Cycads are the only early seed plants that have evolved a specialized root to host endophytic bacteria that fix nitrogen. To provide evolutionary and functional insights into this million-year old symbiosis, we investigate endophytic bacterial sub-communities isolated from coralloid roots of species from Dioon (Zamiaceae) sampled from their natural habitats. We employed a sub-community co-culture experimental strategy to reveal both predominant and rare bacteria, which were characterized using phylogenomics and detailed metabolic annotation. Diazotrophic plant endophytes, including Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium, and Nostoc species, dominated the epiphyte-free sub-communities. Draft genomes of six cyanobacteria species were obtained after shotgun metagenomics of selected sub-communities. These data were used for whole-genome inferences that suggest two Dioon-specific monophyletic groups, and a level of specialization characteristic of co-evolved symbiotic relationships. Furthermore, the genomes of these cyanobacteria were found to encode unique biosynthetic gene clusters, predicted to direct the synthesis of specialized metabolites, mainly involving peptides. After combining genome mining with detection of pigment emissions using multiphoton excitation fluorescence microscopy, we also show that Caulobacter species co-exist with cyanobacteria, and may interact with them by means of a novel indigoidine-like specialized metabolite. We provide an unprecedented view of the composition of the cycad coralloid root, including phylogenetic and functional patterns mediated by specialized metabolites that may be important for the evolution of ancient symbiotic adaptations.

Cycads are the only early seed plants that have evolved a specialized coralloid root to host endophytic bacteria that fix nitrogen for the plant. To provide evolutionary insights into this million-year old symbiosis, we investigate the phylogenetic and functional diversity of its endophytic bacterial community, based on the (meta)genomic characterization of the coralloid root of Dioon merolae collected from natural populations. We employed a co-culture-based metagenomics experimental strategy, termed EcoMining, to reveal both predominant and rare bacteria that were analyzed through phylogenomics and detailed metabolic annotation. Most of the characterized bacteria were identified as diazotrophic plant endophytes belonging to at least 18 different bacterial families. The draft genomes of three Cyanobacteria strains were obtained, and after whole-genome inferences they were found to form a monophyletic group, suggesting a level of specialization characteristic of co-evolved symbiotic relationships. In agreement with their large size over 8 Mbp, the draft genomes of these organisms were found to encode for biosynthetic gene clusters predicted to direct the synthesis of specialized metabolites present only in these symbionts. Overall, we provide a new notion of the composition and evolution of the cycad coralloid root that contributes to studies on phylogenetic and functional patterns in plant-bacteria symbiotic systems.

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