Unveiling the potential of eDNA/eRNA approaches for monitoring the critically endangered Yangtze finless porpoise

Assessing the population distribution and dynamics of the critically endangered Yangtze finless porpoise ( Neophocaena asiaeorientalis ) is essential for informing conservation strategies. Traditional monitoring techniques, while valuable, are labor-intensive and time-consuming. Environmental nucleic acids (eNAs) offer a non-invasive alternative, yet prior research has predominantly relied on single-locus eDNA analysis, limiting resolution. Here, we developed a dual-marker eDNA/eRNA approach targeting mitochondrial COX3 (Cytochrome C Oxidase subunit 3) and CYTB (Cytochrome B) genes, coupled with droplet digital PCR (ddPCR) quantification, to enhance detection sensitivity and reliability. Serial dilution experiments established stringent detection limits (LOD: 0.076 copies/μL; LOQ: 0.35 copies/μL), while degradation trials at the Tongling ex situ conservation site revealed eDNA persistence for 65.24 ( COX3 ) and 50.13 ( CYTB ) hours, compared to rapid eRNA degradation (~30 hours). Field surveys across 20 Yangtze River Estuary locations detected porpoise eDNA at 13 sites and eRNA at 11 sites, with 5 sites showing concurrent detection via both markers—predominantly near the Dongfeng Xisha Reservoir. Detection zones overlapped significantly with passive acoustic monitoring records, yet eNAs revealed broader spatial coverage. Our findings demonstrate that multi-locus eNA analysis provides a sensitive, scalable tool for monitoring critically endangered aquatic species, offering actionable insights to optimize conservation efforts. • Establishment of standardized LOD/LOQ thresholds combined with degradation experiments in natural water bodies enhances field detection reliability. • Integration of eRNA analysis with passive acoustic monitoring confirms real-time species presence for timely conservation actions. • Dual-marker eDNA/eRNA enables sensitive spatiotemporal tracking of Critically Endangered Yangtze finless porpoise, establishing a scalable non-invasive monitoring paradigm.