Genome-based reassessment of Sphingobacterium reveals the limitation of the fixed dDDH threshold for prokaryotic species delineation

Accurate species delimitation in prokaryotes increasingly relies on genome-scale comparisons, yet fixed genomic thresholds can be unreliable in lineages shaped by extensive gene flux. In this study, we revisited the taxonomy of genus <i>Sphingobacterium</i> using phylogenomic reconstruction and comprehensive whole-genome comparisons. The genus displays a highly open pan-genome, with only 22 universally conserved genes and nearly 20,000 cloud genes, indicating pronounced genomic plasticity. Within this complex evolutionary context, we detected a rare non-transitive paradox in digital DNA-DNA hybridization (dDDH) within the <i>Sphingobacterium siyangense</i> group. All strains share average nucleotide identity (ANI) values above the accepted species boundary (95%), yet some strain pairs exhibit dDDH values below the species threshold (70%), resulting in a conflict restricted to this metric. Phylogenomic analyses, core genome variation, average amino acid identity (AAI) patterns, and functional gene profiles consistently support the monophyly and genomic cohesion of these strains, showing that dependence on dDDH alone may lead to ambiguous species boundaries. Based on the combined evidence, we treat all members of the <i>S. siyangense</i> cluster as a single species and propose additional taxonomic revisions. <i>Sphingobacterium ginsenosidimutans</i> is recognized as a heterotypic synonym of <i>Sphingobacterium detergens</i>. The species <i>Rhinopithecimicrobium faecis</i> is proposed for reclassification as <i>Sphingobacterium faecis</i> comb. nov. These findings demonstrate that rigid dDDH cutoffs cannot fully capture evolutionary relationships and highlight the value of integrating phylogenomic and pan-genomic evidence for resolving complex species level classifications in prokaryotes.