Is the nitrogen-fixing root nodule symbiosis polyphyletic?

Root nodule symbiosis is known to be limited to four plant orders Fabales, Fagales, Cucurbitales, and Rosales which are grouped in the rosid I clade of angiosperms. However, several articles have reported ambiguous root nodule formation in Zygophyllaceae and Roystonea regia (Arecaceae) but have remained unexplained for decades.We collected root samples of Tribulus terrestris and Roystonea regia from West Asia and the Caribbean, respectively. Nodule-like structures were observed in roots of both plant species.Clusters of vesicles resembling those found in Frankia were observed in T. terrestris root nodules. Fixation thread like structures containing few bacterial cells were observed in R. regia root nodules. Metagenome sequence data suggest a possible association of T. terrestris and R. regia with nodulating Frankia and Bradyrhizobium respectively. Phylogenomic analyses indicate that the isolated bacteria from T. terrestris and R. regia are nested within the Gammaproteobacteria and Bacilli which are not typically known as nodulating bacteria.We conclude that T. terrestris and R. regia evolved to produce nodules in association with nitrogen-fixing bacteria, which is challenging the existing theories regarding the evolution of root nodule symbiosis. Although the bacteria identified in silico can fix nitrogen, their endosymbiotic association with these plant species remains unclear.