Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis

Genomic traces of symbiosis loss A symbiosis between certain bacteria and their plant hosts delivers fixed nitrogen to the plants. Griesmann et al. sequenced several plant genomes to analyze why nitrogen-fixing symbiosis is irregularly scattered through the evolutionary tree (see the Perspective by Nagy). Various genomes carried traces of lost pathways that could have supported nitrogen-fixing symbiosis. It seems that this symbiosis, which relies on multiple pathways and complex interorganismal signaling, is susceptible to selection and prone to being lost over evolutionary time. Science, this issue p. eaat1743; see also p. 125 Genome-wide comparative analysis across species reveals the fragility of the plant-bacterial symbiosis needed for nitrogen fixation. INTRODUCTION Access to nutrients such as nitrogen is required for plant growth. Legumes and nine additional plant families benefit from the nitrogen-fixing root nodule (NFN) symbiosis, in which roots develop nodules that intracellularly host nitrogen-fixing bacteria. In this mutually beneficial symbiosis, the bacteria convert atmospheric nitrogen into ammonium and deliver it to the host plant. NFN symbiosis thus enables plant survival under nitrogen-limiting conditions in terrestrial ecosystems. In agriculture, this symbiosis reduces reliance on nitrogen fertilizer, thus reducing the costs, ecological impact, and fossil fuel consumption attendant on large-scale application of fertilizers. RATIONALE Molecular phylogenies show that NFN symbiosis is restricted to four angiosperm orders—Fabales, Fagales, Cucurbitales, and Rosales—that together form the monophyletic NFN clade. However, only 10 of the 28 plant families within this clade contain species engaged in the NFN symbiosis. Even within these 10 families, most genera do not form this symbiosis. The NFN symbiosis requires the coordinated function of more than 30 essential genes. Presence of this symbiosis in related families suggests that a genetic change in the ancestor of the NFN clade enabled evolution of NFN symbiosis in this clade. The scattered distribution of functional NFN symbiosis across the clade has led to the question of whether NFN symbiosis evolved multiple times independently in a convergent manner or was lost multiple times regardless of the number of times it arose. Fossil data have been unable to answer this question. Here we used molecular evidence to ask how the current pattern of plant species with NFN symbiosis evolved. RESULTS We sequenced the genomes of seven nodulating species belonging to the Fagales, Rosales, and Cucurbitales orders and the legume subfamily Caesalpinioideae. We complemented this dataset by sequencing three genomes of nonnodulating species from the Cucurbitales and from the legume subfamilies Cercidoideae and Papilionoideae. Using a genome-wide phylogenomic approach, we found that all legume genes with a characterized role in NFN symbiosis are conserved in nodulating species with one exception. We observed larger numbers of order-specific gene family expansions that, solely because of their phylogenetic distribution, may include genes contributing to multiple gains or subsequent refinements of the symbiosis. In parallel, we discovered signatures of multiple independent loss-of-function events for the gene encoding the indispensable NFN symbiosis regulator NODULE INCEPTION (NIN) in 10 of 13 genomes of nonnodulating species within the NFN clade. The pattern suggests at least eight independent losses of NFN symbiosis. CONCLUSION We found that multiple independent losses of NFN symbiosis occurred in the four orders of the NFN clade. These results suggest that NFN symbiosis has previously been more common than currently evident and that this symbiosis is subject to an underestimated adverse selection pressure. Phylogenomics and evolution of NFN symbiosis. Genome sequencing of nodulating and nonnodulating species combined with 27 previously available genomes resulted in a dataset spanning the NFN clade and species outside the NFN clade as an outgroup. Orthogroups were identified and filtered following three phylogenetic patterns. This genome-wide analysis identified two genes involved in NFN symbiosis, NIN and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG), that were lost in most nonnodulating species. The occurrence of multiple losses (red crosses) of NFN symbiosis suggests an adverse selection pressure. The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator NODULE INCEPTION in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.