DNA analysis of archaeological sheep remains from China

Recent research has thrown considerable light on the history of the domestic sheep, but has not extended to ancient sheep specimens. In the present study, ancient DNA analysis was carried out on eight archaeological sheep remains recovered from Erlitou archaeological site in Henan Province (ca. 2100–1800 B.C.) to explore the genetic structure of ancient sheep and the phylogenetic relationship between ancient and modern sheep. We analyzed the control region sequences and coding regions of mitochondrial DNA from the remains by direct sequencing and restriction fragment length polymorphism analysis, respectively. Our results reveal that all ancient sheep belong to lineage A defined by modern sheep sequences. Phylogenetic analysis shows that neither argali (Ovis ammon) nor urial (Ovis vignei) mtDNA is closely related to Erlitou ancient sheep. In addition, our results suggest that ancient DNA analysis can serve as a powerful tool in tracing prehistoric population movement.     

Distinguishing between archaeological sheep and goat bones using a single collagen peptide

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.     

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.     

Characterising the potential of sheep wool for ancient DNA analyses

The use of wool derived from sheep (Ovis aries) hair shafts is widespread in ancient and historic textiles. Given that hair can represent a valuable source of ancient DNA, wool may represent a valuable genetic archive for studies on the domestication of the sheep. However, both the quality and content of DNA in hair shafts are known to vary, and it is possible that common treatments of wool such as dyeing may negatively impact the DNA. Using quantitative real-time polymerase chain reaction (PCR), we demonstrate that in general, short fragments of both mitochondrial and single-copy nuclear DNA can be PCR-amplified from wool derived from a variety of breeds, regardless of the body location or natural pigmentation. Furthermore, although DNA can be PCR-amplified from wool dyed with one of four common plant dyes (tansy, woad, madder, weld), the use of mordants such as alum or iron leads to considerable DNA degradation. Lastly, we demonstrate that mtDNA at least can be PCR-amplified, cloned and sequenced from a range of archaeological and historic Danish, Flemmish and Greenlandic wool textile samples. In summary, our data suggest that wool offers a promising source for future ancient mitochondrial DNA studies.     

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.     

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.     

Repeat silica extraction: a simple technique for the removal of PCR inhibitors from DNA extracts

Polymerase chain reaction (PCR) inhibitors are often co-extracted with ancient DNA (aDNA) and when present make the analysis of aDNA difficult, if not impossible. In this study we review previous research on PCR inhibitors and techniques that address their co-extraction with DNA from sub-optimal samples. Additionally, we introduce a simple extraction technique, “repeat silica extraction,” that effectively removed PCR inhibitors from extracts of 7000–8000-year-old human skeletal remains from the Windover archaeological site in Florida and 700–2000-year-old human coprolites excavated from Fish Slough Cave in southern California. A series of tests on these same samples demonstrates that N-phenacylthiazolium bromide is largely ineffective, despite previously reported success using this compound as part of the DNA extraction process. We also describe a method for demonstrating the presence as well as successful removal of PCR inhibitors by use of a “positive aDNA control,” a test necessary to conclude that negative PCR amplification is the result of the absence of preserved DNA.     

The molecular palaeoecology of geese: identification of archaeological goose remains using ancient DNA analysis

The remains of six species of geese are commonly recovered from archaeological sites in Britain dating from the Saxon and later periods. However, identification of this material to species level is hampered by a lack of morphological variation and a large overlap in size. To address this issue we obtained DNA sequence data for a section of the mitochondrial cytochrome b gene from modern samples of each species, and successfully identified several DNA markers for Branta species. No markers were found within the cytochrome b gene for the genus Anser. Ancient DNA techniques were then used to recover DNA from goose bones excavated from two archaeological sites. The DNA sequences enabled identification of Barnacle goose (Branta leucopsis) from one site and confirmed the presence of Anser species at another. © 1998 John Wiley & Sons, Ltd.     

Independent confirmation of a diagnostic sheep/goat peptide sequence through DNA analysis and further exploration of its taxonomic utility within the Bovidae

Buckley et al. (2010) recently identified a Type I collagen (COL1A2) peptide sequence that discriminates between sheep and goat species. The exact location of this peptide sequence on the genome was not reported. We identified the sequence's location and developed a PCR based approach that amplifies the diagnostic sequence from exon 41 of the COL1A2 gene. Using DNA analysis, we confirmed that this COL1A2 peptide discriminates between goat and sheep species reliably, but is limited as a more general phylogenetic marker.     

PROVENANCE OF ANCIENT TEXTILES—A PILOT STUDY EVALUATING THE STRONTIUM ISOTOPE SYSTEM IN WOOL*

Strontium isotopes are used in archaeology to reconstruct human and animal migration routes. We present results of a pilot study applying strontium isotope analyses to modern sheep hair as a basis for its potential use as a provenance tracer for ancient woollen textiles. Our hydrofluoric acid‐based, lipid soluble analytical protocol, also tested on a number of ancient textile fibres, allows for contamination‐free, low blank strontium isotope analysis of minimal amounts of archaeological material. ⁸⁷Sr/⁸⁶Sr ratios of decontaminated sheep hair agree well with the compositions of biologically available (soluble) strontium fractions from the respective feeding ground soils, a translatable requirement for any potentially successful provenance tracing applied to wool textiles.     

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.     

Rice archaeological remains and the possibility of DNA archaeology: examples from Yayoi and Heian periods of Northern Japan

Japanese rice cultivation in paddy fields has 2,400∼3,000 years of history. Most of modern Japanese rice varieties are classified as Temperate-japonica (Tm-J). Few landraces are recognized as Tropical-japonica (Tr-J) only in southwestern Japan. However, ancient DNA studies and phytolith analysis suggest that Tr-J strains were more popular in the past than now. Maekawa is a complex archaeological site composed of paddies dated from the Yayoi (2,100 years BP) to the Heian (1,100 years BP) periods. Phytolith analysis indicates that intensive rice cultivation was practiced in both periods, but there was no cultivation in the intervening period. Morphological features of bulliform phytoliths suggest that Tr-J was cultivated during both periods. Locally, rice cultivation during the Heian period was brought to a close by a flood event, in which immature rice plants were pulled down and buried in silt to be preserved in a quasi-carbonized/ waterlogged state. Ancient DNA (aDNA) analysis of the carbonized plant culm from Heian Maekawa recovered chloroplast DNA sequences of the 6C7A plastid subtype, which is common to both Tr-J and Tm-J, whereas two plastid subtypes, such as 6C7A and also 7C6A, were found in aDNA of carbonized grains from the Tareyanagi site of the Yayoi period. The latter plastid subtype was specific only to Tr-J. In order to better characterize the past rice populations, modern landraces collected in the local area were classified with morphophysiological traits. Some of the landraces were found to carry several traits of Tr-J, including bulliform phytolith types, but mixed with Tm-J traits. Based on the discontinuous distribution of rice phytoliths between the Yayoi and the Heian period, the early introduction of rice cultivation may have been discontinuous and locally reintroduced after a ∼1,000-year hiatus, but with a genetically different rice population. Such populations were composed from Tr-J like strains as shown by landraces but with reduced diversity in plastid types. Through such changes, since the Yayoi era, Tr-J was largely replaced by Tm-J, although ancient Tr-J continued to participate in the genetic makeup of later rice populations and may have aided the local adaptation of introduced Tm-J.     

Ancient molecules and human prehistory

Abstract

DNA and other molecules found in ancient remains are yielding new information about the origins, spread, interaction, and culture of early humans. Molecules of animal fats preserved in archaeological pottery have shown that in medieval societies people ate non-ruminant animals, such as pigs, but burned tallow from ruminants, such as sheep and cattle. Geochemical analysis of bitumen in the Middle East has documented ancient trade routes. Studies of molecular genetic diversity have shed light on when and how cattle were domesticated. Analysis of human hair from remains up to 5200 years old has revealed the diets of those ancient people. DNA recovered from other remains has also provided evidence for theories about the origins and spread of agriculture, and human migration into the Pacific.     

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.     

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.     

Camelid Gastrointestinal Parasites from the Archaeological Site of Huanchaquito (Peru): First Results

ABSTRACT

Palaeoparasitological investigation was conducted on a first set of samples from 13 sacrificed domestic camelids recovered from the pre-Hispanic Chimú culture site of Huanchaquito-Las Llamas, Peru. The aim was to establish the animals’ gastrointestinal parasite diversity and enlighten on their health status at the time of their death. To this end, 20 samples of coprolites and intestinal contents were analysed to check for the presence of parasite markers, i.e. preserved eggs and oocysts. Microscopic examinations revealed the presence of five taxa of helminths and protozoans in a majority of the tested animals (61%). Our analysis revealed the presence in some animals of protozoan oocysts belonging to the species Eimeria macusaniensis (phylum Apicomplexa). Our study is the first report of the possible presence of a parasite egg attributed to the order Plagiorchiida (family Fasciolidae) in ancient camelids. This preliminary study shows that there is interesting potential for conducting palaeoparasitological analysis at the site and that such analysis is promising for answering questions about the health status of the Huanchaquito-Las Llamas camelids.     

Addressing seasonal site use through ancient DNA species identification of Pacific salmon at Dionisio Point,Galiano Island,British Columbia

We use ancient DNA analysis to identify Pacific salmon vertebrae to species in order to provide an important line of evidence that helps to establish the timing of seasonal residence at a Pacific Northwest Coast village site. Ancient DNA results from House 2 at Dionisio Point allow a characterization of the salmon fishery. Ten of eleven randomly selected smaller-sized salmon vertebrae were positively identified as sockeye salmon (Oncorhynchus nerka) while only a single pink salmon (Oncorhynchus gorbuscha) was identified. Of the 322 whole salmon vertebrae identified from House 2 occupation deposits during zooarchaeological analysis, 58 percent measure less than 8.0 mm and 70 percent are less than 8.5 mm in maximum transverse diameter. Together with documented aspects of the material record from Dionisio Point, most notably the vertebrate fauna from House 2, the indication that sockeye was the primary focus of the Dionisio Point salmon fishery suggests the site was inhabited during the spring and summer. This approach to the identification of season-specific site occupation has the potential for application over much of the Northeast Pacific.     

DNA of Mycobacterium leprae detected by PCR in ancient bone

We report the extraction and amplification of DNA of Mycobacterium leprae, from ancient skeletal material. The significance of the extraction of ancient bacterial DNA is discussed, as are future directions of research into ancient disease.     

Comparing the survival of osteocalcin and mtDNA in archaeological bone from four European sites

The small mineral-binding bone protein, osteocalcin, has been applied in a number of studies on ancient bone due to predictions of its long-term stability. However, the intact protein has not been shown to survive in ancient bone devoid of DNA, which is a much more phylogenetically informative biomolecule. In this investigation, the survival of osteocalcin is directly compared to the amplification of mtDNA in a set of 34 archaeological samples from four sites throughout Europe. We also present unpublished osteocalcin sequences of seven mammalian species in addition to the 19 published sequences to highlight phylogenetic limitations of this protein. The results indicate that the intact osteocalcin molecule survives less in archaeological samples than mtDNA and is more subject to the temperature of the archaeological site. Amino acid analyses show the persistence of the dominant protein collagen in samples that failed both osteocalcin and mtDNA analyses. The implications these findings present for biomolecular species identification in archaeological and palaeontological material are that, although proteins do survive beyond ancient DNA, osteocalcin does not appear to be the most ideal target.