Elemental and isotopic analysis of leaves predicts nitrogen-fixing phenotypes.

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 (δN and δC), 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 δN-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.