Archaeology meets Environmental Genomics: implementing sedaDNA in the study of the human past

doi:10.21203/rs.3.rs-3568244/v1

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Archaeology meets Environmental Genomics: implementing sedaDNA in the study of the human past

https://doi.org/10.21203/rs.3.rs-3568244/v1

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published 27 Jun, 2024

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Sedimentary ancient DNA (sedaDNA) has become one of the standard applications in the field of paleogenomics in recent years. It has been used for paleoenvironmental reconstructions, detecting the presence of prehistoric species in the absence of macro remains and even investigating the evolutionary history of a few species. However, its application in archaeology has been limited and primarily focused on humans. This article argues that sedaDNA holds a significant potential in addressing key archaeological questions concerning the origins, lifestyles and environments of past human populations. Our aim is to facilitate the integration of sedaDNA into the standard workflows in archaeology as a transformative tool and thereby unleashing its full potential for studying the human past. Ultimately, we not only underscore the challenges inherent in the sedaDNA field but also provide a research agenda for essential enhancements needed for implementing sedaDNA into the standard workflows of archaeologists.

sedaDNA

ancient DNA

archaeogenemics

ancient metagenomics

bioarchaeology

environmental archaeology

Ancient DNA or shortly aDNA refers to all DNA fragments of once lived organisms. Most ancient DNA studies that address archeological questions focus on the use of human remains to study past human ancestry, mostly testing the effect of migration in historical events. Yet, the studies that also explore the evolutionary and population history of other taxa are becoming more abundant (Kistler et al., 2020; MacHugh et al., 2017).

Despite most paleogenomic studies using macroscopic organic remains, an increasing number of studies look for ancient DNA in environmental samples. Modern environmental DNA (eDNA) might be preserved in water (Thomsen et al., 2012), soil (Taberlet et al., 2012) or air (de Groot et al., 2021). However, eDNA in archaeological context is typically recovered from sediments (either from terrestrial or aquatic sites), in which case it is referred to as sedimentary ancient DNA or sedaDNA. SedaDNA samples hold genetic information of all living organisms that have interacted with a given layer or context. In paleoenvironmental research, sedaDNA has been embraced as the main methods for reconstructing past environments.

Studies using permafrost, cave, and lake sediments have helped answer questions about extinct hominins and large mammal species in prehistory (Gelabert et al., 2021; Parducci et al., 2017; Vernot et al., 2021; Wang et al., 2021). Yet, these studies have so far mostly studied a few distinct species, thus not making use of the full potential that sedaDNA has to offer, e.g. the recovery of a broad biodiversity. Furthermore, archaeological sedaDNA studies to date focused mainly on prehistoric periods, leaving biodiversity assessments from historical periods largely untouched.

However, before sedaDNA becomes a standard method in archaeology, concerted action is required. The sedaDNA field is in continuous development; new lab methods are being developed, along with tailor-made bioinformatic approaches to process the big amount of genomic data. Both developments require highly specialized skills and as a result, debates on advantages and disadvantages of sedaDNA applications are highly technical, which makes the field difficult to explore for archaeologists, perhaps even more so than any new method in archaeology. Moreover, it is highly tempting to become enchanted with the technique and have too much trust in it. If we want to avoid the boom-and-bust cycle typical for many methods (Jones & Bösl, 2021; Lebrasseur et al., 2018) we need to collectively develop a research agenda. To make sedaDNA a cornerstone in archaeology, we need to bridge the gap between on the one side archaeology and on the other side (ancient) environmental metagenomics.

Table 1

Common terms used in sedaDNA studies.
Term	Meaning
Ancient DNA (aDNA)	DNA fragments obtained from fossils and ancient sediments
Environmental DNA	DNA fragments obtained from environmental sources
Ancient environmental DNA (aeDNA)	Ancient DNA obtained from ancient environmental sources
Sedimentary ancient DNA (sedaDNA)	Ancient DNA obtained from ancient sediment samples
Paleogenomics	The scientific field of reconstructing and analyzing ancient genomic material
Metagenomics	The study of genetic material recovered from samples contains multiple organisms.
Ancient metagenomics	Metagenomics of ancient DNA samples
Permafrost	Surface materials (e.g. soil, subsoil) that remain consistently frozen regardless of seasonal variations.
Pathogen	A general term for microorganisms that cause diseases
Mitochondrial DNA (mtDNA)	Maternally inherited DNA that is present in a separate organelle, i.e mitochondria, rather than nucleus of the cell
Phylogenetic tree	A tree-shaped graph that shows evolutionary relationship between organisms
Polymerase chain reaction (PCR)	A commonly method used for enzymatic amplification of DNA out of cell
Shotgun sequencing	A method for sequencing random fragments from a DNA library
Target enrichment / target capture	A laboratory method for selecting DNA fragments belonging chosen taxa in a DNA library
Contamination	Undesired DNA fragments from outside the sample, mostly modern DNA
Authentication	Determining whether the recovered DNA is ancient or not
Damage pattern	Specific chemical alterations occurred in DNA molecules in the post-mortem period that are used for authenticating ancient DNA
Cross-contamination	Contamination occurred between samples

Based on a structured literature review, the authors of this paper will outline the fundamental principles of sedaDNA and their successful applications. We will, then, identify on which fronts it can contribute to archaeology and on which not. Subsequently, we will offer a research agenda highlighting the needed developments and approaches for efficiently implementing sedaDNA into archaeological research.

2.1. Sources

The field of paleogenomics reconstructs and analyzes genomic information from any sample type and uses it for identifying taxa and acquiring deeper information about past organisms. However, ancient DNA for most archaeogenomic research is extracted directly from larger preserved organic artifacts and ecofacts such as human or animal bones, and macro-botanical remnants. In well-preserved contexts (e.g. permafrost) soft tissues of organisms may be preserved that can be sampled directly (Maixner et al., 2016).

Sedimentary ancient DNA, however, is most likely found in micro remains or coprolites (Massilani et al., 2022) or bound to minerals in the soil (Kjær et al., 2022). Due to the natural decomposition of organic materials, sedaDNA is damaged, degraded, fragmented, and intermixed (Orlando et al., 2021). Furthermore, a single ancient sediment sample most likely contains ancient DNA from a broad range of species, animal, vegetal, bacteria and fungi, as well as -in most cases- DNA of recent origin of a suite of organisms from the same range of taxonomic groups. These characteristics heavily determine and restrict the methodological workflow for sedaDNA analysis.

2.2. Workflow

Similar to aDNA studies for sedaDNA follow strict protocols and workflow consisting of (1) sampling, (2) laboratory-based data generation, and (3) bioinformatic processing (see Fig. 1). Per step, multiple protocols can be available and choices in this respect have direct implications for the types of questions that can be tackled.

2.2.1. Sampling

Although there are protocols throughout the whole workflow to prevent, detect and filter out contamination with DNA molecules from present-day sources, such contamination is to be avoided as much as possible already in the first steps of the workflow to maximize the amount of useful information gained per sample. Therefore, sedaDNA sampling requires sterile disposable materials, specialized protective clothing and careful handling including multiple steps to clean materials and sediment surfaces. To avoid not only the influence of present-day DNA but also cross-contamination with actual ancient DNA from other contexts or nearby soil layers, sedaDNA samples are to be taken either from the inside of soil sampling cores (Murchie et al., 2021) or directly from archeological sections after removing the air-exposed top layer (Wang et al., 2021). Since aDNA fragments are highly degraded and fragmented, signals of DNA degradation and sequenced fragment lengths can be used to authenticate aDNA and to detect contamination (Peyrégne & Prüfer, 2020).

2.2.2. Data generation: Laboratory process

For the same reasons, the extraction of aDNA from sedimentary samples happens in an ultra-clean (i.e., contamination-free) aDNA lab (Fulton & Shapiro, 2019). Since ancient DNA is heavily fragmented, extraction methods are designed to target only those small fragments of aDNA. Thus, most modern contaminants are eliminated during the extraction (Dabney et al., 2013). Sediment samples are prone to disruption of lab analysis mainly due to humic acids, heavy metals and complex proteins naturally occurring in organically rich sediment which is called inhibition. A series of additional protocols have been developed to minimize the impact of such inhibitors (Armbrecht et al., 2020; Murchie et al., 2021; Rohland et al., 2018; Slon et al., 2017; Wang et al., 2021).

For the analysis of the DNA pool of a whole community of organisms, a specific workflow is imperative, that is capable of parallel identification of individual taxa. In principle, various approaches are available. One of the approaches, DNA metabarcoding zooms in on one or a few regions of the genome which are known to show variation among the taxa studied (Armbrecht et al., 2020). Available fragments of those ‘barcode regions’ are then multiplied via PCR amplification and the DNA code of all resulting copies is translated into a letter code in a digital file (i.e., a ‘sequence read’). This translation is done in parallel for all copies in a process called ‘high-throughput sequencing’ (Illumina Cooperation, 2023a). While DNA metabarcoding secures data for informative parts of the genome (enhancing chances for species-level identifications) and downstream data analysis is relatively straightforward, a huge downside of this method is the inability to screen the resulting dataset for any remaining modern contaminants. While tools are available to assess that detected taxa is either modern or ancient (see below), such tools cannot be applied on the products of the metabarcoding approach.

Another method, referred to as metagenomics, requires amplification of all DNA fragments in the original DNA extract regardless of their position in the genome. This so-called ‘shotgun metagenomics’ approach results in a much more complex dataset yet does allow an authenticity check for each single taxon.

DNA metabarcoding has been the method of choice in the majority of recent sedaDNA-based palaeoenvironmental studies. Authenticity checks can be less crucial for the type of research questions explored there, which are mainly general shifts in community composition across time and space. Yet, applications of sedaDNA in an archeological setting are often driven by questions of the presence or absence of particular taxa at certain locations of moments in time. In such cases, avoiding false-positive detection of a taxon in a particular sample is crucial. For archeological applications, metagenomics is therefore the method of choice. Hence, it becomes evident that informatic techniques centered on the comparative analysis of multiple taxa, that integrate robust statistical discrimination methods, emerge as the most dependable and effective means to assess the presence of taxa (Vogel et al., 2023).

The untargeted data collection via metagenomics may require a deeper sequencing (i.e., collection of DNA codes for a larger subset of fragments from the DNA extract) to gather sufficient data for parts of the genome that a discriminatory among species or genetic variants within species. To reduce costs and/or enhance data volume especially for more detailed studies at the level of e.g., populations, strains or individuals, new approaches are developed to enrich the DNA pool for discriminatory fragments prior to sequencing. Such target enrichment works by capturing regions of interest by hybridization to tailor-made probes attached to magnetic beads, which can then be isolated from the pool using magnetic pulldown (Illumina Cooperation, 2023b).Hence, apart from tailoring the resolution to meet project-specific inquiries, the method chosen is also contingent upon the budget.

Ultimately, the resolution of generated data is a vital element in sedaDNA studies and can be defined in two layers: i) covering more diverse organisms in the sample or ii) gaining deeper knowledge on relevant organisms. While the former corresponds to metagenomics or metabarcoding, the latter can be achieved by using the targeted enrichment approach. The choice of the method depends on the archaeological research question.

2.2.3. Data processing: Bioinformatics

Bioinformatics, which represents the analytical step of these kinds of studies, is a crucial step, especially now that advanced sequencing and extraction techniques are yielding vast amounts of data. Bioinformatic tools help researchers to (1) detect contamination, (2) perform the authentication, and (3) taxonomic classification. An aspect of bioinformatics specific to metagenomic analysis as a tool for biodiversity screening is the assignment of DNA sequences to specific taxa (species, or higher taxonomic levels). This process requires matching each individual sequence read against databases containing reference sequences of known taxa. Different bioinformatic tools have been published that automate this process (Hübler et al., 2019; Pochon et al., 2023; Yates et al., 2021).

After determining which organisms are present in a sample and authenticating them, it is possible to do further deeper analyses on the sequences of a selected taxon, depending on the quantity and quality of the recovered data. For instance, the co-occurrence of certain organisms can be used as an indicator of environmental dynamics (Wang et al., 2021), the co-occurrence of two closely related species in the same sample might be disentangled (Pedersen et al., 2021) or the population history of a species from a specific site (Vernot et al., 2021) may be determined.

When high-quality genomes are available for particular species, combined with the tools offered by population genetics, it is possible to explore not only the presence/absence of species but also profound changes in genome structure and function. This sheds light on the underlying evolutionary pressures.

2.3. Application of sedaDNA in paleoenvironmental studies & archaeology

A key feature of environmental samples, in contrast to aDNA samples from macro remains, is that it contains traces of DNA from multiple taxa preserved at the same time and space. Therefore, data yielded from sedaDNA studies offers a groundbreaking advancement in the field of evolutionary research, by enriching our understanding not only in terms of spatial distribution but also by adding a crucial temporal dimension.

In the existing literature, researchers have employed various sources for isolating sedaDNA, as shown in Table 2. Source material varies considerably due to the diverse environmental conditions found on Earth's surface, such as permafrost, aquatic and terrestrial sediments, among others. Whilst the choice of the source material is based on the specific research objectives.

Table 2

Selected articles that used various types of sediments as aDNA source.
Sources of sedaDNA	Literature
Lake sediment	Pedersen et al. 2016, Graham et al., 2016, Wang et al. 2017, Ahmed et al. 2018, Parducci et al., 2019, Schulte et al., 2020, Moguel et al., 2021, Lammers et al., 2021, Rijal et al., 2021, Wang et al. 2021, Hebda et al., 2022,
Marine sediment	Smith et al., 2015, Gaffney et al., 2020, Armbrecht et al., 2020
Rock shelter sediment	Braadbaart et al., 2020
Midden sediment	Seersholm et al., 2016, Moore et al., 2020, Borry et al., 2020
Cave sediment	Slon et al., 2017, Ardelean et al., 2020, Zhang et al., 2020, Gelebert et al., 2021, Zavala et al., 2021, Pedersen et al., 2021, Vernot et al., 2021
Swamp forest sediment	Dommain et al. 2020
Permafrost	Liang et al. 2021, Wang et al. 2021, Murchie et al. 2021, Murchie et al., 2022
Latrine/pond/well sediment	Søe et al. 2018, Tams et al., 2018, Sabin et al., 2020,

Aquatic sediments have provided valuable information on past ecosystems through both bioarcheological and genetic analyses (Parducci et al., 2019). By using lake sediments, researchers recovered ancient DNA from organisms belonging to a variety of taxa (Capo et al., 2021), and marine sediments have been used to analyze floral and faunal changes through time (Armbrecht et al., 2020; Gaffney et al., 2020; Smith et al., 2015). Furthermore, several studies showed that it is possible to secure and reconstruct chloroplast and mitochondrial genomes, efficiently enough to perform population genomic analyses by using aquatic sediments (Lammers et al., 2021; Wang et al., 2021).

Permafrost sediments have also been used for reconstructing quaternary environments mostly from high latitudes of the northern hemisphere (Fernandez-Guerra et al., 2023; Kjær et al., 2022; Murchie et al., 2021, 2022; Wang et al., 2021). Recent studies showed that DNA could be preserved at least 2 million years in permafrost. This provides a unique opportunity to reconstruct ancient environments starting from the Late Pliocene and to uncloak the alterations of climate, vegetation and other living communities in time and space (Kjær et al., 2022). Thanks to its ability to reconstruct genomes, this type of data allows to reveal evolutionary histories of species (Murchie et al., 2021, 2022; Wang et al., 2021).

Meanwhile, sedaDNA has also been used for the recovery of ancient DNA, present in archaeological sites. It allows us to determine the presence of human groups and study temporal changes such as the extinction of Neanderthals. While it could be collected from alongside any retrieved bone (thus limiting the need to damage the bone), human ancient DNA can in some cases also be retrieved from soil in the absence of bones, potentially tremendously enlarging sample sizes and spatiotemporal coverage of future population genetic studies (Gelabert et al., 2021; Pedersen et al., 2021; Slon et al., 2017; Zavala et al., 2021). The same, in principle, applies to other faunal, as well as floral taxa.

We can name two main applications of this technique in the Paleolithic contexts: 1) First, the determination of presence or absence of an specific taxon such as humans in a space and time and 2) second, while more recent and/or well-preserved contexts sedaDNA has the potential to yield more detail (higher taxonomic resolution), and may detect additional organisms that are usually not observed in current methods due to bad preservation conditions. It can potentially enable us to gain access to more sample locations for a given period, a development that we now see in the application of sedaDNA to paleoenvironmental reconstructions.

SedaDNA holds promise in addressing recent historical questions. Faunal, floral or microbial compositions of a given environment change with human activity in some historical cornerstones (i.e. agricultural activities). Series of temporal sampling from the archaeological cores and investigation of compositional alterations in these samples may allow, for example, the understanding of the history of urbanization and its effects on the environment or temporal differences in vegetation due to various effects.

Within the subfields of geoarchaeology, zooarchaeology, and archaeobotany, ecofacts are used to tackle fundamental archaeological research questions. Most studies to date use macro- or microscopic visual classification to identify past environments, species, and subsistence practices. Consequently, these methods are time-consuming and costly. Furthermore, morphological data is sometimes inadequate to distinguish closely related species. The principles of sedaDNA and the first trials of the method on archaeological contexts highlight metagenomic approaches that have the potential to transform those subfields. Yet, like any method, sedaDNA has limitations and visual-based inspection of ecofacts may yield more or better information in some cases. Therefore, the potential of sedaDNA in comparison to other ancient DNA sources (i.e., macro remains) or conventional archaeological methods depends on the type of question at hand and the type and resolution of data required to provide answers to that question. This potential is summarized in Table 3 and justified in more detail below.

Table 3

Potential of sedaDNA for answering different types of archaeologically relevant research questions (* ‘-’: limited, ‘+’: promising, ‘++’: significant).
Main questions	Specific topics	Conventional methods		Ancient DNA
Main questions	Specific topics	Microscopic inspection (e.g. palynology)	Macroscopic inspection	Macro remains	Sediments
What did they eat?	Diet composition (plants and animals)	++	++	++	++
	Storage and transportation of food products	+	+	+	++
	Food preparation procedures	+	++	-	-
How healthy were they?	Detection of illness and disease	-	++	-	-
	Presence of pathogens and parasites (including zoonoses)	+	-	++	+
	Hygiene and healthcare indicators	++	++	++	++
How did they control their environment?	Land management and spatial planning	++	+	++	++
	Agricultural practices	++	+	++	++
	Usage of wild animals (hunting and gathering)	-	++	++	++
	Domestication practices	-	++	++	+
	Effects on wild populations and natural ecosystems	+	+	+	++
	Movement of domestic livestock and plants during migrations	++	++	++	++
	Interbreeding of domestic varieties	-	-	++	+
What did the environment look like?	Ecosystem status and complexity	++	-	-	++
	Indicators of climatic fluctuations through biodiversity	++	-	-	++
	Indicators of past landforms	++	-	-	++
	Indicators of natural disasters (e.g. volcanic eruptions)	+	-	-	+
Who were they and where did they come from?	Mobility of people during their lifetimes	-	-	-	-
	Individual and population level migrations	-	-	++	+
	Diversity within the population	-	-	++	+
	Relatedness of given individuals	-	-	++	-

3.1. What did they eat?

Organic remains are important indicators to reconstruct past subsistences, diets and cultural preferences. Micro- and macroscopic methods based on morphological characteristics currently define archaeozoological and archaeobotanical research. Increasingly, isotope ratios are also used for reconstructing diets (DeNiro, 1987; Hedges & Reynard, 2007; Katzenberg, 2008; Larsen et al., 2019; Richards et al., 2001).

While off-site contexts such as ponds, and ditches can provide important insights into the fauna and flora consumed during a period (Reitz & Shackley, 2012) especially latrines, occupation layers and trash contexts as are often rich in organic remains and provide evidence about the consumption of plants and animals. For the study of animal products, macrofaunal assemblages remain the main source of information. Microscopic analyses of e.g. pollen, and phytoliths provide the most important line of evidence for plants (Larsen et al., 2019; Santini et al., 2022; Warnock & Reinhard, 1992).

Ancient DNA analysis of micro and macro remains can increase the taxonomic resolution to which they can be identified (Hagan et al., 2020; Kuch & Poinar, 2012; Maixner et al., 2021; Nodari et al., 2021; Oskam et al., 2012). Also ancient DNA analysis of human remains can provide evidence for adaptations to consumption changes. For example, the presence or diversity of genes coding for certain enzymes may signal certain diets (Fan et al., 2016; Rees et al., 2020).

As ancient DNA theoretically can be recovered from almost any archaeological context containing organic material, sedaDNA can be an efficient way in identifying key biomarkers. It may provide a faster way to scan pollen deposits without first extracting pollen. Furthermore, sedaDNA can provide a high level of taxonomic detail as DNA extracted from ecofacts. It may especially be valuable to allow the identification of species of which micro- or macro-remains are notoriously absent from the archeological records due to preservation conditions. Likewise, sedaDNA may help to identify unrecognizable fragments and remains of food products in storage vessels (Drieu et al., 2020).

While sedaDNA holds potential for consumption practices, its potential to answer questions on food preparation procedures is more limited. Here, biochemical techniques can make a difference (Hendy, 2021). Furthermore, DNA cannot differentiate between different parts of the same species. Likewise, it is always likely a consumption plant is present in a context because its pollen was blown in from the surroundings (de Groot et al., 2021).

Contexts such as latrines that are less influenced by external factors and abundantly rich in organic remains are especially suited for or sedaDNA research into consumption practices. Clearly, sedaDNA and archaeozoological/botanical studies are not mutually exclusive, but complementary. Not only can such methods provide final proof for the presence of a taxon, visual inspection also provides more final proof of culinary processing (Martín et al., 2014; Medina et al., 2012; Yravedra et al., 2012).

3.2. How was their health?

Osteological approaches are essential in studying individual life histories. Macroscopic inspection of human remains enables us to identify diseases (Ortner, 2011). Traumas and healing patterns give insights into the daily life of individuals and the social and medical institutions around them (Altınışık et al., 2022; Spikins et al., 2018). Dental inspection (development, caries, wear etc.) enables the reconstruction of oral health and dietary habits (Larsen et al., 2019).

While human remains are an important source, they are not often available due to preservation conditions and burial practices, hampering systematic analysis. Findings of ancient pathogens can provide additional information and can be recovered from sediments rich in human feces. These contain microfossil remains associated with bacteria and viruses such as fungi and parasites. (Sabin et al., 2020; Warnock & Reinhard, 1992). Eggs of intestinal parasites (e.g. helminths) are generally well preserved in latrines and coprolites and have been the focus of paleoparasitological research for decades (Reinhard et al., 1986). While techniques for extraction and examination have been refined, overlap in morphological characters often limits identification beyond genus level (Søe et al., 2018). The integration of techniques from genetics into paleoparasitology has advanced abilities to identify eggs extracted from latrines or coprolites to the species level (Loreille et al., 2001e et al., 2015).

Recent exploratory studies have shown that sedaDNA has the potential to be a versatile method for the recovery of ancient parasite DNA from soil (Søe et al., 2018). Depending on DNA preservation, direct extraction of sedaDNA could be a cost-effective alternative for sieving eggs from the sediment. Although larger numbers of eggs can be inspected by sieving them from the sediment, metagenomic analysis of a small sediment sample may already yield a large diversity of soil- and meat-borne parasites (Søe et al., 2018).

Over the past decade dental calculus, bone tissues, and dentine have been subject to aDNA analysis for identifying ancient protists, and bacterial and viral pathogens (de-Dios et al., 2023; Enard & Petrov, 2018; Greenbaum et al., 2019; Guzmán-Solís et al., 2021; Margaryan et al., 2018). However, ancient pathogen DNA from environmental sources remains understudied but presents itself as an important domain for future innovation (Malyarchuk et al., 2022).

3.3. How did they domesticate fauna and flora?

A central research question in archaeology remains the domestication of the fauna and flora. Macrofaunal and archaeobotanical remains have been used to distinguish domestic species from their wild counterparts and identify markers of domestication. Visual comparison has been central in pinpointing the origin of domestication events of different species (Price & Hongo, 2020). Unfortunately, for many species, it is not always possible to distinguish domestic and wild species since they are still physically close to each other. In such cases, tracing domestication events by using ancient DNA from organic remains becomes a more reliable alternative. There have been numerous genetic studies to detect evolutionary splits of wild and domestic species, as well as hybridization events afterward (Bergström et al., 2022). However, these ancient DNA studies still require well-preserved macroscopic organic remains.

Here, sedaDNA has the potential to fill in the gaps on the map between sites where such remains have been found, and thereby help researchers figure out the geographical distribution of domesticated species and, given sufficient quantity and quality of data, even the origins of those species. Likewise, sedaDNA may help to increase spatial coverage of historical distribution maps of wild species, as well as -on a more local scale- assessment of their presence in the surroundings of humans. A drawback of using sedaDNA for the detection of specific domesticated or wild variants of a taxon is that such variants may be hard to distinguish in case of low DNA yields from a certain taxon, quality or availability of references, postmortem damage (Atağ et al., 2022) and in some cases when samples have DNA from multiple closely related taxa.

3.4. What was their environment?

Humans not only alter plants and animals, but human action is also prescribed by the environment. Biotic and abiotic factors define livelihood practices and are thus crucial for the interpretation of sites. A suite of faunal and botanic processes enables us to directly reconstruct both the biodiversity around a site and indirectly abiotic factors such as climate.

Certain archaeological contexts can also act as catchments where organic materials from the local or regional environment slowly accumulate (e.g. wells, deep canals, peat bogs or lakes). Microbotanical (pollen) and microfaunal (diatoms and insects) remain valuable sources for identifying key biomarkers that yield important insights into past landscapes and climate (Chevalier et al., 2020; Reitz & Shackley, 2012).

Therefore, in many paleoenvironmental studies, sedaDNA has already been embraced as a fast, cost-effective and detailed alternative to palynology (Armbrecht et al., 2020; Gaffney et al., 2020; Murchie et al., 2021, 2022; Smith et al., 2015; Wang et al., 2021). These studies mostly use samples from permafrost or lake sediments. Archaeological contexts that are now used for environmental reconstruction through traditional methods, thus, can easily be used with the same methods.

However, cautionary selection and interpretation of contexts are needed as sedaDNA does not discriminate between material originating from wild populations and material originating from human practices, unless a clear signal of domestication is found. Furthermore, with traditional methods pollen can be quantified, providing insights into the dominance and divergence between species. Metagenomic analysis of pollen may provide trustworthy relative abundances among the observed taxa but fails to provide absolute densities, making it more difficult to make quantitative distribution assessments per species.

3.5. Who were they and where did they come from?

Human mobility is proven to have been considerable throughout history (Lazaridis et al., 2014). While moving, people spread ideas, norms, and livelihood strategies. In the past, material culture was a central source for tracing human mobility. Important advances in the natural sciences have ensured that for the last two decades, DNA and isotopic ratios collected from human remains have been the main information sources for studying paleo-mobility (Bentley et al., 2003; Brettell et al., 2012; Duxfield et al., 2020; Pospieszny et al., 2023; Shaw et al., 2016).

As contemporary human DNA also enables us to map migrations routes and the genetic proximity of populations to each other (Gilbert et al., 2022), DNA from ancient individuals provides information about when and how population movements occurred. Therewith, answering important archaeological questions became possible, such as the distribution of certain languages or cultures (Fernandes et al., 2020; Wang et al., 2021).

Ancient human DNA from sediments might be advantageous when there are no human remains available because of several reasons, such as preventing destructive sampling, ethical concerns, the type of burial, or the absence of human remains in a site due to preservation levels. Despite the lower quality of data, studies have shown that it is possible to study the ancestry of individual genomes found from soil, enabling the performance of population genetics analyses with the absence of bone material (Gelabert et al., 2021; Pedersen et al., 2022). One of the limitations of this type of study, however, is that the recovered mtDNA or genomic data might originate from multiple individuals. This adds a layer of complexity to the population genetics analyses, which rely on the comparison of diverse genomic positions between individuals. A composite sequence that originates from multiple biological sources will present more variable positions than a real single genome, adding artificial divergence to the recovered consensus sequences (Gelabert et al., 2021).

3.6. Summary: potential and position of sedaDNA in future archaeology

SedaDNA has the capacity to recover genetic material from unidentified taxa, or from taxa with scarce remains. Therefore, it presents itself as an essential method that can truly revolutionize our study of past consumption practices. Also, for the reconstruction of past landscapes and mapping of environmental change, it holds great potential. SedaDNA also has the potential to tackle other research questions, especially when certain organic remains are unavailable, have not been preserved, or are too costly for the excavators. However, sedaDNA is not the magic solution and single method that can solve all research questions.

Despite such limitations, we contend that sedaDNA has the potential to contribute to archaeology on three fronts:

Because of its versatility in cost-effectively identifying taxa from a given sample, it can add unseen details to archaeobotany, archaeozoology and geoarchaeology, and identify taxa which are impossible to classify due to taphonomy or lack of morphological distinctiveness.

Since it can map changes in consumption practices and the environment when zooming in on specific biomarkers, it can make existing models more fine-grained and provide us with detailed insights into changes over time.

It has the potential to straightforwardly map the absence or presence of key species, the technique presents itself as an ideal scanning or preliminary evaluation tool to assess the potential of a context or site for further analysis with traditional techniques.

Possibility to recover genomes of key species, including humans with absence of bones which provides a broader temporal and spatial distribution patterns of those taxa.

Clearly, sedaDNA is not to replace existing methods. Rather it should become part of the toolkit of those allied subfields studying organic remains. Perhaps its biggest potential lies in its role as a scanning method preceding further analysis.

With the decreasing sequencing costs, and improving laboratory and computational methods, sedaDNA is on track to become a standard method in archaeology. However, at this moment, sedaDNA remains an innovative application that has only been tested on a limited range of archeological contexts and research questions. We need to ensure that we do not end up in a “hype cycle” where everybody is blindly enchanted by the potential of sedaDNA and the technique is widely applied without focus and criticism (Jones & Bösl, 2021).

We argue that concerted collaborative research across international labs is needed to ensure sedaDNA becomes a trustworthy cornerstone in the study of the human past. Thus, a shared research agenda is imperative to focus methodological developments and thereby arrive at the right conditions so fundamental research questions in archaeology can be addressed with sedaDNA. We argue that in order to become adopted as a standard method by archaeologists, five main topics require concerted attention.

(1) Clean sampling strategies more easily integrated into the archaeological workflow need to be developed. The current workflow requires trained DNA experts. However, archaeological (rescue) excavations often are ad hoc, making it difficult to plan and consistently sample those context rich in organics. Therefore, archaeologists would need to have the tools and knowledge at hand to sample those high potential contexts. This requires a standardized protocol executable for archaeologists that is both versatile and sterile enough for further extraction in the lab.

(2) Increasing the data resolution for specific archaeological research questions: The resolution layer and correspondingly, data generation strategies in sedaDNA depends on the research question as mentioned in the workflow section (see 2.2.) above. A comprehensive research plan based on research questions and budget should be made before data generation to increase the feasibility and efficiency of the method. To explore the full biodiversity of an environmental community, one should aim to encompass the widest range of different organisms, typically achieved through metabarcoding or when authentication is crucial, shotgun metagenomics. On the other hand, for a more in-depth exploration of a specific organism, targeted enrichment is often the preferred method for a high-resolution analysis. The efficiency of targeted enrichment depends significantly on prior knowledge of the genomic diversity of the relevant organism. Well-studied species (e.g., humans and domesticates), generally possess relatively high-quality reference genomes and diversity panels. However, for most, the scarcity of data results in limited prior knowledge. In the latter case, capture probe design can be challenging and requires competent know-how. This could potentially decrease the resolution of the data. The long-term solution is accumulating data from a wide variety of organisms in time, while a more immediate approach lies in openly releasing designed probes along with publications.

(3) More comprehensive reference databases to increase the quality of taxonomic and evolutionary estimations. Since the taxonomic classifications heavily depend on the reference genomes available, the lack thereof creates false positives and negatives during the analysis. To tackle it, more reference genomes from different taxa are needed. Existing initiatives beyond archaeology show that sequencing genomes of relevant species can increase the quality of bioinformatic species determination.

(4) Fostering an open science perspective where raw data and methodological protocols are shared: Such an approach helps other scientists use the similar methods for their own studies or help them avoid double work for certain aspects of their studies. Next to those, when the shared data is raw and unprocessed sequencing data, it allows other scientists to re-use it for comparison or certain types of analyses that were not done during the actual study, due to different focus or a limited time. Once the raw data is shared it becomes a good resource for other scientists and contributes to the growth of the scientific field.

(5) Multi-proxy comparisons between sedaDNA and other archaeological methods: The comparison of results of sedaDNA with e.g., pollen analysis enables us to explore the limits and blind spots of sedaDNA. This information will help us in positioning sedaDNA in the archaeological workflow and thus solve the important question: at which stage can sedaDNA bring added value and be efficiently integrated into the current workflow.

SedaDNA has become an essential tool in the field of paleogenomics while its applications for archaeology remain limited. This article highlighted the vast potential of sedaDNA to address pressing archaeological questions. Especially, the research questions related to diets, origins, and environments of ancient human populations are yet to unleash this potential. However, it is evident that future studies need to overcome several key challenges. These are mainly about its applicability in archaeological workflows, data resolution and quality and comprehensive reference databases. Our primary goal is to facilitate a broader use and to unlock the full potential of sedaDNA in archaeological research. The optimization of sedaDNA’s use in archaeological research will provide newly found perspectives in the pursuit of a deeper understanding of the human past. Therefore, a concerted collaboration between sedaDNA labs is needed if it would become a cornerstone of the archaeological practice.

Acknowledgements

This paper derives from the project ‘Constructing the Limes: Employing citizen science to understand borders and border systems from the Roman period until today’ (C-Limes), funded by the Dutch Research Council (NWO) as part of the Dutch Research Agenda. (2021–2026, Project number: NWA.1292.19.364). Pere Gelabert was funded by FWF under project P–36433: “Social genomics in Late Antique and Early-Medieval Societies”’. Ezgi Altınışık was funded by TÜBİTAK Project No: 121Z485. Lastly, we would like to extend our appreciation to Irene Mena for creating the illustration on sedaDNA workflow (see Fig. 1).

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Archaeology meets Environmental Genomics: implementing sedaDNA in the study of the human past

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Abstract

Figures

1. Introduction

2. Current approaches and applications of sedaDNA

2.1. Sources

2.2. Workflow

2.2.1. Sampling

2.2.2. Data generation: Laboratory process

2.2.3. Data processing: Bioinformatics

2.3. Application of sedaDNA in paleoenvironmental studies & archaeology

3. Added Value of sedaDNA in Archaeology

3.1. What did they eat?

3.2. How was their health?

3.3. How did they domesticate fauna and flora?

3.4. What was their environment?

3.5. Who were they and where did they come from?

3.6. Summary: potential and position of sedaDNA in future archaeology

4. Towards a research agenda for sedaDNA in Archaeology

5. Conclusion

Declarations

Acknowledgements

References

Additional Declarations

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