Ancient Bone Research Articles

Ancient and modern bone diagnosis: Towards a better understanding of chemical and structural feature alterations

Chemical and structural alterations hold great importance in the field of diagenesis. Attenuated Total Reflectance − Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a valuable method for examining bio-apatite composition changes. Infrared spectroscopy (IR) and X-ray diffraction (XRD) were employed to analyze both modern and archaeological bone specimens. Organic and mineral component changes in modern and ancient samples were investigated. Ancient bone samples were collected from two archaeological sites in Jordan, dating back to the Iron and Byzantine ages. IR results indicated that collagen cross-links and mineral maturity are higher in modern bones compared to ancient bones. Additionally, the crystallinity index is higher in modern bones than in archaeological bones, while the carbonate to phosphate ratio (C/P) is lower in modern bones than in ancient ones. Curve fitting was applied to reveal the carbonate substitution inside the hydroxyapatite lattice within the IR region from 850 to 890 cm−1. A2-type carbonates (identified as υ2 of CO32−) denote hydroxyl site substitution, and B-type carbonates represent a substitution to the phosphate site. The full width at half maximum (FWHM) of the peak at 604 cm−1 from IR spectra reveals that crystallinity is higher in modern bones, as confirmed by the FWHM of the (002)-apatite pattern in XRD. Statistical analysis was conducted to validate these findings, ensuring the robustness of the results. Finally, the results obtained in this investigation align with previous literature reports regarding ATR-FTIR ratios. This suggests that modern bones have better crystallinity compared to ancient bones. Furthermore, the ATR-FTIR ratios indicate that the hydroxyl and phosphate sites of modern bones undergo more substitution than older bones. The findings of this study not only enhance our understanding of the diagnostic processes in archaeological bone specimens but also have broader implications for fields such as archaeology, anthropology, and forensics, where the analysis of bone composition and structural changes can provide valuable insights into the history and characteristics of ancient remains.

Multi-proxy investigations of Bronze Age diet and environment in the hyper-arid eastern Tarim Basin (Lop Nur), northwest China

Humans have a long history of occupying ecologically marginal regions, and the ancient cultural mechanisms of adaptation to these challenging environments remain poorly understood. We examine archaeological deposits from a Bronze Age settlement in the hyper-arid eastern Tarim Basin (Lop Nur), identifying well-preserved plant remains and bone fragments that shed light on the palaeodiet and past environmental setting. We use the newly developed peptide mass fingerprinting method of ZooMS (zooarchaeology by mass spectrometry) and TimsTOF MS (trapped ion mobility time of flight mass spectrometry) to identify fragments of ancient fish bones as Cyprinidae and possible Salmonidae, indicating both their consumption and the former presence of intra-settlement fresh water-bodies. Remains of littoral and aquatic plants attest to a marshy palaeo-delta at the confluence of the Tarim and Kongque rivers, testifying to a former biodiverse oasis in the Lop Desert when humans occupied the site. We conclude that the use of both aquatic and desert peripheral resources, combined with the intentional management of local environments, were all essential prehistoric adaptations. In addition, we show that radiocarbon dates of aquatic remains have an offset spanning hundreds to thousands of years, due to a freshwater reservoir effect, requiring careful consideration of ages for archaeological bones in the broader region.

A New Technique for Age Assessment of Ancient Bones by Using Cr-39 Track Detector

A new highly sensitive technique is proposed to estimate the age of ancient bones by the specific activity of 226Ra, which is determined by the registration of 222Rn α–particles using a CR-39 track detector in an isolated chamber. Comparison of specific activity of 226Ra in the bones of archanthropus, southern mammoths, and dinosaurs between standard bones showed that there is a direct correlation between age and specific activity of 226Ra in these bones. The work also shows the specific activity of 226Ra in soils taken from the sites of detection of the studied skeletons.

Nanoscopic imaging of ancient protein and vasculature offers insight into soft tissue and biomolecule fossilization

Fossil bones have been studied by paleontologists for centuries. Despite this, empirical knowledge regarding the progression of biomolecular (soft) tissue diagenesis within ancient bone is limited; this is particularly the case for specimens spanning Pleistocene directly into pre-Ice Age strata. A nanoscopic approach is reported herein that facilitates direct imaging, and thus empirical observation, of soft tissue preservation state. Presented data include the first extensive nanoscopic (up to 150,000× magnification), three-dimensional (3D) images of ancient bone protein and vasculature; chemical signals consistent with collagen protein and membrane lipids, respectively, are also localized to these structures. These findings support the analyzed permafrost bones are not fully fossilized but rather represent subfossil bone tissue as they preserve an underlying collagen framework. Extension of these methods to specimens spanning the geologic record will help reveal changes biomolecular tissues undergo during fossilization and is a potential proxy approach for screening specimen suitability for molecular sequencing.

Tracking of Infectious Diseases in Shahr-i Sokhta (Burnt City) during the Bronze Age (ca. 3200-2200 BCE) through Anemic Signs Observed in Excavated Human Skeletons

Background: The intriguing area of paleopathology merges the disciplines of archeology and biological studies. Using this line of research, it is possible to identify diseases that have left skeletal traces in the past. In addition, diseases such as various anemia that occur in childhood, when bone tissue is soft and retains evidence, can be identified in ancient bones. Cribra orbitalia (Co), cribra cranii (Cc), and porotic hyperostosis (Ph) were ancient skeletal remains' most common degenerative anomalies. Methods: Shahr-i Sokhta dated back to 3200-1800 BCE, is the subject of our research; it is located in Sistan and Baluchistan province (Iran). The research was done on the archaeological data collected during the MAIPS expeditions at Shahr-i Sokhta (2017–2021) kept at the storage of the excavated materials on the site. The skeletal remains were examined for bone abnormalities such as Co, Cc, and Ph. These symptoms were analyzed to obtain traces of anemia-related diseases at this site. Data has been utilized following the Data Collection Codebook Results: Ninety-six adults were studied while the anemic signs of CC and Co are respectively seen in 27/72 (37.5 %) and 10/57 (17; 5 %), and these samples have been kept for future analysis. Conclusion: Bones may narrate a person's life, their gender and how old they were when they died besides the diseases they had. Some of the skeletons show signs of anemia, Classical paleopathology lets us to reconfirm studying diseases by further targeted sampling using molecular methods.

From the grave to the lab: evaluation of archaeological human bone preservation based on micro-computed tomography analysis

Timely understanding of the preservation status of archaeological human bones is the foundation for conducting scientific archaeological work. This paper applies Micro-CT technology to analyze the microscopic preservation status of ancient human femora unearthed from the Rui State site in Liangdai Village, Hancheng, Shaanxi, the Chejiasi Cemetery in Baoji, and the Ouerping Cemetery in Xizhou Village, Yushe, Shanxi, and obtained imaging results and cortical bone porosity (Ct.Po), bone volume fraction (BV/TV), and bone density (BMD) parameters for 9 samples. The results show that the poorly preserved fragile group has lower Ct.Po, BV/TV, and BMD, and macroscopically presents fragile and porous features; while the well-preserved dense group has relatively higher Ct.Po, BV/TV, and BMD, and macroscopically appears solid and dense. This study employs Micro-CT technology to analyze the micro-preservation status of human bones from historical periods, confirming the effectiveness of this technology in revealing the microstructure of ancient human bones, and providing a reference for establishing a human bone preservation status evaluation system.

"View Wisps of Smoke Rising from The Stove Again"

Chinese calligraphy is the carrier of cultural heritage and has profound cultural connotations. The origin of Chinese calligraphy can be traced back to the ancient oracle bone inscriptions and Chinese bronze inscriptions period, which has a history of several thousand years. It is a key element of Chinese culture and an important means to inherit China's excellent traditional culture in the new era. Under the impact of information technology and “fast food ” culture, various computer fonts and personalized fonts have emerged, and people tend to use some "odd" fonts, and their cultural awareness is gradually weakened. In the new era, Chinese calligraphy culture is full of vigor, which requires us to deeply understand the essence of calligraphy culture, respect history and culture better, enhance cultural identity, and firmly believe in culture. We should adapt to the development of science and technology, using artificial intelligence technology to add new energy to traditional culture, inherit the historical context better, create an atmosphere where everyone strives to be a cultural inheritor, and carry forward the spirit of writing.

A fresh perspective on infrared spectroscopy as a prescreening method for molecular and stable isotopes analyses on ancient human bones

Following the development of modern genome sequencing technologies, the investigation of museum osteological finds is increasingly informative and popular. Viable protocols to help preserve these collections from exceedingly invasive analyses, would allow greater access to the specimens for scientific research. The main aim of this work is to survey skeletal tissues, specifically petrous bones and roots of teeth, using infrared spectroscopy as a prescreening method to assess the bone quality for molecular analyses. This approach could overcome the major problem of identifying useful genetic material in archaeological bone collections without resorting to demanding, time consuming and expensive laboratory studies. A minimally invasive sampling of archaeological bones was developed and bone structural and compositional changes were examined, linking isotopic and genetic data to infrared spectra. The predictive model based on Infrared parameters is effective in determining the occurrence of ancient DNA (aDNA); however, the quality/quantity of aDNA cannot be determined because of the influence of environmental and local factors experienced by the examined bones during the burial period.

Collagen of ancient bones gives an indication of endogenous DNA preservation based on next-generation sequencing technology

Collagen of ancient bones gives an indication of endogenous DNA preservation based on next-generation sequencing technology

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Comparing extraction method efficiency for high-throughput palaeoproteomic bone species identification

High-throughput proteomic analysis of archaeological skeletal remains provides information about past fauna community compositions and species dispersals in time and space. Archaeological skeletal remains are a finite resource, however, and therefore it becomes relevant to optimize methods of skeletal proteome extraction. Ancient proteins in bone specimens can be highly degraded and consequently, extraction methods for well-preserved or modern bone might be unsuitable for the processing of highly degraded skeletal proteomes. In this study, we compared six proteomic extraction methods on Late Pleistocene remains with variable levels of proteome preservation. We tested the accuracy of species identification, protein sequence coverage, deamidation, and the number of post-translational modifications per method. We find striking differences in obtained proteome complexity and sequence coverage, highlighting that simple acid-insoluble proteome extraction methods perform better in highly degraded contexts. For well-preserved specimens, the approach using EDTA demineralization and protease-mix proteolysis yielded a higher number of identified peptides. The protocols presented here allowed protein extraction from ancient bone with a minimum number of working steps and equipment and yielded protein extracts within three working days. We expect further development along this route to benefit large-scale screening applications of relevance to archaeological and human evolution research.

Evaluating methods for determining mercury concentrations in ancient marine fish and mammal bones as an approach to assessing millennial-scale fluctuations in marine ecosystems

Millennial-scale datasets of heavy metals in biota are difficult to obtain but are important for determining patterns and underlying drivers of toxicant concentrations. This is particularly important to better discriminate contemporary natural and anthropogenic sources. Globally mercury is a contaminant of concern. Post-industrial increases in mercury in arctic biota have been documented and monitoring of Steller sea lions (Eumetopias jubatus) in the Aleutian Islands, Alaska, has revealed a high proportion of pups with fur mercury concentrations above thresholds of concern in some regions. As bone is a tissue that is well preserved in archeological middens, it may prove useful for developing long-term mercury data sets under appropriate conditions. The goal of this study was to evaluate methodologies for measuring mercury concentration in Steller sea lion bone using a direct mercury analyzer, considering sample preparation methods and variability among bone tissue types (e.g., compact versus spongy bone). Finally, we directly compare sensitivity and precision of two different direct mercury analyzer models. Based on the methods presented here, direct mercury analysis using the Nippon MA-3000 can quantify small (ppb) quantities of Hg accurately and precisely in 20 to 60mg of bone with minimal specimen processing. The described method is efficient, relatively inexpensive, and requires minimal bone, conserving rare and valuable specimens. Hydrogen peroxide cleaning and collagen extraction were not required, and may be detrimental for optimal Hg quantification in bone. Further, while homogenization of distinct compact and spongy bone did not impact concentration determination, variance of technical replicates was lower improving quantitation precision. Most importantly, significant differences between compact and spongy bone exist within some individual specimen; however, the difference is not consistent and may indicate differential Hg exposure windows influenced by turnover rate of bone types. We conclude bone provides a natural archive for mercury ecosystem dynamics over millennial time scales in regions where appropriate samples are available. Compact bone has lower and less variable [THg] simplifying analysis and interpretation of data; however, the more dynamic concentrations observed in spongy bone should not be dismissed as invaluable due to their variability in [THg]. Comparisons of [THg] between bone type within individual may provide insight into more acute changes in mercury exposure within an individual’s lifetime.

Revealing an ancient injury: Ulna fracture in a Late Pleistocene Myrmecophaga tridactyla Linnaeus, 1758 juvenile individual

Analysis of ancient animal bone fractures can provide insights into ecological and behavioral aspects, as well as death processes and bone preservation modes. This paper describes and interprets the cause of a bone fracture observed in the fossil remains of a juvenile Myrmecophaga tridactyla (giant anteater) recovered from a limestone cave in Brazil. We identified an indirect bone trauma with a curved or spiral fracture line and a well-formed bone callus in the left ulna through a macroscopic and radiological examination. These features indicate that the giant anteater fell from a great height with an outstretched hand.

A highly efficient scheme for library preparation from single-stranded DNA

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.

The first maternal genetic study of hunter-gatherers from Vietnam.

The current limitation of ancient DNA data from Vietnam led to the controversy surrounding the prehistory of people in this region. The combination of high heat and humidity damaged ancient bones that challenged the study of human evolution, especially when using DNA as study materials. So far, only 4kyears of history have been recorded despite the 65kyears of history of anatomically modern human occupations in Vietnam. Here we report, to our knowledge, the oldest mitogenomes of two hunter-gatherers from Vietnam. We extracted DNA from the femurs of two individuals aged 6.2kcal BP from the Con Co Ngua (CCN) site in Thanh Hoa, Vietnam. This archeological site is the largest cemetery of the hunter-gatherer population in Southeast Asia (SEA) that was discovered, but their genetics have not been explored until the present. We indicated that the CCN haplotype belongs to a rare haplogroup that was not detected in any present-day Vietnamese individuals. Further matrilineal analysis on CCN mitogenomes showed a close relationship with ancient farmers and present-day populations in SEA. The mitogenomes of hunter-gatherers from Vietnam debate the "two layers" model of peopling history in SEA and provide an alternative solution for studying challenging ancient human samples from Vietnam.

Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology.

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period.

Ancient human DNA recovered from a Palaeolithic pendant

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited2–5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000–25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.

Genome secrets of extinct hominids, or can paleogenomics answer the question: humankind, who we are? Nobel Prize in Physiology or Medicine 2022

The Nobel Prize in Physiology or Medicine 2022 was awarded to Professor Svante Pääbo, a Swedish­ paleogenetic researcher, specialist in the field of evolutionary genetics, Director of the Department of Evolutionary Genetics at the Max Planck Institute for Evolutionary Anthropology of Leipzig (Germany), for “discoveries concerning the genomes of extinct hominids and human evolution”. Explaining the significance of S. Pääbo’s work, the Nobel Committee noted that “he pioneered the methods to isolate and analyze DNA from archaic bone remains. In ancient bones, DNA has decayed, been chemically damaged and massively contaminated with DNA from bacteria and contemporary humans who work with the samples. Having utilized modern genetic methods, S. Pääbo created his own methods of sequencing the ancient DNA”. Keywords: genome, hominids, human evolution, Nobel Prize in Physiology or Medicine 2022, Svante Pääbo

Differentiating present-day from ancient bones by vibrational spectroscopy upon acetic acid treatment

Acetic acid treatment for an accurate differentiation between ancient and recent human bones was assessed using Raman and FTIR-ATR spectroscopies. Each set of skeletal samples was analysed by these techniques, prior and after chemical washing, in order to determine the variations in bone´s chemical composition and crystallinity. Bone samples were collected from several independent sources: recent bones burned under controlled experimental conditions or cremated, and archaeological (XVII century and Iron Age). The effect of acetic acid, expected to impact mostly on carbonates, was clearly evidenced in the spectra of all samples, particularly in FTIR-ATR, mainly through the bands typical of A- and B-carbonates. Furthermore, as seen for crematoria and archaeological samples, acetic acid was found to remove contaminants such as calcium hydroxide. Overall, acetic acid treatment can be an effective method for removing carbonates (exogenous but possibly also endogenous) and external contaminants from bone. However, these effects are dependent on the skeletal conditions (e.g. post-mortem interval and burning settings). In addition, this chemical washing was shown to be insufficient for an unequivocal discrimination between recent and archaeological skeletal remains. Based on the measured IR indexes, only cremated bones could be clearly distinguished.

Near-infrared hyperspectral imaging to map collagen content in prehistoric bones for radiocarbon dating

Many of the rarest prehistoric bones found by archaeologists are enormously precious and are considered to be part of our cultural and historical patrimony. Radiocarbon dating is a well-established technique that estimates the ages of bones by analysing the collagen still present. However, this method is destructive, and its use must be limited. In this study, we used imaging technology to quantify the presence of collagen in bone samples in a non-destructive way to select the most suitable samples (or sample regions) to be submitted to radiocarbon dating analysis. Near-infrared spectroscopy (NIR) that was connected to a camera with hyperspectral imaging (HSI) was used along with a chemometric model to create chemical images of the distribution of collagen in ancient bones. This model quantifies the collagen at every pixel and thus provides a chemical mapping of collagen content. Our results will offer significant advances for the study of human evolution as we will be able to minimise the destruction of valuable bone material, which is under the protection and enhancement of European cultural heritage and thus allow us to contextualise the valuable object by providing an accurate calendar age.