, with fixed parameters t I~1 2h and s I~1 h. Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation. Bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to B. subtilis. For more than six decades, this organism has played an integral role in the biodefense community as a simulant for biological warfare and bioterrorism events (BW) and is commonly referred to by its military two-letter designation ''BG'' [1, 2] . B. atrophaeus has served in studies of agent dispersal [3] , decontamination simulations [4, 5] and large-scale process development [6] . In addition to its historical use as a BW simulant, it is currently in widespread commercial use as a surrogate for spore-forming bacteria [5, 7] and is the basis of numerous assays for spore inactivation [8, 9] . In addition to its role as a simulant, the organism plays an important role in the biotechnology industry as a source of restriction endonucleases and of the glycosylation inhibitor nojirimycin [10] .