Molecular identification and characterization of Mycobacterium tuberculosis complex in ancient Egyptian mummies

A considerable number of molecular studies have provided evidence for the presence of Mycobacterium tuberculosis complex (MTB) DNA in ancient skeletal and mummified material. Moreover first studies on the differentiation of sub‐types of the MTB (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii) have successfully been performed on ancient tissue samples. In our present study we extend previous analyses and investigate bone and soft tissue samples from 118 ancient Egyptian mummies and skeletons from the Pre‐ to Early Dynastic site of Abydos and different tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (c. 2050–500 BC). The samples were tested for the presence of MTB DNA and further identified by spoligotyping. Twenty‐six samples provided molecular evidence for the presence of ancient mycobacterial DNA by amplification of a 123 base pair fragment of the repetitive element IS6110. Out of the 26 positive samples, 12 provided a complete spoligotyping signature, which was compared to an international database. Ten further cases showed an incomplete, patchy hybridization pattern, while in four cases no spoligotyping signature could be obtained. Interestingly, they all show either a M. tuberculosis or M. africanum pattern, but none revealed a M. bovis specific pattern. In the material from a Middle Kingdom tomb (used exclusively between c. 2050–1650 BC) several samples revealed a M. africanum type specific spoligotyping signature, while samples from later periods provided patterns typical for M. tuberculosis. This study clearly shows that spoligotyping can be used for the characterization of members of the MTB in historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type. Copyright © 2004 John Wiley & Sons, Ltd.     

Tuberculosis among Iron Age individuals from Tyva,South Siberia: palaeopathological and biomolecular findings

The study focuses on the evidence for tuberculosis apparent in an Iron Age population recovered from the cemetery of Aymyrlyg, Tyva (Tuva), South Siberia. A recent wholly molecular study of five of the cases confirmed the presence of Mycobacterium tuberculosis (MTB) complex DNA in four of the individuals. In all cases the disease was caused by strains of Mycobacterium bovis rather than Mycobacterium tuberculosis and represents the first positive identification of the bovine form of the disease in archaeological human remains. Details of the palaeopathological characteristics of the cases are provided in the current paper, while the molecular observations are extended to include a quantitative evaluation of the surviving mycobacterial DNA using real-time PCR. The observation that bovine tuberculosis was the pathogen responsible is discussed in terms of current understanding of the evolution of the MTB complex as well as the implications for future ancient DNA studies in this area.     

MICROSCOPIC IDENTIFICATION AND SOURCING OF ANCIENT EGYPTIAN PLANT FIBRES USING LONGITUDINAL THIN SECTIONING*

Plant fibres and the artefacts constructed from them often remain overlooked in the archaeological record because of their poor survival and the problems related to the precise identification of the species to which the fibres belong. The goal of this study was to design a simple and accurate method of identifying archaeological plant fibre sources. Twenty‐two fibre samples from two sets of ancient Egyptian botanical artefacts were examined under both a stereomicroscope and a compound microscope, and compared to a large reference collection and to previously published research. By examining longitudinal thin sections of the ancient plant specimens, we identified plant fibres from the following species: Hyphaene thebeica, Cyperus papyrus, Desmostachya bipinnata, Imperata cylindrica, Phragmites australis and Linum usitatissimum. Our identification of these plant fibres reveals essential information about the materials used for producing ropes, baskets, sandals, mats and fabric. The results of this study demonstrate the value of longitudinal thin sectioning and light microscopy as a major means of identifying the source material of botanical artefacts, and advance our knowledge of ancient Egyptian plant exploitation as well as the associated technologies involved in constructing these types of artefacts.     

The application of ancient DNA analysis to identify neolithic caprinae: a case study from the site of Hatoula,Israel

The evidence for wild sheep (Ovis orientalis) in archaeological sites from the southern Levant is examined through the application of ancient DNA analysis to specimens from the Pre‐Pottery Neolithic A (8300–7500 uncal. BC) site of Hatoula, Israel. The results indicate that at least one of the bones from this site, previously identified as sheep, is in fact goat. To date this is the earliest faunal sample to have yielded DNA in the region. This study highlights the problems in applying morphological criteria to distinguish between caprine species, and illustrates how ancient DNA analysis can serve as a powerful tool in resolving questions of species attribution. Copyright © 2003 John Wiley & Sons, Ltd.     

Wild or domesticated: DNA analysis of ancient water buffalo remains from north China

Recent zooarchaeological studies on water buffalo (Bubalus sp.) remains from China and south Asia question the traditional view that water buffalo were first domesticated in Neolithic China over 7000 years ago. The results from several recent population genetic studies of modern domesticated buffalo (Bubalus bubalis) are not consistent with each other, placing the original center of buffalo's domestication in south Asia, southeast Asia, or China. This paper reports a study using an ancient DNA approach to analyze water buffalo remains from Neolithic sites in north China to investigate their affinities with modern domesticated water buffalo, and to shed light on the origin of modern domesticated water buffalo in China.A 169 bp fragment of D-loop mitochondrial DNA was successfully amplified and verified for 13 of 24 bone samples obtained from seven archaeological sites along the Wei River valley in Shaanxi Province, China. The bone samples which yielded positive DNA can be dated to 8000–3600 cal. BP. The phylogenetic analysis of the obtained DNA sequences along with modern water buffalo sequences indicated that the ancient water buffalos were not the direct ancestor of modern domesticated water buffalo. However, the phylogenetic analysis, along with BLAST searches of these ancient DNA sequences, did demonstrate their relatedness to water buffalo more so than to any other bovid species, confirming the existence of indigenous wild (but now extinct) water buffalo species (B. mephistopheles) in ancient China.The DNA analysis of these ancient remains failed to establish direct links between modern domesticated water buffalo (B. bubalis) and indigenous water buffalo (B. mephistopheles) from ancient China. If further DNA studies of more ancient remains from other regions of China confirm the observation of solely indigenous water buffalo species in ancient China, it would suggest modern water buffalo might not have been first domesticated in China.     

The Problem of the Missing Harbour of Evagoras at Salamis,Cyprus: a review of the evidence and pointers to a solution

This article summarises the archaeological evidence for the existence of Evagoras' naval harbour at Salamis in North Cyprus, which ancient texts credit him with building c.410–400 BC. Based on a critical examination of previous surveys and his own on‐site observations, the author concludes there is indicative evidence of a constructed harbour c.800 m long, which was divided into two basins by a stone jetty, separated from the city by a stone wall and with some evidence of ship‐sheds at its north end. © 2012 The Author     

Ancient DNA Analysis of Anatolian Goat Remains Excavated from a Urartian Castle in Eastern Turkey

In recent years, mitochondrial diversity of goats has been extensively studied, in order to shed light on domestication processes. There are limited studies on genetic diversity and demographic history of Anatolian goat breeds, and these studies have focused only on modern goats. Until today, no research has been conducted on DNA analysis of ancient Anatolian goats. In the present study, seven ancient DNA samples of goat remains from Van‐Yoncatepe archaeological site in Eastern Anatolia, Turkey, dating 1000 BC, were successfully extracted and amplified for the first time. A 230‐bp fragment of mitochondrial DNA Hypervariable Region 1 of the samples was analyzed. All of the ancient sequences belonged to haplogroup A, which is the most widely distributed and frequently encountered haplogroup in modern goats. The results also pointed out that Anatolian ancient goats might have contributed to the genetic structure of modern goats in the Near East, the Mediterranean Region and South‐Southeastern Asia. Copyright © 2014 John Wiley & Sons, Ltd.     

BYZANTINE CERAMIC PRODUCTION FROM CUMA (CAMPI FLEGREI,NAPOLI)*

Samples of table and cooking ware, dating back to the Byzantine period of the ancient Greek colony of Cuma, were analysed by optical microscopy, X‐ray diffraction, X‐ray fluorescence and scanning electron microscopy, in order to reconstruct the main technological properties of these manufactures. The comparison of minero‐petrographical data from these samples with those of some kiln wastes allowed us to hypothesize a local manufacture for most of the investigated specimens and to confirm the relative reference groups. A restricted number of samples did not link with the main groups, indicating a regional (or maybe extra‐regional) production, in agreement with the widespread circulation of this high‐medieval ceramic production in southern Italy.     

Animal DNA in PCR reagents plagues ancient DNA research

Molecular archaeology brings the tools of molecular biology to bear on fundamental questions in archaeology, anthropology, evolution, and ecology. Ancient DNA research is becoming widespread as evolutionary biologists and archaeologists discover the power of the polymerase chain reaction (PCR) to amplify DNA from ancient plant and animal remains. However, the extraordinary susceptibility of PCR to contamination by extraneous DNA is not widely appreciated. We report the independent observation of DNA from domestic animals in PCR reagents and ancient samples in four separate laboratories. Since PCR conditions used in ancient DNA analyses are extremely sensitive, very low concentrations of contaminating DNA can cause false positives. Previously unidentified animal DNA in reagents can confound ancient DNA research on certain domestic animals, especially cows, pigs, and chickens.     

Mitochondrial DNA haplotypes of Devensian hyaenas from Creswell Crags,England

Spotted hyaena (Crocuta crocuta) remains have been recovered from British Middle and Upper Pleistocene sites at intervals within the period 700–730 ka BP. Morphological studies have suggested that hyaenas of the Last Interglacial sensu stricto (Ipswichian: Marine Isotope Stage [MIS] 5e, 130–115 ka BP) and Last Glacial (Devensian: MIS 3, 61–24 ka BP) were two distinct populations, the Ipswichian hyaenas becoming extinct in Britain during MIS 5 and the Devensian ones arriving via a subsequent migration from continental Europe. However, the apparent presence of hyaenas in later MIS 5 deposits has led to the alternative suggestion that there was a southern relict population from which the Devensian hyaenas originated. We obtained ancient DNA (aDNA) sequences from four Devensian hyaena specimens from Creswell Crags, Derbyshire, dated to around 45 ka ¹⁴C BP. Each of these four specimens belonged to the mitochondrial DNA (mtDNA) clade A. This clade is not thought to have been present in Europe until ~360 ka BP, after the initial arrival of hyaenas in Britain. The DNA results, therefore, suggest that there were at least two waves of hyaena dispersals into Britain. The results are consistent with the repeated dispersals into Britain of another Pleistocene social carnivore, Homo sapiens.     

Molecular history of tuberculosis from ancient mummies and skeletons

The origin and evolution of the infectious disease tuberculosis (TB) and its pathogens is still not fully understood. An important effort for a better understanding of the underlying mechanisms of TB evolution lies within the investigation of skeletal and mummified material dating back several thousand of years. In this work, molecular data from mummified and skeletal material from different time periods of the Old World are compared, and the current status of ancient mycobacterial DNA analysis in ancient human remains is discussed, with particular reference to the genetic evolution of human TB. The molecular analysis of material from southern Germany (1400–1800 AD), Hungary (600–1700 AD) and Egypt (3500–500 BC) revealed high frequencies of TB in all time periods. In several individuals from ancient Egypt the mycobacterial DNA could be further characterised by spoligotyping. Thereby, evidence for ancestral M. tuberculosis strains was found in the pre‐ to early dynastic material from Abydos (3500–2650 BC), while typical M. africanum signatures were detected in the Middle Kingdom tomb in Thebes‐West (2050–1650 BC). Samples from the New Kingdom to Late Period tombs (1500–500 BC) were characterised as modern M. tuberculosis strains. In concordance with other studies on ancient skeletal and mummified samples, no evidence for the presence of M. bovis was found. These results contradict the theory that M. tuberculosis evolved from M. bovis during domestication, but supports the new scenario that M. tuberculosis probably derived from an ancestral progenitor strain. Copyright © 2007 John Wiley & Sons, Ltd.     

GAMBLING WITH ETRUSCAN DICE: A TALE OF NUMBERS AND LETTERS

The graphical and linguistic interpretation of the first six Etruscan numerals has long been confronted with the ambiguous assignment of the words huth and sa to either 4 or 6. Here, we show how the systematic combinatorial analysis of the numerals appearing on ancient southern Etrurian dice dated from the eighth to the third centuries bc , together with the careful comparison of the results with the only two existing dice carrying the alphabetical translations of the numerals conserved at the Bibliothèque Nationale de France, Paris, finally allows unambiguous mathematical resolution of the linguistic riddle, allowing the firm attribution of the numeral 6 to the graphical value huth and 4 to sa. Combinatorial analysis of the numerals distribution on the six faces of the die shows that only two of the 15 possible numerical combinations were actually in use in southern Etruria, and that during the fifth century bc there was a marked shift from the typical (1–2, 3–4, 5–6) combination used in the early seventh‐ to fifth‐century bc dice to the (1–6, 2–5, 3–4) combination used at later times and still largely adopted today. The largest body of archaeometric data on dice specimens from Etruria is presented, based on macroscopic examination, X‐ray diffraction, DRIFT spectroscopy and density measurements.     

DNA FROM HUMAN ANCIENT BACTERIA: A NOVEL SOURCE OF GENETIC EVIDENCE FROM ARCHAEOLOGICAL DENTAL CALCULUS

We report a molecular methodology to obtain and analyse ancient bacterial DNA from archaeological dental calculus. Recent and archaeological DNA samples, as old as 4000 bp , were successfully extracted and amplified with species‐specific PCR primers. We propose this approach in order to: detect the presence of specific bacterial species infecting past human populations; compare the composition of ancient oral microbiomes among human populations; and analyse the genetic variability and covariation of bacteria and human host populations. Genomic analysis of bacteria from dental calculus is a promising source of evidence for palaeopathological and micro‐evolutionary studies, focused either on micro‐organisms or their human hosts.     

Dama Dentition: A New Tooth Eruption and Wear Method for Assessing the Age of Fallow Deer (Dama dama)

Reliable ageing techniques for wild animals are notoriously challenging to develop because of the scarcity of sizeable collections of known‐age specimens. Without such techniques it is difficult to reconstruct hunting patterns, which is a significant problem for the examination of assemblages from pre‐farming cultures. This paper presents a new method, based on mandibular tooth eruption and wear, for assessing the age of fallow deer. The method was developed from a large collection (n = 156) of known‐age Dama dama specimens, has been blind tested by members of the zooarchaeological community and represents a user‐friendly system with the potential to generate large compatible datasets through which the dynamics of human–Dama relationships can be examined. Copyright © 2016 John Wiley & Sons, Ltd.     

Osteometric Variation in Domestic Dogs (Canis familiaris) from the Kodiak Archipelago,Alaska

The research presented here is a re‐examination of domestic dog (Canis familiaris) remains from the Uyak site on Kodiak Island, Alaska. Previous analyses suggest there were two breeds of dog represented in this archaeological assemblage, primarily on the basis of dog cranial size. Here, we use a series of metric and nonmetric traits to test the argument that these ‘breeds’ actually represent a population of male and female dogs. On the basis of the results presented here, we argue the metric and nonmetric data produced by this study suggest that the dogs in this sample are male and female specimens, rather than two distinct breeds. The Uyak assemblage is the largest collection of domestic dog from Kodiak, and these results have the potential to contribute to our understanding of human–dog relationships in the archipelago. Copyright © 2012 John Wiley & Sons, Ltd.     

Sex identification of ancient DNA samples using a microfluidic device

A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis.     

Gastrointestinal parasites in ancient South American camelid feces from the Atacama Desert (Pampa del Tamarugal,Tarapacá, northern Chile)

Faunal remains from archeological sites worldwide, especially feces and regurgitated pellets, are usually subjected to paleoparasitological examination, allowing the identification of past animal parasites. In this study, we analyzed 10 samples of South American camelid dung, which played an important role for the ancient human groups in the South-Central Andes, dated between 341 and 1635 calCE, from the Iluga Túmulos site (Pampa del Tamarugal, Tarapacá region, Atacama Desert, Chile). Microscopy examination revealed parasite remains in seven samples, in which oocysts of Eimeria macusaniensis and eggs of Lamanema chavezi/Nematodirus lamae, Trichuris sp., Moniezia sp., capillariids, strongylids, and unidentified nematodes were found. Although some of these species pose a potential health risk to camelids, most are not considered to be of major zoonotic importance, which could explain the absence of these parasites on human coprolites from this site analyzed previously. This first paleoparasitological study on camelid fecal remains from northern Chile sheds light on the occurrence of gastrointestinal parasites and its relationship with Andean ancient human populations and the environment.     

Methods for the Study of Ancient Hair: Radiocarbon Dates and Gene Sequences from Individual Hairs

Genetic data from archaeological specimens provides a potent new approach for addressing questions in prehistory. Hair from archaeological sites is an important but overlooked data source amenable to molecular analysis. To date DNA identification from hair is limited to modern samples. Our study is the first to report DNA recovery from a 9800-year-old specimen. We report methods used to extract mitochondrial DNA and obtain accelerator mass spectrometry radiocarbon dates (AMS-¹⁴C) from hairs plucked from a piece of ancient bighorn sheep (Ovis canadensis nelsoni) skin found at Smith Creek Cave, Nevada. The ability to obtain reliable radiocarbon dates and DNA sequences from single ancient hairs opens new possibilities for addressing biological questions in prehistory.     

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.     

Mitochondrial DNA Analysis of Ancient Domestic Goats in the Southern Caucasus: A Preliminary Result from Neolithic Settlements at Göytepe and Hacı Elamxanlı Tepe

This study presents preliminary results of mitochondrial DNA analyses of modern and ancient domestic goats in the southern Caucasus in order to examine their phylogenetic relationship with modern and ancient goats. For this purpose, seven ancient samples were obtained from two early agricultural villages in west Azerbaijan (Göytepe and Hacı Elamxanlı tepe, dated to ca. 6000–5500 cal bc , the Pottery Neolithic period), in addition to five modern goat samples in the same region. In the study, mitochondrial DNA segments of the control region (216 bp for the Neolithic samples and 481 bp for the modern samples) were amplified, and phylogenetic analyses were performed using previously published reference DNA sequences. As a result, all the haplotypes found in this study were grouped in the haplogroup A of goats. The finding of the haplogroup A among domestic goats in the southern Caucasus in the early sixth millennium bc can be interpreted as part of the geographic expansion of this lineage from the areas of initial domestication to surrounding areas that include also South and Southeast Europe. In the southern Caucasus, the haplogroup A probably continued to be a major lineage among domestic goats since their emergence in this area to the present. In contrast, this lineage has not been detected among local wild goats including Capra aegagrus , indicating the external origin of domestic goats. This possibility is consistent with archaeological records that indicate sudden appearance of agricultural lifeways in the southern Caucasus and cultural connections with northern Mesopotamia. Copyright © 2016 John Wiley & Sons, Ltd.