‘Old wood’ effect in radiocarbon dating of prehistoric cremated bones?

Numerous reports of successful radiocarbon dating of cremated bones have emerged during the last decade. The success of radiocarbon dating cremated bones depends on the temperature during burning and the degree of recrystallisation of the inorganic bone matrix. During cremation bones undergo major morphological and mineralogical changes which have raised some interesting questions and discussion on the origin of the carbon source in archaeologically cremated bones. Recent laboratory experiments reveal that the properties of the combustion atmosphere play a significant role regarding the source carbon in cremated bones. Thus radiocarbon dating cremated bones is potentially dating the wood used for the cremation fire. Here we compare a high precision radiocarbon dated human bone with an associated dendrochronological age from an oak coffin. We find that the age discrepancy between the dendrochronological age and the cremated bone of 73 ± 26 ¹⁴C yr is best accounted for by the so called ‘old wood’ effect.     

Distinguishing Remains of Human Cremations from Burned Animal Bones

Abstract

Archaeological remains of human cremations, often consisting of a few calcined bone fragments unassociated with any other indicators of human mortuary behavior, are sometimes difficult to distinguish from mammalian remains representing ancient garbage. Although experimental and archaeological studies of human cremation may facilitate recognition of cremated or otherwise burned mammal bone, they do not provide the necessary criteria to distinguish cremation from other kinds of burning and, therefore, to recognize, immediately, a human cremation in archaeological context. Experiments were conducted to provide criteria for distinguishing the remains of cremated whole large mammals from intensively burned fresh, anhydrous, archaeological, boiled, and baked bones of large mammals. Results indicate that distinctive patterns of cracking, fracturing, and warping of calcined bone, and especially the absence of evidence of perimortem fracture, may alert the excavator to the possibility of a human cremation.     

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.     

Characterisation and blind testing of radiocarbon dating of cremated bone

The success of radiocarbon dating of burned or cremated bones depends on the exposed temperature during burning and the degree of re-crystallisation of the inorganic bone matrix. We present a method for characterisation of likely cremated bones by employing visual inspection, infrared spectrometry and carbon stable isotope analysis on the bio-apatite fraction. The method of radiocarbon dating of cremated bones was tested by dating paired samples of bone and associated context materials such as pitch, charcoal and a dendrochronologically dated oak coffin. The dating of these paired test samples were largely performed as blind tests and showed excellent agreement between pitch and bone. The weighted mean age difference of all test samples is observed to −9 ± 60 ¹⁴C yr. To test the indicators and the effects of the degree of burning, a Late-Neolithic human individual has been studied, as this individual exhibits the full spectrum from low temperature burning (charred) to high temperature (“cremated”) from one end of a single bone to the other. This is reflected as a marked step in numerous parameters as well as in a significant difference in ¹⁴C age between the charred and the cremated bone samples.     

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.     

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.     

Biological mineral content in Iberian skeletal cremains for control of diagenetic factors employing multivariate statistics

The aim of this study was to define a strategy for a correct selection of bone samples by employing inductively coupled plasma optical emission spectroscopy (ICP-OES) for reconstructing the biological mineral content in bones through the determination of major elements, trace elements and Rare Earth Elements (REE, lanthanides) in skeletal cremains of ancient Iberians (III–II B.C), discovered in the Necropolis of Corral de Saus (Moixent, Valencia) between 1972 and 1979. The biological mineral content was determined taking into account diagenetic factors. A control method for a better reading of results was applied. To explore large geochemical datasets and to reduce the number of variables, Principal Component Analysis (PCA) was used, thus, providing a deeper insight into the structure of the variance of the dataset. PCA shows that the elemental profiles of bone and soil samples are clearly different. Bone samples obtained from the outer bone layer were shown to have a different elemental composition; more similar to soil samples than samples of the inner bone layer. PCA scores and loadings plots were preferred to dendrograms obtained using Cluster Analysis, due to the limits of the latter one to appreciate the spatial ordering of samples. Partial least squares discriminant analysis (PLS-DA), a frequently used supervised classification method, was applied to differentiate between degradation states of bone samples. PLS-DA results obtained in this study confirmed that changes derived from different burning conditions were associated with transformations in the mineral part of the bones. Accordingly, carbonized bones can be differentiated from cremated bones. Class assignment of bone samples with uncertain thermal conditions in dependence on their elemental composition has shown to be feasible. Consequently, for biochemical-archaeological studies the analysis and statistical classification of carbonized and cremated archaeological bones, as well as those exposed to unknown thermal conditions together with experiments in modern bones, are recommended.     

The effect of cremation on the study of trace elements

Cremation was a frequent practice during the Bronze and Iron Ages in Europe. The destruction caused by this rite has meant that there are few anthropological studies of this period and that the amount of information that has been obtained is limited. Most studies deal with the number of individuals and the temperature of cremation. At present, however, studies are under way to determine the possibility of carrying out analyses of trace elements on cremated individuals in order to determine their diet. The present study, carried out at a necropolis with two kinds of burial (S'Illot des Porros, Mallorca, Iron Age), presents new data regarding the diagenetic effects differentiating cremated from buried bones. The levels of Sr, Ca, Ba, Zn, Cu and Mg in the spongy and cortical tissue of 197 femurs are analysed. The results obtained at this necropolis demonstrate that only the levels of calcium and magnesium in the cortical tissue of cremated bones increase, so diagenesis acts in the same way in cremated and non-cremated bones in this necropolis.     

Analysis of ancient Japanese society through mitochondrial DNA sequencing

In this study, DNA was extracted from human bones recovered from a 2000-year-old archaeological site located in northern Kyushu in southwestern Japan. Part of the mitochondrial control region was amplified by a polymerase chain reaction. Mitochondrial DNA sequences determined from 55 individuals were classified into 16 different types. Comparing the location of the burial site and the sequence types, people buried at separate sites were shown to have different maternal lineages. Our palaeomolecular biological findings strengthen the opinion that social differentiation began during this period in Japan, a fact that is generally accepted among archaeologists. The results of this study show that intensive analysis of ancient DNA from archaeological sites is a useful tool for investigating the social systems of vanished populations.     

A Mediaeval Case of Lepromatous Leprosy from 13–14th Century Orkney, Scotland

Erosion in the 1960s resulted in exposure of human skeletal remains from a Norse Christian cemetery at Newark Bay, Orkney, Scotland. One set of remains showed osteological evidence of advanced lepromatous leprosy, but the absence of bones from the lower limbs precluded definitive diagnosis. The aim of the present study was to determine whether Mycobacterium leprae could be detected in bone extracts, as a means of confirming the diagnosis of leprosy. Bone samples were examined from the suspected leprosy case and from a second contemporary burial thought to be free of disease. DNA was amplified by polymerase chain reaction (PCR) using primers specific for a repetitive element (RLEP) characteristic of M. leprae. Additional PCR tests specific for Mycobacterium tuberculosis and for amelogenin (a human gene suitable for sex determination) were also applied to the samples. M. leprae DNA was detected only in the skull sample from the suspected leprosy case. The DNA sequence was identical to that found in present day isolates of M. leprae. Positive results were obtained only using a PCR reaction designed to amplify relatively short stretches of DNA (<175 bp), suggesting the microbial DNA had undergone extensive fragmentation. There was no evidence of M. tuberculosis DNA in bones from the leprosy suspect or control individual. The ability to recover ancient samples of DNA provides an opportunity to study long-term evolutionary changes that may affect the epidemiology of microbial pathogens.     

Skeletal analysis and mortuary practice in an Early Roman chamber tomb at Kenchreai,Greece

Recent exploration of the site of Kenchreai, the eastern port of Corinth in southern Greece, has focussed on a cemetery of subterranean chamber tombs dating chiefly to the Early Roman period (middle‐late 1st to 3rd centuries AD). The copious but fragmentary human bones and teeth found in Tomb 10 have been disturbed since burial by natural processes, including bedrock erosion and the infiltration of moisture, roots and basic sediment, and by destructive looters. Nonetheless, the remains furnish considerable information about the mortuary practices of wealthy residents. Analysis of the remains in their archaeological and taphonomic contexts reveals that Tomb 10 contained at least 23 individuals, 15 inhumed in loculi and 8 cremated and placed in niches, sometimes in urns. The identification of males, females and subadults among the burned and unburned bones and teeth suggests that spouses, parents and children probably from several generations of the same lineage or household were buried here. The nature of the cremated fragments reflects a laborious process during which mourners burned bodies on substantial pyres at extremely high temperatures for a long time. Then they carefully extracted a representative sample of small fragments from throughout the reduced skeleton for burial at the tomb. This study contributes to a better understanding of mortuary practices in the Roman East, particularly the Greek world, where the chamber tomb was a common sepulchral type. Copyright © 2009 John Wiley & Sons, Ltd.     

Bone preservation and DNA amplification

The use of ancient DNA has increased during the past two decades in several scientific disciplines. However, the underlying mechanisms of DNA degradation in bone tissue are poorly understood. Here we address the importance of hydroxyapatite and collagen for DNA preservation in bone. We used two series of bones and teeth, one set of modern experimentally degraded bovid bones and one set of ancient horse bones/teeth. From these samples, we measured crystallinity, DNA presence and extracted collagen. The mtDNA fragments, parts of cytochrome b and the D–loop were amplified and sequenced. Our results show that presence of DNA was strongly related to the crystallinity in the hydroxyapatite and to the amount of collagen. This suggests that the hypothesis that hydroxyapatite has a crucial role in DNA preservation in calcified tissue is valid; and hydroxyapatite and collagen can be used to indicate whether DNA is present in the material. This is what would be expected if DNA is adsorbed to and stabilized by hydroxyapatite in calcified tissue, and collagen is part of the complex system that preserves DNA in bone tissue. Further, since collagen is the preferred material for radiocarbon dating, such bones may be a starting–point for a DNA analysis.     

A DNA test for sex identification in cattle confirms osteometric results

It is of vital importance to be able to sex identify cattle remains to understand the strategies and importance of cattle husbandry in an ancient society. This is usually done from osteoarchaeological assemblages and often relies on measurements of metapodials. The breadth measurement of the distal trochlea is considered an easy way to identify the sex. Bones from males appears to be easily distinguishable from female counterparts, although it has been complicated to find an external control for the morphological results. Here we investigate the reliability of these particular morphometrics for sex identifying cattle bones with molecular genetics. We use a sex discriminating single nucleotide polymorphism in the ZFXY gene and we apply it to DNA from the bones. To keep the fragment size short and suitable for ancient DNA we base the test on a SNP. The test confirms the osteological sex identification in all cases were DNA could be retrieved. This molecular method can also be used when no fragments suitable for osteological sex identification can be found or when the measurements are non-conclusive.     

Mitochondrial DNA from 3000-year old chickens at the Teouma site,Vanuatu

Chickens were part of the Lapita cultural complex, transported into and through the Pacific by prehistoric colonists; as such they can be used as a proxy for tracking prehistoric migration and interaction. The Lapita site of Teouma in Vanuatu is well known for the recovery of complete dentate stamped pots and a cemetery containing the largest collection of Lapita period skeletons ever found. Chicken bones recovered from these excavations provide the first ancient DNA sequences from any commensal organism directly associated with a Lapita context. The ancient mtDNA sequences obtained from two Teouma chicken bones are compared with previously published archaeologically derived ancient DNA sequences to extend our understanding of the spread of chickens in Pacific prehistory. The results also show that the haplogroup E signature was present in very early populations of chickens transported into Remote Oceania. This study also adds to the suite of available data relating to isotopic signatures for commensal animals during the early settlement of Vanuatu and may reveal a different diet for Teouma chickens than those from other early prehistoric assemblages in the Pacific.     

A new calibration of the XRD technique for the study of archaeological burned human remains

A new calibration of human bones as a function of programmed temperature (200–1000 °C) and time (0, 18 and 60 min) is presented and discussed in order to investigate the issues related to the study of cremated bone remains by using the powder X-ray diffraction approach. The experimental results confirm the growth of hydroxylapatite crystallites as a function of the applied temperature, with a sigmoid behaviour that has been parameterized to the experimental data points. Particularly, it was observed that the thermal treatments for 60 min anticipate of about 100 °C the effects that are otherwise observed after the treatments for 0 min. The developed procedure was subsequently applied to cremated remains of various archaeological sites of Spain and supplied precise information not only about the temperature reached during the funerary rites, but also on the presumed duration for the cremation.     

Bone Preservation and Ancient DNA: The Application of Screening Methods for Predicting DNA Survival

Given the technical difficulties associated with ancient DNA research, any methods that help to identify samples that will yield amplifiable DNA will be of great value. This study examined the relationships between gross preservation, histological preservation, bone size and the ability to amplify short fragments of mitochondrial DNA in 323 goose humeri from the Anglo-Saxon site at Flixborough. Bone size was not a good predictor of the presence of amplifiable DNA, but there was a significant association between both gross and histological preservation and DNA survival. This suggests that it is worthwhile to preferentially select morphologically well-preserved bones for ancient DNA studies. Our results with ancient avian bone mirror those previously obtained with mammalian archaeological bone, although the relationship between DNA survival and histological preservation was stronger for the latter.     

Arsenic accumulation on the bones in the Early Bronze Age İkiztepe Population,Turkey

In this study, arsenic, copper and lead content of a group of human and animal bones recovered from the Early Bronze Age ?kiztepe site have been analyzed using ICP-MS method. Average arsenic value of 90 femur bones of a human was found to be 15.0 ± 5.79 ppm which was varied among age and sex groups, and among species. Origin of arsenic accumulation in bones was diagenetic because overall the groups were highly variable. The distribution of metallic items among the burials had a big affect on the arsenic uptake of the bones. Copper and lead values supported the diagenetic arsenic accumulation in the bones as well. Their values on the bones were not as high as those for the individuals involved in metal working activities in the ancient world. Judging from these data, it is concluded that ?kiztepe people did not produce the manufactured metallic items using arsenical copper but might have been imported from the other sites.     

The Protohistoric ‘Quicklime Burials’ from the Balearic Islands: Cremation or Inhumation

Traditionally, the Balearic so‐called ‘quicklime burials’ of the Iron Age have been considered to be inhumations in quicklime. The general appearance of the bones, however, resembles more closely that of cremated bones. Laboratory tests reveal that the observed features of the bones from these burials, including cracks, thumbnail fractures and warping, cannot be explained by an inhumation in quicklime. The δ ¹³C value, Fourier transform infrared spectra, SF values and the low carbon content of the apatite moreover indicate a thermal manipulation of the bones. The ¹⁴C content is depleted with regard to the accepted archaeological age of the sample, which can best be explained by carbon exchange between bio‐apatite and fossil CO₂ released during the heating of limestone. This implies that the Balearic ‘quicklime burials’ must be interpreted as an elaborate cremation practice in presence of limestone. Copyright © 2013 John Wiley & Sons, Ltd.     

Stable carbon- and nitrogen-isotope ratios and electron spin resonance (ESR) g-values of charred bones: changes with heating and a critical evaluation of the utility of g-values for reconstructing thermal history and original isotope ratios

Changes in the stable carbon and nitrogen isotope ratios of modern bone samples heated to a variety of times and temperatures were used to determine the effect of heating on isotope ratios and the retention of organic matter in charred bones. For organic extracts produced by slow demineralization in weak acid, δ¹³C values were unchanged, while δ¹⁵N values increased by up to 5‰ and were primarily determined by heating temperature. Changes in the electron spin resonance (ESR) g-value of whole bone and organic extracts were also measured. For organic extracts from charred bones, the g-value was well-correlated with δ¹⁵N and temperature, suggesting that g-values could be used to estimate the charring temperature and original δ¹⁵N values of charred bones. Thus, g-values from demineralized extracts could be very useful in forensic investigations where it would important to reconstruct the thermal history of burned bones. Isotope ratios and g-values of demineralized extracts from four prehistoric components at three sites that produced cremated human bone were used to test whether the same approach can be applied to archaeological materials. While carbon isotope ratios of the prehistoric samples were similar to those of uncharred specimens, nitrogen isotope ratios were increased and the g-value corrections for nitrogen isotope ratios were not effective.