Ancient DNA Sequences Research Articles

Targeted PCR Amplification and Multiplex Sequencing of Ancient DNA for SNP Analysis.

The analysis of single-nucleotide polymorphisms (SNPs) has proven to be advantageous for addressing variation within samples of highly degraded or low-quality DNA samples. This is because only short fragments need to be amplified to analyze SNPs, and this can be achieved by multiplex PCR. Here, we present a sensitive method for the targeted sequencing of SNP loci that requires only small amounts of template DNA. The approach combines multiplex amplification of very short fragments covering SNP positions followed by sample barcoding and next-generation sequencing. This method allows generation of data from large sample sets of poorly preserved specimens, such as fossil remains, forensic samples, and museum specimens. The approach is cost-effective, rapid, and applicable to forensics, population genetics, and phylogenetic research questions.

Double-Stranded Library Preparation for Ancient and Other Degraded Samples.

High-throughput sequencing (HTS) allows fast and cost-efficient sequencing of ancient DNA (aDNA) without prior information about what sequences should be targeted. One necessary step for HTS is the preparation of a sequencing library. Commercial kits are available for this purpose, but many of these are not suitable for aDNA or other types of damaged DNA. Here, we outline a protocol for HTS library preparation that is optimized for ancient DNA. We report the library conversion rate for a range of input template and adapter concentrations. Our results show that the protocol performs at a high efficiency.

Sexing Viking Age horses from burial and non-burial sites in Iceland using ancient DNA

Horses are the most common grave goods found in Icelandic Viking Age graves. Horse skeletons have previously been sexed based on pelvis shape and the presence of canine teeth in male horses over 4–5 years of age. Morphological data has shown that all horses from Icelandic burials that were amenable to sexing were male. Yet the use of morphological methods to determine sex is problematic since they rely on finding a well-preserved pelvis and/or robust canine teeth. Furthermore, quantitative data underlying the features of the horse pelvis used for sexing is lacking and canine teeth have been reported to occur in mares. In this study we build upon and extend recently developed methodologies to make use of shotgun sequencing of ancient DNA (aDNA) for molecular sexing of Viking Age horse remains. With minimal sequencing effort we identified the sex of the largest collection (n = 22) of Viking Age Icelandic horses studied to date, sourced from both burial (n = 19) and non-burial (n = 3) sites. Our results revealed a male to female sex bias ratio of 18:1 in burial sites, versus 0:3 in non-burial sites. These findings support the significant over-representation of male horses in Viking Age graves in Iceland, yet show that –albeit rare– mares could also be selected for ritual burial in Viking Age Iceland. This cost-effective method provides statistical confidence to allow for sexing of highly fragmented archaeological specimens with low endogenous DNA content.

Rites of Passage: Mortuary Practice, Population Dynamics, and Chronology at the Carrowkeel Passage Tomb Complex, Co. Sligo, Ireland

The first detailed investigation of the human remains from the Carrowkeel passage tomb complex since their excavation in 1911 has revealed several new and important insights about life, death, and mortuary practice in Neolithic Ireland. Osteological analysis provides the first conclusive proof for the occurrence of dismemberment of the dead at Irish passage tombs, practised contemporarily with cremation as one of a suite of funerary treatments. The research also highlights changes in burial tradition at the complex over the course of the Neolithic. Providing a chronology for these changes allows them to be linked to wider trends in monument construction, which may relate to changes in both land use and climate during the period. Multi-isotope analysis hints at the presence of non-local individuals among the interred and the possible existence of different food sourcing areas at the onset of the later Neolithic period. Preliminary results from ancient DNA sequencing of six individuals from Carrowkeel provide evidence for the genetic ancestry of Irish Neolithic populations, demonstrating their Anatolian origins and links along the Atlantic façade.

Scaffolding of Ancient Contigs and Ancestral Reconstruction in a Phylogenetic Framework

Ancestral genome reconstruction is an important task to analyze the evolution of genomes. Recent progress in sequencing ancient DNA led to the publication of so-called paleogenomes and allows the integration of this sequencing data in genome evolution analysis. However, the de novo assembly of ancient genomes is usually fragmented due to DNA degradation over time among others. Integrated phylogenetic assembly addresses the issue of genome fragmentation in the ancient DNA assembly while aiming to improve the reconstruction of all ancient genomes in the phylogeny simultaneously. The fragmented assembly of the ancient genome can be represented as an assembly graph, indicating contradicting ordering information of contigs. In this setting, our approach is to compare the ancient data with extant finished genomes. We generalize a reconstruction approach minimizing the Single-Cut-or-Join rearrangement distance towards multifurcating trees and include edge lengths to improve the reconstruction in practice. This results in a polynomial time algorithm that includes additional ancient DNA data at one node in the tree, resulting in consistent reconstructions of ancestral genomes.

Ancient DNA of crested penguins: Testing for temporal genetic shifts in the world’s most diverse penguin clade

Human impacts have substantially reduced avian biodiversity in many parts of the world, particularly on isolated islands of the Pacific Ocean. The New Zealand archipelago, including its five subantarctic island groups, holds breeding grounds for a third of the world’s penguin species, including several representatives of the diverse crested penguin genus Eudyptes. While this species-rich genus has been little studied genetically, recent population estimates indicate that several Eudyptes taxa are experiencing demographic declines. Although crested penguins are currently limited to southern regions of the New Zealand archipelago, prehistoric fossil and archaeological deposits suggest a wider distribution during prehistoric times, with breeding ranges perhaps extending to the North Island. Here, we analyse ancient, historic and modern DNA sequences to explore two hypotheses regarding the recent history of Eudyptes in New Zealand, testing for (1) human-driven extinction of Eudyptes lineages; and (2) reduced genetic diversity in surviving lineages. From 83 prehistoric bone samples, each tentatively identified as ‘Eudyptes spp.’, we genetically identified six prehistoric penguin taxa from mainland New Zealand, including one previously undescribed genetic lineage. Moreover, our Bayesian coalescent analyses indicated that, while the range of Fiordland crested penguin (E. pachyrhynchus) may have contracted markedly over the last millennium, genetic DNA diversity within this lineage has remained relatively constant. This result contrasts with human-driven biodiversity reductions previously detected in several New Zealand coastal vertebrate taxa.

Quantifying and reducing spurious alignments for the analysis of ultra-short ancient DNA sequences

BackgroundThe study of ancient DNA is hampered by degradation, resulting in short DNA fragments. Advances in laboratory methods have made it possible to retrieve short DNA fragments, thereby improving access to DNA preserved in highly degraded, ancient material. However, such material contains large amounts of microbial contamination in addition to DNA fragments from the ancient organism. The resulting mixture of sequences constitutes a challenge for computational analysis, since microbial sequences are hard to distinguish from the ancient sequences of interest, especially when they are short.ResultsHere, we develop a method to quantify spurious alignments based on the presence or absence of rare variants. We find that spurious alignments are enriched for mismatches and insertion/deletion differences and lack substitution patterns typical of ancient DNA. The impact of spurious alignments can be reduced by filtering on these features and by imposing a sample-specific minimum length cutoff. We apply this approach to sequences from four ~ 430,000-year-old Sima de los Huesos hominin remains, which contain particularly short DNA fragments, and increase the amount of usable sequence data by 17–150%. This allows us to place a third specimen from the site on the Neandertal lineage.ConclusionsOur method maximizes the sequence data amenable to genetic analysis from highly degraded ancient material and avoids pitfalls that are associated with the analysis of ultra-short DNA sequences.

Parasitic infections and resource economy of Danish Iron Age settlement through ancient DNA sequencing.

In this study, we screen archaeological soil samples by microscopy and analyse the samples by next generation sequencing to obtain results with parasites at species level and untargeted findings of plant and animal DNA. Three separate sediment layers of an ancient man-made pond in Hoby, Denmark, ranging from 100 BC to 200 AD, were analysed by microscopy for presence of intestinal worm eggs and DNA analysis were performed to identify intestinal worms and dietary components. Ancient DNA of parasites, domestic animals and edible plants revealed a change in use of the pond over time reflecting the household practice in the adjacent Iron Age settlement. The most abundant parasite found belonged to the Ascaris genus, which was not possible to type at species level. For all sediment layers the presence of eggs of the human whipworm Trichuris trichiura and the beef tapeworm Taenia saginata suggests continuous disposal of human faeces in the pond. Moreover, the continuous findings of T. saginata further imply beef consumption and may suggest that cattle were living in the immediate surrounding of the site throughout the period. Findings of additional host-specific parasites suggest fluctuating presence of other domestic animals over time: Trichuris suis (pig), Parascaris univalens (horse), Taenia hydatigena (dog and sheep). Likewise, alternating occurrence of aDNA of edible plants may suggest changes in agricultural practices. Moreover, the composition of aDNA of parasites, plants and vertebrates suggests a significant change in the use of the ancient pond over a period of three centuries.

Carriers of mitochondrial DNA macrohaplogroup L3 basal lineages migrated back to Africa from Asia around 70,000 years ago

BackgroundThe main unequivocal conclusion after three decades of phylogeographic mtDNA studies is the African origin of all extant modern humans. In addition, a southern coastal route has been argued for to explain the Eurasian colonization of these African pioneers. Based on the age of macrohaplogroup L3, from which all maternal Eurasian and the majority of African lineages originated, the out-of-Africa event has been dated around 60-70 kya. On the opposite side, we have proposed a northern route through Central Asia across the Levant for that expansion and, consistent with the fossil record, we have dated it around 125 kya. To help bridge differences between the molecular and fossil record ages, in this article we assess the possibility that mtDNA macrohaplogroup L3 matured in Eurasia and returned to Africa as basal L3 lineages around 70 kya.ResultsThe coalescence ages of all Eurasian (M,N) and African (L3 ) lineages, both around 71 kya, are not significantly different. The oldest M and N Eurasian clades are found in southeastern Asia instead near of Africa as expected by the southern route hypothesis. The split of the Y-chromosome composite DE haplogroup is very similar to the age of mtDNA L3. An Eurasian origin and back migration to Africa has been proposed for the African Y-chromosome haplogroup E. Inside Africa, frequency distributions of maternal L3 and paternal E lineages are positively correlated. This correlation is not fully explained by geographic or ethnic affinities. This correlation rather seems to be the result of a joint and global replacement of the old autochthonous male and female African lineages by the new Eurasian incomers.ConclusionsThese results are congruent with a model proposing an out-of-Africa migration into Asia, following a northern route, of early anatomically modern humans carrying pre-L3 mtDNA lineages around 125 kya, subsequent diversification of pre-L3 into the basal lineages of L3, a return to Africa of Eurasian fully modern humans around 70 kya carrying the basal L3 lineages and the subsequent diversification of Eurasian-remaining L3 lineages into the M and N lineages in the outside-of-Africa context, and a second Eurasian global expansion by 60 kya, most probably, out of southeast Asia. Climatic conditions and the presence of Neanderthals and other hominins might have played significant roles in these human movements. Moreover, recent studies based on ancient DNA and whole-genome sequencing are also compatible with this hypothesis.

Fox parasites in pre-Columbian times: Evidence from the past to understand the current helminth assemblages

This work aims to increase the information on the entero-parasitism in Holocene carnivores, by examining coprolites found in Patagonia. Molecular analysis was conducted following the Authenticity Criteria to Determine Ancient DNA sequences. The nucleotide sequences showed 99% of identity with the Control Region sequences of Lycalopex culpaeus (culpeo fox). Coprolites were positive for gastrointestinal parasites. The presence of Alaria sp. and Clonorchis sp. represents the first record for pre-Columbian America. The parasitological findings suggest the importance of these carnivores for the dissemination of their own parasites and those to their prey in rockshelters, areas with high re-use of space.

A comparison of methods for estimating substitution rates from ancient DNA sequence data

BackgroundPhylogenetic analysis of DNA from modern and ancient samples allows the reconstruction of important demographic and evolutionary processes. A critical component of these analyses is the estimation of evolutionary rates, which can be calibrated using information about the ages of the samples. However, the reliability of these rate estimates can be negatively affected by among-lineage rate variation and non-random sampling. Using a simulation study, we compared the performance of three phylogenetic methods for inferring evolutionary rates from time-structured data sets: regression of root-to-tip distances, least-squares dating, and Bayesian inference. We also applied these three methods to time-structured mitogenomic data sets from six vertebrate species.ResultsOur results from 12 simulation scenarios show that the three methods produce reliable estimates when the substitution rate is high, rate variation is low, and samples of similar ages are not all grouped together in the tree (i.e., low phylo-temporal clustering). The interaction of these factors is particularly important for least-squares dating and Bayesian estimation of evolutionary rates. The three estimation methods produced consistent estimates of rates across most of the six mitogenomic data sets, with sequence data from horses being an exception.ConclusionsWe recommend that phylogenetic studies of ancient DNA sequences should use multiple methods of inference and test for the presence of temporal signal, among-lineage rate variation, and phylo-temporal clustering in the data.

Palaeoproteomics for human evolution studies

The commonplace sequencing of Neanderthal, Denisovan and ancient modern human DNA continues to revolutionize our understanding of hominin phylogeny and interaction(s). The challenge with older fossils is that the progressive fragmentation of DNA even under optimal conditions, a function of time and temperature, results in ever shorter fragments of DNA. This process continues until no DNA can be sequenced or reliably aligned. Ancient proteins ultimately suffer a similar fate, but are a potential alternative source of biomolecular sequence data to investigate hominin phylogeny given their slower rate of fragmentation. In addition, ancient proteins have been proposed to potentially provide insights into in vivo biological processes and can be used to provide additional ecological information through large scale ZooMS (Zooarchaeology by Mass Spectrometry) screening of unidentifiable bone fragments. However, as initially with ancient DNA, most ancient protein research has focused on Late Pleistocene or Holocene samples from Europe. In addition, only a limited number of studies on hominin remains have been published. Here, an updated review on ancient protein analysis in human evolutionary contexts is given, including the identification of specific knowledge gaps and existing analytical limits, as well as potential avenues to overcome these.

Implications and future prospects for evolutionary analyses of DNA in historical herbarium collections

Global herbarium collections house a vast number of plant specimens stretching back over centuries. They include rare and extinct species, important historical collections, and valuable type specimens that could help to resolve long-standing taxonomic issues. Historical plant collections also provide unique windows into past evolutionary processes and population histories that do not exist with modern data alone. However, because the DNA in such specimens is often degraded and scarce, manipulation and analysis of their genetic material can be challenging. Recent developments in non-destructive genetic sampling and working with very small quantities of genomic DNA, especially in next-generation sequencing and bioinformatic analysis of ancient DNA, now make the majority of herbarium specimens potentially accessible to phylogenetic, population genetic, and barcoding studies. For example, studies of DNA from long time series of Ipomoea batatas and Anacamptis palustris collections revealed linkages between anthropogenic activity and changes in haplotype distribution. These time series also enable the identification of genes under recent selection in the genomes of both the plants and their pathogens, as was shown for Phytophthora infestans, a microbial pathogen of Solanum tuberosum. Here, we summarize the major challenges in using historical plant DNA in evolutionary studies and review genetic studies integrating herbarium specimens. We expect future genetic studies of historical herbarium specimens to use genomic, metagenomic, and population genetic approaches to: investigate how plant populations respond to environmental change; infer temporal changes in genetic diversity; identify genes under recent selection; and investigate past plant pathogen epidemics.

Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene.

The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cytb and 12s rRNA, partial 16s rRNA and ND1, and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential.

A combined method for DNA analysis and radiocarbon dating from a single sample

Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given specimen, thereby increasing the extent of undesirable damage to precious archaeological material. Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories. Using a set of 12 bones of different ages and preservation conditions we demonstrate that on average 89% of the DNA can be released from sample powder with minimal, or 38% without any, detectable collagen loss. We also detect no skews in radiocarbon dates compared to untreated samples. Given the different material demands for radiocarbon dating (500 mg of bone/dentine) and DNA analysis (10–100 mg), combined DNA and collagen extraction not only streamlines the sampling process but also drastically increases the amount of DNA that can be recovered from limited sample material.

Coprolites reveal ecological interactions lost with the extinction of New Zealand birds

Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ's natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses.

The kinship of two 12th Dynasty mummies revealed by ancient DNA sequencing

We resolve a longstanding question regarding the kinship of two high-status Egyptians from the 12th Dynasty, Nakht-Ankh and Khnum-Nakht, whose mummies were discovered in 1907 by Egyptian workmen directed by Flinders Petrie and Ernest Mackay. Although their coffin inscriptions indicate that Nakht-Ankh and Khnum-Nakht were brothers, when the mummies were unwrapped in 1908 the skeletal morphologies were found to be quite different, suggesting an absence of family relationship. We extracted ancient DNA from the teeth of the two mummies and, following hybridization capture of the mitochondrial and Y chromosome fractions, sequenced the DNA by a next generation method. Analysis of single nucleotide polymorphisms showed that both Nakht-Ankh and Khnum-Nakht belonged to mitochondrial haplotype M1a1, suggesting a maternal relationship. The Y chromosome sequences were less complete but showed variations between the two mummies, indicating that Nakht-Ankh and Khnum-Nakht had different fathers. Our study emphasizes the importance of kinship in ancient Egypt, and represents the first successful typing of both mitochondrial and Y chromosomal DNA in Egyptian mummies.

Updated: First big efforts to sequence ancient African DNA reveal how early humans swept across the continent

Updated: First big efforts to sequence ancient African DNA reveal how early humans swept across the continent

Ancient Ascaris DNA Sequences of Cytochrome B, Cytochrome C Oxidase Subunit 1, NADH Dehydrogenase Subunit 1, and Internal Transcribed Spacer 1 Genes from Korean Joseon Mummy Feces.

We analyzed Ascaris ancient DNA of cytochrome b, cytochrome c oxidase subunit 1, NADH dehydrogenase subunit 1, and internal transcribed spacer 1 genes extracted from the feces or precipitates of 15- to 18th-century Korean mummies. After multiple Ascaris genes in ancient samples were successfully amplified by polymerase chain reaction (PCR), consensus sequences could be determined by the alignment of the sequences of cloned PCR products. The obtained sequences of each gene were highly similar to those of Ascaris spp. reported thus far but were genetically distinct from Baylisascaris, Parascaris, and Toxascaris spp. The current report establishes that the genetic characteristics of the Ascaris spp. infecting pre-modern Korean societies were not uniform but were diverse to some degree.

Did interaction between human pressure and Little Ice Age drive biological turnover in New Zealand?

AbstractAimTo test for simultaneous Holocene biogeographic turnover events in the New Zealand region. Specifically, we synthesize ancient DNA, radiocarbon data and archaeological data to assess the chronologies of late Holocene lineage extinction and replacement.LocationCool‐temperate coastal ecosystems of New Zealand and the subantarctic.MethodsWe present new ancient DNA and radiocarbon data for New Zealand sea lions, and synthesize existing climatic, genetic and archaeological data, to test for synchronous megafaunal extinction and replacement events. The collated data include ancient DNA sequences from over 200 ancient sea lion and penguin specimens, in addition to 150 modern genetic samples.ResultsOur temporal genetic analyses show that, following human‐driven extinction events, synchronous marine megafaunal replacement events occurred at around 1500 AD, coinciding with the Little Ice Age onset and an associated drastic human demographic decline in southern New Zealand.ConclusionsA combination of climatic and human demographic shifts likely facilitated northward expansion of subantarctic sea lion and penguin lineages, replacing extirpated mainland New Zealand marine megafauna. Broadly, the interaction between human pressure and late Holocene climatic change may explain multiple biological turnover events in the Southern Hemisphere.