Environmental DNA concentrations vary greatly across productive and degradative conditions, with implications for the precision of population estimates.

Population size is an important metric to inform the conservation and management of species. For aquatic species, environmental DNA (eDNA) concentration has been suggested for non-invasively estimating population size. However, many biotic and abiotic factors simultaneously influence the production and degradation of eDNA which can alter the relationship between population size and eDNA concentration. We investigated the influence of temperature, salinity, and ranavirus infection on eDNA concentrations using tadpole mesocosms. Using linear regression models, we tested the influence of each experimental treatment on eDNA concentrations at three time points before and during epidemics. Prior to infection, elevated temperatures lowered eDNA concentrations, indicating that degradation was the driving force influencing eDNA concentrations. During early epidemics, no treatments strongly influenced eDNA concentrations and in late epidemics, productive forces dominated as ranavirus intensity and dead organisms increased eDNA concentrations. Finally, population size was only an important predictor of eDNA concentration in late epidemics and we observed high levels of variation between samples of replicate mesocosms. We demonstrate the complexities of several interacting factors influencing productive and degradative forces, variation in influences on eDNA concentration over short time spans, and examine the limitations of estimating population sizes from eDNA with precision in semi-natural conditions.

Environmental RNA can distinguish life stages in amphibian populations.

Applications of environmental DNA (eDNA) analysis methods for biomonitoring have grown exponentially over the last decade and provide a wealth of new information on the distribution of species. However, eDNA methods have limited application for estimating population-level metrics. Environmental RNA (eRNA) has the potential to address ecological questions by gathering population demographic information from environmental media but may be challenging to detect and analyze. We developed gene-specific eRNA assays targeting keratin-associated genes in two focal species, American bullfrogs (Lithobates catesbeianus) and tiger salamanders (Ambystoma mavortium) to answer an important question in amphibian management: whether species detections represent breeding populations versus transitory adults. We performed an extensive laboratory validation with amphibians housed across development stages, where we collected 95 and 127 environmental samples for bullfrogs and salamanders, respectively. Both assays were highly specific to the larval stage and amplified with high sensitivity (90% in bullfrog and 88.4% in tiger salamander samples). We then applied our validated assays to multiple natural systems. When larvae were present, we found 74.1% overall detection in bullfrog field samples and 70.8% and 48.5% overall detection in field samples from ponds with A. macrodactylum and A. californiense larvae, correlating with eDNA detection rates. When only adults were present, we did not detect larvae-specific eRNA in A. macrodactylum ponds, despite high eDNA detection rates. Although much work is ahead for optimizing assay design, sampling and filtering methods, we demonstrate that eRNA can successfully be used to discern life stages with direct application for ecology and conservation management.