What lurks beneath the surface? The hidden Frankia biodiversity in Casuarina nodules across continents

Background and Aims Actinorhizal root nodule symbioses are formed between a diverse group of mostly woody dicotyledonous plants and nitrogen-fixing soil Actinomycetota of the genus Frankia . One of the most ecologically relevant actinorhizal plants are (Allo-)Casuarina species, used widely in shelter belts and phytoremediation due to their high tolerance to abiotic stresses and ability to thrive on marginal soils. All sequenced Frankia strains isolated from (Allo-)Casuarina nodules via traditional techniques show high sequence identity and belong to a single species, Frankia casuarinae . This lack of diversity in nodules is unusual in actinorhizal symbioses. We hypothesised that (Allo-)Casuarina nodules are colonized by Frankia strains that cannot be cultivated and exhibit genome erosion. Methods To test this, we directly sequenced nodule metagenomes from four countries, followed by reconstruction of metagenome-assembled genomes (MAGs). Results Our findings show that the dominant Frankia strains in field samples were far more diverse than the isolated strains and included MAGs with substantial genome reduction – one exhibiting over 25% reduction compared to F. casuarinae . Notably, we observed erosion of two types of [NiFe] hydrogenases, a phenomenon linked to evolution toward obligate symbiosis in other Frankia groups. Conclusion These results suggest that potentially obligate symbionts may dominate nodules in nature but had gone undetected by conventional approaches. For applications such as reforestation or tsunami shelter belts, crushed, nodule-derived strains may offer superior ecological compatibility. We speculate that Frankia strains followed two different evolutionary trajectories; one, towards obligate symbiosis accompanied by strong genome erosion, and two, towards rhizosphere colonization involving limited genome erosion.