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.

Population Genetics and Signatures of Selection in Early Neolithic European Farmers.

Human expansion in the course of the Neolithic transition in western Eurasia has been one of the major topics in ancient DNA research in the last 10 years. Multiple studies have shown that the spread of agriculture and animal husbandry from the Near East across Europe was accompanied by large-scale human expansions. Moreover, changes in subsistence and migration associated with the Neolithic transition have been hypothesized to involve genetic adaptation. Here, we present high quality genome-wide data from the Linear Pottery Culture site Derenburg-Meerenstieg II (DER) (N = 32 individuals) in Central Germany. Population genetic analyses show that the DER individuals carried predominantly Anatolian Neolithic-like ancestry and a very limited degree of local hunter-gatherer admixture, similar to other early European farmers. Increasing the Linear Pottery culture cohort size to ∼100 individuals allowed us to perform various frequency- and haplotype-based analyses to investigate signatures of selection associated with changes following the adoption of the Neolithic lifestyle. In addition, we developed a new method called Admixture-informed Maximum-likelihood Estimation for Selection Scans that allowed us test for selection signatures in an admixture-aware fashion. Focusing on the intersection of results from these selection scans, we identified various loci associated with immune function (JAK1, HLA-DQB1) and metabolism (LMF1, LEPR, SORBS1), as well as skin color (SLC24A5, CD82) and folate synthesis (MTHFR, NBPF3). Our findings shed light on the evolutionary pressures, such as infectious disease and changing diet, that were faced by the early farmers of Western Eurasia.

Massive migration from the steppe was a source for Indo-European languages in Europe.

We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost 400,000 polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies and to obtain new insights about the past. We show that the populations of Western and Far Eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ∼8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ∼24,000-year-old Siberian. By ∼6,000-5,000 years ago, farmers throughout much of Europe had more hunter-gatherer ancestry than their predecessors, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but also from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ∼4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ∼75% of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ∼3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for a steppe origin of at least some of the Indo-European languages of Europe.

Pottery spilled the beans: Patterns in the processing and consumption of dietary lipids in Central Germany from the Early Neolithic to the Bronze Age.

The need to better understand economic change and the social uses of long-ago established pottery types to prepare and consume food has led to the study of 124 distinct ceramic vessels from 17 settlement and funerary sites in Central Germany (present day Saxony-Anhalt). These, dated from the Early Neolithic (from 5450 cal. BCE onwards) to the Late Bronze Age (1300-750 cal. BCE; youngest sample ca. 1000 BCE), include vessels from the Linear Pottery (LBK), Schiepzig/Schöningen groups (SCHIP), Baalberge (BAC), Corded Ware (CWC), Bell Beaker (BBC), and Únetice (UC) archaeological cultures. Organic residue analyses performed on this assemblage determined the presence of vessel contents surviving as lipid residues in 109 cases. These were studied in relation to the changing use of settlement and funerary pottery types and, in the case of burials, to the funerary contexts in which the vessels had been placed. The obtained results confirmed a marked increase in the consumption of dairy products linked to innovations in pottery types (e.g., small cups) during the Funnel Beaker related Baalberge Culture of the 4th millennium BCE. Although the intensive use of dairy products may have continued into the 3rd millennium BCE, especially amongst Bell Beaker populations, Corded Ware vessels found in funerary contexts suggest an increase in the importance of non-ruminant products, which may be linked to the production of specific vessel shapes and decoration. In the Early Bronze Age circum-Harz Únetice group (ca. 2200-1550 BCE), which saw the emergence of a highly hierarchical society, a greater variety of animal and plant derived products was detected in a much more standardised but, surprisingly, more multifunctional pottery assemblage. This long-term study of lipid residues from a concise region in Central Europe thus reveals the complex relationships that prehistoric populations established between food resources and the main means to prepare, store, and consume them.

Rib lesions in skeletons from early neolithic sites in Central Germany: on the trail of tuberculosis at the onset of agriculture.

As an infectious disease, tuberculosis (TB) is one of the major causes of death worldwide. Paleopathological and paleomicrobiological studies indicate a long standing association of the causative agent Mycobacterium tuberculosis and its human host. Since the occurrence and the epidemic spread of this pathogen seem to be closely linked to social and biological factors, it is of particular interest to understand better the role of TB during periods of social and nutritional change such as the Neolithic. In this study, 118 individuals from three sites in Saxony-Anhalt (Germany) dating to the Linear Pottery Culture (5400-4800 BC) were examined macroscopically to identify TB related bone lesions. In two individuals, Pott's disease was detected. In addition, periosteal reactions of varying degrees and frequency were observed mainly along the neck of the ribs in 6.5% (2/31) of subadults and 35.1% (20/57) of adults, with one site standing out markedly. Rib lesions, however, are not specific indicators of TB as they can also be caused by other diseases; so additional investigations were undertaken using histology and micro-CT scans to say more about the disease process. Supplementary molecular analyses indicate the presence of pathogens belonging to the Mycobacterium tuberculosis complex in individuals of all sites. Furthermore, we discuss the occurrence and spread of TB during the Neolithic with regard to nutritional aspects and possible risks of infection. The data presented provide important insights into the health status of Early Neolithic populations in Central Germany.

Dental pulp calcifications in prehistoric and historical skeletal remains.

BACKGROUND: The prevalence of hard tissue formations in the dental pulp varies considerably. Beside ageing processes and irritations of the dental pulp, etiological associations with cardiovascular disease and dietary habits have been discussed, which are of particular research interest. The aim of this pilot study is to provide new insights on structural and etiological factors involved in the development of pulp calcifications by investigating skeletal remains from different (pre)historic periods. METHODS: The jaws of 46 skeletons excavated in central Germany, were examined for the presence of pulp stones using digital volume tomography (DVT). A total of 1122 teeth were examined with all tooth types considered. To obtain information about the three-dimensional structure of pulp calcifications, micro-CT images were taken. Thin sections of three molars were histologically analysed. Potential dietary effects were studied by analysing stable isotope ratios of nitrogen (δ15N) in bone samples collected from each individual. RESULTS: The analysis indicates that pulp stones affect molars in particular and increase slightly with age and dental wear. The micro-CT scans and the histological analysis show that the structures are much more complex than presumed on the basis of DVT imaging. Individuals with lower δ15N-isotope values and thus with a potentially lower proportion of animal protein in their diet appear to be less affected by pulp stones. CONCLUSION: When comparing between archaeological and recent data, DVT analysis provides qualitatively comparable results. Micro-CT and histological images illustrate the excellent preservation of pulp calcifications and their complex structure. Differences in prevalence rates and δ15N-isotope values of Neolithic and historical individuals support the assumption that dietary habits and living conditions could have an influence on the development of pulp calcifications. Due to the small sample size these results require further validation.

Using Y-chromosome capture enrichment to resolve haplogroup H2 shows new evidence for a two-path Neolithic expansion to Western Europe.

Uniparentally-inherited markers on mitochondrial DNA (mtDNA) and the non-recombining regions of the Y chromosome (NRY), have been used for the past 30 years to investigate the history of humans from a maternal and paternal perspective. Researchers have preferred mtDNA due to its abundance in the cells, and comparatively high substitution rate. Conversely, the NRY is less susceptible to back mutations and saturation, and is potentially more informative than mtDNA owing to its longer sequence length. However, due to comparatively poor NRY coverage via shotgun sequencing, and the relatively low and biased representation of Y-chromosome variants on capture assays such as the 1240 k, ancient DNA studies often fail to utilize the unique perspective that the NRY can yield. Here we introduce a new DNA enrichment assay, coined YMCA (Y-mappable capture assay), that targets the "mappable" regions of the NRY. We show that compared to low-coverage shotgun sequencing and 1240 k capture, YMCA significantly improves the mean coverage and number of sites covered on the NRY, increasing the number of Y-haplogroup informative SNPs, and allowing for the identification of previously undiscovered variants. To illustrate the power of YMCA, we show that the analysis of ancient Y-chromosome lineages can help to resolve Y-chromosomal haplogroups. As a case study, we focus on H2, a haplogroup associated with a critical event in European human history: the Neolithic transition. By disentangling the evolutionary history of this haplogroup, we further elucidate the two separate paths by which early farmers expanded from Anatolia and the Near East to western Europe.

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.

A systematic investigation of human DNA preservation in medieval skeletons.

Ancient DNA (aDNA) analyses necessitate the destructive sampling of archaeological material. Currently, the cochlea, part of the osseous inner ear located inside the petrous pyramid, is the most sought after skeletal element for molecular analyses of ancient humans as it has been shown to yield high amounts of endogenous DNA. However, destructive sampling of the petrous pyramid may not always be possible, particularly in cases where preservation of skeletal morphology is of top priority. To investigate alternatives, we present a survey of human aDNA preservation for each of ten skeletal elements in a skeletal collection from Medieval Germany. Through comparison of human DNA content and quality we confirm best performance of the petrous pyramid and identify seven additional sampling locations across four skeletal elements that yield adequate aDNA for most applications in human palaeogenetics. Our study provides a better perspective on DNA preservation across the human skeleton and takes a further step toward the more responsible use of ancient materials in human aDNA studies.

Author Correction: Spotted phenotypes in horses lost attractiveness in the Middle Ages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ancient genome-wide DNA from France highlights the complexity of interactions between Mesolithic hunter-gatherers and Neolithic farmers.

Starting from 12,000 years ago in the Middle East, the Neolithic lifestyle spread across Europe via separate continental and Mediterranean routes. Genomes from early European farmers have shown a clear Near Eastern/Anatolian genetic affinity with limited contribution from hunter-gatherers. However, no genomic data are available from modern-day France, where both routes converged, as evidenced by a mosaic cultural pattern. Here, we present genome-wide data from 101 individuals from 12 sites covering today's France and Germany from the Mesolithic (N = 3) to the Neolithic (N = 98) (7000-3000 BCE). Using the genetic substructure observed in European hunter-gatherers, we characterize diverse patterns of admixture in different regions, consistent with both routes of expansion. Early western European farmers show a higher proportion of distinctly western hunter-gatherer ancestry compared to central/southeastern farmers. Our data highlight the complexity of the biological interactions during the Neolithic expansion by revealing major regional variations.

Decline of genetic diversity in ancient domestic stallions in Europe.

Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.

4000 years of human dietary evolution in central Germany, from the first farmers to the first elites.

Investigation of human diet during the Neolithic has often been limited to a few archaeological cultures or single sites. In order to provide insight into the development of human food consumption and husbandry strategies, our study explores bone collagen carbon and nitrogen isotope data from 466 human and 105 faunal individuals from 26 sites in central Germany. It is the most extensive data set to date from an enclosed geographic microregion, covering 4,000 years of agricultural history from the Early Neolithic to the Early Bronze Age. The animal data show that a variety of pastures and dietary resources were explored, but that these changed remarkably little over time. In the human δ15N however we found a significant increase with time across the different archaeological cultures. This trend could be observed in all time periods and archaeological cultures (Bell Beaker phenomenon excluded), even on continuously populated sites. Since there was no such trend in faunal isotope values, we were able largely to exclude manuring as the cause of this effect. Based on the rich interdisciplinary data from this region and archaeological period we can argue that meat consumption increased with the increasing duration of farming subsistence. In δ13C, we could not observe any clear increasing or decreasing trends during the archaeological time periods, either for humans or for animals, which would have suggested significant changes in the environment and landscape use. We discovered sex-related dietary differences, with males of all archaeological periods having higher δ15N values than females, and an age-related increasing consumption of animal protein. An initial decrease of δ15N-values at the age of 1-2 years reveals partial weaning, while complete weaning took place at the age of 3-4 years.

Neolithic and medieval virus genomes reveal complex evolution of hepatitis B.

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.

The face of war: Trauma analysis of a mass grave from the Battle of Lützen (1632).

Contemporary accounts of battles are often incomplete or even erroneous because they reflect the-often biased-viewpoints of the authors. Battlefield archaeology faces the task of compiling an historical analysis of a battle and of gathering all the available facts. Besides cultural historical evidence and artefacts, the human remains of those who have fallen in battle also provide invaluable information. In studying mass graves from a military context, the injury types and patterns are significant. They allow us to reconstruct the circumstances surrounding the soldiers' deaths and provide information on the hostilities that occurred on the battlefield. One such mass grave was discovered in 2011 at Lützen, Saxony-Anhalt (Germany). Based on its geographical location and on the results obtained from archaeological examinations carried out in the area, the grave could be dated to the Thirty Years War (1618-1648). Further archaeological research confirmed that the dead had been soldiers from the Battle of Lützen (1632). The mass grave was block-lifted and then comprehensively examined at the State Museum of Prehistory in Halle (Saale). As well as osteological examinations to determine age, sex, height, state of health, i.e. diseases or injuries, imaging methods were also employed and histological and isotopic analyses carried out. The focus of this study was on the injuries sustained by the soldiers both prior to and during the battle. The results revealed that the 47 deceased had been between the ages of 15 and 50 when they died. Numerous healed injuries showed that the men had often been involved in violent encounters. Approximately three in every four soldiers had injuries that could have been fatal. Wounds inflicted by handguns, particularly to the skull, were predominant. The integrative analysis of the archaeological and anthropological data allowed us to conclude that the majority had been killed during a cavalry attack.

The maternal genetic make-up of the Iberian Peninsula between the Neolithic and the Early Bronze Age.

Agriculture first reached the Iberian Peninsula around 5700 BCE. However, little is known about the genetic structure and changes of prehistoric populations in different geographic areas of Iberia. In our study, we focus on the maternal genetic makeup of the Neolithic (~ 5500-3000 BCE), Chalcolithic (~ 3000-2200 BCE) and Early Bronze Age (~ 2200-1500 BCE). We report ancient mitochondrial DNA results of 213 individuals (151 HVS-I sequences) from the northeast, central, southeast and southwest regions and thus on the largest archaeogenetic dataset from the Peninsula to date. Similar to other parts of Europe, we observe a discontinuity between hunter-gatherers and the first farmers of the Neolithic. During the subsequent periods, we detect regional continuity of Early Neolithic lineages across Iberia, however the genetic contribution of hunter-gatherers is generally higher than in other parts of Europe and varies regionally. In contrast to ancient DNA findings from Central Europe, we do not observe a major turnover in the mtDNA record of the Iberian Late Chalcolithic and Early Bronze Age, suggesting that the population history of the Iberian Peninsula is distinct in character.

Holes in teeth - Dental caries in Neolithic and Early Bronze Age populations in Central Germany.

This study provides diachronic insight into the epidemiology of carious defects in teeth of Neolithic and Early Bronze Age populations in Central Germany over a period of 4000 years. The data were retrieved from skeletal remains uncovered at 21 sites throughout the Middle Elbe-Saale region (MES), comprising a total of 494 individuals with preserved teeth. The data generated were examined for age- and sex-related differences in order to gain information about the dietary habits and socio-economic structures of the period with the goal of identifying potential diachronic changes. The results indicated that dietary habits changed over the course of the Neolithic period: the prevalence of caries significantly decreased between the Early and Late Neolithic. The adults from the Early Neolithic sample, particularly those from the LBK bore the highest rate of caries. This highlights the essential importance of cereals in the diet of the early farmers in the Middle Elbe-Saale region. As time went on, meat and dairy products became more and more important, which had a positive impact on dental health. The data also show sex-specific differences: women were more often affected by caries than men and female jaws also generally exhibited greater numbers of carious teeth than their male counterparts. Dental health is a reflection of both biological factors and of economic and sociocultural structures.

Spotted phenotypes in horses lost attractiveness in the Middle Ages.

Horses have been valued for their diversity of coat colour since prehistoric times; this is especially the case since their domestication in the Caspian steppe in ~3,500 BC. Although we can assume that human preferences were not constant, we have only anecdotal information about how domestic horses were influenced by humans. Our results from genotype analyses show a significant increase in spotted coats in early domestic horses (Copper Age to Iron Age). In contrast, medieval horses carried significantly fewer alleles for these phenotypes, whereas solid phenotypes (i.e., chestnut) became dominant. This shift may have been supported because of (i) pleiotropic disadvantages, (ii) a reduced need to separate domestic horses from their wild counterparts, (iii) a lower religious prestige, or (iv) novel developments in weaponry. These scenarios may have acted alone or in combination. However, the dominance of chestnut is a remarkable feature of the medieval horse population.

Ancient DNA reveals key stages in the formation of central European mitochondrial genetic diversity.

The processes that shaped modern European mitochondrial DNA (mtDNA) variation remain unclear. The initial peopling by Palaeolithic hunter-gatherers ~42,000 years ago and the immigration of Neolithic farmers into Europe ~8000 years ago appear to have played important roles but do not explain present-day mtDNA diversity. We generated mtDNA profiles of 364 individuals from prehistoric cultures in Central Europe to perform a chronological study, spanning the Early Neolithic to the Early Bronze Age (5500 to 1550 calibrated years before the common era). We used this transect through time to identify four marked shifts in genetic composition during the Neolithic period, revealing a key role for Late Neolithic cultures in shaping modern Central European genetic diversity.

Neolithic mitochondrial haplogroup H genomes and the genetic origins of Europeans.

Haplogroup H dominates present-day Western European mitochondrial DNA variability (>40%), yet was less common (~19%) among Early Neolithic farmers (~5450 BC) and virtually absent in Mesolithic hunter-gatherers. Here we investigate this major component of the maternal population history of modern Europeans and sequence 39 complete haplogroup H mitochondrial genomes from ancient human remains. We then compare this 'real-time' genetic data with cultural changes taking place between the Early Neolithic (~5450 BC) and Bronze Age (~2200 BC) in Central Europe. Our results reveal that the current diversity and distribution of haplogroup H were largely established by the Mid Neolithic (~4000 BC), but with substantial genetic contributions from subsequent pan-European cultures such as the Bell Beakers expanding out of Iberia in the Late Neolithic (~2800 BC). Dated haplogroup H genomes allow us to reconstruct the recent evolutionary history of haplogroup H and reveal a mutation rate 45% higher than current estimates for human mitochondria.