Next generation sequencing of DNA in 3300-year-old charred cereal grains

We investigated whether ‘next generation’ methods can be used to sequence ancient DNA molecules in charred cereal grains. We prepared a DNA extract from a mixed sample of barley, einkorn, emmer and broomcorn millet, taken from a 3300-year-old assemblage of charred cereal grains from Assiros Toumba, Greece. Using the SOLiD 5500 system, we obtained 21,112,844 unique sequence reads. Of these, 178,779 had a 75% or greater nucleotide sequence similarity with one or more entries in the full nucleotide sequence database; 496 of these matches were to previously reported sequences from barley, einkorn, emmer, broomcorn millet or related species such as hexaploid wheat. The unique reads were also compared with a database comprising only wheat sequences. This analysis identified 1658 charred grain sequences that had 90% or greater similarity with segments of the wheat genome. The presence of barley, wheat and millet sequences in the next generation dataset confirms the presence of ancient DNA in this charred grain assemblage. Enrichment of extracts by hybridization capture or equivalent methods is likely to enable sequences to be obtained for entire genes and other genomic regions of interest.     

Improved methodology for extraction and amplification of DNA from single grains of charred wheat

Comparisons were made between two commonly used methods for the extraction of ancient DNA from charred plant remains. Using artificially charred wheat seeds, we show that silica-binding is the most efficient method for extraction of DNA. We describe a improved silica-binding procedure, including pre-incubation with N-phenacylthiazolium bromide and increased washing of bound DNA, which yields amplifiable DNA from seeds heated at 200 °C for up to 8 h, conditions which promote the formation of Maillard products which often copurify with aDNA and inhibit subsequent PCRs. We believe that this method will be effective in ancient DNA extraction with most types of charred archaeobotanical material. Both cold- and hot-start PCR procedures gave good amplicon yields with extracts prepared in this way, but cold-start PCRs also resulted in synthesis of short artefact products. Addition of bovine serum albumin to PCRs, an inert carrier substance thought to enhance amplification efficiency by binding contaminants, had no advantageous effect and in fact reduced amplicon synthesis.     

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.     

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.     

Ancient DNA in archaeological wheat grains: preservation conditions and the study of pre-Hispanic agriculture on the island of Gran Canaria (Spain)

The recovery of ancient DNA from archaeological wheat samples under different preservation conditions was assessed using a number of genetic markers. It was possible to amplify nuclear DNA from desiccated grains but not from charred. The desiccated grain was from a pre-Hispanic grain silo in Gran Canaria and showed excellent DNA preservation, enabling the amplification of the ribosomal DNA markers IGS and ITS, the upstream region of the HMW-glutenin locus and single-locus nuclear microsatellites. Our results demonstrated the presence of both durum and bread wheat in an assemblage of naked grain. We were also able to identify different genotypes in durum wheat and compare these with extant landraces, providing insights into the agrarian practices of the ancient Canarians and the origin of their crops.     

Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii

The persistent uncertainty on the classification of the “new” glume wheat found in Neolithic and Bronze Age sites from Greece and other European settlements might be resolved only through analysis of its ancient DNA. Tools able to discriminate among different Triticum species on the basis of scarce, very damaged DNA, are therefore essential. While current attempts concentrate on DNA fragments sequencing and comparison, in some instances PCR-based selective amplification techniques might offer a cheaper and quicker alternative. The purpose of this research was therefore the identification of species-specific primers, able to distinguish caryopses of Triticum timopheevii subsp. timopheevii from those of Triticum turgidum subsp. dicoccum. Primers and their working conditions were defined and optimized using DNA from modern accessions. The ribosomal primers ITS1 tim and ITS2 tim, and the nuclear primer acetyl-coenzyme A tim clearly discriminated the sequences of Triticum timopheevii from other species. Finally, Neolithic charred wheat grains found in the sites of Sammardenchia (Pozzuolo del Friuli, Udine) and La Marmotta (Lago di Bracciano, Roma), belonging to the “new” wheat type or to emmer, were tested with the three selected primers. However, the results were not conclusive, because the samples analysed were apparently too degraded to yield useful DNA.     

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.     

The effect of charring and burial on the biochemical composition of cereal grains: investigating the integrity of archaeological plant material

Stable isotope analysis of charred archaeobotanical cereal grains has the potential to provide direct evidence of crop growing conditions in the past and to refine palaeodietary predictions. If isotope values of archaeobotanical material are to be considered robust, it is necessary to characterise the compositional changes associated with their charring and burial. This study used a suite of analytical techniques, including FT-IR and solid state ¹³C NMR, to characterise changes in the biochemical composition of modern einkorn grains with heating at 230 °C for 2 h, 4 h, 8 h and 24 h, encompassing conditions that replicate their undistorted ancient counterparts. The biochemical composition of archaeobotanical charred einkorn grains was also investigated by FT-IR and solid state ¹³C NMR in order to assess the changes in composition which occur during burial. Results of FT-IR and solid-state ¹³C NMR show that heating of modern einkorn grains resulted in Maillard reactions between cereal proteins and starch, forming high molecular weight melanoidins, which contain both alkyl and aromatic carbon. Loss of low molecular weight carbon and nitrogen-containing volatiles resulted in a slight but non-systematic increase in the δ¹³C values and a systematic increase of 0.8‰ in the δ¹⁵N values of the charred einkorn grains. Solid-state ¹³C NMR shows that the ancient charred einkorn grains consisted entirely of aromatic carbon and retained a similar proportion of nitrogen to their modern 24 h charred counterparts, despite a significantly lower concentration of amino acids. This indicates that the amino acid nitrogen in the ancient charred grains was retained in the stable melanoidins whose polymeric structure makes them resistant to subsequent degradation.     

THE POTENTIAL OF CARBONIZED GRAIN TO PRESERVE BIOGENIC 87SR/86SR SIGNATURES WITHIN THE BURIAL ENVIRONMENT*

Carbonized grains survive for millennia in many archaeological contexts. Their stable structure raises the possibility that they preserve biogenic strontium isotope signatures. This hypothesis was investigated using short‐term, laboratory experiments with modern grain immersed in Chalk solution. HCl leaching removed > 95% of secondary alteration from charred grain, and isotope ratios close to the starting value were recovered. This could not be achieved with uncharred grains. HCl leaching of archaeological carbonized grains produced comparable levels of decontamination. Although preliminary, these results suggest that strontium isotope analysis of archaeological carbonized grains from calcareous burial contexts could be used to investigate ancient trade and agriculture.     

Archaeobotanical inference of Bronze Age land use and land cover in the eastern Mediterranean

Charcoal and charred seeds at five Bronze Age archaeological sites discern ancient land use in the eastern Mediterranean. Seed frequencies of orchard crops, annual cereals and pulses, and wild or weedy plants are used to characterize plant utilization at different archaeological sites on the island of Cyprus, in the Rift Valley of Jordan, and in the Jabbul Plain and along the upper Euphrates River valley in Syria. Seed to charcoal ratios provide proxies to determine the relative usage of dung versus wood for fuel across the ancient Mediterranean landscape. Greater charcoal and lower charred seed values are interpreted to represent a wooded environment, while higher amounts of charred seeds and minimal wood charcoal suggest a much great use of dung as a fuel source. Interestingly, Politiko-Troullia (Cyprus, Cypriot archaeological sites are, by convention, named for the nearest modern village (Politiko), followed by an italicized toponym (Troullia) referring to the plot of land that incorporates the site) has the lowest seed to charcoal ratio, suggesting its residents primarily burned wood and that the landscape surrounding Troullia remained relatively wooded during the Bronze Age. In contrast, villagers at Tell el-Hayyat (Jordan) utilized a mixture of wood and dung, in contrast to Tell Abu en-Ni’aj (Jordan), and especially Umm el-Marra and Tell es-Sweyhat (Syria), where inhabitants relied solely on dung fuel. Comparative analysis and interpretation of seed and charcoal evidence thus illustrates the variety of fuel use strategies necessitated by the dynamic and diverse Bronze Age landscapes of the Eastern Mediterranean.     

From the modern to the archaeological: starch grains from millets and their wild relatives in China

Starch grains from 31 modern samples of millets derived from the seeds of 7 species within the genus Setaria and 2 species within the genus Panicum were analyzed to determine diagnostic morphological characteristics. Ancient starch grains recovered from a sample of broomcorn millet (Panicum miliaceum) excavated from the Cishan site (10.0-7.6 cal yr BP) in the North China Plain were then subjected to the same analyses to determine the differences in morphologies, if any, between modern and ancient samples. The data indicate that morphological features, and particularly surface and fissure features, will allow for solid identifications of ancient millet starches, while size classes will be helpful, but will not be dependable taxonomic indicators.     

The origins of agriculture in North-West Africa: macro-botanical remains from Epipalaeolithic and Early Neolithic levels of Ifri Oudadane (Morocco)

This research aims to shed light on the early stages of agricultural development in Northern Africa through the analysis of the rich macro-botanical assemblages obtained from Ifri Oudadane, an Epipalaeolithic–Early Neolithic site from North-East Morocco. Results indicate the presence of domesticated plants, cereals (Hordeum vulgare, Triticum monococcum/dicoccum, Triticum durum and Triticum aestivum/durum) and pulses (Lens culinaris and Pisum sativum) in the Early Neolithic. One lentil has been dated to 7611 ± 37 cal BP representing the oldest direct date of a domesticated plant seed in Morocco and, by extension, in North Africa. Similarities in both radiocarbon dates and crop assemblages from Early Neolithic sites in Northern Morocco and the Iberian Peninsula suggest a simultaneous East to West maritime spread of agriculture along the shores of the Western Mediterranean. Wild plants were abundantly collected in both the Epipalaeolithic and the Early Neolithic periods pointing to the important role of these resources during the two periods. In addition to fruits and seeds that could have been consumed by both humans and domesticated animals, fragments of esparto grass (Stipa tenacissima) rhizomes have been identified. This is a western Mediterranean native plant that may have been used as a source of fibres for basketry.     

Presence of the genus Cuon in upper Pleistocene and initial Holocene sites of the Iberian Peninsula: new remains identified in archaeological contexts of the Mediterranean region

The dhole (Cuon alpinus) has been documented on the Iberian Peninsula throughout the Middle and Upper Pleistocene in archaeological and palaeontological contexts. However, this is an uncommon species among the Iberian quaternary faunae as a whole. This paper focuses on the remains of the genus Cuon recovered in three different sites of the Iberian Mediterranean region and their morphological and osteometrical study. By comparing these with fourteen complete skeletons of Canis lupus, as well as with other remains of C. alpinus, we intend to highlight some anatomical and metrical differences between Cuon and Canis that, added to those already published, could be a functional base-line for the identification of this genus. The identification of this species is complex, especially in archaeological contexts, and in some cases the dhole has been identified as Canis or Canidae. Furthermore, this paper explores the chronology of the presence of the dhole in the Iberian Peninsula and the different contexts where these animals have been recovered from.     

山东高青陈庄遗址炭化种子果实研究

通过对山东高青陈庄遗址灰坑等遗迹中出土的炭化植物种子果实进行鉴定和统计分析,探讨了该聚落人类与植物的关系:(1)遗址中出土的丰富的粟、黍和草木犀属植物遗存,可能与马的喂养有关;(2)陈庄聚落西周时期农业是主要的食物来源,农作物包括了文献记载的"五谷",就是粟、黍、小麦、大豆和稻,粟和小麦等旱地作物占的比重最高,水稻可能较少,牛筋草等杂草可能是收获等过程中带入聚落内的;(3)常见的野生植物如野大豆、枣、葡萄以及动物遗骸等可能反映狩猎捕捞仍然作为食物的补充。     

Short-chain n-alkanes (C16–20) in ancient soil are useful molecular markers for prehistoric biomass burning

The incorporation of plant biomass into soil usually leads to long-chain n-alkanes with a relative predominance of odd carbon numbered homologues. Contrastingly, an increase in short-chain even carbon numbered n-alkanes was found in charred biomass with progressing temperatures. We applied lipid analysis to buried ancient topsoils that contained charred organic matter and to corresponding control soils, which were characterized by a lighter color and lower contents of charred materials. Commonly, the proportion of lipid extracts was found to be lower in the ancient soil than in the control samples, which indicated an enhanced degradation of organic matter, e.g., by thermal degradation. All samples displayed a particular pattern of short-chain and even carbon numbered n-alkanes (maximum at n-C₁₆ or n-C₁₈). The ratios CPI (carbon preference index) and ACL (average chain length) for the investigated soil samples matched the ratios found for maize and rye straw charred at 400 °C or 500 °C, respectively. These molecular ratios indicate the presence of charred biomass. The predominance of short-chain and even carbon numbered n-alkanes was a result of thermal degradation of biomass. The degradation products were preserved in ancient soils and could be applied as molecular markers in archaeological or palaeoenvironmental research.     

The oldest mongoose of Europe

The Egyptian mongoose, Herpestes ichneumon, has no Pleistocene record in Europe. Consequently, it has been suggested that this animal was introduced to the Iberian Peninsula by the Arabs, who employed it for eliminating rodents and reptiles, as in the case of the genet, Genetta genetta. Here we describe a subfossil mongoose skull from the Cave of Nerja, southern Spain, which has been AMS dated 885 ± 40 years BP. This specimen constitutes the oldest record of mongooses in Europe, providing evidence that these animals were introduced during historical times in the South of the Iberian Peninsula.     

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.     

Taxonomic reassignment of the Paleolithic human navicular from Cueva de los Torrejones (Guadalajara,Spain)

Evidence of human activity and hominin remains are very scarce inland on the Iberian Peninsula. This fact raises the issue of the scarcity of evidence that Paleolithic Homo sapiens occupied this area outside of the littoral margins (Atlantic, Cantabrian, and Mediterranean coasts). Here, we comparatively describe a human right adult navicular bone recovered in the Cueva de los Torrejones site, located in the village of Tamajón (Guadalajara, Spain). This fossil was preliminarily established as belonging to Homo cf. neanderthalensis, due to the late Pleistocene faunal association, mainly because of the presence of Crocuta crocuta and Panthera pardus. The metrical and morphological study of the navicular T93-S3.27 from Cueva de los Torrejones clearly differentiates it from Neandertals and their ancestors, the hominins from Sima de los Huesos, allowing for this fossil to be taxonomically assigned with confidence as H. sapiens. The navicular from the Cueva de los Torrejones is absolutely and relatively medio-laterally narrow with a low wedging index as those of fossil and modern H. sapiens, and clearly different of Neandertals. The increased discoveries and publications of new naviculars belonging to genus Homo, together with the findings of P. pardus and C. crocuta in more recent chronologies in the Iberian Peninsula, are compatible with this reevaluation. We propose a probable chronology for this fossil between 12 and 15 ka and ca. 25 ka, based on the biostratigraphy and the oldest presence of H. sapiens in the Iberian Peninsula. This work confirms the human presence within the Iberian Peninsula during the Upper Paleolithic and reopens the question of the peopling of the inner Peninsula during this period.     

Miliacin in palaeosols from an Early Iron Age in Ukraine reveal in situ cultivation of broomcorn millet

During the archaeobotanical investigation of Scythian–Sarmatian period (Early Iron Age), pits with crop processing waste, discovered in the floodplain of Donets River, eastern Ukraine, and charred remains of cereal grains, dominated by broomcorn millet, were recorded. The grains from the pits were radiocarbon dated to the fifth to first century BC. Those pits are distant from any known contemporaneous settlement. The apparent disconnection of these pits from any local settlement suggests that (1) millet was brought from other locations by mobile groups, or (2) millet was cultivated locally by populations whose settlements have left no discernible archaeological trace. The analysis of molecular biomarkers preserved in palaeosols that are stratigraphically connected to the pits revealed high levels of miliacin, a molecule that can be preserved in ancient soils and sediments, and that is consistent with broomcorn millet (Panicum miliaceum). High levels in miliacin in soils stratigraphically connected to the pits are interpreted as the result of a large biomass of P. miliaceum produced at time of soil formation. Our biogeochemical results applied to a palaeosol thus attest to the in situ cultivation of crops dominated by the broomcorn millet during the early Iron Age in the floodplain of Donets River. Biochemical examination of soils and palaeosols can thus provide useful information on past dynamics of land-use by ancient population, especially when settlements or macrobotanical remains are absent.     

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.