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Combined visual and biochemical analyses confirm depositor and diet for Neolithic coprolites from Skara Brae

Combined visual and biochemical analyses confirm depositor and diet for Neolithic coprolites from... Coprolites (fossilized faeces) can provide valuable insights into species’ diet and related habits. In archaeozoological contexts, they are a potential source of information on human-animal interactions as well as human and animal subsistence. However, despite a broad discussion on coprolites in archaeology, such finds are rarely subject to detailed examination by researchers, perhaps due to the destructive nature of traditional analytical methods. Here, we have examined coprolitic remains from the Neolithic (third millennium BCE) settlement at Skara Brae, Orkney, using a range of modern methods: X-ray computed tomography, scanning electron microscopy, lipid and protein analysis (shotgun proteomics of the coprolite matrix as well as collagen peptide mass fingerprinting of isolated bone fragments). This combined approach minimised destructiveness of sampling, leaving sufficient material for subse- quent study, while providing more information than traditional morphological examination alone. Based on gross visual examination, coprolites were predominantly attributed to domestic dogs (Canis familiaris), with morphologi- cally identified bone inclusions derived from domestic sheep (Ovis aries) and common voles (Microtus arvalis). Partial dissection of a coprolite provided bone samples containing protein markers akin to those of domestic sheep. Considering the predominance of vertebral and distal limb bone fragments, Skara Brae dogs were probably consum- ing human butchery or meal refuse, either routinely fed to them or scavenged. The presumably opportunistic consumptionofrodents mayalsohave played a role in pest control. . . . . . Keywords Neolithic Coprolite Diet X-ray computed tomography Mass spectrometry Scanning electron microscopy Supplementary Information The online version of this article (https:// doi.org/10.1007/s12520-020-01225-9) contains supplementary material, which is available to authorized users. * Andrzej A. Romaniuk School of Natural Sciences, University of Manchester, Andrzej.Romaniuk@ed.ac.uk Manchester M1 7DN, UK School of Biological Sciences, University of Edinburgh, School of History, Classics and Archaeology, University of Edinburgh EH9 3FL, UK Edinburgh, Edinburgh EH8 9AG, UK Department of Collections Services, National Museums Scotland, Department of Natural Sciences, National Museums Scotland, Edinburgh EH1 1JF, UK Edinburgh EH1 1JF, UK Skara Brae Publication Project, 509 King Street, Aberdeen AB24 School of Geosciences, University of Edinburgh, Edinburgh EH9 3BT, UK 3FE, UK 4 9 Oxford University Museum of Natural History, Parks Road, Department of Scottish History and Archaeology, National Museums Oxford OX1 3PW, UK Scotland, Edinburgh EH1 1JF, UK 274 Page 2 of 15 Archaeol Anthropol Sci (2020) 12:274 Introduction alongside many heavily fragmented finds, were retrieved from the settlement core (Trench I). The settlement periphery A serious concern when using finite remains to study the past (Trench II) and off-site Trench III provided only a few finds, is the destructive nature of many widely adopted methods. It is in each case confined to a single context. Assuming domestic an especially serious concern in the case of archaeological dogs, Canis familiaris, as likely depositors, a parasitological remains, which are at best a finite resource and often unique study by Hopkins (Hopkins J pers. comm.)examined 58 sam- (Maschner and Chippindale 2005; Renfrew and Bahn 2012; ples in search of transmission stages of parasites. While the Frank et al. 2015). Coprolites are a prime example of this parasitological results were negative, “rehydration” of the problem. Beyond examination of their external appearance coprolites revealed that most contained large numbers of bone and the identification of visible parts of inclusions, the pre- fragments. Alongside the general absence of plant material dominant method used to analyse coprolites involves dissec- other than microscopic pollen (Clarke DV and Shepherd AN tion, usually after dissolving (“rehydrating”) the coprolite ma- pers. comm.), this supports the interpretation that they were trix in a specific solution (Callen 1963), or dry-pulverizing its deposited by dogs. However, among rehydrated material, contents (Heizer 1963), in order to isolate and visually identify eleven samples showed solution colours more similar to ones any inclusions. However, such approaches narrow the retriev- obtainable from human coprolites, raising the question of able data strictly to the inclusions and preclude further exam- whether humans were also responsible for the creation of ination, for example of the internal arrangement of the copro- Skara Braecoproliticmaterial. lite content or its chemical composition. Moreover, it pre- The study herein was designed to provide the maximum cludes the further assessment of those finds in the future with data from a series of the available finds, while limiting direct other methods. On archaeological sites where coprolitic finds physical impact, to enable their re-examination in the future. are relatively common, this problem can be mitigated, for The objectives were to identify depositor species and provide example by utilising subsampling and leaving some coprolites data about the depositors’ diet. In contrast to other studies, or parts of them for future research. However, many sites which generally rely on a single approach, four distinct provide only a sparse number of coprolites, often as singular methods were employed: (1) traditional visual examination; finds, and the potential loss of information is too important for (2) scanning electron microscopy (SEM); (3) high-resolution a dissection method to be applied. X-ray micro computed tomography (μCT); (4) lipid and pro- Because of these drawbacks, in recent decades, there has tein analysis via mass spectrometry. Only the last of these been a surge in publications exploring potential non- methods required any invasive and destructive measures, in destructive approaches towards archaeological material (e.g. the form of partial coprolite dissection and drilling of several Biró 2005; Borgwardt and Wells 2017). In the case of copro- coprolite specimens to obtain samples for analysis. lites, X-ray computed tomography (μCT) scanning has been The study is a part of a bigger research effort of multiple utilised for the past two decades to avoid destructive analysis, research groups from different research institutions to provide facilitate replicability and create raw data for future research. a comprehensive overview of the Skara Brae site and life of its Initially used only to generate 2-dimensional cross-sectional inhabitants during the Orcadian Neolithic period (Clarke and data (e.g. Farlow et al. 2010), it has more recently been com- Shepherd in prep). bined with 3-dimensional (3D) digital imaging techniques for more comprehensive analysis of content and structure (Milàn et al. 2012a, b; Bravo-Cuevas et al. 2017;Wang etal. 2018). Materials and methods This has permitted identification of the coprolite depositor as well as its prey and other food items. Meanwhile, in destruc- As the Skara Brae assemblages contained predominantly tive sampling, one can see a trend towards standardisation of heavily fragmented coprolites, only contexts containing intact sampling protocols and reduction of sample numbers, which finds or fragments with identifiable inclusions visible on the is important to allow replication and therefore reproducibility, surface were selected for this study. Samples from 13 con- and to leave material for analysis with subsequently developed texts, along with any related bone fragments, were selected techniques (see Wood and Wilmshurst 2016). Multiple ap- for the first stage of analysis (Table 1). Materials from the proaches are rarely combined in the study of coprolites; re- contexts had been retrieved by sieving with a standard 3-cm searchers often prefer to use one specific method, and even if mesh. The first nine contexts, containing most of the intact this does not destroy a sample, it constrains the diversity of coprolites, represented the second phase of occupation of the data obtained. main settlement at Skara Brae, dating from around mid- A number of coprolites were found during the excavations twenty-eighth to mid-twenty-fifth century cal BCE (Sheridan of the Neolithic settlement of Skara Brae (Orkney, UK) in et al. 2013; Shepherd 2016; Bayliss et al. 2017), and corre- 1972-3 (Clarke 1976a, 1976b) and in 1977 (Clarke DV and sponding to the bulk of the occupational remains currently Shepherd AN pers. comm.). The majority of intact coprolites, visible on the site. In contrast, earlier phases contained far Archaeol Anthropol Sci (2020) 12:274 Page 3 of 15 274 Table 1 Sampled contexts, including phasing and presence of canid bones (Clarke DV and Shepherd A pers.comm.), and material selected from them for specific methods. Weight (g), intact coprolites with > 75% surface/content present, partial coprolites with 25–75% surface/content present and coprolite fragments with less than 25% surface/content present. Specific methods include computed tomography (CT), protein (Proteins) and lipid (Lipids) analysis, and scanning electron microscopy (SEM) General context data Sampled material Specific methods selection Context Phase Dog bones present? Fox bones present? Amount Weight Intact Partial Fragm. 102 2 Yes No Whole 9.89 1 2 4 110 2 Yes No Sampled 38.39 2 6 39 CT: Selected coprolite fragments (20, 7.01 g) SEM: 2 samples Proteins: 3 coprolite fragments 170 2 Yes No Whole 37.86 7 4 1 CT: Single intact coprolite (6.48 g) 113 2 No No Sampled 55.27 5 5 12 CT: Single intact coprolite (14.76 g, after dissection: 6.92) Proteins: 10 bone fragments (1.07 g) 1surface sample Lipids: 4 coprolite content samples (1.37 g) 122 2 No No Whole 7.17 1 0 0 CT: Single intact coprolite (7.17 g) 126 2 Yes No Sampled 11.07 0 0 34 CT: Selected coprolite fragments (20, 7.71 g) Proteins: 3 coprolite fragments 132 2 No No Sampled 6.02 0 0 11 132.2 2 No No Whole 20.36 1 1 54 134 2 Yes No Sampled 38.21 2 18 30 139 Int. No No Sampled 18.71 3 2 3 Single intact coprolite (7.83 g) Proteins: 1 surface sample 142 1 No No Whole 4.50 0 0 6 Proteins: 2 coprolite fragments 157 0 No Yes Whole 42.56 0 0 102 213 1 Yes Yes Sampled 102.34 8 13 31 274 Page 4 of 15 Archaeol Anthropol Sci (2020) 12:274 fewer finds and the remaining four contexts represented the Proteins were then ultrafiltered using 10 kDa Vivaspin (UK) rest of the site stratigraphy (intermediate phase: 139, phase 1: ultrafiltration units, into 50 mM ammonium bicarbonate, and 142, 213; phase 0: 157; details in Shepherd 2016). the retentate reduced and alkylated with dithiothreitol and The first stage of the research consisted of visual examina- iodoacetamide respectively following previously used tion of the selected coprolites and associated bone fragments, methods (Wadsworth et al. 2017), prior to tryptic digestion and further subsampling for subsequent μCT, SEM and pro- overnight at 37 °C. The digests were then acidified to 0.1% teomics analysis. Bone or teeth inclusions visible on a copro- trifluoroacetic acid (TFA), zip tipped with OMIX C18 pipette lite’s surface, as well as bone remains with coprolite matrix tips into 50% acetonitrile/0.1% TFA solutions and dried to remains on them, were assessed visually and identification of completion. After re-suspension in 5% acetonitrile/0.1% skeletal element and species was attempted. Vertebrate skele- formic acid, the digests were analysed by LC-Orbitrap Elite tal material in the National Museums Scotland (NMS) collec- mass spectrometry following Buckley et al. (2015). Searches tions was utilised as a source of comparative references for were carried out using Mascot (Perkins et al. 1999) against the identification, alongside widely used identification books for SwissProt database containing 556,568 sequences with fixed large (Schmid 1972) and small (Lawrence and Brown 1973; carbamidomethyl C modifications and variable oxidations of Hillson 2005) mammals. References for taphonomic changes P, K and M, as well as allowance for deamidations of N and Q were also used (Andrews 1990; Fernández-Jalvo and residues. Only proteins with 2 or more peptides above the Andrews 2016). Intact coprolites and unique finds were homology threshold were considered. ZooMS analyses on photographed and, where advantageous, multiple photo stack- the 10 bone fragments were carried out following van der ing utilised. Following visual analysis, four intact coprolites Sluis et al. (2014), in which the 0.6 M hydrochloric acid- between 3 and 5 cm in length from contexts 113, 170, 122 and soluble fraction following overnight decalcification was 139, and two sets of fragmented coprolites from contexts 110 ultrafiltered into 50 mM ammonium bicarbonate and digested and 126, were selected for μCT scanning. with sequencing grade trypsin overnight at 37 °C. The sam- X-ray micro-computed tomographic data on the internal ples were then ziptipped into 10% and 50% acetonitrile frac- structure of the intact coprolites were obtained at the tions, dried completely and resuspended then spotted onto a University of Edinburgh School of Geosciences stainless steel matrix assisted laser desorption ionization Experimental Geoscience Facility. Their in-house, custom- (MALDI) target plate. The fingerprints were acquired using built μCT system comprises a Feinfocus 10–160 kV dual a Bruker Ultraflex II MALDI Time of Flight mass spectrom- transmission/reflection source, MICOS UPR-160-AIR ultra- eter collecting over the m/z range of 700–3,700 with up to high precision air bearing table, PerkinElmer XRD0822 amor- 2,000 laser acquisitions and compared to reference spectra phous silicon X-ray flat panel detector and terbium-doped biomarkers presented by Buckley et al. (2017). gadolinium oxysulfide scintillator. Data were acquired using An additional four samples of internal coprolite matrix > in-house software, reconstructed using filtered back projection 0.3 g were taken from different parts of the coprolite for po- in Octopus 8.9 software, and then segmented and visualised tential taxonomic identification via lipid analysis (e.g. using Mimics 19.0. The scan resolution for the larger copro- Harrault et al. 2019). The lipids were extracted following lites was 64 μm per voxel, and the smaller fragments 26 μm. established methods (Evershed et al. 1990; Charters et al. Three-dimensional digital reconstructions of the copro- 1993) and to maximize the amount available for analysis, lites and their contents were generated to permit analy- three of the coprolite samples were combined (0.74 g in total). sis of their spatial orientation. Identification of inclu- The sample was extracted by ultrasonication, after the addition sions was also attempted using these reconstructions, of an internal standard (20 μgoftetracosane-d ), with a as in the initial observation. 10-mL chloroform-methanol mixture (2:1 v/v) and the super- The largest intact coprolite, from context 113, was partially natant liquid was collected after centrifugation. The extraction dissected to obtain samples for further proteomics analysis. steps were repeated three times, and the combined total lipid Dissection included 7.8 g of the coprolite, approximately extract (TLE) obtained was evaporated using a rotary evapo- 53% of its whole weight, and provided 10 bone fragments rator and redissolved in 3 ml of chloroform-methanol mixture. ranging in weight from 0.02 to 0.24 g; these bone fragments An aliquot (1 ml) of the TLE was taken, dried under nitrogen were analysed by collagen peptide mass fingerprinting (also and 2 ml of 5% methanolic sodium hydroxide solution (9:1 called ZooMS: Zooarchaeology by Mass Spectrometry; MeOH: H O) was added. After heating at 70 °C for 1 h, with Buckley et al. 2009). An additional set of 10 samples of cop- regular mixing, the mixture was allowed to cool, acidified to rolite matrix were also taken by drilling two intact coprolites pH ~ 3 with 1 M HCl and the organic fraction was extracted (from contexts 113 and 139) and powdering coprolite frag- using hexane (2 ml, three times). This fraction was dried under ments from three contexts (see Table 1). A standard proteomic nitrogen, 100 μLof a BF -CH OH complex was added and 3 3 method was used for all 20 samples, in which 6 M GuHCl was heated at 75 °C for 1 h. The solution was cooled, 2 mL of added to 100 mg sample and incubated at 4 °C overnight. dichloromethane washed double distilled water was added, Archaeol Anthropol Sci (2020) 12:274 Page 5 of 15 274 and the organic fraction was extracted using chloroform (1 Micromammal inclusions occasionally retrieved from frag- mL, three times), dried under nitrogen and frozen until GC- mentary coprolites during drilling for samples were assessed MS analysis. For GC-MS analysis, the residue was dissolved using a MX 2500 CamScan scanning electron microscope work- in 100 μL of hexane. A second aliquot (1 ml) of the TLE was ing with a backscattered detector (SEM-BSC) in Envac mode dried under nitrogen, 50 μLof N,O- (50 Pa). The samples were observed without any surface prepa- Bis(trimethylsilyl)trifluoroacetamide (BSFTA) was added ration at the working distance of 20 mm using 20 kV accelerating and the mixture was heated at 60 °C (1 h). The excess voltage. The scans were used to examine the effects of digestion BSFTA was evaporated to dryness under nitrogen, and the on bone and tooth surfaces and its impact on the overall preser- residue was dissolved in 100 μl of hexane and immediately vation of elements, similarly to the examples in available litera- analysed by GC-MS. ture (Andrews 1990; Fernández-Jalvo and Andrews 2016). The samples were analysed using an Agilent 7890A gas All coprolite materials (apart from fragments completely pow- chromatograph fitted with a Zebron ZB-5MS capillary col- dered for proteomic analysis) remain accessible in the research umn (30 m, 0.25 mm i.d., 0.25-μm film thickness) coupled collections of the National Museums of Scotland and all datasets to an Agilent 5975C MSD single quadrupole mass spectrom- generated during this research are available online. eter operated in electron ionization (EI) mode in scan/SIM −1 mode (scanning a range of m/z 50–650 at 1 scan s with a 4-min solvent delay; ionization energy 70 eV) and Agilent 7683 autosampler. The injector port temperatures were set at Results 280 °C, the heated interface at 280 °C, the EI source at 230 °C and the MS quadrupole at 150 °C. Helium was used as the The external shape of the coprolites from Skara Brae corre- carrier gas with a flow rate of 1 ml/minute and the samples lates with mid-size canid species (see Fig. 1). An external were introduced in the pulsed splitless injection mode. The typology of carnivoran faeces was developed by Diedrich −1 oven was programmed from 50 to 130 °C at 20 °C min , (2012, Figs. 4 and 6) based on modern African spotted hy- followed by a rate of 6 °C/min to 310 °C and held at this aenas (Crocuta crocuta crocuta, Erxleben, 1777), in order to temperature for 15 min. Compounds were identified by com- study coprological remains from European sites attributed to parison with spectra from the literature. Crocuta crocuta spelaea (Goldfuss, 1823). Applying Fig. 1 a Comparison of four intact coprolites to the line of hyaena droppings (on the left, after Diedrich 2012); b Digital photographs of selected coprolites (scale bar 10 mm); c Modern ex- ample of dog faeces (below). Images copyright National Museums Scotland 274 Page 6 of 15 Archaeol Anthropol Sci (2020) 12:274 Diedrich’s hyaena typology, the larger Skara Brae coprolites contexts 170, 113, 126, 132.2 and 139. Evidence for plant that were sampled for μCT are identified as form A (conical, remains were scarce. Only one possible example was identi- beginning of the dropping line; context 122), C (oval, mid- fied among the intact coprolites included in the μCT analysis. dropping line; context 139) and D (long-oval; context 110 and This comprised a hollow, cuboid depression in the outer shell 113) (Fig. 1a). Smaller coprolites resembled types E (round), of a coprolite from context 122, with its surface covered in F (irregular) and G (so-called drop, fragmented end of a com- regular linear impressions, akin to plant fibres (Online plete dropping). Resource 1). Inorganic inclusions were occasionally present Inclusions exposed on coprolite surfaces were almost ex- in the form of fine gravel or lumps of sand, covering up to clusively fragmented bone and teeth. The majority of bone 10% of the coprolite surface in the case of the intact coprolite inclusions were either small fragments of cancellous (trabec- from context 139. ular or spongy) bone, sometimes partially covered by remains Identification of larger remains attributed to ungulates was of the cortical bone layer. This is similar to those reported hampered by the degree of fragmentation, with only a few from Links of Noltland (Carrot 2011) aswellasinprevious morphological features remaining intact. Four fragments of Skara Brae research (Hopkins J pers. comm.). Bone fragments ungulate thoracic vertebra were retrieved from contexts 113 composed only of cortical bone were also common. Larger and 126, including a pedicle fragment and anterior vertebral and more complete skeletal remains were rarely present and body with unfused epiphyses (Fig. 2a, b) as well as a matching only accessible within fragmented coprolites retrieved from vertebral body and epiphyseal plate encased in two separate Fig. 2 Identified skeletal fragments found within Skara Brae coprolite assemblages. Contexts are 113 (a, b), 126 (c, d, f), 170 (e), 110 (g, i) and 139 (h). In the case of B and F, two perspectives were included for the sake of clarity. All scale bars 10 mm. Images copyright National Museums Scotland Archaeol Anthropol Sci (2020) 12:274 Page 7 of 15 274 coprolite fragments (Fig. 2c, d). In addition, two distal limb vertebral body from context 126, anterior parts of superior and fragments were found in contexts 170 and 126 (Fig. 2e, f): the inferior ridges appeared to have been chewed off in a manner distal and unfused part of an ungulate metapodial shaft, and similar to one from context 113. Another taphonomic change the proximal end of an intermediate phalanx of a sheep. visible on the bone surface is heavy digestive corrosion. Additionally, context 126 contained three bone fragments, Digestion appears to have penetrated the cortical layer of two of which could be identified as the epiphysis of a long bone, creating a wavy cracking pattern as well as thinning it bone and the shaft of a rib. considerably and partially exposing the trabecular structure Identification of rodent remains was more straightforward beneath. In some cases, such as the metapodial fragment from due to the presence of relatively intact teeth and bones. Molar context 170, cortical bone was removed to the point of reveal- teeth from voles were found in four coprolite fragments, three ing trabeculae on the whole surface. from context 110 and one from context 139. In context 110, SEM micrographs of micromammal bones also revealed two teeth were still located in the sockets of an almost com- digestion characteristic of diurnal raptors or carnivorous mam- plete maxillary bone (Fig. 2g), and a third tooth was found mals (Fig. 4a–d; see Andrews 1990; Fernández-Jalvo and separately. In context 139, a complete molar tooth row was Andrews 2016). In contrast to the larger inclusions, these present in anatomical sequence within the coprolite matrix, bones were not broken, thus permitting the study of digestion but without any bone remaining (Fig. 2h). Two complete vole marks without the obstruction caused by fragmentation. Vole vertebrae were found in context 110 (Fig. 2i), and one group molars were altered considerably, with enamel on the salient of vole metapodials were found in context 126. edges heavily thinned or chipped away and cementum irreg- The majority of identified inclusions exhibited taphonomic ularly fragmented between them (Fig. 4a). Exposed dentine changes on their surface related to bone breakage during in- was also partially eroded, creating a surface sloping towards gestion or digestion (Fig. 3). Alterations to the ungulate ver- the eroded enamel. Similarly to the larger remains, the surface tebrae suggest a depositor species trying to bite through, or of the micromammal bones exhibited either wavy cracking (a bite off, their parts. This was especially clear in the case of maxilla, Fig. 4b), or thinning and exposure of trabeculae context 113, where a vertebral body was fragmented roughly (Fig.4c, d). along the sagittal plane (Fig. 2b), with the resulting exposure The μCT data provided more information on bone inclu- of trabecular bone forming a surprisingly straight layer, even sions present within the sample. All four intact coprolites pre- after digestion. Signs of bone fragmentation due to chewing dominantly contained small fragments of cancellous (trabec- could also be seen on a proximal phalanx from context 126 ular or spongy) bone densely packed within the matrix (Fig. (Fig. 2f), of which the shaft was also crushed. On the complete 5). Only four bone fragments were > 2 cm (largest 29.37 mm), Fig. 3 Example of a bone with various taphonomic marks present (surface) layer of the bone thinner or even removed, leaving trabecular (vertebra from context 113, see Fig. 2b). Several minor tooth marks (spongy) bone structure inside clearly visible. Chewing of the ridges itself (highlighted in green) are present on both cranial and caudal side of the could also help in digestive acids to penetrate the bone to a degree cur- vertebral body, suggesting that the vertebra was at some point chewed rently visible. See Fernández-Jalvo and Andrews (2016, Fig.A.152,355 through, leaving half of vertebral body and most of vertebral arch missing & 816) for comparisons. Scale bar 10 mm. Images copyright National (highlighted in blue). Remaining surfaces, especially around ridges of Museums Scotland caudal and cranial end, were further altered by digestion, leaving cortical 274 Page 8 of 15 Archaeol Anthropol Sci (2020) 12:274 Fig. 4 SEM micrographs of two micromammal finds retrieved from context 110 assemblage: Orkney vole maxilla (a, b)and rodent (vole?) vertebral body (c, d). Areas in (a)showchipping (1) or sloping digestion (2) of molar enamel, with exposed dentine be- neath also showing sloping loss towards enamel outline. Area 3 in (b) in turn shows digestive changes on bone tissue, in a form of a wavy cracking on even cor- tical surface. In case of area 4 in (c) (seen in detail in d), cortical layer erosion alongside vertebral epiphyseal line can be seen. Thinning of the bone in several cases leads to the creation of large perforations, exposing trabecula beneath. Epiphyseal line itself may be visible only due to ero- sion. Images copyright National Museums Scotland 2 2 which is similar to the size of finds identified from visual assess- the coprolite from context 113 (r =0.30, r = 0.41) were gener- ment. Of the 73 fragments within the coprolites, 30 fragments ally low and the removal of outliers did not change the outcome rangedbetween1and2cminlength, andthe remaining43were significantly. Weight data obtained from 10 bone fragments re- < 1 cm in length. The percentage of bone in the coprolite, by trieved from an intact coprolite from context 113 showed the volume, ranged from 11.56% (coprolite from context 139) to highest coefficient with width (r = 0.88), with the other two 21.65% (coprolite from context 170). The number of bone frag- measurements providing moderate values (r = 0.46 for length, ments within each coprolite correlated with its overall size, the r = 0.43 for depth). largest coprolite (113) containing 32 fragments, the smallest Only one bone, fragment 23 from context 113, was mor- (139) containing only nine and the intermediate-sized coprolites phologically intact within the coprolites (Fig. 5c). It was lo- containing 21 (122) and 15 (170). The coefficient of determina- cated using the digital 3D reconstructions and later dissection. tion between the length and width of the inclusions was relatively Considering its size (length of ~ 1 cm), shape and the location high, with a combined r for all four coprolites of 0.74 (correla- of an articular surface on only one side of the bone, it is tion = 0.86, for df = 76 significant over 0.2). For three coprolites, identified as a carpal, most likely a pisiform from a small to the r was higher (0.85–0.89), whereas for the coprolite from medium-sized ungulate. The most similar bone among the context 113, it was only 0.61, possibly due to the length to width reference material was the left pisiform of a domestic sheep, ratio of the largest fragment within it (no. 13, see Fig. 5 and which is of approximately the same length, shape and facet Online Resource 3), which differed from the rest of the fragments orientation. The only morphological difference in fragment 23 present. Bone was most likely crushed into fragments of similar was a depression visible in the 3D digital reconstruction, run- size as a result of mastication or later digestion. Larger bone ning from the end of the articular facets to a narrow ridge on fragments were located deeper within the coprolite matrix. The top of the bone, and possibly representing loss of cortical mean depth of the 10 largest bone fragments was 4.84 mm, as bone. Once retrieved during the dissection (Online opposed to 2.99 mm for all of the other fragments studied. This is Resource 2), the assessment based on the digital recon- best seen in coprolites from contexts 170 (r = 0.70 for length/ struction was confirmed, both with regard to the identi- 2 2 2 depth and r = 0.84 for width/depth) and 139 (r =0.66and r = fication as a pisiform and the presence of an area with 0.78). In the case of a coprolite from context 122, the results (r = trabecular bone exposed. The perforation of the cortical 0.31, r = 0.33) were skewed towards the largest fragment (no. 2, bone near the articular facet seems to have been created see Fig. 5) and rose significantly when it was omitted from the by chewing, and the surrounding areas of exposed tra- 2 2 sample pool (r =0.57, r = 0.81). In contrast, the coefficients for becula the result of the subsequent digestive processes. Archaeol Anthropol Sci (2020) 12:274 Page 9 of 15 274 Fig. 5 Digital reconstructions of intact coprolites from microCT data. Two perspectives are shown for each coprolite, including external surface and internal composition of bone fragments, and inclusion numbers (for more data about specific inclusions see Online Resource 3. All scale bars 10 mm. Images copyright National Museums Scotland Other inclusions from the coprolite from context 113 could from context 139 contained fragment 5, a disc-like structure not easily be identified visually. From the digital reconstruc- reminiscent of a small (< 1 cm in diameter) epiphyseal plate. tions, it is possible to infer high levels of bone fragmentation, One side of fragment 5 was smooth and slightly convex, the and inclusions with trabecular bone exposed were reminiscent other was covered in billowing formations not uncommon on of epiphyses of limb bones, especially metapodials or phalan- epiphyseal surfaces. Among the two sets of fragmented cop- ges. But, the identity of other bone fragments, notably the thin rolites, inclusions could only be found in those from context elements reminiscent of flat bones rather than long ones, could 126, and these were identified as the proximal tibial epiphysis not be established. Fragments obtained during dissection and a segment of a sacrum from a rodent. showed extreme alteration caused by digestion, with many While biochemical analysis of coprolite matrix did not re- exposed trabeculae visible through the mostly digested corti- turn conclusive results, proteomics on bone inclusions from a cal layer. Although exact anatomical or taxonomical prove- dissected coprolite provided additional information about spe- nance could not be established, the most likely source was cies provenance (Table 2). Proteomic analysis of the coprolite smaller distal limb bones, possibly of an ungulate. matrix samples from multiple coprolites yielded very few in- Remains within the other large coprolites were less infor- formative peptides, with some appearing devoid of peptide mative, but suggest the same interpretation as for the coprolite matches (samples 17 and 19) and others either containing from context 113. The coprolite from context 170 contained single peptides per protein ‘match’ or multiple from keratins, fragment 12, a large chunk of trabecular bone covered on one especially human. Such proteins could derive from various side by a thin and slightly concave cortical bone layer. forms of contamination. Lipid analysis of the coprolite matrix Considering its shape and size, it could be a part of a proximal also did not provide depositor or prey identification. The first phalange or similar skeletal element. Similarly, the coprolite fraction (Aliquot 1) was dominated by a homologous series of 274 Page 10 of 15 Archaeol Anthropol Sci (2020) 12:274 Table 2 Details of samples extracted from coprolites through dissection (bone fragment and matrix) and drilling (matrix), showing most likely species based on identified proteins/lipids. * indicates likely contaminations. Test samples excluded Sample type Context Sample no. Bone fr. No. Weight (g) Result Bone fragment (dissection) 113 1 1 0.14 Sheep (collagen) Bone fragment (dissection) 113 2 3 0.13 –//– Bone fragment (dissection) 113 3 6 0.02 –//– Bone fragment (dissection) 113 4 18 0.03 –//– Bone fragment (dissection) 113 5 22 0.22 –//– Bone fragment (dissection) 113 6 23 0.05 –//– Bone fragment (dissection) 113 7 24 0.01 –//– Bone fragment (dissection) 113 8 28 0.06 –//– Bone fragment (dissection) 113 9 29 0.18 –//– Bone fragment (dissection) 113 10 30 0.24 –//– Matrix (dissection) 113 A – 0.33 Unknown (saturated & unsaturated fatty acids & alkohols, unknown sterols, plastic derivatives*) Matrix (dissection) 113 B – 0.36 –//– Matrix (dissection) 113 C – 0.33 –//– Matrix (dissection) 113 D – 0.36 –//– Matrix (drilling) 110 11 –– Human* (keratin) Matrix (drilling) 110 12 –– –//– Matrix (drilling) 110 13 –– Human* (multiple skin proteins) Matrix (drilling) 113 14 – Human* (keratin) Matrix (drilling) 126 15 ––//– Matrix (drilling) 126 16 ––//– Matrix (drilling) 126 17 – ?(trypsinonly) Matrix (drilling) 139 18 – Human* (keratin) Matrix (drilling) 142 19 – ?(trypsinonly) Matrix (drilling) 142 20 – Human* (keratin) C -C n-alkanoic acids, with the C (hexadecenoic acid) intact bone described above), returned the same identification. 12 28 16 and C (octadecanoic acid) member being the most abundant. It is likely that most bones within the coprolite came not only The high molecular weight (HMW) n-alkanoic acids (>C20) from the same species, but also the same animal. showed a clear even-over-odd carbon number predominance The μCT scan data revealed differences between the inner in line with a plant derived origin. Also present were a series and outer layers of three of the four intact coprolites. A of the monounsaturated C -C alkanoic acids. The second mineralised outer layer, denser than the coprolitic matrix and 12 18 fraction (Aliquot 2) was dominated by homologous series of of similar greyscale values to the dense cortical bone content, C -C n-alkanols, with the C member being the most was especially evident in the coprolite from context 122. 10 32 26 abundant. The HMW n-alkanols showed a clear even-over- Although one coprolite from 110 also exhibited such a layer, odd carbon number predominance in line with a plant derived it did not encase the entire contents, notably being absent origin. Also present were some contaminants, including the where the bone content was visible at the surface or the cop- alkyl phthalates and alkyl phenol derivatives, as well as minor rolite itself was apparently eroded. The coprolite from context amounts of cholesterol and cholestanol. However, in contrast 113 also had a mineralised outer layer, but it was thinner and to previous studies, no faecal stanols, such as coprostanol, less visible in the μCT data. The coprolite from context 139 epicoprostanol, 24-ethylcoprostanol or 24- did not show any outer layer mineralisation. ethylepicoprostanol, could be identified (Bull et al. 2002; CT and visual data from the Skara Brae coprolites were Gill et al. 2009; Harrault et al. 2019). In turn, collagen finger- similar to previous observations of the contents of the faeces printing of the bone samples showed a strong signal for the of dogs (Payne and Munson 1985), but differed somewhat. In presence of sheep collagen. Moreover, all bone samples, in- their experiment, a dog was fed parts of specific animals of cluding fragment 23 from context 113 (the morphologically three different body sizes (the largest being domestic goat, Archaeol Anthropol Sci (2020) 12:274 Page 11 of 15 274 Capra hircus; intermediate being eastern cottontail rabbit, identified to species, more closely resembling the data from Sylvilagus floridanus and the smallest fox squirrel, Sciurus smaller species’ ingestion. Among the identified remains, vole niger and grey squirrel, S. carolinensis) and surviving inclu- bones smaller than 1 cm dominated, followed by far larger sions in faecal matter, as well as uneaten remains, were ungulate remains, 2–3 cm in diameter. Only two remains were analysed in a variety of ways. As in the results of that study, identified within the range of 1–2 cm, leaving a distinct gap the Skara Brae material shows a predominance of small frag- between the size of the two ingested species. ments (Table 3), although not to the degree reported by Payne and Munson (80–90%). The Skara Brae material comprised 56% fragments < 1 cm in the four μCT scanned intact copro- Discussion lites, and 61% including the scanned smaller fragments and visually assessed material. Payne and Munson also found only Although some non-food elements may accidentally be a minor part of the assemblage was identifiable, with the size ingested or attached to faeces after deposition, such as gravel of identifiable fragments related to the size of the prey species or small particulates, the majority of coprolite contents should to which they belong (1–2 cm for goat, and 0.01–1cmfor faithfully represent the diet of the depositor (Hunt et al. 1994). lagomorphs and rodents). In the case of Skara Brae however, The best known coprolite assemblages are predominantly of the situation is more complex due to the presence of species of canid origin, and have been studied since the nineteenth cen- both sizes within the same coprolite matrix. Only 1.3% of the tury (Buckland 1823). More recently, canid coprolites have content of the four intact coprolites could be identified, which been examined using μCT scanning to analyse their internal is similar to the proportion of goat remains identifiable in structure (Bravo-Cuevas et al. 2017;Wang et al. 2018). The Payne and Munson (1985). However, when all the data are internal composition of the four intact coprolites from Skara considered together, 14.7% of bone inclusions could be Brae resembles that of finds examined in Wang et al. (2018), Table 3 Comparison of greatest length for bone fragments from Skara range of size classes (Payne and Munson 1985), data presented in size Brae (intact coprolites only versus all gathered data) with those from classes (1 cm intervals) coprolites of dogs fed on three mammal species, selected to represent a Maximal length Coprolite fragments from Skara Brae, Coprolite fragments from Skara Brae, all finds (visual + CT) major finds (CT, 4 major coprolites) Size class Overall as% Identified as% Overall as% Identified as% 4.00–4.99 0 0.00 0 0 0.00 0 3.00–3.99 0 0.00 0 0 0.00 0 2.00–2.99 4 5.19 0 0.00 9 7.76 5 29.41 1.00–1.99 30 38.96 1 100.00 36 31.03 2 11.76 To 0.99 43 55.84 0 0.00 71 61.21 10 58.82 Overall 77 – 11.30 116 – 17 14.66 Maximal length Capra remains in dog faeces (Payne and Munson 1985 t.5) Sylvilagus remains in dog faeces (Payne and Munson 1985 t.5) Size class Overall as% Identified as% Overall as% Identified as% 4.00–4.99 1 0.01 0 0.00 0 0.00 0 3.00–3.99 28 0.36 6 5.61 0 0.00 0 2.00–2.99 221 2.82 33 30.84 4 0.65 1 2.33 1.00–1.99 1127 14.37 64 59.81 44 7.20 4 9.30 To 0.99 6466 82.44 4 3.74 563 92.14 38 88.37 Overall 7843 – 107 1.36 611 – 43 7.04 Maximal length Sciurus remains in dog faeces (Payne and Munson 1985 t.5) Size class Overall as% Identified as% 4.00–4.99 0 0.00 0 3.00–3.99 0 0.00 0 2.00–2.99 2 0.40 2 1.54 1.00–1.99 67 13.45 32 24.62 To 0.99 429 86.14 96 73.85 Overall 498 – 130 26.10 274 Page 12 of 15 Archaeol Anthropol Sci (2020) 12:274 attributed to the extinct bear-dog Borophagus parvus,with a millennia after the deposition of the Skara Brae remains. number of fragmented bones entrapped completely or almost However, as for the pine marten (Martes martes) remains completely within the fossilized faecal matter. However, in from the Neolithic Pierowall Quarry on Westray, Orkney, contrast to the bear-dog, or unidentified canids in other studies such finds could be a sign of a short-lived local population (e.g. Bravo-Cuevas et al. 2017), the Skara Brae dog faeces (Sharples 1984). Evidence for a third carnivore found at Skara contain fewer identifiable remains, most being too finely Brae was the single find of an otter (Lutra lutra) metacarpal. fragmented to permit the recognition of anatomical and taxo- Otters are generally considered to be intrusive at archaeolog- nomical provenance. Bone inclusions from Skara Brae were ical sites, and produce highly characteristic faeces (‘spraint’), also tightly condensed, often with only a small amount of which are clearly morphologically different from the Skara coprolite matrix between the pieces of trabecular bone. Brae coprolitic finds. These differences may be attributed to Borophagus parvus The presence of a hard outer coating is considered a typical being a bone-crushing canid, with specialisations in dentition feature of fossilized carnivore faeces (Bryant 1974). This coat- and digestion to chew and ingest most of the skeletal structure ing corresponds to the mineralised outer layer present on the of their adult prey (Wang et al. 2018). On the other hand, Skara Brae coprolites. As first suggested by Bradley (1946), modern wolves (Canis lupus) and dogs do not exhibit this and confirmed by recent studies (Hollocher et al. 2010; specialism, relying on chewing easily modifiable elements, Pesquero et al. 2014), calcium phosphate and other bone min- often from younger prey, and often smaller bones and long eral content in coprolites contributes to their long-term pres- limb epiphyses (Fosse et al. 2012). ervation. The only intact Skara Brae coprolite without such a The visual methods and proteomics both confirmed the coating still contained bone, but a smaller amount than the prey species as being sheep, whose main skeletal elements other three finds. This appears to confirm the relationship cannot be ingested whole by dogs. As a result, they are poten- between bone content in the creation of an outer coating. It tially not identifiable when sufficiently fragmented to be may also point towards differential diagenetic processes be- swallowed. However, minor skeletal elements, for example tween contexts, although it is currently not known exactly phalanges or vertebrae, could be ingested either complete or how this may contribute to coprolite preservation and mineral in fragments that are large enough to be identifiable. content. Considering that some bones came from immature individuals Considering the findings of the visual and μCT analyses, (as evidenced by the lack of epiphyseal fusion), dogs from Neolithic humans, as omnivores, are unlikely to be depositors Skara Brae were most likely fed by humans; low meat- of the studied coprolites. Human faeces are usually diverse in bearing distal limbs and vertebral spines would have originat- their contents (containing bones, fibres, seeds, etc.) and the ed from butchery products and meal waste. mineralised coating attributable to carnivores does not usually The abundance of highly fragmented and digested bones occur on their surface (Callen 1963; Heizer 1963; Bryant within the Skara Brae coprolites points unmistakably towards 1974; Reinhard and Bryant 1992; Reinhard 2000). However, a typical carnivorous species fed by humans or actively scav- research on excavated coprolites of possible human origin enging human refuse. Such a species is represented at Skara almost exclusively relies on DNA-based identifications (e.g. Brae by domestic dogs (Clarke 1976B; Clarke and Sharples Gilbert et al. 2008; Jenkins et al. 2012; Petrigh and Fugassa 1985; Clarke DV and Shepherd AN pers. comm.). Scattered 2017) or microbiological studies (Cano et al. 2014), with only dog remains were found in multiple contexts from the middle a marginal interest in bone inclusions. As a consequence, there stage of phase 1 until the late stage of phase 2, including the is a lack of visual and μCT data on human coprolites to pro- intermediate period between both phases. Many of these re- vide a comparative dataset for our analysis. Regular human mains were isolated teeth, suggesting deposition from natural coprolite deposition would also be unlikely from the contex- tooth loss rather than death of the animal. tual perspective. Skara Brae had a form of drainage system Although the remains of two other carnivore species were which could be used regularly for removing human faeces also found in Skara Brae (Clarke DV and Shepherd AN pers. (Shepherd 2016; Clarke and Sharples 1985), leading to such comm.), they are less likely to have deposited the coprolites. material being transported outside of the site or gathered as a Red fox (Vulpes vulpes) finds were located in contexts belong- manure for nearby fields (Clarke and Sharples 1985). The ing to the earliest two phases (0 and early/middle 1), and these majority of coprolite finds came from deposits behind and do not correlate with the main bulk of the coprolite finds between settlement structures, areas more likely to be (phase 2). Their remains mainly comprised long limb bones frequented by animals rather than humans. and innominates, along with a single jaw. This suggests the The presence of micromammal remains provides evidence processing of foxes to obtain their pelts. It is also debateable of other processes taking place at the site. Orkney vole re- whether red foxes were living wild on Orkney at that time; mains from Skara Brae coprolites are much smaller than the foxes were most likely present from the Early Iron age until North American pocket gophers (Pappogeomys/ the Late Norse period (Fairnell and Barrett 2006), two Cratogeomys) in canid coprolites studied by Bravo-Cuevas Archaeol Anthropol Sci (2020) 12:274 Page 13 of 15 274 et al. (2017), but show a similar level of preservation, with uncontaminated material from coprolites. Drilled material af- fragments being easily identifiable anatomically and in many fected a smaller area of coprolites but showed too much con- cases also taxonomically. Previous research on the Skara Brae tamination by human proteins. micromammal assemblages found significant concentrations of rodents within the site centre (Trench I) and periphery (TrenchII),possiblyrelated to humanoccupation Conclusions (Romaniuk et al. 2016). Two likely explanations were that these are the remains produced as a by-product of pest The combination of visual assessment, μCT scanning with dig- control, or processing of micromammals as a food source. Dogs may have been fed voles in the absence of addition ital reconstruction, scanning electron microscopy and protein/ to the routine meal or butchery waste; possibly, they were lipid analysis all in one study is presently rare. This approach has proven especially useful in identifying coprolite depositors trained to hunt such animals as a form of pest control, or simply caught and ate them of their own volition. Modern and providing data about their possible diet. A number of bone fragments embedded on or within the coprolite matrix were in- dogs on Orkney are known to hunt rodents, but are rarely reported as eating them (Rose 1975). Micromammal vestigated and identified as the distal limb bones or vertebrae of an ungulate, possibly sheep. Proteomics confirmed the presence bones are also known to appear in human coprolites (Reinhard and Bryant 1992;Reinhard et al. 2007), but of sheep collagen within bone inclusions sampled during the dissection. Some finds also included relatively complete remains in the case of Skara Brae, the coprolite fragments in which inclusions were found did not differ substantially of Orkney voles. Many coprolites had a thick, mineralised outer layer, consistent with a typically carnivorous diet and the inges- from the rest of the coprolite assemblage, established in previous paragraphs to resemble canid deposition. tion of large quantities of skeletal remains. The majority of finds, However, it is unlikely that deposition by dogs, even over including all μCT scanned intact coprolites, were likely deposit- ed by dogs. As the depositor diet shows consumption of the low a longer period of time, was responsible for all of the micromammal finds from the excavations at Skara Brae. meat-bearing elements of domesticated species in the anthropic environment, it is likely that these dogs were routinely fed on the The micromammal inclusions show severe taphonomic alterations of bone and tooth surfaces characteristic of refuse of butchery or meals refuse, or scavenged them. Additional micromammal material could come from occasional carnivore digestion, especially the loss of enamel or den- tine and the thinning or cracking of bone. Previous anal- catches, either as intentional pest-control measures, or subsis- tence practices. ysis of the micromammal skeletal assemblage from Skara Brae (Romaniuk et al. 2016) did not provide such finds. X-ray computed tomography proved to be useful for the study of intact coprolites, rather than fragmented remains. Signs of digestion on the micromammal remains were Study of proteins was useful for assessing bone inclusions scarce and did not follow a pattern that has previously been identified as indicative of any mammalian species. embedded within coprolites, but samples of coprolite matrix itself did not yield significant results. This may indicate the From a methodological perspective, the present study demonstrates the value of a combined approach, successfully necessity of hard tissues for the preservation of prey proteins, as well as the effect of carnivore digestion in removing traces limiting destructive analysis while providing more useful data than reliance on a single method alone. From over 392 g of of depositor proteins. Lipid analysis proved to be more useful in the case of coprolite matrix; however, the method needs to assemblage, only 5.5 g of content was fragmented during the dissection, and only ~ 2.5–3.0 g was destroyed during the be refined, especially with regard to identification and proce- dures for avoidance of contamination. This case study high- proteomics analysis. Enough material remains to replicate this study in the future while X-ray computed tomography en- lights the benefits of non-destructive and comparative studies on coprolitic finds from archaeological sites, and provides a sured non-direct replicability though reinvestigation of the template for future research methodology. μCT data. Of equal importance, the combination of methods provided complementary data and further support for the re- Acknowledgements We would like to thank the National Museums of sults obtained by individual methods. Anatomical identifica- Scotland for access to the Skara Brae remains and vertebrate skeletal tions were based on visual examination combined with digital reference material. Thanks to Dr Stig Walsh, who (along with EP) co- imaging, while taxonomic identifications benefitted from supervised CW during her dissertation. these methods together with proteomics. Taphonomic chang- es could be assessed visually, using μCT, and using SEM Author’s contributions The study was conceived by AAR, EP and JSH. Data collection and analyses were performed by AAR (traditional visual imaging. The molecular analyses were more indicative of analysis), LT (SEM), IB, CW and EP (μCT scanning and digital recon- the dietary component rather than the depositor species, con- struction), MB, BEvD and MPC (proteins & lipids). Figures were com- trary to the reported ability to do the latter. Moreover, dissec- piled by AAR and EP. All authors contributed to the writing of the paper tion proved to be the optimal method to obtain and gave final approval for publication. 274 Page 14 of 15 Archaeol Anthropol Sci (2020) 12:274 Funding AAR was in receipt of a School Doctoral Scholarship from the Buckley M, Collins M, Thomas-Oates J, Wilson JC (2009) Species iden- School of History, Classics and Archaeology, University of Edinburgh. tification by analysis of bone collagen using matrix-assisted laser EP was funded by NERC, grant number NE/L002558/1. CW carried out desorption/ionisation time-of-flight mass spectrometry. Rapid μCT work as part of her undergraduate final dissertation at the University Communications in Mass Spectrometry 23: 3843–3854. https:// of Edinburgh, funded by The School of Biological Sciences, University doi.org/10.1002/rcm.4316 of Edinburgh. Buckley M, Farina RA, Lawless C, Tambusso PS, Varela L, Carlini AA, Powell JE, Martinez JG (2015) Collagen sequence analysis of the extinct giant ground sloths Lestodon and Megatherium. PLoS One Data availability Available data supporting this paper include Excel data 10(11):e0139611. https://doi.org/10.1371/journal.pone.0139611 file (Online Resource 3), R script file with short statistical analysis eCollection 2015 (Online Resource 4), compressed file with X-ray computed tomographic Buckley M, Harvey VL, Chamberlain AT (2017) Species identification data and files containing coprolite 3D digital reconstructions (Online and decay assessment of Late Pleistocene fragmentary vertebrate Resource 5), video showing the internal structure of the intact coprolite remains from Pin Hole Cave (Creswell Crags, UK) using collagen from context 113 (Online Resource 6) and two additional figures (Online fingerprinting. Boreas 46: 402–411. https://doi.org/10.1111/bor. Resource 1 and 2). 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Electrophoresis 20:3551–3567. https://doi. org/10.1002/(SICI)1522-2683(19991201)20:18<3551::AID- Publisher’snote Springer Nature remains neutral with regard to jurisdic- ELPS3551>3.0.CO;2-2 tional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archaeological and Anthropological Sciences Springer Journals

Combined visual and biochemical analyses confirm depositor and diet for Neolithic coprolites from Skara Brae

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10.1007/s12520-020-01225-9
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Abstract

Coprolites (fossilized faeces) can provide valuable insights into species’ diet and related habits. In archaeozoological contexts, they are a potential source of information on human-animal interactions as well as human and animal subsistence. However, despite a broad discussion on coprolites in archaeology, such finds are rarely subject to detailed examination by researchers, perhaps due to the destructive nature of traditional analytical methods. Here, we have examined coprolitic remains from the Neolithic (third millennium BCE) settlement at Skara Brae, Orkney, using a range of modern methods: X-ray computed tomography, scanning electron microscopy, lipid and protein analysis (shotgun proteomics of the coprolite matrix as well as collagen peptide mass fingerprinting of isolated bone fragments). This combined approach minimised destructiveness of sampling, leaving sufficient material for subse- quent study, while providing more information than traditional morphological examination alone. Based on gross visual examination, coprolites were predominantly attributed to domestic dogs (Canis familiaris), with morphologi- cally identified bone inclusions derived from domestic sheep (Ovis aries) and common voles (Microtus arvalis). Partial dissection of a coprolite provided bone samples containing protein markers akin to those of domestic sheep. Considering the predominance of vertebral and distal limb bone fragments, Skara Brae dogs were probably consum- ing human butchery or meal refuse, either routinely fed to them or scavenged. The presumably opportunistic consumptionofrodents mayalsohave played a role in pest control. . . . . . Keywords Neolithic Coprolite Diet X-ray computed tomography Mass spectrometry Scanning electron microscopy Supplementary Information The online version of this article (https:// doi.org/10.1007/s12520-020-01225-9) contains supplementary material, which is available to authorized users. * Andrzej A. Romaniuk School of Natural Sciences, University of Manchester, Andrzej.Romaniuk@ed.ac.uk Manchester M1 7DN, UK School of Biological Sciences, University of Edinburgh, School of History, Classics and Archaeology, University of Edinburgh EH9 3FL, UK Edinburgh, Edinburgh EH8 9AG, UK Department of Collections Services, National Museums Scotland, Department of Natural Sciences, National Museums Scotland, Edinburgh EH1 1JF, UK Edinburgh EH1 1JF, UK Skara Brae Publication Project, 509 King Street, Aberdeen AB24 School of Geosciences, University of Edinburgh, Edinburgh EH9 3BT, UK 3FE, UK 4 9 Oxford University Museum of Natural History, Parks Road, Department of Scottish History and Archaeology, National Museums Oxford OX1 3PW, UK Scotland, Edinburgh EH1 1JF, UK 274 Page 2 of 15 Archaeol Anthropol Sci (2020) 12:274 Introduction alongside many heavily fragmented finds, were retrieved from the settlement core (Trench I). The settlement periphery A serious concern when using finite remains to study the past (Trench II) and off-site Trench III provided only a few finds, is the destructive nature of many widely adopted methods. It is in each case confined to a single context. Assuming domestic an especially serious concern in the case of archaeological dogs, Canis familiaris, as likely depositors, a parasitological remains, which are at best a finite resource and often unique study by Hopkins (Hopkins J pers. comm.)examined 58 sam- (Maschner and Chippindale 2005; Renfrew and Bahn 2012; ples in search of transmission stages of parasites. While the Frank et al. 2015). Coprolites are a prime example of this parasitological results were negative, “rehydration” of the problem. Beyond examination of their external appearance coprolites revealed that most contained large numbers of bone and the identification of visible parts of inclusions, the pre- fragments. Alongside the general absence of plant material dominant method used to analyse coprolites involves dissec- other than microscopic pollen (Clarke DV and Shepherd AN tion, usually after dissolving (“rehydrating”) the coprolite ma- pers. comm.), this supports the interpretation that they were trix in a specific solution (Callen 1963), or dry-pulverizing its deposited by dogs. However, among rehydrated material, contents (Heizer 1963), in order to isolate and visually identify eleven samples showed solution colours more similar to ones any inclusions. However, such approaches narrow the retriev- obtainable from human coprolites, raising the question of able data strictly to the inclusions and preclude further exam- whether humans were also responsible for the creation of ination, for example of the internal arrangement of the copro- Skara Braecoproliticmaterial. lite content or its chemical composition. Moreover, it pre- The study herein was designed to provide the maximum cludes the further assessment of those finds in the future with data from a series of the available finds, while limiting direct other methods. On archaeological sites where coprolitic finds physical impact, to enable their re-examination in the future. are relatively common, this problem can be mitigated, for The objectives were to identify depositor species and provide example by utilising subsampling and leaving some coprolites data about the depositors’ diet. In contrast to other studies, or parts of them for future research. However, many sites which generally rely on a single approach, four distinct provide only a sparse number of coprolites, often as singular methods were employed: (1) traditional visual examination; finds, and the potential loss of information is too important for (2) scanning electron microscopy (SEM); (3) high-resolution a dissection method to be applied. X-ray micro computed tomography (μCT); (4) lipid and pro- Because of these drawbacks, in recent decades, there has tein analysis via mass spectrometry. Only the last of these been a surge in publications exploring potential non- methods required any invasive and destructive measures, in destructive approaches towards archaeological material (e.g. the form of partial coprolite dissection and drilling of several Biró 2005; Borgwardt and Wells 2017). In the case of copro- coprolite specimens to obtain samples for analysis. lites, X-ray computed tomography (μCT) scanning has been The study is a part of a bigger research effort of multiple utilised for the past two decades to avoid destructive analysis, research groups from different research institutions to provide facilitate replicability and create raw data for future research. a comprehensive overview of the Skara Brae site and life of its Initially used only to generate 2-dimensional cross-sectional inhabitants during the Orcadian Neolithic period (Clarke and data (e.g. Farlow et al. 2010), it has more recently been com- Shepherd in prep). bined with 3-dimensional (3D) digital imaging techniques for more comprehensive analysis of content and structure (Milàn et al. 2012a, b; Bravo-Cuevas et al. 2017;Wang etal. 2018). Materials and methods This has permitted identification of the coprolite depositor as well as its prey and other food items. Meanwhile, in destruc- As the Skara Brae assemblages contained predominantly tive sampling, one can see a trend towards standardisation of heavily fragmented coprolites, only contexts containing intact sampling protocols and reduction of sample numbers, which finds or fragments with identifiable inclusions visible on the is important to allow replication and therefore reproducibility, surface were selected for this study. Samples from 13 con- and to leave material for analysis with subsequently developed texts, along with any related bone fragments, were selected techniques (see Wood and Wilmshurst 2016). Multiple ap- for the first stage of analysis (Table 1). Materials from the proaches are rarely combined in the study of coprolites; re- contexts had been retrieved by sieving with a standard 3-cm searchers often prefer to use one specific method, and even if mesh. The first nine contexts, containing most of the intact this does not destroy a sample, it constrains the diversity of coprolites, represented the second phase of occupation of the data obtained. main settlement at Skara Brae, dating from around mid- A number of coprolites were found during the excavations twenty-eighth to mid-twenty-fifth century cal BCE (Sheridan of the Neolithic settlement of Skara Brae (Orkney, UK) in et al. 2013; Shepherd 2016; Bayliss et al. 2017), and corre- 1972-3 (Clarke 1976a, 1976b) and in 1977 (Clarke DV and sponding to the bulk of the occupational remains currently Shepherd AN pers. comm.). The majority of intact coprolites, visible on the site. In contrast, earlier phases contained far Archaeol Anthropol Sci (2020) 12:274 Page 3 of 15 274 Table 1 Sampled contexts, including phasing and presence of canid bones (Clarke DV and Shepherd A pers.comm.), and material selected from them for specific methods. Weight (g), intact coprolites with > 75% surface/content present, partial coprolites with 25–75% surface/content present and coprolite fragments with less than 25% surface/content present. Specific methods include computed tomography (CT), protein (Proteins) and lipid (Lipids) analysis, and scanning electron microscopy (SEM) General context data Sampled material Specific methods selection Context Phase Dog bones present? Fox bones present? Amount Weight Intact Partial Fragm. 102 2 Yes No Whole 9.89 1 2 4 110 2 Yes No Sampled 38.39 2 6 39 CT: Selected coprolite fragments (20, 7.01 g) SEM: 2 samples Proteins: 3 coprolite fragments 170 2 Yes No Whole 37.86 7 4 1 CT: Single intact coprolite (6.48 g) 113 2 No No Sampled 55.27 5 5 12 CT: Single intact coprolite (14.76 g, after dissection: 6.92) Proteins: 10 bone fragments (1.07 g) 1surface sample Lipids: 4 coprolite content samples (1.37 g) 122 2 No No Whole 7.17 1 0 0 CT: Single intact coprolite (7.17 g) 126 2 Yes No Sampled 11.07 0 0 34 CT: Selected coprolite fragments (20, 7.71 g) Proteins: 3 coprolite fragments 132 2 No No Sampled 6.02 0 0 11 132.2 2 No No Whole 20.36 1 1 54 134 2 Yes No Sampled 38.21 2 18 30 139 Int. No No Sampled 18.71 3 2 3 Single intact coprolite (7.83 g) Proteins: 1 surface sample 142 1 No No Whole 4.50 0 0 6 Proteins: 2 coprolite fragments 157 0 No Yes Whole 42.56 0 0 102 213 1 Yes Yes Sampled 102.34 8 13 31 274 Page 4 of 15 Archaeol Anthropol Sci (2020) 12:274 fewer finds and the remaining four contexts represented the Proteins were then ultrafiltered using 10 kDa Vivaspin (UK) rest of the site stratigraphy (intermediate phase: 139, phase 1: ultrafiltration units, into 50 mM ammonium bicarbonate, and 142, 213; phase 0: 157; details in Shepherd 2016). the retentate reduced and alkylated with dithiothreitol and The first stage of the research consisted of visual examina- iodoacetamide respectively following previously used tion of the selected coprolites and associated bone fragments, methods (Wadsworth et al. 2017), prior to tryptic digestion and further subsampling for subsequent μCT, SEM and pro- overnight at 37 °C. The digests were then acidified to 0.1% teomics analysis. Bone or teeth inclusions visible on a copro- trifluoroacetic acid (TFA), zip tipped with OMIX C18 pipette lite’s surface, as well as bone remains with coprolite matrix tips into 50% acetonitrile/0.1% TFA solutions and dried to remains on them, were assessed visually and identification of completion. After re-suspension in 5% acetonitrile/0.1% skeletal element and species was attempted. Vertebrate skele- formic acid, the digests were analysed by LC-Orbitrap Elite tal material in the National Museums Scotland (NMS) collec- mass spectrometry following Buckley et al. (2015). Searches tions was utilised as a source of comparative references for were carried out using Mascot (Perkins et al. 1999) against the identification, alongside widely used identification books for SwissProt database containing 556,568 sequences with fixed large (Schmid 1972) and small (Lawrence and Brown 1973; carbamidomethyl C modifications and variable oxidations of Hillson 2005) mammals. References for taphonomic changes P, K and M, as well as allowance for deamidations of N and Q were also used (Andrews 1990; Fernández-Jalvo and residues. Only proteins with 2 or more peptides above the Andrews 2016). Intact coprolites and unique finds were homology threshold were considered. ZooMS analyses on photographed and, where advantageous, multiple photo stack- the 10 bone fragments were carried out following van der ing utilised. Following visual analysis, four intact coprolites Sluis et al. (2014), in which the 0.6 M hydrochloric acid- between 3 and 5 cm in length from contexts 113, 170, 122 and soluble fraction following overnight decalcification was 139, and two sets of fragmented coprolites from contexts 110 ultrafiltered into 50 mM ammonium bicarbonate and digested and 126, were selected for μCT scanning. with sequencing grade trypsin overnight at 37 °C. The sam- X-ray micro-computed tomographic data on the internal ples were then ziptipped into 10% and 50% acetonitrile frac- structure of the intact coprolites were obtained at the tions, dried completely and resuspended then spotted onto a University of Edinburgh School of Geosciences stainless steel matrix assisted laser desorption ionization Experimental Geoscience Facility. Their in-house, custom- (MALDI) target plate. The fingerprints were acquired using built μCT system comprises a Feinfocus 10–160 kV dual a Bruker Ultraflex II MALDI Time of Flight mass spectrom- transmission/reflection source, MICOS UPR-160-AIR ultra- eter collecting over the m/z range of 700–3,700 with up to high precision air bearing table, PerkinElmer XRD0822 amor- 2,000 laser acquisitions and compared to reference spectra phous silicon X-ray flat panel detector and terbium-doped biomarkers presented by Buckley et al. (2017). gadolinium oxysulfide scintillator. Data were acquired using An additional four samples of internal coprolite matrix > in-house software, reconstructed using filtered back projection 0.3 g were taken from different parts of the coprolite for po- in Octopus 8.9 software, and then segmented and visualised tential taxonomic identification via lipid analysis (e.g. using Mimics 19.0. The scan resolution for the larger copro- Harrault et al. 2019). The lipids were extracted following lites was 64 μm per voxel, and the smaller fragments 26 μm. established methods (Evershed et al. 1990; Charters et al. Three-dimensional digital reconstructions of the copro- 1993) and to maximize the amount available for analysis, lites and their contents were generated to permit analy- three of the coprolite samples were combined (0.74 g in total). sis of their spatial orientation. Identification of inclu- The sample was extracted by ultrasonication, after the addition sions was also attempted using these reconstructions, of an internal standard (20 μgoftetracosane-d ), with a as in the initial observation. 10-mL chloroform-methanol mixture (2:1 v/v) and the super- The largest intact coprolite, from context 113, was partially natant liquid was collected after centrifugation. The extraction dissected to obtain samples for further proteomics analysis. steps were repeated three times, and the combined total lipid Dissection included 7.8 g of the coprolite, approximately extract (TLE) obtained was evaporated using a rotary evapo- 53% of its whole weight, and provided 10 bone fragments rator and redissolved in 3 ml of chloroform-methanol mixture. ranging in weight from 0.02 to 0.24 g; these bone fragments An aliquot (1 ml) of the TLE was taken, dried under nitrogen were analysed by collagen peptide mass fingerprinting (also and 2 ml of 5% methanolic sodium hydroxide solution (9:1 called ZooMS: Zooarchaeology by Mass Spectrometry; MeOH: H O) was added. After heating at 70 °C for 1 h, with Buckley et al. 2009). An additional set of 10 samples of cop- regular mixing, the mixture was allowed to cool, acidified to rolite matrix were also taken by drilling two intact coprolites pH ~ 3 with 1 M HCl and the organic fraction was extracted (from contexts 113 and 139) and powdering coprolite frag- using hexane (2 ml, three times). This fraction was dried under ments from three contexts (see Table 1). A standard proteomic nitrogen, 100 μLof a BF -CH OH complex was added and 3 3 method was used for all 20 samples, in which 6 M GuHCl was heated at 75 °C for 1 h. The solution was cooled, 2 mL of added to 100 mg sample and incubated at 4 °C overnight. dichloromethane washed double distilled water was added, Archaeol Anthropol Sci (2020) 12:274 Page 5 of 15 274 and the organic fraction was extracted using chloroform (1 Micromammal inclusions occasionally retrieved from frag- mL, three times), dried under nitrogen and frozen until GC- mentary coprolites during drilling for samples were assessed MS analysis. For GC-MS analysis, the residue was dissolved using a MX 2500 CamScan scanning electron microscope work- in 100 μL of hexane. A second aliquot (1 ml) of the TLE was ing with a backscattered detector (SEM-BSC) in Envac mode dried under nitrogen, 50 μLof N,O- (50 Pa). The samples were observed without any surface prepa- Bis(trimethylsilyl)trifluoroacetamide (BSFTA) was added ration at the working distance of 20 mm using 20 kV accelerating and the mixture was heated at 60 °C (1 h). The excess voltage. The scans were used to examine the effects of digestion BSFTA was evaporated to dryness under nitrogen, and the on bone and tooth surfaces and its impact on the overall preser- residue was dissolved in 100 μl of hexane and immediately vation of elements, similarly to the examples in available litera- analysed by GC-MS. ture (Andrews 1990; Fernández-Jalvo and Andrews 2016). The samples were analysed using an Agilent 7890A gas All coprolite materials (apart from fragments completely pow- chromatograph fitted with a Zebron ZB-5MS capillary col- dered for proteomic analysis) remain accessible in the research umn (30 m, 0.25 mm i.d., 0.25-μm film thickness) coupled collections of the National Museums of Scotland and all datasets to an Agilent 5975C MSD single quadrupole mass spectrom- generated during this research are available online. eter operated in electron ionization (EI) mode in scan/SIM −1 mode (scanning a range of m/z 50–650 at 1 scan s with a 4-min solvent delay; ionization energy 70 eV) and Agilent 7683 autosampler. The injector port temperatures were set at Results 280 °C, the heated interface at 280 °C, the EI source at 230 °C and the MS quadrupole at 150 °C. Helium was used as the The external shape of the coprolites from Skara Brae corre- carrier gas with a flow rate of 1 ml/minute and the samples lates with mid-size canid species (see Fig. 1). An external were introduced in the pulsed splitless injection mode. The typology of carnivoran faeces was developed by Diedrich −1 oven was programmed from 50 to 130 °C at 20 °C min , (2012, Figs. 4 and 6) based on modern African spotted hy- followed by a rate of 6 °C/min to 310 °C and held at this aenas (Crocuta crocuta crocuta, Erxleben, 1777), in order to temperature for 15 min. Compounds were identified by com- study coprological remains from European sites attributed to parison with spectra from the literature. Crocuta crocuta spelaea (Goldfuss, 1823). Applying Fig. 1 a Comparison of four intact coprolites to the line of hyaena droppings (on the left, after Diedrich 2012); b Digital photographs of selected coprolites (scale bar 10 mm); c Modern ex- ample of dog faeces (below). Images copyright National Museums Scotland 274 Page 6 of 15 Archaeol Anthropol Sci (2020) 12:274 Diedrich’s hyaena typology, the larger Skara Brae coprolites contexts 170, 113, 126, 132.2 and 139. Evidence for plant that were sampled for μCT are identified as form A (conical, remains were scarce. Only one possible example was identi- beginning of the dropping line; context 122), C (oval, mid- fied among the intact coprolites included in the μCT analysis. dropping line; context 139) and D (long-oval; context 110 and This comprised a hollow, cuboid depression in the outer shell 113) (Fig. 1a). Smaller coprolites resembled types E (round), of a coprolite from context 122, with its surface covered in F (irregular) and G (so-called drop, fragmented end of a com- regular linear impressions, akin to plant fibres (Online plete dropping). Resource 1). Inorganic inclusions were occasionally present Inclusions exposed on coprolite surfaces were almost ex- in the form of fine gravel or lumps of sand, covering up to clusively fragmented bone and teeth. The majority of bone 10% of the coprolite surface in the case of the intact coprolite inclusions were either small fragments of cancellous (trabec- from context 139. ular or spongy) bone, sometimes partially covered by remains Identification of larger remains attributed to ungulates was of the cortical bone layer. This is similar to those reported hampered by the degree of fragmentation, with only a few from Links of Noltland (Carrot 2011) aswellasinprevious morphological features remaining intact. Four fragments of Skara Brae research (Hopkins J pers. comm.). Bone fragments ungulate thoracic vertebra were retrieved from contexts 113 composed only of cortical bone were also common. Larger and 126, including a pedicle fragment and anterior vertebral and more complete skeletal remains were rarely present and body with unfused epiphyses (Fig. 2a, b) as well as a matching only accessible within fragmented coprolites retrieved from vertebral body and epiphyseal plate encased in two separate Fig. 2 Identified skeletal fragments found within Skara Brae coprolite assemblages. Contexts are 113 (a, b), 126 (c, d, f), 170 (e), 110 (g, i) and 139 (h). In the case of B and F, two perspectives were included for the sake of clarity. All scale bars 10 mm. Images copyright National Museums Scotland Archaeol Anthropol Sci (2020) 12:274 Page 7 of 15 274 coprolite fragments (Fig. 2c, d). In addition, two distal limb vertebral body from context 126, anterior parts of superior and fragments were found in contexts 170 and 126 (Fig. 2e, f): the inferior ridges appeared to have been chewed off in a manner distal and unfused part of an ungulate metapodial shaft, and similar to one from context 113. Another taphonomic change the proximal end of an intermediate phalanx of a sheep. visible on the bone surface is heavy digestive corrosion. Additionally, context 126 contained three bone fragments, Digestion appears to have penetrated the cortical layer of two of which could be identified as the epiphysis of a long bone, creating a wavy cracking pattern as well as thinning it bone and the shaft of a rib. considerably and partially exposing the trabecular structure Identification of rodent remains was more straightforward beneath. In some cases, such as the metapodial fragment from due to the presence of relatively intact teeth and bones. Molar context 170, cortical bone was removed to the point of reveal- teeth from voles were found in four coprolite fragments, three ing trabeculae on the whole surface. from context 110 and one from context 139. In context 110, SEM micrographs of micromammal bones also revealed two teeth were still located in the sockets of an almost com- digestion characteristic of diurnal raptors or carnivorous mam- plete maxillary bone (Fig. 2g), and a third tooth was found mals (Fig. 4a–d; see Andrews 1990; Fernández-Jalvo and separately. In context 139, a complete molar tooth row was Andrews 2016). In contrast to the larger inclusions, these present in anatomical sequence within the coprolite matrix, bones were not broken, thus permitting the study of digestion but without any bone remaining (Fig. 2h). Two complete vole marks without the obstruction caused by fragmentation. Vole vertebrae were found in context 110 (Fig. 2i), and one group molars were altered considerably, with enamel on the salient of vole metapodials were found in context 126. edges heavily thinned or chipped away and cementum irreg- The majority of identified inclusions exhibited taphonomic ularly fragmented between them (Fig. 4a). Exposed dentine changes on their surface related to bone breakage during in- was also partially eroded, creating a surface sloping towards gestion or digestion (Fig. 3). Alterations to the ungulate ver- the eroded enamel. Similarly to the larger remains, the surface tebrae suggest a depositor species trying to bite through, or of the micromammal bones exhibited either wavy cracking (a bite off, their parts. This was especially clear in the case of maxilla, Fig. 4b), or thinning and exposure of trabeculae context 113, where a vertebral body was fragmented roughly (Fig.4c, d). along the sagittal plane (Fig. 2b), with the resulting exposure The μCT data provided more information on bone inclu- of trabecular bone forming a surprisingly straight layer, even sions present within the sample. All four intact coprolites pre- after digestion. Signs of bone fragmentation due to chewing dominantly contained small fragments of cancellous (trabec- could also be seen on a proximal phalanx from context 126 ular or spongy) bone densely packed within the matrix (Fig. (Fig. 2f), of which the shaft was also crushed. On the complete 5). Only four bone fragments were > 2 cm (largest 29.37 mm), Fig. 3 Example of a bone with various taphonomic marks present (surface) layer of the bone thinner or even removed, leaving trabecular (vertebra from context 113, see Fig. 2b). Several minor tooth marks (spongy) bone structure inside clearly visible. Chewing of the ridges itself (highlighted in green) are present on both cranial and caudal side of the could also help in digestive acids to penetrate the bone to a degree cur- vertebral body, suggesting that the vertebra was at some point chewed rently visible. See Fernández-Jalvo and Andrews (2016, Fig.A.152,355 through, leaving half of vertebral body and most of vertebral arch missing & 816) for comparisons. Scale bar 10 mm. Images copyright National (highlighted in blue). Remaining surfaces, especially around ridges of Museums Scotland caudal and cranial end, were further altered by digestion, leaving cortical 274 Page 8 of 15 Archaeol Anthropol Sci (2020) 12:274 Fig. 4 SEM micrographs of two micromammal finds retrieved from context 110 assemblage: Orkney vole maxilla (a, b)and rodent (vole?) vertebral body (c, d). Areas in (a)showchipping (1) or sloping digestion (2) of molar enamel, with exposed dentine be- neath also showing sloping loss towards enamel outline. Area 3 in (b) in turn shows digestive changes on bone tissue, in a form of a wavy cracking on even cor- tical surface. In case of area 4 in (c) (seen in detail in d), cortical layer erosion alongside vertebral epiphyseal line can be seen. Thinning of the bone in several cases leads to the creation of large perforations, exposing trabecula beneath. Epiphyseal line itself may be visible only due to ero- sion. Images copyright National Museums Scotland 2 2 which is similar to the size of finds identified from visual assess- the coprolite from context 113 (r =0.30, r = 0.41) were gener- ment. Of the 73 fragments within the coprolites, 30 fragments ally low and the removal of outliers did not change the outcome rangedbetween1and2cminlength, andthe remaining43were significantly. Weight data obtained from 10 bone fragments re- < 1 cm in length. The percentage of bone in the coprolite, by trieved from an intact coprolite from context 113 showed the volume, ranged from 11.56% (coprolite from context 139) to highest coefficient with width (r = 0.88), with the other two 21.65% (coprolite from context 170). The number of bone frag- measurements providing moderate values (r = 0.46 for length, ments within each coprolite correlated with its overall size, the r = 0.43 for depth). largest coprolite (113) containing 32 fragments, the smallest Only one bone, fragment 23 from context 113, was mor- (139) containing only nine and the intermediate-sized coprolites phologically intact within the coprolites (Fig. 5c). It was lo- containing 21 (122) and 15 (170). The coefficient of determina- cated using the digital 3D reconstructions and later dissection. tion between the length and width of the inclusions was relatively Considering its size (length of ~ 1 cm), shape and the location high, with a combined r for all four coprolites of 0.74 (correla- of an articular surface on only one side of the bone, it is tion = 0.86, for df = 76 significant over 0.2). For three coprolites, identified as a carpal, most likely a pisiform from a small to the r was higher (0.85–0.89), whereas for the coprolite from medium-sized ungulate. The most similar bone among the context 113, it was only 0.61, possibly due to the length to width reference material was the left pisiform of a domestic sheep, ratio of the largest fragment within it (no. 13, see Fig. 5 and which is of approximately the same length, shape and facet Online Resource 3), which differed from the rest of the fragments orientation. The only morphological difference in fragment 23 present. Bone was most likely crushed into fragments of similar was a depression visible in the 3D digital reconstruction, run- size as a result of mastication or later digestion. Larger bone ning from the end of the articular facets to a narrow ridge on fragments were located deeper within the coprolite matrix. The top of the bone, and possibly representing loss of cortical mean depth of the 10 largest bone fragments was 4.84 mm, as bone. Once retrieved during the dissection (Online opposed to 2.99 mm for all of the other fragments studied. This is Resource 2), the assessment based on the digital recon- best seen in coprolites from contexts 170 (r = 0.70 for length/ struction was confirmed, both with regard to the identi- 2 2 2 depth and r = 0.84 for width/depth) and 139 (r =0.66and r = fication as a pisiform and the presence of an area with 0.78). In the case of a coprolite from context 122, the results (r = trabecular bone exposed. The perforation of the cortical 0.31, r = 0.33) were skewed towards the largest fragment (no. 2, bone near the articular facet seems to have been created see Fig. 5) and rose significantly when it was omitted from the by chewing, and the surrounding areas of exposed tra- 2 2 sample pool (r =0.57, r = 0.81). In contrast, the coefficients for becula the result of the subsequent digestive processes. Archaeol Anthropol Sci (2020) 12:274 Page 9 of 15 274 Fig. 5 Digital reconstructions of intact coprolites from microCT data. Two perspectives are shown for each coprolite, including external surface and internal composition of bone fragments, and inclusion numbers (for more data about specific inclusions see Online Resource 3. All scale bars 10 mm. Images copyright National Museums Scotland Other inclusions from the coprolite from context 113 could from context 139 contained fragment 5, a disc-like structure not easily be identified visually. From the digital reconstruc- reminiscent of a small (< 1 cm in diameter) epiphyseal plate. tions, it is possible to infer high levels of bone fragmentation, One side of fragment 5 was smooth and slightly convex, the and inclusions with trabecular bone exposed were reminiscent other was covered in billowing formations not uncommon on of epiphyses of limb bones, especially metapodials or phalan- epiphyseal surfaces. Among the two sets of fragmented cop- ges. But, the identity of other bone fragments, notably the thin rolites, inclusions could only be found in those from context elements reminiscent of flat bones rather than long ones, could 126, and these were identified as the proximal tibial epiphysis not be established. Fragments obtained during dissection and a segment of a sacrum from a rodent. showed extreme alteration caused by digestion, with many While biochemical analysis of coprolite matrix did not re- exposed trabeculae visible through the mostly digested corti- turn conclusive results, proteomics on bone inclusions from a cal layer. Although exact anatomical or taxonomical prove- dissected coprolite provided additional information about spe- nance could not be established, the most likely source was cies provenance (Table 2). Proteomic analysis of the coprolite smaller distal limb bones, possibly of an ungulate. matrix samples from multiple coprolites yielded very few in- Remains within the other large coprolites were less infor- formative peptides, with some appearing devoid of peptide mative, but suggest the same interpretation as for the coprolite matches (samples 17 and 19) and others either containing from context 113. The coprolite from context 170 contained single peptides per protein ‘match’ or multiple from keratins, fragment 12, a large chunk of trabecular bone covered on one especially human. Such proteins could derive from various side by a thin and slightly concave cortical bone layer. forms of contamination. Lipid analysis of the coprolite matrix Considering its shape and size, it could be a part of a proximal also did not provide depositor or prey identification. The first phalange or similar skeletal element. Similarly, the coprolite fraction (Aliquot 1) was dominated by a homologous series of 274 Page 10 of 15 Archaeol Anthropol Sci (2020) 12:274 Table 2 Details of samples extracted from coprolites through dissection (bone fragment and matrix) and drilling (matrix), showing most likely species based on identified proteins/lipids. * indicates likely contaminations. Test samples excluded Sample type Context Sample no. Bone fr. No. Weight (g) Result Bone fragment (dissection) 113 1 1 0.14 Sheep (collagen) Bone fragment (dissection) 113 2 3 0.13 –//– Bone fragment (dissection) 113 3 6 0.02 –//– Bone fragment (dissection) 113 4 18 0.03 –//– Bone fragment (dissection) 113 5 22 0.22 –//– Bone fragment (dissection) 113 6 23 0.05 –//– Bone fragment (dissection) 113 7 24 0.01 –//– Bone fragment (dissection) 113 8 28 0.06 –//– Bone fragment (dissection) 113 9 29 0.18 –//– Bone fragment (dissection) 113 10 30 0.24 –//– Matrix (dissection) 113 A – 0.33 Unknown (saturated & unsaturated fatty acids & alkohols, unknown sterols, plastic derivatives*) Matrix (dissection) 113 B – 0.36 –//– Matrix (dissection) 113 C – 0.33 –//– Matrix (dissection) 113 D – 0.36 –//– Matrix (drilling) 110 11 –– Human* (keratin) Matrix (drilling) 110 12 –– –//– Matrix (drilling) 110 13 –– Human* (multiple skin proteins) Matrix (drilling) 113 14 – Human* (keratin) Matrix (drilling) 126 15 ––//– Matrix (drilling) 126 16 ––//– Matrix (drilling) 126 17 – ?(trypsinonly) Matrix (drilling) 139 18 – Human* (keratin) Matrix (drilling) 142 19 – ?(trypsinonly) Matrix (drilling) 142 20 – Human* (keratin) C -C n-alkanoic acids, with the C (hexadecenoic acid) intact bone described above), returned the same identification. 12 28 16 and C (octadecanoic acid) member being the most abundant. It is likely that most bones within the coprolite came not only The high molecular weight (HMW) n-alkanoic acids (>C20) from the same species, but also the same animal. showed a clear even-over-odd carbon number predominance The μCT scan data revealed differences between the inner in line with a plant derived origin. Also present were a series and outer layers of three of the four intact coprolites. A of the monounsaturated C -C alkanoic acids. The second mineralised outer layer, denser than the coprolitic matrix and 12 18 fraction (Aliquot 2) was dominated by homologous series of of similar greyscale values to the dense cortical bone content, C -C n-alkanols, with the C member being the most was especially evident in the coprolite from context 122. 10 32 26 abundant. The HMW n-alkanols showed a clear even-over- Although one coprolite from 110 also exhibited such a layer, odd carbon number predominance in line with a plant derived it did not encase the entire contents, notably being absent origin. Also present were some contaminants, including the where the bone content was visible at the surface or the cop- alkyl phthalates and alkyl phenol derivatives, as well as minor rolite itself was apparently eroded. The coprolite from context amounts of cholesterol and cholestanol. However, in contrast 113 also had a mineralised outer layer, but it was thinner and to previous studies, no faecal stanols, such as coprostanol, less visible in the μCT data. The coprolite from context 139 epicoprostanol, 24-ethylcoprostanol or 24- did not show any outer layer mineralisation. ethylepicoprostanol, could be identified (Bull et al. 2002; CT and visual data from the Skara Brae coprolites were Gill et al. 2009; Harrault et al. 2019). In turn, collagen finger- similar to previous observations of the contents of the faeces printing of the bone samples showed a strong signal for the of dogs (Payne and Munson 1985), but differed somewhat. In presence of sheep collagen. Moreover, all bone samples, in- their experiment, a dog was fed parts of specific animals of cluding fragment 23 from context 113 (the morphologically three different body sizes (the largest being domestic goat, Archaeol Anthropol Sci (2020) 12:274 Page 11 of 15 274 Capra hircus; intermediate being eastern cottontail rabbit, identified to species, more closely resembling the data from Sylvilagus floridanus and the smallest fox squirrel, Sciurus smaller species’ ingestion. Among the identified remains, vole niger and grey squirrel, S. carolinensis) and surviving inclu- bones smaller than 1 cm dominated, followed by far larger sions in faecal matter, as well as uneaten remains, were ungulate remains, 2–3 cm in diameter. Only two remains were analysed in a variety of ways. As in the results of that study, identified within the range of 1–2 cm, leaving a distinct gap the Skara Brae material shows a predominance of small frag- between the size of the two ingested species. ments (Table 3), although not to the degree reported by Payne and Munson (80–90%). The Skara Brae material comprised 56% fragments < 1 cm in the four μCT scanned intact copro- Discussion lites, and 61% including the scanned smaller fragments and visually assessed material. Payne and Munson also found only Although some non-food elements may accidentally be a minor part of the assemblage was identifiable, with the size ingested or attached to faeces after deposition, such as gravel of identifiable fragments related to the size of the prey species or small particulates, the majority of coprolite contents should to which they belong (1–2 cm for goat, and 0.01–1cmfor faithfully represent the diet of the depositor (Hunt et al. 1994). lagomorphs and rodents). In the case of Skara Brae however, The best known coprolite assemblages are predominantly of the situation is more complex due to the presence of species of canid origin, and have been studied since the nineteenth cen- both sizes within the same coprolite matrix. Only 1.3% of the tury (Buckland 1823). More recently, canid coprolites have content of the four intact coprolites could be identified, which been examined using μCT scanning to analyse their internal is similar to the proportion of goat remains identifiable in structure (Bravo-Cuevas et al. 2017;Wang et al. 2018). The Payne and Munson (1985). However, when all the data are internal composition of the four intact coprolites from Skara considered together, 14.7% of bone inclusions could be Brae resembles that of finds examined in Wang et al. (2018), Table 3 Comparison of greatest length for bone fragments from Skara range of size classes (Payne and Munson 1985), data presented in size Brae (intact coprolites only versus all gathered data) with those from classes (1 cm intervals) coprolites of dogs fed on three mammal species, selected to represent a Maximal length Coprolite fragments from Skara Brae, Coprolite fragments from Skara Brae, all finds (visual + CT) major finds (CT, 4 major coprolites) Size class Overall as% Identified as% Overall as% Identified as% 4.00–4.99 0 0.00 0 0 0.00 0 3.00–3.99 0 0.00 0 0 0.00 0 2.00–2.99 4 5.19 0 0.00 9 7.76 5 29.41 1.00–1.99 30 38.96 1 100.00 36 31.03 2 11.76 To 0.99 43 55.84 0 0.00 71 61.21 10 58.82 Overall 77 – 11.30 116 – 17 14.66 Maximal length Capra remains in dog faeces (Payne and Munson 1985 t.5) Sylvilagus remains in dog faeces (Payne and Munson 1985 t.5) Size class Overall as% Identified as% Overall as% Identified as% 4.00–4.99 1 0.01 0 0.00 0 0.00 0 3.00–3.99 28 0.36 6 5.61 0 0.00 0 2.00–2.99 221 2.82 33 30.84 4 0.65 1 2.33 1.00–1.99 1127 14.37 64 59.81 44 7.20 4 9.30 To 0.99 6466 82.44 4 3.74 563 92.14 38 88.37 Overall 7843 – 107 1.36 611 – 43 7.04 Maximal length Sciurus remains in dog faeces (Payne and Munson 1985 t.5) Size class Overall as% Identified as% 4.00–4.99 0 0.00 0 3.00–3.99 0 0.00 0 2.00–2.99 2 0.40 2 1.54 1.00–1.99 67 13.45 32 24.62 To 0.99 429 86.14 96 73.85 Overall 498 – 130 26.10 274 Page 12 of 15 Archaeol Anthropol Sci (2020) 12:274 attributed to the extinct bear-dog Borophagus parvus,with a millennia after the deposition of the Skara Brae remains. number of fragmented bones entrapped completely or almost However, as for the pine marten (Martes martes) remains completely within the fossilized faecal matter. However, in from the Neolithic Pierowall Quarry on Westray, Orkney, contrast to the bear-dog, or unidentified canids in other studies such finds could be a sign of a short-lived local population (e.g. Bravo-Cuevas et al. 2017), the Skara Brae dog faeces (Sharples 1984). Evidence for a third carnivore found at Skara contain fewer identifiable remains, most being too finely Brae was the single find of an otter (Lutra lutra) metacarpal. fragmented to permit the recognition of anatomical and taxo- Otters are generally considered to be intrusive at archaeolog- nomical provenance. Bone inclusions from Skara Brae were ical sites, and produce highly characteristic faeces (‘spraint’), also tightly condensed, often with only a small amount of which are clearly morphologically different from the Skara coprolite matrix between the pieces of trabecular bone. Brae coprolitic finds. These differences may be attributed to Borophagus parvus The presence of a hard outer coating is considered a typical being a bone-crushing canid, with specialisations in dentition feature of fossilized carnivore faeces (Bryant 1974). This coat- and digestion to chew and ingest most of the skeletal structure ing corresponds to the mineralised outer layer present on the of their adult prey (Wang et al. 2018). On the other hand, Skara Brae coprolites. As first suggested by Bradley (1946), modern wolves (Canis lupus) and dogs do not exhibit this and confirmed by recent studies (Hollocher et al. 2010; specialism, relying on chewing easily modifiable elements, Pesquero et al. 2014), calcium phosphate and other bone min- often from younger prey, and often smaller bones and long eral content in coprolites contributes to their long-term pres- limb epiphyses (Fosse et al. 2012). ervation. The only intact Skara Brae coprolite without such a The visual methods and proteomics both confirmed the coating still contained bone, but a smaller amount than the prey species as being sheep, whose main skeletal elements other three finds. This appears to confirm the relationship cannot be ingested whole by dogs. As a result, they are poten- between bone content in the creation of an outer coating. It tially not identifiable when sufficiently fragmented to be may also point towards differential diagenetic processes be- swallowed. However, minor skeletal elements, for example tween contexts, although it is currently not known exactly phalanges or vertebrae, could be ingested either complete or how this may contribute to coprolite preservation and mineral in fragments that are large enough to be identifiable. content. Considering that some bones came from immature individuals Considering the findings of the visual and μCT analyses, (as evidenced by the lack of epiphyseal fusion), dogs from Neolithic humans, as omnivores, are unlikely to be depositors Skara Brae were most likely fed by humans; low meat- of the studied coprolites. Human faeces are usually diverse in bearing distal limbs and vertebral spines would have originat- their contents (containing bones, fibres, seeds, etc.) and the ed from butchery products and meal waste. mineralised coating attributable to carnivores does not usually The abundance of highly fragmented and digested bones occur on their surface (Callen 1963; Heizer 1963; Bryant within the Skara Brae coprolites points unmistakably towards 1974; Reinhard and Bryant 1992; Reinhard 2000). However, a typical carnivorous species fed by humans or actively scav- research on excavated coprolites of possible human origin enging human refuse. Such a species is represented at Skara almost exclusively relies on DNA-based identifications (e.g. Brae by domestic dogs (Clarke 1976B; Clarke and Sharples Gilbert et al. 2008; Jenkins et al. 2012; Petrigh and Fugassa 1985; Clarke DV and Shepherd AN pers. comm.). Scattered 2017) or microbiological studies (Cano et al. 2014), with only dog remains were found in multiple contexts from the middle a marginal interest in bone inclusions. As a consequence, there stage of phase 1 until the late stage of phase 2, including the is a lack of visual and μCT data on human coprolites to pro- intermediate period between both phases. Many of these re- vide a comparative dataset for our analysis. Regular human mains were isolated teeth, suggesting deposition from natural coprolite deposition would also be unlikely from the contex- tooth loss rather than death of the animal. tual perspective. Skara Brae had a form of drainage system Although the remains of two other carnivore species were which could be used regularly for removing human faeces also found in Skara Brae (Clarke DV and Shepherd AN pers. (Shepherd 2016; Clarke and Sharples 1985), leading to such comm.), they are less likely to have deposited the coprolites. material being transported outside of the site or gathered as a Red fox (Vulpes vulpes) finds were located in contexts belong- manure for nearby fields (Clarke and Sharples 1985). The ing to the earliest two phases (0 and early/middle 1), and these majority of coprolite finds came from deposits behind and do not correlate with the main bulk of the coprolite finds between settlement structures, areas more likely to be (phase 2). Their remains mainly comprised long limb bones frequented by animals rather than humans. and innominates, along with a single jaw. This suggests the The presence of micromammal remains provides evidence processing of foxes to obtain their pelts. It is also debateable of other processes taking place at the site. Orkney vole re- whether red foxes were living wild on Orkney at that time; mains from Skara Brae coprolites are much smaller than the foxes were most likely present from the Early Iron age until North American pocket gophers (Pappogeomys/ the Late Norse period (Fairnell and Barrett 2006), two Cratogeomys) in canid coprolites studied by Bravo-Cuevas Archaeol Anthropol Sci (2020) 12:274 Page 13 of 15 274 et al. (2017), but show a similar level of preservation, with uncontaminated material from coprolites. Drilled material af- fragments being easily identifiable anatomically and in many fected a smaller area of coprolites but showed too much con- cases also taxonomically. Previous research on the Skara Brae tamination by human proteins. micromammal assemblages found significant concentrations of rodents within the site centre (Trench I) and periphery (TrenchII),possiblyrelated to humanoccupation Conclusions (Romaniuk et al. 2016). Two likely explanations were that these are the remains produced as a by-product of pest The combination of visual assessment, μCT scanning with dig- control, or processing of micromammals as a food source. Dogs may have been fed voles in the absence of addition ital reconstruction, scanning electron microscopy and protein/ to the routine meal or butchery waste; possibly, they were lipid analysis all in one study is presently rare. This approach has proven especially useful in identifying coprolite depositors trained to hunt such animals as a form of pest control, or simply caught and ate them of their own volition. Modern and providing data about their possible diet. A number of bone fragments embedded on or within the coprolite matrix were in- dogs on Orkney are known to hunt rodents, but are rarely reported as eating them (Rose 1975). Micromammal vestigated and identified as the distal limb bones or vertebrae of an ungulate, possibly sheep. Proteomics confirmed the presence bones are also known to appear in human coprolites (Reinhard and Bryant 1992;Reinhard et al. 2007), but of sheep collagen within bone inclusions sampled during the dissection. Some finds also included relatively complete remains in the case of Skara Brae, the coprolite fragments in which inclusions were found did not differ substantially of Orkney voles. Many coprolites had a thick, mineralised outer layer, consistent with a typically carnivorous diet and the inges- from the rest of the coprolite assemblage, established in previous paragraphs to resemble canid deposition. tion of large quantities of skeletal remains. The majority of finds, However, it is unlikely that deposition by dogs, even over including all μCT scanned intact coprolites, were likely deposit- ed by dogs. As the depositor diet shows consumption of the low a longer period of time, was responsible for all of the micromammal finds from the excavations at Skara Brae. meat-bearing elements of domesticated species in the anthropic environment, it is likely that these dogs were routinely fed on the The micromammal inclusions show severe taphonomic alterations of bone and tooth surfaces characteristic of refuse of butchery or meals refuse, or scavenged them. Additional micromammal material could come from occasional carnivore digestion, especially the loss of enamel or den- tine and the thinning or cracking of bone. Previous anal- catches, either as intentional pest-control measures, or subsis- tence practices. ysis of the micromammal skeletal assemblage from Skara Brae (Romaniuk et al. 2016) did not provide such finds. X-ray computed tomography proved to be useful for the study of intact coprolites, rather than fragmented remains. Signs of digestion on the micromammal remains were Study of proteins was useful for assessing bone inclusions scarce and did not follow a pattern that has previously been identified as indicative of any mammalian species. embedded within coprolites, but samples of coprolite matrix itself did not yield significant results. This may indicate the From a methodological perspective, the present study demonstrates the value of a combined approach, successfully necessity of hard tissues for the preservation of prey proteins, as well as the effect of carnivore digestion in removing traces limiting destructive analysis while providing more useful data than reliance on a single method alone. From over 392 g of of depositor proteins. Lipid analysis proved to be more useful in the case of coprolite matrix; however, the method needs to assemblage, only 5.5 g of content was fragmented during the dissection, and only ~ 2.5–3.0 g was destroyed during the be refined, especially with regard to identification and proce- dures for avoidance of contamination. This case study high- proteomics analysis. Enough material remains to replicate this study in the future while X-ray computed tomography en- lights the benefits of non-destructive and comparative studies on coprolitic finds from archaeological sites, and provides a sured non-direct replicability though reinvestigation of the template for future research methodology. μCT data. Of equal importance, the combination of methods provided complementary data and further support for the re- Acknowledgements We would like to thank the National Museums of sults obtained by individual methods. Anatomical identifica- Scotland for access to the Skara Brae remains and vertebrate skeletal tions were based on visual examination combined with digital reference material. Thanks to Dr Stig Walsh, who (along with EP) co- imaging, while taxonomic identifications benefitted from supervised CW during her dissertation. these methods together with proteomics. Taphonomic chang- es could be assessed visually, using μCT, and using SEM Author’s contributions The study was conceived by AAR, EP and JSH. Data collection and analyses were performed by AAR (traditional visual imaging. The molecular analyses were more indicative of analysis), LT (SEM), IB, CW and EP (μCT scanning and digital recon- the dietary component rather than the depositor species, con- struction), MB, BEvD and MPC (proteins & lipids). Figures were com- trary to the reported ability to do the latter. Moreover, dissec- piled by AAR and EP. All authors contributed to the writing of the paper tion proved to be the optimal method to obtain and gave final approval for publication. 274 Page 14 of 15 Archaeol Anthropol Sci (2020) 12:274 Funding AAR was in receipt of a School Doctoral Scholarship from the Buckley M, Collins M, Thomas-Oates J, Wilson JC (2009) Species iden- School of History, Classics and Archaeology, University of Edinburgh. tification by analysis of bone collagen using matrix-assisted laser EP was funded by NERC, grant number NE/L002558/1. CW carried out desorption/ionisation time-of-flight mass spectrometry. Rapid μCT work as part of her undergraduate final dissertation at the University Communications in Mass Spectrometry 23: 3843–3854. https:// of Edinburgh, funded by The School of Biological Sciences, University doi.org/10.1002/rcm.4316 of Edinburgh. Buckley M, Farina RA, Lawless C, Tambusso PS, Varela L, Carlini AA, Powell JE, Martinez JG (2015) Collagen sequence analysis of the extinct giant ground sloths Lestodon and Megatherium. PLoS One Data availability Available data supporting this paper include Excel data 10(11):e0139611. https://doi.org/10.1371/journal.pone.0139611 file (Online Resource 3), R script file with short statistical analysis eCollection 2015 (Online Resource 4), compressed file with X-ray computed tomographic Buckley M, Harvey VL, Chamberlain AT (2017) Species identification data and files containing coprolite 3D digital reconstructions (Online and decay assessment of Late Pleistocene fragmentary vertebrate Resource 5), video showing the internal structure of the intact coprolite remains from Pin Hole Cave (Creswell Crags, UK) using collagen from context 113 (Online Resource 6) and two additional figures (Online fingerprinting. Boreas 46: 402–411. https://doi.org/10.1111/bor. Resource 1 and 2). All files are available online. Bull ID, Lockheart MJ, Elhmmali MM, Roberts DJ, Evershed RP (2002) Compliance with ethical standards The origin of faeces by means of biomarker detection. Environ Int 27(8):647–654 Competing interests The authors declare that they have no competing Callen EO (1963) Diet as Revealed by Coprolites. In: Brothwell D, Higgs interests. E (eds) Science in Archaeology: a survey of progress and research. Thames & Hudson, London, pp 235–243 Open Access This article is licensed under a Creative Commons Cano RJ, Rivera-Perez J, Toranzos GA, Santiago-Rodriguez TM, Attribution 4.0 International License, which permits use, sharing, Narganes-Storde YM, Chanlatte-Baik L, García-Roldán E, adaptation, distribution and reproduction in any medium or format, as Bunkley-Williams L, Massey SE (2014) Paleomicrobiology: long as you give appropriate credit to the original author(s) and the Revealing fecal microbiomes of ancient indigenous cultures. PLoS source, provide a link to the Creative Commons licence, and indicate if One 9(9):E106833. https://doi.org/10.1371/journal.pone.0106833 changes were made. The images or other third party material in this article Carrot J (2011) Assessment of possible coprolites. In: Moore H, Wilson are included in the article's Creative Commons licence, unless indicated G (eds) Shifting sands: links of Noltland, Westray: interim report on otherwise in a credit line to the material. If material is not included in the Neolithic and Bronze Age Excavations 2007-2009. Historic article's Creative Commons licence and your intended use is not Scotland, Edinburgh, p 108 permitted by statutory regulation or exceeds the permitted use, you will Charters S, Evershed RP, Goad LJ, Leyden A, Blinkhorn PW, Denham V need to obtain permission directly from the copyright holder. 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