Identification of animal skin of historical parchments by polymerase chain reaction (PCR)-based methods

This study deals with establishing of a PCR-based strategy with the aim to recognize the animal origin of different historical parchments. This is one of relatively rare studies on the analysis of ancient DNA from parchments. Robustness of the PCR technology is demonstrated by successful identification of the animal species using only a small amount of DNA isolated from 12 parchment samples. Ten PCR-based assays specific for the detection of different animal species (Bos taurus, Ovis aries, Capra hircus, Sus scrofa, Oryctolagus domestica, Cervus elaphus, Capreolus capreolus, Dama dama) and two PCR assays utilizing universal primers were evaluated and optimized with the aim to find a rapid parchment identification method, which would be more reliable than the classical microscopic examination. The optimized PCR methods produced satisfactory results. Out of 12 investigated parchments, 9 items were unambiguously identified, DNA from 2 samples could not be amplified with any of the species-specific PCR assays, and only one parchment produced controversial results. The species-specific PCR results were confirmed by direct sequencing and PCR cloning with consequent sequencing. Our approach, including isolation of parchment DNA by chaotropic solid-phase extraction, optimization of the PCR programs and high-stringency annealing temperatures, demonstrated to be effective, easy and reliable for the analysis of historical parchment DNA. We consider this PCR-based strategy potentially useful also for investigation of other types of animal items conserved in museums, galleries or libraries.     

A flock of sheep,goats and cattle: ancient DNA analysis reveals complexities of historical parchment manufacture

Parchments comprise one of the most common and valuable sources of archaeological and historical data. Previous studies have shown that parchment also preserves genetic data. These data could be valuable for population studies, to understand past animal husbandry, the development of breeds and varieties and to comment on the provenance of parchments. To improve our understanding of DNA contained in parchments, we analysed genetic data, including both mitochondrial and autosomal loci, from 18th to 19th century English parchments which stable isotope analysis had indicated were well-preserved. DNA results were unexpected. All but one of the parchments produced multiple sequences matching several different species. Ion beam analysis ruled out surface treatments of the parchments (including ink and animal glues) as the origin of these multiple sequences. Our results suggest that the DNA content of parchment is more complex than previous research has suggested and that multiple stages of parchment manufacture, treatment and storage are preserved in parchment DNA extracts.     

A study of the composition and microstructure of silver hoards from Tel Beth-Shean,Tel Dor,and Tel Miqne,Israel

The chemical composition and microstructure of objects excavated in three hoards from the end of the Late Bronze and Iron Age sites in Israel were studied using energy-dispersive x-ray fluorescence analysis and optical metallography. The objects are industrial in origin (scrap or jewelry pieces) and include differently shaped ingots, mainly so-called hacksilber, in various sizes. The primary composition is Ag with typically several percent of Cu and Au and less than 1 % of Pb. The concentrations of these metals in each hoard differ significantly. The microstructure of the hacksilber ingots shows a typical dendritic as-cast structure and a surface cold work deformation, probably caused by chiseling the ingot piece off a larger cast bar. Relatively high concentrations of Au, as in the above samples, were observed previously in Egyptian ancient silver which differs from the ancient Greek silver that contains a significantly lower Au content.     

Aspects of ancient Greek trade re-evaluated with amphora DNA evidence

Ancient DNA trapped in the matrices of ceramic transport jars from Mediterranean shipwrecks can reveal the goods traded in the earliest markets. Scholars generally assume that the amphora cargoes of 5th-3rd century B.C. Greek shipwrecks contained wine, or to a much lesser extent olive oil. Remnant DNA inside empty amphoras allows us to test that assumption. We show that short ∼100 nucleotides of ancient DNA can be isolated and analyzed from inside the empty jars from either small amounts of physical scrapings or material captured with non-destructive swabs. Our study material is previously inaccessible Classical/Hellenistic Greek shipwreck amphoras archived at the Ministry of Culture and Tourism Ephorate of Underwater Antiquities in Athens, Greece. Collected DNA samples reveal various combinations of olive, grape, Lamiaceae herbs (mint, rosemary, thyme, oregano, sage), juniper, and terebinth/mastic (genus Pistacia). General DNA targeting analyses also reveal the presence of pine (Pinus), and DNA from Fabaceae (Legume family); Zingiberaceae (Ginger family); and Juglandaceae (Walnut family). Our results demonstrate that amphoras were much more than wine containers. DNA shows that these transport jars contained a wide range of goods, bringing into question long-standing assumptions about amphora use in ancient Greece. Ancient DNA investigations open new research avenues, and will allow accurate reconstruction of ancient diet, medicinal compounds, value-added products, goods brought to market, and food preservation methods.     

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.     

First ancient bovine DNA evidence from India: difficult but not impossible

Ancient DNA isolation from the tropical countries has been shown to be very difficult in the past. Here for the first time we have been successful in isolating ancient DNA from Indian cattle samples. We were able to obtain DNA and sequence the partial mitochondrial D-loop in 3 of the 15 bovine fossil samples ranging in age from 2000 BC to 1000 AD, and were able to further identify the most recent sample as being of Bos indicus origin. Our results on ancient DNA extraction from India will encourage other researchers in this field to carry out further studies of ancient DNA from Indian bovine samples. Our results represent the first successful extraction and amplification of bovine ancient DNA from India, and thus may pave the road for a better understanding of demographic and historical processes of cattle domestication that has taken place in this region.     

Early history of Chinese domestic sheep indicated by ancient DNA analysis of Bronze Age individuals

China has a long history of sheep husbandry, and has several indigenous sheep breeds. However, the exact geographic origin of Chinese domestic sheep remains unclear. To provide valuable genetic information for origin of Chinese domestic sheep, we performed an ancient DNA study on 22 sheep excavated from four Bronze Age archaeological sites in Northern China. Two lineages (A and B) were observed in ancient Chinese sheep, of which lineage A was predominant reaching a frequency of 95.5%. Furthermore, phylogenetic network showed that the most frequent haplotype in ancient sheep was the founder of lineage A. These results suggest that Lineage A may hold the key to understanding the origin of Chinese domestic sheep. Sequence sharing and principal component analysis showed that the ancient Chinese sheep had a close affinity to modern Chinese sheep. However, there was no significant breed structure among three modern Chinese sheep groups, making it difficult to determine their relationship to ancient Chinese sheep. Lastly, our results imply that ancient DNA analysis could provide a new way to investigate prehistoric East-West contact.     

Ancient DNA provides new insights into the origin of the Chinese domestic horse

Domestic horses played a pivotal role in ancient China, but their exact origin remains controversial. To investigate the origin of Chinese domestic horses, we analyzed mitochondrial DNA (mtDNA) from 35 horse remains, aged between 4000 and 2000 years, excavated from nine archaeological sites in northern China. The Chinese ancient horses exhibited high matrilineal diversity, falling into all the seven haplogroups (A–G) observed in modern horses. These results suggest that several maternal lines were introduced into the gene pool of Chinese horses in the past. Haplogroups A and F were more prevalent in ancient horses than the other haplogroups. Interestingly, only haplogroups A and F were present in the samples older than 4000 years, while the more recent horses (between 2000 and 3000 years BP) fell into all seven haplogroups. Comparison with DNA data of present-day horses suggests that haplogroup F is like to be an ancient haplogroup of East Asian origin. These analyses also suggest that the origin of Chinese domestic horses is complex, and external mtDNA input occurred after initial domestication. Our results indicate that the Chinese ancient horses are more related to the modern Mongolian horses. Lastly, our results cannot support the previous hypothesis that early Chinese domestic horses were derived from the Przewalski horse.     

DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.     

A Newly Acquired Ancient Ship-model in Kassel, Germany

Study of the small clay ship-model T832 in the Kassel Museum suggests that it is an instructive document bearing on ancient Greek maritime history and folklore. A preliminary dating that has had to rely on iconographic and literary sources gives an origin in the 6th or 5th centuries BC and a tentative identification as a small coastal warship. Some suggestions as to the purpose and use of the model are discussed.
© 2004 The Nautical Archaeology Society     

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.     

阿波罗崇拜的起源与传播路线

阿波罗被认为是最具希腊性的神。然而,他最初并不是一个希腊本土的神。依据古代典籍的记载、结合语源学和考古学的成果来考察,可以得出的结论是:阿波罗是一个多种文化元素融合后塑造成的希腊本族神。阿波罗崇拜形成发展过程中所体现的文化交流和融合,正是希腊文明吸收周边文明的具体体现。对阿波罗崇拜起源和传播路线的考察,一方面是在文明交流的母题下进行的微观考证,另一方面又为文明交流史的研究提供了具体史料的佐证。     

DNA analysis of archaeological rabbit remains from the American Southwest

Ancient DNA analysis was carried out on 20 archaeological rabbit remains from an early Pueblo II period site in Colorado (circa 1000 A.D.) to explore the possibility of obtaining accurate rabbit genus and species identifications. The presence of abundant rabbit remains at archaeological sites in the American Southwest indicates the importance of rabbit species in the subsistence economy and ritual activities of early aboriginal populations. The study of these remains is hindered by the difficulty of accurate identification due to the fragmentary nature of the bones and the lack of genus- and species-specific morphological features.A short cytochrome b gene fragment was amplified and sequenced to produce a genetic profile for each bone sample. At the genus level, the DNA identifications were consistent with those based on the analysis of mandible morphology for the majority of specimens. When compared to species-specific reference DNA sequences, Lepus americanus and Lepus californicus samples were easily identified. Identification of an unexpected L. americanus (snowshoe hare) from the remains provided new information concerning hunting ranges or exchange between groups in the region. Sylvilagus nuttallii and Sylvilagus audubonii, however, could not be confidently differentiated at this point due to the difficulty in obtaining accurate species-specific reference sequences.The inability to obtain such reference sequences can be a serious problem for DNA species identification of non-domestic animals that lack population-level genetic data and have few sequences available in GenBank. The lack of the DNA data increases the possibility that inappropriate reference sequences could be applied, resulting in false species identification even when authentic DNA is retrieved and amplified from ancient remains.     

Wild or domesticated? An ancient DNA approach to canid species identification in South Africa's Western Cape Province

The fragmentary remains typical of archaeological fauna can prove impossible to identify when confronted with closely related sympatric species. In southern Africa domesticated dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) pose just such a problem, rendering difficult an accurate assessment of the timing of arrival of domesticated dogs in the region. Contextual evidence has suggested that canid remains from four Later Stone Age archaeological sites were likely to be domesticated dogs rather than jackals, and were subjected to ancient DNA analyses to determine species. Every tested specimen proved to derive from black-backed jackals. These data provide not only an unexpected window on southern African prehistory, but also highlight the value in applying ancient DNA techniques to archaeological species identification.     

Variation in tooth mark frequencies on long bones from the assemblages of all three extant bone-collecting hyaenids

Tooth mark frequencies on long bones are examined from the assemblages of all three extant bone-collecting hyaenids. Comparisons are made with a recent study examining tooth mark frequencies and possible sources of variation from a single spotted hyaena (Crocuta crocuta) assemblage (Faith, J.T., 2007. Sources of variation in carnivore tooth-mark frequencies in a modern spotted hyena (Crocuta crocuta) den assemblage, Amboseli Park, Kenya. Journal of Archaeological Science 34 (10), 1601–1609). The factors that may influence tooth mark frequencies are fragment size, fragments from different sized animals, region of skeletal element and bone density. All four factors are examined in the present study and compared across species and with previous results. The results indicate that there is a great deal of variation in tooth mark frequencies not only between the species but also from the same species.     

Ancient DNA investigation of prehistoric salmon resource utilization at Keatley Creek,British Columbia,Canada

This study applied ancient DNA techniques to achieve accurate species identifications for the archaeological salmon remains recovered from the prehistoric pithouse village of Keatley Creek in British Columbia, Canada. Previous archaeological studies indicate that economic stratification within the community might have resulted in differential access to some preferred salmon species, such as sockeye and chinook. Unambiguous ancient DNA species identification now makes it possible to more accurately address the issue of early salmon resource utilization in the region. This study analyzed 60 salmon remains from two specialized structures and two residential structures in order to identify any species differences among bony salmon remains found within the structure. Although high success rates (over 90%) were obtained for ancient DNA tests, only three species (chinook, sockeye and coho salmon) were identified from the remains. Pink salmon was not identified among the tested sample, despite the fact that it was originally assumed to be a staple species for the site's native inhabitants. The absence of pink salmon in our sample significantly altered the picture of early salmon fishing activities in the region. As a result, the effects of economic stratification on differential access to the remaining so-called preferred species of sockeye and chinook within the four structures studied were not as dramatic as previously thought, although differences among the structures could still be observed.     

Rice chaff in ceramics from the archaeological site of Sumhuram (Dhofar,Southern Oman)

The identification of the chaff used as temper in ancient ceramics represents a possible source of information about the area of origin of the pottery. This paper studies the occurrence of rice (Oryza sp.) chaff in potsherds from the archaeological site of Sumhuram (Dhofar, Sultanate of Oman). The information gathered at the site offers insight into commerce routes and networks to which the town belonged in the first centuries AD. The analysis of the plant material in the pottery and potsherds can also furnish interesting data on the ancient technologies of pottery production.     

Ancient Iberian horses: a method to recover DNA from archaeological samples buried under sub-optimal conditions for preservation

Existing methods to extract, amplify, and sequence ancient DNA (aDNA) from horse bone and teeth were optimized to recover DNA from a depositional environment of highly permeable acidic soil. DNA was successfully retrieved using 0.10g of bone powder from horse (Equus sp.) remains dating to 25 K years utilizing the methods optimized for this archaeological material. The genetic analyses were performed in a facility that is dedicated to ancient DNA research (Paleo-DNA Laboratory, Thunder Bay, ON, Canada) and has not been previously used to analyse modern or ancient horse DNA. Research was replicated to obtain reliable sequencing results for six samples from the Iberian Peninsula that were consistent with published sequences of Equus caballus. The archaeological sequence data obtained support hypotheses that promote the significance that the Iberian Peninsula has had to the multi-focal centres of origin for horse domestication and distribution of modern horse breeds. The data presented may provide evidence of the existence of an Iberian refugium for Equus during the last glacial period, 10 K years BP. Further molecular data analyses will enhance the ideas presented by this data and our understanding of horse domestication and phylogeny. The optimization of molecular techniques to successfully obtain DNA using minimally destructive, cosmetically sensitive techniques from archaeological remains endeavours to foster further cooperation between museums and researchers.     

Firing technique characterization of black-slipped pottery in Praeneste by low field 2D NMR relaxometry

This study was aimed at individuating characteristics related to the pottery’s firing technique of Classical Roman producers, through the low field single-sided NMR relaxometry. The approach is based on a recent method of analysis, the correlation of the longitudinal and transverse NMR relaxation times of a liquid probe (in this case distilled water) filling the porous system of ceramics. The analyzed findings form two homogeneous classes with dating and place of production well established through the producer trademarks. Pottery findings belong to the black-slipped pottery productions of the Latin ancient city of Praeneste between third and second century B.C. and have been attributed to two different ”patriziae” production families: gens Samiaria and gens Trebonia. A further fragment with Greek kylix style, found in the same archaeological area, has also been analyzed, in order to understand if it had been imported from Greece, or made in Praeneste in imitation of the Greek style.