Multi-tool monitoring: integrating non-invasive methods to assess terrestrial vertebrate diversity and trophic complexity

This project investigates how different environmental DNA (eDNA) substrates capture vertebrate diversity within a terrestrial rewilding landscape and evaluates how eDNA complements other non-invasive monitoring methods. The study was carried out across a 40 ha rewilding site in the Scottish Highlands, United Kingdom, where eDNA samples were collected in June 2023 from four substrate types: water, soil, tree-surface roller swabs, and mammal scat. Sampling formed part of a multi-tool biodiversity monitoring framework that integrates eDNA, camera traps, and passive acoustic recorders to assess vertebrate diversity and trophic complexity.DNA was extracted using the Mu-DNA protocol (Sellers et al., 2018), which enables parallel extraction across diverse environmental substrates. The vertebrate mitochondrial 12S-V5 fragment was amplified using primers 12SV5F (5'-TAGAACAGGCTCCTCTAG-3') and 12SV5R (5'-TTAGATACCCCACTATGC-3') following Riaz et al. (2011). Library preparation followed the nested two-step PCR workflow described by Griffiths et al. (2023). Ten pooled libraries (Lib1-Lib10), each containing 24 uniquely tagged eDNA samples, were sequenced on an Illumina MiSeq 2 x 300 bp platform.The raw FASTQ files deposited here represent pooled amplicon libraries prior to bioinformatic demultiplexing. Tag-mapping files and bioinformatic pipelines are documented in the associated protocols.io workflow: https://www.protocols.io/view/multi-tool-monitoring-edna-sample-collection-and-p-g3xfbypjp.Additional processed data and materials are archived at Zenodo: https://zenodo.org/records/15723455. This dataset supports the development of multi-tool approaches for monitoring vertebrate diversity and ecosystem recovery in terrestrial rewilding and restoration projects.