Ancient mitogenomes from Pre-Pottery Neolithic Central Anatolia and the effects of a Late Neolithic bottleneck in sheep (Ovis aries).

Occupied between ~10,300 and 9300 years ago, the Pre-Pottery Neolithic site of Asikli Höyük in Central Anatolia went through early phases of sheep domestication. Analysis of 629 mitochondrial genomes from this and numerous sites in Anatolia, southwest Asia, Europe, and Africa produced a phylogenetic tree with excessive coalescences (nodes) around the Neolithic, a potential signature of a domestication bottleneck. This is consistent with archeological evidence of sheep management at Asikli Höyük which transitioned from residential stabling to open pasturing over a millennium of site occupation. However, unexpectedly, we detected high genetic diversity throughout Asikli Höyük's occupation rather than a bottleneck. Instead, we detected a tenfold demographic bottleneck later in the Neolithic, which caused the fixation of mitochondrial haplogroup B in southwestern Anatolia. The mitochondrial genetic makeup that emerged was carried from the core region of early Neolithic sheep management into Europe and dominates the matrilineal diversity of both its ancient and the billion-strong modern sheep populations.

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.

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.

Detection of Yersinia pestis DNA in two early medieval skeletal finds from Aschheim (Upper Bavaria, 6th century A.D.).

In the course of a molecular genetic investigation of a double inhumation, presumably a mother/child burial from Aschheim (Upper Bavaria, 6th century A.D.), which included analysis of mitochondrial DNA, molecular sexing, and polymorphic nuclear DNA, Yersinia pestis-specific DNA was detected. Molecular analyses were performed on DNA extracts obtained from two teeth of one skeleton and four teeth of the other. The use of the primer pair YP12D/YP11R (Raoult et al. [2000] Proc. Natl. Acad. Sci. 97:12800-12803), able to amplify part of the Y. pestis plasmid pPCP1 pla sequence, resulted in amplification products of the expected fragment size. Using BLASTN 2.2.2, the sequences of these amplification products shared 100% identity with that of the modern Y. pestis pla sequence in GenBank, with the exception of one amplification product which revealed a single base substitution. The application of a "suicide PCR" with the independent primer pair YP11D/YP10R (Raoult et al. [2000] Proc. Natl. Acad. Sci. 97:12800-12803) resulted in amplification products which shared a 96-98% homology with that of the modern Y. pestis pla sequence in GenBank. The observed deviations were presumably due to miscoding lesions in the template DNA. No modern Y. pestis DNA was introduced into the institute, and thus no positive controls were carried along. All extraction and PCR controls remained negative. The identification of Y. pestis-specific DNA sequences in these two skeletons, buried in the second half of the 6th century A.D., constitutes molecularly supported evidence for the presence of Y. pestis, the causative agent of plague, during the first pandemic recorded.