Bacterial endosymbionts that provide nutrients to hosts often have genomes that are extremely stable in structure and gene content. In contrast, the genome of the endosymbiont has fractured into multiple distinct lineages in some species of the cicada genus To better understand the frequency, timing, and outcomes of lineage splitting throughout this cicada genus, we sampled cicadas over three field seasons in Chile and performed genomics and microscopy on representative samples. We found that a single ancestral lineage has split at least six independent times in over the last 4 million years, resulting in complexes of between two and six distinct lineages per host. Individual genomes in these symbiotic complexes differ dramatically in relative abundance, genome size, organization, and gene content. Each lineage retains a small set of core genes involved in genetic information processing, but the high level of gene loss experienced by all genomes suggests that extensive sharing of gene products among symbiont cells must occur. In total, complexes that consist of multiple lineages encode nearly complete sets of genes present on the ancestral single lineage and presumably perform the same functions as symbionts that have not undergone splitting. However, differences in the timing of the splits, along with dissimilar gene loss patterns on the resulting genomes, have led to very different outcomes of lineage splitting in extant cicadas.

Phylogenetic asymmetry is common throughout the tree of life and results from contrasting patterns of speciation and extinction in the paired descendant lineages of ancestral nodes. On the depauperate side of a node, we find extant ‘relict’ taxa that sit atop long, unbranched lineages. Here, we show that a tiny, pale green, inconspicuous and poorly known cicada in the genus Derotettix, endemic to degraded salt-plain habitats in arid regions of central Argentina, is a relict lineage that is sister to all other modern cicadas. Nuclear and mitochondrial phylogenies of cicadas inferred from probe-based genomic hybrid capture data of both target and non-target loci and a morphological cladogram support this hypothesis. We strengthen this conclusion with genomic data from one of the cicada nutritional bacterial endosymbionts, Sulcia, an ancient and obligate endosymbiont of the larger plant-sucking bugs (Auchenorrhyncha) and an important source of maternally inherited phylogenetic data. We establish Derotettiginae subfam. nov. as a new, monogeneric, fifth cicada subfamily, and compile existing and new data on the distribution, ecology and diet of Derotettix. Our consideration of the palaeoenvironmental literature and host-plant phylogenetics allows us to predict what might have led to the relict status of Derotettix over 100 Myr of habitat change in South America.

Mitochondrial genomes can provide valuable information on the biology and evolutionary histories of their host organisms. Here, we present and characterize the complete coding regions of 107 mitochondrial genomes (mitogenomes) of cicadas (Insecta: Hemiptera: Auchenorrhyncha: Cicadoidea), representing 31 genera, 61 species, and 83 populations. We show that all cicada mitogenomes retain the organization and gene contents thought to be ancestral in insects, with some variability among cicada clades in the length of a region between the genes nad2 and cox1, which encodes 3 tRNAs. Phylogenetic analyses using these mitogenomes recapitulate a recent 5-gene classification of cicadas into families and subfamilies, but also identify a species that falls outside of the established taxonomic framework. While protein-coding genes are under strong purifying selection, tests of relative evolutionary rates reveal significant variation in evolutionary rates across taxa, highlighting the dynamic nature of mitochondrial genome evolution in cicadas. These data will serve as a useful reference for future research into the systematics, ecology, and evolution of the superfamily Cicadoidea.

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