Amresh Kumar Sharma Anup Som Department of Bioinformatics, Kalinga University, Naya Raipur, Chhattisgarh-492101, India Centre of Bioinformatics, Institute of Interdisciplinary Studies, University of Allahabad, Prayagraj-211002, India Network Biology ISSN 2220-8879 http://www.iaees.org/publications/journals/nb/online-version.asp 2026 16 1 15 30 International Academy of Ecology and Environmental Sciences Hong Kong 3 August 2025 10 September 2025 1 March 2026 Wolbachia evolution phylogenomics average nucleotide identity dDDH recombination Wolbachia is an alpha-proteobacteria and endosymbiont, causing infection in arthropods as parasites and in nematodes as mutualists. Unraveling the evolution of Wolbachia supergroups is the most fascinating topic among the scientific community. In this quest, we analyzed 30 Wolbachia genomes that belong to 11 supergroups (A, B, C, D, E, F, J, L, M, S, and T). We also performed average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis to understand the supergroup demarcation. Furthermore, we carried out multi-locusphylogenetic analysis using 189 single-copy orthologs followed by recombination analysis. We found that ANI values for each strain belonging to the same supergroups are supported by a threshold value of not less than 95% except for two strains wCtub and wDcau (ANI value is 82%). dDDH analysis finds that most supergroups follow species boundary thereshold except supergroup J. Further, a phylogenomic tree was reconstructed for supergroup analysis and found that the strains wCtub and wDcau were monophyletic and belong to the same supergroup J. Further, strains from supergroup A and supergroup B were monophyletic. Supergroups J and C were monophyletic, and supergroup S was an outgroup to them. Supergroup T was an outgroup to supergroups C, D, F, J, and S. Supergroups E, L and M were at the base of other supergroups radiation (i.e., supergroups A, B, C, D, F, J, S, and T). Recombination analysis finds 8 genes (out of 189 genes) showed genetic recombination, which infers the role of recombination has minimal effect in Wolbachia supergroup evolution. Overall, this study concludes that besides the 16S rRNA-based phylogeny, ANI analysis, and dDDH test, phylogenomic study indispensable for unraveling the evolution of Wolbachia supergroups. http://www.iaees.org/publications/journals/nb/articles/2026-16(1)/evolution-of-Wolbachia-supergroup.pdf