| Capturing environmental deoxyribonucleic acid (eDNA), or genetic material shed into water, could help overcome the challenge of determining the presence of species that are difficult to find using traditional surveys. To determine the most effective eDNA sampling protocol in a stream system, we conducted a survey for two hard-to-find species, the Sierra Nevada yellow-legged frog (Rana sierrae) and the foothill yellow-legged frog (R. boylii). We collected samples at 100-meter intervals in two different volumes at seven points along several streams in Plumas National Forest in California. We collected samples at four time points over the summer of 2016 using single-use funnels with 0.45-micrometer filters and a vacuum pump for a total of 286 eDNA samples. We analyzed samples in triplicate using species-specific quantitative polymerase chain reaction (qPCR) assays we developed and validated. Detection results were analyzed using generalized linear mixed effect models using a binomial response. Filtering double the volume of water (2 liters) increased odds of detection five times and sampling later in the season increased odds of detection by 1.016 per day. Over a 100-meter distance, the number of wells with a positive eDNA signal decreased by 28%, indicating that sampling at close intervals is important for detecting these secretive amphibians. |