Ancient DNA: Using molecular biology to explore the past

Ancient DNA has been discovered in many types of preserved biological material, including bones, mummies, museum skins, insects in amber and plant fossils, and has become an important research tool in disciplines as diverse as archaeology, conservation biology and forensic science. In archaeology, ancient DNA can contribute both to the interpretation of individual sites and to the development of hypotheses about past populations. Site interpretation is aided by DNA-based sex typing of fragmentary human bones, and by the use of genetic techniques to assess the degree of kinship between the remains of different individuals. On a broader scale, population migrations can be traced by studying genetic markers in ancient DNA, as in recent studies of the colonisation of the Pacific islands, while ancient DNA in preserved plant remains can provide information on the development of agriculture.     

The boylston street fishweir: Revisited

Excavations for the Boston subway system early in this century and later for building sites revealed the presence of waterlogged wood in the peat and silt deep beneath the present surface. Beginning more than 50 yr ago, the investigation of geological and biological materials recovered from these sites, especially the wood remains, believed to have been set in place by prehistoric Native Americans, became a benchmark for the multidisciplinary application of scientific methods in archaeology and environmental reconstruction. Recent excavations for building foundations in Boston have exposed additional wooden materials. We have analyzed 216 specimens recently recovered from an excavation at 500 Boylston Street where older office buildings were demolished to make way for a new commercial structure. Although some of our findings differ from those of the previous investigators we find support for the earlier supposition that the remains represent an ancient Native American fishweir, a fencelike barrier and trap for fish on an ancient shoreline.     

方兴未艾的古代DNA的研究

保留在古代生物遗骸中的遗传物质DNA是一种重要的遗传资源。古代DNA的研究对于了解包括人类在内的各种生物的起源、进化和迁徙有重要意义。古代DNA的研究有其自身的特点,并且已经取得一系列重要成就。本文综述古代DNA研究的历史、方法和进展。 Abstract：DNA present in ancient samples can be recovered,amplified and analysed.It opens a new window for genetic analysis in many different disciplines,such as anthropology,archaeology,human population genetics,animal and plant evolutionary taxonomy and forensic science.In general,ancient DNA is rare in quantity,damaged in quality.To ensure the reproducibility and reliability of the results,great cares should be taken,such as various measurements against contamination and phylogenetic analysis of ancient DNA sequences.In this paper we review recovery,amplification and analysis of ancient DNA,also discuss the guidelines to ensure the authenticity of ancient DNA and the recent advances in ancient DNA study.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

A cost‐effective high‐throughput metabarcoding approach powerful enough to genotype ~44 000 year‐old rodent remains from Northern Africa

We present a cost‐effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next‐generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human‐ and/or climate‐induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high‐throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour‐intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.     

古DNA 及其在生物系统与进化研究中的应用

古DNA是指从已经死亡的古代生物的遗体和遗迹中得到的DNA。本文回顾了近20年古DNA研究所经历的3个阶段, 从早期参与研究的科学家较少并主要利用克隆技术, 到后来由于PCR技术的出现以及提取化石DNA技术的成熟从而出现大量有关古DNA的报道; 近几年由于发现不少问题, 并引起激烈的争论, 科学家们因此而开始考虑古DNA的真实性问题, 并且提出了开展古DNA研究的严格标准。本文还讨论了古DNA在人类起源、系统发育重建、动植物驯化及考古研究中的重要意义以及现状, 表明古DNA的研究给某些原先的观点如人类的非洲起源说提供了重要证据, 也对某些观点提出了挑战; 古DNA研究还提供了某些已经灭绝生物的形态学和分子资料, 为从序列上确定古代材料的系统位置并有效地补充仅用现代DNA建立起来的谱系提供了来自古生物的依据。在动植物驯化及考古方面, 古DNA证据也为科学家提供了许多有价值的信息。最后, 本文还对古DNA研究的应用前景进行了展望。     

古DNA及其在生物系统与进化研究中的应用

古DNA是指从已经死亡的古代生物的遗体和遗迹中得到的DNA.本文回顾了近20年古DNA研究所经历的3个阶段,从早期参与研究的科学家较少并主要利用克隆技术,到后来由于PCR技术的出现以及提取化石DNA技术的成熟从而出现大量有关古DNA的报道;近几年由于发现不少问题,并引起激烈的争论,科学家们因此而开始考虑古DNA的真实性问题,并且提出了开展古DNA研究的严格标准.本文还讨论了古DNA在人类起源、系统发育重建、动植物驯化及考古研究中的重要意义以及现状,表明古DNA的研究给某些原先的观点如人类的非洲起源说提供了重要证据,也对某些观点提出了挑战;古DNA研究还提供了某些已经灭绝生物的形态学和分子资料,为从序列上确定古代材料的系统位置并有效地补充仅用现代DNA建立起来的谱系提供了来自古生物的依据.在动植物驯化及考古方面,古DNA证据也为科学家提供了许多有价值的信息.最后,本文还对古DNA研究的应用前景进行了展望.     

Analysis of ancient bone DNA: techniques and applications.

The analysis of DNA from ancient bone has numerous applications in archaeology and molecular evolution. Significant amounts of genetic information can be recovered from ancient bone: mitochondrial DNA sequences of 800 base pairs have been amplified from a 750-year-old human femur by using the polymerase chain reaction. DNA recovery varies considerably between bone samples and is not dependent on the age of the specimen. We present the results of a study on a small number of bones from a mediaeval and a 17th-century cemetery in Abingdon showing the relation between gross preservation, microscopic preservation and DNA recovery.     

试论动物考古中的小哺乳动物研究

小哺乳动物种类及其数量,在哺乳动物中占据大多数。它们是自然界生态链中非常重要的、不可缺少的环节。它们体质进化较快,大部分种类居住区域选择性强,种群活动范围比较小。所以研究小哺乳动物种群面貌及其生态特征,可以帮助我们判断古遗址相对时代,解析历史时期的生态演变。小哺乳动物的生态指示性,能够帮助我们准确复原和研究古遗址环境背景。它们的分布状况及很多生态规律,在帮助我们研究古遗址环境卫生及古居民住房条件等方面有独特作用。小哺乳动物种群变化与人类经济生产的互动性,以及它们与人类的伴栖关系、人类对小哺乳动物资源的开发和利用等,均是动物考古工作中应该重视和积极开展研究的内容。开展小哺乳动物研究,需要必要的工作计划、专业技术和实验设备。我国旧石器时代的小哺乳动物考古已取得丰硕成果,新石器时代及其以后的小哺乳动物考古尚有待加强。小哺乳动物研究工作的拓展,将会有力促进动物考古学进展,促使动物考古在考古学实践中发挥出更大作用。     

Checking the geographical origin of oak wood: molecular and statistical tools

New methods for better identification of timber geographical origin would constitute an important technical element in the forest industry, for phytosanitary certification procedures or in the chain of custody developed for the certification of timber from sustainably managed forests. In the case of the European white oaks, a detailed reference map of chloroplast (cp) DNA variation across the range exists, and we propose here to use the strong geographical structure, characterized by a differentiation of western vs. eastern populations, for the purpose of oak wood traceability. We first developed cpDNA markers permitting the characterization of haplotype on degraded DNA obtained from wood samples. The techniques were subsequently validated by confirming the full correspondence between genotypes obtained from living tissues (buds) and from wood collected from the same individual oak. Finally, a statistical procedure was used to test if the haplotype composition of a lot of wood samples is consistent with its presumed geographical origin. Clearly, the technique cannot permit the unambiguous identification of wood products of unknown origin but can be used to check the conformity of genetic composition of wood samples with the region of alleged origin. This could lead to major applications not only in the forest industry but also in archaeology or in palaeobotany.     

Novel perspectives in wood certification and forensics: dry wood as a source of DNA

The importance of wood for human societies can hardly be understated. If dry wood were amenable to molecular genetic investigations, this could lead to major applications in wood forensics, certification, archaeology and palaeobotany. To evaluate the potential of wood for molecular genetic investigations, we have attempted to isolate and amplify, by PCR, DNA fragments of increasing size corresponding to all three plant genomes from different regions of 10 oak logs. Stringent procedures to avoid contamination with external DNA were used in order to demonstrate the authenticity of the fragments amplified. This authenticity was further confirmed by demonstrating genetic uniformity within each log using both nuclear and chloroplast microsatellites. For most wood samples DNA was degraded, and the sequences that gave the best results were those of small size and present in high copy number (chloroplast, mitochondrial, or repeated nuclear sequences). Both storage conditions and storage duration play a role in DNA conservation. Overall, this work demonstrates that molecular markers from all three plant genomes can be used for genetic analysis on dry oak wood, but outlines some limitations and the need for further evaluation of the potential of wood for DNA analysis.     

Unravelling migrations in the steppe: mitochondrial DNA sequences from ancient central Asians

This study helps to clarify the debate on the Western and Eastern genetic influences in Central Asia. Thirty-six skeletal remains from Kazakhstan (Central Asia), excavated from different sites dating between the fifteenth century BC to the fifth century AD, have been analysed for the hypervariable control region (HVR-I) and haplogroup diagnostic single nucleotide polymorphisms (SNPs) of the mitochondrial DNA genome. Standard authentication criteria for ancient DNA studies, including multiple extractions, cloning of PCR products and independent replication, have been followed. The distribution of east and west Eurasian lineages through time in the region is concordant with the available archaeological information: prior to the thirteenth-seventh century BC, all Kazakh samples belong to European lineages; while later an arrival of east Eurasian sequences that coexisted with the previous west Eurasian genetic substratum can be detected. The presence of an ancient genetic substratum of European origin in West Asia may be related to the discovery of ancient mummies with European features in Xinjiang and to the existence of an extinct Indo-European language, Tocharian. This study demonstrates the usefulness of the ancient DNA in unravelling complex patterns of past human migrations so as to help decipher the origin of present-day admixed populations.     

Analysis of the fuel wood used in Late Bronze Age and Early Iron Age copper mining sites of the Schwaz and Brixlegg area (Tyrol,Austria)

Charcoal analysis was carried out as part of an interdisciplinary project focusing on the copper mining history of the former mining area of Schwaz and Brixlegg, a region pivotal as a copper source in prehistoric Europe. The goal was to use remains of carbonised wood to investigate environmental implications of prehistoric mining, as well as to gain new insight about the ancient mining technique of fire-setting. Charcoal samples from seven copper mining sites (Late Bronze Age to Early Iron Age) were analysed. The results reveal a strong preference for coniferous wood as fuel in fire-setting, but not in ore smelting/roasting processes. Species composition at the ore-processing sites indicates moderate forest degradation processes caused by human intervention.     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

Optimization of sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees

Aims: To develop fast and reliable sampling procedures for DNA‐based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4‐mm‐diameter, 43‐cm‐long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1–4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56·6% for the scheme based on a single drill to 96·8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0·05) from those based on two and three drillings, whose efficiency was 72·6% and 83·9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of the Study: Fast and reliable sampling procedures make DNA‐based diagnosis more suitable for tree inspection procedures.     

How ancient DNA may help in understanding the origin and spread of agriculture

The origin and spread of agriculture have been central questions in archaeology for the last 75 years and are increasingly being addressed by a multidisciplinary approach involving biologists, ecologists, geographers and anthropologists as well as archaeologists. Molecular genetics has the potential to make an important contribution, especially by enabling the number of times that a crop or animal was domesticated to be determined. Molecular genetics can also assign approximate dates to domestication events, identify the wild progenitor of a domesticate, and provide new forms of evidence relevant to agricultural spread. With wheat, molecular genetical studies of modern plants have suggested that einkorn was domesticated just once but that emmer might have been domesticated more than once. Ancient DNA studies of animal remains have benefited from progress made with equivalent analyses of human bones, and with plant material there have been clear demonstrations of DNA preservation in desiccated seeds. Charred remains have also been shown to contain ancient DNA but this finding is unexpected in view of the high temperatures to which these seeds have supposedly been exposed. Ancient DNA studies of wheat remains have been used in taxonomic identification and in assessment of the possible bread-making quality of the wheat grown at an Early Bronze Age site in Greece.     

Mitochondrial DNA variation in the Viking age population of Norway

The medieval Norsemen or Vikings had an important biological and cultural impact on many parts of Europe through raids, colonization and trade, from about AD 793 to 1066. To help understand the genetic affinities of the ancient Norsemen, and their genetic contribution to the gene pool of other Europeans, we analysed DNA markers in Late Iron Age skeletal remains from Norway. DNA was extracted from 80 individuals, and mitochondrial DNA polymorphisms were detected by next-generation sequencing. The sequences of 45 ancient Norwegians were verified as genuine through the identification of damage patterns characteristic of ancient DNA. The ancient Norwegians were genetically similar to previously analysed ancient Icelanders, and to present-day Shetland and Orkney Islanders, Norwegians, Swedes, Scots, English, German and French. The Viking Age population had higher frequencies of K*, U*, V* and I* haplogroups than their modern counterparts, but a lower proportion of T* and H* haplogroups. Three individuals carried haplotypes that are rare in Norway today (U5b1b1, Hg A* and an uncommon variant of H*). Our combined analyses indicate that Norse women were important agents in the overseas expansion and settlement of the Vikings, and that women from the Orkneys and Western Isles contributed to the colonization of Iceland.     

A Statistical Approach to Identify Ancient Template DNA

One of the key problems in the study of ancient DNA is that of authenticating sequences obtained from PCR amplifications of highly degraded samples. Contamination of ancient samples and postmortem damage to endogenous DNA templates are the major obstacles facing researchers in this task. In particular, the authentication of sequences obtained from ancient human remains is thought by many to be rather challenging. We propose a novel approach, based on the c statistic, that can be employed to help identify the sequence motif of an endogenous template, based on a sample of sequences that reflect the nucleotide composition of individual template molecules obtained from ancient tissues (such as cloned products from a PCR amplification). The c statistic exploits as information the most common form of postmortem damage observed among clone sequences in ancient DNA studies, namely, lesion-induced substitutions caused by cytosine deamination events. Analyses of simulated sets of templates with miscoding lesions and real sets of clone sequences from the literature indicate that the c-based approach is highly effective in identifying endogenous sequence motifs, even when they are not present among the sampled clones. The proposed approach is likely to be of general use to researchers working with DNA from ancient tissues, particularly from human remains, where authentication of results has been most challenging. [Reviewing Editor: Dr. Magnus Nordborg]     

Putative ancient microorganisms from amber nuggets.

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified.     

Paleoethnobotany: Modern Research Connecting Ancient Plants and Ancient Peoples

Paleoethnobotany is a growing subdiscipline of archaeology that utilizes information from numerous other disciplines to show the relationships between ancient plants and ancient peoples. The two primary disciplines that underlie paleoethnobotanical research are archaeology and botany. As such, the results of ongoing botanical research on taphonomic processes, genetic identification of ancient plant types, pollen analysis, phytoliths analysis, and seed identification directly affect the strength of paleoethnobotanical models of past human behavior. Preserved seeds form a significant portion of the archaeobotanical record. They represent not only the environment that was present when they were deposited but also a connection to the activity and culture of ancient people. Using the type of archaeobotanical remains and the archeological context of the remains, paleoethnobotanists study a diverse range of topics. These topics include, but are not limited to, the use of plants in ancient cultures, the development and rise of agriculture, and the relationship between agriculture and settlement patterns.