Environmental (e)RNA advances the reliability of eDNA by predicting its age.

Environmental DNA (eDNA) analysis has advanced conservation biology and biodiversity management. However, accurate estimation of age and origin of eDNA is complicated by particle transport and the presence of legacy genetic material, which can obscure accurate interpretation of eDNA detection and quantification. To understand the state of genomic material within the environment, we investigated the degradation relationships between (a) size of fragments (long vs short), (b) genomic origins (mitochondrial vs nuclear), (c) nucleic acids (eDNA vs eRNA), and (d) RNA types (messenger (m)RNA vs ribosomal (r)RNA) from non-indigenous Dreissena mussels. Initial concentrations of eRNA followed expected transcriptional trends, with rRNAs found at > 1000 × that of eDNA, and a mitosis-associated mRNA falling below detection limits within 24 h. Furthermore, the ratio of eRNA:eDNA significantly decreased throughout degradation, potentially providing an estimate for the age of genomic material. Thus, eRNA quantification can increase detection due to the high concentrations of rRNAs. Furthermore, it may improve interpretation of positive detections through the eRNA:eDNA ratio and/or by detecting low abundant mitosis-associated mRNAs that degrade within ~ 24 h.