The indigenous populations of inner Eurasia, a huge geographic region covering the central Eurasian steppe and the northern Eurasian taiga and tundra, harbor tremendous diversity in their genes, cultures and languages. In this study, we report novel genome-wide data for 763 individuals from Armenia, Georgia, Kazakhstan, Moldova, Mongolia, Russia, Tajikistan, Ukraine, and Uzbekistan. We furthermore report additional damage-reduced genome-wide data of two previously published individuals from the Eneolithic Botai culture in Kazakhstan (~5,400 BP). We find that present-day inner Eurasian populations are structured into three distinct admixture clines stretching between various western and eastern Eurasian ancestries, mirroring geography. The Botai and more recent ancient genomes from Siberia show a decrease in contribution from so-called “ancient North Eurasian” ancestry over time, detectable only in the northern-most “forest-tundra” cline. The intermediate “steppe-forest” cline descends from the Late Bronze Age steppe ancestries, while the “southern steppe” cline further to the South shows a strong West/South Asian influence. Ancient genomes suggest a northward spread of the southern steppe cline in Central Asia during the first millennium BC. Finally, the genetic structure of Caucasus populations highlights a role of the Caucasus Mountains as a barrier to gene flow and suggests a post-Neolithic gene flow into North Caucasus populations from the steppe.

The two living species of bison (European and American) are among the few terrestrial megafauna to have survived the late Pleistocene extinctions. Despite the extensive bovid fossil record in Eurasia, the evolutionary history of the European bison (or wisent, Bison bonasus) before the Holocene (<11.7 thousand years ago (kya)) remains a mystery. We use complete ancient mitochondrial genomes and genome-wide nuclear DNA surveys to reveal that the wisent is the product of hybridization between the extinct steppe bison (Bison priscus) and ancestors of modern cattle (aurochs, Bos primigenius) before 120 kya, and contains up to 10% aurochs genomic ancestry. Although undetected within the fossil record, ancestors of the wisent have alternated ecological dominance with steppe bison in association with major environmental shifts since at least 55 kya. Early cave artists recorded distinct morphological forms consistent with these replacement events, around the Last Glacial Maximum (LGM, ∼21–18 kya).

The hepatitis B virus (HBV) is one of the most widespread human pathogens known today, yet its origin and evolutionary history are still unclear and controversial. Here, we report the analysis of three ancient HBV genomes recovered from human skeletons found at three different archaeological sites in Germany. We reconstructed two Neolithic and one medieval HBV genome by de novo assembly from shotgun DNA sequencing data. Additionally, we observed HBV-specific peptides using paleo-proteomics. Our results demonstrated that HBV has circulated in the European population for at least 7000 years. The Neolithic HBV genomes show a high genomic similarity to each other. In a phylogenetic network, they do not group with any human-associated HBV genome and are most closely related to those infecting African non-human primates. The ancient viruses appear to represent distinct lineages that have no close relatives today and possibly went extinct. Our results reveal the great potential of ancient DNA from human skeletons in order to study the long-time evolution of blood borne viruses.

Highlights d Genomic and isotopes data suggest an African origin for the three individuals d One 14X Treponema pallidum sub. pertenue genome was recovered d One 1,500X hepatitis B virus genome was recovered d Both pathogen genomes cluster together with present day pathogens from Africa

34The hepatitis B virus (HBV) is one of the most widespread human pathogens known today, 35 yet its origin and evolutionary history are still unclear and controversial. Here, we report the 36 analysis of three ancient HBV genomes recovered from human skeletons found at three 37 different archaeological sites in Germany. We reconstructed two Neolithic and one medieval 38 HBV genomes by de novo assembly from shotgun DNA sequencing data. Additionally, we 39 observed HBV-specific peptides using paleo-proteomics. Our results show that HBV 40 circulates in the European population for at least 7000 years. The Neolithic HBV genomes 41show a high genomic similarity to each other. In a phylogenetic network, they do not group 42 with any human-associated HBV genome and are most closely related to those infecting 43African non-human primates. These ancient virus forms appear to represent distinct lineages 44 that have no close relatives today and went possibly extinct. Our results reveal the great 45 potential of ancient DNA from human skeletons in order to study the long-time evolution of 46 blood borne viruses. 47 48 49

Sub-fossilised remains may still contain highly degraded ancient DNA (aDNA) useful for palaeogenetic investigations. Whether X-ray computed [micro-] tomography ([μ]CT) imaging of these fossils may further damage aDNA remains debated. Although the effect of X-ray on DNA in living organisms is well documented, its impact on aDNA molecules is unexplored. Here we investigate the effects of synchrotron X-ray irradiation on aDNA from Pleistocene bones. A clear correlation appears between decreasing aDNA quantities and accumulating X-ray dose-levels above 2000 Gray (Gy). We further find that strong X-ray irradiation reduces the amount of nucleotide misincorporations at the aDNA molecule ends. No representative effect can be detected for doses below 200 Gy. Dosimetry shows that conventional μCT usually does not reach the risky dose level, while classical synchrotron imaging can degrade aDNA significantly. Optimised synchrotron protocols and simple rules introduced here are sufficient to ensure that fossils can be scanned without impairing future aDNA studies.

Ancient genomic studies have identified Yersinia pestis (Y. pestis) as the causative agent of the second plague pandemic (fourteenth-eighteenth century) that started with the Black Death (1,347-1,353). Most of the Y. pestis strains investigated from this pandemic have been isolated from western Europe, and not much is known about the diversity and microevolution of this bacterium in eastern European countries. In this study, we investigated human remains excavated from two cemeteries in Riga (Latvia). Historical evidence suggests that the burials were a consequence of plague outbreaks during the seventeenth century. DNA was extracted from teeth of 16 individuals and subjected to shotgun sequencing. Analysis of the metagenomic data revealed the presence of Y. pestis sequences in four remains, confirming that the buried individuals were victims of plague. In two samples, Y. pestis DNA coverage was sufficient for genome reconstruction. Subsequent phylogenetic analysis showed that the Riga strains fell within the diversity of the already known post-Black Death genomes. Interestingly, the two Latvian isolates did not cluster together. Moreover, we detected a drop in coverage of the pPCP1 plasmid region containing the pla gene. Further analysis indicated the presence of two pPCP1 plasmids, one with and one without the pla gene region, and only one bacterial chromosome, indicating that the same bacterium carried two distinct pPCP1 plasmids. In addition, we found the same pattern in the majority of previously published post-Black Death strains, but not in the Black Death strains. The pla gene is an important virulence factor for the infection of and transmission in humans. thus, the spread of pla-depleted strains may, among other causes, have contributed to the disappearance of the second plague pandemic in eighteenth century Europe. Yersinia pestis (Y. pestis) is the causative agent of plague that evolved from a relatively benign pathogen-Yersinia pseudotuberculosis-thousands of years ago 1-4. Whilst plague is a zoonotic disease with rodents being the primary hosts of the pathogen 5-7 , it has also afflicted humans for at least 5,000 years 1. In the last two millennia, Y. pestis was responsible for three major epidemics, of which the second was the most infamous. This pandemic began with the Black Death in the fourteenth century (1,346-1,353). It is thought to have originated in Asia from where it quickly spread to and across the European continent killing approximately 30-50% of the population within a few years 8,9. The second pandemic persisted for over 400 years 10 causing, among others, the Great Plague of London (1,665-1,666) and Marseille (1,720-1,722) 11. In contrast to western Europe, the first major outbreaks of plague in the northeastern part of the continent took place mainly in the post-Black Death period (after 1,353),

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 suggests 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.