Comparative genomics of the nonlegume <i>Parasponia</i> reveals insights into evolution of nitrogen-fixing rhizobium symbioses

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete <i>Frankia</i> as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using <i>Parasponia</i> species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between <i>Parasponia andersonii</i> and its nonnodulating relative <i>Trema tomentosa</i> demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of <i>P. andersonii</i> and the legume <i>Medicago truncatula</i> revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including <i>NODULE INCEPTION</i> (<i>NIN</i>) and <i>RHIZOBIUM-DIRECTED POLAR GROWTH</i> (<i>RPG</i>). Comparative analysis of genomes from three <i>Parasponia</i> species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of <i>NIN</i>, <i>RPG</i>, and <i>NOD FACTOR PERCEPTION</i> Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.