Glyphosate-based restoration of a degraded grassland threatens soil health and the diversity of nematode communities

Invasive species have become a dominant component of native grasslands, leading to a reduction in biodiversity and ecosystem functioning. Grasslands hold the potential to sequester more carbon than forests, yet few large-scale intact grasslands remain on Earth. Consequently, the restoration of degraded grasslands is critical to resurrect the ecological and economic services they once provided. Although glyphosate-based herbicide restoration projects are among the most effective strategies to remove invasive plants and allow native species to reestablish, their off-target effects on belowground diversity are unclear. Nematodes are the most abundant and diverse animals on Earth and have been recognized as indicators of soil health due to their positioning at all trophic levels and ability to respond to environmental change such as pollution, disturbance, and climate change. The goal of this study was to examine the response of nematode communities to a glyphosate-based grassland restoration project. We applied different frequencies of Roundup ProMax (R) to field plots in the first year of the project and characterized the impact on nematode communities over the following four years using morphological counts and 18S rRNA metabarcoding. To test the effects of the herbicide on nematode community diversity, composition, and structure, we utilized a modelling approach with general linear models, PERMANOVAs, distance-based redundancy and co -occurrence network analyses. We show persistent negative effects on nematode diversity across all four years. In addition, compositional shifts and changes in nematode-specific functional indices indicated less healthy soils in herbicide-treated plots. Co -occurrence networks further confirmed less structured and more disturbed communities. No recovery of nematode communities in glyphosate treated plots was observed after four years, demonstrating the longevity of effects. In conclusion, this study reports negative off-target effects of glyphosate herbicide restoration projects on belowground diversity and the need to consider these factors in evaluating the long -term success of herbicide-based grassland restoration.