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ABSTRACT Environmental DNA (eDNA) has evolved into a valuable asset of the ecologists' toolkit, enabling time‐ and cost‐efficient biodiversity assessments in a wide variety of ecosystems. Since eDNA can be isolated from a broad range of environmental substrates, its persistence times in those media are of decisive importance for drawing inferences about species presence. For the first time, we characterize eDNA persistence in water and sediment samples of seminatural waterholes in a savanna system in South Africa to gain a better understanding of eDNA decay in these waterbodies. Using mesocosm experiments, we tracked eDNA decay in two different DNA states (extracellular and membrane bound), during two different seasons (wet and dry), and from two different substrates (surface water and sediment). Extracellular DNA degraded rapidly in a first‐order exponential decay fashion and membrane‐bound DNA exhibited a slower decline with more intricate patterns, involving initial reduction followed by a subsequent increase in measured DNA concentrations. The latter we attribute to cell disassociation and cell lysis at 24–48 h after introduction into the environment. Higher stochasticity of membrane‐bound DNA capture in the dry season highlights the need for higher sampling efforts in natural systems in which eDNA is presumably more patchily distributed. Additionally, we observed longer eDNA persistence in sediments than in water samples, presumably due to better protection from nucleases. We could not reveal any effects of environmental parameters on eDNA decay, emphasizing that further research is needed to better understand eDNA dynamics in those waterbodies in order to exploit their full potential for eDNA‐based bioassessments in those systems.

Abstract Environmental DNA (eDNA) methods are increasingly applied in the marine environment to identify species and community structure. To establish widely applicable eDNA techniques for elasmobranchs, we used the Critically Endangered largetooth sawfish (Pristis pristis Linnaeus, 1758) as a model species for: (1) assessing eDNA particle size distribution; (2) assessing the efficiency of long‐term preservation of water samples; and (3) comparing the efficiency and detection sensitivity of filtration and precipitation methods. Water samples (1 L) collected from a tank containing one largetooth sawfish specimen were sequentially filtered through five filter membranes of decreasing pore size (20, 10, 5, 1.2, and 0.45 μm). The proportion of sawfish eDNA retained within each size class was determined through quantitative real‐time PCR (qPCR) using a species‐specific TaqMan probe assay. A linear mixed‐effects model (lme) showed that the 1.2 and 20 µm filters captured most of the eDNA particles present in the sampled water. Additionally, whole water samples (0.375 L) were preserved in Longmire's buffer, stored at tropical ambient temperatures (26.3°C ± 3.0 SD) and extracted at five time points: immediately, one, two, and three months after collection, as well as frozen and extracted three months later, to assess the preservation efficiency of Longmire's buffer via qPCR analysis. A linear mixed‐effects model showed that samples maintained maximal eDNA yield for at least three months after collection at ambient storage. Lastly, when comparing the filtration and precipitation methods, filtration using 0.45 µm pore size was more sensitive to capture of largetooth sawfish eDNA than filtration with 20 µm filter or water precipitation. However, water precipitation was more efficient when accounting for volume of water processed. These results provide options for best capture and preservation of elasmobranch eDNA.

Environmental DNA (eDNA) has seen a massive increase in application in freshwater systems with a concurrent growth in protocol developments and a drive to gain a better understanding of the 'ecology' of eDNA. This raises the question of whether we are currently still in an early, developmental phase of eDNA-based assessments or already transitioning into a more applied stage for biomonitoring. I conducted a systematic literature review on 381 eDNA-focused studies in freshwater systems targeting macro-organisms over the last 5 years, assessing study goals, methods, target systems and taxa and study design aspects. The results show an increase of biomonitoring-focused studies throughout the years, while the fraction of studies investigating the 'ecology' of eDNA decreased. The application of metabarcoding significantly increased while studies applying qPCRs tentatively declined. A geographic inequality was observed concerning study numbers and study goals biased towards the global North. Descriptive studies increased, but the fraction of in-field studies and studies applying eDNA and conventional methods combined revealed no trend. These results show a shift towards application-focused work for eDNA-based assessments but also reveal this field to still be developing. In this transitional phase, practitioners need to ensure consistency and data comparability for long-term monitoring programmes. [ABSTRACT FROM AUTHOR]

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra‐organismal DNA from macro‐organisms. We agree with this view. However, we are concerned that their proposed two‐level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra‐organismal) from which ecological interpretations are derived. [ABSTRACT FROM AUTHOR]

Environmental DNA (eDNA) is gaining traction in conservation ecology as a powerful tool for detecting species at risk. We developed a quantitative polymerase chain reaction assay to detect a DNA amplicon fragment of the mitochondrial nicotinamide adenine dinucleotide locus of the Blanding's turtle ( Emydoidea blandingii ) for detecting overwintering individuals. Seventy-eight water samples were collected from 17 wetland sites in Ontario, Canada. We used traditional field data to identify a priori positive and negative control sites. Fifty percent of positive control sites amplified. Detection was related to the number of individuals estimated from field observations in at least one region surveyed. Positive control sites had lower total dissolved solids and electrical conductivity in relation to negative control sites. Shedding rates were within the same order of magnitude for brumating and active turtles. We recommend collecting additional samples at a larger number of locations to maximize detection. Recommended sampling design changes may overshadow the additional effects of water chemistry and low eDNA shedding rates. eDNA offers tremendous potential to practitioners conducting species at risk assessments in environmental consulting by providing a faster, more efficient method of detection compared with traditional surveys.