Lateral gene transfer shapes the distribution of nitrogen fixation within a cosmopolitan clade of marine Thalassolituus

Biological nitrogen fixation converts dinitrogen gas into ammonia, supplying new bioavailable nitrogen to marine ecosystems, but the evolutionary processes shaping its distribution among heterotrophic bacteria remain unresolved. Thalassolituus, a genus within the family Oceanospirillaceae (order Oceanospirillales), is best known for hydrocarbon degradation, yet nitrogen fixation has been confirmed in only one cultured isolate. We analyzed 74 quality-filtered genomes assigned to Thalassolituus within a broader dataset of 421 Oceanospirillaceae genomes to reconstruct the distribution and evolutionary history of the minimal nifHDKENB gene set. Twenty-five genomes encoded complete or near-complete nif loci and occurred in four well-supported clades interspersed with genomes lacking the pathway. Statistical topology tests rejected the species-tree topology for concatenated NifHDK and NifHDKENB protein alignments, and eleven recombination events across nif loci were supported by at least four detection methods. The core nifHDK gene order remained broadly conserved, but accessory neighborhoods differed among clades, and structural nifHDK genes showed stronger codon adaptation than biosynthesis nifENB genes. Clade 2 combined species-gene tree congruence, conserved gene neighborhoods, and comparatively high nifH codon adaptation, whereas Clades 1 and 4 showed greater phylogenetic discordance, more recombination, and weaker codon adaptation. These results support a reticulate history in Thalassolituus, in which lateral acquisition introduced nitrogen fixation into distinct lineages, vertical inheritance preserved it within some clades, and homologous recombination continued to reshape nif loci. These processes help explain why nitrogen fixation is unevenly distributed among closely related marine heterotrophic bacteria.