Abstract
This paper presents the first phase in the development and validation of a simple and reliable environmental (e) DNA method using conventional PCR to detect four species of non‐native freshwater fish: pumpkinseed Lepomis gibbosus , sunbleak Leucaspius delineatus , fathead minnow Pimephales promelas and topmouth gudgeon Pseudorasbora parva . The efficacy of the approach was demonstrated in indoor tank (44 l) trials in which all four species were detected within 24 h. Validation was through two field trials, in which L. gibbosus was detected 6–12 h after its introduction into outdoor experimental ponds and P. parva was successfully detected in disused fish rearing ponds where the species was known to exist. Thus, the filtration of small (30 ml) volumes of pond water was sufficient to capture fish eDNA and the approach emphasised the importance of taking multiple water samples of sufficient spatial coverage for detecting species of random or patchy distribution.