Differential decay of multiple environmental nucleic acid components.

Detections of environmental nucleic acids (eNA), such as DNA and RNA, are powerful tools for monitoring biodiversity. Yet, precise interpretation of these indirect detections requires understanding of eNAs persistence. We conducted a decay experiment to track degradation of six eNA components derived from the bottlenose dolphin Tursiops truncatus: mitochondrial eDNA of varying lengths, ribosomal eRNA, and messenger eRNA. Target eNAs were quantified over seven days via digital droplet PCR (ddPCR). Decay followed a biphasic exponential model with rapid initial loss (~ 24 h at 15 °C), followed by slower degradation. Mitochondrial messenger eRNA was least stable, disappearing within four hours. Ribosomal eRNA persisted longer but degraded slightly faster than its eDNA counterpart (decay rate λ₁ = 0.236 vs. 0.165 h⁻¹). Longest eDNA fragments decayed more rapidly (λ₁ = 0.190 h(-1)) than shorter ones (λ₁ = 0.114 h(-1)). These findings support using eDNA fragment length as a proxy for degradation and reinforce that combining multiple eNA components with distinct stabilities can provide a molecular clock to infer eNA age. This approach improves the spatiotemporal resolution of eNA-based monitoring, particularly for rare cetaceans that act as point sources. We also emphasize the importance of explicitly distinguishing between RNA types (ribosomal vs. messenger) in environmental studies, given their divergent stability and interpretability.