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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. 

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies. 

Abstract AimNitrogen (N)‐fixing plants are an important component of global plant communities, but the drivers of N‐fixing plant diversity, especially in temperate regions, remain underexplored. Here, we examined broad‐scale patterns of N‐fixing and non‐fixing plant phylogenetic diversity (PD) and species richness (SR) across a wide portion of temperate North America, focusing on relationships with soil N and aridity. We also tested whether exotic species, with and without N‐fixing symbiosis, have fewer abiotic limitations compared with native species. LocationUSA and Puerto Rico. Time periodCurrent. Major taxa studiedVascular plants, focusing on N‐fixing groups (orders Fabales, Fagales, Rosales and Cucurbitales). MethodsWe subset National Ecological Observatory Network (NEON) plant plot data from all sites along two axes (N fixing–non‐N fixing and native–exotic), calculating plot‐level SR, PD and mean pairwise phylogenetic distance (MPD). We then used linear mixed models to investigate relationships between diversity values and key soil measurements, along with aridity, temperature and fire frequency. ResultsAridity was the sole predictor of proportional phylogenetic diversity of N fixers. The SR of N fixers still decreased marginally in arid regions, whereas native N‐fixer MPD increased with aridity, indicative of unique lineages of N fixers in the driest conditions, in contrast to native non‐N fixers. The SR of both native N fixers and non‐N fixers increased in low‐N soils. Aridity did not affect SR of exotic non‐N fixers, unlike other groups, whereas exotic N fixers showed lower MPD in increasingly high‐N soils, suggesting filtering, contrary what was found for native N fixers. Main conclusionsOur results suggest that it is not nitrogen, or any soil nutrient, that has the strongest effect on the relative success of N fixers in plant communities. Rather, aridity is the key driver, at least for native species, in line with empirical results from other biomes and increased understanding of N fixation as a key mechanism to avoid water loss.