Archaeol Anthropol Sci (2011) 3:271–280 DOI 10.1007/s12520-011-0066-z

ORIGINAL PAPER

Collagen fingerprinting of archaeological and teeth remains from Domuztepe, South Eastern Turkey

Mike Buckley & Sarah Whitcher Kansa

Received: 7 December 2010 /Accepted: 21 March 2011 /Published online: 7 May 2011 # Springer-Verlag 2011

Abstract We applied a recently developed method of collagen fingerprinting. We also show that the collagen collagen peptide mass fingerprinting for taxonomic identi- extracted from dentine is equally amenable to the proposed fication in faunal remains to 111 specimens from the methodology as bone collagen. site of Domuztepe in southeastern Turkey. Advances on the published technique allow us to move Keywords Collagen fingerprinting . MALDI . beyond the key domesticated fauna (sheep, goat, pig and Archaeological proteomics . ZooMS cattle) to include non-domesticates (deer and gazelle, as well as humans). As the economy of Domuztepe was dominated by domestic sheep and goats, a large Introduction number of the samples tested were morphologically indistinguishable sheep/goat remains. A number of samples The domestication and exploitation of represents represented elements that provide information on age (i.e., one of the critical stages in the changing human articulation mandibles) or sex (pelvis); thus, this represents the first with the living world (Barker et al. 1988). Sheep and goat major study to attempt to improve kill-off profiles using were two of the first animals domesticated in the Near East and, by the 8th millennium BC, became the primary animals exploited by Neolithic people in this region (Peters et al. 2005). However, as sheep and goat are Electronic supplementary material The online version of this article notoriously difficult to distinguish, particularly with young (doi:10.1007/s12520-011-0066-z) contains supplementary material, individuals or fragmented assemblages, zooarchaeologists which is available to authorized users. frequently aggregate them into a single ‘sheep/goat’ M. Buckley (*) category. While aggregation increases sample size and Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, provides a general impression of the age and sex compo- 131 Princess Street, sition of the combined sheep/goat population, it is much Manchester M1 7DN, UK e-mail: [email protected] more desirable to separate the sheep from the goats in order to reveal differential exploitation patterns that can be M. Buckley reflected in the harvest profiles of sheep and goats BioArCh, Department of Biology, Archaeology and Chemistry, separately. Though their bones are difficult to distinguish, University of York, Heslington, domestic sheep ( aries) and goats ( hircus) are York YO10 5YW, UK dissimilar in many ways: they have different feeding patterns and requirements with regards to husbandry S. W. Kansa practices, they tolerate different environmental conditions The Alexandria Archive Institute, 125 El Verano Way, and they provide a variety of products. The ability to San Francisco, CA 94127, USA distinguish the archaeological bones of sheep and goats, 272 Archaeol Anthropol Sci (2011) 3:271–280 thus, has profound implications for understanding prehis- peptides are fractionated using solid-phase extraction (SPE) toric economies and the differential utilisation of these C18 pipette tips to a lesser extent than the method used species by humans. previously (Buckley et al. 2010), resulting in a greater Zooarchaeologists working with material from the Near number of peptides being utilised as markers. A previously East tend to rely on a few key studies that present identified set of collagen peptide markers for a wide range of morphological distinctions between sheep and goat bones mammalian species (Buckley et al. 2009) to which peptide (e.g. Boessneck 1969; Payne 1969; Payne 1985; Prummel markers for gazelle (Gazella gazella) have been added, are and Frisch 1986), although more recent studies have both used to identify challenging samples (i.e. highly fragmented expanded and tested these earlier works (Halstead et al. specimens or those with few distinguishing morphological 2002; Helmer 2000; Zeder and Lapham 2010; Zeder and criteria present) including bones and teeth from Domuztepe, a Pilaar 2010). The methodologies described in many of large settlement in south east Turkey, where excavations to these early studies have become commonplace in Near date have produced material culture dating from ca. 5,800 to Eastern faunal analysis. Nevertheless, the reliability of an ca. 5,450 cal. BC; (Campbell et al. 1999). individual researcher’s determination will largely be depen- The animal economy of Domuztepe was dominated by dent on the preservation of the specimen, the age of the domestic sheep and goats, whose bones make up more than animal at death (where very young animals can lack clear 50% of the entire analysed assemblage to date (Kansa In morphological markers) and, finally, the analyst’s skill and prep.). Management of sheep and goat herds varies depend- confidence in making the determination. Recent studies ing on the needs of the communities keeping the animals. have demonstrated that certain morphological criteria on While both sheep and goat certainly provided a reliable long bones are, indeed, more reliable than others and can be source of meat, sinews, and hides to the people of used to confidently distinguish between sheep and goat, Domuztepe, they also may have been used to provide milk, particularly for complete, adult specimens where multiple hair, wool and dung. These ‘secondary products’, which criteria are present (Buckley et al. 2010; Zeder and Lapham can be taken from the animal while it is still alive (Sherratt 2010). In the absence of clear morphological markers on 1983), differ between taxa, and herds were managed in fragmented specimens, however, there is a common desire for specific ways to target certain products. However, the a cheap and reliable molecular method that still succeeds in difficulty of distinguishing sheep and goats often obscures thermally degraded samples such as those found in the Near some of the finer-grained differences in the harvesting East, and one such method is presented here. profiles of these two taxa. Other methods of differentiation have focussed on dietary In our previous report (Buckley et al. 2010), we signatures such as stable carbon isotopes (Balasse and demonstrated the ability to distinguish between sheep and Ambrose 2005) and tooth wear (Grant 1982; Grine et al. goat using a single collagen peptide marker. This report 1987), but due to influences in dietary response cannot be seeks to show the potential for an alternative method, using considered objective approaches. Ancient DNA analysis does a more informative collagen fingerprint that not only offer a clearly objective approach to species identification but distinguishes between sheep and goats, but also between rarely succeeds in Near Eastern samples of such antiquity many other taxa including humans. A human signature has (Bar-Gal et al. 2003; Loreille et al. 1997). Proteins on the been included because in some circumstances, such as the other hand, have long been known as the dominant ‘Death Pit’ at Domuztepe, where >10,000 remains from biomolecules in modern and ancient bone (Ambler and humans and domestic food animals were highly fragmented Daniel 1991), where collagen (type 1) is by far the most and frequently mixed, distinction between human and non- abundant, taking up approximately 90% by weight of the human vertebrate remains is extremely difficult. However, proteins in bone and known to persist with extraordinary the focus of this paper, the collagen fingerprinting method- longevity under these conditions. We have previously ology, is applied to a much larger number of morpholog- reported a method (ZooMS; Buckley et al. 2010) whereby a ically unidentifiable sheep/goat specimens, some with single collagen peptide that is capable of distinguishing estimated age and known sex, in order to derive support between sheep and goat collagen can be reproducibly isolated for the proposed methods of animal husbandry for the site. via their interactions with standard sample preparation C18 columns, and analysed by matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) mass spectrometry. Experimental Here, we report the application of a simple alternative that produces peptide markers diagnostic of a range of taxa Materials (Buckley et al. 2009), not only key domesticate fauna such as sheep, goat, pig and cattle, but also non-domesticates Hydrochloric acid (HCl), ammonium bicarbonate, and including deer and gazelle, as well as humans. In this variant, acetonitrile (ACN) were obtained from BDH (UK). Archaeol Anthropol Sci (2011) 3:271–280 273

Sequencing grade trypsin, trifluoroacetic acid (TFA), mass TFA, followed by one bed volume of 0.1% TFA. Following spectrometric standards and α-cyano-4-hydroxycinnamic enzymatic digestion, the samples were centrifuged at acid were purchased from Sigma-Aldrich (UK). C18 SPE 13,000×g for 10 min and the supernatant acidified to 0.1% pipette tips were purchased from OMIX (UK). The six TFA and applied to the pipette tip. Following two wash steps MALDI-ToF-MS calibration peptides were purchased from (100 μL 0.1% TFA), a stepped gradient of increasing ACN Applied Biosystems. concentration (100 μL fractions of 10% and 50% ACN in 111 archaeological samples from Neolithic Domuztepe 0.1% aqueous TFA) was applied to the pipette tip and the

(thermal age of >40 kacollagen@10˚C (where the thermal age of eluting peptides were collected. These fractions were then a sample incorporates other factors such as temperate and dried to completion using a centrifugal evaporator and burialdepthinadditiontotheageinyears(Smithetal.2003)) resuspended with 10 μL of 0.1% TFA. Sample solution were selected. The specimens were selected from a variety of (1 μL) from each fraction was spotted onto a Bruker ultraflex elements that had been morphologically identified by SWK as II target plate, mixed together with 1 μLofα-cyano-4- sheep, goat, deer, gazelle, pig or simply to an animal size hydroxycinnamic acid matrix solution (1% in ACN/H2O1:1 category and are archived in Open Context (see ESM 1; (v/v)) and allowed to dry. The fractions of each collagen Buckley and Kansa 2011). Many of the selected specimens digest were analysed in triplicate by MALDI-MS in were highly fragmented or came from very young animals reflectron mode using a Bruker ultraflex II MALDI-ToF/ that were difficult to distinguish to taxon because of a lack of ToF mass spectrometer equipped with a Nd/YAG smart clear morphological markers. Thus, over half (64) of the beam laser. Mass spectra were acquired over the m/z range specimens were identified only to the broader sheep/goat 800–4,000. Mass spectra were externally calibrated against an category or had other speculative identifications such as adjacent spot containing six peptides (des-Arg1-Bradykinin, ‘deer?’ (ESM 1). The samples also included a selection of M+H+ at m/z of 904.681; Angiotensin I, M+H+ at m/z of sheep and goat mandibles in order to test the reliability of 1,296.685; Glu1-Fibrinopeptide B, M+H+ at m/z of morphological criteria for distinguishing mandibles to taxon 1,750.677; ACTH (1–17 clip), M+H+ at m/z of 2,093.086; (Halstead et al. 2002). To demonstrate that the presence of ACTH (18–39 clip), M+H+ at m/z of 2,465.198; ACTH (7– human-derived bone fragments can easily be distinguished 38 clip), M+H+ at m/z of 3,657.929). Monoisotopic masses from those of animal remains, four securely identified human were obtained using a SNAP averagine algorithm (C 4.9384, bone samples also from Domuztepe were included in this N 1.3577, O 1.4773, S 0.0417, H 7.7583). In addition to the study. Other securely identified modern and ancient bone peptide markers previously identified for a wide variety of samples were also analysed to serve as references including , analogous peptide markers for gazelle were modern gazelle (G. gazella) from the Natural History identified by manual interpretation of MS spectra and added Museum of Scotland; balearic mouse-goat (Myotragus to the dataset. Analogous peptide markers for human collagen balearicus) from Cova des Gorgs (Escorca, Mallorca; 6480– were identified using published sequence data and supported 6390 cal BC); and modern mouflon (Ovis aries orientalis), with mass spectrometric analysis of morphologically identi- ibex (Capra ibex), bharal ( nayaur), chamois fied archaeological human bone samples from Domuztepe. ( rupicapra) and the (Hemitragus jemlahicus) from the Oxford Museum of Natural History. Results Collagen peptide fingerprinting Collagen peptide markers Collagen peptide fingerprints were obtained following meth- ods adapted from Buckley et al. (2009).Samplesofbone Those m/z values representing previously published peptide were powdered and approximately 20 mg demineralised with markers of known type I collagen sequence that from their 500 μL 0.6 M HCl over 4 h. Following centrifugation at sequences are clearly useful for taxonomic discrimination 13,000×g and removal of the supernatant, the acid-insoluble are labelled representing their position in the tropocollagen residue was then gelatinised by heating at 65°C in 300 μL molecule (e.g. A1T55/56 denotes a peptide spanning the 50 mM ammonium bicarbonate (pH 7.8) for 3 h. Following 55th and 56th tryptic peptides from the start of the alpha 2 gelatinisation, the samples were centrifuged at 13,000×g for (I) chain whereas A2T85 denotes the 85th tryptic peptide 15 min to precipitate ungelatinised protein and the superna- from the start of the alpha 2 (I) chain)(Table 1). Because tant digested with trypsin. 2 μLof1μg/μL trypsin solution none of these markers could be used to differentiate was added to the sample, which was then incubated at 37°C between cervine (fallow and red) deer and gazelle, for 18 h and the digestion terminated by the addition of additional markers were identified in analyses of modern 30 µL 1% TFA. 100 μL (P-100) C18 OMIX pipette tips standards that could be used for such differentiation were prepared with one bed volume of 50% ACN/0.1% (Fig. 1). Without sequence data, further sampling would 274 Table 1 The presence/absence of designated peptide markers in the major domesticate species analysed in this study plus additional ancestral caprine species m/z value of M+H+ion taurus reference sequence (PO2453 & PO2465) Cattle Sheep Goat Pig Fallow Roe Gazelle Human

1,180.6+1,196.6 A2T85 A2T85 A2T85 A2T85 A2T85 A2T85 1,192.6+1,208.6 IGQPGAVGPAGIR A2T85 1,119.6+1,235.6 A2T85 1,299.7 X 1,427.7 GIPGEFGLPGPAGAR A2T43 A2T43 A2T43 A2T43 A2T43 A2T43 1,453.7 A2T43 1,477.7 A2T43 1,550.8 A2T45 A2T45 A2T45 A2T45 1,580.8 GPPGESGAAGPTGPIGSR A2T45 A2T45 A2T45 A2T45 1,604.8 XX 2,115.1 A2T69 2,131.1 GLPGVAGSVGEPGPLGIAGPPGAR A2T69 A2T69 A2T69 A2T69 A2T69 A2T69 A2T69 2,261.2/2,283.1 X X X 2,263.1/2,285.1 XX 2,293.1 XX 2,853.5+2,869.5 GLTGPIGPPGPAGAPGDKGEAGPSGPAGPTGAR A1T55/56 2,883.5+2,899.5 A1T55/56 A1T55/56 A1T55/56 A1T55/56 2,869.5+2,899.5 A1T55/56 2,957.5 A2T67 3,017.4+3,033.4 GPSGEPGTAGPPGTPGPQGL LGAPGFLGLPGSR A2T67 A2T67 A2T67 A2T67 A2T67 3,043.4+3,059.4 A2T67 3,077.4+3,093.4 A2T67

Collagen alpha 1 (I) and alpha 2 (I) chain peptide markers with known sequence information are labelled A2T85, A2T43, A2T45, A2T69, A1T55/56 and A2T67 denoting the tryptic peptide

position from the alpha 1 (A1) or alpha 2 (A2) chain. (X) indicates markers not described previously 3:271 (2011) Sci Anthropol Archaeol – 280 Archaeol Anthropol Sci (2011) 3:271–280 275

1500 1427.7 1560.8 3059.4 B 1545.8 Α2Τ45 1000 2131.1 1550.8 1568.8 Α2Τ43 1562.8 898.5 2705.2 1560.8 500 1299.7 1726.8 Α2Τ69 2578.1 3651.4 Α2Τ45 1546.8 1580.8 0 A 1560.8 3033.7

15000 2705.4 Α2Τ45 2131.2 1580.8 1427.8

10000 Α2Τ67 Α2Τ67 1560.8 1550.8 1560.9 Α2Τ45 5000 898.5 Α2Τ43 1832.9 2578.2 1574.8 2883.5 3651.7 1544.8 1274.7 0 1560.8 1299.7 2000 2131.1 Α2Τ45 1550.8 3093.5 1544.8 1576.8 1427.7 1540 1545 1550 1555 1560 1565 1570 1575 1580

1000 2705.2 Α2Τ67 1726.9 898.5 2293.2

1189.6 Α2Τ43 2562.2 3651.7 C 0 Intensity (a.u.) 2131.1 2263.2 2285.1 75000 1427.7 1726.9 3033.4 2705.2 50000

1560.8 1888.9 Α2Τ67 2263.1 25000 1105.6 Α2Τ43 2883.4 898.5 2261.1 3227.6 3651.3 2285.2 0 2131.2 8000 2261.2 1560.8 2307.2 6000 1427.7 2705.3 2283.1 2300.1 Α2Τ69 Α2Τ69 Α2Τ69 Α2Τ69 Α2Τ67 4000 1726.9 3033.5 898.51105.6 2293.2 2000 Α2Τ43 1976.1 2883.5 2329.1 2562.2 2300.1 0 2307.2 1000 1500 2000 2500 3000 3500 2260 2270 2280 2290 2300 2310 m/z

Fig. 1 MALDI-ToF mass spectra showing some of the collagen detected in the 10% acetonitrile fractions (b, c), where others are much peptide markers from modern bone samples of (top to bottom) gazelle, more intense in the 50% acetonitrile fractions (a) goat, sheep, roe deer and fallow deer. Some markers were only be required to confirm the taxonomic-specificity of the 1974). There is also a noticeable decrease in the relative other observed m/z values reported in Table 1. amounts of the larger molecular weight peptides, likely resulting from chemical hydrolysis in the burial environment Analysis of problematica (Fig. 2). After allowing for deamidation (+1 Da), the combination One particular attraction of protein-based identification is of peaks at m/z values of 1,180.6 and 1,196.6 (COL1A2T85 its robustness relative to methods using nucleic acids with and without a hydroxylated proline), 1,427.7 (Nielsen-Marsh 2002). In order to explore this further, we (COL1A2T43), 1,580.8 (COL1A2T45), 2,883.5 tested samples known to prove problematic for DNA-based (COL1A1T55/56) and 3,033.4 (COL1A2T62) clearly iden- identification, archaeological bone with a thermal age of tify 10 of the unknown ovicaprid samples as sheep (Ovis >40,000 at 10°C (Smith et al. 2003). The 111 sp.). Similarly, 21 of the unknown archaeological samples archaeological samples from Domuztepe (ESM 1) were were identified as goat (Capra sp.) from the presence of analysed by mass spectrometry (e.g. Fig. 2) and show that, peaks corresponding to m/z values of 1,180.6 and 1,196.6, despite ~7,500 years of burial in a warm climate, peptide 1,427.7, 1,580.8, 2,883.5 and 3,093.4 (COL1A2T67). mass spectra are of good quality. Differences in the These samples included those taken from tooth dentine recorded mass of some peptides due to the deamidation is collagen, which were of similar quality to those from bone observed; deamidation of asparagine and glutamine residues collagen (Fig. 3). The 27 morphologically identified goat is known to occur in proteins by hydrolytic cleavage of the specimens and 14 of the 16 morphologically identified amide side chain, particularly at low pH and high temper- sheep specimens were supported by biochemical analysis. atures, although deamidation of glutamine occurs at much One specimen (DT56) tentatively identified morphological- slower rates than that of asparagine (Robinson and Rudd ly as ‘wild sheep’ was identified as cervine (red or fallow 276 Archaeol Anthropol Sci (2011) 3:271–280

Fig. 2 MALDI-ToF mass 0.04 Human DTH6 spectra of the 50% acetonitrile 1477.7 fractions eluted from C18 2115.2 pipette tip following tryptic 0.02 A2T67 digestion of collagen from A2T43 2958.5 archaeological bone samples A2T69 showing examples of (top to 0 bottom) human, sheep, pig, goat 0.4 1427.7 Sheep DT76 and roe deer. Note that each representative of marker peptide 0.2 A2T69 – A2T67 A2T67 (m/z, 2,958.5 3,060.5) is A2T43 1560.8 2131.1 deamidated resulting in a shift 1848.9 2706. 3 3034.5 by one mass unit 0

4 1453.8 Pig DT103 2 A2T69 A2T67 1 A2T43 2131.2 1586.8 1848.9 2706.3 3034.6 0 Goat DT94 1427.7 2 A2T69 Intensity (a.u.)x10 A2T43 2131.2 A2T67 1 1648.9 2411.2 2883.4 3094.5 0 Roe Deer DT97 0.2 1427.7 2131.1 3060.5 0.1 A2T43 A2T67 1562.8 A2T69 2706.2 0 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z

deer), another confidently identified ‘sheep’ specimen identification in the field. Less common taxa such as (DT73) identified as goat and another confidently identified gazelle and various species of deer are not well represented ‘sheep/goat’ (DT98) identified as cervine deer. The five by in-field reference materials at Domuztepe, so these suspect goat specimens were correctly identified as goat. suspected specimens were set aside for future identification Three of the four suspect sheep specimens were identified in the laboratory. The lower confidence and higher rate of as sheep from collagen fingerprinting but the other (DT29) error on deer and gazelle highlights the importance of the identified as cervine deer. One sample (DT49) was use of high-quality reference books or, ideally, skeletal suspected as either sheep or gazelle, in this case confirmed collections of the taxa in question. If comparative speci- to be sheep. The two confidently identified deer specimens, mens are not available, then the identification should be left and more accurately suspected as being fallow deer (DT6 intentionally broad (such as ‘sheep or deer’) until adequate and DT46) were identified as cervine (red or fallow deer) comparative materials can be referenced. rather than roe deer (the only other cervid likely present at this site, by collagen fingerprints). However, the only suspect deer specimens were identified as sheep (DT70) Discussion and pig (DT66). One specimen thought to be either gazelle or deer (DT97) was identified as roe deer (Capreolus Specificity of selected collagen peptide markers capreolus; Fig. 3) and another (DT62) as sheep. The only ‘confidently’ identified gazelle (DT57) was identified as As described elsewhere, the taxonomic resolution of this sheep and one of the six confidently identified deer collagen fingerprinting technique is generally at the (DT106) identified as goat from the collagen fingerprints. level in most large mammals, with divergence times ~5– The four morphologically identified pig specimens were all 7 Ma (Buckley et al. 2009). We included the set of closely supported by biochemical analysis. The higher rate of error related caprines analysed by Buckley et al. (2010) to further on the ‘suspected’ specimens reflects the fact that they were investigate the variations in collagen through evolution. selected for this study because of the lack of confidence in These included mouflon (ESM 2), bharal (ESM 3), Archaeol Anthropol Sci (2011) 3:271–280 277

Fig. 3 MALDI-ToF mass 4 1706.8 spectra of the 10% and 50% 3060.4

1560.8 10% 50% 3 acetonitrile fractions eluted from 3059.4 2020.0 C18 pipette tip following tryptic 2 3018.2 A2T43 1848.9 3043.3 3075.3 digestion of collagen from 2216.1 1267.7 1 2883.4 archaeological samples showing 4 2582.2 roe deer bone (DT97), and 0 1.50 archaeological dentine from goat 1427.7 1.25 2131.1 (DT115) and sheep (DT116)

1.00 A2T43 both previously identified as DT97 3060.4 Intensity x10 0.75 sheep/goat. A close up of the 0.50 A2T69 1560.8 high m/z markers from the 0.25 A2T67 1267.628 1848.8 2706.2 50% acetonitrile fractions 0.00 (inset) are shown to highlight 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z that instrumental accuracy to 1427.7 at least ~0.2 Da is required to 6 2131.1 distinguish the roe deer marker A2T43 1562.8 at m/z 3,059.4 (and its deami- 4 DT115

4 1727.9 dated form at m/z 3,060.4) 1848.9 A2T69 2 from a peptide at m/z 3,060.8 1267.7 2411.1 2899.4 A2T67 commonly observed in most 0 1427.7 1.2 mammalian collagen 1586.8 3094.4 1.0 analyses to date Intensity x10 1706.8

A2T43 3060.8 0.8

0.6 1261.6 3019.3 1848.9 2131.1 3093.4 0.4 0.2 A2T69 2513.1 2899.3 0.0 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z 1586.8 1.25 3034.5 1427.8 3033.5 1.00 1277.6 3060.8 3018.4 0.75 A2T43 1848.9 3045.0

4 0.50 0.25 2513.2 2883.4 0.00 1427.8 3.0

2.5 2131.2 50% Intensity x10

2.0 A2T43 1560.8 1.5 1706.8 DT116 A2T69 1.0 A2T67 1272.6 1848.9 2411.2 0.5 2706.2 3034.5 0.0 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z

Himalayan tahr (ESM 4), balearic mouse-goat (ESM 5), a priori knowledge of the specimen being analysed (i.e. chamois (ESM 5) and ibex (ESM 7). The previously ovicaprid in the previous method; Buckley et al. 2010). identified sheep/goat peptide marker (Buckley et al. 2010) was routinely identified as a diagnostic peptide amongst Survival in archaeological samples these taxa, with the goat version of the peptide originating at the base of Capra (Buckley et al. 2010). However, a Discrimination between archaeological sheep and goat bones potential marker at m/z 1299 was only observed in goat using bone morphology is difficult and these two species are specimens and none of the other caprine specimens. currently often grouped together simply as ‘ovicaprids’ or Although this latter marker is observed in all of the ‘caprines’. When gazelle are added to this the problems become archaeological goat and none of the archaeological sheep much more significant. A reliable method to differentiate these specimens analysed here (ESM 1), the homologous sheep species in highly fragmented archaeological assemblages or marker could not be confirmed. This paper presents assemblages containing large numbers of young individuals markers that not only separate gazelle from caprines (m/z could have a profound impact on the understanding of past value of 1,550), but ones that separate them from deer (m/z husbanding practices. The amalgamation of sheep and goats value of 2,263). Representatives of other species, such as into a single taxon risks the loss of significant archaeological pig and human, were also included to show the capability information, necessitating the development of new unambigu- of this more advanced technique that no longer requires any ous means of differentiating the species in poorly preserved 278 Archaeol Anthropol Sci (2011) 3:271–280 assemblages. Biomolecular methods offer unambiguous as- morphological criteria from a single specimen. A recent study signment, and the proposed collagen peptide marker method by Zeder and Lapham (2010) demonstrated that many of the benefits a number of advantages over DNA-based assignment, common morphological criteria used by zooarchaeologists to such as the greater resistance of collagen to decay in the distinguish the long bones of sheep and goat are highly archaeological burial environment, reduced risk of contamina- reliable, with the exception of only a few less reliable criteria. tion and greatly reduced analytical costs. The degradation of Buckley et al. (2010) tested morphological identifications collagen occurs at much slower rates than DNA (Nielsen- against single collagen peptide markers and demonstrated, Marsh 2002) and we have recently presented collagen likewise, that the morphological criteria were highly reliable, fingerprints in Lower Palaeolithic and older samples (Buckley improving our confidence in morphological criteria (which and Collins, Submitted). Collagen fingerprinting analysis are frequently the only tools available to the zooarchaeologist (ZooMS) is direct from the bone, making it less susceptible in the field). However, when a wider range of possible taxa to the contamination problems that beset ancient DNA are included, such as the deer within this study, a greater analyses, requires small amounts of sample (4–20 mg of bone number of misidentifications may occur (two of the seven depending on preservation) and is not as affected by the burial confidently identified ‘deer’ or ‘gazelle’ specimens in this environmental conditions as demonstrated by low success study actually deriving from sheep or goat influenced by rates of DNA in early sites in regions with hot climates, such diagenetic alterations to the bone specimen). as PPNB in the Near East (Bar-Gal et al. 2003). In this follow- The modified ZooMS method allows the differentiation up study, we have focused on testing the developed method of immature sheep and goat bones, overcoming the problem against a much larger archaeological sample set (115) of of underdeveloped or ambiguous morphological criteria, which more than half could not be morphologically identified especially on fragmented specimens. At the site of (Fig. 4). The high level of correspondence between the Domuztepe, mortality patterns based on both tooth erup- morphological and chemical determinations for many of the tion/wear and fusion data suggest that the inhabitants may samples suggests that when confident (~50% of the 115 have preferentially killed goats at a very young age, specimens selected above, including the 4 human specimens) favouring adult sheep for their wool (Kansa In prep.). the identifications are usually adequate and would not require There were 51 uncertain caprine specimens, three of which additional biochemical support (with four exceptions in this were suspected sheep, four suspected goat, three suspected case study). In this case the ZooMS methodology was able to sheep or deer and one suspected sheep/goat/deer. The identify all 31 of the ‘ovicaprid’ specimens. To minimize remaining 40 specimens were all indistinguishable sheep/ misidentifications, analysts are constrained by the need for goat remains, 10 of which were from young (unfused) tested and reliable criteria, and the need to apply multiple sheep/goat individuals. However, we note that seven of

Fig. 4 Species composition with and without biochemical analyses. Note that speculative morphological identifications are indicated by ‘question marks’ Archaeol Anthropol Sci (2011) 3:271–280 279 these ten specimens that were too young to morphologically ages. Although studies of this nature would benefit from the determine the species came from goats, compared with only analysis of much larger numbers of specimens, the sample in three sheep, which is consistent with the general observa- this study supports previous analysis (based on morphological tion that goats were killed younger than sheep. In the other distinctions and combined sheep/goat mandibular data) that cases of uncertainty, the specimens were either mandibles sheep at Domuztepe were maintained to adulthood (probably from older individuals or too fragmented to make the for wool), while goats served more as meat providers and were morphological distinction. This method could be used to killed at much younger ages than sheep. test conclusions about differential sheep and goat kill-off patterns because of the ability to reliably test immature caprine bones and better understand culling practices Conclusions involving immature individuals. We have also shown that the method works equally well with tooth dentine, which is Collagen persists in mineralized tissues, antler, teeth and bone, essentially dominated by the same collagen type as bone is. where it is the dominant protein and has extraordinary Twenty one of the specimens analysed in this study were longevity within this environment. The ability to discriminate aged using common methods (Payne 1973; Zeder 1991) between bone fragments from different taxa using a simple, and distinguished (if possible) as sheep or goat based on the rapid and robust mass spectrometric analysis of collagen criteria presented in Halstead et al. (2002). Of these, seven tryptic peptides has many obvious applications in archaeo- were correctly identified as either sheep or goat using logical studies throughout the world. That archaeological morphological criteria. These seven were all from young bone and teeth can be collagen fingerprinted without and prime age individuals (under 4–6 years). Eleven contamination complications (such as occurs in ancient specimens, all from individuals older than 4–6 years, were DNA analyses during the amplification process) add to the not differentiated morphologically due to ambiguities in the feasibility and potential of such collagen-based species tooth wear patterns. One specimen, belonging to a 6–8- identification. The discrimination between morphologically old goat, was misidentified using morphological criteria. similar species need not be restricted to mammals where This implies that the morphological distinction is accurate species from other classes such as birds, amphibians and fish for younger animals, before the teeth become too worn could also be routinely studied using the methodology down. As the animals age, it becomes more difficult to described here. In addition to testing the reliability of specific distinguish the two species. Indeed, a recent study that morphological criteria, the main applications of the method tested morphological criteria including those proposed for would be to identify morphologically undiagnostic bones. mandibles found that mandibular criteria were less reliable Many sites would find this method useful, either because than originally thought, particularly among very young (0– material is too fragmented to distinguish between these 6 months) and older individuals (Zeder and Lapham 2010; particular taxa or because there are immature animal remains. Zeder and Pilaar 2010). The applied collagen fingerprinting Using a set of 115 archaeological bones (including the 4 method, thus is helpful to establish a better understanding human specimens) of varying amenability to traditional of the older end of the spectrum, i.e. helping identify older morphological identification methods from a site with a individuals (beyond the age of maturity/bone fusion) as thermal age of 40 kacollagen@10°C we have demonstrated that well as with the very young end of the spectrum. Thus, the method is accurate and can be 100% successful in though long bones are common in the archaeological record assemblages from early agricultural Near Eastern sites. We and often provide abundant fusion data and reliable have also confirmed that many of the widely used morphological distinctions between sheep and goats, they morphological criteria for distinguishing sheep and goat only provide mortality information up to the point of fusion bones are reliable, though distinction should only be made (adulthood). Mandibular teeth, which erupt and wear down with a high degree of confidence when more than one in a predictable way throughout a sheep or goat’s lifetime, marker is present. The method further promises to augment provide a much more complete picture of harvest profiles investigations into animal management by providing a more (not one focused only on the animals up to the age of reliable means of distinguishing between taxa at various maturity, but well beyond). Because current morphological ages, from very young to very old. criteria for distinguishing sheep and goat mandibles have been shown to be unreliable, the collagen fingerprinting method presented here is extremely promising for helping Acknowledgements The authors would like to thank Malgosia understand harvest profiles across the full life of a herd. As Nowak-Kemp (Oxford Natural History Museum; samples 19437 and the fusion data suggest, in the Domuztepe sample used in 19945) and Jerry Herman (Scotland Natural History Museum) for – supplying modern specimens to sample and to Suellen Gauld for this study, only goats were killed at young ages (less than 1 archaeological human and Josep Antoni Alcover for Myotragus samples. 2 years). After 2 years, sheep and goats both survive to older We would also like to acknowledge the Natural Environment Research 280 Archaeol Anthropol Sci (2011) 3:271–280

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