Background Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays.

Bighead Carp Hypophthalmichthys nobilis, Silver Carp H. molitrix, and Grass Carp Ctenopharyngodon idella (collectively termed “Asian carp”) were introduced into North America during the 1960s and 1970s and have become established in the lower Mississippi River basin. Previously published evidence for spawning of these species in the upper Mississippi River has been limited to an area just downstream of Dam 22 (near Saverton, Missouri). In 2013 and 2014, we sampled ichthyoplankton at 18 locations in the upper Mississippi River main stem from Dam 9 through Dam 19 and in four tributaries of the Mississippi River (Des Moines, Skunk, Iowa, and Wisconsin rivers). We identified eggs and larvae by using morphological techniques and then used genetic tools to confirm species identity. The spawning events we observed often included more than one species of Asian carp and in a few cases included eggs that must have been derived from more than one upstream spawning event. The upstream extent of genetically confirmed Grass Carp ichthyoplankton was the Wisconsin River, while Bighead Carp and Silver Carp ichthyoplankton were observed in Pool 16. In all these cases, ichthyoplankton likely drifted downstream for several hours prior to collection. Higher water velocities (and, to a lesser extent, higher temperatures) were associated with an increased likelihood of observing eggs or larvae, although the temperature range we encountered was mostly above 17°C. Several major spawning events were detected in 2013, but no major spawning events were observed in 2014. The area between Dam 15 and Dam 19 appears to be the upstream edge of spawning activity for both Silver Carp and Bighead Carp, suggesting that this area could be a focal point for management efforts designed to limit further upstream movement of these species. Received July 18, 2016; accepted May 4, 2017Published online July 19, 2017

Freshwater organisms of North America have had their contemporary genetic structure shaped by vicariant events, especially Pleistocene glaciations. Life history traits promoting dispersal and gene flow continue to shape population genetic structure. Cumberlandia monodonta, a widespread but imperiled (IUCN listed as endangered) freshwater mussel, was examined to determine genetic diversity and population genetic structure throughout its range. Mitochondrial DNA sequences and microsatellite loci were used to measure genetic diversity and simulate demographic events during the Pleistocene using approximate Bayesian computation (ABC) to test explicit hypotheses explaining the evolutionary history of current populations. A phylogeny and molecular clock suggested past isolation created two mtDNA lineages during the Pleistocene that are now widespread. Two distinct groups were also detected with microsatellites. ABC simulations indicated the presence of two glacial refugia and post-glacial admixture of them followed by simultaneous dispersal throughout the current range of the species. The Ouachita population is distinct from others and has the lowest genetic diversity, indicating that this is a peripheral population of the species. Gene flow within this species has maintained high levels of genetic diversity in most populations; however, all populations have experienced fragmentation. Extirpation from the center of its range likely has isolated remaining populations due to the geographic distances among them.

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Techniques for obtaining DNA noninvasively or nonlethally are highly desirable in molecular genetic studies of protected species, and several advances have been made in these types of sampling and extraction techniques. Insects present a unique set of difficulties in this regard that are not present when working with most vertebrates. This study evaluated the effectiveness of several nonlethal sampling techniques for larval and adults of the federally listed endangered dragonfly Somatochlora hineana (Williamson) (Odonata: Corduliidae). Fecal pellets and shed exuviae from captive S. hineana larvae did not provide high enough quality DNA for microsatellite analyses. Invasive, but nonlethal, wing clips from adults and tarsi from larvae provided high-quality DNA that amplified 10 microsatellite markers for this species. Ten loci were polymorphic in 94 specimens with four to 14 alleles per locus. Two populations in WI had average observed heterozygosity of 0.47, which is within the range reported for other odonates. Our sampling techniques and these new microsatellite markers provide an essential tool for determining the genetic structure of S. hineana populations throughout its range.

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Natural resource managers use data on the spatial range of species to guide management decisions. These data come from survey or monitoring efforts that use a wide variety of tools. Environmental DNA (eDNA) is a surveillance tool that uses genetic markers for detecting species and holds potential as a tool for large‐scale monitoring programs. Two challenges of eDNA‐based studies are uncertainties created by imperfect capture of eDNA in collection samples (e.g., water field samples) and imperfect detection of eDNA using molecular methods (e.g., quantitative PCR). Occurrence models can be used to address these challenges, thus we use an occurrence model to address two objectives: first, to determine how many samples were required to detect species using eDNA; second, to examine when and where to take samples. We collected water samples from three different habitat types in the Upper Mississippi River when both Bighead Carp and Silver Carp were known to be present based on telemetry detections. Each habitat type (backwater, tributary, and impoundment) was sampled during April, May, and November. Detections of eDNA for both species varied across sites and months, but were generally low, 0–19.3% of samples were positive for eDNA. Overall, we found that eDNA‐based sampling holds promise to be a powerful monitoring tool for resource managers; however, limitations of eDNA‐based sampling include different biological and ecological characteristics of target species such as seasonal habitat usage patterns as well as aspects of different physical environments that impact the implementation of these methods such as water temperature.

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