Summary1. Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed. 2. Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms. 3. Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA. 4. We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

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Environmental DNA (eDNA) analysis is an effective method of determining the presence of aquatic organisms such as fish, amphibians, and other taxa. This publication is meant to guide researchers and managers in the collection, concentration, and preservation of eDNA samples from lentic and lotic systems. A sampling workflow diagram and three sampling protocols are included as well as a list of suggested supplies. Protocols include filter and pump assembly using: (1) a hand-driven vacuum pump, ideal for sample collection in remote sampling locations where no electricity is available and when equipment weight is a primary concern; (2) a peristaltic pump powered by a rechargeable battery-operated driver/drill, suitable for remote sampling locations when weight consideration is less of a concern; (3) a 120-volt alternating current (AC) powered peristaltic pump suitable for any location where 120-volt AC power is accessible, or for roadside sampling locations. Images and detailed descriptions are provided for each step in the sampling and preservation process.

The costs of invasive species in the United States alone are estimated to exceed US$100 billion per year, so a critical tactic in minimizing the costs of invasive species is the development of effective, early-detection systems. To this end, we evaluated the efficacy of adding environmental (e)DNA surveillance to the U.S. Geological Survey (USGS) streamgage network, which consists of >8200 streamgages nationwide systemically visited by USGS hydrologic technicians. Incorporating strategic eDNA sample collection during routine streamgage visits could provide early-detection surveillance of aquatic invasive species with minimal additional cost. For this evaluation, USGS hydrologic technicians collected monthly eDNA water samples, May-September 2018, from streamgages downstream of reservoirs in the Columbia River Basin thought to be vulnerable to invasive dreissenid mussel (Dreissenidae spp.) establishment. We tested water samples for dreissenid mussel DNA and also for kokanee (Oncorhynchus nerka) and yellow perch (Perca flavescens) DNA; the two fishes were used to assess if streamgages are adequately located to provide early-detection eDNA surveillance of taxa known to be present in upstream reservoirs. No Columbia River Basin streamgage samples met our criteria for being scored as positive for dreissenid DNA. We did detect kokanee and yellow perch DNA at all streamgages downstream of reservoirs where these species are known to occur. Field collection, laboratory analyses, and personnel time required for collection of four eDNA samples at a streamgage site cost US$500-US$600 (net). Given these results, incorporating eDNA biosurveillance into routine streamgage visits might decrease costs associated with an invasion since early detection maximizes the potential for eradication, containment, and mitigation.