UDK 903'i2/'i5(4/5)"633/634">575-i7; 903'i2/'i5(4/5)"633/634">574-9i Documenta Praehistorica XXXV (2008) Early herding practices revealed through organic residue analysis of pottery from the early Neolithic rock shelter of Mala Triglavca, Slovenia Lucija {oberl1, Andreja ?ibrat Gaspari;2, Mihael Budja2 and Richard P. Evershed1* 1 Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol, UK 2 Department of Archaeology, Faculty of Arts, University of Ljubljana, Ljubljana, SI *author for correspondence> r.p.evershed@bristol.ac.uk ABSTRACT - A collection of pottery from the early Neolithic site of Mala Triglavca was analysed with the aim of obtaining insights into vessel use and early animal domestication and husbandry prac- tices in the Adriatic region. Total lipid extracts were submitted to gas chromatography (GC), GC-mass spectrometry (GC-MS) and GC-combustion-isotope ratio MS (GC-C-IRMS) in order to obtain molecu- lar and stable carbon isotope signatures as the basis for determining the nature and origins of the residues. The extracts were dominated by degraded animal fats. The majority (70%) of the total lipid extracts displayed intact triacylglycerol distributions attributable to ruminant adipose and dairy fats, which were subsequently confirmed through Cm and C1&0 fatty acid S13C values. IZVLEČEK - Izbor keramičnih vzorcev iz zgodnje neolitskega najdišča Mala Triglavca je bil analiziran z namenom, da bi pridobili dodatne informacije o uporabi keramičnih posod ter značaju zgodnje ži- vinoreje na Jadranskem prostoru. Lipidne ekstrakte vzorcev smo analizirali s pomočjo plinske kroma- tografije (GC), plinske kromatografije sklopljene z masno spektrometrijo (GC/MS) in plinske kroma- tografije sklopljene s sežigno masno spektrometrijo razmerij izotopov (GC-C-IRMS). V lipidnih eks- traktih so prevladovale živalske maščobe. V večini (70%) lipidnih ekstraktov so bile prisotne nespre- menjene trigliceridne distribucije, ki jih lahko pripišemo tolsčnim ter mlečnim maščobam prežveko- valcev. Lipidni izvor je bil nadalje potrjen z S13C vrednostmi prostih maščobnih kislin C160 in C1&0. KEY WORDS - Neolithic, Adriatic, organic residues, fatty acyl lipids, S13C values Introduction Organic residues survive in two principal forms in association with archaeological pottery, namely as: (i) surface residues appearing as visible residues on the exterior or interior of vessels, and (ii) as ab- sorbed residues preserved within the vessel wall; in- visible to the naked eye. The first class of residues, occurs on the exterior surfaces of and cor- respond to either sooting derived from heating on a fire or seemingly rather rare instances of applied decorations (Urem-Kotsou et al. 2002; Connan et al. 2004). Together with exterior sooting, the inte- rior surface residues are probably the group of resi- dues most familiar to pottery analysts. These are often carbonised, and presumed to be residues of 'cooking' failures, while post-firing treatments are also seen, particularly as copious linings associated with Roman amphorae (Beck et al. 1989). Absorbed residues are by far the most common in pottery and probably the most widely occurring residue type. Analyses performed to date suggest that absorbed organic residues survive in >80% of domestic cook- ing pottery assemblages worldwide. Lipids residues of cooking and the processing of other organic commodities have been found to sur- vive in archaeological pottery vessels as components of surface and absorbed residues for several millen- nia. The most successful analytical approaches in- volve solvent extraction, then using a combination of instrumental analytical techniques, including: high temperature-gas chromatography (HTGC), GC/mass spectrometry (GC/MS; Evershed et al. 1990) and GC- combustion-isotope ratio MS (GC-C-IRMS; Evershed et al. 1994), to identify and quantify the components of the lipid extracts of such residues. Characterisa- tion of lipid extracts to commodity type is only pos- sible through detailed knowledge of diagnostic com- pounds and their associated degradation products formed during vessel use and/or burial. An increas- ing range of commodities is being detected in pot- tery vessels, including animals products (e.g. Ever- shed et al. 1992; Copley et al. 2003), leafy vegeta- bles (Evershed et al. 1991; Evershed et al. 1994), specific plant oils (Condamin et al. 1976; Copley et al. 2005a) and beeswax (Heron et al. 1994; Ever- shed et al. 1997; Regert et al. 2001). Animal fats are by far the most common residue identified from archaeological pottery, characterised by high abundances of free fatty acids, particularly palmitic (Ci6:o) and stearic acid (Ci8:o). Triacylglyce- rols (TAGs) are the major constituents of modern animal fats, however, they are degraded to diacylgly- cerols (DAGs), monoacylglycerols (MAGs) and free fatty acids during vessel use and burial, such that in archaeological pottery the free fatty acids tend to predominate. This has been observed in numerous pottery vessels (Evershed et al. 2001) and verified through laboratory degradation experiments (e.g. Charters et al. 1997; Dudd and Evershed 1998; Evershed 2008). Precise assigning of the origins of animal fats is only possible through the use of com- pound-specific stable carbon isotope analysis. GC-C- IRMS allows the carbon stable isotope (813C) values of individual compounds (within a mixture) to be determined. It has been previously observed that the 513C values for the principal fatty acids (Ci6:o and Ci8:o) are crucial in distinguishing between diffe- rent animal fats, e.g. ruminant and non-ruminant adipose fats and dairy fats (Evershed et al. 1997a, Dudd and Evershed 1998), as well as in the identi- fication of the mixing of commodities (Evershed et al. 1999; Copley et al. 2001). Recently it has been demonstrated that dairy products were important commodities in Early Neolithic at various archaeolo- gical sites throughout Europe and Near East, as illu- strated through the persistence of dairy fats in ar- chaeological pottery vessels (Copley et al. 2003; 2005b; Evershed et al. 2008). The aim of this investigation was to apply organic residue analysis to prehistoric pottery from the Neo- lithic rock shelter Mala Triglavca in order to deter- mine the nature and origin of preserved lipids and thereby provide new insights into food preparation and consumption of the inhabitants. As a conse- quence of the wider interest in the use of rock shel- ters in the early Neolithic, the organic analysis of the pottery from this site offers an important opportu- nity to explore aspects of animal husbandry, particu- larly dairying. Sites and samples The Neolithic rock shelter site of Mala Triglavca is situated in the Dinaric karst in south-western Slo- venia. There is evidence that the site has been con- tinuously occupied from the Mesolithic until the Mid- dle ages. Rock shelter sites in the region have most- ly been interpreted in two different ways: (i) as sea- sonal camps for hunters/shepherds, or (ii) as places for long-term settlement. Archaeozoological remains discovered on the site mainly belong to domestica- ted animals (cattle, sheep, goat, dog) as well as wild animals (wild boar, red deer, roe deer). A total of 36 potsherds were selected for analysis from earliest Neolithic phase. Materials and methods Lipid analyses were performed using established pro- tocols which are described in detail in earlier publi- cations (Evershed et al. 1990; Charters et al. 1993b). Briefly, analyses proceeded as follows: Solvent extraction of lipid residues Lipid analyses of potsherds involved taking c. 2g samples from area of the the sherd that had been surface-cleaned using a modelling drill to remove any exogenous lipids (e.g. soil or finger lipids due to handling). The sub-samples were then ground to a fine powder, accurately weighed and a known amount (20mg) of internal standard (n-tetratriacon- tane) added, to enable determination of the lipid concentration. The lipids were extracted with a mix- ture of chloroform and methanol (2:1 v/v). Follo- wing separation from the ground potsherd the sol- vent was evaporated under a gentle stream of nitro- gen to obtain the total lipid extract (TLE). Portions (generally one third aliquots) of the extracts were then trimethylsilylated and submitted directly to ana- lysis by HTGC. Where necessary combined GC/MS analyses were also performed on trimethylsilylated aliquots of the lipid extracts to enable the elucidation of structures of components not identifiable on the basis of HTGC retention time alone. Preparation of trimethylsilyl derivatives Portions of the total lipid extracts were derivatised using jV,0-bis(trimethylsilyl)trifluoroacetamide (40^1; 70°C; 60 min; Sigma-Aldrich Company Ltd., Gilling- ham, UK) and analysed by HTGC and GC-MS. Saponification of total lipid extracts Methanolic sodium hydroxide (5% v/v) was added to the TLE and heated at 70°C for 1 h. Following neutralisation, lipids were extracted into chloroform and the solvent reduced under gentle stream of ni- trogen. Preparation of methyl ester derivatives (FAMEs) FAMEs were prepared by reaction with BF3-metha- nol (14% w/v; 100ml; Sigma-Aldrich, Gillingham, UK) at 70°C for 1 h. The methyl ester derivatives were extracted with chloroform and the solvent re- moved under nitrogen. FAMEs were re-dissolved in hexane for analysis by GC and GC-C-IRMS. MAGs, DAGs and TAGs, which are indicative of de- graded animal fat were detected in 7 of the TLEs to- gether with relatively high abundances of the pal- mitic (C160) and stearic (C180) free fatty acids, which as discussed above, are the terminal products of TAG hydrolysis. Previous work has shown that the TAG distributions can be linked to different sources, thereby allowing preliminary differentiation of fats of the two major classes of domestic animals (rumi- nant and non-ruminant) and ruminant dairy fats. However, laboratory experiments have shown that TAG distributions can be skewed by degradation; the wide TAG distribution characteristic of fresh rumi- nant dairy fat is considerably narrowed, and thus comes to resemble the narrower distribution seen in ruminant adipose fat (Dudd et al. 1998; Dudd and Evershed 1998). Therefore conclusions drawn from TAG distributions have to be made with caution. The TAG distributions preserved in the extracts from the Mala Triglavca sherds are shown in Figure 3. Results The HTGC and GC/MS analyses serve to quantify and identify compounds in the TLE, revealing the possible presence of: (i) animal fat or plant oil, and/ or (ii) plant epicuticular waxes, and/or (iii) beeswax or other sealants, and/or (iv) mid-chain ketones that indicate that the vessel has been heated (Evershed et al. 1995; Raven et al. 1997). Further analyses by GC-C-IRMS analyses can distinguish between rumi- nant and non-ruminant adipose fats and dairy fats by investigating the 613C16 ö and 613C18 ö values. Tab- le 1 lists the sample designations, the concentrations of lipids detected and the assignments of the broad commodity groups present in individual sherds based on the molecular and isotopic compositions of the components of the TLEs. Ten of the sherds (28%) yielded significant concentrations of lipid (i.e. >5mgg-1) sufficient for further analysis by GC-MS and GC-C-IRMS. Figure 1 shows a typical partial gas chromatogram for the TLE of sample 08MT, revealing the presence of free fatty acids, with high abundances of C160 and C18:0 components, mono-, di- and triacylglycerols (MAGs, DAGs, TAGs). The chromatogram also shows presence of odd carbon number saturated fatty acids, iso- and anteiso-branched odd carbon number fatty acids (C15:0br, C170br), which may indicate a ruminant source (Mottram et al. 1999; Evershed et al. 2001). Traces of wax esters were also present eluting in the region of the TAGs. The total lipid extracts (TLEs) of samples 08MT, 18MT, 78MT, 79MT and 159MT displayed relatively broad TAG distributions with acyl carbon number range of C44 to C54, maximising at C50/C52. Such dis- tributions are characteristic of reference ruminant adipose fat, or degraded milk fat. In contrast, the ex- tract of 13MT displayed quite a narrow TAG distribu- Fig. 1. Partial HTGC profile of the trimethylsilylated total lipid extract from sample 08MT, showing the distribution of components characteristic of degra- ded animal fat. Key: Cx.0 are saturated free fatty acids of carbon length x, br stand for branched fatty acids, IS is the internal standard (C34 n-alka- ne). MAGs are monoacylglycerols; DAGs are dia- cylglycerols; TAGs are triacylglycerols. Fig. 2. Histograms showing the typical acyl carbon number distributions expected for triacylglycerols deriving from degraded lipid residues obtained from: (a) ruminant dairy fat, (b) ruminant adipose, and (c) pig adipose (Berstan 2002). tion, with an acyl carbon number range of C50 to C54, maximising at C52, which is identical to reference ru- minant adipose fats (Fig. 2). The 8 TLEs that yielded appreciable lipid concentra- tions analysed further by GC-C-IRMS to determine the 813C values for the major fatty acids (C1&0 and C18:0); these values are plotted in Figure 4. The 813C values obtained for modern reference animal fats from the major domesticated animals exploited in prehistory are grouped within confidence ellipses, onto which the values from archaeological pottery are plotted. The 813C values for the C18:0 fatty acid are more depleted in milk fats than in ruminant adi- pose fats, thus enabling distinctions to be drawn be- tween milk and adipose fats from ruminant animals (Dudd and Evershed 1998). This is witnessed in the c. 2.5 %o shift between centroids of the reference ru- minant adipose fat and ruminant dairy fat ellipses. The less depleted 813C values seen for the fatty acids in non-ruminant fats compared to equivalent compo- nents in ruminant fat are to be due to differences in diet and in the metabolic and biochemical processes involved in the formation of body fats in ruminant and non-ruminant animals. The 813C values for the C16« and C18:0 fatty acids from 18MT, 79MT, 87MT, 88MT and 161MT plot within or adjacent to the dairy fat reference confi- dence ellipse, while that from 75MT plots within the ruminant adipose reference fat ellipse. Values from 08MT and 13MT plot between the porcine adipose fat and ruminant adipose fat ellipses. These 813C va- lues are most likely indicative of mixing of commodi- ties in the vessels, which may have occurred through multiple use of the vessel or through the contempo- raneous mixing of animal products. The modern fats used to construct the reference iso- tope plot were reared on a strict C3 diet of fodders and cereals. The slight displacement of some of the 813C values to the right of the mixing curves may be Fig. 3. The distributions of TAGs detected in the Mala Triglavca total lipid extracts. Lab. sample no. Lipid concentration (mg g-1) Lipids detected 813CI6:O ± 0.3 (%o) S13Ci8:O ± 0.3 (%o) Predominant commodity type 08MT 173.35 FA (16<18; 14, 15, 15br,17, 17br, 19, 20), MAG, DAG, TAG, WE -26.5 -29.9 mixture of animal fats 09MT 1.90 n/a n/a n/a n/a 11MT 0.81 n/a n/a n/a n/a 12MT 0.42 n/a n/a n/a n/a 13MT 11.56 FA (16<18), MAG, A, OH, DAG, WE, TAG -26.7 -28.8 ruminant adipose fat 14MT 0.24 n/a n/a n/a n/a 15MT 0.00 n/a n/a n/a n/a 16MT 0.00 n/a n/a n/a n/a 17MT 1.01 n/a n/a n/a n/a 18MT 88.09 FA (16<18; 14, 15, 15br,17, 17br, 18:1, 19, 20, 21, 22, 23, 24), MAG, DAG, TAG -27.7 -33.3 dairy fat 75MT 90.54 FA (16<18; 14, 15, 15br,17, 17br, 18:1, 19, 20), MAG, DAG, TAG -29.0 -31.5 ruminant adipose fat 76MT 2.92 n/a n/a n/a n/a 77MT 0.00 n/a n/a n/a n/a 78MT 2.58 n/a n/a n/a n/a 79MT 27 23 FA (16>18; 14,17, 17br, 18:1, 19, 20), MAG, OH, A, DAG, TAG, P -27.8 -32.9 dairy fat 80MT 3.45 n/a n/a n/a n/a 81MT 1.34 n/a n/a n/a n/a 82MT 0.00 n/a n/a n/a n/a 83MT 3.12 n/a n/a n/a n/a 84MT 1.10 n/a n/a n/a n/a 85MT 1.34 n/a n/a n/a n/a 86MT 1.38 n/a n/a n/a n/a 87MT 21.93 FA (16>18; 14,17, 17br, 18:1, 19, 20), OH, A, P -27.3 -32.2 dairy fat 88MT 9.93 FA (16>18; 14, 20), P -27.0 -31.9 dairy fat 89MT 0.00 n/a n/a n/a n/a 156MT 10.06 FA (16>18; 18:1), P n/a n/a | 157MT 4.06 n/a n/a n/a n/a 158MT 0.98 n/a n/a n/a n/a 159MT 12.65 FA (16>18), MAG, DAG, TAG, P n/a n/a dairy fat 1 160MT 5.53 n/a n/a n/a n/a 161MT 43.81 FA (16<18), MAG, DAG -29.5 -34.1 dairy fat 162MT 2.77 n/a n/a n/a n/a 163MT 4.02 n/a n/a n/a n/a 164MT 2.47 n/a n/a n/a n/a 165MT 1.53 n/a n/a n/a n/a 166MT 4.67 n/a n/a n/a n/a Tab. 1. Summary of the results of the organic residue analyses of Mala Triglavca early Neolithic pot- sherds. Key: FA refers to free fatty acids, MAG to monoacylglycerols; DAG to diacylglycerols; TAG to triacylglyc- erols; A are n-alkanes, K are mid chain ketones, WE are wax esters, P are plasticizers and nd = none detected. Annotation 18:1 refers to the level of unsaturation and 17br to branched free fatty acids. 1 1 Pig adipose fats _ 1 » 1 -i edominantly C3 diet Increasing marine diet 1 Ruminant adipose fats ' •13MT • 75MT Ruminant dairy fats • 08MT > 161MT • 87MT 79MT* 88MT 18MT* 1 1 -34 -32 513C16:0(%o) -30 -28 -26 -24 -22 > O -3 -7 -40 -30 513C -20 16:0 (%o) Fig. 5. A plot showing the difference between A13C values (Sl3Ci8.o - St3Ci6.o) and &3C values obtai- ned from the C16.0 fatty acids extracted from the Mala Triglavca potsherds. The ranges for the mo- dern reference fats are plotted to the left of the dia- gram. due to the fact that the animals in prehistory were reared on diets, which varied in d13C values compa- red to today's values today environmental influen- ces. D13C values (613Ci6 o - 513Ci8 o) are also a use- ful indicator of lipid origin where such variations exist. Figure 5 displays the D13C values plotted against S13C16 o values for the Mala Triglavca pot- sherd fatty acids. The ranges on the left side of the graph are from the modern reference fats. 1 1 1 1 1 1 1 1 1 1 ~~ Porcine adipose fat---- 1 f •13MT - - / •08MT - - Ruminant 75MT/ / #88MT adipose fat( —?87MT •J79MT 9l8MT — Ruminant / — _ dairy fat /16IMT/ 1,1, 1,1,1 - -22 -24 -26 00 -28 0 OO -30 0 0 -32 -34 -36 Fig. 6. A plot showing the correlation between success rate (which is number of TLEs with appreciable lipid concentration divided by to- tal number of samples analysed) and mean lipid concentration for Mala Triglavca and other regions with evidences for early Neolithic milk use. Fig. 4. Scatter plot showing the St3C values of C16.0 and C18.0 fatty acids prepared from total lipid extracts of Mala Triglavca potsherds. The values of modern reference fats are represented by confi- dence ellipses (1 standard deviation). Lines con- necting the ellipses represent theoretical &3C val- ues obtained through the mixing of these fats. Using Figure 5, it was possible to more securely attri- bute the Mala Triglavca residues to their potential lipid sources. Sample 13MT, which was plotted on Figure 4 in between the ruminant dairy and adipose reference ellipse, can now be more accurately attri- buted to the latter, together with 75MT. Unfortuna- tely, the same could not be achieved for TLE of sam- ple o8MT, which remains plotted on the boarder of two ranges and most likely the consequence of mix- ing of different types of fat during the pottery use. Discussion The lipid components of the orga- nic residues preserved in the early Neolithic vessels from Mala Triglav- ca displayed reasonable preserva- tion given their age, with appreci- able TLEs being detected in 28% of the sherds analysed. The high de- gree of preservation overall was also reflected in the survival of acyl- glycerol components (MAGs, DAGs and TAGs) in a significant propor- tion (70%) of TLEs. Although some- what later age the lipid residues from Mala Triglavca show similar rate of recovery and mean concen- trations to those observed in early Neolithic pottery from SE Europe, Turkey and Near East (Fig. 6; Evershed et al. 2008). Interestingly, dairy fats dominate the preserved lipids at Mala Tri- glavca, and display a mean lipid concentration of 15mgg-1, which is comparable to the concentration seen in pottery from the other regions where early Neolithic milk use has been demonstrated. The con- centrations and rate of recovery of lipid from British Neolithic pottery are both significantly higher than the more southerly located sites and likely reflect preservational differences related to climate and age (Copley et al. 2005b). Returning to the Mala Triglavca residues there is also a good correlation between the triacylglycerol distributions preserved and interpretations of rumi- nant dairy and adipose fats in pottery based upon stable carbon isotope values. None of the total lipid extracts contained porcine adipose fat, which agrees with the low percentages of pigs in faunal assem- blage from the site, which is dominated by small cat- tle and sheep/goat. The latter clearly correlates with the fat type detected in the pottery, although the fats from the different species cannot be separated. In- terestingly, Mlekuž has recently managed to partial- ly reconstruct herd structures using faunal remains from early Neolithic sites on the Adriatic coast. The earliest animal domestication and husbandry ap- pears to have involved exploitation of both animal meat as well as dairy products (Mlekuž 2006). Ana- lyses of absorbed lipid residues of pottery from Mala Triglavca confirm this interpretation - the Neolithic inhabitants of the site were using diverse domestica- ted animal products in every day food preparation and consumption. Since no mid-chain ketones were present in any of the extracts it appears that the ves- sels were not heated to high temperatures (>300° C) during use (Raven et al. 1997). In summary, the results obtained from lipid analy- ses of the Mala Triglavca pottery is consistent with on-going debate concerning the integration of ani- mal domestication into early farming as part of the Neolithisation process along the Adriatic coast. The results concur with recent findings from organic re- sidue analyses of Neolithic pottery from the SE Eu- rope and Near East, where it has been shown that the early use of dairy products dates back at least to the 7th millennium BC and rather than being a fixed package, likely developed in different ways and in different geographical regions (Evershed et al. 2008; Mlekuž et al. 2008). -ACKNOWLEDGEMENTS- This research was undertaken while LS was in receipt of a EU Leonardo da Vinci Scholarship. Drs R. Ber- stan and I. D. Bull are thanked for technical support. The NERC are thanked for mass spectrometry facili- ties. 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