N-Deposition and the Rhizobium-legume mutualism: How do rhizobia become less cooperative in N-rich environments?

Background/Question/Methods

Mutualistic interactions may be sensitive to environmental changes including anthropogenic nitrogen (N) deposition. Given their diverse and essential roles in natural communities, quantifying mutualism responses, and understanding the evolutionary mechanisms leading to these responses, will be important. Rhizobium-legume mutualisms in particular are predicted to be sensitive to N-addition, since plants trade costly photosynthates in return for fixed N. We have isolated 64 strains of Rhizobium leguminosarum from N-fertilized plots and non-fertilized control plots from an LTER experiment. Rhizobia from N-fertilized plots have been found to be less-beneficial for Trifolium (clover) hosts. How did the rhizobia in N-plots become less beneficial to Trifolium? Was there a shift in rhizobial species composition or were there underlying microevolutionary processes? We employed a phylogenetic approach. We first sequenced the internal transcribed spacer (ITS) region to ask whether the strains isolated from N and control LTER treatments were significantly clustered on the tree, or even belonged to distinct species. We also sequenced plasmid-borne symbiosis genes in order to compare with the ITS phylogram and test for evidence of horizontal gene transfer among distantly-related lineages.

Results/Conclusions

We found that our 64 experimental strains were nested within what is currently the Rhizobium leguminosarum species complex, though we may have sampled multiple species. Nevertheless, N- versus control-plot rhizobia were intermixed in the best supported maximum likelihood phylogram. These results suggest that the less beneficial phenotype has resulted from microevolutionary processes rather than a shift in community composition (i.e., "species sorting"). The differences in rhizobia mutualistic quality appear to be chiefly the result of microevolutionary processes such as point mutations, deletions of particular symbiosis genes, or the horizontal transmission of symbiosis islands among strains or even from other species. We also present the results of phylogenetic congruence tests between the ITS phylogram and those of three symbiosis genes, which give insight into the potential for horizontal gene transmission among distantly-related ITS types to contribute to mutualism degradation in rhizobia from N-fertilized plots. Given the immense ecological role of mutualisms and their apparent sensitivity to anthropogenic change, the evolutionary origins of less-beneficial mutualists might have profound impacts on ecosystems that are vulnerable to N-deposition.