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.

The Anglo-Saxon migration and the formation of the early English gene pool.

The history of the British Isles and Ireland is characterized by multiple periods of major cultural change, including the influential transformation after the end of Roman rule, which precipitated shifts in language, settlement patterns and material culture1. The extent to which migration from continental Europe mediated these transitions is a matter of long-standing debate2-4. Here we study genome-wide ancient DNA from 460 medieval northwestern Europeans-including 278 individuals from England-alongside archaeological data, to infer contemporary population dynamics. We identify a substantial increase of continental northern European ancestry in early medieval England, which is closely related to the early medieval and present-day inhabitants of Germany and Denmark, implying large-scale substantial migration across the North Sea into Britain during the Early Middle Ages. As a result, the individuals who we analysed from eastern England derived up to 76% of their ancestry from the continental North Sea zone, albeit with substantial regional variation and heterogeneity within sites. We show that women with immigrant ancestry were more often furnished with grave goods than women with local ancestry, whereas men with weapons were as likely not to be of immigrant ancestry. A comparison with present-day Britain indicates that subsequent demographic events reduced the fraction of continental northern European ancestry while introducing further ancestry components into the English gene pool, including substantial southwestern European ancestry most closely related to that seen in Iron Age France5,6.

Geographically dispersed zoonotic tuberculosis in pre-contact South American human populations.

Previous ancient DNA research has shown that Mycobacterium pinnipedii, which today causes tuberculosis (TB) primarily in pinnipeds, infected human populations living in the coastal areas of Peru prior to European colonization. Skeletal evidence indicates the presence of TB in several pre-colonial South and North American populations with minimal access to marine resources- a scenario incompatible with TB transmission directly from infected pinnipeds or their tissues. In this study, we investigate the causative agent of TB in ten pre-colonial, non-coastal individuals from South America. We reconstruct M. pinnipedii genomes (10- to 15-fold mean coverage) from three contemporaneous individuals from inland Peru and Colombia, demonstrating the widespread dissemination of M. pinnipedii beyond the coast, either through human-to-human and/or animal-mediated routes. Overall, our study suggests that TB transmission in the pre-colonial era Americas involved a more complex transmission pathway than simple pinniped-to-human transfer.

Ancient Metagenomic Studies: Considerations for the Wider Scientific Community.

Like modern metagenomics, ancient metagenomics is a highly data-rich discipline, with the added challenge that the DNA of interest is degraded and, depending on the sample type, in low abundance. This requires the application of specialized measures during molecular experiments and computational analyses. Furthermore, researchers often work with finite sample sizes, which impedes optimal experimental design and control of confounding factors, and with ethically sensitive samples necessitating the consideration of additional guidelines. In September 2020, early career researchers in the field of ancient metagenomics met (Standards, Precautions & Advances in Ancient Metagenomics 2 [SPAAM2] community meeting) to discuss the state of the field and how to address current challenges. Here, in an effort to bridge the gap between ancient and modern metagenomics, we highlight and reflect upon some common misconceptions, provide a brief overview of the challenges in our field, and point toward useful resources for potential reviewers and newcomers to the field.

The origin and legacy of the Etruscans through a 2000-year archeogenomic time transect.

The origin, development, and legacy of the enigmatic Etruscan civilization from the central region of the Italian peninsula known as Etruria have been debated for centuries. Here we report a genomic time transect of 82 individuals spanning almost two millennia (800 BCE to 1000 CE) across Etruria and southern Italy. During the Iron Age, we detect a component of Indo-European­associated steppe ancestry and the lack of recent Anatolian-related admixture among the putative non­Indo-European­speaking Etruscans. Despite comprising diverse individuals of central European, northern African, and Near Eastern ancestry, the local gene pool is largely maintained across the first millennium BCE. This drastically changes during the Roman Imperial period where we report an abrupt population-wide shift to ~50% admixture with eastern Mediterranean ancestry. Last, we identify northern European components appearing in central Italy during the Early Middle Ages, which thus formed the genetic landscape of present-day Italian populations.

Community-curated and standardised metadata of published ancient metagenomic samples with AncientMetagenomeDir.

Ancient DNA and RNA are valuable data sources for a wide range of disciplines. Within the field of ancient metagenomics, the number of published genetic datasets has risen dramatically in recent years, and tracking this data for reuse is particularly important for large-scale ecological and evolutionary studies of individual taxa and communities of both microbes and eukaryotes. AncientMetagenomeDir (archived at https://doi.org/10.5281/zenodo.3980833 ) is a collection of annotated metagenomic sample lists derived from published studies that provide basic, standardised metadata and accession numbers to allow rapid data retrieval from online repositories. These tables are community-curated and span multiple sub-disciplines to ensure adequate breadth and consensus in metadata definitions, as well as longevity of the database. Internal guidelines and automated checks facilitate compatibility with established sequence-read archives and term-ontologies, and ensure consistency and interoperability for future meta-analyses. This collection will also assist in standardising metadata reporting for future ancient metagenomic studies.

Multi-omic detection of Mycobacterium leprae in archaeological human dental calculus.

Mineralized dental plaque (calculus) has proven to be an excellent source of ancient biomolecules. Here we present a Mycobacterium leprae genome (6.6-fold), the causative agent of leprosy, recovered via shotgun sequencing of sixteenth-century human dental calculus from an individual from Trondheim, Norway. When phylogenetically placed, this genome falls in branch 3I among the diversity of other contemporary ancient strains from Northern Europe. Moreover, ancient mycobacterial peptides were retrieved via mass spectrometry-based proteomics, further validating the presence of the pathogen. Mycobacterium leprae can readily be detected in the oral cavity and associated mucosal membranes, which likely contributed to it being incorporated into this individual's dental calculus. This individual showed some possible, but not definitive, evidence of skeletal lesions associated with early-stage leprosy. This study is the first known example of successful multi-omics retrieval of M. leprae from archaeological dental calculus. Furthermore, we offer new insights into dental calculus as an alternative sample source to bones or teeth for detecting and molecularly characterizing M. leprae in individuals from the archaeological record. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

A seventeenth-century Mycobacterium tuberculosis genome supports a Neolithic emergence of the Mycobacterium tuberculosis complex.

BACKGROUND: Although tuberculosis accounts for the highest mortality from a bacterial infection on a global scale, questions persist regarding its origin. One hypothesis based on modern Mycobacterium tuberculosis complex (MTBC) genomes suggests their most recent common ancestor followed human migrations out of Africa approximately 70,000 years before present. However, studies using ancient genomes as calibration points have yielded much younger dates of less than 6000 years. Here, we aim to address this discrepancy through the analysis of the highest-coverage and highest-quality ancient MTBC genome available to date, reconstructed from a calcified lung nodule of Bishop Peder Winstrup of Lund (b. 1605-d. 1679). RESULTS: A metagenomic approach for taxonomic classification of whole DNA content permitted the identification of abundant DNA belonging to the human host and the MTBC, with few non-TB bacterial taxa comprising the background. Genomic enrichment enabled the reconstruction of a 141-fold coverage M. tuberculosis genome. In utilizing this high-quality, high-coverage seventeenth-century genome as a calibration point for dating the MTBC, we employed multiple Bayesian tree models, including birth-death models, which allowed us to model pathogen population dynamics and data sampling strategies more realistically than those based on the coalescent. CONCLUSIONS: The results of our metagenomic analysis demonstrate the unique preservation environment calcified nodules provide for DNA. Importantly, we estimate a most recent common ancestor date for the MTBC of between 2190 and 4501 before present and for Lineage 4 of between 929 and 2084 before present using multiple models, confirming a Neolithic emergence for the MTBC.

Emergence of human-adapted Salmonella enterica is linked to the Neolithization process.

It has been hypothesized that the Neolithic transition towards an agricultural and pastoralist economy facilitated the emergence of human-adapted pathogens. Here, we recovered eight Salmonella enterica subsp. enterica genomes from human skeletons of transitional foragers, pastoralists and agropastoralists in western Eurasia that were up to 6,500 yr old. Despite the high genetic diversity of S. enterica, all ancient bacterial genomes clustered in a single previously uncharacterized branch that contains S. enterica adapted to multiple mammalian species. All ancient bacterial genomes from prehistoric (agro-)pastoralists fall within a part of this branch that also includes the human-specific S. enterica Paratyphi C, illustrating the evolution of a human pathogen over a period of 5,000 yr. Bacterial genomic comparisons suggest that the earlier ancient strains were not host specific, differed in pathogenic potential and experienced convergent pseudogenization that accompanied their downstream host adaptation. These observations support the concept that the emergence of human-adapted S. enterica is linked to human cultural transformations.

A 5700 year-old human genome and oral microbiome from chewed birch pitch.

The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007-0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003-70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Salmonella enterica genomes from victims of a major sixteenth-century epidemic in Mexico.

Indigenous populations of the Americas experienced high mortality rates during the early contact period as a result of infectious diseases, many of which were introduced by Europeans. Most of the pathogenic agents that caused these outbreaks remain unknown. Through the introduction of a new metagenomic analysis tool called MALT, applied here to search for traces of ancient pathogen DNA, we were able to identify Salmonella enterica in individuals buried in an early contact era epidemic cemetery at Teposcolula-Yucundaa, Oaxaca in southern Mexico. This cemetery is linked, based on historical and archaeological evidence, to the 1545-1550 CE epidemic that affected large parts of Mexico. Locally, this epidemic was known as 'cocoliztli', the pathogenic cause of which has been debated for more than a century. Here, we present genome-wide data from ten individuals for Salmonella enterica subsp. enterica serovar Paratyphi C, a bacterial cause of enteric fever. We propose that S. Paratyphi C be considered a strong candidate for the epidemic population decline during the 1545 cocoliztli outbreak at Teposcolula-Yucundaa.

Population resequencing of European mitochondrial genomes highlights sex-bias in Bronze Age demographic expansions.

Interpretations of genetic data concerning the prehistory of Europe have long been a subject of great debate, but increasing amounts of ancient and modern DNA data are now providing new and more informative evidence. Y-chromosome resequencing studies in Europe have highlighted the prevalence of recent expansions of male lineages, and focused interest on the Bronze Age as a period of cultural and demographic change. These findings contrast with phylogeographic studies based on mitochondrial DNA (mtDNA), which have been interpreted as supporting expansions from glacial refugia. Here we have undertaken a population-based resequencing of complete mitochondrial genomes in Europe and the Middle East, in 340 samples from 17 populations for which Y-chromosome sequence data are also available. Demographic reconstructions show no signal of Bronze Age expansion, but evidence of Paleolithic expansions in all populations except the Saami, and with an absence of detectable geographical pattern. In agreement with previous inference from modern and ancient DNA data, the unbiased comparison between the mtDNA and Y-chromosome population datasets emphasizes the sex-biased nature of recent demographic transitions in Europe.

Parallel detection of ancient pathogens via array-based DNA capture.

DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone.