Ancient DNA Research Articles

Ancient dog DNA reveals the human livelihood mode transitions during the late Neolithic in Northeastern China

The Honghe site is located in the middle reaches of the Nenjiang River in northeastern China and contains remains from the late Neolithic through the Ming and Qing dynasties. Among these remains, dogs were the only species domesticated in the Neolithic period. In this study, we analyzed the ancient DNA of 29 canine remains from the late Neolithic to the Bronze Age at the Honghe site, and the results showed that two new A subhaplogroups (A3 and A4) were found in the canines from the Honghe site compared to the ancient canines from the Yellow River Basin and the Yangtze River Basin in China. At the same time, the sex ratio of domestic dogs at the site changed between the different historical phases, which may have been influenced by a number of factors, possibly including changes in livelihood mode transitions. In addition, the discovery of dog haplotypes B and C at the site suggests that the influence of European domestic dogs reached northeast China during this period.

Millennial processes of population decline, range contraction and near extinction of the European bison.

European bison (Bison bonasus) were widespread throughout Europe during the late Pleistocene. However, the contributions of environmental change and humans to their near extinction have never been resolved. Using process-explicit models, fossils and ancient DNA, we disentangle the combinations of threatening processes that drove population declines and regional extinctions of European bison through space and across time. We show that the population size of European bison declined abruptly at the termination of the Pleistocene in response to rapid environmental change, hunting by humans and their interaction. Human activities prevented populations of European bison from rebounding in the Holocene, despite improved environmental conditions. Hunting caused range loss in the north and east of its distribution, while land use change was responsible for losses in the west and south. Advances in hunting technologies from 1500 CE were needed to simulate low abundances observed in 1870 CE. While our findings show that humans were an important driver of the extinction of the European bison in the wild, vast areas of its range vanished during the Pleistocene-Holocene transition because of post-glacial environmental change. These areas of its former range have been climatically unsuitable for millennia and should not be considered in reintroduction efforts.

An ancient genome perspective on the dynamic history of the prehistoric Jomon people in and around the Japanese archipelago

The Jomon people were prehistoric residents of the Japanese archipelago who occupied the region from ca. 16,500 to 2,300 years before present (BP). While recent accumulation of ancient genomes and genome-wide data of the Jomons has substantially enhanced our understanding of their genetic profiles and contribution to present-day populations, their genetic history in the Jomon-period archipelago, spanning over 14,000 years in time and 2,000 km in distance, remains scarcely investigated. Here we report multiple findings illuminating the Jomon genetic history based on the analysis of the genetic relationship between published ancient genome-wide data of 23 Jomon and Jomon-like individuals. First, the Initial Jomon individual from Shikoku, dated to ca. 9,000 BP, forms a common outgroup to the remaining later Jomon individuals, suggesting a population turnover in western Japan. Second, genetically Jomon-like individuals outside the Jomon archaeological context, found in the Miyako Island in Ryukyu and the Yokjido island in the southern coast of Korea, show the closest genetic affinity with the Late Jomon individual from Shikoku, narrowing down their sources in space and time. This study highlights a dynamic history of the Jomon people in and out of the Japanese archipelago and calls for a large-scale investigation of the ancient Jomon genomes.

Search for differentially methylated regions in ancient and modern genomes

Currently, active research is focused on investigating the mechanisms that regulate the development of various pathologies and their evolutionary dynamics. Epigenetic mechanisms, such as DNA methylation, play a significant role in evolutionary processes, as their changes have a faster impact on the phenotype compared to mutagenesis. In this study, we attempted to develop an algorithm for identifying differentially methylated regions associated with metabolic syndrome, which have undergone methylation changes in humans during the transition from a hunter­gatherer to a sedentary lifestyle. The application of existing whole­genome bisulfite sequencing methods is limited for ancient samples due to their low quality and fragmentation, and the approach to obtaining DNA methylation profiles differs significantly between ancient hunter­gatherer samples and modern tissues. In this study, we validated DamMet, an algorithm for reconstructing ancient methylomes. Application of DamMet to Neanderthal and Denisovan genomes showed a moderate level of correlation with previously published methylation profiles and demonstrated an underestimation of methylation levels in the reconstructed profiles by an average of 15–20 %. Additionally, we developed a new Python­based algorithm that allows for the comparison of methylomes in ancient and modern samples, despite the absence of methylation profiles in modern bone tissue within the context of obesity. This analysis involves a two­step data processing approach, where the first step involves the identification and filtration of tissue­specific methylation regions, and the second step focuses on the direct search for differentially methylated regions in specific areas associated with the researcher’s target condition. By applying this algorithm to test data, we identified 38 differentially methylated regions associated with obesity, the majority of which were located in promoter regions. The pipeline demonstrated sufficient efficiency in detecting these regions. These results confirm the feasibility of reconstructing DNA methylation profiles in ancient samples and comparing them with modern methylomes. Furthermore, possibilities for further methodological development and the implementation of a new step for studying differentially methylated positions associated with evolutionary processes are discussed.

Identification of DNA Viruses in Ancient DNA from Herbarium Samples.

Herbaria encompass millions of plant specimens, mostly collected in the nineteenth and twentieth centuries that can represent a key resource for investigating the history and evolution of phytopathogens. In the last years, the application of high-throughput sequencing technologies for the analysis of ancient nucleic acids has revolutionized the study of ancient pathogens including viruses, allowing the reconstruction of historical genomic viral sequences, improving phylogenetic based molecular dating, and providing essential insight into plant virus ecology. In this chapter, we describe a protocol to reconstruct ancient plant and soil viral sequences starting from highly fragmented ancient DNA extracted from herbarium plants and their associated rhizospheric soil. Following Illumina high-throughput sequencing, sequence data are de novo assembled, and DNA viral sequences are selected, according to their similarity with known viruses.

Benchmarking software tools for trimming adapters and merging next-generation sequencing data for ancient DNA.

Ancient DNA is highly degraded, resulting in very short sequences. Reads generated with modern high-throughput sequencing machines are generally longer than ancient DNA molecules, therefore the reads often contain some portion of the sequencing adaptors. It is crucial to remove those adaptors, as they can interfere with downstream analysis. Furthermore, overlapping portions when DNA has been read forward and backward (paired-end) can be merged to correct sequencing errors and improve read quality. Several tools have been developed for adapter trimming and read merging, however, no one has attempted to evaluate their accuracy and evaluate their potential impact on downstream analyses. Through the simulation of sequencing data, seven commonly used tools were analyzed in their ability to reconstruct ancient DNA sequences through read merging. The analyzed tools exhibit notable differences in their abilities to correct sequence errors and identify the correct read overlap, but the most substantial difference is observed in their ability to calculate quality scores for merged bases. Selecting the most appropriate tool for a given project depends on several factors, although some tools such as fastp have some shortcomings, whereas others like leeHom outperform the other tools in most aspects. While the choice of tool did not result in a measurable difference when analyzing population genetics using principal component analysis, it is important to note that downstream analyses that are sensitive to wrongly merged reads or that rely on quality scores can be significantly impacted by the choice of tool.

Ancient genome analyses shed light on the origin and kinship among humans of a cliff tomb from southwestern China

The cliff tomb was a distinctive regional burial form that lasted for centuries in the southwestern region of China. However, the origin of cliff tombs and the genetic relatedness among the co-buried individuals remain elusive. Here, we successfully identified four individuals from the scatted bone fragments in a single cliff tomb in the Sichuan Basin by using ancient DNA and physical anthropology analyses, and retrieved genomic data for the four individuals, to our best knowledge, the first ancient genomic data from the region. Our findings provide direct evidence supporting the Central Plains of China hypothesis for the people who practiced cliff tombs and further suggest that cliff tombs burial practices may also originate from the Central Plains of China, as supported by ancient DNA, archaeological and historical attestations. We successfully reconstructed the family tree of the four individuals and provided concrete evidence that individuals buried in the same cliff tomb are from a nuclear family rather than the long-hypothesized extended family, suggesting that genetic kinship plays a significant role for burial customs in the Sichuan Basin.

Genomic landscape of introgression from the ghost lineage in a gobiid fish uncovers the generality of forces shaping hybrid genomes.

Extinct lineages can leave legacies in the genomes of extant lineages through ancient introgressive hybridization. The patterns of genomic survival of these extinct lineages provide insight into the role of extinct lineages in current biodiversity. However, our understanding on the genomic landscape of introgression from extinct lineages remains limited due to challenges associated with locating the traces of unsampled 'ghost' extinct lineages without ancient genomes. Herein, we conducted population genomic analyses on the East China Sea (ECS) lineage of Chaenogobius annularis, which was suspected to have originated from ghost introgression, with the aim of elucidating its genomic origins and characterizing its landscape of introgression. By combining phylogeographic analysis and demographic modelling, we demonstrated that the ECS lineage originated from ancient hybridization with an extinct ghost lineage. Forward simulations based on the estimated demography indicated that the statistic γ of the HyDe analysis can be used to distinguish the differences in local introgression rates in our data. Consistent with introgression between extant organisms, we found reduced introgression from extinct lineage in regions with low recombination rates and with functional importance, thereby suggesting a role of linked selection that has eliminated the extinct lineage in shaping the hybrid genome. Moreover, we identified enrichment of repetitive elements in regions associated with ghost introgression, which was hitherto little known but was also observed in the re-analysis of published data on introgression between extant organisms. Overall, our findings underscore the unexpected similarities in the characteristics of introgression landscapes across different taxa, even in cases of ghost introgression.

Haplotype-based inference of recent effective population size in modern and ancient DNA samples

Individuals sharing recent ancestors are likely to co-inherit large identical-by-descent (IBD) genomic regions. The distribution of these IBD segments in a population may be used to reconstruct past demographic events such as effective population size variation, but accurate IBD detection is difficult in ancient DNA data and in underrepresented populations with limited reference data. In this work, we introduce an accurate method for inferring effective population size variation during the past ~2000 years in both modern and ancient DNA data, called HapNe. HapNe infers recent population size fluctuations using either IBD sharing (HapNe-IBD) or linkage disequilibrium (HapNe-LD), which does not require phasing and can be computed in low coverage data, including data sets with heterogeneous sampling times. HapNe shows improved accuracy in a range of simulated demographic scenarios compared to currently available methods for IBD-based and LD-based inference of recent effective population size, while requiring fewer computational resources. We apply HapNe to several modern populations from the 1,000 Genomes Project, the UK Biobank, the Allen Ancient DNA Resource, and recently published samples from Iron Age Britain, detecting multiple instances of recent effective population size variation across these groups.

The genetic legacy of the expansion of Bantu-speaking peoples in Africa

The expansion of people speaking Bantu languages is the most dramatic demographic event in Late Holocene Africa and fundamentally reshaped the linguistic, cultural and biological landscape of the continent1–7. With a comprehensive genomic dataset, including newly generated data of modern-day and ancient DNA from previously unsampled regions in Africa, we contribute insights into this expansion that started 6,000–4,000 years ago in western Africa. We genotyped 1,763 participants, including 1,526 Bantu speakers from 147 populations across 14 African countries, and generated whole-genome sequences from 12 Late Iron Age individuals8. We show that genetic diversity amongst Bantu-speaking populations declines with distance from western Africa, with current-day Zambia and the Democratic Republic of Congo as possible crossroads of interaction. Using spatially explicit methods9 and correlating genetic, linguistic and geographical data, we provide cross-disciplinary support for a serial-founder migration model. We further show that Bantu speakers received significant gene flow from local groups in regions they expanded into. Our genetic dataset provides an exhaustive modern-day African comparative dataset for ancient DNA studies10 and will be important to a wide range of disciplines from science and humanities, as well as to the medical sector studying human genetic variation and health in African and African-descendant populations.

Ancient DNA sheds light on the origin and migration patterns of the Xianbei confederation

Ancient DNA sheds light on the origin and migration patterns of the Xianbei confederation

Temporal variation in introgressed segments' length statistics computed from a limited number of ancient genomes sheds light on past admixture pulses.

Hybridization is recognized as an important evolutionary force, but identifying and timing admixture events between divergent lineages remains a major aim of evolutionary biology. While this has traditionally been done using inferential tools on contemporary genomes, the latest advances in paleogenomics have provided a growing wealth of temporally distributed genomic data. Here, we used individual-based simulations to generate chromosome-level genomic data for a two-population system and described temporal neutral introgression patterns under a single- and two-pulse admixture model. We computed six summary statistics aiming to inform the timing and number of admixture pulses between interbreeding entities: lengths of introgressed sequences and their variance within-genomes, as well as genome-wide introgression proportions and related measures. The first two statistics could confidently be used to infer inter-lineage hybridization history, peaking at the beginning and shortly after an admixture pulse. Temporal variation in introgression proportions and related statistics provided more limited insights, particularly when considering their application to ancient genomes still scant in number. Lastly, we computed these statistics on Homo sapiens paleogenomes and successfully inferred the hybridization pulse from Neanderthal that occurred approximately 40 to 60 kya. The scarce number of genomes dating from this period prevented more precise inferences, but the accumulation of paleogenomic data opens promising perspectives as our approach only requires a limited number of ancient genomes.

Guinea pig remains from the XVIth century Ojców castle (southern Poland)

The mandible of a guinea pig (Cavia porcellus) was found in osteological material collected during archaeological excavations at Ojców Castle (southern Poland). The bone fragment was radiocarbon dated and subjected to ancient DNA analysis. The age of this guinea pig mandible fragment was directly dated between the late 16th and the early 17th century. The radiocarbon dating results are compatible with dating based on stratigraphic position. The ancient DNA analysis confirmed the taxonomic identification and indicated possible routes of introduction of this species into Europe. Given the high-status of the castle’s possessors in the 16–17th centuries, it can be assumed that the single guinea pig bone in Ojców belonged to a pet and was probably not acquired as a source of food, which also may have been the case in the few other European archaeological sites with guinea pig remains.

Disentangling archaic introgression and genomic signatures of selection at human immunity genes

Pathogens and infectious diseases have imposed exceptionally strong selective pressure on ancient and modern human genomes and contributed to the current variation in many genes. There is evidence that modern humans acquired immune variants through interbreeding with ancient hominins, but the impact of such variants on human traits is not fully understood. The main objectives of this research were to infer the genetic signatures of positive selection that may be involved in adaptation to infectious diseases and to investigate the function of Neanderthal alleles identified within a set of 50 Lithuanian genomes. Introgressed regions were identified using the machine learning tool ArchIE. Recent positive selection signatures were analysed using iHS. We detected high-scoring signals of positive selection at innate immunity genes (EMB, PARP8, HLAC, and CDSN) and evaluated their interactions with the structural proteins of pathogens. Interactions with human immunodeficiency virus (HIV) 1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified. Overall, genomic regions introgressed from Neanderthals were shown to be enriched in genes related to immunity, keratinocyte differentiation, and sensory perception.

Vikings and Migrants: Unravelling Scandinavia's Genetic Mosaic in the Viking Era

We analysed ancient Scandinavian genomes over 2,000 years, revealing Viking-era influx from southern Europe, British-Irish Isles, and the east Baltic. Genetic diversity increased during that time, but later Scandinavians have less non-local ancestry. Uralic ancestry found 1,000 years ago still impacts northern Scandinavians.

Delayed postglacial colonization of Betula in Iceland and the circum North Atlantic

As the Arctic continues to warm, woody shrubs are expected to expand northward. This process, known as ‘shrubification,’ has important implications for regional biodiversity, food web structure, and high-latitude temperature amplification. While the future rate of shrubification remains poorly constrained, past records of plant immigration to newly deglaciated landscapes in the Arctic may serve as useful analogs. We provide one new postglacial Holocene sedimentary ancient DNA (sedaDNA) record of vascular plants from Iceland and place a second Iceland postglacial sedaDNA record on an improved geochronology; both show Salicaceae present shortly after deglaciation, whereas Betulaceae first appears more than 1000 y later. We find a similar pattern of delayed Betulaceae colonization in eight previously published postglacial sedaDNA records from across the glaciated circum North Atlantic. In nearly all cases, we find that Salicaceae colonizes earlier than Betulaceae and that Betulaceae colonization is increasingly delayed for locations farther from glacial-age woody plant refugia. These trends in Salicaceae and Betulaceae colonization are consistent with the plant families’ environmental tolerances, species diversity, reproductive strategies, seed sizes, and soil preferences. As these reconstructions capture the efficiency of postglacial vascular plant migration during a past period of high-latitude warming, a similarly slow response of some woody shrubs to current warming in glaciated regions, and possibly non-glaciated tundra, may delay Arctic shrubification and future changes in the structure of tundra ecosystems and temperature amplification.

Delayed postglacial colonization of Betula in Iceland and the circum North Atlantic.

As the Arctic continues to warm, woody shrubs are expected to expand northward. This process, known as 'shrubification,' has important implications for regional biodiversity, food web structure, and high-latitude temperature amplification. While the future rate of shrubification remains poorly constrained, past records of plant immigration to newly deglaciated landscapes in the Arctic may serve as useful analogs. We provide one new postglacial Holocene sedimentary ancient DNA (sedaDNA) record of vascular plants from Iceland and place a second Iceland postglacial sedaDNA record on an improved geochronology; both show Salicaceae present shortly after deglaciation, whereas Betulaceae first appears more than 1000 y later. We find a similar pattern of delayed Betulaceae colonization in eight previously published postglacial sedaDNA records from across the glaciated circum North Atlantic. In nearly all cases, we find that Salicaceae colonizes earlier than Betulaceae and that Betulaceae colonization is increasingly delayed for locations farther from glacial-age woody plant refugia. These trends in Salicaceae and Betulaceae colonization are consistent with the plant families' environmental tolerances, species diversity, reproductive strategies, seed sizes, and soil preferences. As these reconstructions capture the efficiency of postglacial vascular plant migration during a past period of high-latitude warming, a similarly slow response of some woody shrubs to current warming in glaciated regions, and possibly non-glaciated tundra, may delay Arctic shrubification and future changes in the structure of tundra ecosystems and temperature amplification.

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P <0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

Limited historical admixture between European wildcats and domestic cats

Domestic cats were derived from the Near Eastern wildcat (Felis lybica), after which they dispersed with people into Europe. As they did so, it is possible that they interbred with the indigenous population of European wildcats (Felis silvestris). Gene flow between incoming domestic animals and closely related indigenous wild species has been previously demonstrated in other taxa, including pigs, sheep, goats, bees, chickens, and cattle. In the case of cats, a lack of nuclear, genome-wide data, particularly from Near Eastern wildcats, has made it difficult to either detect or quantify this possibility. To address these issues, we generated 75 ancient mitochondrial genomes, 14 ancient nuclear genomes, and 31 modern nuclear genomes from European and Near Eastern wildcats. Our results demonstrate that despite cohabitating for at least 2,000 years on the European mainland and in Britain, most modern domestic cats possessed less than 10% of their ancestry from European wildcats, and ancient European wildcats possessed little to no ancestry from domestic cats. The antiquity and strength of this reproductive isolation between introduced domestic cats and local wildcats was likely the result of behavioral and ecological differences. Intriguingly, this long-lasting reproductive isolation is currently being eroded in parts of the species' distribution as a result of anthropogenic activities.

Exploring different methodological approaches to unlock paleobiodiversity in peat profiles using ancient DNA

Natural and human-induced environmental changes deeply affected terrestrial ecosystems throughout the Holocene. Paleoenvironmental reconstructions provide information about the past and allow us to predict/model future scenarios. Among potential records, peat bogs are widely used because they present a precise stratigraphy and act as natural archives of highly diverse organic remains. Over the decades, several techniques have been developed to identify debris occurring in peat, including their morphological description. However, this is strongly constrained by the researcher's ability to distinguish residues at the species level, which typically requires many years of experience. In addition, potential contamination hampers using these techniques to obtain information from organisms such as fungi or bacteria. Environmental DNA metabarcoding and shotgun metagenome sequencing could represent a solution to detect specific groups of organisms without any a priori knowledge of their characteristics and/or to identify organisms that have rarely been considered in previous investigations. Moreover, shotgun metagenomics may allow the identification of bacteria and fungi (including both yeast and filamentous life forms), ensuring discrimination between ancient and modern organisms through the study of deamination/damage patterns. In the present review, we aim to i) present the state-of-the-art methodologies in paleoecological and paleoclimatic studies focusing on peat core analyses, proposing alternative approaches to the classical morphological identification of plant residues, and ii) suggest biomolecular approaches that will allow the use of proxies such as invertebrates, fungi, and bacteria, which are rarely employed in paleoenvironmental reconstructions.