Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii

The persistent uncertainty on the classification of the “new” glume wheat found in Neolithic and Bronze Age sites from Greece and other European settlements might be resolved only through analysis of its ancient DNA. Tools able to discriminate among different Triticum species on the basis of scarce, very damaged DNA, are therefore essential. While current attempts concentrate on DNA fragments sequencing and comparison, in some instances PCR-based selective amplification techniques might offer a cheaper and quicker alternative. The purpose of this research was therefore the identification of species-specific primers, able to distinguish caryopses of Triticum timopheevii subsp. timopheevii from those of Triticum turgidum subsp. dicoccum. Primers and their working conditions were defined and optimized using DNA from modern accessions. The ribosomal primers ITS1 tim and ITS2 tim, and the nuclear primer acetyl-coenzyme A tim clearly discriminated the sequences of Triticum timopheevii from other species. Finally, Neolithic charred wheat grains found in the sites of Sammardenchia (Pozzuolo del Friuli, Udine) and La Marmotta (Lago di Bracciano, Roma), belonging to the “new” wheat type or to emmer, were tested with the three selected primers. However, the results were not conclusive, because the samples analysed were apparently too degraded to yield useful DNA.     

Two cases of leprosy from Žatec (Bohemia), dated to the turn of the 12th century and confirmed by DNA analysis for Mycobacterium leprae

The leprosy known today primarily from tropical areas was a relatively common disease in European Middle Ages. This article describes two skeletons bearing palaeopathological indications of leprosy from a cemetery at ?atec in North-West Bohemia (Czech Republic). The archaeological context clearly shows that these individuals were buried prior to the second decade of the 12th century, and most probably in the second half of 11th century. This rules out the possibility that these individuals might have contracted the disease in connection with the Crusades, in which a Bohemian contingent under Prince Vladislav II participated from 1141 to 1142. Molecular genetic methods were applied to detect specific DNA fragments of the causative agent of leprosy, Mycobacterium leprae. The nasal concha of one individual yielded DNA that could be directly sequenced after isolation and amplification. The vertebral body of the second individual, on the other hand, did not provide DNA of sufficient quality for direct sequencing and only weak amplification was detectable. The morphological and genetic analyses both indicate that leprosy existed prior to the Crusades in medieval Bohemia, albeit that its prevalence was probably not as great as in northern or western Europe.     

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.     

Ancient DNA supports lineage replacement in European dog gene pool: insight into Neolithic southeast France

We report palaeogenetic analysis of domesticated dog (Canis familiaris) remains excavated from three archaeological sites from southeast France and dating from Middle Neolithic. Ancient DNA analysis was attempted on teeth and bone samples taken from 11 dogs. Three 266-base-pair fragments of the mitochondrial genome Hypervariable Region I (HVR-I) could be retrieved and revealed two haplotypes belonging to HVR-I lineage C. These three sequences were compared to the sequences of Swedish and Italian Neolithic dogs and permitted to confirm that clade C was largely represented all over Western Europe during this period. One haplotype defined in Neolithic French dog was observed for the first time in Canis mtDNA, underlining the loss of mitochondrial diversity in Europe since the Neolithic. Finally, these results point out mitochondrial lineage replacement in Europe, since lineage C represents only 5% of extant European dogs. Altogether, these results support the proposition that palaeogenetic studies are essential for the reconstruction of the past demographic history and the domestication process of dogs.     

A pilot study into the effects of X-ray and computed tomography exposure on the amplification of DNA from bone

This research investigates the intersection of radiography and aDNA, two commonly used methods in bioarchaeology. The goal of this project was to investigate the effects of radiation on the ability to amplify DNA from bone. Bones (n = 124) from domestic pig (Sus scrofa) feet were randomly sorted into a control group and four treatment groups: (1) single exposure X-ray; (2) single exposure CT; (3) multiple exposures X-ray; and (4) multiple exposures CT. Number of PCR cycles required to amplify DNA in 100 bp, 200 bp and 400 bp segments were used as a proxy for the amount of available DNA. In the 200 bp CT sample, distal phalanges required significantly more amplification cycles than did the other bones and were removed from analysis. Results suggest that in general radiation exposure fragments DNA thereby decreasing the amount that is amplifiable. While these results are suggestive, further research is required to elucidate the degree to which radiation fragments DNA in archaeological specimens.     

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.     

First ancient bovine DNA evidence from India: difficult but not impossible

Ancient DNA isolation from the tropical countries has been shown to be very difficult in the past. Here for the first time we have been successful in isolating ancient DNA from Indian cattle samples. We were able to obtain DNA and sequence the partial mitochondrial D-loop in 3 of the 15 bovine fossil samples ranging in age from 2000 BC to 1000 AD, and were able to further identify the most recent sample as being of Bos indicus origin. Our results on ancient DNA extraction from India will encourage other researchers in this field to carry out further studies of ancient DNA from Indian bovine samples. Our results represent the first successful extraction and amplification of bovine ancient DNA from India, and thus may pave the road for a better understanding of demographic and historical processes of cattle domestication that has taken place in this region.     

Dysentery in the crusader kingdom of Jerusalem: an ELISA analysis of two medieval latrines in the City of Acre (Israel)

Dysentery was a significant cause of ill health during medieval times according to written sources of the period. However, it has been very difficult to detect the organisms that cause dysentery in archaeological samples from latrines and cesspools. This is because the cysts of the enteric parasites that cause dysentery are very small and usually fragment in the soil, in contrast to the robust ova of parasitic intestinal worms. This study uses a monoclonal enzyme-linked immunosorbent assay (ELISA) technique to identify the fragments of those enteric parasites that cause diarrhoea. Samples were taken from two excavated medieval latrines in the city of Acre. Coins and radiocarbon dating suggest that the latrines were in use during the thirteenth century CE. Positive results were noted for Entamoeba histolytica and Giardia duodenalis, while Cryptosporidium parvum was negative. This is the first time that either of these parasites have been identified in the Middle East using these methods.     

Chemical analysis of 17th century Millefiori glasses excavated in the Monastery of Sta. Clara-a-Velha,Portugal: comparison with Venetian and façon-de-Venise production

A set of ten Millefiori glass fragments dating from the 17th century, originated from archaeological excavations carried out at the Monastery of Sta. Clara-a-Velha (Coimbra, Portugal), were characterized by X-ray electron probe microanalysis (EPMA), Raman microscopy and UV–Visible absorption spectroscopy. All glasses are of soda-lime-silica type. The use of coastal plant ash is suggested by the relatively high content of MgO, K₂O and P₂O₅, as well as by the presence of chlorine. Tin oxide or calcium antimonate were the opacifiers used in the opaque glasses, cobalt in the blue glasses, copper in the turquoise glasses, iron in the yellow and greenish glasses, and iron and copper were found in the opaque red and aventurine glasses. Based on the concentrations of alumina and silica four different sources of silica were identified, allowing the classification of the glasses into the following compositional groups: low alumina (<2 wt%), which includes a sub-group of cristallo samples with SiO₂ > 70 wt%, medium alumina (2–3 wt%), high alumina (3–6 wt%) and very high alumina (>6 wt%). Comparison with genuine Venetian and façon-de-Venise compositions showed that two fragments are of Venetian production, one of Venetian or Spanish production and the remaining are of unknown provenance. In two fragments the glass of the decoration is probably Venetian or Spanish but the glass used in the body is also of unknown provenance.     

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.     

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.     

Ancient olive DNA in pits: preservation,amplification and sequence analysis

The olive tree (Olea europaea) was domesticated by vegetative propagation of selected wild individuals with superior fruit. Later, new cultivars were established repeatedly from feral trees or from crosses between wild, feral, and domesticated trees. Thus the genetic background of many contemporary domesticated lines is a mixture of ancient cultivars and local wild trees. Ancient DNA may illuminate the complicated process of olive domestication because such DNA sequences provide data about ancient genomes that existed closer to the domestication events. Well preserved DNA must be available for such studies, even though in the Mediterranean region, where olive cultivation took place, the climatic conditions are not favorable for DNA preservation. To select for well preserved pits we measured their proportions of lignin by IR spectroscopy, and correlated this with parameters of DNA quality such as template length in an olive-specific repeat array, and template quantity as determined by real-time PCR amplification. Archaeological pits that passed these tests did contain high quality ancient DNA. We present the first ancient olive DNA sequences and compare them to modern wild, feral and domesticated lines.     

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 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.     

Hemp in ancient rope and fabric from the Christmas Cave in Israel: talmudic background and DNA sequence identification

The “Christmas Cave”, a cave in the Qidron Valley near the Dead Sea and Qumran, has yielded a complex collection of plant-derived rope and fabric artifacts. Using polymerase chain reaction (PCR) to amplify DNA of the samples, we estimated the sizes and determined restriction patterns and base sequences of chloroplast genes, primarily rbcL (gene for the large subunit of ribulose bisphosphate carboxylase). DNA was successfully extracted from all samples, but was limited to sizes of approximately 200–300 base pairs. As expected, the DNA extracted from the samples was identified as coming primarily from flax (Linum usitatissamum L.), but two samples had a significant fraction, and all samples had at least a trace, of hemp (Cannabis sativa L.) DNA. Artifacts from the Christmas Cave were thought to date from Roman times, but it was thought possible that some could be much older. Accelerator mass spectrometry (AMS)-based ¹⁴C dating confirmed that the samples contained representatives from both the Roman and Chalcolithic periods. This paper provides a synthesis of DNA, isotope, and literary analysis to illuminate textile history at the Christmas Cave site.     

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.     

Wild or domesticated? An ancient DNA approach to canid species identification in South Africa's Western Cape Province

The fragmentary remains typical of archaeological fauna can prove impossible to identify when confronted with closely related sympatric species. In southern Africa domesticated dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) pose just such a problem, rendering difficult an accurate assessment of the timing of arrival of domesticated dogs in the region. Contextual evidence has suggested that canid remains from four Later Stone Age archaeological sites were likely to be domesticated dogs rather than jackals, and were subjected to ancient DNA analyses to determine species. Every tested specimen proved to derive from black-backed jackals. These data provide not only an unexpected window on southern African prehistory, but also highlight the value in applying ancient DNA techniques to archaeological species identification.     

The Temple of Venus (Pompeii): a study of the pigments and painting techniques

We report here on a study of 57 fragments of wall painting excavated from the Temple of Venus (Pompeii). These samples were characterised by a wide range of analytical methods. Data showed that the palette is varied, although not so broad as that found in other buildings in Pompeii, and is consistent with pigments used elsewhere in Pompeii and in the Roman Empire. It is composed of: natural earths, minerals and rare artificial pigments. Paintings are made up of thin paint layers (0.01–0.10 mm thick) strongly adhering to the underlying preparation layer. Nonetheless, in some cases thicker layers (up to 0.40 mm) were recognised, often spread on other previous painting layers. Samples were also compared with the microstratigraphic criteria developed in Piovesan (2009) to distinguish wall painting techniques. This comparison demonstrated that both fresco and lime painting techniques were adopted.     

Hydraulic structures of the Roman Mithraeum house in Augusta emerita, Spain

This paper deals with some of the hydraulic structures of Augusta Emerita (Mérida, Spain), specifically those found in the Mithraeum House. In particular, we describe and characterise the hydraulic mortars and coatings of the viridarium water channel. The recipient of the channel was covered with two hydraulic mortars and a finishing coating. Hydrated lime was used as binder. Calcite grains with different morphology have been observed in the mortars studied. The siliceous aggregate was composed of quartz, mica and feldspars. Ceramic fragments, which were added to the mortar to improve its hydraulic properties, were composed of quartz, mica, iron oxides, anorthite and an amorphous phase; aluminium-iron silicates were used as raw materials for their manufacture. We discuss the interactions between the hydrated lime and the surface of the ceramics in the mortars.