Concurrent ecological and evolutionary processes contribute to mutualism breakdown between legumes and rhizobia

Ecology and evolution jointly shape community responses to environmental perturbations, yet these processes are often examined separately, even in microorganisms where both occur over short time-scales. For example, during mutualism breakdown, shifts in diverse microbial communities might coincide with evolutionary changes in mutualistic traits within key symbionts. Here we study the legume-rhizobium mutualism after 33 years of nitrogen fertilization. Pairing a manipulative inoculation study with full-length 16S rRNA amplicon sequencing provides incomparable resolution across biological scales: whole bacterial community, genus Rhizobium, Rhizobium ASVs, and symbiosis plasmids. Clover host plant growth is first limited by symbiont availability; clovers preferred partner declines in N-addition soils, while a diverse, largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary decline in the quality of the preferred symbiont through changing frequencies of symbiotic plasmids. These shifts make mutualists both rarer and inferior, reducing the benefits hosts receive from mutualism.