Legionella pelazae sp. nov. and Legionella navarrensis sp. nov., two novel Legionella species, reveal deep genomic diversification within engineered water systems.

Members of the genus Legionella are Gram-negative aquatic bacteria widely distributed in natural and engineered aquatic environments, yet the genomic diversity of environmental representatives remains incompletely resolved. During routine surveillance of two independent engineered water systems in Spain, two strains were recovered that could not be assigned to any described species using conventional molecular markers. To determine their taxonomic position, we applied a polyphasic approach integrating 16S rRNA and mip gene sequencing, whole-genome sequencing, phylogenomic reconstruction based on conserved core proteins, genome-based relatedness indices, and phenotypic and chemotaxonomic characterization. Phylogenetic analyses based on 16S rRNA and mip gene sequences placed both isolates within the genus Legionella while clearly separating them from recognized taxa. Genome-scale analyses confirmed deep divergence, with average nucleotide identity values of 85.01 % for Legionella pelazae sp. nov. and 70.37 % for Legionella navarrensis sp. nov. relative to their closest described species. The strains exhibited distinct genome architectures, including G+C contents of 35.52 and 48.72 mol%, and showed reproducible physiological and membrane lipid differences consistent with ecological differentiation. No acquired antimicrobial resistance determinants were detected. Taken together, these results reveal substantial intrageneric diversification among environmental Legionella populations inhabiting engineered water systems. On the basis of combined genomic, phylogenetic and phenotypic evidence, the strains represent two novel species within the genus, for which the names Legionella pelazae sp. nov. (CNM-1927-20T = CECT 31282=CCUG 78397T) and Legionella navarrensis sp. nov. (CNM-4043-24T = CECT 31283 = CCUG 78398T) are proposed.