Life on Earth comprises prokaryotes and a broad assemblage of endosymbioses. The pages of Molecular Biology and Evolution and Genome Biology and Evolution have provided an essential window into how these endosymbiotic interactions have evolved and shaped biological diversity. Here, we provide a current perspective on this knowledge by drawing on decades of revelatory research published in Molecular Biology and Evolution and Genome Biology and Evolution, and insights from the field at large. The accumulated work illustrates how endosymbioses provide hosts with novel phenotypes that allow them to transition between adaptive landscapes to access environmental resources. Such endosymbiotic relationships have shaped and reshaped life on Earth. The early serial establishment of mitochondria and chloroplasts through endosymbioses permitted massive upscaling of cellular energetics, multicellularity, and terrestrial planetary greening. These endosymbioses are also the foundation upon which all later ones are built, including everything from land–plant endosymbioses with fungi and bacteria to nutritional endosymbioses found in invertebrate animals. Common evolutionary mechanisms have shaped this broad range of interactions. Endosymbionts generally experience adaptive and stochastic genome streamlining, the extent of which depends on several key factors (e.g. mode of transmission). Hosts, in contrast, adapt complex mechanisms of resource exchange, cellular integration and regulation, and genetic support mechanisms to prop up degraded symbionts. However, there are significant differences between endosymbiotic interactions not only in how partners have evolved with each other but also in the scope of their influence on biological diversity. These differences are important considerations for predicting how endosymbioses will persist and adapt to a changing planet.

Many insects depend on ancient associations with intracellular bacteria for essential nutrition. The genomes of these bacteria are often highly reduced. Although drift is a major driver of symbiont evolution, other evolutionary forces continue to influence them. To understand how ongoing molecular evolution and gene loss shape symbiont genomes, we sequenced two of the most ancient symbionts known, Sulcia and Nasuia, from 20 Hawaiian Nesophrosyne leafhoppers. We leveraged the parallel divergence of Nesophrosyne lineages throughout Hawaii as a natural experimental framework. Sulcia and Nasuia experience ongoing—but divergent—gene loss, often in a convergent fashion. Although some genes are under relaxed selection, purifying and positive selection are also important drivers of genome evolution, particularly in maintaining certain nutritional and cellular functions. Our results further demonstrate that symbionts experience dramatically different evolutionary environments, as evidenced by the finding that Sulcia and Nasuia have one of the slowest and fastest rates of molecular evolution known.

Insects associate with a diversity of microbes that can shape host ecology and diversity by providing essential biological and adaptive services. For most insect groups, the evolutionary implications of host–microbe interactions remain poorly understood. Geographically discrete areas with high biodiversity offer powerful, simplified model systems to better understand insect–microbe interactions. Hawaii boasts a diverse endemic insect fauna (~6000 species) characterized by spectacular adaptive radiations. Despite this, little is known about the role of bacteria in shaping this diversity. To address this knowledge gap, we inaugurate the Native Hawaiian Insect Microbiome Initiative (NHIMI). The NHIMI is an effort intended to develop a framework for informing evolutionary and biological studies in Hawaii. To initiate this effort, we have sequenced the bacterial microbiomes of thirteen species representing iconic, endemic Hawaiian insect groups. Our results show that native Hawaiian insects associate with a diversity of bacteria that exhibit a wide phylogenetic breadth. Several groups show predictable associations with obligate microbes that permit diet specialization. Others exhibit unique ecological transitions that are correlated with shifts in their microbiomes (e.g., transition to carrion feeding from plant-feeding in Nysius wekiuicola). Finally, some groups, such as the Hawaiian Drosophila, have relatively diverse microbiomes with a conserved core of bacterial taxa across multiple species and islands.

The agricultural pest known as the glassy-winged sharpshooter (GWSS) or Homalodisca vitripennis (Hemiptera: Cicadellidae) harbors two bacterial symbionts, “Candidatus Sulcia muelleri” and “Ca. Baumannia cicadellinicola,” which provide the 10 essential amino acids (EAAs) that are limited in the host plant-sap diet. Although they differ in origin and symbiotic age, both bacteria have experienced extensive genome degradation resulting from their ancient restriction to specialized host organs (bacteriomes) that provide cellular support and ensure vertical transmission. GWSS bacteriomes are of different origins and distinctly colored red and yellow. While Sulcia occupies the yellow bacteriome, Baumannia inhabits both. Aside from genomic predictions, little is currently known about the cellular functions of these bacterial symbionts, particularly whether Baumannia in different bacteriomes perform different roles in the symbiosis. To address these questions, we conducted a replicated, strand-specific RNA-seq experiment to assay global gene expression patterns in Sulcia and Baumannia. Despite differences in genomic capabilities, the symbionts exhibit similar profiles of their most highly expressed genes, including those involved in nutrition synthesis and protein stability (chaperonins dnaK and groESL) that likely aid impaired proteins. Baumannia populations in separate bacteriomes differentially express genes enriched in essential nutrient synthesis, including EAAs (histidine and methionine) and B vitamins (biotin and thiamine). Patterns of differential gene expression further reveal complexity in methionine synthesis. Baumannia’s capability to differentially express genes is unusual, as ancient symbionts lose the capability to independently regulate transcription. Combined with previous microscopy, our results suggest that the GWSS may rely on distinct Baumannia populations for essential nutrition and vertical transmission.

Compared to free-living bacteria, endosymbionts of sap-feeding insects have tiny and rapidly evolving genomes. Increased genetic drift, high mutation rates, and relaxed selection associated with host control of key cellular functions all likely contribute to genome decay. Phylogenetic comparisons have revealed massive variation in endosymbiont evolutionary rate, but such methods make it difficult to partition the effects of mutation vs. selection. For example, the ancestor of auchenorrhynchan insects contained two obligate endosymbionts, Sulcia and a betaproteobacterium ( BetaSymb ; called Nasuia in leafhoppers) that exhibit divergent rates of sequence evolution and different propensities for loss and replacement in the ensuing ∼300 Ma. Here, we use the auchenorrhynchan leafhopper Macrosteles sp. nr. severini , which retains both of the ancestral endosymbionts, to test the hypothesis that differences in evolutionary rate are driven by differential mutagenesis. We used a high-fidelity technique known as duplex sequencing to measure and compare low-frequency variants in each endosymbiont. Our direct detection of de novo mutations reveals that the rapidly evolving endosymbiont ( Nasuia ) has a much higher frequency of single-nucleotide variants than the more stable endosymbiont ( Sulcia ) and a mutation spectrum that is even more AT-biased than implied by the 83.1% AT content of its genome. We show that indels are common in both endosymbionts but differ substantially in length and distribution around repetitive regions. Our results suggest that differences in long-term rates of sequence evolution in Sulcia vs. BetaSymb , and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis. SIGNIFICANCE STATEMENT: Two ancient endosymbionts in the same host lineage display stark differences in genome conservation over phylogenetic scales. We show the rapidly evolving endosymbiont has a higher frequency of mutations, as measured with duplex sequencing. Therefore, differential mutagenesis likely drives evolutionary rate variation in these endosymbionts.