A Novel eDNA-Based Approach for Hybrid Detection: Implications for Conservation Management

Abstract Hybridization between invasive and native species poses a hidden but critical threat to biodiversity. While environmental DNA (eDNA) has revolutionized species monitoring, it has lacked the resolution to detect hybrid individuals. Here, we present the first experimental demonstration of hybrid identification using eDNA. Our method isolates a single cell in the environment (hereafter, eCell) and enables cellular-level analysis using multiplex digital PCR targeting nuclear markers from both parental species. Validation with controlled tank experiments using Oncorhynchus masou masou × Salvelinus leucomaenis leucomaenis hybrid individuals confirmed the method’s ability to separately detect hybrid individuals from co-habiting purebred parent individuals. This eCell analysis overcomes the limitations of traditional eDNA methods and offers a scalable, non-invasive tool for detecting cryptic hybridization. By enabling early and accurate detection of hybrid individuals, it supports timely conservation decisions, including management prioritization and the protection of purebred populations. This novel technique bridges a critical gap in conservation genetics and enhances eDNA’s utility for biodiversity management in the face of global change. Competing Interest Statement The authors have declared no competing interest. Footnotes Declarations Availability of data and materials All raw data are included in the result and Supporting Information. Competing interests The authors declare no competing interest. Funding This study was supported by Grants-in-Aid for Scientific Research (Nos. 22K15183 and 23H02556) from the JSPS. This work was partly supported by JSPS Program for Forming Japan’s Peak Research Universities(J-PEAKS)Grant Number JPJS00420230001. Ethics approval statement The experiments were conducted in accordance with the relevant guidelines and regulations, and the tank experiments were approved by Kobe University (Form Nos. 2023-10, 2023-11, and 2023-12).