Clade B consists of the remaining island-living individuals, including those from southern Sumatra. CONCLUSIONS: Overall, we demonstrate that low-coverage sequencing of nDNA from museum specimens provides enough data for examining broad phylogeographic patterns, although greater genome coverage and sequencing depth would be needed to distinguish between very closely related populations, such as those throughout the Philippines.

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation.

Sex estimation of skeletons is fundamental to many archaeological studies. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation.

Rights, interests and expectations: Indigenous perspectives on unrestricted access to genomic data.

Addressing Indigenous rights and interests in genetic resources has become increasingly challenging in an open science environment that promotes unrestricted access to genomic data. Although Indigenous experiences with genetic research have been shaped by a series of negative interactions, there is increasing recognition that equitable benefits can only be realized through greater participation of Indigenous communities. Issues of trust, accountability and equity underpin Indigenous critiques of genetic research and the sharing of genomic data. This Perspectives article highlights identified issues for Indigenous communities around the sharing of genomic data and suggests principles and actions that genomic researchers can adopt to recognize community rights and interests in data.

Accurate Sex Identification of Ancient Elephant and Other Animal Remains Using Low-Coverage DNA Shotgun Sequencing Data.

Sex identification of ancient animal biological remains can benefit our understanding of historical population structure, demography and social behavior. Traditional methods for sex identification (e.g., osteological and morphometric comparisons) may be ineffective when animal remains are not well preserved, when sex distinguishing characteristics have not yet developed, or where organisms do not exhibit sex-associated phenotypic dimorphisms. Here we adapt a method developed for human sex determination so that it can be used to identify the sex of ancient and modern animal taxa. The method identifies sex by calculating the ratio of DNA reads aligning to the X chromosome to DNA reads aligning to autosomes (termed the Rx ratio). We tested the accuracy of this method using low coverage genomes from 15 modern elephants (Loxodonta africana) for which sex was known. We then applied this method to ancient elephant ivory samples for which sex was unknown, and describe how this method can be further adapted to the genomes of other taxa. This method may be especially useful when only low-coverage genomic data are obtainable. Furthermore, because this method relies on only the X and not the Y chromosome, it can be used to determine the sex of organisms for which a reference genome was obtained from a female or for which only the X chromosome is reported. Such taxa include the domestic cat, sheep, goat, and horse; and non-domesticated animals such as the Sumatran orangutan, western lowland gorilla and meerkat.