Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.     

The use (and misuse) of archaeological salmon data to infer historical abundance in North America with a focus on New England

Information about historical animal or plant abundance often either explicitly or implicitly informs current conservation practice. If it can be shown that an organism was not historically abundant in a region, its conservation importance may be downgraded. In contrast to abundant archaeological support for historic importance of salmon in the Pacific Northwest, historic abundance of Atlantic salmon in New England has been called into question based on the rarity of salmon bones in archaeological sites. These data have been used to argue that the importance of salmon to the region has been exaggerated and that expensive restoration efforts in some rivers should be reconsidered. Here, we argue that lack of archaeological bone fragment abundance does not make a convincing case against historical Atlantic salmon abundance in New England for three primary reasons. First, salmon bones were rare or absent at sites that still have large salmon runs. Second, the lack of salmonid bones in general at archaeological sites suggests poor preservation and/or recovery of bone for these species relative to bones of other fishes. Third, given the presence of large numbers of non-salmonid anadromous fish in the site areas where people fished and deposited fish bones, power to detect salmon bones in studies to date may have been generally low. We present reliable historical accounts that help build a convincing case that salmon were historically abundant in New England rivers. We suggest that rarity of salmon bones in the existing archaeological data should not have unwarranted influence on present-day conservation decision-making in New England.     

X射线衍射技术在烧骨实验研究中的初步应用

烧骨作为考古遗址中较为常见的一类特征遗物,对研究古人类用火行为有着重要的意义。过往研究表明,骨骼在加热过程中,其内部晶体会根据加热程度的不同产生不同的变化。骨骼在加热前的状态,能够在一定程度上反映古人类对骨骼进行热处理的动机与目的。为了了解骨骼在焚烧前的初始状态是否会对其内部晶体产生不同的影响,本研究利用56件现生羊骨进行了烧骨实验。实验设置了带肉骨、剔肉骨和干骨三种不同初始状态的骨骼,并在前人的研究基础上进一步细化了焚烧温度和时间参数。焚烧完成后,利用X射线衍射技术对所有样品进行了分析并观察其衍射图的差异。实验结果显示,骨骼有机质含量的多少,骨骼内部元素的不同,在一定的温度和时间条件下,会对骨骼内晶体的形成产生不同的影响。文章最后探讨了这种差异在考古研究中运用的可能性。     

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.     

DNA decay rate in papyri and human remains from Egyptian archaeological sites

The writing sheets made with strips from the stem (caulis) of papyri (Cyperus papyrus) are one of the most ingenious products of ancient technology. We extracted DNA from samples of modern papyri varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The copy number of the plant chloroplast DNA in the sheets was determined using a competitive PCR system designed on the basis of a short (90 bp) tract of the chloroplast's ribulose bisphosphate carboxylase large subunit (rbcL) gene sequence. The results allowed us to establish that the DNA half-life in papyri is about 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being manufactured. In a parallel investigation, we checked the archaeological specimens for the presence of residual DNA and determined the extent of racemization of aspartic (Asp) acid in both modern and ancient specimens, as a previous report (Poinar et al. [1996], Science 272:864-866) showed that racemization of aspartic acid and DNA decay are linked. The results confirmed the complete loss of authentic DNA, even in the less ancient (8th century AD) papyri. On the other hand, when the regression for Asp racemization rates in papyri was compared with that for human and animal remains from Egyptian archaeological sites, it proved, quite surprisingly, that the regressions are virtually identical. Our study provides an indirect argument against the reliability of claims about the recovery of authentic DNA from Egyptian mummies and bone remains.     

Evaluating bacterial pathogen DNA preservation in museum osteological collections

Reports of bacterial pathogen DNA sequences obtained from archaeological bone specimens raise the possibility of greatly improving our understanding of the history of infectious diseases. However, the survival of pathogen DNA over long time periods is poorly characterized, and scepticism remains about the reliability of these data. In order to explore the survival of bacterial pathogen DNA in bone specimens, we analysed samples from 59 eighteenth and twentieth century individuals known to have been infected with either Mycobacterium tuberculosis or Treponema pallidum. No reproducible evidence of surviving pathogen DNA was obtained, despite the use of extraction and PCR-amplification methods determined to be highly sensitive. These data suggest that previous studies need to be interpreted with caution, and we propose that a much greater emphasis is placed on understanding how pathogen DNA survives in archaeological material, and how its presence can be properly verified and used.     

Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants

Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.     

Taphonomic analysis of the early Pleistocene (2.4 Ma) faunal assemblage from A.L. 894 (Hadar, Ethiopia)

The A.L. 894 site (Hadar, Ethiopia) is, together with OGS 7 (Gona, Ethiopia), one of the oldest archaeological sites documenting a spatial association of stone tools and bones retrieved from an in situ excavation. In contrast with OGS 7, the better preservation of the bone assemblage at A.L. 894 allows the identification of taphonomic processes of bone breakage, thanks to abundant green bone fractures. The presence of tooth marks and the lack of hominin-produced bone modifications together argue against hominins as the responsible agents for bone accumulation and modification. This taphonomic study of A.L. 894 shows lack of evidence for functional associations between stone tools and bones, a pattern documented in several other early Pleistocene sites. Such a pattern underscores the complex phenomena involved in site formation processes, especially in the earliest archaeological assemblages     

Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis,molecular hybridization and DNA cloning

Summary Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000–3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the Spring Sanctuary near Metaponto (I–IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.     

Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper‐preserved archaeological bone using size exclusion chromatography

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Degradation of biomolecules: a comparative study of diagenesis of DNA and proteins in human bone tissue

Diagenesis of macromolecules is a not yet fully understood process that can be important for anthropological and forensic research. Trying to elucidate the diagenesis of DNA and proteins we investigated the process of fragmentation of DNA and razemisation of aspartic acid in human bone material. We created an in vitro-model of accelerated aging by incubating bone samples in hot water. A comparison of diagenesis of molecules in those artificially aged samples with altogether 30 historical bones from different regions and of different ages was carried out. The in vitro-model showed the expected positive correlation between the increase of razemisation of aspartic acid and DNA fragmentation, while there was a much lesser correlation when investigating historical bones. The in vitro-model showed the expected correlation between the increase of razemisation of aspartic acid and DNA fragmentation and to a much lesser extent in historical bones. This study shows that diagenesis is probably influenced by additional forces affecting different macromolecules in different ways.     

Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus

Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct Bison antiquus, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C(29), C(30) and C(32) mycocerosates and C(27) mycolipenates, typical of the Mycobacterium tuberculosis complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C(34) and C(36) phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.     

Molecular genetic analysis of ancient cattle bones excavated from archaeological sites in Jeju, Korea

Ancient cattle bones were excavated from archaeological sites in Jeju, Korea. We used molecular genetic techniques to identify the species and establish its relationship to extant cattle breeds. Ancient DNA was extracted from four sources: a humerus (Gonae site, A.D. 700-800), two fragments of radius, and a tooth (Kwakji site, A.D. 0-900). The mitochondrial DNA (mtDNA) D-loop regions were cloned, sequenced, and compared with previously reported sequences of various cattle breeds (9 Asian, 8 European, and 3 African). The results revealed that these bones were of the breed, Bos taurus, and a phylogenetic tree indicated that the four cattle bones formed a monophyletic group with Jeju native black cattle. However, the patterns of sequence variation and reports from archaeological sites suggest that a few wild cattle, with a different maternal lineage, may have existed on Jeju Island. Our results will contribute to further studies of the origin of Jeju native cattle and the possible existence of local wild cattle.     

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.     

A method for estimating age of Danish medieval sub-adults based on long bone length

The preferred method for aging archaeological sub-adult skeletons is by dental examination. In cases where no dental records are available, age estimation may be performed according to epiphyseal union, skeletal elements or diaphyseal lengths. Currently no data have been produced specifically for aging archaeological Danish sub-adults from the medieval period based on diaphyseal lengths. The problem with using data on Danish samples, which have been derived from a different population, is the possibility of skewing age estimates. In this study 58 Danish archaeological sub-adults were examined, aged from approximately six years to twenty-one years. The samples were aged according to two dental methods: Haavikko and Ubelaker. Regression formulae were constructed for aging according to their diaphyseal lengths both for individual long bones and combinations of upper and lower long bones. This study indicated that with the regression formulae developed, estimation of age can be done with reasonable results on Danish sub-adults. The Danish data were then compared to data from a different archaeological sample and a modern sample. It showed that the modern data indicated a consistently lower age compared to this sample which increased until reaching a maximum of nearly five years and six months. When comparing the archaeological data to this study, the growth profile crossed over at 12.5 years with a maximum age difference before the cross point of two years and three months lower for the archaeological data. After the cross point there was a maximum difference of three years and four months higher for the archaeological data. This study has shown the importance of using data for age estimation for archaeological material which has been developed specifically for that population. In addition it has presented a possible solution for Danish sub-adult material when dental material is not available.     

Effects of different kinds of cranial deformation on the incidence of wormian bones

Researchers have debated whether the presence and frequency of wormian bones (sutural bones, supernumerary bones, and ossicles) are attributable to genetic factors, environmental factors, or both. This research examines the effects of many different kinds of cranial deformation on the incidence of wormian bones. A sample of 127 deformed and undeformed crania from New World archaeological sites was examined. An undeformed cranial sample (n=35) was compared to the following cranially deformed groups: 1) occipital, 2) lambdoid, 3) annular, 4) fronto-vertico-occipital, 5) parallelo-fronto-occipital, and 6) sagittal synostosis. Three levels of degree of cultural cranial deformation were qualitatively determined. Type and number of wormian bones along each major suture were recorded for each cranium. Group means were analyzed using Kruskal-Wallis one-way ANOVA statistical tests to test the null hypothesis that cranial deformation does not have an effect on wormian bone incidence. Results indicate that all forms of cranial deformation affect the frequency of some types of wormian bones. In particular, all cranially deformed groups exhibited significantly greater frequencies of lambdoid ossicles. Apical, parieto-mastoid, and occipito-mastoid wormian bones also appeared with greater frequency in some groups of culturally deformed crania. Further, varying degrees of cultural deformation all had more lambdoid wormian bones than the undeformed group. These results suggest that wormian bone development in posteriorly placed sutures may be affected more by environmental forces than are their anteriorly placed counterparts.     

Early history of European domestic cattle as revealed by ancient DNA

We present an extensive ancient DNA analysis of mainly Neolithic cattle bones sampled from archaeological sites along the route of Neolithic expansion, from Turkey to North-Central Europe and Britain. We place this first reasonable population sample of Neolithic cattle mitochondrial DNA sequence diversity in context to illustrate the continuity of haplotype variation patterns from the first European domestic cattle to the present. Interestingly, the dominant Central European pattern, a starburst phylogeny around the modal sequence, T3, has a Neolithic origin, and the reduced diversity within this cluster in the ancient samples accords with their shorter history of post-domestic accumulation of mutation.     

古蛋白质分析在东亚古人类演化中的应用前景

东亚古人类演化是学术界关注的热点科学问题,国内外学者对此进行了多学科的相关研究,取得了很多重要进展,但仍然存在许多尚未解决的问题。古蛋白质分析近年来成为古生物演化领域的又一个前沿和热点方向,取得了一系列重要突破。较之古DNA,古蛋白质的保存优势使其可以在时间上和地域上突破古DNA的限制,在古人类演化领域大有可为。东亚古人类化石丰富且时段大致连续,但更新世或更早时期的分子证据非常缺乏。本文从古蛋白质分析的发展史、研究潜力、难点与挑战以及思考与展望等几方面,对古蛋白质分析在东亚古人类演化研究中的应用前景进行梳理与思考。相信随着更多分子证据的积累,古蛋白质分析可为东亚古人类的演化脉络提供更多关键性的线索,极大地促进人类演化研究。     

High Potential for Using DNA from Ancient Herring Bones to Inform Modern Fisheries Management and Conservation

Pacific herring (Clupea pallasi) are an abundant and important component of the coastal ecosystems for the west coast of North America. Current Canadian federal herring management assumes five regional herring populations in British Columbia with a high degree of exchange between units, and few distinct local populations within them. Indigenous traditional knowledge and historic sources, however, suggest that locally adapted, distinct regional herring populations may have been more prevalent in the past. Within the last century, the combined effects of commercial fishing and other anthropogenic factors have resulted in severe declines of herring populations, with contemporary populations potentially reflecting only the remnants of a previously more abundant and genetically diverse metapopulation. Through the analysis of 85 archaeological herring bones, this study attempted to reconstruct the genetic diversity and population structure of ancient herring populations using three different marker systems (mitochondrial DNA (mtDNA), microsatellites and SNPs). A high success rate (91%) of DNA recovery was obtained from the extremely small herring bone samples (often <10 mg). The ancient herring mtDNA revealed high haplotype diversity comparable to modern populations, although population discrimination was not possible due to the limited power of the mtDNA marker. Ancient microsatellite diversity was also similar to modern samples, but the data quality was compromised by large allele drop-out and stuttering. In contrast, SNPs were found to have low error rates with no evidence for deviations from Hardy-Weinberg equilibrium, and simulations indicated high power to detect genetic differentiation if loci under selection are used. This study demonstrates that SNPs may be the most effective and feasible approach to survey genetic population structure in ancient remains, and further efforts should be made to screen for high differentiation markers.This study provides the much needed foundation for wider scale studies on temporal genetic variation in herring, with important implications for herring fisheries management, Aboriginal title rights and herring conservation.     

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.