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

<i>Salinisphaera</i> is a genus of moderately halophilic bacteria within the class <i>Gammaproteobacteria</i>, 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 <i>Salinisphaera halophila</i> YIM 95161^T and <i>Salinisphaera orenii</i> MK-B5^T. Phylogenomic analyses based on core gene alignments revealed that <i>S. halophila</i> YIM 95161^T and <i>S. orenii</i> 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 <i>S. halophila</i> YIM 95161^T be reclassified as <i>S. orenii</i> subsp. <i>halophila</i> subsp. nov., while <i>S. orenii</i> MK-B5ᵀ be retained as <i>S. orenii</i> subsp. <i>orenii</i> 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.