Genome sequencing of cicada obligate and facultative endosymbionts and co-phylogenetic analyses with their hosts
Metagenomic sequencing datasets for ecological diversification of cicadas and their microbial endosymbionts. Many cicada species host two ancient bacterial obligate nutritional endosymbionts, Karelsulcia and Hodgkinia, which together synthesize ten essential amino acids (EAAs) for their host cicadas. In many of these cicada species, Hodgkinia has split into different cell lineages that must cooperate to fulfill the functions of the original single lineage. As a result, in many cicada lineages, Hodgkinia has been replaced by yeast-like fungal symbionts (YLS), and YLS replacement by another fungus or YLS has also occurred repeatedly. However, no such cicadas (in which Karelsulcia, Hodgkinia, and YLS coexist) have been found. Therefore, it is of great interest whether these three symbionts can coexist and, if so, how they interact. Moreover, considerable diversity has been observed in the genome of Hodgkinia among different cicada species, which seemingly suggest that Hodgkinia independently infected the ancestors of different cicada lineages on multiple occasions. In addition, many cicada species have facultative symbionts whose nutritional contributions to their cicada hosts are also unknown. To shed light on such questions, we reconstructed patterns and consequences of bacteriome- and fat-body-associated obligate endosymbiont loss and concordant gains of YLS in 133 cicada species using microscopy, comparative genomics and cophylogenetic analyses. We show that the phylogenetic relationships of the two ancient vertically-transmitted bacterial endosymbionts of cicadas, Hodgkinia and Karelsulcia, mirror the host phylogeny. In particular, phylogenetic analysis indicates a single ancestral infection of cicadas by Hodgkinia with subsequent host-symbiont codiversification before being replaced in some lineages by YLS. Hodgkinia loss has occurred at least eight times, and YLS replacement by another Ophiocordyceps fungus or YLS has occurred repeatedly at least 17 times. Most importantly, we demonstrate a case of co-existence of Hodgkinia with Karelsulcia and a YLS, representing an advanced ongoing symbiont replacement process. In some individuals of the cicada Chremistica ochracea, the Hodgkinia genome is highly degenerated but colonizes (instead of neighboring) its partner Karelsulcia. The physical fusion of these two bacterial endosymbionts yields a nested symbiosis while the new YLS is recruited, probably preserving essential metabolic pathways necessary for host nutrition and facilitating continued vertical symbiont transmission. Such fusion may have provided refuge for the degraded bacterial endosymbiont and delayed the symbiont replacement. Moreover, phylogenetic analysis revealed that such fungal endosymbionts all belong to the genus Ophiocordyceps, in which cicada-associated YLS form a monophyletic clade, and that free-living Ophiocordyceps fungi tend to have more introns than YLS. The results illuminate a very late ongoing stage in the symbiont replacement, the role of symbiotic associations in driving mitogenome intron loss during evolutionary adaptation, and the complexity of co-evolution between symbionts and Cicadidae. And this answers the enduring puzzles in the life sciences of how host insects can break away from their dependence on an ancient and potentially inefficient endosymbiont and what adaptative or nonadaptive evolution may evolve before a symbiont is going to be replaced by another symbiont. The obligate and facultative emdosymbionts associated with bacteriomes and fat bodies collectively synthesize EAAs and B vitamins for their host cicadas. These findings shed light on adaptive and non-adaptive evolutionary mechanisms involved in symbiont loss and replacement, and offer fresh insights into the endosymbiotic origins of cellular organelles. For more details, please see our article entitled: Genome degradation results in nested symbiosis and endosymbiont replacement in cicadas, doi.org/10.1038/s41467-025-65129-9.