Wild legumes maintain beneficial soil rhizobia populations despite decades of nitrogen deposition

Natural landscapes are increasingly impacted by nitrogen enrichment from aquatic and airborne pollution sources. Nitrogen enrichment in the environment can eliminate the net benefits that plants gain from nitrogen-fixing microbes such as rhizobia, potentially altering host-mediated selection on nitrogen fixation. However, we know little about the long-term effects of nitrogen enrichment on this critical microbial service. Here, we sampled populations of the legume Acmispon strigosus and its associated soil microbial communities from sites spanning an anthropogenic nitrogen deposition gradient. We measured the net growth benefits plants obtained from their local soil microbial communities and quantified plant investment into nodules that house nitrogen-fixing rhizobia. We found that plant growth benefits from sympatric soil microbes did not vary in response to local soil nitrogen levels, and instead varied mainly among plant lines. Soil nitrogen levels positively predicted the number of nodules formed on sympatric plant hosts, although this was likely due to plant genotypic variation in nodule formation, rather than variation among soil microbial communities. The capacity of all the tested soil microbial communities to improve plant growth is consistent with plant populations imposing strong selection on rhizobial nitrogen fixation despite elevated soil nitrogen levels, suggesting that host control traits in A. strigosus are stable under long-term nutrient enrichment.

Methods
We sampled soil from six field sites along a nitrogen deposition gradient in California, USA and chemically analyzed each soil (WSI_soil_data.csv). We prepared slurries of each soil and used these to inoculate Acmispon seedlings in a greenhouse. To check for the presence of microbes in the soil slurries, we plated several dilutions of the soil slurries onto selective media and counted the colonies that formed (WSI_culture_data.csv). For our greenhouse experiment, half of the Acmispon plants were inoculated with live soil slurries (containing soil nutrients and microbes), and half were inoculated with sterilized soil slurries (containing soil nutrients only). We grew the inoculated plants for several weeks and then collected plant biomass data (WSI_plant_data.csv). We also examined the root nodules that formed on the plants inoculated with live soil slurries and scored the colors of these nodules, which can indicate nitrogen-fixing activity (WSI_nodcolor_data.csv).