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Predation is an important species interaction to monitor when assessing an invasive species' impact on a particular ecosystem, but it can be difficult to observe and thus, fully understand. On Kaua'i island, invasive Barn Owls (Tyto alba) predate native seabirds, but difficult terrain in this region and the cryptic nature of owl predation make traditional monitoring of predation quite challenging. Using Barn Owls collected as part of removal efforts on Kaua'i and Lehua islands, we conducted DNA metabarcoding of owl digestive tracts to detect and determine seabird species they predate. We used a seabird-targeted 12s marker to sequence 112 swabs from 55 owls and detected 6 seabird taxa, including 2 ESA-listed seabirds—Hawaiian Petrel (Pterodroma sandwichensis) and Newell's Shearwater (Puffinus newelli), in 12 swabs from 11 owls (20% of sampled owls). Corresponding morphological assessment of owl stomach contents detected seabird species as prey items in only 2% (1/55) of sampled owls, highlighting the utility of molecular approaches for detecting diet items, especially degraded or visually absent items. Additionally, this approach has proven very useful in revealing cryptic trophic interactions in inaccessible seabird populations. For the most comprehensive analysis of diet, the use of both esophageal and cloacal swabs for metabarcoding is recommended. Supplementing metabarcoding with other methods that can provide complementary prey information, such as stable isotope analysis, would help to characterize trophic interactions more fully. The method described here has proven to be a reliable tool for investigating diet in invasive owls and may be used to investigate cryptic predation in living birds as a minimally invasive technique, as well. [ABSTRACT FROM AUTHOR]

Taxon‐specific quantitative PCR (qPCR) assays are commonly used for environmental DNA sampling‐based inference of animal presence. These assays require thorough validation to ensure that amplification truly indicates detection of the target taxon, but a thorough validation is difficult when there are potentially many non‐target taxa, some of which may have incomplete taxonomies. Here, we use a previously published, quantitative model of cross‐amplification risk to describe a framework for assessing qPCR assay specificity when there is missing information and it is not possible to assess assay specificity for each individual non‐target confamilial. In this framework, we predict assay specificity against unsampled taxa (non‐target taxa without sequence data available) using the sequence information that is available for other confamilials. We demonstrate this framework using four case study assays for: (1) An endemic, freshwater arthropod (meltwater stonefly; Lednia tumana), (2) a globally distributed, marine ascidian (Didemnum perlucidum), (3) a continentally distributed freshwater crustacean (virile crayfish; Faxonius virilis, deanae and nais species complex) and (4) a globally distributed freshwater teleost (common carp; Cyprinus carpio and its close relative C. rubrofuscus). We tested the robustness of our approach to missing information by simulating application of our framework for all possible subsamples of 20—all non‐target taxa. Our results suggest that the modelling framework results in estimates which are largely concordant with observed levels of cross‐amplification risk using all available sequence data, even when there are high levels of data missingness. We explore potential limitations and extensions of this approach for assessing assay specificity and provide users with an R Markdown template for generating reproducible reports to support their own assay validation efforts. [ABSTRACT FROM AUTHOR]

Environmental DNA (e DNA) detection has emerged as a powerful tool for monitoring aquatic organisms, but much remains unknown about the dynamics of aquatic e DNA over a range of environmental conditions. DNA concentrations in streams and rivers will depend not only on the equilibrium between DNA entering the water and DNA leaving the system through degradation, but also on downstream transport. To improve understanding of the dynamics of e DNA concentration in lotic systems, we introduced caged trout into two fishless headwater streams and took e DNA samples at evenly spaced downstream intervals. This was repeated 18 times from mid-summer through autumn, over flows ranging from approximately 1-96 L/s. We used quantitative PCR to relate DNA copy number to distance from source. We found that regardless of flow, there were detectable levels of DNA at 239.5 m. The main effect of flow on e DNA counts was in opposite directions in the two streams. At the lowest flows, e DNA counts were highest close to the source and quickly trailed off over distance. At the highest flows, DNA counts were relatively low both near and far from the source. Biomass was positively related to e DNA copy number in both streams. A combination of cell settling, turbulence and dilution effects is probably responsible for our observations. Additionally, during high leaf deposition periods, the presence of inhibitors resulted in no amplification for high copy number samples in the absence of an inhibition-releasing strategy, demonstrating the necessity to carefully consider inhibition in e DNA analysis. [ABSTRACT FROM AUTHOR]