The nitrogen-fixing fern Azolla has a complex microbiome characterized by multiple modes of transmission

Azolla is a floating fern that has closely evolved with a vertically transmitted obligate cyanobacterium endosymbiont--Anabaena azollae--that performs nitrogen fixation in specialized Azolla leaf pockets. This cyanobac-terium has a greatly reduced genome and appears to be in the "advanced" stages of symbiosis, potentially evolving into a nitrogen-fixing organelle. However, there are also other lesser-known inhabitants of the leaf pocket whose role and mode of transmission are unknown. We sequenced 112 Azolla specimens collected across the state of California and characterized their metagenomes in order to identify the common bacterial endosymbionts of the leaf pocket and assess their patterns of co-diversification. Four taxa were found across all samples, establishing that there are multiple endosymbionts that consistently inhabit the Azolla leaf pocket. We found varying degrees of co-diversification across these taxa as well as varying degrees of isolation by distance and of pseudogenation, which implies that the endosymbiotic community is transmitted by a mix of horizontal and vertical mechanisms, and that some members of the microbiome are more facultative symbionts than others. These results show that the Azolla symbiotic community is complex, featuring members at potentially different stages of symbiosis evolution, further supporting the utility of the Azolla microcosm as a system for studying the evolution of symbioses.