Faecal biomarkers can distinguish specific mammalian species in modern and past environments

RESEARCH ARTICLE Faecal biomarkers can distinguish specific mammalian species in modern and past environments Loïc Harrault ID1,2,3¤*, Karen Milek ID1,2☯, Emilie Jardé4, Laurent Jeanneau4, Morgane Derrien ID5, David G. Anderson ID6☯ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Archaeology, Durham University, Durham, United Kingdom, 2 Department of Archaeology, University of Aberdeen, Aberdeen, United Kingdom, 3 The James Hutton Institute, Craigiebuckler, Aberdeen, United Kingdom, 4 Université Rennes, CNRS, Géosciences Rennes, UMR 6118, Rennes, France, 5 Department of Environment and Energy, Sejong University, Seoul, South Korea, 6 Department of Anthropology, University of Aberdeen, Aberdeen, United Kingdom ☯ These authors contributed equally to this work. ¤ Current address: Sorbonne Université, CNRS, EPHE, PSL, UMR METIS, France. * Abstract OPEN ACCESS Citation: Harrault L, Milek K, Jardé E, Jeanneau L, Derrien M, Anderson DG (2019) Faecal biomarkers can distinguish specific mammalian species in modern and past environments. PLoS ONE 14(2): e0211119. https://doi.org/10.1371/journal. pone.0211119 Editor: Juan J. Loor, University of Illinois, UNITED STATES Received: June 5, 2018 Accepted: January 8, 2019 Identifying the presence of animals based on faecal deposits in modern and ancient environments is of primary importance to archaeologists, ecologists, forensic scientists, and watershed managers, but it has proven difficult to distinguish faecal material to the species level. Until now, four 5β-stanols have been deployed as faecal biomarkers to distinguish between omnivores and herbivores, but they cannot distinguish between species. Here we present a database of faecal signatures from ten omnivore and herbivore species based on eleven 5β-stanol compounds, which enables us to distinguish for the first time the faecal signatures of a wide range of animals. We validated this fingerprinting method by testing it on modern and ancient soil samples containing known faecal inputs and successfully distinguished the signatures of different omnivores and herbivores. Published: February 7, 2019 Copyright: © 2019 Harrault et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research is part of the HUMANOR project funded by the JPI Climate Consortium by the Economic and Social Reasearch Council (ESRC, ES/M011054/1) obtained by D. A. and K. M. and by the European Reasearch Council (ERC) Advanced Grant 295458 Arctic Domus obtained by D. A., both based at the University of Aberdeen. ESRC: https://esrc.ukri.org/ ERC: https://erc. Introduction The signatures of animals in the environment, or on an archaeological site, can be detected by the faecal material they leave behind. Archaeologists, forensic scientists, ecologists, watershed managers and others make use of the organic residues derived from faecal inputs in the environment to determine the presence of animals and/or human activities [1–10] or to pinpoint whether animal faeces were a source of organic nutrients (e.g. in arable soils, [11–18]) or pollutants in catchment basins (e.g. a source of pathogenic bacteria, viruses and protozoa, [19– 31]). Particularly useful are a class of lipids known as 5β-stanols, which are direct biomarkers of animal faeces, and have the important advantage of long-term preservation in soils and sediments due to their low solubility in water and their ability to bind to particulate organic matter [25]. Their distribution in faecal material, called a stanol fingerprint, identifies a particular PLOS ONE | https://doi.org/10.1371/journal.pone.0211119 February 7, 2019 1 / 26 Faecal biomarkers identify mammal species europa.eu/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. mammalian species on the basis of its diet (main sterol uptake), its ability to biosynthesize endogenous sterols (secondary sterol uptake) and the way it biohydrogenates sterols and converts them into stanols with the help of digestive tract bacteria (intestinal flora) [19]. The most common techniques used for stanol fingerprinting are currently able to identify whether faeces belong to carnivores, omnivores or herbivores, but do not allow identification of the genus or species. Mainly because of their respective diets, cholesterol-derived 5β-stanols are found in high proportions in the faeces of omnivores and carnivores (coprostanol and epicoprostanol), while 5β-stanols derived from β-sitosterol, a phytosterol (plant sterol), are found in high proportions in herbivore faeces (24-ethylcoprostanol and 24-ethylepicoprostanol) [1]. In order to improve the distinction between the faecal signature of different mammal species in modern [16, 17, 20, 21, 24, 28, 29] and ancient [2, 3, 5, 7, 9, 11–15, 18, 32–34] environments, efforts have been made to develop the technique of faecal steroid biomarker analysis, mainly focused on the ratios of the four above-mentioned important 5β-stanols, sometimes in combination with the analysis of another group of faecal steroids, bile acids. However, the use of ratios calculated from four compounds has significant limitations. Ratio thresholds can and frequently do overlap, making it impossible to distinguish between species [22]. The recent introduction of new 5β-stanol ratios, used in combination with bile acids, has improved the ability to identify some species on the basis of their faeces (e.g. sheep and goats), but even this new method fails in contexts where several species have potentially mingled [10]. A small number of previous studies have applied multivariate analyses of a wider range of steroids, including sterols, 5α- and 5β-stanols and stanones, to deepen the investigation of variations between the faecal fingerprints of different species [4, 19, 23, 26, 27, 31]. However, the sterols and 5α-stanols used in these studies are naturally found in the environment. Sterols and to a lesser extent 5α-stanols may be direct components of soil fauna and vegetation, and 5α-stanols and stanones can also be microbially-mediated degradation products of sterol precursors, which limits their use for faecal fingerprinting [10]. On the other hand, 5β-stanols found in soils almost entirely come from the endogenous biohydrogenation of sterols within the gastrointestinal tract of higher animals, making them useful as biomarkers to identify faecal inputs in soils ([10] and references therein). For faecal fingerprinting with stanols, it is therefore important to restrict the analysis to 5β-stanols alone. We h (...truncated)