Plant remains

Text by Ingrid Bertin, Jonica Doliente, Oya Inanli, Alice Cao, and Deborah Roversi.

Header image from J. Dunne.

Macrofossils (>0.1-0.2 mm) are the major group of plant remains from archaeological contexts. These are woods, roots and tubers, fruits and seeds (e.g. cereals, pulses, and oil crops), fungi and mosses, fibres, and plant materials modified into tools i.e. baskets, and textiles. They are mostly found in waterlogged sites, food storage rooms (e.g. silos and warehouses), wooden containers (e.g. barrels and textile packings), kitchen tools, farming implements, weapons, and their hilts. The analysis of biomolecules - DNA, proteins, and lipids found from these plant remains has the potential to provide detailed information on (1) past diets (Lee-Thorp et al., 2012); (2) culinary practices (Colonese et al., 2017); (3) plant domestication (Larsen et al., 2019); (Ramos-Madrigal et al., 2019); (Kabukcu et al., 2021), (4) plant management (Sadori, Zanchetta and Giardini, 2008; Cortese et al., 2022); and (5) provenance (This, Lacombe and Thomas, 2006). The sampling is currently limited to destructive methods, however, a small quantity of the sample is enough for biomolecular analysis. The challenges that researchers have faced are the contamination and poor preservation of the sample mainly due to detrimental environmental conditions, such as high temperature or water exposure (Briggs, 2020). Aside from the abovementioned challenges, poor sampling strategy should be avoided as it can result in a lack of representativeness and interpretability of plant remains.

In general, the mode of sampling depends on the research objectives, the type of remains, and the type of site. There are various sampling methods - total, random, judgmental, horizontal, and vertical sampling - that can be used and each of them has its own pros and cons. It is then necessary for researchers to be knowledgeable about these sampling methods. In order to overcome these challenges, the sampling has to be carried out under controlled conditions and a suitable sampling strategy must be used. Therefore, we suggest the following protocol for biomolecular analysis of plant remains.

Infographic: Plant Remains 101

Sampling

Before sampling

1- Decide on the sampling strategy:

a. Preliminary survey of the archaeological site

b. Establish the best sampling strategy with a specialist

c. Plan times and modes of collection

2- Be sure to have the required equipment listed (see the table below):

Wear nitrile (not latex) gloves during all handling. Organic molecules derived from skin, as well as latex, can contaminate samples and interfere with downstream analysis. Therefore, change them if you touch uncleaned surfaces or your face.

Carefully clean the trowels and digging tools immediately before collecting each new sample.

Sample one sediment layer at a time where applicable.

Avoid blowing, washing or any liquid exposure to plant remains.

Recording and photography

The following information should be recorded where possible:

The artefact’s site and location where the object was found if known
The sample type of plant remains: e.g. wooden objects/fragments, seeds, fruits
The state of the sample: e.g., mineralised, wet, immersed in water, charred, dried
Take notes on the dimensions/outer appearance of the plant remain and any observations.
Weight of the subsample obtained (weigh on-site or in the lab).

Sample Photography:

Place the plant remain, scale bar and sample label on a clean, plain background. If the object is particularly fragile, place a foam mat.
Photograph the plant remains on all sides when they were discovered in the archaeological site (it is important for keeping a track of its burial environment).
When sampling is complete in the laboratory, re-photograph the object with a scale bar and label it. It is important to make a record of sampling.

Sampling and handling on site

Collect samples in the least destructive way (ideally following the instructions of archaeologists and restorers)
1. If samples are dispersed in sediments, take bulk material (1-2 kg) from the stratigraphical layers.
2. If samples are inside vases/ceramics, collect the ceramic. Sampling will be done by a specialist in the lab. If possible, take samples from the base and the extremities (rim-neck) of the container.
3. If found in silos or warehouses, collect samples of a standard size from all layers (statistical sampling).

Minimum amount of material required
1. For DNA : 5-25 mg
2. For proteomic : 5-20 mg
3. For lipidomic : ≥ 5 mg
4. For isotopes : ≥ 1 mg

Place samples in sterile tubes (glass or plastic), plastic bags, or rigid containers. Be aware that the type of packaging can lead to contamination (e.g. polymers from plastic in lipids studies).
1. If wet, keep the samples wet.
2. If immersed in water, place samples in rigid containers or plastic bags with water inside. Keep them in a cold place (cooler, fridge)
3. If charred, wrap it in aluminum foil first before placing it in the container.

Label them (recording date, site, layer, and archaeological unit)

Record and sketch in your notebook any other detail that may be useful (e.g., preservation state, location in the archaeological structure, and dimensions)

Storage

Open the collected samples only in a sterile laboratory.

If the weighing of samples is not possible during sampling, weigh the subsamples when back in the laboratory.

Samples can be shipped to different locations immediately after sampling without ice blocks. If longer storage is needed, store the samples in a cold, dry place.

Recommendation: If the samples for DNA need to be stored for a longer period (weeks or months), the ideal preservation method is freezing. Please note that repeated freezing/thawing cycles will negatively impact biomolecules. For lipidomic studies, keep the samples in clean (ideally combusted) glass vials or aluminum foil, preferably in cold conditions. Samples can be then stored in cloth bags, sterile containers, or unused plastic bags. If stored in plastic, monitor the humidity and integrity of the aluminum foil. For proteins, keep the sample in the fridge and in water if they are waterlogged, otherwise, samples can be stored at room temperature prior to biomolecular analyses. In general, cold conditions are preferable.

Limitations

Consolidation practices: Preservatives overlay with archaeological biomarkers and are not always totally reversible. It is important to avoid consolidants made out of animal or vegetal (e.g. beeswax) in organic artefacts as it introduces a modern contamination of biomolecules. Polymers can also hide the biomarker signals in the mass spectrometer and prevent biomolecules identification (Rageot, 2015).

When possible, select non restored samples for biomolecular analyses. If the object needs to be consolidated, sample it before to have reliable samples that can be used for a range of analysis.

If the analyses are performed on old discoveries, it is important to know the post excavation history of the object.

Degradation: When an inappropriate sampling strategy is employed, fragile plant remains can be destroyed due to overly harsh cleaning strategies. When food crusts or plant remains are present in a container or ceramic, they should not be washed; this applies for any type of residue.

The conservation of biomolecules depends on the burial environment, the age of the objects, the temperature, the humidity and so on. Therefore, it is very important to have appropriate sample recordings for interpreting the analysis results (see Recording and Photography).

It might be useful to first select a representative number of samples to screen the preservation of biomolecules including control samples.

Contaminations: Take a sample of soil sediment surrounding the plant remains/artefacts to identify contamination due to the soil burial environment (mainly for lipids & DNA).

Pay attention to possible contaminations due to handling in the site (e.g. sunscreen, lotion or finger lipids) (Whelton et al., 2021).

For DNA & proteomics, it is best to avoid wearing animal derived clothes (leather, wool, silk) or latex gloves while handling the object. In addition, human exogenous proteins can be recovered in the samples as keratin or dermcidin (Briggs, 2020); (Hendy et al., 2018).