Major ongoing declines in global biodiversity necessitate biomonitoring strategies that enable precise estimates of community diversity on a fine spatial and temporal scale. While environmental DNA (eDNA) has been established as a powerful tool for biodiversity assessment, studies investigating the comparative performance of environmental RNA (eRNA) are limited. Here, we performed eDNA/eRNA metabarcoding of zooplankton communities in outdoor freshwater mesocosms subject to a dynamic range of pH conditions. We comparatively assessed i) the sensitivity of eRNA metabarcoding relative to eDNA and traditional survey methods in capturing zooplankton diversity, ii) the influence of pH on eDNA/eRNA detectability, and iii) the propensity of eRNA to capture contemporary biological assemblages (i.e., rapid species turnover) with high spatial and temporal acuity. Zooplankton richness was similar amongst eDNA/eRNA metabarcoding and traditional survey methods; however, the composition of zooplankton communities detected was more analogous between eDNA and eRNA metabarcoding than with traditional methods. Both eDNA and eRNA captured similar ZOTU richness and frequency of false negative detections (irrespective of site-specific pH); however, eRNA captured species turnover more rapidly than eDNA. Collectively, our findings suggest that i) relative to traditional methods, eDNA and eRNA metabarcoding may provide users with complementary rather than congruent estimates of biodiversity, ii) eDNA and eRNA provide comparable estimates of species richness irrespective of site-specific pH conditions, and iii) eRNA is able to capture short-term community responses with higher spatial and temporal acuity than eDNA. Overall, our findings support the use of eRNA for characterizing contemporary biodiversity in complex and dynamic aquatic environments.

Paz-Vinas et al. (2021) comment on methodological and data-related limits of our paper (Millette et al. 2020), which affect a small proportion of our datasets and analyses. These points do not refute our conclusions. We address their comments and support the call for the development of best practices for future macrogenetics research.

Almost 50 years ago, Michael Rosenzweig pointed out that nutrient addition can destabilize food webs, leading to loss of species and reduced ecosystem function through the paradox of enrichment. Around the same time, David Tilman demonstrated that increased nutrient loading would also be expected to cause competitive exclusion leading to deleterious changes in food web diversity. While both concepts have greatly illuminated general diversity-stability theory, we currently lack a coherent framework to predict how nutrients influence food web stability across a landscape. This is a vitally important gap in our understanding, given mounting evidence of serious ecological disruption arising from anthropogenic displacement of resources and organisms. Here, we combine contemporary theory on food webs and meta-ecosystems to show that nutrient additions are indeed expected to drive loss in stability and function in human-impacted regions. However, this loss in stability occurs not just from wild oscillations in population abundance, but more frequently from the complete loss of an equilibrium due to edible plant species being competitively excluded. In highly modified landscapes, spatial nutrient transport theory suggests that such instabilities can be amplified over vast distances from the sites of nutrient addition. Consistent with this theoretical synthesis, the empirical frequency of these distant propagating ecosystem imbalances appears to be growing. This synthesis of theory and empirical data suggests that human modification of the Earth’s ecological connectivity is “entangling” once distantly separated ecosystems, causing rapid, expansive, and costly nutrient-driven instabilities over vast areas of the planet. The corollary to this spatial nutrient theory, though – akin to weak interaction theory from food web networks – is that slow spatial nutrient pathways can be potent stabilizers by moderating flows across a landscape