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

Though they jointly shape community responses to environmental perturbations, ecology and evolution are often examined separately, even in microorganisms where both occur over short timescales. Here, we examine ecological and evolutionary responses to 33 years of nitrogen fertilization using the legume-rhizobium mutualism. Pairing a manipulative inoculation study with full-length 16S rRNA gene amplicon sequencing and structural equation modeling allows us to synthesize across biological scales: whole bacterial community, genus Rhizobium, Rhizobium amplicon sequence variants (ASVs), and symbiosis plasmids. Clover's preferred partner decreases in N-addition soils, limiting host growth, while a diverse and largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary degradation of symbiont partner quality via changing frequencies of symbiotic plasmids. Ecological (rarer symbionts) and evolutionary (inferior symbionts) processes each accounted for roughly half of this loss of host benefit, revealing that ecology and evolution jointly shape mutualism breakdown over the short timescales typical of microbial systems.