Validation of salivary DNA extraction for non-invasive genetic sampling of Castor fiber birulai and assessment of DNA persistence on Salix turanica twigs

Genetic monitoring of the endangered Castor fiber birulai is hindered by its elusive behavior and by the logistical challenges of collecting fecal samples in aquatic environments. This proof-of-concept validation assessed salivary DNA retrieved from feeding traces on Salix turanica twigs as a non-invasive source for species-level genetic detection. Using a controlled rescue setting with one individual, we evaluated DNA persistence across a 0–12 h post-feeding timeline under low-temperature conditions (< 10 °C). To reduce PCR inhibition caused by willow bark secondary metabolites (e.g., tannins and polyphenols), we used a soil-based DNA extraction protocol selected for inhibitor removal. The mitochondrial cytochrome b ( cytb ) fragment was amplified successfully from all samples collected within 0–10 h, whereas amplification failed after 12 h. Spearman’s rank correlation revealed significant negative relationships between exposure time and both DNA concentration and purity. These findings define a practical freshness threshold for field sampling under favorable low-temperature conditions and indicate that feeding trace-derived saliva can provide a complementary, species-specific source for non-invasive genetic monitoring of this endangered subspecies.