How do Less-expensive Nitrogen Alternatives Affect Legume Sanctions on Rhizobia?

Mutualistic interactions involving multiple partners require ‘sanctioning’ - the ability to influence the fitness of each partner based on its respective contribution. Sanctions must be sensitive to even small differences if even slightly less-beneficial partners could gain a fitness advantage by diverting resources away from the mutualistic service towards their own reproductive fitness. Here, we test whether legume hosts sanction even mediocre N2-fixing rhizobial strains by influencing either its nodule growth or carbon accumulation (polyhydroxybutryate or PHB) per rhizobia cell. We also test if sanctions depend on the availability of less-expensive nitrogen alternatives, either as nitrate or co-inoculation with a more-efficient isogenic strain. We found that nitrate eliminated differences in nodule size between the mediocre and more-efficient strains, suggesting that host sanctions were compromised. However, nitrate additions also decreased PHB accumulation by the mediocre strain, which may eliminate any fitness advantages of lower fixation by this strain. Co-inoculation with a more-efficient strain could also compromise host sanctions if reduction in fitness from smaller nodules does not offset the potential fitness gain from greater PHB accumulation that we observed in the mediocre strain. Hence, a host’s ability to sanction mediocre strains depends not only on alternative sources of nitrogen but also the relative importance of different components of rhizobial fitness.

Methods
Dataset was collected by two undergraduate students, first into a lab notebook, and then added to Microsoft Excel. That data are then accessed for analyses by exporting to .csv and importing into R Studio. We provide the main dataset .csv file and multiple .R files.