Quality control for modern bone collagen stable carbon and nitrogen isotope measurements

Methods in Ecology and Evolution published by John Wiley & Sons Ltd on behalf of British Ecological Society Isotopic analyses of collagen, the main protein preserved in subfossil bone and tooth, has long provided a powerful tool for the reconstruction of ancient diets and environments. Although isotopic studies of contemporary ecosystems have typically focused on more accessible tissues (e.g. muscle, hair), there is growing interest in the potential for analyses of collagen because it is often available in hard tissue archives (e.g. scales, skin, bone, tooth), allowing for enhanced long-term retrospective studies. The quality of measurements of the stable carbon and nitrogen isotopic compositions of ancient samples is subject to robust and well-established criteria for detection of contaminants and diagenesis. Among these quality control (QC) criteria, the most widely utilized is the atomic C:N ratio (C:NAtomic), which for ancient samples has an acceptable range between 2.9 and 3.6. While this QC criterion was developed for ancient materials, it has increasingly being applied to collagen from modern tissues. Here, we use a large survey of published collagen amino acid compositions (n = 436) from 193 vertebrate species as well as recent experimental isotopic evidence from 413 modern collagen extracts to demonstrate that the C:NAtomic range used for ancient samples is not suitable for assessing collagen quality of modern and archived historical samples. For modern tissues, collagen C:NAtomic falling outside 3.00–3.30 for fish and 3.00–3.28 for mammals and birds can produce systematically skewed isotopic compositions and may lead to significant interpretative errors. These findings are followed by a review of protocols for improving C:NAtomic criteria for modern collagen extracts. Given the tremendous conservation and environmental policy-informing potential that retrospective isotopic analyses of collagen from contemporary and archived vertebrate tissues have for addressing pressing questions about long-term environmental conditions and species behaviours, it is critical that QC criteria tailored to modern tissues are established

Seaweed‐eating sheep show that δ 34 S evidence for marine diets can be fully masked by sea spray effects

RATIONALE: Stable sulfur isotope compositions (δ34 S values) are a useful marker of terrestrial (lower δ34 S) versus marine (higher δ34 S) diets. In coastal areas, 34 S-enriched sea spray can obscure these marine/terrestrial differences. We sought to establish whether δ34 S values of sea spray-affected terrestrial fauna can be distinguished from those of marine-feeding terrestrial fauna. METHODS: We measured bone and dentine collagen δ34 S values, as well as stable carbon (δ13 C) and nitrogen (δ15 N) isotope compositions via continuous flow-elemental analysis/isotope ratio mass spectrometry of 21 sheep (Ovis aries) raised on < 7 km2 island (North Ronaldsay, UK) that had widely divergent access to marine (seaweed) and heavily sea spray-affected terrestrial (grass) food sources. We also analyzed the bone collagen of marine and terrestrial fauna from this island. RESULTS: Sheep bone collagen showed well-defined trends with highly significant correlations between δ13 C and δ15 N values indicative of feeding along a continuum of fully terrestrial to fully marine diets, consistent with other modern baseline data from marine and terrestrial animals in the same area. In contrast, the δ34 S value was generally elevated for all sheep and was not significantly correlated with either δ13 C or δ15 N. CONCLUSIONS: Our findings demonstrate that δ34 S values are poorly suited to differentiate marine and terrestrial diets in terrestrial animals in areas with pronounced sea spray effects. Care must be taken to characterize the isotopic compositions of potential food items before the δ34 S value is used as a marker of the reliance on marine protein in modern and ancient contexts

Improved quality control criteria for stable carbon and nitrogen isotope measurements of ancient bone collagen

The carbon (δ13C) and nitrogen (δ15N) isotopic compositions of bone and tooth collagen provide a powerful tool for studying past biological, environmental, and cultural phenomena. Collagen has a well-understood chemical composition that has enabled the development of invaluable quality control (QC) criteria for isotopic data – something that is extremely rare among biogeochemical research on ancient biomolecules as a whole. The most important of these collagen QC criteria is atomic C:N ratio (ratio of carbon to nitrogen atoms), which provides an indicator of the extent to which the amount of carbon and nitrogen present in a sample matches the known composition of pure collagen, thereby indicating whether contamination or diagenesis may be influencing a sample's isotopic compositions. We present a model describing the relationship between the carbon and nitrogen isotopic and elemental compositions that accounts for the isotopic composition of the endogenous collagen and exogenous contaminants as well as taxon-specific information about the collagen's amino acid composition. In some cases the traditional C:N QC parameters are applicable, while in others they can result in the inclusion of unreliable (altered) isotopic data primarily due to contamination from humic substances. Using new and previously published data on taxa commonly encountered in ancient studies, we further illustrate how using traditional C:N QC parameters may lead to the inclusion of altered isotopic compositions in real archaeological and paleontological scenarios. We argue that the traditional ‘one size fits all’ approach to the C:N QC criterion should be avoided and we outline new collagen QC criteria specific to certain taxa and environments on the basis of the results of our model. These revised criteria will help to improve the interpretation of isotopic data by more accurately identifying samples with isotopic compositions altered by contamination

Descriptive statistics for C, N and Zn stable isotopes.

<p>SD = standard deviation, n = number of samples, TL = trophic level.</p

ISOTOPIC ANALYSES REVEAL GEOGRAPHICAL AND SOCIOECONOMIC PATTERNS IN HISTORICAL DOMESTIC ANIMAL TRADE BETWEEN PREDOMINANTLY WHEAT- AND MAIZE-GROWING AGRICULTURAL REGIONS IN EASTERN NORTH AMERICA

Historical zooarchaeologists have made significant contributions to key questions about the social, economic, and nutritional dimensions of domestic animal use in North American colonial contexts; however, techniques commonly employed in faunal analyses do not offer a means of assessing many important aspects of how animals were husbanded and traded. We apply isotopic analyses to faunal remains from archaeological sites to assess the social and economic importance of meat trade and consumption of local and foreign animal products in northeastern North America. Stable carbon and nitrogen isotope analyses of 310 cattle and pigs from 18 rural and urban archaeological sites in Upper Canada (present-day southern Ontario, Canada; ca. A.D. 1790-1890) are compared with livestock from contemporary American sources to quantify the importance of meat from different origins at rural and higher- and lower-status urban contexts. Results show significant differences between urban and rural households in the consumption of local animals and meat products acquired through long-distance trade. A striking pattern in urban contexts provides new evidence for the social significance of meat origins in historical Upper Canada and highlights the potential for isotopic approaches to reveal otherwise-hidden evidence for social and economic roles of animals in North American archaeology

Compar. Study of the Health Stat of Czech Resid. and the Resid.of Finland

<p>A. Relationship between the Zn concentration and δ⁶⁶Zn values of cancellous (black circles) and cortical part of bones of walruses (circles) and other mammals (crosses), found in surface (green colors) and subsurface deposits (black colors). All the cancellous bone samples fall on a regression line. B. δ⁶⁶Zn values of cancellous and cortical fractions of walrus bones. Lines define the average and two standard deviation area of cortical values. Green rectangles frame values for same individuals.</p

Cortical bone isotopic values of arctic mammals for C, N, and Zn.

<p><i>A</i>) δ⁶⁶Zn_bioapatite vs. δ¹³C_collagen values for four species of marine mammals (B) δ⁶⁶Zn_bioapatite vs. δ¹⁵N_collagen values, (<i>C</i>) δ¹⁵N_collage vs. δ¹³C_collagen and (<i>D</i>) 3D plot showing that Zn, N and C isotopic compositions of marine mammals enables a distinguishing of the different species. Measurement uncertainties are given by the white boxes.</p

Expected distribution of δ⁶⁶Zn values in animal tissues based on values obtained in modern terrestrial food webs and experimental animals [14,15,16].

<p>Expected distribution of δ⁶⁶Zn values in animal tissues based on values obtained in modern terrestrial food webs and experimental animals [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152299#pone.0152299.ref014" target="_blank">14</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152299#pone.0152299.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152299#pone.0152299.ref016" target="_blank">16</a>].</p

Map showing the location of the site, Zn-Pb deposits (green circles) and the Polaris mine.

<p>The map is adapted from the one of Le Moine et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152299#pone.0152299.ref029" target="_blank">29</a>].</p

Effects of lipid extraction and ultrafiltration on stable carbon and nitrogen isotopic compositions of fish bone collagen

RATIONALE: Fish bone collagen isotopic measurements are increasingly important in palaeodietary and paleoenvironmental studies yet differences in the chemical and physical properties of fish relative to other vertebrate bones are rarely considered. Lipid content in fish bone, which can exceed 50%, may underlie the poor collagen integrity criteria typically observed in archaeological studies. METHODS: We compare stable carbon and nitrogen isotopic and elemental compositions of bone collagen prepared using four different methods from a wide range of modern fish species to: (1) assess the extent to which lipid content influences bone collagen δ13C and δ15N values, and (2) evaluate the relative efficacy of chemical (2:1 chloroform/methanol) and physical (30 kDa ultrafilters) methods for removing lipids from bones. RESULTS: Lower δ13C values were observed when the lipid content exceeded 5% of the initial bone mass. The lipid content did not influence the δ15N values. 30 kDa ultrafiltration, a common pretreatment for purifying archaeological collagen, removed fewer lipids and was associated with reduced collagen yields (37% loss) as well as altered amino acid compositions. In contrast, collagen prepared using a 2:1 chloroform/methanol lipid extraction step resulted in significantly improved collagen yields, elemental compositions, and isotopic measurements relative to a control treatment. CONCLUSIONS: The chemical lipid extraction method (2:1 chloroform/methanol) performed significantly better than the physical lipid extraction method (30 kDa ultrafilters). Given the high quantities of lipids in fish bones we recommend the inclusion of a chemical lipid extraction step when isolating collagen from modern and archaeological fish bones.</div