Ancient pathogen DNA in human teeth and petrous bones

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petrous bone which has become the most desired skeletal element in ancient DNA research due to its high endogenous DNA content. To compare the potential for pathogenic aDNA retrieval from teeth and petrous bones, we sampled these elements from five ancient skeletons, previously shown to be carrying Yersinia pestis. Based on shotgun sequencing data, four of these five plague victims showed clearly detectable levels of Y. pestis DNA in the teeth, whereas all the petrous bones failed to produce Y. pestis DNA above baseline levels. A broader comparative metagenomic analysis of teeth and petrous bones from 10 historical skeletons corroborated these results, showing a much higher microbial diversity in teeth than petrous bones, including pathogenic and oral microbial taxa. Our results imply that although petrous bones are highly valuable for ancient genomic analyses as an excellent source of endogenous DNA, the metagenomic potential of these dense skeletal elements is highly limited. This trade‐off must be considered when designing the sampling strategy for an aDNA project.     

Ancient Human Genomes and Environmental DNA from the Cement Attaching 2,000-Year-Old Head Lice Nits

Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500–2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history.     

Combined hybridization capture and shotgun sequencing for ancient DNA analysis of extinct wild and domestic dromedary camel

The performance of hybridization capture combined with next‐generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient‐domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187‐fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient‐domestic dromedaries with 17–95% length coverage and 1.27–47.1‐fold read depths for the covered regions. Using whole‐genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1–1.06‐fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

Shotgun microbial profiling of fossil remains

Millions to billions of DNA sequences can now be generated from ancient skeletal remains thanks to the massive throughput of next‐generation sequencing platforms. Except in cases of exceptional endogenous DNA preservation, most of the sequences isolated from fossil material do not originate from the specimen of interest, but instead reflect environmental organisms that colonized the specimen after death. Here, we characterize the microbial diversity recovered from seven c. 200‐ to 13 000‐year‐old horse bones collected from northern Siberia. We use a robust, taxonomy‐based assignment approach to identify the microorganisms present in ancient DNA extracts and quantify their relative abundance. Our results suggest that molecular preservation niches exist within ancient samples that can potentially be used to characterize the environments from which the remains are recovered. In addition, microbial community profiling of the seven specimens revealed site‐specific environmental signatures. These microbial communities appear to comprise mainly organisms that colonized the fossils recently. Our approach significantly extends the amount of useful data that can be recovered from ancient specimens using a shotgun sequencing approach. In future, it may be possible to correlate, for example, the accumulation of postmortem DNA damage with the presence and/or abundance of particular microbes.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

河南灵井许昌人遗址动物骨骼表面人工改造痕迹

河南灵井许昌人遗址发掘出土了距今约10—8万年前的古人类头骨化石, 与头骨化石同层发现的还有大量哺乳动物化石及石制品等文化遗物。本文是对该遗址2005—2006年出土动物化石骨骼表面人工改造痕迹的观察分析结果。灵井遗址中13%的动物骨骼表面有人工切割痕的产生, 其中切割痕位于长骨骨干部位的约占此类标本总数的98.45%; 同时,在具切割痕的长骨类化石材料中,属于食草动物上部和中部肢骨的分别为34%和41%, 属于下部肢骨的则仅为25%。此外, 灵井动物群中具敲击痕、火烧痕迹、人工使用痕迹的骨骼标本分别占全部观察例数的4.2%、1%、1.32%。总之, 通过对动物骨骼表面保留的上述人工改造痕迹的观察与统计分析, 并与埋藏学实验及其他考古遗址相关属性的对比, 表明古人类是这一遗址中大量脊椎动物肉食资源的初级获取者和利用者, 他们是导致这一动物群聚集形成的主要埋藏学因素。同时, 许昌人遗址中大量破碎动物骨骼的出现可能也与古人类敲骨取髓的取食行为有着非常紧密的联系。     

Lateral angle: a method for sexing using the petrous bone

We report on the results of applying the so-called lateral angle method for sex determination on skeletal remains. The lateral angle denotes the angle of the internal auditory canal in relation to the medial surface of the petrous part of the temporal bone. The method involves making a small cast of the proximal part of the internal acoustic canal and determining the angle at which the canal opens up to the surface of the petrous bone. The method has the great advantage of utilizing one of the sturdiest bone elements of the human skeleton, and may thus be especially suited for analyses of very fragmented skeletal remains or cremated bones, where the petrous bone may still be readily recognizable. The method was tested using a forensic sample of 113 petrous bones with known sex. Intra- and interobserver testing was also performed. We found a statistically significant difference in angle size between males and females (mean angle size of males, 39.3 degrees ; mean angle size of females, 48.2 degrees ; P < 0.001). There was no bilateral difference in angle size. In blind trials, 83.2% of petrous bones were assigned to the correct sex. We also tested the lateral angle method against an archaeological skeletal sample. True sex was not known for this sample; instead, sexing had been carried out by assessing pelvic and cranial morphology in independent trials. We found a higher concordance between the lateral angle and "pelvic" sex than for lateral angle and "cranial" sex. Finally, we note that subadult sexing may also be possible with this method.     

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.     

Recognizing hominoid-modified bones: the taphonomy of colobus bones partially digested by free-ranging chimpanzees in the Kibale Forest, Uganda

We present a taphonomic study of bones that have passed though the digestive tracts of free-ranging chimpanzees from the Kibale Forest in Uganda. The bone assemblage can be characterized as having a very low species diversity; low number of identifiable specimens (NISP) per scat; bones extremely broken up (very small size range); skeletal part frequencies similar in some ways to those resulting from carnivore partial digestion; and sometimes articulated specimens. Modifications to the bones include corrosion, tiny tooth scores and pits, cracking, and fraying of bone edges. Together, these characteristics suggest that hominoid bone digestion may be recognizable, despite some similarities with leopard-, canid-, and eagle-modified bone. Chimpanzees are well-documented hunters of medium-sized vertebrates such as monkeys. This is significant in the study of human evolution if, as it seems, the last common ancestor of chimpanzees and humans was chimpanzee-like. It suggests there was a pre-stone-tool-using hunting phase in human evolution, perhaps by australopiths or the last common ancestor. Taphonomically, pre-stone tool meat eating has been very difficult to detect in the fossil record. However, if chimpanzees leave a recognizable taphonomic signature on the bones of their prey, we will be able to look for analogous signatures in fossil bones associated with fossil hominoids and hominids.     

Choosing the Best Plant for the Job: A Cost-Effective Assay to Prescreen Ancient Plant Remains Destined for Shotgun Sequencing

DNA extracted from ancient plant remains almost always contains a mixture of endogenous (that is, derived from the plant) and exogenous (derived from other sources) DNA. The exogenous ‘contaminant’ DNA, chiefly derived from microorganisms, presents significant problems for shotgun sequencing. In some samples, more than 90% of the recovered sequences are exogenous, providing limited data relevant to the sample. However, other samples have far less contamination and subsequently yield much more useful data via shotgun sequencing. Given the investment required for high-throughput sequencing, whenever multiple samples are available, it is most economical to sequence the least contaminated sample. We present an assay based on quantitative real-time PCR which estimates the relative amounts of fungal and bacterial DNA in a sample in comparison to the endogenous plant DNA. Given a collection of contextually-similar ancient plant samples, this low cost assay aids in selecting the best sample for shotgun sequencing.     

Virtual reconstruction of the Neandertal lower limbs with an estimation of hamstring muscle moment arms

A major problem of fossil hominid analysis is a lack of complete specimens. Many individual specimens have been damaged by the effects of diagenesis and excavation. Significant advances in the field of three dimensional image processing (3D) have enabled the creation of accurately scaled reconstructions of individual fossil bones using mirrored parts of the same fossil bone or human/fossil hominid equivalents. This study presents, for the first time, a method to reconstruct a 3D virtual model of the lower limb of the Neandertal using different bones from different fossil remains (Spy II, Neandertal 1 and Kebara 2) and integrating them into a single model of the Neandertal lower limb. A biomechanical analysis of the model was performed, including computer graphics visualization of the results, motion displacement graphs and muscle moment arms. The overall method has been implemented into an open-source customized software (lhpFusionBox) developed for the biomechanical study of the musculoskeletal system.     

Palaeogenetics of cattle domestication: Methodological challenges for the study of fossil bones preserved in the domestication centre in Southwest Asia

Recently, palaeogenetics encountered enormous success when parts of the nuclear genomes of mammoth and Neanderthal man were analysed. Their bones, however, had been preserved in environments favourable to DNA preservation, i.e., permafrost regions and caves in temperate regions. Few studies have tackled archaeological bones from hot, arid regions, although they bear great significance for the study of evolution of humans and the precursors of modern societies. According to archaeological evidence, a key event in neolithisation, the domestication of cattle, took place around 10,000 years ago in Southwest Asia. Genetic data from prehistoric bovine bones preserved in this region might shed light on this process, but the palaeogenetic approach has been hampered by poor DNA preservation. Here, I discuss various aspects of DNA preservation in fossils and the production of reliable palaeogenetic data and present methodological improvements that have enabled us to shed light on the process of cattle domestication in Southwest Asia and its spread into western Europe.     

Comparing rates of recrystallisation and the potential for preservation of biomolecules from the distribution of trace elements in fossil bones

Preservation of intact macromolecules and geochemical signals in fossil bones is mainly controlled by the extent of post-mortem interaction between bones and sediment pore waters. Trace elements such as lanthanum are added to bone post-mortem from pore waters, and where uptake occurs via a simple process of diffusion and adsorption, the elemental distribution can be used to assess the relative extent of bone-pore water interaction and rate of recrystallisation. Distribution profiles can be parameterised effectively using simple exponential equations, and the extent of bone–water interaction compared within and between sites. In this study, the distribution of lanthanum within bone was determined by laser ablation ICP–MS in 60 archaeological and fossil bones from Pleistocene and Cretaceous sites. The rates of recrystallisation and potential for preservation of intact biogeochemical signals vary significantly within and between sites. Elemental profiles within fossil bones hold promise as a screening technique to prospect for intact biomolecules and as a taphonomic tool.     

古DNA实时荧光定量PCR实验中标准品的制备

实时荧光定量PCR技术通过对PCR每一循环扩增产物的实时检测,可对模板的精确拷贝数进行绝对定量,从而用于古DNA实验中提取和扩增条件的比较和优化.本研究采用异硫氰酸胍碱裂解-SiO2吸附的方法,从采自黑龙江省的晚更新世斑鬣狗化石材料中提取得到了斑鬣狗线粒体基因组古DNA.经常规PCR扩增后,将纯化的扩增产物克隆到微生物体内使其大量复制,再用M13通用引物扩增出含少量外源DNA的古DNA目标片段,从而建立了适用于古DNA荧光定量PCR扩增的标准品的制备方法.经检测分析,运用该方法制备的标准品性质稳定,能够准确地指示反应体系中较为精确的古DNA模板拷贝数,从而反映古DNA的提取和扩增效率,用于比较并优化古DNA提取和扩增条件.     

The use of protein characteristics to assess the retrievability of ancient DNA from ancient bones

The ability to retrieve DNA from ancient specimens has been one of the greatest achievements of the past decade, and has opened a totally new field of research with applications in seemingly distant domains such as archeobotany, the molecular phylogeny of extinct genomes, human paleopathology and the genetic of ancient human populations. However, extraction of ancient DNA has often a very low rate of success, prompting researchers to develop screening methods for the selection of promising specimens. With this goal in mind, we studied the amino acid content of nine human bones of ancient origin. We demonstrate that a single HPLC chromatogram is indicative of the integrity of ancient bone proteins. Among five specimens containing amplifiable DNA, four exhibited a protein content similar to that of contemporary bone protein content. Three of the four specimens, from which we were unable to extract any amplifiable DNA, had an amino acid content strikingly different from that of contem-porary bone. A non-parametric statistical test, Kendall's tau, was used to show that protein content and PCR products, are probably correlated (at a 95% confidence level). In addition, the D/L Asp and D/L Glu racemization ratios obtained are indicative of the presence of ancient organic compounds. We propose that protein analysis should be systematically performed in studies where there are many samples in order to select the specimens that are most likely to contain retrievable ancient DNA.     

Analysis of fossil bone organic matrix by transmission electron microscopy

A transmission electron microscope (TEM) study was initiated on samples of geological ages ranging from Devonian to Jurassic to analyse the ultrastructure of the organic matrix in fossil bones that have preserved a histological structure after demineralisation. All samples show a network of variably well-preserved fibrils. Within the sampling, the best results were obtained in two specimens: the scales of the Devonian sarcopterygian tetrapodomorph Eustenopteron foordi, and the humerus of Jurassic dinosaur Lappentosaurus madagascariensis. Despite an extended time difference between both specimens, their fossil bone is composed of a plywood-like structure in which the fibrils are very closely packed. These observations support the hypothesis that dense initial packing of collagen fibrils favours the preservation of the fossil bone.     

Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

Despite the abundance of fossil remains for the extinct steppe bison (Bison priscus), an animal that was painted and engraved in numerous European Paleolithic caves, a complete mitochondrial genome sequence has never been obtained for this species. In the present study we collected bone samples from a sector of the Trois-Frères Paleolithic cave (Ariège, France) that formerly functioned as a pitfall and was sealed before the end of the Pleistocene. Screening the DNA content of the samples collected from the ground surface revealed their contamination by Bos DNA. However, a 19,000-year-old rib collected on a rock apart the pathway delineated for modern visitors was devoid of such contaminants and reproducibly yielded Bison priscus DNA. High-throughput shotgun sequencing combined with conventional PCR analysis of the rib DNA extract enabled to reconstruct a complete mitochondrial genome sequence of 16,318 bp for the extinct steppe bison with a 10.4-fold coverage. Phylogenetic analyses robustly established the position of the Bison priscus mitochondrial genome as basal to the clade delineated by the genomes of the modern American Bison bison. The extinct steppe bison sequence, which exhibits 93 specific polymorphisms as compared to the published Bison bison mitochondrial genomes, provides an additional resource for the study of Bovinae specimens. Moreover this study of ancient DNA delineates a new research pathway for the analysis of the Magdalenian Trois-Frères cave.     

Preservation of key biomolecules in the fossil record: current knowledge and future challenges

We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record.     

Taphonomy of the south Brazilian Triassic herpetofauna: fossilization mode and implications for morphological studies

Skulls and limb bones of reptiles from Mid-Triassic rocks in southern Brazil show striking morphological and volumetric differences among specimens from the same taxonomic group; this is caused by early calcite cementation. Petrographic analysis of 40 thin sections of selected fossil bones demonstrates that the main agent of fossilization was precipitation of calcite and minor hematite in the pores of bones during early burial mineralization. The framework of the bones has been broken and replaced by calcite (and hematite), beginning in the spongy inner part of the bone (the cancellous bone) and gradually extending into the compact external layers. Three taphonomic groups of fossil bones are recognized: I – those almost entirely retaining their original structure and shape; II – those whose internal structure is destroyed, but which still have solid outer 'bone surface', and III – those composed of fragments of bone apatite 'floating' in a carbonate matrix. Destruction of the bone structure was caused by the displacive force of calcite crystallization, which typically occurs at shallow burial, during early diagenesis. This process occurs in the vadose zone and requires markedly alternating wet and dry seasons, which indicates a semi-arid paleoclimate for the south Brazilian Mid-Triassic, an inference consistent with other paleoclimatic data. Diagenesis can significantly alter the shape and size of bones and cause considerable morphological variation within the same fossil group, possibly leading to taxonomic misinterpretation.