ANI-based species boundaries and functional divergence in the core proteome of human-associated Bifidobacteria

Background . Over the last decade, the field of microbiology has been revolutionised by genome-wide genetic data and advanced genomic tools. This progress has been particularly instrumental in discerning hidden ecological traits across various taxonomic groups and in reclassifying species through genome-to-genome comparisons, offering a more accurate representation of microbial phylogenetic relationships. Here, we conducted a comprehensive analysis of the Bifidobacterium genus (N = 785 genomes) to investigate the phylogenetic relationships within this diverse bacterial group, focusing on species primarily inhabiting the human intestinal tract, representing a large-scale repertoire of genomic information, a wide diversity in terms of well-known species, and well-annotated genome entries for unambiguous analysis. Results . By exhaustively evaluating more than 600K comparisons based on genome-wide alignments, using Average Nucleotide Identity (ANI) metrics at both intra- and inter-species levels, we identified different boundaries for species demarcation compared with those typically accepted for bacteria. Applying robust ANI thresholds helped to resolve conflicts and ambiguities in the sub-speciation of certain Bifidobacterium biotypes. As a result, we propose reclassifying several subspecies of Bifidobacterium longum , Bifidobacterium animalis , and Bifidobacterium catenulatum as new species. Conclusions . This reclassification directly impacts our understanding of the ecology of this diverse bacterial group, the signals of genome divergence across certain biotypes, and on how their speciation traits influence their interactions with humans. Furthermore, by comprehensively analysing the core proteome, evidence of molecular evolution and selection pressure in several sets of functional genes is intuited in this bacterial group of broad interest. This has direct implications for how these bacteria adapt to their specific niches and compete with other microbes.