Identification of animal skin of historical parchments by polymerase chain reaction (PCR)-based methods

This study deals with establishing of a PCR-based strategy with the aim to recognize the animal origin of different historical parchments. This is one of relatively rare studies on the analysis of ancient DNA from parchments. Robustness of the PCR technology is demonstrated by successful identification of the animal species using only a small amount of DNA isolated from 12 parchment samples. Ten PCR-based assays specific for the detection of different animal species (Bos taurus, Ovis aries, Capra hircus, Sus scrofa, Oryctolagus domestica, Cervus elaphus, Capreolus capreolus, Dama dama) and two PCR assays utilizing universal primers were evaluated and optimized with the aim to find a rapid parchment identification method, which would be more reliable than the classical microscopic examination. The optimized PCR methods produced satisfactory results. Out of 12 investigated parchments, 9 items were unambiguously identified, DNA from 2 samples could not be amplified with any of the species-specific PCR assays, and only one parchment produced controversial results. The species-specific PCR results were confirmed by direct sequencing and PCR cloning with consequent sequencing. Our approach, including isolation of parchment DNA by chaotropic solid-phase extraction, optimization of the PCR programs and high-stringency annealing temperatures, demonstrated to be effective, easy and reliable for the analysis of historical parchment DNA. We consider this PCR-based strategy potentially useful also for investigation of other types of animal items conserved in museums, galleries or libraries.     

Digging deeper into the limits of ancient DNA research on syphilis

The search for the origins of syphilis has a long history in the medical and anthropological literatures. If we know more about the emergence of the pathogen that causes the disease in humans we will understand its evolution through time and space as well as shed light on its current state in living populations. Ancient DNA techniques used to isolate Treponema pallidum subsp. pallidum DNA from archaeological human specimens provide direct evidence of its existence in the past. However to date, only Kolman et al. (1999) have been successful in this endeavour, while other attempts have failed (e.g., Barnes and Thomas, 2006; Bouwman and Brown, 2005). Why has there been little success? This paper serves to compliment and add relevant information to Bouwman and Brown's and Barnes and Thomas' discussion concerning our inability to apply ancient DNA techniques to study venereal syphilis in past human populations.Our approach utilized 15 different human specimens from different geographies and different temporal periods: eight samples come from medically diagnosed individuals archived during the American Civil War period; six originate from the United Kingdom and predate 1492 with four of these samples having been previously analyzed by Bouwman and Brown and one sample comes from historic Canada. Human mitochondrial and amelogenin DNA, as well as several genes from the Treponema organism were analyzed revealing the relatively good preservation of human multi-copy and single copy DNA but not treponemal DNA. This study also incorporates a unique molecular experiment using rabbits infected with venereal syphilis to help illustrate that treponemal DNA disseminates to bone early during the first stages of infection but is not present in later stages of the disease using the techniques presented in this study.     

A flock of sheep,goats and cattle: ancient DNA analysis reveals complexities of historical parchment manufacture

Parchments comprise one of the most common and valuable sources of archaeological and historical data. Previous studies have shown that parchment also preserves genetic data. These data could be valuable for population studies, to understand past animal husbandry, the development of breeds and varieties and to comment on the provenance of parchments. To improve our understanding of DNA contained in parchments, we analysed genetic data, including both mitochondrial and autosomal loci, from 18th to 19th century English parchments which stable isotope analysis had indicated were well-preserved. DNA results were unexpected. All but one of the parchments produced multiple sequences matching several different species. Ion beam analysis ruled out surface treatments of the parchments (including ink and animal glues) as the origin of these multiple sequences. Our results suggest that the DNA content of parchment is more complex than previous research has suggested and that multiple stages of parchment manufacture, treatment and storage are preserved in parchment DNA extracts.     

Pig diet in medieval York: carbon and nitrogen stable isotopes

This study investigates bone stable isotopes from pigs from medieval York, to characterise the pigs' diet and to explore their contribution to isotopic values from contemporary human bones. Pig bones from the Swinegate (N?=?9) and Coppergate (N?=?14) sites were used for carbon and nitrogen stable isotope analysis to test the hypothesis that the majority of pigs in medieval York were yard-kept and fed on scraps and fish waste, elevating their nitrogen ratios. The results show that the Swinegate and Coppergate pigs gave nitrogen isotope values similar to contemporary sheep and therefore that animal protein made little or no dietary contribution. One sample showed C and N results consistent with more animal protein in the diet, and we propose that this could have been a yard-kept pig consuming human refuse. The majority of the data indicate that the pigs were eating a largely herbivorous diet and that pigs in medieval York may have been raised in rural or woodland locations rather than in the city.     

Variable nucleotide tandem repeat (VNTR) typing of two palaeopathological cases of lepromatous leprosy from Mediaeval England

Using ancient DNA methods, we have examined in detail two archaeological cases of leprosy from Mediaeval England. The first was a child skeleton with rhino-maxillary changes typical of lepromatous leprosy (LL). The second case was the skeleton of a male adult who showed both typical rhino-maxillary changes and osteitis/periostitis on the leg and foot bones. Bone powder was sampled from both cases and DNA extracts were prepared. These were subjected to a series of polymerase chain reactions (PCRs) specific for regions on the Mycobacterium leprae genome. The repetitive element RLEP was used for confirmation of M. leprae DNA and then three polymorphic regions were successfully amplified and sequenced to determine the number of variable nucleotide tandem repeats (vntr) at these loci. These were the microsatellite regions ML2344 and ML2172 and the minisatellite region ML0058. Genotyping data from the strains preserved within the skeletal remains were compared with those obtained for a reference strain of M. leprae. Variation at these three loci was found between both burials and the reference strain, indicating that vntr typing of LL cases from the archaeological record is a useful way of confirming disease and an additional means of authenticating aDNA data. This demonstrates the feasibility of targeting multiple loci for phylogenetic studies of leprosy strains from archival sources.     

Conflicts in natural and cultural resource management: Archaeological site disturbances by seals and sea lions on California's Northern Channel Islands

Abstract

California's Channel Islands currently have around 150,000 breeding seals and sea lions (pinnipeds). Driven to near extinction by 20th-century exploitation, many pinniped populations have recovered dramatically under federal and state management and continue to expand in number and distribution. Some of these pinniped populations are damaging or destroying coastal archaeological sites as they establish new breeding and haul-out areas—places occupied between periods of foraging activity—on upland landforms. We use archaeological excavations from a prehistoric village on San Miguel Island to illustrate the adverse effects pinnipeds can have on archaeological sites. Estimates based on excavations at Otter Point suggest that in one year nearly 10,000 kg of shellfish remains, 840,000 animal bones, and 1700 formal artifacts were lost to erosion caused by the activities of seals and sea lions. Our study documents potential conflicts between natural and cultural resource management suggesting the need for collaborative efforts between archaeologists and biologists to balance the conservation of both resources.     

Biofilm growth in human skeletal material from ancient Mesopotamia

Investigators have long recognised the effects of microbial activity on archaeological bone. These investigators, however, have focused on single or groups of microbes rather than on complex microbial aggregates such as biofilms, a focus that has affected our understanding of archaeological bone biodeterioration. In this paper, we report on the investigation of a biofilm in archaeological human bone from the site of Tell Leilan, Syria (2900–1900 BCE). Scanning electron microscopy indicated that the biofilm is characterised by single cells and microcolonies of bacteria and fungi, as well as calcite crystals that were all embedded within extracellular polymeric substances. Using culture techniques and DNA sequencing, we isolated and identified several microbes from the biofilm including Amycolatopsis sp., Penicillium chrysogenum, Aspergillus sp., Chaetomium sp., and Cladosporium sp. Having characterised the Leilan biofilm, we are now closer to understanding the complex process of bone biodeterioration in archaeological bone collections.     

DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.     

On the elimination of extraneous DNA in fossil human teeth with hypochlorite

Elimination of extraneous DNA in fossil specimens is of paramount importance for the successful isolation and analysis of authentic DNA; this is especially true when the specimens are of human origin. Bones and teeth are commonly decontaminated with bleach containing the powerful oxidising hypochlorite ion. The procedures involve either submersion in or wiping with the chlorine agent. Using the radioactive isotope Cl³⁶ we showed that submersion of fossil teeth in solutions of small ions such as Cl⁻ or hypochlorite, ClO⁻, cause that they migrate right into the pulp. This may lead to the unwanted destruction of authentic DNA. However, using pairs of teeth from the remains of four ancient Europeans (1000–2000 YBP) as well as tooth and hair from an Inuit skull (>300 YBP) we provide evidence that at least some endogenous human fossil DNA survives in powdered pulp/dentin that has been submersed in 2% hypochlorite. Further, we show that powdered pulp/dentin deliberately contaminated with huge amounts of a 414 bp PCR product is effectively decontaminated by suspension in 2% hypochlorite for 5 min. Decontamination of fossil material from teeth may therefore be accomplished by a short direct action of hypochlorite on the powdered specimen rather than less controllable and less efficient external treatments of the whole specimen.     

Is X-ray diffraction able to distinguish between animal and human bones?

The possibility of determining the human or animal origin of bones from the lattice parameters of their inorganic bioapatite phase, when subjected to a high temperature treatment using the powder X-ray diffraction (XRD) technique, has been explored on a wide number of specimens. Forty-two animal bones were treated in a furnace at 1100 °C for 36 min and compared to 53 cremated human bones from a range of ancient necropolises. The X-ray diffraction patterns of bioapatite were simulated using both monoclinic P21/b and hexagonal P63/m structures to verify any occurrence of phase transformation and any difference in the lattice parameters due to the model. It was determined that the differences between the a-axis and c-axis of the monoclinic and hexagonal lattice were unimportant. Some outlying values were revealed to be caused by the presence of chlorine ions diffused into the apatite structure increasing its average unit cell values. Nevertheless, our results clearly show that in terms of lattice parameters the variability of human specimens are completely overlapped by the non-human variability making the use of XRD in order to distinguish animal from human bones questionable.     

Evidence for Differential Ancient DNA Survival in Human and Pig Bones from the Norse North Atlantic

Current models of DNA degradation and previous research on Icelandic human skeletons predict ancient DNA preservation in the Norse North Atlantic faunal remains to be excellent. In contrast, we found that DNA preservation in Viking‐Age pig remains was poor. We posit that this discrepancy in DNA survival between human and faunal remains is due to differing taphonomies. Our results highlight that DNA degradation is strongly dictated by micro‐environmental taphonomic processes even in regions where the climate is conducive to DNA survival. Due to these differences, DNA preservation in animal remains may not be suitable proxies for DNA preservation in associated human remains. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

The Archaeological Site of Persepolis (Iran): Study of the Finishing Technique of the Bas‐Reliefs and Architectural Surfaces

One of the aims of the 5‐year Iranian/Italian project for Persepolis, called ‘From Palace to Town’ was to contribute to the conservation of the stone monuments of the imperial site. As part of the activities dedicated to this purpose, a diagnostic study was carried out. Various aspects were considered: petrographic characterization of the stone, forms and factors of decay, and in situ testing of suitable conservation treatments. The present paper reports on the unexpected results of the study on the finishing of architectural surfaces. The results obtained on a limited, but nevertheless significant, number of samples collected from the monuments of the imperial Terrace, allow us to state that the dark grey limestone used for several (or many?) monuments was covered on purpose with a thin, fine whitish layer containing fluorapatite, as major component, and calcite. It is highly probable that the fluorapatite was obtained from calcined animal bones and that slaked lime was used as a binder. Further evidence for this is the discovery of a kiln with the remains of calcined bones and, nearby, a waste pit with animal bones containing fluorapatite. A second white layer, obtained with barium sulphate, was detected in one of the samples beneath the external, earthy encrustation. It could be perhaps interpreted as the remnants of a polychrome finishing.     

Deficiencies and challenges in the study of ancient tuberculosis DNA

We explore the standards of research and reporting needed to justify the destructive analysis of archaeological human bone for biomolecular studies of ancient tuberculosis (TB). Acceptable standards in osteological interpretation have been met in some biomolecular papers, but there are also cases where insufficient care has been taken in distinguishing between pathognomonic lesions and those that are ‘consistent with’ a diagnosis of TB. Some biomolecular studies have failed to recognize that archaeological bones might be contaminated with environmental mycobacteria whose DNA could give rise to false positives in polymerase chain reactions directed at members of the Mycobacterium tuberculosis complex. The difficulties of applying spoligotyping to ancient DNA have also been underestimated and conclusions drawn from such analyses are often weakly supported. Assumptions that mycobacterial DNA preserves better than human DNA, and that contamination with modern DNA is less of a problem, has led in some cases to a laxity in research standards with insufficient attention paid to the need to authenticate ancient DNA results. We illustrate our concerns by reference to a recent paper reporting biomolecular detection of ancient TB DNA in skeletons from the eastern Mediterranean Neolithic settlement of Atlit-Yam. We are unconvinced that the skeletal evidence presented in this paper gives sufficient indication of TB to warrant destructive analysis, and we are concerned that during the biomolecular part of the project inadequate attention was paid to the possibility that results might be due to laboratory cross-contamination or to amplification of environmental mycobacterial DNA present in the bones.     

Biochemical and physical correlates of DNA contamination in archaeological human bones and teeth excavated at Matera,Italy

The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In this study the levels of endogenous contamination (i.e. present within the sample prior to sampling for the DNA analysis) are assessed within human bone and teeth specimens sampled from the cemetery of Santa Lucia alle Malve, Matera, Italy. This site is of exceptional interest, because the samples have been assayed for 18 measures of biochemical and physical preservation, and it is the only one identified in a study of more than 107 animal and 154 human bones from 43 sites across Europe, where a significant number of human bones was well preserved. The findings demonstrate several important issues: (a) although teeth are more resilient to contamination than bone, both are readily contaminated (presumably through handling or washing), and (b) once contaminated in this way, both are difficult (if not impossible) to decontaminate. Furthermore, although assessed on bone samples, several of the specific biochemical and physical characteristics that describe overall sample preservation, levels of microbial attack and related increases in sample porosity directly correlate with the presence of observable contamination in both bone and teeth samples from individual samples. While we can only speculate on the cause of this relationship, we posit that they provide useful guides for the assessment of whether samples are likely to be contaminated or not.     

The impact of manuring on nitrogen isotope ratios in cereals: archaeological implications for reconstruction of diet and crop management practices

Recent archaeological studies of human diet have used stable nitrogen isotope ratios (δ¹⁵N) from human bone collagen to infer the relative importance of terrestrial plant and animal foods. This approach is based on widely observed enrichment of δ¹⁵N up the food chain, plants having distinctly lower values than the herbivores that consume them. Studies of early farming diets in Britain, Denmark and Germany have tended to detect relatively high δ¹⁵N values (e.g. c. +9‰), interpreted as evidence of a diet largely based on animal products, though archaeobotanical evidence for crop cultivation (e.g. carbonised cereal grain and chaff) is widespread. This paper investigates the impact of manuring on δ¹⁵N values in modern cereals, and of charring on these cereal values. The results from two long-term experiments demonstrate that manuring significantly raises δ¹⁵N in cereal grain and chaff. Depending on manuring levels and frequency, it appears that human diets with a major component of such grain would conventionally be interpreted as indicating a largely animal-based diet or a mixed plant/animal diet. Moreover, preliminary analyses of experimentally charred grain and chaff from manured and unmanured conditions are promising for the extraction of reliable ancient δ¹⁵N values from archaeobotanical cereal remains. The wider implications of these results, and the need for further work, are discussed.     

Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA

A principal problem facing human DNA studies that use old and degraded remains is contamination from other sources of human DNA. In this study we have attempted to contaminate deliberately bones and teeth sampled from a medieval collection excavated in Trondheim, Norway, in order to investigate this poorly understood phenomenon. Five pairs of teeth and bone samples were bathed in water containing various concentrations (from 10⁻⁹ and 10⁻²¹ g/l) of purified ΦX174 DNA. Subsequently the samples were subjected to a routine decontamination protocol involving a bleach bath followed by exposure to λ=254 nm ultraviolet light, prior to DNA extraction and analysis for evidence of the persistence of the contaminant. The results support previous speculation that bone is more susceptible to water‐borne sources of contaminant DNA, although both bone and teeth are readily contaminated and are difficult to decontaminate using the tested protocol. We believe that this is largely due to the porous nature of bone and teeth facilitating the deep penetration of the contaminant DNA. To simulate a more realistic handling situation, 27 further teeth were directly handled and washed, then decontaminated, prior to assaying for the residual presence of the handler's DNA. Surprisingly, although our results suggest that a large proportion of the teeth were contaminated with multiple sources of human DNA prior to our investigation, we were unable to contaminate the samples with further human DNA. One potential explanation may be the deposition of sediment or other structural changes that occur within the samples as they desiccate post‐excavation, which may protect samples from subsequent contamination, but also prevent the efficacy of bleach baths in decontaminating specimens. Copyright © 2006 John Wiley & Sons, Ltd.     

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.