The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis

To determine whether ancient DNA (aDNA) can be used to study the palaeopathology of venereal syphilis, we carried out a comprehensive analysis of the preservation of human and pathogen DNA in a set of 46 bones of various ages, most of which displayed osteological indications of the disease. Bones came from seven English cemetery sites that were in use during the 9th–19th centuries. Twelve of the 46 bones consistently yielded mitochondrial DNA (mtDNA) sequences after replicate polymerase chain reactions (PCRs), and a further 13 bones yielded mtDNA sequences with less reproducibility. The sequence data enabled tentative mitochondrial haplogroups to be assigned to nine of the bones, and the identities and frequencies of these haplogroups were compatible with the geographical origins of the bones. Twenty-one bones consistently gave negative results with all mtDNA PCRs, indicating that at least these bones were not contaminated with modern human DNA, and those bones that gave positive results only yielded one sequence each, again suggesting that widespread modern contamination had not occurred. Mycobacterium tuberculosis sequences were obtained from seven bones, including three of five bones with tuberculous lesions. The cloned and direct sequences obtained from both the mtDNA and M. tuberculosis PCR products showed features typical of degraded aDNA. All of these results suggest that at least some of the 46 bones that we studied were suitable for aDNA analysis. All 46 bones were tested with nine different treponemal PCRs, each optimised to give a detection limit of ≤5 genomes. Although various bones gave PCR products of the expected size with one or more of these PCRs, sequencing showed that none of these products were authentic treponemal amplicons. Our failure to detect treponemal DNA in bones that were suitable for aDNA analysis, using highly sensitive PCRs, suggests that treponemal DNA is not preserved in human bone and that it is therefore not possible to use aDNA analysis to study venereal syphilis. Any past or future paper claiming detection of treponemal aDNA should therefore be accompanied by a detailed justification of the results.     

Ancient DNA in human bones from Neolithic and Bronze Age sites in Greece and Crete

Attempts were made to detect ancient DNA (aDNA) in samples of 88 human skeletons from eight Neolithic and Bronze Age sites in Greece and Crete. Ancient DNA was absent in specimens from Nea Nikomedia, Lerna, Karaviádena (Zakro), Antron Grave Circle A and Mycenae Grave Circle A. For each of three skeletons from Antron Grave Circle B that were sampled, polymerase chain reactions (PCRs) gave products for nuclear but not mitochondrial DNA, but amplicon yield was low and inconsistent with replicate PCRs failing to give reproducible results. With specimens from Mycenae Grave Circle B, evidence for mitochondrial aDNA was obtained for four of the 22 skeletons that were studied, and at Kouphovouno evidence for mitochondrial and/or nuclear aDNA was obtained with eight of the 20 skeletons that were examined. We conclude that, although aDNA might be present in some Eastern Mediterranean skeletons from later centuries of the Bronze Age, it is not commonly found in material from this period and is likely to be absent from older material.     

Using ancient DNA analysis in palaeopathology: a critical analysis of published papers,with recommendations for future work

Despite an observable increase in the number of studies using ancient DNA analysis to diagnose disease in human remains, there remain issues to be addressed about the quality of the resulting publications. This paper describes the qualitative analysis of published papers that describe the detection of pathogenic DNA in human skeletal and mummified remains from archaeological sites. Its ultimate goal is to provide an overview of the main problematic issues in relationship to standards developed in molecular biology and to make recommendations for future work. Sixty‐five papers published between 1993 and 2006 were surveyed and the quality of each was assessed using 15 criteria. Interesting results emerged. Of particular note was the high number of papers that did not acknowledge the use of even basic contamination control (90%) or procedures to validate results independently (85%). This study illustrates that attention to contamination control and authentication of results is needed in future research, if confidence in aDNA analysis in palaeopathology is to be increased. Additionally, methods of analysis must be described in published papers to ensure transparency in processes utilised to generate the data. Copyright © 2008 John Wiley & Sons, Ltd.     

Post-mortem DNA damage hotspots in Bison (Bison bison) provide evidence for both damage and mutational hotspots in human mitochondrial DNA

Post-mortem damage-driven mutations are a phenomena associated with ancient DNA (aDNA) studies. A previous study has demonstrated that the distribution of such mutations in human mitochondrial DNA is not random, but is concentrated in ‘hotspots’ that correlate with sites of elevated mutation rate in vivo. However, as the previous study was undertaken on human samples, it is possible for a critic to argue that the results might be biased through the presence of modern contaminant DNA sequences among the ancient DNA extracts. In this study we confirm the phenomena of DNA damage hotspots using a data set that is unlikely to be affected by contamination – cloned mitochondrial control region sequences extracted from 81 ancient bison (Bison bison). Furthermore, using published data from modern bovid specimens, we confirm that the damage hotspots correlate with sites of in vivo hypermutation. In conclusion, the aDNA sequences from archaeological specimens provide evidence that structural elements of mitochondrial DNA confer a degree of in vivo and post-mortem protection from sequence modification. This in turn provides useful insights into the debate as to whether mutational hotspots or mitochondrial recombination might best explain homoplasies observed on phylogenetic trees of human mitochondrial sequences.     

Kinship between burials from Grave Circle B at Mycenae revealed by ancient DNA typing

The richness of the burials in Grave Circle B at Mycenae, Greece indicates that the 35 people interred there held elite status during their lifetimes 3500 years ago. It has been speculated that the groups of burials represent different dynasties or branches of the same family. To test this hypothesis, we carried out an exhaustive ancient DNA (aDNA) study of 22 of the skeletons. We were unable to identify nuclear aDNA in any specimen, but we obtained authentic mitochondrial aDNA sequences for four individuals. The results were compared with facial reconstructions and interpreted within the archaeological context represented by the organisation of the graves and the positions of the burials within the graves. We conclude that the contemporaneous male Γ55 and female Γ58 skeletons, which both possess the UK mitochondrial haplogroup, were brother and sister. The implication is that Γ58 was buried in Grave Circle B not because of a marital connection but because she held a position of authority by right of birth. The results illustrate the difficulty in using aDNA to study kinship relationships between archaeological specimens, but also show that aDNA can advance understanding of kinship when used to test hypotheses constructed from other evidence.     

Repeat silica extraction: a simple technique for the removal of PCR inhibitors from DNA extracts

Polymerase chain reaction (PCR) inhibitors are often co-extracted with ancient DNA (aDNA) and when present make the analysis of aDNA difficult, if not impossible. In this study we review previous research on PCR inhibitors and techniques that address their co-extraction with DNA from sub-optimal samples. Additionally, we introduce a simple extraction technique, “repeat silica extraction,” that effectively removed PCR inhibitors from extracts of 7000–8000-year-old human skeletal remains from the Windover archaeological site in Florida and 700–2000-year-old human coprolites excavated from Fish Slough Cave in southern California. A series of tests on these same samples demonstrates that N-phenacylthiazolium bromide is largely ineffective, despite previously reported success using this compound as part of the DNA extraction process. We also describe a method for demonstrating the presence as well as successful removal of PCR inhibitors by use of a “positive aDNA control,” a test necessary to conclude that negative PCR amplification is the result of the absence of preserved DNA.     

Determination of a kinship system using ancient DNA,mortuary practice,and historic records in an upper Canadian pioneer cemetery

Recovered and amplified ancient DNA (aDNA), from a historically documented 19th century Upper Canadian pioneer cemetery produced genotypes that were used to infer a past societal kinship system. While the results from multiplex short tandem repeat (STR) amplifications showed an unreliable polymerase chain reaction (PCR) product, a single locus HUMTH01 analysis yielded reproducible data and an allelic frequency pattern not statistically different from modern populations. Mitochondrial (mt) DNA HVR II data showed that a combined cemetery database exhibited reduced haplotype diversity indicators, as well as clusters of probable maternally related burials. The chronological persistence and replacement of mtDNA clusters approximately every two generations suggests a patrilineal/patrilocal kinship structure from a virilocal burial program for the Harmony Road cemetery. Through the integration of the aDNA analysis with archaeological material culture, historic records, and other ethnohistoric sources of information, this conclusion is supported. In this study persisting patrilineally inherited surnames act as a surrogate for aDNA Y‐chromosome haplotype analysis. These results suggest that aDNA applications on aggregate skeletal collections where sparse, or no ethnological or historical documentation exists, may result in incorrect population history inferences if the presence of a kinship interment bias is not considered. Copyright © 2003 John Wiley & Sons, Ltd.     

Ancient Iberian horses: a method to recover DNA from archaeological samples buried under sub-optimal conditions for preservation

Existing methods to extract, amplify, and sequence ancient DNA (aDNA) from horse bone and teeth were optimized to recover DNA from a depositional environment of highly permeable acidic soil. DNA was successfully retrieved using 0.10g of bone powder from horse (Equus sp.) remains dating to 25 K years utilizing the methods optimized for this archaeological material. The genetic analyses were performed in a facility that is dedicated to ancient DNA research (Paleo-DNA Laboratory, Thunder Bay, ON, Canada) and has not been previously used to analyse modern or ancient horse DNA. Research was replicated to obtain reliable sequencing results for six samples from the Iberian Peninsula that were consistent with published sequences of Equus caballus. The archaeological sequence data obtained support hypotheses that promote the significance that the Iberian Peninsula has had to the multi-focal centres of origin for horse domestication and distribution of modern horse breeds. The data presented may provide evidence of the existence of an Iberian refugium for Equus during the last glacial period, 10 K years BP. Further molecular data analyses will enhance the ideas presented by this data and our understanding of horse domestication and phylogeny. The optimization of molecular techniques to successfully obtain DNA using minimally destructive, cosmetically sensitive techniques from archaeological remains endeavours to foster further cooperation between museums and researchers.     

SEX IDENTIFICATION OF SLAVE SACRIFICE VICTIMS FROM QIN STATE TOMBS IN THE SPRING AND AUTUMN PERIOD OF CHINA USING ANCIENT DNA

Ancient DNA (aDNA) analysis was employed to determine the sex of slave sacrifice victims from Qin State tombs during the Spring and Autumn Period of China. It is difficult to obtain sex information from fragmentary skeleton samples with the aid of skeletal morphology methods. aDNA was extracted from the dentine in selected tooth samples of sacrificial slaves using a modified traditional method, which combines together phenol/chloroform extraction, silicon dioxide adsorption and ultrafiltration concentration. Based on the sequence differences between the amelogenin homologous gene in the X and that in the Y chromosome, a pair of specific primers was designed to identify the sex of the selected samples. In the selected eight typical samples, the aDNA analytical results revealed that three were males and two were females. These findings indicate that molecular sex identification might provide more valuable information for archaeological research on the institution of slave sacrifice in the Spring and Autumn Period of China.     

THE TWILIGHT OF THE GODS? GENOMIC HISTORY AND THE RETURN OF RACE IN THE STUDY OF THE ANCIENT MEDITERRANEAN

This article discusses the impact of genomic history, a subdiscipline that emerged in the study of the ancient Mediterranean in the 2010s. In 2014, scientists first published a method for extracting genetic material, which they christened aDNA (ancient DNA), from ancient human remains in hot climates. After a decade of research, genomic history is now poised to transform our understanding of Mediterranean premodernity, centering migration and conflict as the key mechanisms for cultural change. Despite years of critique, aDNA researchers have failed to seriously examine the bioessentialist assumptions implicit in their work—a failure that has led many to deploy language that is strikingly evocative of pre-World War II racialism. Even worse, some genomic historians continue to make troubling overtures toward the ethnonationalist Right, which has been ascendant across Europe and North America since the 2010s. This article traces the intellectual genealogy of genomic history from World War II to the present, examines recent attempts to answer criticism from the humanities and social sciences, and suggests paths for responsible use of aDNA in historical and prehistorical scholarship.     

Molecular and morphological analyses of avian eggshell excavated from a late thirteenth century earth oven

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) – one of New Zealand’s most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts.     

Improved methodology for extraction and amplification of DNA from single grains of charred wheat

Comparisons were made between two commonly used methods for the extraction of ancient DNA from charred plant remains. Using artificially charred wheat seeds, we show that silica-binding is the most efficient method for extraction of DNA. We describe a improved silica-binding procedure, including pre-incubation with N-phenacylthiazolium bromide and increased washing of bound DNA, which yields amplifiable DNA from seeds heated at 200 °C for up to 8 h, conditions which promote the formation of Maillard products which often copurify with aDNA and inhibit subsequent PCRs. We believe that this method will be effective in ancient DNA extraction with most types of charred archaeobotanical material. Both cold- and hot-start PCR procedures gave good amplicon yields with extracts prepared in this way, but cold-start PCRs also resulted in synthesis of short artefact products. Addition of bovine serum albumin to PCRs, an inert carrier substance thought to enhance amplification efficiency by binding contaminants, had no advantageous effect and in fact reduced amplicon synthesis.     

Sex Identification in Some Putative Infanticide Victims from Roman Britain Using Ancient DNA

Previous study of infant burials has suggested that infanticide was routinely practised during the Roman period in Britain. This, together with the observation that there is an adult sex imbalance in favour of males at many Romano-British cemetery sites, has raised the question of female infanticide. We attempted to investigate this possibility by identifying sex in some infant skeletons from Romano-British contexts using ancient DNA (aDNA) techniques. Of 31 individuals sampled, sex identification was successful in 13, of which nine were males and four females. These results are discussed in the light of previous work on DNA-based seeking of infant burials.     

Tuberculosis on the north coast of Peru: skeletal and molecular paleopathology of late pre-Hispanic and postcontact mycobacterial disease

This paper examines skeletal and ancient DNA evidence in the study of suspected tuberculosis infection in the late pre-Hispanic and Colonial-era Lambayeque Valley Complex, north coast Peru (A.D. 900–1750). We integrate information on macroscopic lesion characteristics and distribution, radiographic and CT scan imagery, and analysis of Mycobacterium tuberculosis complex rpoB and IS6110 ancient DNA (aDNA) sequences. Destructive lesions were observed in the vertebral bodies of three precontact indigenous adult males, one colonial adolescent female, and in the cranium of a Colonial-period subadult. Assessment of lesion morphology and distribution led us to consider multiple diseases, but tuberculosis represents the most likely diagnostic option in all individuals. DNA was poorly preserved in all samples, but an IS6110 sequence was amplified in one precontact individual consistent with macroscopic diagnosis. These findings expand the geographic and temporal extent of tuberculosis to the late pre-Hispanic and Colonial north coast of Peru to highlight potential synergisms between diet, settlement patterns, and the evolution of Andean tuberculosis before and after European conquest. Moreover, this study helps focus several key questions in Andean tuberculosis research, including possible reassessment of the presence of the IS6110 sequence in the pre-Columbian Americas. Methodological considerations include differential diagnosis – especially with incomplete skeletons – and limitations of aDNA studies underscoring an approach integrating macroscopic, radiographic, and molecular lines of evidence in the paleopathological investigation of one of humankind’s most devastating and destabilizing diseases.     

Characterising the potential of sheep wool for ancient DNA analyses

The use of wool derived from sheep (Ovis aries) hair shafts is widespread in ancient and historic textiles. Given that hair can represent a valuable source of ancient DNA, wool may represent a valuable genetic archive for studies on the domestication of the sheep. However, both the quality and content of DNA in hair shafts are known to vary, and it is possible that common treatments of wool such as dyeing may negatively impact the DNA. Using quantitative real-time polymerase chain reaction (PCR), we demonstrate that in general, short fragments of both mitochondrial and single-copy nuclear DNA can be PCR-amplified from wool derived from a variety of breeds, regardless of the body location or natural pigmentation. Furthermore, although DNA can be PCR-amplified from wool dyed with one of four common plant dyes (tansy, woad, madder, weld), the use of mordants such as alum or iron leads to considerable DNA degradation. Lastly, we demonstrate that mtDNA at least can be PCR-amplified, cloned and sequenced from a range of archaeological and historic Danish, Flemmish and Greenlandic wool textile samples. In summary, our data suggest that wool offers a promising source for future ancient mitochondrial DNA studies.     

Rice archaeological remains and the possibility of DNA archaeology: examples from Yayoi and Heian periods of Northern Japan

Japanese rice cultivation in paddy fields has 2,400∼3,000 years of history. Most of modern Japanese rice varieties are classified as Temperate-japonica (Tm-J). Few landraces are recognized as Tropical-japonica (Tr-J) only in southwestern Japan. However, ancient DNA studies and phytolith analysis suggest that Tr-J strains were more popular in the past than now. Maekawa is a complex archaeological site composed of paddies dated from the Yayoi (2,100 years BP) to the Heian (1,100 years BP) periods. Phytolith analysis indicates that intensive rice cultivation was practiced in both periods, but there was no cultivation in the intervening period. Morphological features of bulliform phytoliths suggest that Tr-J was cultivated during both periods. Locally, rice cultivation during the Heian period was brought to a close by a flood event, in which immature rice plants were pulled down and buried in silt to be preserved in a quasi-carbonized/ waterlogged state. Ancient DNA (aDNA) analysis of the carbonized plant culm from Heian Maekawa recovered chloroplast DNA sequences of the 6C7A plastid subtype, which is common to both Tr-J and Tm-J, whereas two plastid subtypes, such as 6C7A and also 7C6A, were found in aDNA of carbonized grains from the Tareyanagi site of the Yayoi period. The latter plastid subtype was specific only to Tr-J. In order to better characterize the past rice populations, modern landraces collected in the local area were classified with morphophysiological traits. Some of the landraces were found to carry several traits of Tr-J, including bulliform phytolith types, but mixed with Tm-J traits. Based on the discontinuous distribution of rice phytoliths between the Yayoi and the Heian period, the early introduction of rice cultivation may have been discontinuous and locally reintroduced after a ∼1,000-year hiatus, but with a genetically different rice population. Such populations were composed from Tr-J like strains as shown by landraces but with reduced diversity in plastid types. Through such changes, since the Yayoi era, Tr-J was largely replaced by Tm-J, although ancient Tr-J continued to participate in the genetic makeup of later rice populations and may have aided the local adaptation of introduced Tm-J.     

Mitochondrial DNA haplotypes of Devensian hyaenas from Creswell Crags,England

Spotted hyaena (Crocuta crocuta) remains have been recovered from British Middle and Upper Pleistocene sites at intervals within the period 700–730 ka BP. Morphological studies have suggested that hyaenas of the Last Interglacial sensu stricto (Ipswichian: Marine Isotope Stage [MIS] 5e, 130–115 ka BP) and Last Glacial (Devensian: MIS 3, 61–24 ka BP) were two distinct populations, the Ipswichian hyaenas becoming extinct in Britain during MIS 5 and the Devensian ones arriving via a subsequent migration from continental Europe. However, the apparent presence of hyaenas in later MIS 5 deposits has led to the alternative suggestion that there was a southern relict population from which the Devensian hyaenas originated. We obtained ancient DNA (aDNA) sequences from four Devensian hyaena specimens from Creswell Crags, Derbyshire, dated to around 45 ka ¹⁴C BP. Each of these four specimens belonged to the mitochondrial DNA (mtDNA) clade A. This clade is not thought to have been present in Europe until ~360 ka BP, after the initial arrival of hyaenas in Britain. The DNA results, therefore, suggest that there were at least two waves of hyaena dispersals into Britain. The results are consistent with the repeated dispersals into Britain of another Pleistocene social carnivore, Homo sapiens.     

Ancient DNA analysis on Clonorchis sinensis eggs remained in samples from medieval Korean mummy

In the present study, Clonorchis sinensis (C. sinensis) ancient DNA (aDNA) was successfully extracted from human remains discovered in a tomb dating to the medieval Joseon dynasty of Korea. The presence of C. sinensis eggs was confirmed by microscopic observation, after which a PCR-based aDNA analysis was performed using primer sets designed for the amplification of nuclear ribosomal internal transcribed spacer 1 (ITS1), 2 (ITS2) and mitochondrial cytochrome c oxidase 1 (CO1) genes. The sequences obtained were 100% homologous to some contemporary C. sinensis gene sequences reported from Korea and other East Asian countries. We believe that the results of our analysis expand the temporal and geographical scopes of research on the history of C. sinensis infection in different human populations.     

Tuberculosis among Iron Age individuals from Tyva,South Siberia: palaeopathological and biomolecular findings

The study focuses on the evidence for tuberculosis apparent in an Iron Age population recovered from the cemetery of Aymyrlyg, Tyva (Tuva), South Siberia. A recent wholly molecular study of five of the cases confirmed the presence of Mycobacterium tuberculosis (MTB) complex DNA in four of the individuals. In all cases the disease was caused by strains of Mycobacterium bovis rather than Mycobacterium tuberculosis and represents the first positive identification of the bovine form of the disease in archaeological human remains. Details of the palaeopathological characteristics of the cases are provided in the current paper, while the molecular observations are extended to include a quantitative evaluation of the surviving mycobacterial DNA using real-time PCR. The observation that bovine tuberculosis was the pathogen responsible is discussed in terms of current understanding of the evolution of the MTB complex as well as the implications for future ancient DNA studies in this area.