Early contact between late farming and pastoralist societies in southeastern Europe.

Archaeogenetic studies have described two main genetic turnover events in prehistoric western Eurasia: one associated with the spread of farming and a sedentary lifestyle starting around 7000-6000 BC (refs. 1-3) and a second with the expansion of pastoralist groups from the Eurasian steppes starting around 3300 BC (refs. 4,5). The period between these events saw new economies emerging on the basis of key innovations, including metallurgy, wheel and wagon and horse domestication6-9. However, what happened between the demise of the Copper Age settlements around 4250 BC and the expansion of pastoralists remains poorly understood. To address this question, we analysed genome-wide data from 135 ancient individuals from the contact zone between southeastern Europe and the northwestern Black Sea region spanning this critical time period. While we observe genetic continuity between Neolithic and Copper Age groups from major sites in the same region, from around 4500 BC on, groups from the northwestern Black Sea region carried varying amounts of mixed ancestries derived from Copper Age groups and those from the forest/steppe zones, indicating genetic and cultural contact over a period of around 1,000 years earlier than anticipated. We propose that the transfer of critical innovations between farmers and transitional foragers/herders from different ecogeographic zones during this early contact was integral to the formation, rise and expansion of pastoralist groups around 3300 BC.

14th century Yersinia pestis genomes support emergence of pestis secunda within Europe.

Pestis secunda (1356-1366 CE) is the first of a series of plague outbreaks in Europe that followed the Black Death (1346-1353 CE). Collectively this period is called the Second Pandemic. From a genomic perspective, the majority of post-Black Death strains of Yersinia pestis thus far identified in Europe display diversity accumulated over a period of centuries that form a terminal sub-branch of the Y. pestis phylogeny. It has been debated if these strains arose from local evolution of Y. pestis or if the disease was repeatedly reintroduced from an external source. Plague lineages descended from the pestis secunda, however, are thought to have persisted in non-human reservoirs outside Europe, where they eventually gave rise to the Third Pandemic (19th and 20th centuries). Resolution of competing hypotheses on the origins of the many post-Black Death outbreaks has been hindered in part by the low representation of Y. pestis genomes in archaeological specimens, especially for the pestis secunda. Here we report on five individuals from Germany that were infected with lineages of plague associated with the pestis secunda. For the two genomes of high coverage, one groups within the known diversity of genotypes associated with the pestis secunda, while the second carries an ancestral genotype that places it earlier. Through consideration of historical sources that explore first documentation of the pandemic in today's Central Germany, we argue that these data provide robust evidence to support a post-Black Death evolution of the pathogen within Europe rather than a re-introduction from outside. Additionally, we demonstrate retrievability of Y. pestis DNA in post-cranial remains and highlight the importance of hypothesis-free pathogen screening approaches in evaluations of archaeological samples.

Improving the extraction of ancient Yersinia pestis genomes from the dental pulp.

Ancient DNA preserved in the dental pulp offers the opportunity to characterize the genome of some of the deadliest pathogens in human history. However, while DNA capture technologies help, focus sequencing efforts, and therefore, reduce experimental costs, the recovery of ancient pathogen DNA remains challenging. Here, we tracked the kinetics of ancient Yersinia pestis DNA release in solution during a pre-digestion of the dental pulp. We found that most of the ancient Y. pestis DNA is released within 60 min at 37°C in our experimental conditions. We recommend a simple pre-digestion as an economical procedure to obtain extracts enriched in ancient pathogen DNA, as longer digestion times release other types of templates, including host DNA. Combining this procedure with DNA capture, we characterized the genome sequences of 12 ancient Y. pestis bacteria from France dating to the second pandemic outbreaks of the 17th and 18th centuries Common Era.

Ancient Yersinia pestis and Salmonella enterica genomes from Bronze Age Crete.

During the late 3rd millennium BCE, the Eastern Mediterranean and Near East witnessed societal changes in many regions, which are usually explained with a combination of social and climatic factors.1-4 However, recent archaeogenetic research forces us to rethink models regarding the role of infectious diseases in past societal trajectories.5 The plague bacterium Yersinia pestis, which was involved in some of the most destructive historical pandemics,5-8 circulated across Eurasia at least from the onset of the 3rd millennium BCE,9-13 but the challenging preservation of ancient DNA in warmer climates has restricted the identification of Y. pestis from this period to temperate climatic regions. As such, evidence from culturally prominent regions such as the Eastern Mediterranean is currently lacking. Here, we present genetic evidence for the presence of Y. pestis and Salmonella enterica, the causative agent of typhoid/enteric fever, from this period of transformation in Crete, detected at the cave site Hagios Charalambos. We reconstructed one Y. pestis genome that forms part of a now-extinct lineage of Y. pestis strains from the Late Neolithic and Bronze Age that were likely not yet adapted for transmission via fleas. Furthermore, we reconstructed two ancient S. enterica genomes from the Para C lineage, which cluster with contemporary strains that were likely not yet fully host adapted to humans. The occurrence of these two virulent pathogens at the end of the Early Minoan period in Crete emphasizes the necessity to re-introduce infectious diseases as an additional factor possibly contributing to the transformation of early complex societies in the Aegean and beyond.

The source of the Black Death in fourteenth-century central Eurasia.

The origin of the medieval Black Death pandemic (AD 1346-1353) has been a topic of continuous investigation because of the pandemic's extensive demographic impact and long-lasting consequences1,2. Until now, the most debated archaeological evidence potentially associated with the pandemic's initiation derives from cemeteries located near Lake Issyk-Kul of modern-day Kyrgyzstan1,3-9. These sites are thought to have housed victims of a fourteenth-century epidemic as tombstone inscriptions directly dated to 1338-1339 state 'pestilence' as the cause of death for the buried individuals9. Here we report ancient DNA data from seven individuals exhumed from two of these cemeteries, Kara-Djigach and Burana. Our synthesis of archaeological, historical and ancient genomic data shows a clear involvement of the plague bacterium Yersinia pestis in this epidemic event. Two reconstructed ancient Y. pestis genomes represent a single strain and are identified as the most recent common ancestor of a major diversification commonly associated with the pandemic's emergence, here dated to the first half of the fourteenth century. Comparisons with present-day diversity from Y. pestis reservoirs in the extended Tian Shan region support a local emergence of the recovered ancient strain. Through multiple lines of evidence, our data support an early fourteenth-century source of the second plague pandemic in central Eurasia.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

Endometriosis: A Retrospective Analysis on Diagnostic Data in a Cohort of 4,401 Patients.

BACKGROUND/AIM: Endometriosis is a gynecological estrogen-dependent inflammatory disease due to ectopic endometrial tissue and often associated with pelvic pain. Despite its high prevalence, there are still uncertainties about its pathogenesis, diagnosis, and therapy. PATIENTS AND METHODS: This study presents a retrospective study conducted on 4,401 endometriosis patients, 584 of which underwent laparoscopic procedures. The archived data about clinical signs, magnetic resonance imaging (MRI) results, topography of the endometriosis lesions (obtained via laparoscopy) associated diseases, sample analysis and histological findings were analyzed. Next, the statistical associations between the information for each case, provided by these diagnostic tools were determined. RESULTS: MRI is the most sensitive and specific diagnostic system for ovarian lesions, but poor in sensitivity and specificity for deep endometriosis lesions and not indicated for peritoneal lesions which remain the exclusive prerogative of laparoscopy. Clinical signs are essential for diagnosing deep lesions. The Ca125 and Ca19.9 markers have a poor reliability and their negativity in symptomatic patients has no clinical value, while in positive cases it could probably be used as a monitoring parameter. CONCLUSION: The results generated will help provide an accurate picture of the topography and distribution of endometriotic lesions. Correlation analyses between the data generated by the clinical-instrumental examinations and those on the site of the disease identified by laparoscopy, allow to define the predictive value of the clinical-instrumental signs in the diagnosis and localization of endometriotic disease.

Ten millennia of hepatitis B virus evolution.

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic.

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.

Dynamic changes in genomic and social structures in third millennium BCE central Europe.

Europe's prehistory oversaw dynamic and complex interactions of diverse societies, hitherto unexplored at detailed regional scales. Studying 271 human genomes dated ~4900 to 1600 BCE from the European heartland, Bohemia, we reveal unprecedented genetic changes and social processes. Major migrations preceded the arrival of "steppe" ancestry, and at ~2800 BCE, three genetically and culturally differentiated groups coexisted. Corded Ware appeared by 2900 BCE, were initially genetically diverse, did not derive all steppe ancestry from known Yamnaya, and assimilated females of diverse backgrounds. Both Corded Ware and Bell Beaker groups underwent dynamic changes, involving sharp reductions and complete replacements of Y-chromosomal diversity at ~2600 and ~2400 BCE, respectively, the latter accompanied by increased Neolithic-like ancestry. The Bronze Age saw new social organization emerge amid a ≥40% population turnover.

Analysis of Genomic DNA from Medieval Plague Victims Suggests Long-Term Effect of Yersinia pestis on Human Immunity Genes.

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes, we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggest that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe.

A genome sequence from a modern human skull over 45,000 years old from Zlatý kun in Czechia.

Modern humans expanded into Eurasia more than 40,000 years ago following their dispersal out of Africa. These Eurasians carried ~2-3% Neanderthal ancestry in their genomes, originating from admixture with Neanderthals that took place sometime between 50,000 and 60,000 years ago, probably in the Middle East. In Europe, the modern human expansion preceded the disappearance of Neanderthals from the fossil record by 3,000-5,000 years. The genetic makeup of the first Europeans who colonized the continent more than 40,000 years ago remains poorly understood since few specimens have been studied. Here, we analyse a genome generated from the skull of a female individual from Zlatý kun, Czechia. We found that she belonged to a population that appears to have contributed genetically neither to later Europeans nor to Asians. Her genome carries ~3% Neanderthal ancestry, similar to those of other Upper Palaeolithic hunter-gatherers. However, the lengths of the Neanderthal segments are longer than those observed in the currently oldest modern human genome of the ~45,000-year-old Ust'-Ishim individual from Siberia, suggesting that this individual from Zlatý kun is one of the earliest Eurasian inhabitants following the expansion out of Africa.

Ancient genomic time transect from the Central Asian Steppe unravels the history of the Scythians.

The Scythians were a multitude of horse-warrior nomad cultures dwelling in the Eurasian steppe during the first millennium BCE. Because of the lack of first-hand written records, little is known about the origins and relations among the different cultures. To address these questions, we produced genome-wide data for 111 ancient individuals retrieved from 39 archaeological sites from the first millennia BCE and CE across the Central Asian Steppe. We uncovered major admixture events in the Late Bronze Age forming the genetic substratum for two main Iron Age gene-pools emerging around the Altai and the Urals respectively. Their demise was mirrored by new genetic turnovers, linked to the spread of the eastern nomad empires in the first centuries CE. Compared to the high genetic heterogeneity of the past, the homogenization of the present-day Kazakhs gene pool is notable, likely a result of 400 years of strict exogamous social rules.

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.

A treponemal genome from an historic plague victim supports a recent emergence of yaws and its presence in 15th century Europe.

Developments in techniques for identification of pathogen DNA in archaeological samples can expand our resolution of disease detection. Our application of a non-targeted molecular screening tool for the parallel detection of pathogens in historical plague victims from post-medieval Lithuania revealed the presence of more than one active disease in one individual. In addition to Yersinia pestis, we detected and genomically characterized a septic infection of Treponema pallidum pertenue, a subtype of the treponemal disease family recognised as the cause of the tropical disease yaws. Our finding in northern Europe of a disease that is currently restricted to equatorial regions is interpreted within an historical framework of intercontinental trade and potential disease movements. Through this we offer an alternative hypothesis for the history and evolution of the treponemal diseases, and posit that yaws be considered an important contributor to the sudden epidemic of late 15th century Europe that is widely ascribed to syphilis.

Paleolithic to Bronze Age Siberians Reveal Connections with First Americans and across Eurasia.

Modern humans have inhabited the Lake Baikal region since the Upper Paleolithic, though the precise history of its peoples over this long time span is still largely unknown. Here, we report genome-wide data from 19 Upper Paleolithic to Early Bronze Age individuals from this Siberian region. An Upper Paleolithic genome shows a direct link with the First Americans by sharing the admixed ancestry that gave rise to all non-Arctic Native Americans. We also demonstrate the formation of Early Neolithic and Bronze Age Baikal populations as the result of prolonged admixture throughout the eighth to sixth millennium BP. Moreover, we detect genetic interactions with western Eurasian steppe populations and reconstruct Yersinia pestis genomes from two Early Bronze Age individuals without western Eurasian ancestry. Overall, our study demonstrates the most deeply divergent connection between Upper Paleolithic Siberians and the First Americans and reveals human and pathogen mobility across Eurasia during the Bronze Age.

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.

Mannan detecting C-type lectin receptor probes recognise immune epitopes with diverse chemical, spatial and phylogenetic heterogeneity in fungal cell walls.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the ß-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.

Phylogeography of the second plague pandemic revealed through analysis of historical Yersinia pestis genomes.

The second plague pandemic, caused by Yersinia pestis, devastated Europe and the nearby regions between the 14th and 18th centuries AD. Here we analyse human remains from ten European archaeological sites spanning this period and reconstruct 34 ancient Y. pestis genomes. Our data support an initial entry of the bacterium through eastern Europe, the absence of genetic diversity during the Black Death, and low within-outbreak diversity thereafter. Analysis of post-Black Death genomes shows the diversification of a Y. pestis lineage into multiple genetically distinct clades that may have given rise to more than one disease reservoir in, or close to, Europe. In addition, we show the loss of a genomic region that includes virulence-related genes in strains associated with late stages of the pandemic. The deletion was also identified in genomes connected with the first plague pandemic (541-750 AD), suggesting a comparable evolutionary trajectory of Y. pestis during both events.

Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens.

The last century has witnessed progress in the study of ancient infectious disease from purely medical descriptions of past ailments to dynamic interpretations of past population health that draw upon multiple perspectives. The recent adoption of high-throughput DNA sequencing has led to an expanded understanding of pathogen presence, evolution, and ecology across the globe. This genomic revolution has led to the identification of disease-causing microbes in both expected and unexpected contexts, while also providing for the genomic characterization of ancient pathogens previously believed to be unattainable by available methods. In this review we explore the development of DNA-based ancient pathogen research, the specialized methods and tools that have emerged to authenticate and explore infectious disease of the past, and the unique challenges that persist in molecular paleopathology. We offer guidelines to mitigate the impact of these challenges, which will allow for more reliable interpretations of data in this rapidly evolving field of investigation.