Diversity of symbiotic cyanobacteria in cycad coralloid roots using a short-read rbcL-X amplicon

Cycads are the only gymnosperms forming a symbiosis with nitrogen-fixing cyanobacteria in a specialized organ: the coralloid root. This paper investigates the endophytic bacterial community inhabiting the coralloid roots of two cycads from Panama. We sampled coralloid roots from Zamia nana (terrestrial) and Zamia pseudoparasitica (epiphytic). Then, we used the 16S rRNA amplicon marker to describe the entire bacterial community. We also designed a new marker to amplify the rbc L- rbc X spacer and around 100 bp of the rbc X gene, targeting cyanobacteria. We found that using 16S, endophytic bacteria diversity is represented mainly by the phyla Actinobacteria, Cyanobacteria, and Proteobacteria. In addition, 16S analyses showed that Zamia species do not share a core cyanobacterial community (using stringent 75% and 90% thresholds), while the two species shared 4 ASVs at a 50% threshold. The newly developed rbc L- rbc X marker revealed that both species share a core cyanobacterial community represented by a single amplicon sequence variant (ASV1) ( Nostoc sp. ) at 90% threshold that is found in the same phylogenetic clade of that contain mostly Panamanian symbiotic cyanobacteria. Using a 75% threshold, only three ASVs (ASV1, ASV2, ASV3) were present across samples, and five ASVs at 50% threshold. This new marker can effectively identify cyanobacteria ASVs and provide a better resolution for microbial analyses in autotroph cyanobacterial symbioses.