Phylogenomics, population structure, and symbiont diversity refine the systematics of Southeast Asian rice leafhoppers (Hemiptera: Cicadellidae).

Leafhoppers of the genus Nephotettix (Hemiptera: Cicadellidae) are major vectors of rice tungro disease in Southeast Asia. Despite their economic significance, their evolutionary history, genomic adaptations, and associations with microbial symbionts remain poorly understood. Here, we aim to resolve the phylogenomic placement of Nephotettix, characterize patterns of gene family evolution, assess population connectivity and document the prevalence of bacterial symbionts. Using transcriptomes from 30 species, we reconstructed a robust phylogeny of Cicadellidae, recovering Nephotettix as a strongly supported monophyletic clade in our Cicadellidae phylogeny. Comparative analyses revealed lineage-specific expansions and contractions of gene families, including calcium-binding proteins and membrane-trafficking factors, with several genes showing signatures of positive selection potentially linked to host adaptation. Sequences from individuals sampled across 8 Thai localities indicated shallow haplotype networks and low nucleotide diversity in Nephotettix virescens (Distant 1908) and Nephotettix nigropictus (Stål 1870), patterns consistent with recent expansion or high dispersal. Screening for symbionts revealed universal infections with the obligate symbionts Karelsulcia and Nasuia, while facultative symbionts, Wolbachia and Spiroplasma, occurred at low prevalence across species and sites. This integrative approach refines Nephotettix systematics, identifies candidate genes underlying adaptation, and provides insights into dispersal and symbiont associations. These findings expand genomic resources for rice leafhoppers and offer a framework for improved diagnostics and evidence-based management of tungro disease vectors.