Predicting provenance of forensic soil samples: Linking soil to ecological habitats by metabarcoding and supervised classification

RESEARCH ARTICLE Predicting provenance of forensic soil samples: Linking soil to ecological habitats by metabarcoding and supervised classification Camilla Fløjgaard ID1☯*, Tobias Guldberg Frøslev ID2☯, Ane Kirstine Brunbjerg1‡, Hans Henrik Bruun3‡, Jesper Moeslund ID1, Anders Johannes Hansen2, Rasmus Ejrnæs1‡ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Section for Biodiversity, Department of Bioscience, Aarhus University, Rønde, Denmark, 2 Natural History Museum of Denmark, Copenhagen, Denmark, 3 Department of Biology, University of Copenhagen, Copenhagen, Denmark ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * Abstract OPEN ACCESS Citation: Fløjgaard C, Frøslev TG, Brunbjerg AK, Bruun HH, Moeslund J, Hansen AJ, et al. (2019) Predicting provenance of forensic soil samples: Linking soil to ecological habitats by metabarcoding and supervised classification. PLoS ONE 14(7): e0202844. https://doi.org/10.1371/ journal.pone.0202844 Editor: Ulrich Melcher, Oklahoma State University, UNITED STATES Received: August 8, 2018 Accepted: June 19, 2019 Published: July 8, 2019 Copyright: © 2019 Fløjgaard 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 data can be found on GitHub (github.com/tobiasgf/provenancing). Funding: We consider this a contribution to a national research project to develop soil forensic methods for Denmark supported by Innovation Fund Denmark Grand Solutions (grant no. 615100002B, https://innovationsfonden.dk/, awarded to AJH) based on data collected in Biowide, a nationwide survey of biodiversity in Denmark supported by the VILLUM foundation (grant no. Environmental DNA (eDNA) is increasingly applied in ecological studies, including studies with the primary purpose of criminal investigation, in which eDNA from soil can be used to pair samples or reveal sample provenance. We collected soil eDNA samples as part of a large national biodiversity research project across 130 sites in Denmark. We investigated the potential for soil eDNA metabarcoding in predicting provenance in terms of environmental conditions, habitat type and geographic regions. We used linear regression for predicting environmental gradients of light, soil moisture, pH and nutrient status (represented by Ellenberg Indicator Values, EIVs) and Quadratic Discriminant Analysis (QDA) to predict habitat type and geographic region. eDNA data performed relatively well as a predictor of environmental gradients (R2 > 0.81). Its ability to discriminate between habitat types was variable, with high accuracy for certain forest types and low accuracy for heathland, which was poorly predicted. Geographic region was also less accurately predicted by eDNA. We demonstrated the application of provenance prediction in forensic science by evaluating and discussing two mock crime scenes. Here, we listed the plant species from annotated sequences, which can further aid in identifying the likely habitat or, in case of rare species, a geographic region. Predictions of environmental gradients and habitat types together give an overall accurate description of a crime scene, but care should be taken when interpreting annotated sequences, e.g. due to erroneous assignments in GenBank. Our approach demonstrates that important habitat properties can be derived from soil eDNA, and exemplifies a range of potential applications of eDNA in forensic ecology. Introduction In ecological studies, bioindication is routinely used to infer environmental conditions and ecosystem properties, and to classify vegetation types [1–3]. The link between species and PLOS ONE | https://doi.org/10.1371/journal.pone.0202844 July 8, 2019 1 / 16 Predicting provenance of forensic soil samples VKR-023343, https://veluxfoundations.dk/, awarded to RE). 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. environmental conditions is also the basis of the application of ecology in forensic science [4]. In a wide range of disciplines–such as palynology, botany and entomology–pollen, plant fragments or insect remains are identified or analyzed by experts to impart ecological information to criminal investigations [5–8]. Similarly, forensic geoscience builds on the geological disciplines of inorganic soil analysis, i.e., soil classification, mineralogy, soil chemistry and physics [9]. Soil is commonly encountered as trace evidence in criminal cases, i.e. mud sticking to footwear, tires and shovels, soil splash marks on vehicles, and traces left on clothes, the floor or in the trunk of vehicles. Those soil samples can be compared to samples from known locations, where an offence is thought to have occurred, thereby establishing a link between a suspect or a victim and a crime scene. In an investigative process, where for example the crime scene is unknown, soil trace evidence can also give valuable information on geographic origin or provenance and help narrow the search for a location. However, inorganic soil properties tend to vary at regional scales, which limits the precision of soil sample provenance [10], but recent digital signatures from x-ray fluorescence have demonstrated a potential for high precision prediction accuracy for local scale soil provenance [11]. Soil contains DNA from the living (and dead) biota below and above ground. Plants are rooted in the soil, whereas fungi and other microorganisms live in the soil, and animals live, dwell or dig through the soil, leaving DNA traces behind. Even organisms living entirely above ground leave DNA in the soil as they defecate, exude secretions or die and decay. Extracting DNA from soil has been in use for decades in microbiology for characterization of bacterial soil communities [12]. Also, microbial profiles from DNA analysis have been used as a fingerprinting tool in forensic investigations [13, 14]. With the development of metabarcoding of environmental DNA from soil (from here onwards: soil eDNA; [15]), high-throughput sequencing of marker genes allow simultaneous detection of multiple species in a complex DNA extract from a single sample. These methods have rapidly found application in ecology [16], in which DNA metabarcoding data are used in ecological studies and conservation biology to identify communities or assess biodiversity [17, 18]. Recently, these methods have also been employed in forensic ecology. Examples include a “biological signature” of annotated taxa in soil DNA used to describe vegetation characteristics [19] and “fingerprints” of sequence composition used to match forensic soil samples [20]. Besides the latter app (...truncated)