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Bartonella is a genetically diverse group of vector-borne bacteria. Over 40 species have been characterized to date, mainly from mammalian reservoirs and arthropod vectors. Rodent reservoirs harbor one of the largest Bartonella diversity described to date, and novel species and genetic variants are continuously identified from these hosts. Yet, it is still unknown if this significant genetic diversity stems from adaptation to different niches or from intrinsic high mutation rates. Here, we explored the vertical occurrence of spontaneous genomic alterations in 18 lines derived from two rodent-associated Bartonella elizabethae-like strains, evolved in nonselective agar plates under conditionsmimicking their vector-andmammalian-associated temperatures, and the transmission cycles between them(i.e., 26 degrees C, 37 degrees C, and alterations between the two), using mutation accumulation experiments. After similar to 1,000 generations, evolved genomes revealed few point mutations (average of one-point mutation per line), evidencing conserved single-nucleotide mutation rates. Interestingly, three large structural genomic changes (two large deletions and an inversion) were identified over all lines, associated with prophages and surface adhesin genes. Particularly, a prophage, deleted during constant propagation at 37 degrees C, was associated with an increased autonomous replication at 26 degrees C(the flea-associated temperature). Complementarymolecular analyses of wild strains, isolated from desert rodents and their fleas, further supported the occurrence of structural genomic variations and prophage-associated deletions in nature. Our findings suggest that structural genomic changes represent an effective intrinsic mechanism to generate diversity in slow-growing bacteria and emphasize the role of prophages as promoters of diversity in nature.