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  1. Abstract Nitrogen (N)-fixing symbiosis is critical to terrestrial ecosystems, yet possession of this trait is known for few plant species. Broader presence of the symbiosis is often indirectly determined by phylogenetic relatedness to taxa investigated via manipulative experiments. This data gap may ultimately underestimate phylogenetic, spatial, and temporal variation in N-fixing symbiosis. Still needed are simpler field or collections-based approaches for inferring symbiotic status. N-fixing plants differ from non-N-fixing plants in elemental and isotopic composition, but previous investigations have not tested predictive accuracy using such proxies. Here we develop a regional field study and demonstrate a simple classification model for fixer status using nitrogen and carbon content measurements, and stable isotope ratios (δ15N and δ13C), from field-collected leaves. We used mixed models and classification approaches to demonstrate that N-fixing phenotypes can be used to predict symbiotic status; the best model required all predictors and was 80–94% accurate. Predictions were robust to environmental context variation, but we identified significant variation due to native vs. non-native (exotic) status and phylogenetic affinity. Surprisingly, N content—not δ15N—was the strongest predictor, suggesting that future efforts combine elemental and isotopic information. These results are valuable for understudied taxa and ecosystems, potentially allowing higher-throughput field-based N-fixer assessments. 
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  2. Biological nitrogen fixation is a fundamental part of ecosystem functioning. Anthropogenic nitrogen deposition and climate change may, however, limit the competitive advantage of nitrogen-fixing plants, leading to reduced relative diversity of nitrogen-fixing plants. Yet, assessments of changes of nitrogen-fixing plant long-term community diversity are rare. Here, we examine temporal trends in the diversity of nitrogen-fixing plants and their relationships with anthropogenic nitrogen deposition while accounting for changes in temperature and aridity. We used forest-floor vegetation resurveys of temperate forests in Europe and the United States spanning multiple decades. Nitrogen-fixer richness declined as nitrogen deposition increased over time but did not respond to changes in climate. Phylogenetic diversity also declined, as distinct lineages of N-fixers were lost between surveys, but the “winners” and “losers” among nitrogen-fixing lineages varied among study sites, suggesting that losses are context dependent. Anthropogenic nitrogen deposition reduces nitrogen-fixing plant diversity in ways that may strongly affect natural nitrogen fixation. 
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