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.     

Molecular and morphological analyses of avian eggshell excavated from a late thirteenth century earth oven

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) – one of New Zealand’s most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts.     

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.     

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.     

Archaeogenetic study of prehistoric rice remains from Thailand and India: evidence of early <Emphasis Type="Italic">japonica</Emphasis> in South and Southeast Asia

We report a successful extraction and sequencing of ancient DNA from carbonized rice grains (Oryza sativa) from six archaeological sites, including two from India and four from Thailand, ranging in age from ca. 2500 to 1500 BP. In total, 221 archaeological grains were processed by PCR amplification and primary-targeted fragments were sequenced for comparison with modern sequences generated from 112 modern rice populations, including crop and wild varieties. Our results include the genetic sequences from both the chloroplast and the nuclear genomes, based on four markers from the chloroplast and six from the nuclear genome. These markers allow differentiation of indica rice from japonica rice, the two major subspecies of Asian rice (O. sativa) considered to have separate geographical origins. One nuclear marker differentiates tropical and temperate forms of subspecies japonica. Other markers relate to phenotypic variation selected for under domestication, such as non-shattering, grain stickiness (waxy starch) and pericarp colour. Recovery and identification of sequences from nuclear markers was generally poor, whereas recovery of chloroplast sequences was successful, with at least one of four markers recovered in 61 % of archaeological grains. This allowed for successful differentiation of indica or japonica subspecies variety, with japonica identified in all the Thai material and a mixture of indica and japonica chloroplasts in the two Indian assemblages. Rice subspecies was also assessed through conventional archaeobotanical methods relying on grain metrics, based on measurements from 13 modern populations and 499 archaeological grains. Grain metrics also suggest a predominance of japonica-type grains in the Southeast Asian sites and a mixture of japonica and indica in the Indian sites with indica in the minority. The similar results of grain metrics and ancient DNA (aDNA) affirm that grain measurements have some degree of reliability in rice subspecies identification. The study also highlights the great potential of ancient DNA recovery from archaeological rice. The data generated in the present study adds support to the model of rice evolution that includes hybridization between japonica and proto-indica.     

Archaeological and Historical Materials as a Means to Explore Finnish Crop History

ABSTRACT

In Northern Europe, barley (Hordeum vulgare L.) has been cultivated for almost 6000 years. Thus far, 150-year-old grains from historical collections have been used to investigate the distribution of barley diversity and how the species has spread across the region. Genetic studies of archaeobotanical material from agrarian sites could potentially clarify earlier migration patterns and cast further light on the origin of barley landraces. In this study, we aimed to evaluate different archaeological and historical materials with respect to DNA content, and to explore connections between Late Iron Age and medieval barley populations and historical samples of barley landraces in north-west Europe. The material analysed consisted of archaeological samples of charred barley grains from four sites in southern Finland, and historical material, with 33 samples obtained from two herbaria and the seed collections of the Swedish museum of cultural history.

The DNA concentrations obtained from charred archaeological barley remains were too low for successful KASP genotyping confirming previously reported difficulties in obtaining aDNA from charred remains. Historical samples from herbaria and seed collection confirmed previously shown strong genetic differentiation between two-row and six-row barley. Six-row barley accessions from northern and southern Finland tended to cluster apart, while no geographical structuring was observed among two-row barley. Genotyping of functional markers revealed that the majority of barley cultivated in Finland in the late nineteenth and early twentieth century was late-flowering under increasing day-length, supporting previous findings from northern European barley.     

Ectopic paragonimiasis from 400-year-old female mummy of Korea

Of the parasite eggs discovered in ancient samples from Korean archaeological sites, Paragonimus spp. are of particular importance in that they are regarded as one of the most insidious trematode infection sources. Although their infection prevalence decreased rapidly in the early 20th century, archaeoparasitological studies on the species are still required, as historical knowledge of paragonimiasis remains far from comprehensive. Fortunately, we recently were given a chance to examine a medieval Korean mummy in a good state of preservation. Using morphological techniques, we discovered evidence of ectopic paragonimiasis in the sample from the 17th-century female. When the ITS 2 gene was extracted, amplified and sequenced from Paragonimus eggs, it showed 100% homology to the sequences of modern Paragonimus westermani reported from Korea and Japan, forming a cluster distinct from South Asian P. westermani. Our report is the first-ever analysis of ancient Paragonimus DNA from any archaeological field in the world. The ectopic paragonimiasis diagnosis made in this study, especially involving the liver sample, also is the first of its kind in archaeoparasitology.     

New data on relevant ancient Egyptian wooden artifacts: Identification of wooden species and study of the state of conservation with multidisciplinary analyses

Wood species identification and characterization of its weathering processes are crucial steps in the scientific approach of conservation of wooden cultural heritage. Many precious wooden objects of ancient Egypt are largely present in museums, nevertheless relatively little information is available concerning the nature of timber used and on their status of conservation. To address this gap, the wooden species of three relevant archaeological wood objects (statue, box, and coffin) arising from different Egyptian archaeological sites dated from the Old Kingdom (2,686–2,181 BC) to New Kingdom (1,550–1,069 BC) were deeply studied. Five hardwood and softwood species were identified belonging to Tamarix mannifera, T. gennessarensis, Ficus sycomorus, Vachellia nilotica, and Cedrus sp. Such data confirmed the recurrence of Vachellia and Tamarix among the most common timbers found in ancient Egypt. Scanning electron microscope, Fourier transform spectroscopy, and synchrotron x-ray radiation diffraction were conducted to evaluate the archaeological wood deterioration. The formation of microcracks, biological degradation patterns (fungal colonization), or chemical characterization (accumulation of salts on and in-between wooden cells) were detected. SEM micrographs showed the presence of fungal hyphae and conidial spores on the wooden cells. Significant changes in the chemical wood composition and decrease in the crystallinity index were detected.     

Avian egg-shell from archaeological sites

Avian egg-shell occurs fairly commonly on archaeological sites, particularly in alkaline deposits. It is rarely studied in detail. Egg-shell structure is described briefly below and features which may be of value for identification purposes are discussed. Egg-shells from 14 archaeological sites are tentatively identified as goose, domestic fowl, duck and guinea fowl. It seems likely that confident identification should ultimately be possible using a combination of features. This should yield information complementary to osteological evidence for the history of the utilization of birds and their eggs.     

Species identification of archaeological dung remains: A critical review of potential methods

Abstract

Dung, macroscopically recognisable as such or not, can more commonly be found in archaeological contexts than is perhaps realised. Up to now, identification of dung to the species which produced it is usually either tenuous, or is not possible. However, species identification can be very informative and is necessary before any further studies can be conducted on the dung, for example on health and hygiene in the past and palaeoecology. This study presents a review of potential methods by which species identifications of archaeological dung can be undertaken. Criteria for identification can be divided into three broad categories: morphometric features of the dung; the content of dung and contextual evidence. Overall, the chances of a precise identification are high; however, a combination of different criteria and techniques will often be necessary to establish a secure identification. Moreover, preservation issues may exclude the application of some criteria while several criteria require more research and the expansion of reference collections of recent material. The overall aim is to move towards standardised methods for species identification of archaeological dung.     

Amplification and sequencing of Trichuris trichiura ancient DNA extracted from archaeological sediments

Despite high prevalence of Trichuris trichiura infection, PCR-based analysis on T. trichiura from archaeological samples has not been established so far. In the present study, we sought to perform PCR-based amplification of T. trichiura aDNA using the sediments from medieval tomb of Korea. The presence of Trichuris eggs were first detected by microscopic observation; then confirmed by PCR-based aDNA analysis. Obtained sequence showed 100% homology to that of the small subunit ribosomal RNA (SSUrRNA) gene of T. trichiura but distinct from that of other Trichuris species. PCR-based aDNA analysis in this study can serve as effective method to confirm the presence of T. trichiura eggs in the soils or coprolites collected from archaeological sites.     

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.     

Morphological and genetic studies of waterlogged Prunus species from the Roman vicus Tasgetium (Eschenz,Switzerland)

Morphological and genetic studies were performed on waterlogged Prunus fruit stones from the Roman vicus Tasgetium (Eschenz, Switzerland). Some fruit stones could be identified to species level based on morphological and metric criteria. Other fruit stones found were not identifiable to species level. Of the latter, the morphological group Prunus insititia/spinosa represents either native sloe, cultivated primitive plum (damson) or a hybrid of both. In one out of 10 individual fruit stones of this group Prunus specific chloroplast trnL-trnF and nuclear ITS1 markers were verifiably amplified. Sequences of trnL-trnF led to the identification of Prunus spinosa. The presence of authentic DNA was confirmed by phylogenetically meaningful sequences from the archaeobotanical group Prunus avium/cerasus of the same sample using chloroplast rbcL and nuclear ITS1 DNA markers. The results demonstrate the utility of waterlogged plant remains for genetic analysis: as seeds of fruit trees are often preserved waterlogged in the archaeological record. Ancient DNA (aDNA) studies are a promising tool to answer archaeobotanical issues concerning early horticulture, which probably started in the Northern alpine region with the onset of Romanisation.     

Separating Sheep (Ovis aries L.) and Goats (Capra hircus L.) Using Geometric Morphometric Methods: An Investigation of Astragalus Morphology from Late and Final Bronze Age Central Asian Contexts

Many qualitative and quantitative methods for the separation of sheep and goat bones are based upon Middle Eastern, Mediterranean and European specimens. However, these methods may not be as applicable in other geographical contexts due to regional morphological variation. In order to address this, both traditional and geometric morphometric methods were applied to sheep, (Ovis aries L.) and goat (Capra hircus L.) astragali from archaeological sites from Kazakhstan dating to the Late and Final Bronze Age (1900–900 bce ). This exploratory research confirmed that qualitative features remained useful for distinguishing between species, while traditional morphometric methods were unable to conclusively support the field identification of species. Geometric morphometric methods found significant morphological differences between species and confirmed group membership while exploring specific qualitative features that were effective in distinguishing between sheep and goat in Central Asian contexts. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

A Novel Application of Molecular Techniques to Pacific Archaeofish Remains*

Until recently, the identification of fish bone from Pacific archaeological sites generally relied upon morphological analysis of a relatively restricted number of skeletal elements. Limited morphological variation within fish families, amongst other things, has often restricted Pacific archaeofish determinations to the level of family or, in some cases, genus. We suggest that these relatively imprecise determinations may mask important aspects of prehistoric human fishing practices and subsistence patterns. This paper describes a molecular technique that allows for more precise taxonomic determinations, initially applied to the large and diverse family of Serranidae. The technique involves extraction, amplification, sequencing and analysis of mitochondrial DNA variation in the 16s ribosomal RNA gene. Following development of protocols and primers for modern reference materials, the technique was applied to an archaeological faunal assemblage from Aitutaki, southern Cook Islands.     

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.     

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.     

Morphometric analysis of limpets from an Iron-Age shell midden found in northwest Portugal

The present work reports an analysis of a shell midden found in Terroso hillfort, an important Iron-Age oppidum located in northwest Portugal. The midden was dated from the latest phase of occupation of the hillfort, between the Ist century BC and the Ist century AD (Roman period), and contained 684 well-preserved Patella shells. The identification of archaeological shells was carried out comparing them with modern specimens collected in two shores in the neighborhood of the hillfort. The identification of modern shells was based on radula pluricuspid teeth and shell morphology. Relative abundance of Patella species in the Terroso midden was different from modern populations. Archaeological shells were dominated by Patella vulgata, but Patella intermedia was the most abundant species in modern populations. Dimensions of archaeological shells were very different from modern populations. Shell length range and variability in archaeological shells were lower than in the modern populations. Log height vs. length plots for archaeological shells were different from modern populations. Patella shells from Terroso midden was significantly taller then modern specimens. These differences between archaeological shells and modern populations could be due to environmental changes, namely an increase in wave action and intensity. These harsher costal conditions could have resulted from an alteration in the morphology of the coast, caused by a rise in the mean sea level from 2000 years BP to the present. Alternatively or additionally, the observed differences can be related to gathering strategies. Archaeological shells could have been selected by size, and collected preferentially at high shore and in sheltered sites.