Molecular Monitoring of Leiocassis longirostris Using Species-Specific qPCR Assays from Environmental DNA

<i>Leiocassis longirostris</i>, a large benthic catfish, has been classified as Regionally Extinct (RE) in South Korea since the 1970s. Assessing the success of ongoing restocking efforts in the Geum River is challenging due to limitations in traditional capture-based monitoring methods. We developed a highly sensitive, species-specific quantitative real-time PCR (qPCR) assay targeting the mitochondrial cytochrome <i>b</i> gene (<i>mt-cyb</i>) for this restocked fish. Assay validation confirmed high specificity against a panel of 25 co-occurring fish species, including 8 related siluriforms, and an excellent limit of detection of 10 copies per reaction. We applied this assay to environmental DNA (eDNA) extracted from paired water and sediment samples collected from 13 stations across the Geum River Basin. eDNA was detected at three water and five sediment stations. Notably, detection rates and average concentrations were consistently higher in a sediment sample (peaking at 1609.93 copies L^-1), an observation consistent with the species' benthic ecology. These findings provide the first molecular evidence that restocked <i>L. longirostris</i> may disperse and persist in the mid to lower reaches of the Geum River. This validated eDNA qPCR tool is essential for guiding the integrated monitoring and adaptive management strategies required to support the long-term recovery of this ecologically important species. This study is the first species-specific qPCR of the bagrid catfish in South Korea.