Phylogenomic and Comparative Genomic Analyses Support the Monophyly and Genomic Cohesion of the Pseudomonas Fluorescens Lineage but Not the Broader “Pseudomonas_E” Grouping

The genus Pseudomonas comprises a large and diverse group of bacteria of major clinical, ecological, and biotechnological importance. Genome-based studies have shown that species currently assigned to this genus are distributed among multiple distinct evolutionary lineages. In this study, we examined the evolutionary relationships and genomic cohesion of the Fluorescens lineage using genome sequences from more than 450 members of the family Pseudomonadaceae. Phylogenomic trees constructed from multiple datasets, including 987 core proteins, the Bac120 marker set, a six-protein housekeeping dataset, and 16S rRNA gene sequences, consistently recovered the Fluorescens lineage as a large and strongly supported monophyletic clade. Whole-genome similarity analyses based on average amino acid identity, percentage of conserved proteins, and Dice similarity further supported the genomic cohesion of this lineage. Pangenome analyses identified 18 protein families for which homologs sharing a minimum of 50% sequence identity and 50% sequence length were present in all examined members of the Fluorescens lineage but absent from other species in the analyzed dataset. In contrast, no protein family meeting these criteria was identified for the broader GTDB grouping formerly designated “Pseudomonas_E”. Additionally, our analyses identified a molecular signature consisting of a conserved one-amino-acid deletion in the SurA N-terminal domain-containing protein that was uniquely shared by all examined members of this lineage. In the GTDB classification, the Fluorescens lineage is included within the broader “Pseudomonas_E” grouping, more recently designated Aquipseudomonas. However, the composition and boundaries of this broader grouping vary among phylogenetic analyses, and it lacks the comparable genomic cohesion and defining molecular characteristics observed for the Fluorescens lineage. These results provide independent evidence that the Fluorescens lineage represents a stable, monophyletic, and genomically cohesive evolutionary group, whereas the broader GTDB grouping is not supported as a comparably coherent lineage.