RT Journal Article SR Electronic T1 Ground-truthing environmental DNA metabarcoding for ecological hypothesis testing at the pondscape JF bioRxiv FD Cold Spring Harbor Laboratory SP 278309 DO 10.1101/278309 A1 Lynsey R. Harper A1 Lori Lawson Handley A1 Christoph Hahn A1 Neil Boonham A1 Helen C. Rees A1 Erin Lewis A1 Ian P. Adams A1 Peter Brotherton A1 Susanna Phillips A1 Bernd Hänfling YR 2018 UL http://biorxiv.org/content/early/2018/07/25/278309.abstract AB Environmental DNA (eDNA) metabarcoding is revolutionising biodiversity monitoring, but has unrealised potential for ecological hypothesis testing. Here, we ground-truth eDNA metabarcoding for describing vertebrate communities from 532 UK ponds, examine associations between the great crested newt (Triturus cristatus, GCN) and other vertebrates, and investigate factors determining GCN occurrence and vertebrate species richness at the pondscape. GCN occurrence was positively correlated with amphibian and waterfowl species richness, where GCN had strong positive associations with smooth newt (Lissotriton vulgaris), common coot (Fulica atra), and common moorhen (Gallinula chloropus), but a negative association with common toad (Bufo bufo). GCN occurrence was negatively correlated to fish species richness, specifically three-spined stickleback (Gasterosteus aculeatus) and ninespine stickleback (Pungitius pungitius) presence. We reaffirm reported abiotic associations (e.g. GCN preference for smaller, deeper ponds) but also provide novel insights, including a negative effect of pond outflow on GCN. Furthermore, we reveal novel determinants of vertebrate species richness at the pondscape, including pond density, macrophyte cover, and terrestrial habitat. Both GCN occupancy and species richness correlated with the GCN Habitat Suitability Index score, supporting its application to GCN survey. Our findings demonstrate the prospects of eDNA metabarcoding for hypothesis testing at unprecedented scales and enhancement of freshwater monitoring and research.