Environmental RNA reflects seasonal changes in the metabolic activity of stream salamanders.

The conservation of stream salamanders relies on effective monitoring of these cryptic species in freshwater habitats increasingly affected by human activities. Environmental DNA (eDNA) and environmental RNA (eRNA) offer non-invasive alternatives to conventional active search methods. However, knowledge gaps remain regarding how eDNA and eRNA (collectively environmental nucleic acids; eNA) signals vary across space and time under natural field conditions, limiting their integration into monitoring programs. We evaluated eNA signals in small headwater streams in Quebec (Canada), focusing on the northern two-lined salamander (Eurycea bislineata) and the spring salamander (Gyrinophilus porphyriticus). We collected water samples over three consecutive days during fall and winter, and we assessed eNA detection and concentration using species-specific qPCR assays targeting mitochondrial markers. Detection rates of eDNA were consistently high across seasons, highlighting its suitability for rapid presence-absence surveys. In contrast, eRNA detection rates were lower in the fall, but increased with repeated sampling. In winter, eRNA detection rates were near zero, consistent with salamanders' markedly reduced metabolic rate during overwintering, suggesting that mitochondrial eRNA primarily represents metabolically active individuals rather than species presence alone. The concentrations of eNA were positively associated with salamander abundance in fall. Our results demonstrate the value of eNA-based approaches for the monitoring of stream salamanders under natural field conditions.