Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

The ancestry and geographical origins of St Helena's liberated Africans.

The island of St Helena played a crucial role in the suppression of the transatlantic slave trade. Strategically located in the middle of the South Atlantic, it served as a staging post for the Royal Navy and reception point for enslaved Africans who had been "liberated" from slave ships intercepted by the British. In total, St Helena received approximately 27,000 liberated Africans between 1840 and 1867. Written sources suggest that the majority of these individuals came from West Central Africa, but their precise origins are unknown. Here, we report the results of ancient DNA analyses that we conducted as part of a wider effort to commemorate St Helena's liberated Africans and to restore knowledge of their lives and experiences. We generated partial genomes (0.1-0.5×) for 20 individuals whose remains had been recovered during archaeological excavations on the island. We compared their genomes with genotype data for over 3,000 present-day individuals from 90 populations across sub-Saharan Africa and conclude that the individuals most likely originated from different source populations within the general area between northern Angola and Gabon. We also find that the majority (17/20) of the individuals were male, supporting a well-documented sex bias in the latter phase of the transatlantic slave trade. The study expands our understanding of St Helena's liberated African community and illustrates how ancient DNA analyses can be used to investigate the origins and identities of individuals whose lives were bound up in the story of slavery and its abolition.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

The evolutionary history of extinct and living lions.

Lions are one of the world's most iconic megafauna, yet little is known about their temporal and spatial demographic history and population differentiation. We analyzed a genomic dataset of 20 specimens: two ca. 30,000-y-old cave lions (Panthera leo spelaea), 12 historic lions (Panthera leo leo/Panthera leo melanochaita) that lived between the 15th and 20th centuries outside the current geographic distribution of lions, and 6 present-day lions from Africa and India. We found that cave and modern lions shared an ancestor ca. 500,000 y ago and that the 2 lineages likely did not hybridize following their divergence. Within modern lions, we found 2 main lineages that diverged ca. 70,000 y ago, with clear evidence of subsequent gene flow. Our data also reveal a nearly complete absence of genetic diversity within Indian lions, probably due to well-documented extremely low effective population sizes in the recent past. Our results contribute toward the understanding of the evolutionary history of lions and complement conservation efforts to protect the diversity of this vulnerable species.

Plasmodium vivax Malaria Viewed through the Lens of an Eradicated European Strain.

The protozoan Plasmodium vivax is responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania, and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations of P. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a European P. vivax strain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate for P. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonization of the Americas. In addition, we found that some known variants for resistance to antimalarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.

Origins and genetic legacies of the Caribbean Taino.

The Caribbean was one of the last parts of the Americas to be settled by humans, but how and when the islands were first occupied remains a matter of debate. Ancient DNA can help answering these questions, but the work has been hampered by poor DNA preservation. We report the genome sequence of a 1,000-year-old Lucayan Taino individual recovered from the site of Preacher's Cave in the Bahamas. We sequenced her genome to 12.4-fold coverage and show that she is genetically most closely related to present-day Arawakan speakers from northern South America, suggesting that the ancestors of the Lucayans originated there. Further, we find no evidence for recent inbreeding or isolation in the ancient genome, suggesting that the Lucayans had a relatively large effective population size. Finally, we show that the native American components in some present-day Caribbean genomes are closely related to the ancient Taino, demonstrating an element of continuity between precontact populations and present-day Latino populations in the Caribbean.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain.

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe-where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.

Genome-wide ancestry of 17th-century enslaved Africans from the Caribbean.

Between 1500 and 1850, more than 12 million enslaved Africans were transported to the New World. The vast majority were shipped from West and West-Central Africa, but their precise origins are largely unknown. We used genome-wide ancient DNA analyses to investigate the genetic origins of three enslaved Africans whose remains were recovered on the Caribbean island of Saint Martin. We trace their origins to distinct subcontinental source populations within Africa, including Bantu-speaking groups from northern Cameroon and non-Bantu speakers living in present-day Nigeria and Ghana. To our knowledge, these findings provide the first direct evidence for the ethnic origins of enslaved Africans, at a time for which historical records are scarce, and demonstrate that genomic data provide another type of record that can shed new light on long-standing historical questions.

A Common Genetic Origin for Early Farmers from Mediterranean Cardial and Central European LBK Cultures.

The spread of farming out of the Balkans and into the rest of Europe followed two distinct routes: An initial expansion represented by the Impressa and Cardial traditions, which followed the Northern Mediterranean coastline; and another expansion represented by the LBK (Linearbandkeramik) tradition, which followed the Danube River into Central Europe. Although genomic data now exist from samples representing the second migration, such data have yet to be successfully generated from the initial Mediterranean migration. To address this, we generated the complete genome of a 7,400-year-old Cardial individual (CB13) from Cova Bonica in Vallirana (Barcelona), as well as partial nuclear data from five others excavated from different sites in Spain and Portugal. CB13 clusters with all previously sequenced early European farmers and modern-day Sardinians. Furthermore, our analyses suggest that both Cardial and LBK peoples derived from a common ancient population located in or around the Balkan Peninsula. The Iberian Cardial genome also carries a discernible hunter-gatherer genetic signature that likely was not acquired by admixture with local Iberian foragers. Our results indicate that retrieving ancient genomes from similarly warm Mediterranean environments such as the Near East is technically feasible.

Demographic history and genetic structure in pre-Hispanic Central Mexico.

Aridoamerica and Mesoamerica are two distinct cultural areas in northern and central Mexico, respectively, that hosted numerous pre-Hispanic civilizations between 2500 BCE and 1521 CE. The division between these regions shifted southward because of severe droughts ~1100 years ago, which allegedly drove a population replacement in central Mexico by Aridoamerican peoples. In this study, we present shotgun genome-wide data from 12 individuals and 27 mitochondrial genomes from eight pre-Hispanic archaeological sites across Mexico, including two at the shifting border of Aridoamerica and Mesoamerica. We find population continuity that spans the climate change episode and a broad preservation of the genetic structure across present-day Mexico for the past 2300 years. Lastly, we identify a contribution to pre-Hispanic populations of northern and central Mexico from two ancient unsampled "ghost" populations.

The population genomic legacy of the second plague pandemic.

Human populations have been shaped by catastrophes that may have left long-lasting signatures in their genomes. One notable example is the second plague pandemic that entered Europe in ca. 1,347 CE and repeatedly returned for over 300 years, with typical village and town mortality estimated at 10%-40%.1 It is assumed that this high mortality affected the gene pools of these populations. First, local population crashes reduced genetic diversity. Second, a change in frequency is expected for sequence variants that may have affected survival or susceptibility to the etiologic agent (Yersinia pestis).2 Third, mass mortality might alter the local gene pools through its impact on subsequent migration patterns. We explored these factors using the Norwegian city of Trondheim as a model, by sequencing 54 genomes spanning three time periods: (1) prior to the plague striking Trondheim in 1,349 CE, (2) the 17th-19th century, and (3) the present. We find that the pandemic period shaped the gene pool by reducing long distance immigration, in particular from the British Isles, and inducing a bottleneck that reduced genetic diversity. Although we also observe an excess of large FST values at multiple loci in the genome, these are shaped by reference biases introduced by mapping our relatively low genome coverage degraded DNA to the reference genome. This implies that attempts to detect selection using ancient DNA (aDNA) datasets that vary by read length and depth of sequencing coverage may be particularly challenging until methods have been developed to account for the impact of differential reference bias on test statistics.

A genomic exploration of the early evolution of extant cats and their sabre-toothed relatives.

Background: The evolutionary relationships of Felidae during their Early-Middle Miocene radiation is contentious. Although the early common ancestors have been subsumed under the grade-group Pseudaelurus, this group is thought to be paraphyletic, including the early ancestors of both modern cats and extinct sabretooths. Methods: Here, we sequenced a draft nuclear genome of Smilodon populator, dated to 13,182 ± 90 cal BP, making this the oldest palaeogenome from South America to date, a region known to be problematic for ancient DNA preservation. We analysed this genome, together with genomes from other extinct and extant cats to investigate their phylogenetic relationships. Results: We confirm a deep divergence (~20.65 Ma) within sabre-toothed cats. Through the analysis of both simulated and empirical data, we show a lack of gene flow between Smilodon and contemporary Felidae. Conclusions: Given that some species traditionally assigned to Pseudaelurus originated in the Early Miocene ~20 Ma, this indicates that some species of Pseudaelurus may be younger than the lineages they purportedly gave rise to, further supporting the hypothesis that Pseudaelurus was paraphyletic.

Ancient viral genomes reveal introduction of human pathogenic viruses into Mexico during the transatlantic slave trade.

After the European colonization of the Americas, there was a dramatic population collapse of the Indigenous inhabitants caused in part by the introduction of new pathogens. Although there is much speculation on the etiology of the Colonial epidemics, direct evidence for the presence of specific viruses during the Colonial era is lacking. To uncover the diversity of viral pathogens during this period, we designed an enrichment assay targeting ancient DNA (aDNA) from viruses of clinical importance and applied it to DNA extracts from individuals found in a Colonial hospital and a Colonial chapel (16th-18th century) where records suggest that victims of epidemics were buried during important outbreaks in Mexico City. This allowed us to reconstruct three ancient human parvovirus B19 genomes and one ancient human hepatitis B virus genome from distinct individuals. The viral genomes are similar to African strains, consistent with the inferred morphological and genetic African ancestry of the hosts as well as with the isotopic analysis of the human remains, suggesting an origin on the African continent. This study provides direct molecular evidence of ancient viruses being transported to the Americas during the transatlantic slave trade and their subsequent introduction to New Spain. Altogether, our observations enrich the discussion about the etiology of infectious diseases during the Colonial period in Mexico.

The arrival of European colonists to the Americas, beginning in the 15^th century, contributed to the spread of new viruses amongst Indigenous people. This led to massive outbreaks of disease, and millions of deaths that caused an important Native population to collapse. The exact viruses that caused these outbreaks are unknown, but smallpox, measles, and mumps are all suspected. During these times, traders and colonists forcibly enslaved and displaced millions of people mainly from the West Coast of Africa to the Americas. The cruel, unsanitary, and overcrowded conditions on ships transporting these people across the Atlantic contributed to the spread of infectious diseases onboard. Once on land, infectious diseases spread quickly, partly due to the poor conditions that enslaved and ndigenous people were made to endure. Native people were also immunologically naïve to the newly introduced pathogens, making them susceptible to severe or fatal outcomes. The new field of paleovirology may help scientists identify the viruses that were circulating in the first years of colonization and trace how viruses arrived in the Americas. Using next-generation DNA sequencing and other cutting-edge techniques, Guzmán-Solís et al. extracted and enriched viral DNA from skeletal remains dating back to the 16^th century. These remains were found in mass graves that were used to bury epidemic victims at a colonial hospital and chapel in what is now Mexico City. The experiments identified two viruses, human parvovirus B19 and a human hepatitis B virus. These viral genomes were recovered from human remains of first-generation African people in Mexico, as well as an individual who was an Indigenous person. Although the genetic material of these ancient viruses resembled pathogens that originated in Africa, the study did not determine if the victims died from these viruses or another cause. On the other hand, the results indicate that viruses frequently found in modern Africa were circulating in the Americas during the slave trade period of Mexico. Finally, the results provide evidence that colonists who forcibly brought African people to the Americas participated in the introduction of viruses to Mexico. This constant influx of viruses from the old world, led to dramatic declines in the populations of Indigenous people in the Americas.

Hi-C chromosome conformation capture sequencing of avian genomes using the BGISEQ-500 platform.

BACKGROUND: Hi-C experiments couple DNA-DNA proximity with next-generation sequencing to yield an unbiased description of genome-wide interactions. Previous methods describing Hi-C experiments have focused on the industry-standard Illumina sequencing. With new next-generation sequencing platforms such as BGISEQ-500 becoming more widely available, protocol adaptations to fit platform-specific requirements are useful to give increased choice to researchers who routinely generate sequencing data. RESULTS: We describe an in situ Hi-C protocol adapted to be compatible with the BGISEQ-500 high-throughput sequencing platform. Using zebra finch (Taeniopygia guttata) as a biological sample, we demonstrate how Hi-C libraries can be constructed to generate informative data using the BGISEQ-500 platform, following circularization and DNA nanoball generation. Our protocol is a modification of an Illumina-compatible method, based around blunt-end ligations in library construction, using un-barcoded, distally overhanging double-stranded adapters, followed by amplification using indexed primers. The resulting libraries are ready for circularization and subsequent sequencing on the BGISEQ series of platforms and yield data similar to what can be expected using Illumina-compatible approaches. CONCLUSIONS: Our straightforward modification to an Illumina-compatible in situHi-C protocol enables data generation on the BGISEQ series of platforms, thus expanding the options available for researchers who wish to utilize the powerful Hi-C techniques in their research.

Paleogenomic insights into the red complex bacteria Tannerella forsythia in Pre-Hispanic and Colonial individuals from Mexico.

The 'red complex' is an aggregate of three oral bacteria (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) responsible for severe clinical manifestation of periodontal disease. Here, we report the first direct evidence of ancient T.forsythia DNA in dentin and dental calculus samples from archaeological skeletal remains that span from the Pre-Hispanic to the Colonial period in Mexico. We recovered twelve partial ancient T. forsythia genomes and observed a distinct phylogenetic placement of samples, suggesting that the strains present in Pre-Hispanic individuals likely arrived with the first human migrations to the Americas and that new strains were introduced with the arrival of European and African populations in the sixteenth century. We also identified instances of the differential presence of genes between periods in the T. forsythia ancient genomes, with certain genes present in Pre-Hispanic individuals and absent in Colonial individuals, and vice versa. This study highlights the potential for studying ancient T. forsythia genomes to unveil past social interactions through analysis of disease transmission. Our results illustrate the long-standing relationship between this oral pathogen and its human host, while also unveiling key evidence to understand its evolutionary history in Pre-Hispanic and Colonial Mexico. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Genomic Adaptations and Evolutionary History of the Extinct Scimitar-Toothed Cat, Homotherium latidens.

Homotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [1-4]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [5]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [6-8]. However, mitochondrial phylogenies can be misled by hybridization [9], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [10], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [10]. To examine the evolutionary history of Homotherium, we generated a ∼7x nuclear genome and a ∼38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (∼22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [5]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [3, 4, 11-14]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage.

Genomic insights into the early peopling of the Caribbean.

The Caribbean was one of the last regions of the Americas to be settled by humans, but where they came from and how and when they reached the islands remain unclear. We generated genome-wide data for 93 ancient Caribbean islanders dating between 3200 and 400 calibrated years before the present and found evidence of at least three separate dispersals into the region, including two early dispersals into the Western Caribbean, one of which seems connected to radiation events in North America. This was followed by a later expansion from South America. We also detected genetic differences between the early settlers and the newcomers from South America, with almost no evidence of admixture. Our results add to our understanding of the initial peopling of the Caribbean and the movements of Archaic Age peoples in the Americas.

Evolutionary History, Genomic Adaptation to Toxic Diet, and Extinction of the Carolina Parakeet.

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes.

Genetic affinities of an eradicated European Plasmodium falciparum strain.

Malaria was present in most of Europe until the second half of the 20th century, when it was eradicated through a combination of increased surveillance and mosquito control strategies, together with cross-border and political collaboration. Despite the severe burden of malaria on human populations, it remains contentious how the disease arrived and spread in Europe. Here, we report a partial Plasmodium falciparum nuclear genome derived from a set of antique medical slides stained with the blood of malaria-infected patients from Spain's Ebro Delta, dating to the 1940s. Our analyses of the genome of this now eradicated European P. falciparum strain confirms stronger phylogeographical affinity to present-day strains in circulation in central south Asia, rather than to those in Africa. This points to a longitudinal, rather than a latitudinal, spread of malaria into Europe. In addition, this genome displays two derived alleles in the pfmrp1 gene that have been associated with drug resistance. Whilst this could represent standing variation in the ancestral P. falciparum population, these mutations may also have arisen due to the selective pressure of quinine treatment, which was an anti-malarial drug already in use by the time the sample we sequenced was mounted on a slide.