Is the detection of aquatic environmental DNA influenced by substrate type?

RESEARCH ARTICLE Is the detection of aquatic environmental DNA influenced by substrate type? Andrew S. Buxton*, Jim J. Groombridge, Richard A. Griffiths Durrell Institute for Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Marlowe Building, Canterbury, Kent, United Kingdom * Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Buxton AS, Groombridge JJ, Griffiths RA (2017) Is the detection of aquatic environmental DNA influenced by substrate type? PLoS ONE 12 (8): e0183371. https://doi.org/10.1371/journal. pone.0183371 Editor: Hideyuki Doi, University of Hyogo, JAPAN Received: May 26, 2017 Accepted: August 2, 2017 Published: August 16, 2017 Copyright: © 2017 Buxton 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: The project was undertaken as part of a privately funded PhD project, with additional funding provided by the University of Kent on the original project grant. Competing interests: The authors have declared that no competing interests exist. The use of environmental DNA (eDNA) to assess the presence-absence of rare, cryptic or invasive species is hindered by a poor understanding of the factors that can remove DNA from the system. In aquatic systems, eDNA can be transported out either horizontally in water flows or vertically by incorporation into the sediment. Equally, eDNA may be broken down by various biotic and abiotic processes if the target organism leaves the system. We use occupancy modelling and a replicated mesocosm experiment to examine how detection probability of eDNA changes once the target species is no longer present. We hypothesise that detection probability falls faster with a sediment which has a large number of DNA binding sites such as topsoil or clay, over lower DNA binding capacity substrates such as sand. Water removed from ponds containing the target species (the great crested newt) initially showed high detection probabilities, but these fell to between 40% and 60% over the first 10 days and to between 10% and 22% by day 15: eDNA remained detectable at very low levels until day 22. Very little difference in detection was observed between the control group (no substrate) and the sand substrate. A small reduction in detection probability was observed between the control and clay substrates, but this was not significant. However, a highly significant reduction in detection probability was observed with a topsoil substrate. This result is likely to have stemmed from increased levels of PCR inhibition, suggesting that incorporation of DNA into the sentiment is of only limited importance. Surveys of aquatic species using eDNA clearly need to take account of substrate type as well as other environmental factors when collecting samples, analysing data and interpreting the results. 1. Introduction Environmental DNA (eDNA) is a rapidly expanding method for the detection and survey of aquatic organisms. Targeted species detection from samples of water using qPCR is increasingly being used in local and regional assessments of invasive[1], rare[2] or protected species [3]. The method is also being used to assess changes in site occupancy over time[3,4], where the use of traditional methodologies would be logistically onerous. For both national assessments and localised presence-absence surveys of target species it is important that limitations surrounding the technique and sampling strategy are understood. Indeed, where eDNA fails PLOS ONE | https://doi.org/10.1371/journal.pone.0183371 August 16, 2017 1 / 14 eDNA detection, persistence and substrate to detect a species that is known to have been recently present, understanding the persistence of eDNA is crucial for reliable interpretation of results. Three processes contribute to the removal of eDNA from the aquatic environment, influencing the length of time a target organism can be detected. Firstly, transport in water flows in lotic systems[5] or currents in the marine environment[6]. However, this is unlikely in small lentic waterbodies such as ponds. Secondly, eDNA becomes unavailable for survey as the DNA is degraded through a wide variety of processes [7–11]. Thirdly, eDNA can be transported vertically out of suspension by binding to particulate matter, settling and becoming incorporated into substrates such as clay[8]. The number of binding sites and binding mechanisms within the substrate play a role in its capacity to bind with DNA, with sand having a lower capacity than clay due to particle size[12]. This difference in capacity means that substrate type can potentially alter the amount of DNA available in eDNA surveys. The persistence of aquatic eDNA is highly variable, with reports suggesting anything from a few hours[6] to two months [11] depending on environmental conditions. However, when incorporated into soil sediments, eDNA persistence may be in excess of months[13] or even thousands of years[14,15]. Where decreases in eDNA concentration are observed following the removal of the target organism, a pattern similar to a negative exponential decline has been documented[16–18]. In mesocosm experiments with Idaho giant salamanders (Dicamptodon aterimus), Pilliod et al. [17], show eDNA degradation of between 94% and 98% over the first two days, with the last positive samples found after 11 days. Also using mesocosms, Thomsen et al.[16] monitored eDNA persistence of the common spadefoot toad (Pelobates fuscus) and the great crested newt (Triturus cristatus), with detection persisting between 2 and 9 days. Neither study attempted to look at qPCR limits of detection or quantification[19]. It is therefore unclear whether the negative exponential decline is real or the studies merely reached their limits of quantification at the point where concentration decline appeared to slow. Simply using the length of time during which eDNA remains detectable after the target species is removed does not show how the probability of detecting the species declines over time. Imperfect detection is commonplace within ecological studies causing errors within monitoring programs[20–26], and this is true for eDNA as well as conventional monitoring methods. Errors may arise during collection of the water sample, extraction of the DNA or amplification of the DNA. A false negative result (i.e., not detecting a species when in fact it is present) could result from non-uniform eDNA distribution within a waterbody, low concentration within the water body, degradation during sample transport and storage, PCR inhibition or poor affinity of the genetic assay with the target DNA[27,28]. PCR inhibition is common in environmental s (...truncated)