Vaughn S. Cooper, Samuel H. Vohr, Sarah C. Wrocklage, Philip J. Hatcher

In bacterial genomes composed of more than one chromosome, one replicon is typically larger, harbors more essential genes than the others, and is considered primary. The greater variability of secondary chromosomes among related taxa has led to the theory that they serve as an accessory genome for specific niches or conditions. By this rationale, purifying selection should be weaker on genes on secondary chromosomes because of their reduced necessity or usage. To test this hypothesis we selected bacterial genomes composed of multiple chromosomes from two genera, Burkholderia and Vibrio, and quantified the evolutionary rates (dN and dS) of all orthologs within each genus. Both evolutionary rate parameters were faster among orthologs found on secondary chromosomes than those on the primary chromosome. Further, in every bacterial genome with multiple chromosomes that we studied, genes on secondary chromosomes exhibited significantly weaker codon usage bias than those on primary chromosomes. Faster evolution and reduced codon bias could in turn result from global effects of chromosome position, as genes on secondary chromosomes experience reduced dosage and expression due to their delayed replication, or selection on specific gene attributes. These alternatives were evaluated using orthologs common to genomes with multiple chromosomes and genomes with single chromosomes. Analysis of these ortholog sets suggested that inherently fast-evolving genes tend to be sorted to secondary chromosomes when they arise; however, prolonged evolution on a secondary chromosome further accelerated substitution rates. In summary, secondary chromosomes in bacteria are evolutionary test beds where genes are weakly preserved and evolve more rapidly, likely because they are used less frequently.