Discordance Between 16S rRNA Similarity and Genome‐Based Species Boundaries in Gluconobacter

ABSTRACT

The 16S rRNA gene is widely used for bacterial identification, but its resolving power is limited in several taxa. In the genus Gluconobacter , species boundaries remain unclear despite reliance on 16S‐based classification. To examine these limitations, we analyzed all publicly available complete genomes ( n  = 12) together with curated 16S rRNA sequences and compared 16S rRNA sequences similarity with genome‐based relatedness. Within the sampled complete genomes, 16S rRNA sequences were highly conserved and showed substantial overlap between intra‐ and interspecific similarities. In contrast, average nucleotide identity (ANI)‐based divergence was pronounced: ANI values ranged from 79.7% to 100%, and G. oxydans included more than one genomic lineage. Notably, two strains (H24 and 1.637) showed higher ANI values (97.2%–97.4%) to G. thailandicus than to other G. oxydans strains. Even the hypervariable V4, V6, and V8 regions lacked clade‐specific signatures, indicating limited species‐level resolution within this group. Furthermore, type‐strain 16S rRNA sequences of G. oxydans , G. frateurii , and G. japonicus show ≥ 99% similarity and are grouped within the same non‐redundant representative in the SILVA NR99 database, indicating limited species‐level resolution. These results demonstrate a clear mismatch between 16S rRNA sequences similarity and genomic structure in Gluconobacter . Genome‐based criteria, supported by refined reference databases and validated gene markers, are essential for accurate taxonomy and microbiome‐based analyses.