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Sedimentary ancient DNA from caves: Challenges and opportunities
Abstract
Caves are unique depositional environments that hold great potential for long‐term preservation of DNA due to their typically cool and stable internal climates, buffered from external climate extremes. Bones from caves have long been recognised as an excellent source of ancient DNA, yet the potential for DNA preservation in cave sediments themselves has only recently begun to be explored. Here, we discuss features of caves that make them well‐suited to the long‐term preservation of sedimentary ancient DNA (sedaDNA), focussing specifically on sources, taphonomy and preservation of cave sedaDNA. We also highlight opportunities for future work to improve our understanding of the processes influencing long‐term DNA preservation within cave sediments. These recommendations seek to improve our understanding of sedimentary ancient DNA taphonomy within caves and enhance the level of insights about the past that can be gained from it and include: (1) designation of reference sections and sample repositories; (2) routine collection of sediment, mineralogy and geochemistry data; (3) routine collection of cave climate data; (4) increased application of microstratigraphic and spatial analyses; (5) advocating use of multiple proxies when interpreting results and (6) ensuring ethical best practices are followed.
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Caves are primary sites for studying human and animal subsistence patterns and genetic ancestry throughout the Palaeolithic. Iberia served as a critical human and animal refugium in Europe during the Last Glacial Maximum (LGM), 26.5 to 19 thousand years before the present (cal kya). Therefore, it is a key location for understanding human and animal population dynamics during this event. We recover and analyse sedimentary ancient DNA (sedaDNA) data from the lower archaeological stratigraphic sequence of El Mirón Cave (Cantabria, Spain), encompassing the (1) Late Mousterian period, associated with Neanderthals, and (2) the Gravettian (c. 31.5 cal kya), Solutrean (c. 24.5–22 cal kya), and Initial Magdalenian (d. 21–20.5 cal kya) periods, associated with anatomically modern humans. We identify 28 animal taxa including humans. Fifteen of these taxa had not been identified from the archaeozoological (i.e., faunal) record, including the presence of hyenas in the Magdalenian. Additionally, we provide phylogenetic analyses on 70 sedaDNA mtDNA genomes of fauna including the densest Iberian Pleistocene sampling of C. lupus. Finally, we recover three human mtDNA sequences from the Solutrean levels. These sequences, along with published data, suggest mtDNA haplogroup continuity in Iberia throughout the Solutrean/Last Glacial Maximum period.
The emergence of the Middle Palaeolithic, and its variability over time and space are key questions in the field of prehistoric archaeology. Many sites have been documented in the south-eastern margins of the Massif central and the middle Rhône valley, a migration path that connects Northern Europe with the Mediterranean. Well-dated, long stratigraphic sequences are essential to understand Neanderthals dynamics and demise, and potential interactions with Homo sapiens in the area, such as the one displayed at the Maras rock shelter (“Abri du Maras”). The site is characterised by exceptional preservation of archaeological remains, including bones dated using radiocarbon (¹⁴C) and teeth using electron spin resonance combined with uranium series (ESR/U-series). Optically stimulated luminescence was used to date the sedimentary deposits. By combining the new ages with previous ones using Bayesian modelling, we are able to clarify the occupation time over a period spanning 200,000 years. Between ca. 250 and 40 ka, the site has been used as a long-term residence by Neanderthals, specifically during three interglacial periods: first during marine isotopic stage (MIS) 7, between 247 ± 34 and 223 ± 33 ka, and then recurrently during MIS 5 (between 127 ± 17 and 90 ± 9 ka) and MIS 3 (up to 39,280 cal BP).
Ecosystem response to climate change is complex. In order to forecast ecosystem dynamics, we need high-quality data on changes in past species abundance that can inform process-based models. Sedimentary ancient DNA (sedaDNA) has revolutionised our ability to document past ecosystems' dynamics. It provides time series of increased taxonomic resolution compared to microfossils (pollen, spores), and can often give species-level information, especially for past vascular plant and mammal abundances. Time series are much richer in information than contemporary spatial distribution information, which have been traditionally used to train models for predicting biodiversity and ecosystem responses to climate change. Here, we outline the potential contribution of sedaDNA to forecast ecosystem changes. We showcase how species-level time series may allow quantification of the effect of biotic interactions in ecosystem dynamics, and be used to estimate dispersal rates when a dense network of sites is available. By combining palaeo-time series, process-based models, and inverse modelling, we can recover the biotic and abiotic processes underlying ecosystem dynamics, which are traditionally very challenging to characterise. Dynamic models informed by sedaDNA can further be used to extrapolate beyond current dynamics and provide robust forecasts of ecosystem responses to future climate change.
This article is part of the theme issue ‘Ecological novelty and planetary stewardship: biodiversity dynamics in a transforming biosphere’.
The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe¹. Local hybridization between the two groups occurred², but not on all occasions³. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups⁴. One such technocomplex for which the makers are unknown is the Lincombian–Ranisian–Jerzmanowician (LRJ), which has been described in northwestern and central Europe5–8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.
Recent excavations at Ranis (Germany) identified an early dispersal of Homo sapiens into the higher latitudes of Europe by 45,000 years ago. Here we integrate results from zooarchaeology, palaeoproteomics, sediment DNA and stable isotopes to characterize the ecology, subsistence and diet of these early H. sapiens. We assessed all bone remains (n = 1,754) from the 2016–2022 excavations through morphology (n = 1,218) or palaeoproteomics (zooarchaeology by mass spectrometry (n = 536) and species by proteome investigation (n = 212)). Dominant taxa include reindeer, cave bear, woolly rhinoceros and horse, indicating cold climatic conditions. Numerous carnivore modifications, alongside sparse cut-marked and burnt bones, illustrate a predominant use of the site by hibernating cave bears and denning hyaenas, coupled with a fluctuating human presence. Faunal diversity and high carnivore input were further supported by ancient mammalian DNA recovered from 26 sediment samples. Bulk collagen carbon and nitrogen stable isotope data from 52 animal and 10 human remains confirm a cold steppe/tundra setting and indicate a homogenous human diet based on large terrestrial mammals. This lower-density archaeological signature matches other Lincombian–Ranisian–Jerzmanowician sites and is best explained by expedient visits of short duration by small, mobile groups of pioneer H. sapiens.
This article discusses ethical frameworks for planning and implementing composite research in the United States. Composites, defined here as archaeological materials with multiple genetic sources, include materials such as sediment, coprolites, birch pitch, and dental calculus. Although composites are increasingly used in genetic research, the ethical considerations of their use in ancient DNA studies have not been widely discussed. Here, we consider how composites’ compositions, contexts, and potential to act as proxies can affect research plans and offer an overview of the primary ethical concerns of ancient DNA research. It is our view that ethical principles established for analyses of Ancestral remains and related materials can be used to inform research plans when working with composite evidence. This work also provides a guide to archaeologists unfamiliar with genetics analyses in planning research when using composite evidence from the United States with a focus on collaboration, having a clear research plan, and using lab methods that provide the desired data with minimal destruction. Following the principles discussed in this article and others allows for engaging in composite research while creating and maintaining positive relationships with stakeholders.
This book, entitled Tracking Environmental Change Using Lake Sediments: Volume 6 – Sedimentary DNA, provides an overview of the applications of sedimentary DNA-based approaches to paleolimnological studies. These approaches have shown considerable potential in providing information about the long-term changes of overall biodiversity in lakes and their watersheds in response to natural and anthropogenic changes, as well as tracking human migrations over the last thousands of years.
Although the first studies investigating the preservation of these molecular proxies in sediments originate from the late-1990s, the number of scientific publications on this topic has increased greatly over the last five years. Alongside numerous ecological findings, several sedimentary DNA studies have been dedicated to understanding the reliability of this approach to reconstruct past ecosystem changes. Despite the major surge of interest, a comprehensive compilation of sedimentary DNA approaches and applications has yet to be attempted. The overall aim of this DPER volume is to fill this knowledge gap.
The extraction of environmental DNA (eDNA) from sediments is providing ground‐breaking views of past ecosystems and biodiversity. Despite this rich source of information, it is still unclear which sediments favor preservation and why. Here, we used atomic force microscopy and molecular dynamics simulations to explore the DNA‐mineral interaction to assess how mineralogy and interfacial geochemistry play a role in the preservation of environmental DNA on mineral substrates. We demonstrate that mineral composition, surface topography, and surface charge influence DNA adsorption behavior as well as preservation. Modeling and experimental data show that DNA damage can be induced by mineral binding if there is a strong driving force for adsorption. The study shows that knowledge of the mineralogical composition of a sediment and the environmental conditions can be useful for assessing if a deposit is capable of storing extracellular DNA and to what extent the DNA would be preserved. Our data adds to the understanding of eDNA taphonomy and highlights that, for some mineral systems, fragmented DNA may not represent old DNA.
Several studies have reported increased microbial diversity, or distinct microbial community compositions, in the microbiomes of Indigenous peoples around the world. However, there is a widespread failure to include Indigenous cultures and perspectives in microbiome research programmes, and ethical issues pertaining to microbiome research involving Indigenous participants have not received enough attention. We discuss the benefits and risks arising from microbiome research involving Indigenous peoples and analyse microbiome ownership as an ethical concept in this context. We argue that microbiome ownership represents an opportunity for Indigenous peoples to steward and protect their resident microbial communities at every stage of research.
The rise of sedimentary ancient DNA (sedaDNA) studies has opened new possibilities for studying past environments. This groundbreaking area of genomics uses sediments to identify organisms, even in cases where macroscopic remains no longer exist. Managing this substrate in Indigenous Australian contexts, however, requires special considerations. Sediments and soils are often considered as waste by‐products during archaeological and paleontological excavations and are not typically regulated by the same ethics guidelines utilised in mainstream ‘western’ research paradigms. Nevertheless, the product of sedaDNA work—genetic information from past fauna, flora, microbial communities and human ancestors—is likely to be of cultural significance and value for Indigenous peoples. This article offers an opinion on the responsibilities of researchers in Australia who engage in research related to this emerging field, particularly when it involves Indigenous communities. One aspect that deserves consideration in such research is the concept of benefit sharing. Benefit sharing refers to the practice of ensuring that the benefits that arise from research are shared equitably with the communities from which the research data were derived. This practice is particularly relevant in research that involves Indigenous communities, who may have unique cultural and spiritual connections to the research material. We argue that the integration of Traditional Knowledges into sedaDNA research would add enormous value to research and its outcomes by providing genomic outputs alongside and within the rich context of multimillennia oral histories.
In Central Yakutia (Siberia) livelihoods of local communities depend on alaas (thermokarst depression) landscapes and the lakes within. Development and dynamics of these alaas lakes are closely connected to climate change, permafrost thawing, catchment conditions, and land use. To reconstruct lake development throughout the Holocene we analyze sedimentary ancient DNA (sedaDNA) and biogeochemistry from a sediment core from Lake Satagay, spanning the last c. 10,800 calibrated years before present (cal yrs BP). SedaDNA of diatoms and macrophytes and microfossil diatom analysis reveal lake formation earlier than 10,700 cal yrs BP. The sedaDNA approach detected 42 amplicon sequence variants (ASVs) of diatom taxa, one ASV of Eustigmatophyceae (Nannochloropsis), and 12 ASVs of macrophytes. We relate diatom and macrophyte community changes to climate-driven shifts in water level and mineral and organic input, which result in variable water conductivity, in-lake productivity, and sediment deposition. We detect a higher lake level and water conductivity in the Early Holocene (c. 10,700–7000 cal yrs BP) compared to other periods, supported by the dominance of Stephanodiscus sp. and Stuckenia pectinata. Further climate warming towards the Mid-Holocene (7000–4700 cal yrs BP) led to a shallowing of Lake Satagay, an increase of the submerged macrophyte Ceratophyllum, and a decline of planktonic diatoms. In the Late Holocene (c. 4700 cal yrs BP–present) stable shallow water conditions are confirmed by small fragilarioid and staurosiroid diatoms dominating the lake. Lake Satagay has not yet reached the final stage of alaas development, but satellite imagery shows an intensification of anthropogenic land use, which in combination with future warming will likely result in a rapid desiccation of the lake.
Traces of DNA found in sediments are shifting paradigms in the analysis of past and present ecosystems. DNA is an unstable polymer and conditions at which the millennial stabilization is achieved are unclear. Confinement of DNA in nanopores of clay minerals is a promising route for this long-term stabilization and storage. Using smectites with various layer charges, we measured adsorption capacity for DNA using UV spectroscopy and intercalation capacity using X-ray diffraction. We found that while the smectite adsorption capacity is large, the DNA intercalation, i.e. nanoconfinement, decreases as smectite charge increases. We show that low-charge smectites intercalate DNA at concentrations relevant to aqueous environments even at neutral pH but the nanoconfinement is minimal or absent in high-charge smectites. Different intercalation behaviour in NaCl and CaCl2 solutions imply different mechanisms driven by electrostatic forces. Our results demonstrate that DNA nanoconfinement in smectites is likely an important strategy for DNA preservation and that protocols targeting low-charge smectites might improve the success of ancient and modern DNA extraction even in hot and humid climates so far deemed unfavourable for DNA preservation.
The ethics of the scientific study of Ancestors has long been debated by archaeologists, bioanthropologists, and, more recently, ancient DNA (aDNA) researchers. This article responds to the article “Ethics of DNA research on human remains: five globally applicable guidelines” published in 2021 in Nature by a large group of aDNA researchers and collaborators. We argue that these guidelines do not sufficiently consider the interests of community stakeholders, including descendant communities and communities with potential, but yet unestablished, ties to Ancestors. We focus on three main areas of concern with the guidelines. First is the false separation of “scientific” and “community” concerns and the consistent privileging of researcher perspectives over those of community members. Second, the commitment of the guidelines’ authors to open data ignores the principles and practice of Indigenous Data Sovereignty. Further, the authors argue that involving community members in decisions about publication and data sharing is unethical. We argue that excluding community perspectives on “ethical” grounds is convenient for researchers, but it is not, in fact, ethical. Third, we stress the risks of not consulting communities that have established or potential ties to Ancestors, using two recent examples from the literature. Ancient DNA researchers cannot focus on the lowest common denominator of research practice, the bare minimum that is legally necessary. Instead, they should be leading multidisciplinary efforts to create processes to ensure communities from all regions of the globe are identified and engaged in research that affects them. This will often present challenges, but we see these challenges as part of the research, rather than a distraction from the scientific endeavor. If a research team does not have the capacity to meaningfully engage communities, questions must be asked about the value and benefit of their research.
The Anthropocene is characterized by dramatic ecosystem changes driven by human activities. The impact of these activities can be assessed by different geochemical and paleontological proxies. However, each of these proxies provides only a fragmentary insight into the effects of anthropogenic impacts. It is highly challenging to reconstruct, with a holistic view, the state of the ecosystems from the preindustrial period to the present day, covering all biological components, from prokaryotes to multicellular eukaryotes. Here, we used sedimentary ancient DNA (sedaDNA) archives encompassing all trophic levels of biodiversity to reconstruct the two century-natural history in Bagnoli-Coroglio (Gulf of Pozzuoli, Tyrrhenian Sea), one of the most polluted marine-coastal sites in Europe. The site was characterized by seagrass meadows and high eukaryotic diversity until the beginning of the 20th century. Then, the ecosystem completely changed, with seagrasses and associated fauna as well as diverse groups of planktonic and benthic protists being replaced by low diversity biota dominated by dinophyceans and infaunal metazoan species. The sedaDNA analysis revealed a five-phase evolution of the area, where changes appear as the result of a multi-level cascade effect of impacts associated with industrial activities, urbanization, water circulation and land-use changes. The sedaDNA allowed to infer reference conditions that must be considered when restoration actions are to be implemented.
The vast potential of using sediment adsorbed DNA as a window to past and present biodiversity rely on the ability of solid surfaces to adsorb environmental DNA. However, a comprehensive insight into DNA adsorption at surfaces in general is lacking. Soot and charcoal are carbonaceous materials widespread in the environment where they readily can come in contact with extracellular DNA shed from organisms. Using batch adsorption, we measured DNA adsorption capacity at soot and charcoal as a function of solution composition, time and DNA length. We observed that the adsorption capacity for DNA is highest at low pH, that it increases with solution concentration and cation valency and that the activation energy for DNA adsorption at both soot and charcoal is ~50 kJmol-1, suggesting strong binding. We demonstrate how the interaction between DNA and soot and charcoal partly occurs via terminal base pairs, suggesting that, besides electrostatic forces, hydrophobic interactions play an important role in binding. The large adsorption capacities and strong binding of DNA to soot and charcoal are features important for eDNA research and provide a motivation for use of carbonaceous materials from, e.g., anthropogenic pollution or wildfire as sources of biodiversity information.
Late Pliocene and Early Pleistocene epochs 3.6 to 0.8 million years ago¹ had climates resembling those forecasted under future warming². Palaeoclimatic records show strong polar amplification with mean annual temperatures of 11–19 °C above contemporary values3,4. The biological communities inhabiting the Arctic during this time remain poorly known because fossils are rare⁵. Here we report an ancient environmental DNA⁶ (eDNA) record describing the rich plant and animal assemblages of the Kap København Formation in North Greenland, dated to around two million years ago. The record shows an open boreal forest ecosystem with mixed vegetation of poplar, birch and thuja trees, as well as a variety of Arctic and boreal shrubs and herbs, many of which had not previously been detected at the site from macrofossil and pollen records. The DNA record confirms the presence of hare and mitochondrial DNA from animals including mastodons, reindeer, rodents and geese, all ancestral to their present-day and late Pleistocene relatives. The presence of marine species including horseshoe crab and green algae support a warmer climate than today. The reconstructed ecosystem has no modern analogue. The survival of such ancient eDNA probably relates to its binding to mineral surfaces. Our findings open new areas of genetic research, demonstrating that it is possible to track the ecology and evolution of biological communities from two million years ago using ancient eDNA.
Paleogenomic research can elucidate the evolutionary history of human and faunal populations. Although the Levant is a key land-bridge between Africa and Eurasia, thus far, relatively little ancient DNA data has been generated from this region, since DNA degrades faster in warm climates. As sediments can be a source of ancient DNA, we analyzed 33 sediment samples from different sedimentological contexts in the Paleolithic layers of Sefunim Cave (Israel). Four contained traces of ancient Cervidae and Hyaenidae mitochondrial DNA. Dating by optical luminescence and radiocarbon indicates that the DNA comes from layers between 30,000 and 70,000 years old, surpassing theoretical expectations regarding the longevity of DNA deposited in such a warm environment. Both identified taxa are present in the zooarchaeological record of the site but have since gone extinct from the region, and a geoarchaeological study suggests little movement of the sediments after their deposition, lending further support to our findings. We provide details on the local conditions in the cave, which we hypothesize were particularly conducive to the long-term preservation of DNA—information that will be pertinent for future endeavors aimed at recovering ancient DNA from the Levant and other similarly challenging contexts.
Alpine ecosystems on the Tibetan Plateau are being threatened by ongoing climate warming and intensified human activities. Ecological time-series obtained from sedimentary ancient DNA (sedaDNA) are essential for understanding past ecosystem and biodiversity dynamics on the Tibetan Plateau and their responses to climate change at a high taxonomic resolution. Hitherto only few but promising studies have been published on this topic. The potential and limitations of using sedaDNA on the Tibetan Plateau are not fully understood. Here, we (i) provide updated knowledge of and a brief introduction to the suitable archives, region-specific taphonomy, state-of-the-art methodologies, and research questions of sedaDNA on the Tibetan Plateau; (ii) review published and ongoing sedaDNA studies from the Tibetan Plateau; and (iii) give some recommendations for future sedaDNA study designs. Based on the current knowledge of taphonomy, we infer that deep glacial lakes with freshwater and high clay sediment input, such as those from the southern and southeastern Tibetan Plateau, may have a high potential for sedaDNA studies. Metabarcoding (for microorganisms and plants), metagenomics (for ecosystems), and hybridization capture (for prehistoric humans) are three primary sedaDNA approaches which have been successfully applied on the Tibetan Plateau, but their power is still limited by several technical issues, such as PCR bias and incompleteness of taxonomic reference databases. Setting up high-quality and open-access regional taxonomic reference databases for the Tibetan Plateau should be given priority in the future. To conclude, the archival, taphonomic, and methodological conditions of the Tibetan Plateau are favorable for performing sedaDNA studies. More research should be encouraged to address questions about long-term ecological dynamics at ecosystem scale and to bring the paleoecology of the Tibetan Plateau into a new era.
Paleogenomics - the study of ancient genomes - has made significant contributions, especially to our understanding of the evolutionary history of humans. This knowledge influx has been a direct result of the coupling of next-generation sequencing with improved methods for DNA recovery and analysis of ancient samples. The appeal of ancient DNA studies in the popular media coupled with the trend for such work to be published in “high impact” journals has driven the amassing of ancestral human remains from global collections, often with limited to no engagement or involvement of local researchers and communities. This practice in the paleogenomics literature has led to limited representation of researchers from the Global South at the research design and subsequent stages. Additionally, Indigenous and descendant communities are often alienated from popular and academic narratives that both involve and impact them, sometimes adversely. While some countries have safeguards against ‘helicopter science’, such as federally regulated measures to protect their biocultural heritage, there is variable oversight in others with regard to sampling and exportation of human remains for destructive research, and differing requirements for accountability or consultation with local researchers and communities. These disparities reveal stark contrasts and gaps in regional policies that lend themselves to persistent colonial practices. While essential critiques and conversations in this sphere are taking place, these are primarily guided through the lens of US-based heritage legislation such as the Native American Graves and Protection Act (NAGPRA). In this article, we aim to expand the scope of ongoing conversations by taking into account diverse regional contexts and challenges drawing from our own research experiences in the field of paleogenomics. We emphasize that true collaborations involve knowledge sharing, capacity building, mutual respect, and equitable participation, all of which take time and the implementation of sustainable research methods; amass-and-publish strategy is simply incompatible with this ethos.
Central Asian caves with Palaeolithic deposits are few, but they provide a rich record of human fossils and cultural assemblages that has been used to model Late Pleistocene hominin dispersals. However, previous research has not yet systematically evaluated the formation processes that influence the frequency of Palaeolithic cave sites in the region. To address this deficiency, we combined field survey and micromorphological analyses in the piedmont zone of south Kazakhstan. Here, we present our preliminary results focusing on selected sites of the Qaratau mountains. Sediment cover varies among the surveyed caves, and loess‐like sediments dominate the cave sequences. The preservation of cave deposits is influenced by reworking of cave sediments within the caves but also by the broader erosional processes that shape semiarid landscapes. Ultimately, deposits of potentially Pleistocene age are scarce. Our study provides new data in the geoarchaeologically neglected region of Central Asia and demonstrates that micromorphology has great analytical potential even within the limitations of rigorous survey projects. We outline some of the processes that influence the formation and preservation of cave deposits in Kazakhstan, as well as broader implications for the distribution of Palaeolithic cave sites in Central Asia and other semiarid environments.
Natural Trap Cave, located in the Big Horn Mountains of north-central Wyoming, has a history of trapping and preserving a range of North American fauna that plummeted into the deep vertical entrance. These animal remains were buried and preserved within sediments of the main chamber and, in turn, have helped elucidate the procession of faunal dynamics during the latest glacial cycle. The cave location, south of the Laurentide and Cordilleran Ice Sheets, and proximal to Yellowstone, is at an ideal geographical juncture to provide insights to ecological changes in North America. The sediments that the animals are buried in inform us about transport and deposition both inside and outside of the cave that relate to catchment dynamics. We report on a series of optically stimulated luminescence (OSL) ages derived from samples obtained within the cave during excavation work in 2014 and in 2018. We also examine chronology produced by argon, tephrochronology, fission track, and luminescence techniques that have been used for understanding the infilling of the cave. The cave sediment ages and in situ measured gamma spectroscopy as measured in this study helped resolve an improved chronological age model when combined with previous data.
The suite of OSL ages is interpreted through the stratigraphic relationships (and vertebrates contained within) which requires the use of an adequate age model; we use either the central age model or minimum age model where appropriate and with justification. Lowest sediments are dated to ∼150 ka with a hiatus at ∼130 to 52 ka. Above this, sediment deposition and entrainment of paleontological materials are representative of Pleistocene and early Holocene times, between 37 ± 6 ka and 7.6 ± 0.5 ka. The stratigraphic architecture suggests that deposition of materials into the cave is episodic and rapid, followed by quiescent periods where hydrologic scour, heavy overland flow, or possibly a cryo-hydrologic process may have altered unit relationships. Thus, the complementary geochronometers and the characteristics of quartz versus feldspar luminescence signals improve temporal interpretations of these complex deposits. This adapted understanding of mixing also sets the stage for future work with the aim to improve our understanding of ages and sources for ash units within these cave deposits. The three ash units recognized in the cave may represent an in-situ reworking of the same ash or may be representative of previously undocumented eruptions from the Yellowstone Caldera.
In-depth knowledge about spatial and temporal variation in microbial diversity and function is needed for a better understanding of ecological and evolutionary responses to global change. In particular, the study of microbial ancient DNA preserved in sediment archives from lakes and oceans can help us to evaluate the responses of aquatic microbes in the past and make predictions about future biodiversity change in those ecosystems. Recent advances in molecular genetic methods applied to the analysis of historically deposited DNA in sediments have not only allowed the taxonomic identification of past aquatic microbial communities, but also enabled tracing their evolution and adaptation to episodic disturbances and gradual environmental change. Nevertheless, some challenges remain for scientists to take full advantage of the rapidly developing field of paleo-genetics, including the limited ability to detect rare taxa and reconstruct complete genomes for evolutionary studies. Here, we provide a brief review of some of the recent advances in the field of environmental paleomicrobiology and discuss remaining challenges related to the application of molecular genetic methods to study microbial diversity, ecology, and evolution in sediment archives. We anticipate that, in the near future, environmental paleomicrobiology will shed new light on the processes of microbial genome evolution and microbial ecosystem responses to Quaternary environmental changes at an unprecedented level of detail. This information can, for example, aid geological reconstructions of biogeochemical cycles and predict ecosystem responses to environmental perturbations, including in the context of human-induced global changes. This article is protected by copyright. All rights reserved.
Significance
DNA preserved in sediments has emerged as an important source of information about past ecosystems, independent of the discovery of skeletal remains. However, little is known about the sources of sediment DNA, the factors affecting its long-term preservation, and the extent to which it may be translocated after deposition. Here, we show that impregnated blocks of intact sediment are excellent archives of DNA. DNA distribution is highly heterogeneous at the microscale in the cave sediment we studied, suggesting that postdepositional movement of DNA is unlikely to be a common phenomenon in cases where the stratigraphy is undisturbed. Combining micromorphological analysis with microstratigraphic retrieval of ancient DNA therefore allows genetic information to be associated with the detailed archaeological and ecological record preserved in sediments.
The fluctuating climatic conditions of the Saharo-Arabian deserts are increasingly linked to human evolutionary events and societal developments. On orbital timescales, the African and Indian Summer Monsoons were displaced northward and increased precipitation to the Arabian Peninsula which led to favorable periods for human occupation in the now arid interior. At least four periods of climatic optima occurred within the last 130,000 years, related to Marine Isotope Stages (MIS) 5e (128–121 ka BP), 5c (104–97 ka BP), 5a (81–74 ka BP) and 1 (10.5–6.2 ka BP), and potentially early MIS 3 (60–50 ka BP). Stalagmites from Southern Arabia have been key to understanding climatic fluctuations and human-environmental interactions; their precise and high-resolution chronologies can be linked to evidence for changes in human distribution and climate/environment induced societal developments. Here, we review the most recent advances in the Southern Arabian Late Pleistocene and Early Holocene stalagmite records. We compare and contrast MIS 5e and Early Holocene climates to understand how these differed, benchmark the extremes of climatic variability and summarize the impacts on human societal development. We suggest that, while the extreme of MIS 5e was important for H. sapeins dispersal, subsequent, less intense, wet phases mitigate against a simplistic narrative. We highlight that while climate can be a limiting and important factor, there is also the potential of human adaptability and resilience. Further studies will be needed to understand spatio-temporal difference in human-environment interactions in a climatically variable region.
El Sidrón Cave is an archaeological and anthropological reference site of the Neanderthal world. It shows singular activity related to cannibalisation, and all existing processes are relevant to explain the specific behaviour of the concerned individuals. This paper presents geoarchaeological data, primarily based on mineralogical and petrographic techniques, from an investigation of the nature of the encrustations or hard coatings that affect a large part of the Neanderthal bone remains and their relationship with the depositional and post-depositional processes at the archaeological site. Crusts and patina were found to be numerous and diverse, mainly composed of calcite and siliciclastic grains, with different proportions and textures. The analysis indicated different origins and scenarios from their initial post-mortem accumulation to the final deposit recovered during the archaeological work. The presence of micromorphological features, such as clotted-peloidal micrite, needle-fibre calcite (NFC) aggregates, clay coatings, iron–manganese impregnation, and/or adhered aeolian dust may indicate that a significant proportion of the remains were affected by subaerial conditions in a relatively short period of time in a shelter, cave entrance, or shallower level of the karstic system, prior to their accumulation in the Ossuary Gallery.
Ancient DNA (aDNA) has played a major role in our understanding of the past. Important advances in the sequencing and analysis of aDNA from a range of organisms have enabled a detailed understanding of processes such as past demography, introgression, domestication, adaptation and speciation. However, to date and with the notable exception of microbiomes and sediments, most aDNA studies have focused on single taxa or taxonomic groups, making the study of changes at the community level challenging. This is rather surprising because current sequencing and analytical approaches allow us to obtain and analyse aDNA from multiple source materials. When combined, these data can enable the simultaneous study of multiple taxa through space and time, and could thus provide a more comprehensive understanding of ecosystem-wide changes. It is therefore timely to develop an integrative approach to aDNA studies by combining data from multiple taxa and substrates. In this review, we discuss the various applications, associated challenges and future prospects of such an approach.
The Middle to Upper Palaeolithic transition, between 50 000 and 40 000 years ago, is a period of important ecological and cultural changes. In this framework, the Rock Shelter of Uluzzo C (Apulia, southern Italy) represents an important site due to Late Mousterian and Uluzzian evidence preserved in its stratigraphic sequence. Here, we present the results of a multidisciplinary analysis performed on the materials collected between 2016 and 2018 from the Uluzzian stratigraphic units (SUs) 3, 15 and 17. The analysis involved lithic technology, use‐wear, zooarchaeology, ancient DNA of sediments and palaeoproteomics, completed by quartz single‐grain optically stimulated luminescence dating of the cave sediments. The lithic assemblage is characterized by a volumetric production and a debitage with no or little management of the convexities (by using the bipolar technique), with the objective to produce bladelets and flakelets. The zooarchaeological study found evidence of butchery activity and of the possible exploitation of marine resources, while drawing a picture of a patchy landscape, composed of open forests and dry open environments surrounding the shelter. Ancient mitochondrial DNA from two mammalian taxa were recovered from the sediments. Preliminary zooarchaeology by mass spectrometry results are consistent with ancient DNA and zooarchaeological taxonomic information, while further palaeoproteomics investigations are ongoing. Our new data from the re‐discovery of the Uluzzo C Rock Shelter represent an important contribution to better understand the meaning of the Uluzzian in the context of the Middle/Upper Palaeolithic transition in south‐eastern Italy.
Denisova Cave in southern Siberia is the type locality of the Denisovans, an archaic hominin group who were related to Neanderthals1–4. The dozen hominin remains recovered from the deposits also include Neanderthals5,6 and the child of a Neanderthal and a Denisovan7, which suggests that Denisova Cave was a contact zone between these archaic hominins. However, uncertainties persist about the order in which these groups appeared at the site, the timing and environmental context of hominin occupation, and the association of particular hominin groups with archaeological assemblages5,8–11. Here we report the analysis of DNA from 728 sediment samples that were collected in a grid-like manner from layers dating to the Pleistocene epoch. We retrieved ancient faunal and hominin mitochondrial (mt)DNA from 685 and 175 samples, respectively. The earliest evidence for hominin mtDNA is of Denisovans, and is associated with early Middle Palaeolithic stone tools that were deposited approximately 250,000 to 170,000 years ago; Neanderthal mtDNA first appears towards the end of this period. We detect a turnover in the mtDNA of Denisovans that coincides with changes in the composition of faunal mtDNA, and evidence that Denisovans and Neanderthals occupied the site repeatedly—possibly until, or after, the onset of the Initial Upper Palaeolithic at least 45,000 years ago, when modern human mtDNA is first recorded in the sediments. Ancient mitochondrial DNA from sediments reveals the sequence of Denisovan, Neanderthal and faunal occupation of Denisova Cave, and evidence for the appearance of modern humans at least 45,000 years ago.
Ancient DNA analysis of human oral microbial communities within calcified dental plaque (calculus) has revealed key insights into human health, paleodemography, and cultural behaviors. However, contamination imposes a major concern for paleomicrobiological samples due to their low endogenous DNA content and exposure to environmental sources, calling into question some published results. Decontamination protocols (e.g. an ethylenediaminetetraacetic acid (EDTA) pre-digestion or ultraviolet radiation (UV) and 5% sodium hypochlorite immersion treatments) aim to minimize the exogenous content of the outer surface of ancient calculus samples prior to DNA extraction. While these protocols are widely used, no one has systematically compared them in ancient dental calculus. Here, we compare untreated dental calculus samples to samples from the same site treated with four previously published decontamination protocols: a UV only treatment; a 5% sodium hypochlorite immersion treatment; a pre-digestion in EDTA treatment; and a combined UV irradiation and 5% sodium hypochlorite immersion treatment. We examine their efficacy in ancient oral microbiota recovery by applying 16S rRNA gene amplicon and shotgun sequencing, identifying ancient oral microbiota, as well as soil and skin contaminant species. Overall, the EDTA pre-digestion and a combined UV irradiation and 5% sodium hypochlorite immersion treatment were both effective at reducing the proportion of environmental taxa and increasing oral taxa in comparison to untreated samples. This research highlights the importance of using decontamination procedures during ancient DNA analysis of dental calculus to reduce contaminant DNA.
Fungi are ecologically important in several ecosystem processes, yet their community composition, ecophysiological roles, and responses to changing environmental factors in historical sediments are rarely studied. Here we explored ancient fungal DNA from lake Lielais Svētiņu sediment throughout the Holocene (10.5 kyr) using the ITS metabarcoding approach. Our data revealed diverse fungal taxa and smooth community changes during most of the Holocene with rapid changes occurring in the last few millennia. More precisely, plankton parasitic fungi became more diverse from the Late Holocene (2–4 kyr) which could be related to a shift towards a cooler climate. The Latest Holocene (~2 kyr) showed a distinct increase in the richness of plankton parasites, mycorrhizal, and plant pathogenic fungi which can be associated with an increased transfer rate of plant material into the lake and blooms of planktonic organisms influenced by increased, yet moderate, human impact. Thus, major community shifts in plankton parasites and mycorrhizal fungi could be utilized as potential paleo-variables that accompany host-substrate dynamics. Our work demonstrates that fungal aDNA with predicted ecophysiology and host specificity can be employed to reconstruct both aquatic and surrounding terrestrial ecosystems and to estimate the influence of environmental change.
Analysis of ancient environmentalDNA(eDNA) has revolutionized our ability to describe biological communities in space and time, by allowing for parallel sequencing of DNA from all trophic levels. However, because
environmental samples contain sparse and fragmented data from multiple individuals, and often contain closely related species, the field of ancient eDNA has so far been limited to organellar genomes in its contribution to population and phylogenetic studies. This is in contrast to data from fossils, where full-genome studies are routine, despite these being rare and their destruction for sequencing undesirable. Here, we
report the retrieval of three low-coverage (0.033) environmental genomes from American black bear (Ursus americanus) and a 0.043 environmental genome of the extinct giant short-faced bear (Arctodus simus) from cave sediment samples from northern Mexico dated to 16–14 thousand calibrated years before present (cal kyr BP), which we contextualize with a new high-coverage (263) and two lower-coverage giant short-faced bear genomes obtained from fossils recovered from Yukon Territory, Canada, which date to �22–50 cal kyr BP. We show that the Late Pleistocene black bear population in Mexico is ancestrally related to the presentday Eastern American black bear population, and that the extinct giant short-faced bears present in Mexico were deeply divergent from the earlier Beringian population. Our findings demonstrate the ability to separately analyze genomic-scale DNA sequences of closely related species co-preserved in environmental samples,
which brings the use of ancient eDNA into the era of population genomics and phylogenetics.
Marine sedimentary ancient DNA (sedaDNA) is increasingly used to study past ocean ecosystems, however, studies have been severely limited by the very low amounts of DNA preserved in the subseafloor, and the lack of bioinformatic tools to authenticate sedaDNA in metagenomic data. We applied a hybridisation capture ‘baits’ technique to target marine eukaryote sedaDNA (specifically, phyto- and zooplankton, ‘Planktonbaits1’; and harmful algal bloom taxa, ‘HABbaits1’), which resulted in up to 4- and 9-fold increases, respectively, in the relative abundance of eukaryotes compared to shotgun sequencing. We further used the bioinformatic tool ‘HOPS’ to authenticate the sedaDNA component, establishing a new proxy to assess sedaDNA authenticity, “% eukaryote sedaDNA damage”, that is positively correlated with subseafloor depth. We used this proxy to report the first-ever DNA damage profiles from a marine phytoplankton species, the ubiquitous coccolithophore Emiliania huxleyi. Our approach opens new avenues for the detailed investigation of long-term change and evolution of marine eukaryotes over geological timescales.
The use of lake sedimentary DNA to track the long-term changes in both terrestrial and aquatic biota is a rapidly advancing field in paleoecological research. Although largely applied nowadays, knowledge gaps remain in this field and there is therefore still research to be conducted to ensure the reliability of the sedimentary DNA signal. Building on the most recent literature and seven original case studies, we synthesize the state-of-the-art analytical procedures for effective sampling, extraction, amplification, quantification and/or generation of DNA inventories from sedimentary ancient DNA (sedaDNA) via high-throughput sequencing technologies. We provide recommendations based on current knowledge and best practises.
A timeline of cave dwellers in sediment
Two archaic lineages overlapped with modern humans outside of Africa: the well-studied Neanderthals and their more mysterious cousins, the Denisovans. Denisovan remains are rare, being limited to Denisovan Cave in Siberia and a putative, undated jaw from Tibet. However, there is evidence for multiple introgressions from Denisovans into modern-day humans, especially in Australasian populations. By examining the sediment of Baishiya Karst Cave located on a high plateau in Tibet, Zhang et al. identified ancient mitochondrial DNA from Denisovans indicating their presence at about 100 thousand, 60 thousand, and possibly 45 thousand years ago. This finding provides insight into the timing and distribution of Denisovans in Asia and extends the time of occupation of the Tibetan plateau by hominins.
Science , this issue p. 584
DNA preserved in sedimentary materials can be used to study past ecosystem changes, such as species’ colonization and extinction. It is believed that minerals, especially clay minerals, enhance the preservation of DNA. However, the role of minerals, as well as organic matter, on DNA sorption in heterogeneous sediments is still not clear. In this study, we examined the effect of mineral and organic matter on DNA binding in lake sediments. Bulk and size-fractionated sediments (0-4, 4-16, 16-64, and >64 μm), having different mineral and organic composition, were used to test DNA sorption; similar experiments were also run after the removal of sedimentary organic matter. Additionally, diffuse reflectance infrared spectroscopy (DRIFT) was used to determine the chemical changes caused by DNA sorption and subsequently produce a DNA-infrared (IR) fingerprint. Clay minerals were the main minerals to sorb DNA in the different samples. Moreover, mica promoted DNA sorption in all size fractions, while chlorite promoted DNA sorption in size fractions greater than 16 μm; clay-mineral and organo-mineral complexes caused a preference of certain clay minerals over others. Sedimentary organic matter affected DNA sorption by covering as well as by amplifying potential DNA binding sites, yet DNA sorption did not change significantly. DNA sorption showed IR spectral modifications mainly at ∼1640, 1416, and 1231 cm⁻¹. Interestingly, the DNA-IR fingerprint in the heterogeneous sediments was evident by those peaks after spectral subtraction. Finally, we proposed a simple model, based on sediment geochemistry, that can be used to determine potential DNA-hotspots in sediments.
The initial colonization of the Americas remains a highly debated topic1, and the exact timing of the first arrivals is unknown. The earliest archaeological record of Mexico—which holds a key geographical position in the Americas—is poorly known and understudied. Historically, the region has remained on the periphery of research focused on the first American populations. However, recent investigations provide reliable evidence of a human presence in the northwest region of Mexico, the Chiapas Highlands, Central Mexico and the Caribbean coast during the Late Pleistocene and Early Holocene epochs. Here we present results of recent excavations
at Chiquihuite Cave—a high-altitude site in central-northern Mexico—that corroborate previous findings in the Americas of cultural evidence that dates to the Last Glacial Maximum (26,500–19,000 years ago), and which push back dates for human dispersal to the region possibly as early as 33,000–31,000 years ago. The site yielded about 1,900 stone artefacts within a 3-m-deep stratified sequence, revealing a
previously unknown lithic industry that underwent only minor changes over millennia. More than 50 radiocarbon and luminescence dates provide chronological control, and genetic, palaeoenvironmental and chemical data document the changing environments in which the occupants lived. Our results provide new evidence for the antiquity of humans in the Americas, illustrate the cultural diversity of the earliest dispersal groups (which predate those of the Clovis culture) and open
new directions of research
The initial colonization of the Americas remains a highly debated topic¹, and the exact timing of the first arrivals is unknown. The earliest archaeological record of Mexico—which holds a key geographical position in the Americas—is poorly known and understudied. Historically, the region has remained on the periphery of research focused on the first American populations². However, recent investigations provide reliable evidence of a human presence in the northwest region of Mexico3,4, the Chiapas Highlands⁵, Central Mexico⁶ and the Caribbean coast7,8,9 during the Late Pleistocene and Early Holocene epochs. Here we present results of recent excavations at Chiquihuite Cave—a high-altitude site in central-northern Mexico—that corroborate previous findings in the Americas10,11,12,13,14,15,16,17of cultural evidence that dates to the Last Glacial Maximum (26,500–19,000 years ago)¹⁸, and which push back dates for human dispersal to the region possibly as early as 33,000–31,000 years ago. The site yielded about 1,900 stone artefacts within a 3-m-deep stratified sequence, revealing a previously unknown lithic industry that underwent only minor changes over millennia. More than 50 radiocarbon and luminescence dates provide chronological control, and genetic, palaeoenvironmental and chemical data document the changing environments in which the occupants lived. Our results provide new evidence for the antiquity of humans in the Americas, illustrate the cultural diversity of the earliest dispersal groups (which predate those of the Clovis culture) and open new directions of research.
While the use of fire has long been recognised as a crucial innovation in the cultural evolution of humankind, much research has focused on the (debated) chronology of its earliest use and control, and less on the ways in which fire was used in the deep past. At its latest by the Upper Palaeolithic, hunter-gatherers routinely used fire to heat a wide range of materials, adjusting parameters like temperature, exposure time and fuel type to the specific requirements of the treated materials, for instance in food preparation or tool production.
Comparing analyses of the chemical and physical properties of modern materials, heated under a range of controlled conditions in a laboratory, to archaeological ones might allow the reconstruction of the “heating history” of excavated materials and hence to infer the function of particular fires in the past - provided changes affecting the properties of the heated archaeological material during burial time are taken into consideration. To investigate the feasibility of such an approach, heated materials sampled from ~40,000 to 25,000 year old fireplaces (hearths) and their sedimentary matrices from the Upper Palaeolithic Abri Pataud rock shelter in South-Western France are used here to study (1) the fuel type(s) used by the site’s occupants, (2) the temperatures reached in fireplaces and (3) the potential changes in human activities related to fireplaces over time, with the influence of post-depositional processes taken into explicit consideration throughout. For this purpose, we used a range of methods to analyse macroscopically visible as well as “invisible” (microscopic and molecular) heat-altered materials.
The results suggest that charred organic materials (COM) encountered in the samples predominantly result from the fuel used in fireplaces, including the earliest reported use of dung as fuel. Earlier suggestions about the use of bone as fuel at the Abri Pataud are not supported by this study. The heating temperature of COM increased gradually from 350 °C in the Aurignacian to 450 °C in Gravettian levels. Py-GC–MS studies identified a range of organic compounds, biomolecules derived from plant as well as animal sources, still preserved in the sediments after exposure to heat and burial in the rock shelter more than 20,000 years ago. Mammalian mtDNA was identified in sediment samples retrieved from the fireplaces, including ancient mtDNA fragments that originated from one or more modern human-like mitochondrial genome(s). This makes the Abri Pataud the first archaeological site for which ancient modern human mtDNA has been retrieved from sediment samples.
The absence of specific organic compounds (furans) in the Aurignacian levels and their presence in the Gravettian ones, the changes in temperatures reached through the Aurignacian-Gravettian sequence as well as changes in the character of the fireplaces (presence/absence of lining river pebbles) suggest that the functions of hearths changed through time.
These results highlight the potential of multi-proxy analyses of macro- and microscopic traces of ancient fireplaces, and especially of a shift in focus towards molecular traces of such activities. Systematic sampling of fireplaces and their sedimentary matrix should become a standard part of the excavation protocol of such features, to improve our understanding of the activities of humans in the deep past.
Doggerland was a landmass occupying an area currently covered by the North Sea until marine inundation took place during the mid-Holocene, ultimately separating the British landmass from the rest of Europe. The Storegga Event, which triggered a tsunami reflected in sediment deposits in the northern North Sea, northeast coastlines of the British Isles and across the North Atlantic, was a major event during this transgressive phase. The spatial extent of the Storegga tsunami however remains unconfirmed as, to date, no direct evidence for the event has been recovered from the southern North Sea. We present evidence of a tsunami deposit in the southern North Sea at the head of a palaeo-river system that has been identified using seismic survey. The evidence, based on lithostratigraphy, geochemical signatures, macro and microfossils and sedimentary ancient DNA (sedaDNA), supported by optical stimulated luminescence (OSL) and radiocarbon dating, suggests that these deposits were a result of the tsunami. Seismic identification of this stratum and analysis of adjacent cores showed diminished traces of the tsunami which was largely removed by subsequent erosional processes. Our results confirm previous modelling of the impact of the tsunami within this area of the southern North Sea, and also indicate that these effects were temporary, localized, and mitigated by the dense woodland and topography of the area. We conclude that clear physical remnants of the wave in these areas are likely to be restricted to now buried, palaeo-inland basins and incised river valley systems.
Present‐day contamination can lead to false conclusions in ancient DNA studies. A number of methods are available to estimate contamination, which use a variety of signals and are appropriate for different types of data. Here an overview of currently available methods highlighting their strengths and weaknesses is provided, and a classification based on the signals used to estimate contamination is proposed. This overview aims at enabling researchers to choose the most appropriate methods for their dataset. Based on this classification, potential avenues for the further development of methods are discussed. To ensure that ancient DNA studies yield valid results, it is crucial to quantify present‐day DNA contamination. The signals and methods that can be used to estimate the proportion of contamination are surveyed.
Large-scale changes in global climate at the end of the Pleistocene significantly impacted ecosystems across North America. However, the pace and scale of biotic turnover in response to both the Younger Dryas cold period and subsequent Holocene rapid warming have been challenging to assess because of the scarcity of well dated fossil and pollen records that covers this period. Here we present an ancient DNA record from Hall's Cave, Texas, that documents 100 vertebrate and 45 plant taxa from bulk fossils and sediment. We show that local plant and animal diversity dropped markedly during Younger Dryas cooling, but while plant diversity recovered in the early Holocene, animal diversity did not. Instead, five extant and nine extinct large bodied animals disappeared from the region at the end of the Pleistocene. Our findings suggest that climate change affected the local ecosystem in Texas over the Pleistocene-Holocene boundary, but climate change on its own may not explain the disappearance of the megafauna at the end of the Pleistocene.
Ancient environmental DNA (aeDNA) data are close to enabling insights into past global-scale biodiversity dynamics at unprecedented taxonomic extent and resolution. However, achieving this potential requires solutions that bridge bioinformatics and paleoecoinformatics. Essential needs include support for dynamic taxonomic inferences, dynamic age inferences, and precise stratigraphic depth. Moreover, aeDNA data are complex and heterogeneous, generated by dispersed researcher networks, with methods advancing rapidly. Hence, expert community governance and curation are essential to building high-value data resources. Immediate recommendations include uploading metabarcoding-based taxonomic inventories into paleoecoinformatic resources, building linkages among open bioinformatic and paleoecoinformatic data resources, harmonizing aeDNA processing workflows, and expanding community data governance. These advances will enable transformative insights into global-scale biodiversity dynamics during large environmental and anthropogenic changes.
Current knowledge about Paleolithic human plant use is limited by the rare survival of identifiable plant remains as well as the availability of methods for plant detection and identification. By analyzing DNA preserved in cave sediments, we can identify organisms in the absence of any visible remains, opening up new ways to study details of past human behavior, including plant use. Aghitu-3 Cave contains a 15,000-yearlong record (from ∼39,000 to 24,000 cal BP) of Upper Paleolithic human settlement and environmental variability in the Armenian Highlands. Finds from this cave include stone artifacts, faunal remains, bone tools, shell beads, charcoal, and pollen, among others. We applied sedimentary ancient DNA (sedaDNA) metabarcoding to the Aghitu-3 sedimentary sequence and combined this with pollen data to obtain a temporal reconstruction of plant assemblages. Our results reveal a stratification of plant abundance and diversity where sedaDNA reflects periods of human occupation, showing higher diversity in layers with increased human activity. Low pollen concentrations combined with high sedaDNA abundance indicate plant remains may have been brought into the cave by animals or humans during the deposition of the lower two archaeological horizons. Most of the recovered plants are reported to be useful for food, flavor, medicine, and/or technical purposes, demonstrating the potential of the environment around Aghitu-3 Cave to support humans during the Upper Paleolithic. Moreover, we identified several specific plant taxa that strengthen previous findings about Upper Paleolithic plant use in this region (i.e., for medicine and the manufacturing and dyeing of textiles). This study represents the first application of plant sedaDNA analysis of cave sediments for the investigation of potential plant use by prehistoric humans.
Cave sediments have been shown to preserve ancient DNA but so far have not yielded the genome-scale information of skeletal remains. We retrieved and analyzed human and mammalian nuclear and mitochondrial environmental “shotgun” genomes from a single 25,000-year-old Upper Paleolithic sediment sample from Satsurblia cave, western Georgia:first, a human environmental genome with substantial basal Eurasian ancestry, which was an ancestral component of the majority of post-Ice Age people in the Near East, North Africa, and parts of Europe; second, a wolf environmental genome that is basal to extant Eurasian wolves and dogs and represents a previously unknown, likely extinct, Caucasian lineage; and third, a European bison environmental genome that is basal to present-day populations, suggesting that population structure has been substantially reshaped since the Last Glacial Maximum. Our results provide new insights into the Late Pleistocene genetic histories of these three species and demonstrate that direct shotgun sequencing of sediment DNA, without target enrichment methods, can yield genome-wide data informative of ancestry and phylogenetic relationships.
The value of dirty DNA
Environmental DNA can identify the presence of species, even from the distant past. Surveying three cave sites in western Europe and southern Siberia, Vernot et al. identified nuclear DNA and confirmed that it is from the close relatives of anatomically modern humans—Neanderthal and Denisovan individuals. A phylogenetic analysis and modeling show that the DNA in sediment samples from several layers corresponds to previously studied skeletal remains. These results demonstrate that environmental data can be applied to study the population genetics of the extinct Neanderthal and Denisovan lineages, identifying a turnover of Neanderthal populations ∼100,000 years ago.
Science , this issue p. eabf1667
Double-stranded ribonucleic acid (dsRNA) molecules are novel plant-incorporated protectants expressed in genetically modified RNA interference (RNAi) crops. Ecological risk assessment (ERA) of RNAi crops requires a heretofore-missing detailed understanding of dsRNA adsorption in soils, a key fate process. Herein, we systematically study the adsorption of a model dsRNA molecule and of two double-stranded deoxyribonucleic acid (DNA) molecules of varying lengths to three soil iron (oxyhydr-)oxides - goethite, lepidocrocite, and hematite - over a range of solution pH (4.5-10), ionic strength (I = 10-100 mM NaCl) and composition (0.5, 1, and 3 mM MgCl2) and in the absence and presence of phosphate (0.05-5 mM) as co-adsorbate. We hypothesized comparable adsorption characteristics of dsRNA and DNA based on their structural similarities. Consistently, the three nucleic acids (NAs) showed high adsorption affinities to the iron (oxyhydr-)oxides with decreasing adsorption in the order goethite, lepidocrocite, and hematite, likely reflecting a decrease in the hydroxyl group density and positive charges of the oxide surfaces in the same order. NA adsorption also decreased with increasing solution pH, consistent with weakening of NA electrostatic attraction to and inner-sphere complex formation with the iron (oxyhydr-)oxides surfaces as pH increased. Adsorbed NA concentrations increased with increasing I and in the presence of Mg2+, consistent with adsorbed NA molecules adopting more compact conformations. Strong NA-phosphate adsorption competition demonstrates that co-adsorbates need consideration in assessing dsRNA fate in soils. Comparable adsorption characteristics of dsRNA and DNA molecules to iron (oxyhydr-)oxides imply that information on DNA adsorption to soil particle surfaces can inform dsRNA ERA.
With increasing environmental application, biochar (BC) will inevitably interact with and impact environmental behaviors of widely distributed extracellular DNA (eDNA), which however still remains to be studied. Herein, the adsorption/desorption and the degradation by nucleases of eDNA on three aromatized BCs pyrolyzed at 700 °C were firstly investigated. The results show that the eDNA was irreversibly adsorbed by aromatized BCs and the pseudo-second-order and Freundlich models accurately described the adsorption process. Increasing solution ionic strength or decreasing pH below 5.0 significantly increased the eDNA adsorption on BCs. However, increasing pH from 5.0 to 10.0 faintly decreased eDNA adsorption. Electrostatic interaction, Ca ion bridge interaction, and π-π interaction between eDNA and BC could dominate the eDNA adsorption, while ligand exchange and hydrophobic interactions were minor contributors. The presence of BCs provided a certain protection to eDNA against degradation by DNase I. BC-bound eDNA could be partly degraded by nuclease, while BC-bound nuclease completely lost its degradability. These findings are of fundamental significance for the potential application of biochar in eDNA dissemination management and evaluating the environmental fate of eDNA.
Anticipating and addressing the social implications of scientific work is a fundamental responsibility of all scientists. However, expectations for ethically sound practices can evolve over time as the implications of science come to be better understood. Contemporary researchers who work with ancient human remains, including those who conduct ancient DNA research, face precisely this challenge as it becomes clear that practices such as community engagement are needed to address the important social implications of this work. To foster and promote ethical engagement between researchers and communities, we offer five practical recommendations for ancient DNA researchers: (1) formally consult with communities; (2) address cultural and ethical considerations; (3) engage communities and support capacity building; (4) develop plans to report results and manage data; and (5) develop plans for long-term responsibility and stewardship. Ultimately, every member of a research team has an important role in fostering ethical research on ancient DNA.
In the two decades or so since ancient sedimentary DNA ( sed aDNA) took its place as a new Quaternary paleo-proxy, there have been large advances in the scope of its applications and its reliability. The two main approaches, metabarcoding and shotgun sequencing, have contributed exciting insights into areas such as floristic diversity change, plant-herbivore interactions, extinction, conservation baselines and impacts of invasive species. Early doubts as to its potential to contribute novel information have been dispelled; more is now understood about the passage of sed aDNA from the original organism to a component of soil or sediment and about the range of uncertainties that must be addressed in the interpretation of data. With its move into the mainstream, it is now time to develop effective data archives for sed aDNA, refine our understanding of central issues such as taphonomy, and further expand the potential for describing, both qualitatively and quantitatively, the history of past ecosystems.