Organic products in EIA pottery from Vix-Mont Lassois

Molecular biomarkers were preserved in most of the samples investigated (90/99). Interpretations were attempted only for samples containing ≥5μg/g of lipid (n = 85)[29, 30], with the exception of 5 samples whose overall lipid content was low but in which highly diagnostic biomarkers (miliacin; n = 2) and/or insoluble markers in the lipid fraction (short-chain carboxylic acids; n = 3) were identified in the absence of exogenous contamination (see S3 Text for control samples). Several molecular families were identified including fatty acids, n-alcohols, n-alkanes, long chain esters, triglycerides, terpenes and short-chain carboxylic compounds. These could result from over 10 natural organic products such as animal adipose fats, dairy products, beeswax, plant waxes and oils, resins, tars, grape products/wine, cereals and possibly a beverage produced from the fermentation of cereals (barley or millet) as revealed by the presence of bacteriohopanoids and supported by the archaeobotanical and use-wear records. Most of the organic substances identified were present in different combinations and within a broad range of pottery shapes and contexts. Interestingly, some of the substances identified were specific to and/or varied quantitatively according to particular vessel type and/or context (discussed below).

The presence of animal fats (n = 11), namely dairy products, ruminant adipose and porcine fats, was indicated by the distribution of saturated triglycerides (TAGs) containing up to 54 carbon atoms (n = 8), and by compound specific isotopic measurements (δ13C) of the palmitic and stearic fatty acids (n = 11) [25, 31](see S3 Text, S1 Table, S3–S5 Tables).

Long chain palmitic esters with an even number of carbon atoms ranging from C40 up to C50 were identified in a large number of vessels (n = 40) (S1 Table, S3–S5 Tables, and Fig 3). These, together with their hydrolysis products (palmitic acid and even-numbered n-alcohols comprising 22 to 34 carbon atoms), a series of odd-number n-alkanes (C 27 major), and often with saturated long chain even-numbered fatty acids (C 24:0 to C 28:0 ) are indicative of beeswax [32, 33]. The results obtained show an extensive exploitation of beehive products, where beeswax was identified in about 50% of the local pottery tested. Beeswax is a versatile material that can be used for a variety of purposes, including diet, body care, art and technology [32–35]. Furthermore, honey, which comprises mainly fast-decaying saccharides, is unlikely to survive over archaeological timescales. Its use could however be suggested by the presence of beeswax, which is difficult to filter off completely. The high quantities of beeswax identified and the relative ease with which honey can be fermented suggests mead as a potential fermented beverages. However, no direct chemical evidence exists to support this. Hence, the presence of mead is a possibility although it cannot be conclusively determined.

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larger image TIFF original image Download: Fig 3. Chromatogram showing molecular constituents of a mixture containing beeswax, plant wax, millet, bacteriohopanes and animal fat (δ13C and Δ13 signatures confirming a dairy fat) in local fine bowl from Champ Fossé (VIX-CF-015). Cx:y = fatty acid with x carbon atoms and y representing the number of unsaturations; Ax = n-alcohol, ax = n-alkanes and Wx = long chain esters with x carbon atoms. https://doi.org/10.1371/journal.pone.0218001.g003

Other long chain palmitic esters identified together with stearic and arachidic esters suggest plant waxes [36](S3 Text, S1–S5 Tables, and Fig 3) in 36 samples. These are plant by-products found in the leaves of various fruits, vegetables and cereals [36]. The botanical record attests to the possible consumption of oily plants, cereals (especially barley, millet, wheat and rye), legumes (lentil, ervil, pea, broad bean) and fruits [37, 38]. The identification of oleic acid (oleic acid ≥stearic acid; n = 20), linoleic acid (n = 12) and unsaturated TAGs (n = 3) (S3 Text) could suggest oily plants in the vessels from Vix-Mont Lassois. The high oleic to stearic acid ratios as well as the presence of unsaturated TAGs are extremely rare in archaeological samples due to decay mechanisms [29], and their identification has been associated with plant oil (31). The archaeobotanical record for local oily plants in South West Germany [37] shows the exploitation of linseed, opium poppy and camelina in Central Europe. The suite of unsaturated TAGs (54:3, 52:2 and 50:1) was identified in 3 samples (S1 Table, S3 Table and Fig 4). This TAG assemblage is present in various different plant oils. However, only a substance highly concentrated in these specific unsaturated TAGs and favourable preservation contexts will lead to their preservation and identification. Further analysis of modern reference oils was undertaken using the same analytical conditions and concentration range as in our archaeological samples. These tests showed that at least one plant oil identified at Vix-Mont Lassois does not appear to have been grown locally and is mostly consistent with a Mediterranean plant oil, particularly olive oil (Table A in S3 Text, [26]).

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larger image TIFF original image Download: Fig 4. Chromatogram showing molecular markers of plant oil, type olive, in an imported Attic amphora (VIX-PL-11A) from the plateau context. Cx:y = fatty acid with x carbon atoms and y representing the number of unsaturations; Ax = n-alcohol with x carbon atoms; DAG = diglycerides; TAG = triglycerides. Grey: Exogenous contamination (present in the control sample). https://doi.org/10.1371/journal.pone.0218001.g004

Miliacin, a triterpenoid marker of common millet [18, 27] (S1 Table, S3–S5 Tables, and Fig 3), was identified in more than 20% (n = 18) of the local vessels, indicating a significant consumption of this cereal. Millet can be consumed in various ways, as whole grains, porridge and cakes. However, most of these methods of consumption are unlikely to leave a chemical signal. Preparation of millet to produce porridge or a beverage, perhaps beer, is more likely to have led to the absorption of miliacin in the ceramics. The archaeobotanical record shows the presence of millet, wheat, rye and especially barley as the major cereals at Vix-Mont Lassois, and in other parts of the so-called Westhallstattkreis[37, 38]. The absence of wheat, barley and rye alkylresorcinols [39] from the pottery does not exclude these cereals from having been present in the vessels. Their absence could be due to the lower stability of these markers compared to miliacin, leading to an over-representation of millet.

Pinaceae resin/tar diterpenoids from the abietate family were detected in 26 vessels (S1 Table and S2 Table), and a Pinus origin could be specified for 6 of them due to the additional presence of pimarates and seco-abietates [28] (S3 Text). Products made from birch bark or birch sap were identified in 4 vessels through the identification of their major triterpenoid biomarkers (betulin and lupeol) [40, 41]. Additional degradation markers identified in another 3 pots show the presence of birch bark tar [42] (S3 Text).

Short-chain carboxylic acids including succinic, fumaric, malic and at times tartaric acid were identified in 20 vessels and suggest the presence of fruit products. Tartaric acid, identified in 16 vessels, is considered to be a biomarker for grape products/wine because of its higher concentration in grapes [23, 24, 43, 44] compared to other fruits available in Europe during the EIA (Table B in S3 Text, S2–S5 Tables). Grape wine consumed at Vix-Mont Lassois was probably imported from the Mediterranean area since the scant evidence of grape pips does not support the exploitation of the local wild vine. Unlike Mediterranean contexts, there is no evidence for winemaking (e.g. pressed grapes) in the Western Central European EIA [45–47].

A series of compounds characterised by a base peak at m/z 191 and molecular ions (M+.) 398 (C 29 H 50 ) and 412 (C 30 H 52 ), and occasionally 426 (C 31 H 54 ), 440 (C 32 H 56 ), 454 (C 33 H 58 ), 468 (C 34 H 60 ) and 482 (C 35 H 62 ), corresponding to hopanes, was identified in 39 vessels (S1–S3 Tables and Fig 5). These biomarkers have previously been associated with the fermentation of alcoholic beverages other than wine [48] (S3 Text), but they also occur in other substances such as bitumen [49]. Their presence at Vix-Mont Lassois was associated with a specific vessel techno-typology, and they were only identified in lipid extracts taken from the interior surface of vessels, while none were found in the corresponding exterior surfaces and sediment controls (see associated control samples in S3 Text). Our association of hopanoids with fermented beverages rather than bitumen or even plankton is supported by i) bacteriohopanoids were detected mostly in fine ceramics (absent in coarse vessels) whose shape indicates liquid consumption and which have consequently been attributed drinking and serving functions, ii) most of the bacteriohopanoid identifications were made in vessels recovered from the plateau area, which archaeological data associates with elite feasting practices and tableware and iii) use-wear analysis demonstrated pitting as a result of fermentation processes on the inner surface of experimental vessels [50], and similar evidence for pitting was observed on the neck of specific local handmade bottle-shaped vessels (Fig 6) in which bacteriohopanoids were also identified. At Vix-Mont Lassois, bacteriohopanoids were often found in association with plant oil/wax, pinaceae resin, millet and/or beeswax, showing the variety of fermented beverages which could have possibly been produced. The presence, more rarely, of animal fats and fruit/grape products may also be explained by vessel reutilisation. The more plausible interpretation is the production of beer from millet or barley, for which large quantities of botanical evidence was found on the plateau context at Vix-Mont Lassois[38].

A detailed description of the analytical results and interpretations are provided in S3 Text, and these are further contextualised in S1–S5 Tables.