Fish ’n chips: ZooMS peptide mass fingerprinting in a 96 well plate format to identify fish bone fragments

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.     

A new statistical approach for determining the crystallinity of heat-altered bone mineral from FTIR spectra

The crystallinity of bone mineral represents an established method of measuring heat-induced change and is of importance to fields including material science, clinical science, anthropology and archaeology. A commonly used technique involves the calculation of the Crystallinity Index (CI) using selected peaks from Fourier Transform Infrared (FTIR) absorbance spectra. However, the choice of peaks has to date not been statistically justified. In this study a novel application of statistical techniques to the FTIR spectra of bone samples burned in the range 100 °C–1100 °C has been used to identify 5 new spectral indices of heat-induced crystallinity change. The validity of the new indices was tested by using a statistical classification model (LDA) to predict the burning temperature of a set of 108 bone samples burned between 100 °C and 1100 °C. A correct classification rate (CCR) of 97.2% was obtained when a combination of 4 indices including the CI was used. This was significantly better than the CCR of 66.7% which was obtained when the CI was used on its own.     

Brackish water foraging: isotopic landscapes and dietary reconstruction in Suisun Marsh,Central California

While bone stable isotope signatures of humans foraging in open coastal and fully terrestrial environments have been relatively well documented, the signature of foraging near brackish-water estuaries is less well known. This study examines the Central California isotopic landscape, or isoscape, showing how foods in different ecosystems have distinctive isotopic compositions. We present new bone collagen and apatite stable isotope data from two Late Holocene sites (CA-SOL-11 and CA-SOL-69) near Suisun Marsh, a low salinity brackish water estuary upstream from San Francisco Bay. Faunal remains at the sites include significant numbers of aquatic resources including fish and shellfish, in addition to terrestrial mammals, while paleobotanical remains suggest mainly terrestrial nuts and seeds. Stable isotope data at the population level corroborate this finding and show consumption of significant levels of brackish water nutrients, including both dietary protein and dietary carbohydrates and fats, paired with terrestrial foods. This dietary focus generates a unique brackish-water isotopic signature that is distinct from others in Central California. The documentation of an isoscape with strong regional gradients can be of tremendous help in reconstructing ancient patterns in subsistence, migration, and trade.     

Investigation of the ‘canopy effect’ in the isotope ecology of temperate woodlands

Anomalously ¹³C-depleted carbon stable isotope values from closed forest ecosystems have been termed the ‘canopy effect’. Originally this was ascribed to recycling of depleted carbon from forest floor decomposition of organic material, although others have suggested that it is equally likely to be due to variations in leaf-level processes in response to increased shade. This depletion in the heavier carbon isotope is passed on to woodland herbivores feeding within the forest environments. A similar isotopic depletion has also been reported in the archaeological literature from more open temperate woodland settings, but no measurements have been made on the plants at the base of the food chain in order to quantify the effect. In this study we attempt this by examining the carbon and nitrogen stable isotopic values of different species of grasses from a range of open to closed habitat settings within Wytham Wood, Oxfordshire, UK. We find a strong relationship between carbon isotopic depletion of plant tissue and lowered light intensity with an up to 5‰ shift between grass grown in open and closed locations. In order to follow this up the food chain, we also report data on wool from sheep grazing in open pastures near the Wood, and on fallow deer living within the woodland, but which turn out not to show a strong canopy effect, probably related to their feeding strategies. We conclude that there is indeed a strong ‘canopy effect’ in temperate woodland, probably related to differential light levels, but that not all apparently woodland-dwelling mammals show such an effect. We also show considerable isotopic variation at the base of the food chain, which should counsel caution when attempting to interpret dietary isotopes using mixing models.     

Experimental patterns of hammerstone percussion damage on bones: implications for inferences of carcass processing by humans

The common occurrence of hammerstone percussion damage (pits, striae, notches and impact flakes) on the fossil limb bones of ungulates indicates that marrow extraction has been an important component of hominid butchery for over two million years. Beyond this level of basic inference, it would be behaviorally informative if three deeper aspects of marrow harvesting were understood more clearly: (1) whether inter-element patterns of bone fragmentation vary when processing intensity is held constant; (2) whether butcher investment in marrow extraction correlates positively with the number of percussion marks generated; (3) whether taphonomic effectors can be identified based on percussion mark morphology, frequency and placement. Some experimental work has been conducted previously in service of exploring these questions, but we set out here to address them explicitly through the analysis of a large sample of white-tailed deer (Odocoileus virginianus) limb elements fractured by hammerstone percussion. Our results indicate that (1) measures of bone fragmentation, which supposedly reflect processing intensity, are highly contingent on the research question being posed. This stresses the fact that researchers must be explicit in their definition of processing intensity. (2) In addition, hypothesized covariance between number of hammerstone blows and percussion mark frequencies are not met in our sample, corroborating previous conclusions of a lack of covariance between cutting strokes and cutmark frequencies. These results highlight the contingent nature of butchery mark production, and emphasize the need to investigate carcass resource exploitation by posing questions that do not rely on mark frequencies, but instead utilize other zooarchaeological measures. (3) Finally, our results—showing high incidences of impact notches and flakes created by direct anvil contact and “anvil scratches” created by direct hammerstone contact—suggest caution in using specific categories of percussion damage to infer their taphonomic effectors.     

Foddering by Mongolian pastoralists is recorded in the stable carbon (δC) and nitrogen (δN) isotopes of caprine dentinal collagen

Direct identification of fodder provisioning in individual animals is a previously unexplored area that has important implications for determining animal husbandry strategies in prehistory. In this study, foddering practices are examined in modern domestic and wild caprines from Mongolia (Baga Gazar'in Chuluu) through carbon and nitrogen analyses of bulk sampled dentin collagen in molar teeth. Foddered caprines display significantly different δ¹³C values compared to non-provisioned caprines. This pattern reflects the consumption of C₄ plants year-round by provisioned caprines while non-provisioned caprines ingest C₄ plants only during the summer months. Provisioned caprines also display significantly different nitrogen isotopic values relative to non-provisioned animals. The difference in nitrogen isotopes between wild and foddered animals may be due to increased access in quality graze through foddering by domestic caprines, which results in improved nutrition and extended lactation periods, or enriched δ¹⁵N of fodder plants relative to background flora. Differences in tooth wear between sampled wild and foddered animals may also contribute to the observed δ¹⁵N differences.     

Stable isotope evidence for palaeodiets in southern Turkmenistan during Historical period and Iron Age

The subsistence patterns of Iron Age and Historical period humans from south-western Turkmenistan have been reconstructed using the carbon and nitrogen isotopic compositions of archaeological faunal and human bones. A qualitative comparison of the isotopic signatures points to a small proportion of ruminant meat and dairy in human diet for both periods. The ranges of proportions of dietary items yielded by a quantitative approach based on concentration dependent mixing models confirm the high proportions of plant food in the average diet, and show little change in the reconstructed diet for both periods. A comparison of results from zooarchaeological and isotopic approaches illustrates their complementarity in subsistence patterns reconstruction.     

Assessing the distribution of Asian Palaeolithic sites: a predictive model of collagen degradation

Having previously shown that there is a statistically significant relationship between areas of low collagen degradation and the location of archaeological sites in Africa [K.M. Holmes, K.A. Robson Brown, W.P. Oates, M.J. Collins, Assessing the distribution of African Palaeolithic sites: a predictive model of collagen degradation, Journal of Archaeological Science 32 (2005) 157–166], we here test the applicability of the same model on Palaeolithic archaeological remains from Asia.     

Confocal laser scanning microscopy: a flexible tool for simultaneous polarization and three-dimensional fluorescence imaging of archaeological compact bone

Wide-field polarized light and epifluorescence microscopy have been used to enhance analysis of archaeological bone tissue, providing information on bone formation, modeling, pathology, preservation, age estimation, and biomechanics. Though valuable, these techniques are limited by their inability to remove out-of-focus light and view multiple levels of a sample, restricting our understanding of the three-dimensional (3-D) microarchitecture of compact bone. Modern technological advances, such as microscopic computerized tomography, allow increasing resolution in 3-D bone imaging, but do not allow fluorescence labeling or polarized-light analysis. Confocal laser scanning microscopy (CLSM) is a valuable tool for 3-D histology. However, its application to the study of compact bone is lacking, especially in archaeological and forensic sciences. The current study investigated CLSM as a tool for fluorescence and polarized-light microscopy of archaeological compact bone in order to demonstrate its advantages. Standard techniques and CLSM are compared in their suitability for imaging well preserved archaeological bones from the Dakhleh Oasis, Egypt. CLSM's high resolution, multi-channel, two- and three-dimensional capabilities augment the flexibility and creativity of compact bone imaging and have the potential to increase the accuracy of quantitative medical and anthropological histomorphometric techniques. CLSM is specifically suggested as a useful tool for the investigation of ancient bone fluorescence caused by the presence of tetracycline and/or other fluorochromes.     

Palaeoenvironmental and chronological investigations of the Magdalenian sites of Goyet Cave and Trou de Chaleux (Belgium), via stable isotope and radiocarbon analyses of horse skeletal remains

Trou de Chaleux and Goyet are caves situated less than 30 km apart in Belgium that contain stratified Magdalenian artefacts and butchered faunal remains. Published radiocarbon dates suggest that both sites were contemporaneously occupied during to the Late-glacial interstadial. It has previously been suggested that the Trou de Chaleux Late-glacial faunal remains might be slightly older than those at Goyet Cave, and that Trou de Chaleux has two phases of occupation during the Late-glacial interstadial. However, the limited number of radiocarbon dates currently available makes it impossible to determine whether the sites are truly contemporaneous, and the assessment of their absolute chronologies is complicated by a plateau in the calibration curve. In this paper, bone collagen δ¹³C and δ¹⁵N signatures of horse bones from the two sites are used to reconstruct local palaeoenvironments. We hypothesise that if occupations at the two sites were contemporaneous, the horse collagen δ¹³C and δ¹⁵N isotope signatures from both sites will be similar, since comparable carbon and nitrogen isotopic values would reflect similar diets, ecologies and environments. To provide clear dating parameters, new AMS radiocarbon determinations are also presented. Results show that horse collagen δ¹³C and δ¹⁵N isotope signatures at the two sites are different, indicating that the populations were not subject to similar diets, ecologies and environments. The new radiocarbon dates confirm that the horse bones from Trou de Chaleux date to the Late-glacial interstadial, but they also show that in Goyet Cave Horizons 1 and 2, older horse remains dating to ca. 32?000–27?000 ¹⁴C yr BP are mixed with Late-glacial horse remains. The δ¹⁵N values of the Goyet Cave horse bones radiocarbon dated to the Pleniglacial are substantially higher than those that have Late-glacial radiocarbon dates. Therefore, we now hypothesise that the majority of the horse bones isotopically analysed from Goyet Cave Horizon 1 are Pleniglacial in age. Statistical analysis of the radiocarbon dates indicates that the Late-glacial occupation at the two sites was contemporaneous, and that while there may appear to be two phases of occupation at Trou de Chaleux, this may be an artefact of the radiocarbon calibration curve.