Phylogenomic studies and molecular markers clarifying the evolutionary relationships and classification of Pseudalkalibacillus species: proposal for the family Guptibacillaceae fam. nov. harbouring the genera Guptibacillus gen. nov. and Exobacillus g

The genus <i>Pseudalkalibacillus</i>, created by the reclassification of specific deep-branching <i>Alkalihalobacillus</i> species, exhibits polyphyletic branching. The Genome Taxonomy Database (GTDB) also assigns <i>Pseudalkalibacillus</i> species into two families and three genera. To clarify the evolutionary relationships and classification of <i>Pseudalkalibacillus</i> species, we report detailed investigations using phylogenomic and molecular signature-based approaches. In phylogenomic trees, <i>Pseudalkalibacillus</i> species are distributed within two family-level lineages. One of these clades, containing the type species <i>of Pseudalkalibacillus</i> (viz. <i>Pseudalkalibacillus decolorationis</i>), represents the genus <i>Pseudalkalibacillus</i>, groups within the family <i>Fictibacillaceae</i>. Ten novel conserved signature indels (CSIs) identified in this study are specific for this clade, providing a robust means for the differentiation of the emended genus <i>Pseudalkalibacillus</i>. The remaining <i>Pseudalkalibacillus</i> species form a separate family-level clade, designated as f_HBI72195 in the GTDB. Within this clade, all species except <i>Pseudalkalibacillus caeni</i> form a robust clade designated as <i>Pseudalkalibacillus</i> clade -2 in our work and g_<i>Anaerobacillus</i>_A in the GTDB. We have also identified 15 novel CSIs specific to this clade. As the <i>Pseudalkalibacillus</i> clade -2 is distinct from <i>Pseudalkalibacillus</i>, we propose transferring species from this clade into a new genus, <i>Guptibacillus</i> gen. nov. The species <i>P. caeni</i> branches distinctly from other <i>Pseudalkalibacillus</i> species, and the GTDB considers it a novel genus (g_<i>Bacillus_</i>BR). Six newly identified CSIs are specific to this species, and we are proposing the transfer of this species into a new genus, <i>Exobacillus</i> gen. nov. Two additional identified CSIs are shared by members of the novel family-level taxon (f_HBI72195) comprising the proposed genera <i>Guptibacillus</i> and <i>Exobacillus</i>, for which we are proposing the name <i>Guptibacillaceae</i> fam. nov. Lastly, the results presented here also show that '<i>Pseudalkalibacillus hemicentroti</i>' and '<i>Pseudalkalibacillus macyae</i>' are later heterotypic synonyms of <i>Guptibacillus hwajinpoensis</i>. These changes, which reliably depict the evolutionary relationships among <i>Pseudalkalibacillus</i> species, should be helpful in future studies of these organisms.