Animal DNA in PCR reagents plagues ancient DNA research

Molecular archaeology brings the tools of molecular biology to bear on fundamental questions in archaeology, anthropology, evolution, and ecology. Ancient DNA research is becoming widespread as evolutionary biologists and archaeologists discover the power of the polymerase chain reaction (PCR) to amplify DNA from ancient plant and animal remains. However, the extraordinary susceptibility of PCR to contamination by extraneous DNA is not widely appreciated. We report the independent observation of DNA from domestic animals in PCR reagents and ancient samples in four separate laboratories. Since PCR conditions used in ancient DNA analyses are extremely sensitive, very low concentrations of contaminating DNA can cause false positives. Previously unidentified animal DNA in reagents can confound ancient DNA research on certain domestic animals, especially cows, pigs, and chickens.     

Amplification and sequencing of Trichuris trichiura ancient DNA extracted from archaeological sediments

Despite high prevalence of Trichuris trichiura infection, PCR-based analysis on T. trichiura from archaeological samples has not been established so far. In the present study, we sought to perform PCR-based amplification of T. trichiura aDNA using the sediments from medieval tomb of Korea. The presence of Trichuris eggs were first detected by microscopic observation; then confirmed by PCR-based aDNA analysis. Obtained sequence showed 100% homology to that of the small subunit ribosomal RNA (SSUrRNA) gene of T. trichiura but distinct from that of other Trichuris species. PCR-based aDNA analysis in this study can serve as effective method to confirm the presence of T. trichiura eggs in the soils or coprolites collected from archaeological sites.     

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.     

Ancient DNA reveals a major genetic change during the transition from hunting economy to reindeer husbandry in northern Scandinavia

The timing and origin of reindeer domestication has been highly debated. Recent molecular analyses show several mitochondrial lineages of domestic reindeer across Eurasia suggesting different origins of Fennoscandian and Siberian domestic reindeer. In order to investigate the origin of domestic Fennoscandian reindeer, we sequenced a mitochondrial control region fragment of 68 ancient reindeer remains from archaeological sites in Finnmark, the major county for extant reindeer husbandry in Norway, spanning from ca. BC 3400 to AD 1800. The majority of the Stone and Iron Age reindeer assemblages in Finnmark are from settlements in the eastern part of the county, in the Varangerfjord area. The reindeer remains from these settlements show affiliation to the large and complex Beringian haplotype cluster, found in extant reindeer from the Kola Peninsula to north-eastern Russia. A distinct haplotype shift is observed in the late medieval period, when the typical haplotype signatures of extant domestic Fennoscandian reindeer appeared in coastal regions of both eastern and western Finnmark. These haplotypes were not found among the Stone and Iron Age wild reindeer samples of Finnmark, suggesting that this population was not ancestral to extant domestic reindeer of Fennoscandia.     

Ancient DNA analysis of desiccated wheat grains excavated from a Bronze Age cemetery in Xinjiang

Wheat has been one of the most important crop in Eurasia since the Neolithic period. Understanding the spread of wheat cultivation is crucial to understanding the spread of agriculture as a whole and the interactions between prehistoric populations across the Eurasian continent. However, the routes by which wheat cultivation spread eastwards have been poorly understood to date, due to the scarcity of plant remains recovered from archaeological sites. Desiccated wheat grains excavated from the Xiaohe cemetery in Xinjiang, and dated to the early Bronze Age, show excellent DNA preservation. Here we present an ancient DNA (aDNA) analysis of wheat (Triticum sp.) grains excavated from Xiaohe and provide the first definitive evidence for bread wheat in China during the Bronze Age. The nuclear ribosomal DNA internal transcribed spacer regions (ITS1 and ITS2) and the intergenic spacer region (IGS) were amplified. The IGS region within the D genome of wheat has a 71 bp insertion that is absent from corresponding regions in the A and B genomes. The results showed that the Xiaohe wheat showed most sequence similarity to hexaploid bread wheat (Triticum aestivum), including the characteristic insertion into the D genome. The presence of bread wheat at the Xiaohe cemetery is discussed in relation to it having spread into Xinjiang by the Bronze Age, providing new insight into the origins of bread wheat in East Asia.     

Aspects of ancient Greek trade re-evaluated with amphora DNA evidence

Ancient DNA trapped in the matrices of ceramic transport jars from Mediterranean shipwrecks can reveal the goods traded in the earliest markets. Scholars generally assume that the amphora cargoes of 5th-3rd century B.C. Greek shipwrecks contained wine, or to a much lesser extent olive oil. Remnant DNA inside empty amphoras allows us to test that assumption. We show that short ∼100 nucleotides of ancient DNA can be isolated and analyzed from inside the empty jars from either small amounts of physical scrapings or material captured with non-destructive swabs. Our study material is previously inaccessible Classical/Hellenistic Greek shipwreck amphoras archived at the Ministry of Culture and Tourism Ephorate of Underwater Antiquities in Athens, Greece. Collected DNA samples reveal various combinations of olive, grape, Lamiaceae herbs (mint, rosemary, thyme, oregano, sage), juniper, and terebinth/mastic (genus Pistacia). General DNA targeting analyses also reveal the presence of pine (Pinus), and DNA from Fabaceae (Legume family); Zingiberaceae (Ginger family); and Juglandaceae (Walnut family). Our results demonstrate that amphoras were much more than wine containers. DNA shows that these transport jars contained a wide range of goods, bringing into question long-standing assumptions about amphora use in ancient Greece. Ancient DNA investigations open new research avenues, and will allow accurate reconstruction of ancient diet, medicinal compounds, value-added products, goods brought to market, and food preservation methods.     

Clarifying prehistoric parasitism from a complementary morphological and molecular approach

This paper reports an approach to the identification of prehistoric parasitic infection, which integrates traditional morphological methods with molecular methods. The approach includes the strengths of each method while mitigating the limitations. Demonstrating the efficacy of this approach, we provide a case study from a 1400 year old desiccated fecal sample from La Cueva de los Muertos Chiquitos, archaeological site, near Rio Zape, Durango, Mexico. Traditionally prepared microscope slides were processed via microscopy and tentative ascarids were identified. Information regarding the parasites' developmental stage was recorded. DNA was then extracted directly from the slide material. From this DNA extract, a small segment of the 18S ribosomal RNA gene variant that is specific to Ascaris, and its phylogenetically close relatives, was targeted for PCR amplification and sequencing. Phylogenetic analysis of the DNA sequence best matched a member of physalopterids, rather than ascarids, with a single exception of a match to Contracaecum spiculigerum. Subsequent extractions, amplifications and sequencing of the original rehydrated coprolite material confirmed these results. The C. spiculigerum sequence represented a phylogenetic anomaly and subsequent analysis determined the sequence was an error in the BLAST database, likely attributable to misidentification of juvenile specimens prior to sequencing and submission. Physaloptera are a difficult genus to identify morphologically and can carry major health burdens. They may be underreported in humans, in part, because of morphological similarities to the more common human parasites belonging to ascarids. We conclude that integrating traditional morphological methods with molecular methods can help resolve this issue, in both contemporary and prehistoric populations.     

Chemical characterisation of anthro-technosols from Bronze to Middle Age in Bologna (Italy)

The aim of this research was the understanding of a peculiarly expanded soil sequence in Bologna (i.e., Roman Bononia, Etruscan Felsina), one of the most important archaeological poles for Etruscan colonisation and settlement in northern Italy. The uppermost part of the chronological sequence dating from the Bronze Age up to medieval and modern times was analysed for the first time from geochemical and pedological viewpoints. The integration of archaeological, stratigraphical and pedological data supported by soil chemical analysis, a fundamental key for reconstructing a correct evolutionary sequence, made it possible to recognise seventeen buried Soil Units (ancient Technosols, Anthrosols and Cambisols) characterised by different kinds and degrees of intensity of anthropogenic influence. In some cases, the results show a relatively severe element concentration (Cu, Sn) mainly characterising the Iron Age (Etruscan and Villanovan) stratigraphic units, representing an ancient industrial pollution linked to local dispersal of melting activity dump products generated in near-site surrounding areas. Therefore, a comparison was possible between natural blank and anthropogenically polluted geochemical values for some local soils.     

Recovery of DNA from archaeological insect remains: first results,problems and potential

We report the recovery of short fragments of PCR amplifiable ancient DNA from exoskeletal fragments of the grain weevil Sitophilus granarius (L.) extracted from Roman and medieval deposits in Northern England. If DNA preservation in archaeological insect remains is widespread then many applications in the spheres of evolutionary studies and archaeology can be conceived, some of which are outlined.     

Wild or domesticated: DNA analysis of ancient water buffalo remains from north China

Recent zooarchaeological studies on water buffalo (Bubalus sp.) remains from China and south Asia question the traditional view that water buffalo were first domesticated in Neolithic China over 7000 years ago. The results from several recent population genetic studies of modern domesticated buffalo (Bubalus bubalis) are not consistent with each other, placing the original center of buffalo's domestication in south Asia, southeast Asia, or China. This paper reports a study using an ancient DNA approach to analyze water buffalo remains from Neolithic sites in north China to investigate their affinities with modern domesticated water buffalo, and to shed light on the origin of modern domesticated water buffalo in China.A 169 bp fragment of D-loop mitochondrial DNA was successfully amplified and verified for 13 of 24 bone samples obtained from seven archaeological sites along the Wei River valley in Shaanxi Province, China. The bone samples which yielded positive DNA can be dated to 8000–3600 cal. BP. The phylogenetic analysis of the obtained DNA sequences along with modern water buffalo sequences indicated that the ancient water buffalos were not the direct ancestor of modern domesticated water buffalo. However, the phylogenetic analysis, along with BLAST searches of these ancient DNA sequences, did demonstrate their relatedness to water buffalo more so than to any other bovid species, confirming the existence of indigenous wild (but now extinct) water buffalo species (B. mephistopheles) in ancient China.The DNA analysis of these ancient remains failed to establish direct links between modern domesticated water buffalo (B. bubalis) and indigenous water buffalo (B. mephistopheles) from ancient China. If further DNA studies of more ancient remains from other regions of China confirm the observation of solely indigenous water buffalo species in ancient China, it would suggest modern water buffalo might not have been first domesticated in China.     

First evidence of white wine in ancient Egypt from Tutankhamun's tomb

Wine has been considered to be mainly red in ancient Egypt linked with the blood of Osiris, the God of resurrection. No text that refers to white wines from the Dynastic Period (3150–332 BC) exists. The first white wine from ancient Egypt was made near Alexandria during the third century AD. To investigate the presence of white wine in ancient Egypt, dry residue samples from King Tutankhamun's amphorae are studied in this article using the LC/MS/MS method for wine markers. This investigation into the existence of white wines in Tutankhamun's tomb allows us to shed new light on the symbolism of white wine in ancient Egypt.     

Ectopic paragonimiasis from 400-year-old female mummy of Korea

Of the parasite eggs discovered in ancient samples from Korean archaeological sites, Paragonimus spp. are of particular importance in that they are regarded as one of the most insidious trematode infection sources. Although their infection prevalence decreased rapidly in the early 20th century, archaeoparasitological studies on the species are still required, as historical knowledge of paragonimiasis remains far from comprehensive. Fortunately, we recently were given a chance to examine a medieval Korean mummy in a good state of preservation. Using morphological techniques, we discovered evidence of ectopic paragonimiasis in the sample from the 17th-century female. When the ITS 2 gene was extracted, amplified and sequenced from Paragonimus eggs, it showed 100% homology to the sequences of modern Paragonimus westermani reported from Korea and Japan, forming a cluster distinct from South Asian P. westermani. Our report is the first-ever analysis of ancient Paragonimus DNA from any archaeological field in the world. The ectopic paragonimiasis diagnosis made in this study, especially involving the liver sample, also is the first of its kind in archaeoparasitology.     

Aspartic acid racemization variability in ancient human remains: implications in the prediction of ancient DNA recovery

The extent of racemization of aspartic acid (Asp) – expressed as d/l ratio – has been used as a marker of biomolecular degradation in ancient remains. However, Asp racemization rate is highly variable, and depends on biochemical and geochemical factors. In this paper we aim to determine to which extent the fraction analyzed and the kind of sample used may influence the d/l Asp ratios. Other factors, such as burial site and sample preservation conditions, are also considered.     

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.     

Neolithic Hedgehogs (Erinaceus europaeus) from the Island of Gotland show early contacts with the Swedish mainland

Previous research probing early migrations and contacts in the Baltic Sea area is characterized by the analysis of different chronologies and subsistent strategies on all sides of the Sea. Several studies performed on artifact typology, ceramics, grave rituals and physical anthropology ended with varying results. Although the question of human origins remains inconclusive, in this study, we rely on the phylogeography of an animal associated with humans to elucidate findings regarding prehistoric human migration and contacts.Hedgehogs, along with other fauna on Gotland, were brought over to the island by humans. We examined hedgehog mitochondrial DNA from the Pitted Ware Culture (Middle Neolithic). The genetic signatures of the animals on the island were investigated to determine the animal’s origin.From the 23 bones originally examined, twelve bones from all five locations studied yielded reliable results and resembled published extant Erinaceus europaeus sequences from Sweden, Norway and Denmark. We postulate that a western heritage for the Neolithic hedgehogs on Gotland indicates early human contact with the Swedish mainland.     

Tikal timbers and temples: ancient Maya agroforestry and the end of time

Tikal, a major lowland Maya civic-ceremonial center in the heart of the Petén region of Central America, relied heavily on the adjacent lowland rainforest as a resource base for fuel and construction materials. In this study, we analyzed 135 wood samples from timbers used in the construction of all six of the city's major temples as well as two major palaces to determine which tree species were being exploited and to better understand ancient Maya agroforestry practices during the Late Classic period. We found evidence for a change in preference from the large-growing, upland forest species, Manilkara zapota, to a seasonal wetland species Haematoxylon campechianum in A.D. 741 as well as a decrease in lintel beam widths over time. Though M. zapota later returned as the wood species of choice in A.D. 810, beam widths were found to be significantly smaller. These findings concur with models that hypothesize widespread deforestation during the Late Classic period and indicate a declining forest resource base by the 9th century A.D. Because of the many large timbers available for temple construction in the 8th century, some system of forest conservation is indicated for the ancient Maya prior to the Late Classic period.     

Genetic analysis of archaeological wood remains: first results and prospects

A total of 51 ancient oak wood samples originating from various European archaeological sites, dating from the Neolithic period to the 18th century, were assayed for the presence of reproducible chloroplast (cp) DNA sequences. Five polymorphic chloroplast fragments were targeted. Only five of the samples could be fully genetically characterised, revealing four different oak cpDNA haplotypes. In all cases, the haplotypes detected on ancient woods and the haplotypes characterised from fresh samples from the same localities matched. Overall, this congruence is consistent with a genetic continuity between ancient and modern European oaks, confirming the hypothesis that the mapped genetic patterns largely reflect the original structure that established during the post-glacial. This stability of the genetic structure implies that, in the future, the technique could be used to infer or confirm the transport of wood by man, providing interesting perspectives for the genetic analysis of ancient woods.     

Ancient DNA of the Pacific rat (Rattus exulans) from Rapa Nui (Easter Island)

We report analysis of ancient mitochondrial DNA sequences from nine archaeological specimens (8 femura and 1 incissor) of Rattus exulans excavated from Anakena Beach Dune on Rapa Nui. Sequence of a 239-base-pair fragment of the hypervariable mitochondrial control region reveals a single mitochondrial DNA (mtDNA) sequence of all samples corresponding to the R9 haplotype prevalent in East Polynesia. This suggests a single or very limited introduction of Rattus exulans to the island. Rapa Nui, like other remote islands of Polynesia, remained effectively isolated following colonization.     

Cats of the Pharaohs: Genetic Comparison of Egyptian Cat Mummies to their Feline Contemporaries

The ancient Egyptians mummified an abundance of cats during the Late Period (664 - 332 BC). The overlapping morphology and sizes of developing wildcats and domestic cats confounds the identity of mummified cat species. Genetic analyses should support mummy identification and was conducted on two long bones and a mandible of three cats that were mummified by the ancient Egyptians. The mummy DNA was extracted in a dedicated ancient DNA laboratory at the University of California - Davis, then directly sequencing between 246 and 402 bp of the mtDNA control region from each bone. When compared to a dataset of wildcats (Felis silvestris silvestris, F. s. tristrami, and F. chaus) as well as a previously published worldwide dataset of modern domestic cat samples, including Egypt, the DNA evidence suggests the three mummies represent common contemporary domestic cat mitotypes prevalent in modern Egypt and the Middle East. Divergence estimates date the origin of the mummies' mitotypes to between two and 7.5 thousand years prior to their mummification, likely prior to or during Egyptian Predyanstic and Early Dynastic Periods. These data are the first genetic evidence supporting that the ancient Egyptians used domesticated cats, F. s. catus, for votive mummies, and likely implies cats were domesticated prior to extensive mummification of cats.     

Biomolecular archaeology of ancient tuberculosis: response to “Deficiencies and challenges in the study of ancient tuberculosis DNA” by Wilbur et al. (2009)

It is sixteen years since the first detection of Mycobacterium tuberculosis DNA in archaeological specimens, yet the validity of findings continues to be questioned. Rigorous scientific scrutiny and debate is valuable and has led to a coalescence of procedures and precautions amongst those actively engaged in this work. It is disappointing that these good practices are not recognised by certain scientists whose primary expertise is in the related fields of archaeology, palaeopathology, and eukaryote ancient DNA. There is a danger that by constant repetition, disputable and inadequately justified concerns will assume the status of self-perpetuating myths and misunderstandings. We discuss these issues with reference to a recent article in this journal, in which clear peer-reviewed scientific data were specifically targeted as part of a general critique of the field of the palaeomicrobiology of tuberculosis. We believe we have given sufficient evidence and cogent argument to persuade the unbiased reader that the views in the critique by Wilbur et al. are unjustified.