Between Raetia Secunda and the dutchy of Bavaria: Exploring patterns of human movement and diet.

During the transition from Late Antiquity to the Middle Ages, the Roman Empire dissolved in the West and medieval empires were founded. There has been much discussion about the role that migration played in this transition. This is especially true for the formation of the Baiuvariian tribe and the founding of this tribal dukedom, which took place from the 5th to the 6th century in what is now Southern Bavaria (Germany). In this study, we aimed to determine the extent of immigration during the beginning of this transformation and to shed further light on its character. To achieve this goal, we analyzed stable isotope values of strontium, carbon, and nitrogen from the teeth and bones of over 150 human remains from Southern Germany, dating from around 500 AD. This group of individuals included women with cranial modifications (ACD) which can be found sporadically in the burial grounds of this period. Our results showed an above-average migration rate for both men and women in the second half of the 5th century. They also indicate that a foreign background may also be assumed for the women with ACD. The demonstrably different origins of the immigrants from isotopically diverse regions, and the identification of local differences in detectable migration rate, as well as indication for different timing of residential changes, highlight the complexity of immigration processes and the need for more studies at the regional level.

On the premises of mixing models to define local bioavailable 87Sr/86Sr ranges in archaeological contexts.

In archaeological mobility studies, non-local humans and animals can be identified by means of stable strontium isotope analysis. However, defining the range of local 87Sr/86Sr ratios is prerequisite. To achieve this goal, proxy-based mixing models have recently been proposed using 87Sr/86Sr ratios measured in modern local vegetation, water and soil samples. Our study complements earlier efforts by introducing archaeological animal bones as an additional proxy. We then evaluate the different modelling approaches by contrasting proxy-results generated for the county of Erding (Upper Bavaria, Germany) with a comprehensive set of strontium measurements obtained from tooth enamel of late antique and early medieval human individuals (n = 49) from the same micro-region. We conclude that current mixing models based on environmental proxies clearly underestimate the locally bioavailable 87Sr/86Sr ratios due to the limited sample size of modern environmental specimens and a suit of imponderables inherent to efforts modelling complex geobiological processes. In sum, currently available mixing models are deemed inadequate and can therefore not be recommended.

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.

Kinship-based social inequality in Bronze Age Europe.

Revealing and understanding the mechanisms behind social inequality in prehistoric societies is a major challenge. By combining genome-wide data, isotopic evidence, and anthropological and archaeological data, we have gone beyond the dominating supraregional approaches in archaeogenetics to shed light on the complexity of social status, inheritance rules, and mobility during the Bronze Age. We applied a deep microregional approach and analyzed genome-wide data of 104 human individuals deriving from farmstead-related cemeteries from the Late Neolithic to the Middle Bronze Age in southern Germany. Our results reveal individual households, lasting several generations, that consisted of a high-status core family and unrelated low-status individuals; a social organization accompanied by patrilocality and female exogamy; and the stability of this system over 700 years.

Ancient Yersinia pestis genomes from across Western Europe reveal early diversification during the First Pandemic (541-750).

The first historically documented pandemic caused by Yersinia pestis began as the Justinianic Plague in 541 within the Roman Empire and continued as the so-called First Pandemic until 750. Although paleogenomic studies have previously identified the causative agent as Y. pestis, little is known about the bacterium's spread, diversity, and genetic history over the course of the pandemic. To elucidate the microevolution of the bacterium during this time period, we screened human remains from 21 sites in Austria, Britain, Germany, France, and Spain for Y. pestis DNA and reconstructed eight genomes. We present a methodological approach assessing single-nucleotide polymorphisms (SNPs) in ancient bacterial genomes, facilitating qualitative analyses of low coverage genomes from a metagenomic background. Phylogenetic analysis on the eight reconstructed genomes reveals the existence of previously undocumented Y. pestis diversity during the sixth to eighth centuries, and provides evidence for the presence of multiple distinct Y. pestis strains in Europe. We offer genetic evidence for the presence of the Justinianic Plague in the British Isles, previously only hypothesized from ambiguous documentary accounts, as well as the parallel occurrence of multiple derived strains in central and southern France, Spain, and southern Germany. Four of the reported strains form a polytomy similar to others seen across the Y. pestis phylogeny, associated with the Second and Third Pandemics. We identified a deletion of a 45-kb genomic region in the most recent First Pandemic strains affecting two virulence factors, intriguingly overlapping with a deletion found in 17th- to 18th-century genomes of the Second Pandemic.

Population genomic analysis of elongated skulls reveals extensive female-biased immigration in Early Medieval Bavaria.

Modern European genetic structure demonstrates strong correlations with geography, while genetic analysis of prehistoric humans has indicated at least two major waves of immigration from outside the continent during periods of cultural change. However, population-level genome data that could shed light on the demographic processes occurring during the intervening periods have been absent. Therefore, we generated genomic data from 41 individuals dating mostly to the late 5th/early 6th century AD from present-day Bavaria in southern Germany, including 11 whole genomes (mean depth 5.56×). In addition we developed a capture array to sequence neutral regions spanning a total of 5 Mb and 486 functional polymorphic sites to high depth (mean 72×) in all individuals. Our data indicate that while men generally had ancestry that closely resembles modern northern and central Europeans, women exhibit a very high genetic heterogeneity; this includes signals of genetic ancestry ranging from western Europe to East Asia. Particularly striking are women with artificial skull deformations; the analysis of their collective genetic ancestry suggests an origin in southeastern Europe. In addition, functional variants indicate that they also differed in visible characteristics. This example of female-biased migration indicates that complex demographic processes during the Early Medieval period may have contributed in an unexpected way to shape the modern European genetic landscape. Examination of the panel of functional loci also revealed that many alleles associated with recent positive selection were already at modern-like frequencies in European populations ∼1,500 years ago.

Early medieval stone-lined graves in Southern Germany: analysis of an emerging noble class.

OBJECTIVES: Stone-lined graves, which first appear in Bavarian territory during the 7th century AD, are assumed to be tombs of emerging nobility. While previous research on stone-lined grave goods supports their status as elite burials, an important factor defining nobility-kinship-has not been examined so far. MATERIALS AND METHODS: Morphological analysis of the commingled skeletal remains of 21 individuals from three archaeological sites was carried out. Radiocarbon dating was conducted on these individuals to gain information on usage intervals of these graves. To test whether stone-lined graves can be considered family graves, analyses of mitochondrial HVR I, Y-chromosomal and autosomal STRs were carried out. RESULTS: Morphological examination revealed a surplus of males buried in stone-lined graves and radiocarbon dating points to usage of the tombs for several generations. According to aDNA analysis, kinship can be assumed both between and within stone-lined graves. DISCUSSION: Taken together, these results hint at burials of family members with high social status being inhumed at the same site, in some cases even the same grave, for several generations. They also suggest, for the first time, that an early medieval linear cemetery was structured according to biological kinship.

A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim.

The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany.

Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century.

Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/µl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.

United in death-related by blood? Genetic and archeometric analyses of skeletal remains from the neolithic earthwork Bruchsal-Aue.

OBJECTIVES: Straight next to a segment of the outer ditch of the Late Neolithic Michelsberg Culture earthwork of Bruchsal-Aue in SW-Germany (ca. 4250-3650 calBC), a multiple burial of eight individuals (two male adults and six children) plus a subsequent child burial was excavated. In this study, we applied a multidisciplinary approach to elucidate interpersonal relationships and life histories within this collective. MATERIALS AND METHODS: To determine the identity of this collective, we performed aDNA analyses in addition to osteological examination using HVR I plus Y-chromosomal and autosomal STR profiling to find evidence for kinship relations. Strontium isotopic analyses were used to reconsider migrational behavior. To find evidence for a specific social affiliation, the individual diet was reconstructed by performing nitrogen and carbon isotopic analyses. Furthermore, radiocarbon-dating was carried out to integrate the burial context into an absolute timeframe. Two nearby single burials were included in the analyses for comparison. RESULTS: Because of a shared HVR I haplotype, three pairs of individuals were most likely linked by kinship, and statistical testing on autosomal STR profiles shows a high probability for the pair of two men being brothers. Although it cannot be excluded, isotopic data gave no clear proof for migration. A rather poor health status is indicated by skeletal stress markers even though the isotope data attest to a diet rich in meat and fish. DISCUSSION: Although clear kinship relations among the infants remain unconfirmed, a relationship could also be indicated by the positioning of the bodies in the burial pit. Whereas a common cause of death might have been the presupposition for their special treatment, interpersonal relationships were likely the decisive factor for the multiple burial.

Yersinia pestis and the plague of Justinian 541-543 AD: a genomic analysis.

BACKGROUND: Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic. METHODS: Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. FINDINGS: Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics. INTERPRETATION: We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations. FUNDING: McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.

Research potential and limitations of trace analyses of cremated remains.

Human cremation is a common funeral practice all over the world and will presumably become an even more popular choice for interment in the future. Mainly for purposes of identification, there is presently a growing need to perform trace analyses such as DNA or stable isotope analyses on human remains after cremation in order to clarify pending questions in civil or criminal court cases. The aim of this study was to experimentally test the potential and limitations of DNA and stable isotope analyses when conducted on cremated remains. For this purpose, tibiae from modern cattle were experimentally cremated by incinerating the bones in increments of 100°C until a maximum of 1000°C was reached. In addition, cremated human remains were collected from a modern crematory. The samples were investigated to determine level of DNA preservation and stable isotope values (C and N in collagen, C and O in the structural carbonate, and Sr in apatite). Furthermore, we assessed the integrity of microstructural organization, appearance under UV-light, collagen content, as well as the mineral and crystalline organization. This was conducted in order to provide a general background with which to explain observed changes in the trace analyses data sets. The goal is to develop an efficacious screening method for determining at which degree of burning bone still retains its original biological signals. We found that stable isotope analysis of the tested light elements in bone is only possible up to a heat exposure of 300°C while the isotopic signal from strontium remains unaltered even in bones exposed to very high temperatures. DNA-analyses seem theoretically possible up to a heat exposure of 600°C but can not be advised in every case because of the increased risk of contamination. While the macroscopic colour and UV-fluorescence of cremated bone give hints to temperature exposure of the bone's outer surface, its histological appearance can be used as a reliable indicator for the assessment of the overall degree of burning.

Soft tissue removal by maceration and feeding of Dermestes sp.: impact on morphological and biomolecular analyses of dental tissues in forensic medicine.

Maceration techniques remove soft tissue by the destruction of biomolecules, but the applied techniques may also affect the morphology and the molecular integrity of the hard tissue itself. The impact of seven different techniques for soft tissue removal on morphological and biomolecular parameters of teeth and dental tissues was systematically examined. All methods tested showed significant changes in dental morphology and in the molecular integrity of DNA and the dental proteins, as revealed by aspartic acid racemisation (AAR). In forensic casework this may have severe impacts on the results of morphological methods (e.g. age estimation based on root translucency) and of biomolecular analyses (e.g. age estimation based on AAR and DNA analysis). Therefore, age estimation based on AAR should not be applied to tissue treated in such a manner, and it is recommended that teeth for analysis should be extracted before soft tissue removal. DNA in the hard tissue seems to be less susceptible to soft tissue removal than proteins, and several of the tested maceration techniques appear not to have a damaging effect on DNA. Generally, the indication for soft tissue removal demands a careful case management to avoid methodological collisions.

[Degradation of biomolecules in bones: effects of the biological forensics as an example of the stability of isotope ratios in collagen]. / Degradation von Biomolekülen in Knochen: Auswirkung auf die biologische Spurenkunde am Beispiel stabiler Isotopenverhältnisse im Kollagen.

Modern bone samples were experimentally degraded by incubation into water at increased temperature and examined in terms of their collagen content, the stable C and N isotopic ratios, and the molar C/N ratio. The same analyses were carried out with archaeological human bone of varying age (300 up to 8000 years). The experimentally degraded samples exhibited changes of the collagen's integrity, which influence the stable isotope ratios. In the case of the archaeological material, a correlation between stable delta13C- and delta15N-values and collagen content could be demonstrated. The molar C:N ratio was no suitable criterion for the assessment of the state of preservation of extractable collagen.

A simple Duplex-PCR to evaluate the DNA quality of anthropological and forensic samples prior short tandem repeat typing.

Typing of DNA from ancient or otherwise highly degraded material, e.g. formalin fixed tissues, can be difficult, time consuming and costly. Very often, genetic typing is not possible at all. We present an inexpensive and easy to use Duplex-PCR that amplifies a 164 bp fragment specific for nuclear DNA together with a 260 bp mitochondrial DNA fragment and that can be employed as a pretest prior to short tandem repeat (STR) typing. All together, we analyzed DNA from 20 ancient bones, 20 formalin fixed tissues and 20 other forensic samples in different concentrations. Each sample that failed in the presented Duplex-amplification was also negative for STR typing, while samples that showed strong and clear signals in the Duplex-PCR led to reproducible genetic profiles using the multiplex kits AmpFLSTR Identifiler and Powerplex ES. The Duplex-PCR worked as a reliable indicator of DNA quality in the sample.

[Degradation of biomolecules: a comparative study of diagenesis of DNA and proteins in human bone tissue]. / Degradation von Biomolekülen: Eine vergleichende Studie zur Diagenese von DNA und Proteinen in humanem Knochengewebe.

Diagenesis of macromolecules is a not yet fully understood process that can be important for anthropological and forensic research. Trying to elucidate the diagenesis of DNA and proteins we investigated the process of fragmentation of DNA and razemisation of aspartic acid in human bone material. We created an in vitro-model of accelerated aging by incubating bone samples in hot water. A comparison of diagenesis of molecules in those artificially aged samples with altogether 30 historical bones from different regions and of different ages was carried out. The in vitro-model showed the expected positive correlation between the increase of razemisation of aspartic acid and DNA fragmentation, while there was a much lesser correlation when investigating historical bones. The in vitro-model showed the expected correlation between the increase of razemisation of aspartic acid and DNA fragmentation and to a much lesser extent in historical bones. This study shows that diagenesis is probably influenced by additional forces affecting different macromolecules in different ways.

Extraction and amplification of nuclear and mitochondrial DNA from ancient and artificially aged bones.

An experimental design is presented that simulates an accelerated process of DNA degradation in human bone tissues and thus provides a possibility for a systematic investigation of factors hampering DNA extraction and amplification. Equal sized slices of human femoral bones were incubated in 90 degrees C water for 2 h up to 30 days. DNA was extracted and subjected to a human specific Duplex polymerase chain reaction (PCR) and also to a Multiplex short tandem repeat (STR) PCR. Additionally 24 ancient bones representing different age periods were investigated in the same way. The results were compared to those from the artificially aged samples. After just 12 h of incubation, DNA is totally degraded, but still fully typable. After 36 h no reproducible amplification of DNA is possible. Using Multiplex PCR the DNA from artificially aged bones shows the typical STR pattern for ancient samples suggesting that the in vitro approach provides a useful and comparable method to elucidate the DNA degradation process in bones.