Generating and testing ecological hypotheses at the pondscape with environmental DNA metabarcoding: a case study on a threatened amphibian

Abstract

2 eDNA metabarcoding represents a new tool for community biodiversity assessment 22 in a broad range of aquatic and terrestrial habitats. However, much of the existing 23 literature focuses on methodological development rather than testing of ecological 24hypotheses. Here, we use presence-absence data generated by eDNA 25 metabarcoding of over 500 UK ponds to examine: 1) species associations between 26 the great crested newt (Triturus cristatus) and other vertebrates, 2) determinants of 27 great crested newt occurrence at the pondscape, and 3) determinants of vertebrate 28 species richness at the pondscape. The great crested newt was significantly 29 associated with nine vertebrate species. Occurrence in ponds was broadly reduced 30 by more fish species, but enhanced by more waterfowl and other amphibian species. 31Abiotic determinants (including pond area, depth, and terrestrial habitat) were 32 identified, which both corroborate and contradict existing literature on great 33 crested newt ecology. Some of these abiotic factors (pond outflow) also determined 34 species richness at the pondscape, but other factors were unique to great crested 35 newt (pond area, depth, and ruderal habitat) or the wider biological community 36 (pond density, macrophyte cover, terrestrial overhang, rough grass habitat, and 37 overall terrestrial habitat quality) respectively. The great crested newt Habitat 38 Suitability Index positively correlated with both eDNA-based great crested newt 39 occupancy and vertebrate species richness. Our study is one of the first to use eDNA 40 metabarcoding to test abiotic and biotic determinants of pond biodiversity. eDNA 41 metabarcoding provided new insights at scales that were previously unattainable 42 using established methods. This tool holds enormous potential for testing ecological 43 hypotheses alongside biodiversity monitoring and pondscape management. 44Freshwater ecosystems comprise <1% of the Earth's surface but provide vital 45 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/278309 doi: bioRxiv preprint first posted online Mar. 7, 2018; 3 ecosystem services and are hotspots of biodiversity [1][2][3] 4,7,8 . Effective management of pondscapes requires knowledge of abiotic and 55 biotic factors that influence biodiversity, community structure and productivity. 56Moreover, the biodiversity that ponds support individually and in combination must be 57 examined, but can only be maintained if stressors and threats to these systems are 58 understood 4,5,7,8,10 . Exhaustive sampling of pond biodiversity is impeded by the 59 complexity of these species-rich habitats, and numerous tools required for different taxa 60 with associated bias 11 and cost 12 . However, large-scale community-level monitoring, 61 encompassing alpha (site), beta (between-site) and gamma (landscape) diver...

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

“…Notably, no survey method captured semi-aquatic otter despite presence at study sites and successful detection in eDNA metabarcoding studies of UK ponds (Harper et al, 2019) and lakes (Hänfling et al, 2017). Captive otter also had a weaker eDNA signal than other semi-aquatic mammals studied here.…”

“…Our process controls identified low-level contamination at all stages of metabarcoding, but primarily during sampling or PCR (Appendix A1). We applied taxon-specific sequence thresholds to our data to mitigate false positives as in Harper et al (2019). Remnant contaminants were cichlid (laboratory), Gentoo penguin (environment), reindeer (environment), and human (environment/laboratory).…”

“…Bálint et al, 2018; Valentini et al, 2016) communities. However, mammals also leave eDNA signatures in water that metabarcoding can detect (Harper et al, 2019; Klymus et al, 2017; Ushio et al, 2017). Ponds in particular provide drinking, foraging, dispersive, and reproductive opportunities for semi-aquatic and terrestrial mammals (Klymus et al, 2017).…”

“…Ponds in particular provide drinking, foraging, dispersive, and reproductive opportunities for semi-aquatic and terrestrial mammals (Klymus et al, 2017). Samples from these waterbodies could uncover biodiversity present in the wider environment (Deiner et al, 2017; Harper et al, 2019). Drinking is a major source of eDNA deposition due to the release of saliva, but mammals may also swim, wallow, urinate or defecate in water (Rodgers & Mock, 2015; Ushio et al, 2017; Williams et al, 2018).…”

Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds

“…eDNA analysis has since shown potential for estimation of relative abundance and biomass (Takahara et al, 2012;Thomsen et al, 2012;Buxton et al, 2017b), and has begun to outperform conventional counterparts, for example, large-scale sampling and distribution modelling of the threatened great crested newt Triturus cristatus (Laurenti, 1768) (Biggs et al, 2015), and may deepen our understanding of species distribution patterns and activity. This capacity of eDNA analysis to upscale freshwater monitoring and research, particularly ecological hypothesis testing, was reinforced by Harper et al (2018c), where eDNA metabarcoding (see Table 1) was used in over 500 ponds to identify biotic and abiotic determinants of T. cristatus at the UK pondscape. Ponds are often considered to be closed systems, but may receive inputs from inflow, land surface run-off (especially during high rainfall and flood events), and mobile species (e.g.…”