Metabarcoding stem residue as a novel environmental DNA (eDNA) tool to identify spread of Phragmites australis biological control agents

Detection and monitoring of biological control agents are critical for evaluating their establishment and spread yet remain challenging when species are cryptic, and densities are low. We assessed whether environmental DNA (eDNA) metabarcoding of stem residues could be used to detect and distinguish the stem-boring noctuid moths Archanara neurica and Lenisa geminipuncta , biological control agents of introduced Phragmites australis released in Ontario, Canada. Using 16S rDNA metabarcoding supplemented with newly generated reference sequences, we analyzed stem residue samples spanning a gradient of quality, including laboratory culture stems (in which agent larvae were present at the time of sampling), confirmed Canadian release sites, and degraded stems from unmanaged Phragmites stands in western New York, USA. Target species were consistently detected in laboratory samples, where they comprised 76.7–100% ( L. geminipuncta ) and 79.2–100% ( A. neurica ) of Lepidoptera reads. At Canadian release sites, A. neurica was detected in 5 of 11 samples and L. geminipuncta in all 10 samples, with relative read abundances ranging from trace levels to > 90%. Among 27 damaged stems collected in the United States, A. neurica and L. geminipuncta DNA was detected in three samples at very low abundances (0.003–0.12% of total reads), representing the first molecular evidence consistent with trans -border dispersal of these agents. Although detections were rare and do not confirm population establishment, results demonstrate that stem-residue eDNA metabarcoding provides a sensitive, non-invasive tool for early detection and post-release monitoring of biological control agents for invasive plants.