Contrasting bacteriome of the hornwortLeiosporoceros dussiiin two nearby sites with emphasis on the hornwort-cyanobacterial symbiosis

Symbioses between plants and nitrogen-fixing cyanobacteria are benchmark biological systems to understand mutualism and patterns of coevolution. The diversity of cyanobacteria associated to non-vascular land plants is still being unraveled, with a huge gap in knowledge from tropical areas, especially in hornworts. We focus on the Neotropical hornwortLeiosporoceros dussii, which has a semi-permanent association with cyanobacteria. Unlike other hornworts, the peculiarNostoccanals suggest cyanobiont specificity and selectivity. Here, we characterize the first hornwort bacteriome and focus on the endophytic cyanobacteria by describing its morphology, metagenomic diversity and phylogeny between two sampling sites in Panama. First, we use a metagenomic analyses (16S) from gametophytes from Rio El Guayabo and Rio Indio in Panama to identify the endophytic bacteria community ofL. dussii. To provide more information on the cyanobacterial endophytes, we have extracted cyanobacterial endophytes to measure cell size variation and use the markers trnL and rbcLX to determine their phylogenetic relationships. Finally, we use stable isotope delta N-15 to assess nitrogen flow in the plants. We find little diversity inLeiosporocerosbacteriome but observe significant community variations between sites. Results indicate a lack of cyanobacterial specificity of cyanobacteria associated toLeiosporoceros, with six unrelated clades ofNostocforming a polyphyletic assemblage. Four clades are nested within bacteria collected as free-living taxa suggesting that typically non-symbiotic cyanobacteria can be associated with this hornwort. Finally, the little nitrogen fractioning of the plant suggests the role of the cyanobacterial symbionts in nitrogen fixation. This study provides the first hornwort bacteriome and is an important step in the characterization of tropical bryophyte symbioses.