Chromosome-level genome assembly of the aster leafhopper (Macrosteles quadrilineatus) reveals the role of environment and microbial symbiosis in shaping pest insect genome evolution.

Leafhoppers comprise over 20,000 plant-sap feeding species, many of which are important agricultural pests. Most species rely on two ancestral bacterial symbionts, Sulcia and Nasuia, for essential nutrition lacking in their phloem and xylem plant sap diets. To understand how pest leafhopper genomes evolve and are shaped by microbial symbioses, we completed a chromosomal-level assembly of the aster leafhopper's genome (ALF; Macrosteles quadrilineatus). We compared ALF's genome to three other pest leafhoppers, Nephotettix cincticeps, Homalodisca vitripennis, and Empoasca onukii, which have distinct ecologies and symbiotic relationships. Despite diverging ~155 million years ago, leafhoppers have high levels of chromosomal synteny and gene family conservation. Conserved genes include those involved in plant chemical detoxification, resistance to various insecticides, and defence against environmental stress. Positive selection acting upon these genes further points to ongoing adaptive evolution in response to agricultural environments. In relation to leafhoppers' general dependence on symbionts, species that retain the ancestral symbiont, Sulcia, displayed gene enrichment of metabolic processes in their genomes. Leafhoppers with both Sulcia and its ancient partner, Nasuia, showed genomic enrichment in genes related to microbial population regulation and immune responses. Finally, horizontally transferred genes (HTGs) associated with symbiont support of Sulcia and Nasuia are only observed in leafhoppers that maintain symbionts. In contrast, HTGs involved in non-symbiotic functions are conserved across all species. The high-quality ALF genome provides deep insights into how host ecology and symbioses shape genome evolution and a wealth of genetic resources for pest control targets.

Genomic Comparisons Reveal Selection Pressure and Functional Variation Between Nutritional Endosymbionts of Cave-Adapted and Epigean Hawaiian Planthoppers.

Planthoppers in the family Cixiidae (Hemiptera: Auchenorrhyncha: Fulgoromorpha) harbor a diverse set of obligate bacterial endosymbionts that provision essential amino acids and vitamins that are missing from their plant-sap diet. "Candidatus Sulcia muelleri" and "Ca. Vidania fulgoroidea" have been associated with cixiid planthoppers since their origin within the Auchenorrhyncha, whereas "Ca. Purcelliella pentastirinorum" is a more recent endosymbiotic acquisition. Hawaiian cixiid planthoppers occupy diverse habitats including lava tube caves and shrubby surface landscapes, which offer different nutritional resources and environmental constraints. Genomic studies have focused on understanding the nutritional provisioning roles of cixiid endosymbionts more broadly, yet it is still unclear how selection pressures on endosymbiont genes might differ between cixiid host species inhabiting such diverse landscapes, or how variation in selection might impact symbiont evolution. In this study, we sequenced the genomes of Sulcia, Vidania, and Purcelliella isolated from both surface and cave-adapted planthopper hosts from the genus Oliarus. We found that nutritional biosynthesis genes were conserved in Sulcia and Vidania genomes in inter- and intra-host species comparisons. In contrast, Purcelliella genomes retain different essential nutritional biosynthesis genes between surface- and cave-adapted planthopper species. Finally, we see the variation in selection pressures on symbiont genes both within and between host species, suggesting that strong coevolution between host and endosymbiont is associated with different patterns of molecular evolution on a fine scale that may be associated with the host diet.

Erratum: A complex interplay of evolutionary forces continues to shape ancient co-occurring symbiont genomes.

[This corrects the article DOI: 10.1016/j.isci.2022.104786.].

A complex interplay of evolutionary forces continues to shape ancient co-occurring symbiont genomes.

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.

Mutational Pressure Drives Differential Genome Conservation in Two Bacterial Endosymbionts of Sap-Feeding Insects.

Compared with 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 versus 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 novode 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 potentially 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 versus BetaSymb, and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis.