Competition experiments to quantify the fitness differences between the ancestral and novel alleles homozygotes under selective conditions.

a) Pairwise competition experiments between plasmid carrying strains A. baylyi BD413 pTAD-R (GFP, ancestral plasmid allele) and A. baylyi BD413 pTAD-R (nptII, novel plasmid allele); A. baylyi BD4 (tetA, ancestral chromosomal allele) and A. baylyi BD413 (nptII, novel chromosomal allele). The experiments were conducted under selective conditions (kanamycinplasmid = 0.375μg/ml and kanamycinchromosome = 0.4μg/ml) A bigger range of antibiotic concentrations were first tested for the plasmid replicon to determine the range of kanamycin concentrations that would give the desired selective benefit. The chromosomal antibiotic values tested were restricted to those closer to the plasmid replicon concentration values. A transfer regime was employed where cultures were grown at 30°C and serially passaged every 24h for a duration of 120h. Experiments were conducted with 6 biological replicates per replicon. Fitness was calculated relative to the growth as described in refs. [50,51], a lower fitness effect could be observed under selective conditions, where the fitness was not significantly different than 0.9 (pTAD-R H0: w = 0.9, p = 0.4375, n = 6 & Chromosome H0: w = 0.9, p = 5625, n = 6. Wilcoxon test). b) Selection coefficient applicable to the ancestral allele carrying strain under selective conditions. Selection coefficient was calculated by the formula s = w -1[50]. A significant selection coefficient was applicable to the ancestral strain. The antibiotic treatment applied to the plasmid gives a mean selective coefficient of s = -0.1185 ± 0.0415, that was not significantly different from s = -0.1 (p = 0.4375, using Wilcoxon test, n = 6). For the chromosomal replicon the antibiotic treatment gives a mean selective coefficient of s = -0.0984 ± 0.0305, with no significant difference from s = -0.1 (p = 0.5625, using Wilcoxon test, n = 6). Ancestral allele containing strain shows a selective disadvantage (s (TIFF)