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Text by Tabea Koch & Anna White. Header image by Theis Jensen.
Organic matter which is usually derived from living animal and plant organisms, can sometimes be found in the archaeological record as an artefact in itself. This means that instead of adhering to or being absorbed by an external object (e. g. ceramic, bone, stone, among others), organic matter constitutes the substrate. For example, organic lumps of variable size were found in multiple contexts from the Mesolithic to the Iron Age (Binder et al., 1990; Hayek et al., 1990; Aveling and Heron, 1998; Regert et al., 2000, 2003; Peche-Quilichini et al., 2017; Rageot et al., 2021). Through lipid analysis, these artefacts were identified as birch tar. In some cases, additives such as animal fats, plant oils or beeswax could also be detected. Similar lumps were discovered bearing tooth imprints (Heron et al., 1989; Aveling, 1997; Aveling and Heron, 1999; Jensen et al., 2019), and are therefore called chewing gums. These can bear traces of beeswax or fats/oils (Van Gijn and Boon, 2006) that could have been added intentionally. DNA analyses of such chewing gums showed that DNA can be preserved within these materials and can be analysed to provide genetic information on the substance itself as well as the person who chewed the gum (Jensen et al., 2019).
Sampling
Sampling
The preservation of such organic substrates is dependent on the environmental context of the archaeological site and can be influenced by climate, precipitation, soil-pH and bacterial activity. If organic substrates are recovered during an excavation, special attention must be given when handling the object. When possible, the direct handling of the object with bare hands (or any skin contact) should be avoided. Modern contamination of fatty acids can cloud the molecular profile of the sample (for lipid analysis) and can leave traces of excavator's DNA. Precise suggestions for adequate sampling of organic substrates in regards to lipid and DNA analysis are given in Table 1.
Table 1. Guidelines for sampling of organic substrates.
Storage
Storage
Organic substates should be stored in adequant containers after excavation, prior to analysis. For DNA analyses, the organic samples can be stored in sealable plastic bags. However, for lipid analyses, the samples should preferably be stored either wrapped in aluminium foil (careful: kitchen foil often has one plastified side, this should be avoided), but best put into glas vials to avoid any contamination with modern plastifiers. The glas vials can be closed with plastic lids, if these are not in direct contact with the samples.
Applications
Applications
Lipids
The majority of studies with the objective to chemically characterise adhesive samples, especially in recent studies, make use of Gas-Chromatography/ Mass-Spectrometry. The method is destructive and requires sample preparation. Prior to analysis, the archaeological sample needs to be ground and extracted via solvent extraction and is then derivatised. The solution can then be directly injected into the gas chromatograph. The method allows a separation (chromatography) of the different molecules and their identification (mass spectrometry). Previous studies have, as a consequence, been able to identify the plant resources used for adhesive manufacture and intentional mixtures of plant resins, animal fats, and waxes, as well as determine the degree of anthropogenic transformation and alteration.
DNA
In resinous substances, DNA from different origins can be found. An example is birch bark tar, which has been chewed in the past. In these tar pieces, people have left DNA behind from the plants and animals they have been eating and their human- associated oral microbiome (Jensen et al., 2019; Kashuba et al., 2019). In one study, Jensen et al. (2019) were able to identify that the birch bark tar was chewed by a female who likely had dark skin, dark brown hair, and blue eyes. Also, enough DNA was present to study her ancestry and determine that she was lactase non-persistent. All of this information combined shows the value and the wealth of information that aDNA analysis can provide. By determining the origin of the DNA we are able to provide an insight into the use of artefacts and the people that used them. The DNA obtained from ancient artefacts can provide snapshots of past populations, their diets and associated microbiome.
Limitations
Limitations
Lipids
Organic lumps resemble soil aggregates and may be destroyed in routine water sieving or washed off in the routine cleaning of lithic and bone artefacts prior to storage. Even if favourable preservation conditions are present, post-excavation conservation practices, such as use of PEG on organic matter, pose additional analytic challenges. The modern biomarkers associated with conservative and consolidant substances often overlay the (pre)historic resin/tar markers, obfuscating molecular identification (M. Rageot 2015). When directly handling the artefacts, sunscreen or other creams can leave lipid markers that might also overlay the molecular signature in the sample (Whelton et al. 2021).
DNA
DNA that degrades over time. The nucleotides that make up the DNA may change, especially at the ends of the DNA molecules (Dabney, Meyer, and Pääbo 2013). DNA will also fragment into smaller pieces due to hydraulic depurination leading to single stranded breaks. This makes DNA harder to analyse the older it gets. Therefore, the analysis of DNA is limited by its age, with the maximum age of aDNA analysed so far being 1.3 to 1.6 million years old (van der Valk et al. 2021). However, these C to T changes and small fragments are characteristic for aDNA and can thus be used to differentiate between modern and ancient DNA, authenticating the presence of aDNA (Krause et al. 2010).
DNA analysis is further limited by the quantity of endogenous DNA in the sample, which refers to DNA from the organism of interest. This is influenced by the preservation of the sample, which is affected by factors such as the presence of water, higher temperatures, and soil pH. Low DNA content can cause additional challenges due to contamination from nearby (un)related sources of DNA. Contamination may occur in the depositional environment, during excavation, as well as in subsequent sample preparation and analysis. DNA is largely present in all environments, therefore modern DNA can easily contaminate the sample if proper care is not taken. This will result in DNA sequencing being less efficient and therefore more costly. These aspects may limit the analysis of DNA from organic samples.
Further reading
Further reading
Aveling, E., 1997. Chew, chew, that ancient chewing gum. British Archaeology. 21.
Aveling, E., Heron, C., 1998. Identification of Birch Bark Tar at the Mesolithic Site of Star Carr. Ancient Biomolecules. 2.
Aveling, E., Heron, C.P., 1999. Chewing tar in the early Holocene: an archaeological and ethnographic evaluation. Antiquity. 73, 579–584.
Binder, D., Benoist, F., Bourgeois, G., Vitry, C., 1990. Identification de brai de bouleau (Betula) dans le Néolithique de Giribaldi (Nice, France) par la spectrométrie de masse. ArchéoSciences, revue d’Archéométrie. 37–42.
Dabney, Jesse, Matthias Meyer, and Svante Pääbo. 2013. “Ancient DNA Damage.” Cold Spring Harbor Perspectives in Biology 5 (7).
Hayek, E.W.H., Krenmayr, P., Lohninger, H., 1990. Identification of Archaeological and Recent Wood Tar Pitches Using Gas Chromatography/Mass Spectrometry and Pattern Recognition. Analytical Chemistry. 62, 2038–2043.
Heron, C., Evershed, R.P., Chapman, B., Pollard, A.M., 1989. Glue, disinfectant and “chewing gum” in prehistory. Oxford: Oxbow, Bradford.
Jensen, T.Z.T., Niemann, J., Iversen, K.H., Fotakis, A.K., Gopalakrishnan, S., Vågene, Å.J., Pedersen, M.W., Sinding, M.-H.S., Ellegaard, M.R., Allentoft, M.E., Lanigan, L.T., Taurozzi, A.J., Nielsen, S.H., Dee, M.W., Mortensen, M.N., Christensen, M.C., Sørensen, S.A., Collins, M.J., Gilbert, M.T.P., Sikora, M., Rasmussen, S., Schroeder, H., 2019. A 5700 year-old human genome and oral microbiome from chewed birch pitch. Nat Commun. 10, 5520.
Kashuba, N., Kırdök, E., Damlien, H., Manninen, M.A., Nordqvist, B., Persson, P., Götherström, A., 2019. Ancient DNA from mastics solidifies connection between material culture and genetics of mesolithic hunter–gatherers in Scandinavia. Communications Biology. 2.
Krause, Johannes, Adrian W. Briggs, Martin Kircher, Tomislav Maricic, Nicolas Zwyns, Anatoli Derevianko, and Svante Pääbo. 2010. “A Complete mtDNA Genome of an Early Modern Human from Kostenki, Russia.” Current Biology: CB 20 (3): 231–36.
Peche-Quilichini, K., Rageot, M., Regert, M., 2017. Systèmes de réparation, de réutilisation et de recyclage des vaisselles céramiquesprotohistoriques de Corse. Bulletin de la Société préhistorique française. 114, 137–166.
Rageot, M., 2015. Les substances naturelles en Méditerranée nord-occidentale (VIe-Ie Millénaire BCE) : chimie et archéologie des matériaux exploités pour leurs propriétés adhésives et hydrophobes (Doctorat). Université Nice Sophia Antipolis.
Rageot, M., Lepère, C., Henry, A., Binder, D., Davtian, G., Filippi, J.-J., Fernandez, X., Guilaine, J., Jallet, F., Radi, G., Thirault, E., Terradas, X., Regert, M., 2021. Management systems of adhesive materials throughout the Neolithic in the North-West Mediterranean. Journal of Archaeological Science. 126, 105309.
Regert, M., Garnier, N., Binder, D., Pétrequin, P., 2000. Les adhésifs néolithiques : quels matériaux utilisés, quelles techniques de production dans quel contexte social ? L’exemple des adhésifs des sites de Giribaldi et de Chalain. pp. 585–604.
Regert, M., Vacher, S., Moulherat, C., Decavallas, O., 2003. Adhesive Production and Pottery Function During the Iron Age at the Site of Grand Aunay (Sarthe, France)*. Archaeometry. 45, 101–120.
Van Gijn, A., Boon, J., 2006. Birch bark tar. In: Kooijmans, L.P.L., Jongste, P.F.B. (Eds.), Analecta Praehistorica Leidensia 37/38 / Schipluiden : A Neolithic Settlement on the Dutch North Sea Coast c. 3500 CAL BC. Leiden University, pp. 262–266.
Valk, Tom van der, Patrícia Pečnerová, David Díez-Del-Molino, Anders Bergstrˆm, Jonas Oppenheimer, Stefanie Hartmann, Georgios Xenikoudakis, et al. 2021. “Million-Year-Old DNA Sheds Light on the Genomic History of Mammoths.” Nature 591 (7849): 265–69. https://doi.org/10.1038/s41586-021- 03224-9.
Whelton, H.L., Hammann, S., Cramp, L.J.E., Dunne, J., Roffet-Salque, M., Evershed, R.P., 2021. A call for caution in the analysis of lipids and other small biomolecules from archaeological contexts. Journal of Archaeological Science. 132, 105397.
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