Recovery of DNA from archaeological insect remains: first results,problems and potential

We report the recovery of short fragments of PCR amplifiable ancient DNA from exoskeletal fragments of the grain weevil Sitophilus granarius (L.) extracted from Roman and medieval deposits in Northern England. If DNA preservation in archaeological insect remains is widespread then many applications in the spheres of evolutionary studies and archaeology can be conceived, some of which are outlined.     

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.     

Ancient olive DNA in pits: preservation,amplification and sequence analysis

The olive tree (Olea europaea) was domesticated by vegetative propagation of selected wild individuals with superior fruit. Later, new cultivars were established repeatedly from feral trees or from crosses between wild, feral, and domesticated trees. Thus the genetic background of many contemporary domesticated lines is a mixture of ancient cultivars and local wild trees. Ancient DNA may illuminate the complicated process of olive domestication because such DNA sequences provide data about ancient genomes that existed closer to the domestication events. Well preserved DNA must be available for such studies, even though in the Mediterranean region, where olive cultivation took place, the climatic conditions are not favorable for DNA preservation. To select for well preserved pits we measured their proportions of lignin by IR spectroscopy, and correlated this with parameters of DNA quality such as template length in an olive-specific repeat array, and template quantity as determined by real-time PCR amplification. Archaeological pits that passed these tests did contain high quality ancient DNA. We present the first ancient olive DNA sequences and compare them to modern wild, feral and domesticated lines.     

Ancient DNA for the Archaeologist: The Future of African Research

Ancient DNA analyses are increasingly popular in archaeology. With the exception of Egypt, the African continent has been grossly understudied using these techniques. We review the state of ancient DNA research on Africa, ancient DNA analysis techniques, and common pitfalls in these studies. We provide recommendations for archaeologists interested in collecting and interpreting ancient DNA data.     

Neolithic Hedgehogs (Erinaceus europaeus) from the Island of Gotland show early contacts with the Swedish mainland

Previous research probing early migrations and contacts in the Baltic Sea area is characterized by the analysis of different chronologies and subsistent strategies on all sides of the Sea. Several studies performed on artifact typology, ceramics, grave rituals and physical anthropology ended with varying results. Although the question of human origins remains inconclusive, in this study, we rely on the phylogeography of an animal associated with humans to elucidate findings regarding prehistoric human migration and contacts.Hedgehogs, along with other fauna on Gotland, were brought over to the island by humans. We examined hedgehog mitochondrial DNA from the Pitted Ware Culture (Middle Neolithic). The genetic signatures of the animals on the island were investigated to determine the animal’s origin.From the 23 bones originally examined, twelve bones from all five locations studied yielded reliable results and resembled published extant Erinaceus europaeus sequences from Sweden, Norway and Denmark. We postulate that a western heritage for the Neolithic hedgehogs on Gotland indicates early human contact with the Swedish mainland.     

Ancient DNA analysis on Clonorchis sinensis eggs remained in samples from medieval Korean mummy

In the present study, Clonorchis sinensis (C. sinensis) ancient DNA (aDNA) was successfully extracted from human remains discovered in a tomb dating to the medieval Joseon dynasty of Korea. The presence of C. sinensis eggs was confirmed by microscopic observation, after which a PCR-based aDNA analysis was performed using primer sets designed for the amplification of nuclear ribosomal internal transcribed spacer 1 (ITS1), 2 (ITS2) and mitochondrial cytochrome c oxidase 1 (CO1) genes. The sequences obtained were 100% homologous to some contemporary C. sinensis gene sequences reported from Korea and other East Asian countries. We believe that the results of our analysis expand the temporal and geographical scopes of research on the history of C. sinensis infection in different human populations.     

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.     

Evolving ecosystems: ecological data from an Iron Age small mammal accumulation at Alorda Park (Catalonia,Spain)

Small mammals are considered to be good indicators of ancient environments. One particular layer in a fortification tower of a Mediterranean Iberian village, Alorda Park (4th century BC), provided a large amount of well-preserved small mammal bones (about 19,200 remains) probably accumulated by barn owls (Tyto alba). Such an accumulation is rarely available from the Iron Age. It provides not only palaeoenvironmental data, but also further information about other studies such as morphometrics, ancient DNA and zoogeography. This article focuses on the ecological data, and attempts to develop an accurate taphonomic study to assess the reliability and statistical significance of this archaeological sample.     

Clarifying prehistoric parasitism from a complementary morphological and molecular approach

This paper reports an approach to the identification of prehistoric parasitic infection, which integrates traditional morphological methods with molecular methods. The approach includes the strengths of each method while mitigating the limitations. Demonstrating the efficacy of this approach, we provide a case study from a 1400 year old desiccated fecal sample from La Cueva de los Muertos Chiquitos, archaeological site, near Rio Zape, Durango, Mexico. Traditionally prepared microscope slides were processed via microscopy and tentative ascarids were identified. Information regarding the parasites' developmental stage was recorded. DNA was then extracted directly from the slide material. From this DNA extract, a small segment of the 18S ribosomal RNA gene variant that is specific to Ascaris, and its phylogenetically close relatives, was targeted for PCR amplification and sequencing. Phylogenetic analysis of the DNA sequence best matched a member of physalopterids, rather than ascarids, with a single exception of a match to Contracaecum spiculigerum. Subsequent extractions, amplifications and sequencing of the original rehydrated coprolite material confirmed these results. The C. spiculigerum sequence represented a phylogenetic anomaly and subsequent analysis determined the sequence was an error in the BLAST database, likely attributable to misidentification of juvenile specimens prior to sequencing and submission. Physaloptera are a difficult genus to identify morphologically and can carry major health burdens. They may be underreported in humans, in part, because of morphological similarities to the more common human parasites belonging to ascarids. We conclude that integrating traditional morphological methods with molecular methods can help resolve this issue, in both contemporary and prehistoric populations.     

DNA in cremated bones from an early bronze age cemetery cairn

The discovery of polymeric DNA in charred wheat grains and other burnt plant remains prompted us to examine a set of cremated bones for the presence of ancient DNA. Extracts of cremated bones from an early Bronze Age cemetery cairn were analysed by hybridization probing and the polymerase chain reaction. Hybridization with two probes targeting human DNA resulted in strong positive signals. Polymerase chain reactions directed at a multicopy sequence in human nuclear DNA were successful, extracts of five burials giving amplification products of the expected size. Exploitation of ancient DNA in cremated bones could be of great value in archaeology as it may enable information pertaining to sex and kinship to be obtained from fragmented material.     

Genetic analysis of archaeological wood remains: first results and prospects

A total of 51 ancient oak wood samples originating from various European archaeological sites, dating from the Neolithic period to the 18th century, were assayed for the presence of reproducible chloroplast (cp) DNA sequences. Five polymorphic chloroplast fragments were targeted. Only five of the samples could be fully genetically characterised, revealing four different oak cpDNA haplotypes. In all cases, the haplotypes detected on ancient woods and the haplotypes characterised from fresh samples from the same localities matched. Overall, this congruence is consistent with a genetic continuity between ancient and modern European oaks, confirming the hypothesis that the mapped genetic patterns largely reflect the original structure that established during the post-glacial. This stability of the genetic structure implies that, in the future, the technique could be used to infer or confirm the transport of wood by man, providing interesting perspectives for the genetic analysis of ancient woods.     

Cats of the Pharaohs: Genetic Comparison of Egyptian Cat Mummies to their Feline Contemporaries

The ancient Egyptians mummified an abundance of cats during the Late Period (664 - 332 BC). The overlapping morphology and sizes of developing wildcats and domestic cats confounds the identity of mummified cat species. Genetic analyses should support mummy identification and was conducted on two long bones and a mandible of three cats that were mummified by the ancient Egyptians. The mummy DNA was extracted in a dedicated ancient DNA laboratory at the University of California - Davis, then directly sequencing between 246 and 402 bp of the mtDNA control region from each bone. When compared to a dataset of wildcats (Felis silvestris silvestris, F. s. tristrami, and F. chaus) as well as a previously published worldwide dataset of modern domestic cat samples, including Egypt, the DNA evidence suggests the three mummies represent common contemporary domestic cat mitotypes prevalent in modern Egypt and the Middle East. Divergence estimates date the origin of the mummies' mitotypes to between two and 7.5 thousand years prior to their mummification, likely prior to or during Egyptian Predyanstic and Early Dynastic Periods. These data are the first genetic evidence supporting that the ancient Egyptians used domesticated cats, F. s. catus, for votive mummies, and likely implies cats were domesticated prior to extensive mummification of cats.     

Ancient DNA analysis of desiccated wheat grains excavated from a Bronze Age cemetery in Xinjiang

Wheat has been one of the most important crop in Eurasia since the Neolithic period. Understanding the spread of wheat cultivation is crucial to understanding the spread of agriculture as a whole and the interactions between prehistoric populations across the Eurasian continent. However, the routes by which wheat cultivation spread eastwards have been poorly understood to date, due to the scarcity of plant remains recovered from archaeological sites. Desiccated wheat grains excavated from the Xiaohe cemetery in Xinjiang, and dated to the early Bronze Age, show excellent DNA preservation. Here we present an ancient DNA (aDNA) analysis of wheat (Triticum sp.) grains excavated from Xiaohe and provide the first definitive evidence for bread wheat in China during the Bronze Age. The nuclear ribosomal DNA internal transcribed spacer regions (ITS1 and ITS2) and the intergenic spacer region (IGS) were amplified. The IGS region within the D genome of wheat has a 71 bp insertion that is absent from corresponding regions in the A and B genomes. The results showed that the Xiaohe wheat showed most sequence similarity to hexaploid bread wheat (Triticum aestivum), including the characteristic insertion into the D genome. The presence of bread wheat at the Xiaohe cemetery is discussed in relation to it having spread into Xinjiang by the Bronze Age, providing new insight into the origins of bread wheat in East Asia.     

木炭在考古学研究中的应用

利用考古发掘出土的木炭可以重建考古遗址周围的环境,重建植物群,探索遗址的居民与自然界植被的关系,寻找有关古人类的信息。该对木炭在考古学研究中的可行性、必要性及取样与鉴定方法进行了简要的概括,并以研究实例进一步说明木炭的鉴定过程,对拓宽我国科技考古研究方法有一定的意义。     

DNA from ancient stone tools and bones excavated at Bugas-Holding,Wyoming

DNA residues may preserve on ancient stone tools used to process animals. We studied 24 stone tools recovered from the Bugas-Holding site in northwestern Wyoming. Nine tools that yielded DNA included five bifaces, two side scrapers, one end scraper, and one utilized flake. The excavators did not handle three of these tools with bare hands, and they were sealed in separate plastic bags to minimize contact with modern DNA. Five sediment samples did not yield mammalian DNA. One tool contained DNA from three species, and these templates competed during polymerase chain reaction (PCR) amplification. DNA identifications suggested a butchery pattern indicative of a human population under resource stress or a broader range of industrial activities at Bugas-Holding. We describe several improvements in DNA residue analysis, including a more effective DNA recovery protocol, methods to measure sensitivity and inhibition of PCR in each sample, and strategies to surmount competition between templates during PCR.     

Chemical characterisation of anthro-technosols from Bronze to Middle Age in Bologna (Italy)

The aim of this research was the understanding of a peculiarly expanded soil sequence in Bologna (i.e., Roman Bononia, Etruscan Felsina), one of the most important archaeological poles for Etruscan colonisation and settlement in northern Italy. The uppermost part of the chronological sequence dating from the Bronze Age up to medieval and modern times was analysed for the first time from geochemical and pedological viewpoints. The integration of archaeological, stratigraphical and pedological data supported by soil chemical analysis, a fundamental key for reconstructing a correct evolutionary sequence, made it possible to recognise seventeen buried Soil Units (ancient Technosols, Anthrosols and Cambisols) characterised by different kinds and degrees of intensity of anthropogenic influence. In some cases, the results show a relatively severe element concentration (Cu, Sn) mainly characterising the Iron Age (Etruscan and Villanovan) stratigraphic units, representing an ancient industrial pollution linked to local dispersal of melting activity dump products generated in near-site surrounding areas. Therefore, a comparison was possible between natural blank and anthropogenically polluted geochemical values for some local soils.     

Ancient DNA of the Pacific rat (Rattus exulans) from Rapa Nui (Easter Island)

We report analysis of ancient mitochondrial DNA sequences from nine archaeological specimens (8 femura and 1 incissor) of Rattus exulans excavated from Anakena Beach Dune on Rapa Nui. Sequence of a 239-base-pair fragment of the hypervariable mitochondrial control region reveals a single mitochondrial DNA (mtDNA) sequence of all samples corresponding to the R9 haplotype prevalent in East Polynesia. This suggests a single or very limited introduction of Rattus exulans to the island. Rapa Nui, like other remote islands of Polynesia, remained effectively isolated following colonization.     

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.     

Experimental micromorphology in Tierra del Fuego (Argentina): building a reference collection for the study of shell middens in cold climates

Commonly used in archaeological contexts, micromorphology did not see a parallel advance in the field of experimental archaeology. Drawing from early work conducted in the 1990’s on ethnohistoric sites in the Beagle Channel, we analyze a set of 25 thin sections taken from control features and experimental tests. The control features include animal pathways and environmental contexts (beach samples, forest litter, soils from the proximities of archaeological sites), while the experimental samples comprise anthropic structures, such as hearths, and valves of Mytilus edulis (the most important component of shell middens in the region) heated from 200 °C to 800 °C. Their micromorphological study constitutes a modern analogue to assist archaeologists studying site formation and ethnographical settings in cold climates, with particular emphasis on shell midden contexts.