Comprehensive genomic review of the genus Salinisphaera with the proposal of two new subspecies

Salinisphaera is a genus of moderately halophilic bacteria within the class Gammaproteobacteria , commonly isolated from diverse saline environments. Traditional phenotypic methods have proven insufficient to resolve taxonomic boundaries among closely related species in this genus. With the advent of whole-genome sequencing, genome-based analyses have become essential for accurate species and subspecies delineation. This study used comprehensive phenotypic and genomic comparisons to investigate the taxonomic relationship between Salinisphaera halophila YIM 95161 T and Salinisphaera orenii MK-B5 T . Phylogenomic analyses based on core gene alignments revealed that S. halophila YIM 95161 T and S. orenii MK-B5 T form a robust monophyletic clade, consistent with high average nucleotide identity (>96%) and digital DNA–DNA hybridization values (>70%), confirming their membership within the same species. However, notable and consistent differences in growth physiology, enzymatic activity, fatty acid composition and genomic G+C content (69.5 mol% vs. 63.4–63.6 mol%) support the recognition of two distinct subspecies. Carbohydrate-active enzyme (CAZyme) repertoires also revealed divergence in CAZyme profiles, aligning with ecological differentiation. Therefore, we propose that S. halophila YIM 95161 T be reclassified as S. orenii subsp. halophila subsp. nov., while S. orenii MK-B5ᵀ be retained as S. orenii subsp. orenii subsp. nov. This refined classification highlights the value of integrated genomic and phenotypic approaches in resolving intraspecific structure and improving taxonomic resolution within halophilic bacterial genera.