The first eight mutations play a role in the evolution of switching

The work in section 5.3.4 shows that, mechanistically, the carB mutation is the sole cause of the switcher phenotype. Nonetheless, switching took nine rounds of selection to evolve, indicating a possible evolutionary role for the preceding mutations. This possibility was addressed by two evolutionary experiments detailed in the following sections and in Beaumont et al. (2009).

5.3.5.1 Differential evolution of dimorphic genotypes from SBW25 and 1s4

The question of whether the first eight mutations play a role in the evolution of switching was addressed in an evolutionary experiment performed by Dr. Hubertus Beaumont. In this experiment, a single bout of REE selection in the static environment was applied to replicate populations of SBW25 (no mutations) and 1s4 (eight mutations)

(see section 2.2.9), and the frequency of switcher evolution from both genotypes was recorded. Out of 138 replicate SBW25 populations there were zero occurrences of switcher evolution, while switchers ‘won’ the evolutionary round in three out of 36 replicate 1s4 populations. Collectively, these results indicate that SBW25 and 1s4 differed in their capacity to give rise to colony switching (two-tailed Fisher’s exact test,

P=0.0083; see Beaumont et al. (2009)), suggesting that at least some of the early mutations contribute to the evolution of switching. Additionally, ‘losing’ switcher genotypes (i.e. observed but not numerically dominant) were isolated from a further three 1s4 populations, while no non-numerically dominant switcher genotypes were observed in SBW25 replicates. Each of the six switcher genotypes evolved from 1s4 produced translucent and opaque colonies on KB agar, and a mixture of capsulated and non-capsulated cell types (Table 5.5). For further phenotypic and genotypic characterization of these six genotypes, see section 6.3.1.

Phenotype Genotype Numerically dominant Colony Cell Evolution (days)

1w4-reD2 Yes Translucent and opaque colonies cap-/cap+ 9

1w4-reD12 Yes _{Translucent and opaque colonies cap-/cap+ 9}

1w4-reD1.8 Yes _{Translucent and opaque colonies cap-/cap+ 9}

1w4-reN1.2 No _{Translucent and opaque colonies cap-/cap+ 9}

1w4-reN1.4 No _{Translucent and opaque colonies cap-/cap+ 9}

1w4-reN1.5 No Translucent colonies with opaque sectors _{cap-/cap+ 9}

Table 5.5: Numerical dominance, colony morphology and cell phenotype of the six additional

switcher genotypes evolved in a static environment from 1s4. Genotype names contain information

about numerical dominance; D=dominant, N=not dominant. Evolution column gives time required for evolution of genotype in days (three days growth per transfer). See text for details.

5.3.5.2 The effect of preceding mutations on biological fitness of the carB mutation An experiment was devised to further investigate the nature of the evolutionary role played by the first eight mutations. The most parsimonious explanation for the results in

section 5.3.5.1 invokes differential relative fitness of the carB mutation in the SBW25 and 1s4 genetic backgrounds; in order to be observed in the REE, an emerging genotype must rise in frequency, and thus requires a fitness advantage over the dominant (starting) genotype. It is possible that 1w4 (and 1s4-carBmut) has a selective advantage over 1s4 in the static environment, while SBW25-carBmut has no selective advantage over SBW25. In order to test this hypothesis, fitness experiments were performed with the assistance of Dr. Christian Kost (section 2.2.11.5). Four distinct competition experiments were performed: (A) 1s4 and 1w4, (B) 1s4 and 1s4-carBmut, (C) 1w4 and 1w4-carBwt, and (D) SBW25-lacZ and SBW25-carBmut (Table 5.6).

Each experiment involved counting the frequency of competitor genotypes in ten replicate microcosms at the beginning and end of a 72 hour, static incubation (Appendix A3.2, Table 5.6). Most of these frequencies were easily counted using distinct colony morphotypes of competitor genotypes on KB agar. The exception was competition D, where insufficiently distinct morphotypes required the use of a neutral lacZ marker on KB agar containing X-gal (Zhang & Rainey, 2007). As detailed in section 2.2.12.2, the relative fitness of the competing genotypes was calculated for each replicate. From these data, the mean relative fitness of genotypes in each competition was obtained (Table 5.6; Beaumont et al., 2009). The results showed a selective advantage for 1w4 and 1s4-carBmut over 1s4, and a selective advantage for 1w4 over 1w4-carBwt. No fitness difference was detected between SBW25-carBmut and SBW25-lacZ.

Competition Competitor 1 Competitor 2 Mean RF ± 95 % C.I.a d.f.b P-valuec

A 1w4 1s4 _{1.16 ± 0.0269} 9 2.04 x 10-4***

B 1s4-carBmut 1s4 _{1.13 ± 0.0262} 8 1.25x 10-3**

C 1w4 1w4-carBwt _{1.18 ± 0.0296} 8 4.00x 10-4***

D SBW25-carBmut SBW25-lacZ _{1.05 ± 0.0814} 9 0.519

Table 5.6: Relative fitness of indicated genotypes as determined by 72-hour competition

experiments in a static environment. aMean and 95 % confidence interval (C.I.) of relative fitness

(R.F.; competitor 1/competitor 2) calculated from nine or ten replicates. bDegrees of freedom (d.f.) indicate the number of replicates used for calculations (d.f.=number of replicates-1). cP-values calculated for a two-tailed one sample t-test for a significant deviation of relative fitness values from 1.

5.3.5.3 Importance of the biological environment in switcher evolution

During competition D above, it was noted that WS-types evolved from both SBW25-

carBmut (white WS) and SBW25-lacZ (blue WS). This was an intriguing observation as the evolution of WS types, which have a high fitness in the static environment (Rainey & Rainey, 2003), has the potential to influence the relative fitness of the carB

mutation. To further investigate this possibility, the competition between SBW25-

carBmut and SBW25-lacZ was repeated, and relative fitness data collected from ten replicates at each of 24, 48 and 72 hours (Figure 5.12A, Appendix A3.2). No significant fitness difference was found between SBW25-carBmut and SBW25-lacZ at 24 and 72 hours (P-values=0.585 and 0.519, respectively), while at 48 hours SBW25-carBmut was found to have a significant fitness advantage (one-tailed t-test P=3.37 x 10-3). The number of white and blue WS types observed at each time point was recorded, and the proportion of WS in each replicate estimated (Figure 5.12B, Appendix A3.3). The SBW25-carBmut fitness decrease at 72 hours correlates with an increase in the proportion of WS types. These results suggest that the evolution of WS types from SBW25 causes a reduction in the relative fitness of the carB mutation. This reduction is not seen in competitions with 1s4, in which all conventional mutational routes to WS have been sequentially removed. Thus, it is proposed that the evolutionary role of the earlier mutations was to remove competitor WS types from the biological environment.

Figure 5.12: Results of 24, 48 and 72-hour competition experiments between SBW25-carBmut and

SBW25-lacZ in a static environment. (A) Relative fitness (RF) of competitor genotypes at each time

point. An RF of one (solid black line) indicates no difference in fitness; an RF over one (solid black line) indicates a selective advantage for SBW25-carBmut (as seen at 48 hours); an RF below one indicates a selective advantage for SBW25-lacZ.(B) Proportion of observed colonies that are WS at each time point. Both sets of data points are mean values of ten replicates, and all error bars indicate one standard error.

A

B

Length of competition (hours)

**

Length of competition (hours)

Pr o p or ti o n o f W S co lo n ie s Re la ti v e fi tn e ss ( RF )

5.4 Discussion