23Environmental DNA (eDNA) metabarcoding can identify terrestrial taxa utilising aquatic habitats 24 alongside aquatic communities, but terrestrial species' eDNA dynamics are understudied. We 25 evaluated eDNA metabarcoding for monitoring semi-aquatic and terrestrial mammals, 26 specifically nine species of conservation or management concern, and examined 27 spatiotemporal variation in mammal eDNA signals. We hypothesised eDNA signals would be 28 stronger for semi-aquatic than terrestrial mammals, and at sites where individuals exhibited 29 behaviours. In captivity, we sampled waterbodies at points where behaviours were observed 30 ('directed' sampling) and at equidistant intervals along the shoreline ('stratified' sampling). We 31 surveyed natural ponds (N = 6) where focal species were present using stratified water 32 sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-33 specific primers. All focal species were detected in captivity. eDNA signal strength did not differ 34 between directed and stratified samples across or within species, between semi-aquatic or 35 terrestrial species, or according to behaviours. eDNA was evenly distributed in artificial 36 waterbodies, but unevenly distributed in natural ponds. Survey methods deployed at natural 37 ponds shared three species detections. Metabarcoding missed badger and red fox recorded by 38 cameras and field signs, but detected small mammals these tools overlooked, e.g. water vole.
39Terrestrial mammal eDNA signals were weaker and detected less frequently than semi-aquatic 40 mammal eDNA signals. eDNA metabarcoding could enhance mammal monitoring through 41 large-scale, multi-species distribution assessment for priority and difficult to survey species, and 42 provide early indication of range expansions or contractions. However, eDNA surveys need high 43 3 spatiotemporal resolution and metabarcoding biases require further investigation before 44 routine implementation. 45 46