Stable nutritional endosymbiosis across cryptic diversity of a leafhopper species complex

Ancient nutritional symbioses underpin the ecological success of many sap-feeding insects. In 'true hoppers' - the hemipteran suborder Auchenorrhyncha, obligate bacterial partners provide essential amino acids lacking in plant phloem diets. However, the stability and persistence of such associations across the diversity of hoppers are poorly understood, and investigations are often complicated by insufficiently resolved host identity.

Here, we combined multitarget amplicon sequencing, metagenomics, and microscopy to assess the compositional and functional diversity of the microbiota across Polish, Swedish, and Austrian populations of leafhoppers morphologically identified as Verdanus abdominalis. Host COI data revealed pronounced cryptic genetic diversity, indicating several deeply divergent lineages within the characterized collection, but limited microbiota variation among populations. 16S rRNA amplicon data confirmed the consistent presence of the ancient bacterial endosymbionts Candidatus Sulcia muelleri and Candidatus Nasuia deltocephalinicola, and metagenomics showed that their reduced but complementary genomes jointly encode the complete set of essential amino acid biosynthesis pathways required by the host. Other microbes were uncommon in these symbioses. Microscopy corroborated these findings, revealing conserved bacteriome organization and spatial separation of Sulcia and Nasuia within distinct bacteriocytes.

Our results demonstrate that the Sulcia-Nasuia dual symbiosis remains evolutionarily stable across cryptic Verdanus diversity, underscoring the robustness of ancient nutritional partnerships despite ongoing host diversification.