Validation of an eDNA-based workflow for monitoring inter- and intra-specific CytB haplotype diversity of alpine amphibians

Environmental DNA (eDNA) analysis is a promising tool for monitoring wild animal populations and, more recently, their genetic variability. In this study, we used the mitochondrial Cytochrome B gene to develop and apply new eDNA metabarcoding assays targeting amphibian families and genera in order to estimate both inter- and intraspecific genetic diversity. We designed and tested seven new primer pairs (a) in silico against an amphibian reference database based on the target genera; (b) in vitro on tissue samples of the target species; and (c) in situ on water samples from 38 wet- lands in the Province of Trento (Italy). Overall, most target species were amplified successfully, although some markers also amplified non-target amphibian species. In addition, to complete the workflow, we compared the performance of three different bioinformatic pipelines (namely, MICCA with VSEARCH, and OBITools using ecotag or metabinkit), in retrieving reads and exact sequence variants from the metabarcoding datasets. Overall, the MICCA based pipeline retrieved more reads, but less putative haplotypes of amphibians. After comparing these sequences with previously known haplotypes from tissue-based studies, when the aim is to both decrease the probabil- ity of detecting false haplotypes and retrieve the highest number of reads, we suggest using MICCA+VSEARCH, unless a direct comparison with tissue-based genetic data is possible