Major ongoing declines in global biodiversity necessitate biomonitoring strategies that enable precise estimates of community diversity on a fine spatial and temporal scale. While environmental DNA (eDNA) has been established as a powerful tool for biodiversity assessment, studies investigating the comparative performance of environmental RNA (eRNA) are limited. Here, we performed eDNA/eRNA metabarcoding of zooplankton communities in outdoor freshwater mesocosms subject to a dynamic range of pH conditions. We comparatively assessed i) the sensitivity of eRNA metabarcoding relative to eDNA and traditional survey methods in capturing zooplankton diversity, ii) the influence of pH on eDNA/eRNA detectability, and iii) the propensity of eRNA to capture contemporary biological assemblages (i.e., rapid species turnover) with high spatial and temporal acuity. Zooplankton richness was similar amongst eDNA/eRNA metabarcoding and traditional survey methods; however, the composition of zooplankton communities detected was more analogous between eDNA and eRNA metabarcoding than with traditional methods. Both eDNA and eRNA captured similar ZOTU richness and frequency of false negative detections (irrespective of site-specific pH); however, eRNA captured species turnover more rapidly than eDNA. Collectively, our findings suggest that i) relative to traditional methods, eDNA and eRNA metabarcoding may provide users with complementary rather than congruent estimates of biodiversity, ii) eDNA and eRNA provide comparable estimates of species richness irrespective of site-specific pH conditions, and iii) eRNA is able to capture short-term community responses with higher spatial and temporal acuity than eDNA. Overall, our findings support the use of eRNA for characterizing contemporary biodiversity in complex and dynamic aquatic environments.

Although the use and development of molecular biomonitoring tools based on eNAs (environmental nucleic acids; eDNA and eRNA) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site-specific physicochemical parameters on eNA-based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid-base gradient following complete removal of the progenitor organism (Daphnia pulex). Using water samples collected over a 30-day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of D. pulex. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability—predicted by an exponential decay model—for up to 57 hours (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats.