Combining multiple markers significantly increases the sensitivity and precision of <scp>eDNA</scp>‐based single‐species analyses

Abstract

Abstract The analysis of environmental DNA (eDNA) is becoming integrated as an established biomonitoring tool, often characterized by detection limits exceeding those of conventional counterparts. However, further improving the sensitivity of these methods may be invaluable for the early detection of invasive species, or for locating remnant populations of endangered species, and the adequate quantification of their abundances. In this study, we provide empirical evidence showing that the implementation of multiple genetic markers targeting different loci is a surprisingly overlooked strategy to increase the sensitivity of single‐species detections and quantification of their abundance, particularly at the lower end of the species abundance range. We analyzed 45 natural eDNA samples obtained from a wide range of water bodies in Belgium, in which either the invasive American bullfrog ( Lithobates catesbeianus ) or the rare European weather loach ( Misgurnus fossilis ) occurred under variable abundances, and compared detection success and precision of quantification of simplex (single locus) versus multiplex (multilocus) droplet digital PCR (ddPCR) analyses. Multiplexing different primer/probe assays targeting independent loci resulted in a significantly enhanced detection probability compared to the simplex analyses, gaining a twofold reduction in the limit of detection (LOD). Also the precision of eDNA quantification significantly improved in multiplex reactions, especially in low concentration samples. This was reflected in a significant reduction in the coefficient of variation (CV) among technical replicates, resulting in an associated decrease of the limit of quantification (LOQ). We conclude that the use of multiple markers can significantly improve the analytical sensitivity of eDNA‐based single‐species detections and precision of absolute abundance quantifications.