Studying population dynamics and their impact on evolutionary processes in microorganisms such as bacteria is challenging. Evolution is the result of random mutations and selection under given environmental conditions. Environments are composed of abiotic and biotic factors and how an environment changes over time is presumably unpredictable (Lewontin, 1966; Beatty & Desjardins, 2009). The surrounding environment of an organism is dynamic and constantly changing,
with consequences for evolutionary outcomes (Franke et al., 2011). The aim of this study was to obtain information about the impact of population composition on switcher evolution and how population dynamics change during evolution depending on historical genotypes that occurred along the evolutionary path in Line 1.
Earlier genotypes (SBW25, 1s1, 1s2) are less likely to produce a switcher genotype than genotypes that appeared at a later stage along the evolutionary history (1s3, 1s4; Fig. 5.3). In addition to the number of observed switchers, the total number of new types, the proportion of new types in the total population, cell densities and phenotypic diversity (number of different colony morphotypes) within a microcosm were recorded for each replicate to get an estimate of how the population compositions developed within three days of growth and diversification in a static environment. As has been pointed out earlier, SBW25, 1s1 and 1s2 evolved faster than did 1s3 and 1s4. Nearly all replicates of SBW25, 1s1 and 1s2 produced a new phenotype within the first three days in a static microcosm (Fig. 5.4). In addition, new types were able to reach high cell frequencies (Fig. 5.5), indicating that some new variants had a substantial advantage under these conditions. At the same time the likelihood of switcher occurrence decreased (Fig. 5.6). It has been shown that genetic constraints in 1s1 and 1s2 are likely to limit the evolution of a switcher (Fig. 5.1), perhaps due to negative genetic interactions (Khan et al., 2011). In SBW25 however it is possible that the variety of ecological interactions between the many different types that appeared quite quickly decrease the likelihood of switcher occurrence. For example, strong competition for the air-‐liquid interface may have prevented the establishment of a switcher genotype within the population.
The comparison of the different founder populations shows that diversity was lower in 1s3 and 1s4 than in the genotypes SBW25, 1s1 and 1s2 (Fig. 5.7). Low frequency of new types and lower diversity after diversification of genotypes that occurred later along the evolutionary pathway suggest that the supply of beneficial mutations was higher in the beginning and decreased over time (Lenski & Travisano, 1994; Arjan et al., 1999). Due to the increased supply of beneficial mutations many different novel types evolved simultaneously (McDonald et al.,
2009; Sniegowski & Gerrish, 2010) in the beginning and prevented any switcher mutation that might have occurred of becoming established in the population. Later on the rate of beneficial mutations decreased, which may explain the increased number of transfers that were needed to detect a new type in 1s3 and 1s4. The lack of beneficial mutations, on the other hand, might have increased the chance of switcher mutations becoming fixed within populations of 1s3 and 1s4 before another beneficial mutation arose. Here timing of occurrence of the switcher mutation determines the success of the novel phenotype (Hegreness et al., 2006). The lack of beneficial mutations can have different causes. As addressed earlier, the high fitness of 1s3 and 1s4 because of cellulose production might decrease the chances of mutations that imply only a small benefit (e.g. weak biofilm). Alternatively, common evolutionary routes might become depleted over the course of evolution. Other mutational pathways that can improve the performance under present environmental circumstances have to be found, which may take a longer time. This will be discussed in more detail in the next section.
5.4.3.1.1 Depletion of evolutionary pathways with proceeding evolution
The capacity to evolve a switcher based on carB* has been shown to exist in the ancestor SBW25. Here carB* had a positive effect on fitness, indicating that a switcher can increase in frequency within a SBW25 population (Fig. 5.1). This raises the question as to why no switcher was found in the first selection round during the REE in any of the 12 parallel lineages, since they started from the same ancestor.
The evolutionary history of Line 1 (Tab. 5.1) is the result of evolution in two alternating environments, static and shaken, starting from the common ancestor P. fluorescens SBW25. It is known that P. fluorescens SBW25 diversifies quickly when growing in a static KB filled microcosm (Rainey & Travisano, 1998). Within hours usually, WS types arise as a consequence of an oxygen gradient that develops within the media. The WS types are able to colonise the air-‐liquid interface due to biofilm formation, which is the result of the overproduction of a cellulosic polymer
(Spiers et al., 2003; Rainey & Travisano, 2003). The transfer of a WS type into a shaken environment generally causes the reversal to an ancestral-‐like SM type (Koza et al., 2011). Suppressor analysis of novel genotypes that evolved during the course of the REE in the 12 replicate lineages identified important common genetic pathways that underlie the evolution of WS and SM types. In many cases novel types showed mutations in wsp, wss, aws and mws (see Chapter 1, section 1.4.3; Kahn et al., 1998; Bantinaki, 2001; Spiers et al., 2002; Spiers et al., 2003; Gehrig, 2005; McDonald et al., 2009). Noteworthy is the modular nature of mutation occurrences in all 12 lineages, such as that one mutation induces the synthesis of a cellulosic polymer under static conditions followed by a mutation in the same locus that turns off the production under shaken conditions. For example in Line 1 the first mutation appeared in mwsR and led to a WS type (Tab. 5.1). The subsequent mutation took place again in mwsR and caused a SM-‐like phenotype. In the next selection round the WS type was induced by a mutation in aws. After the change to a shaken environment another mutation in aws caused the reversal to the SM type. This modularity was not observed for the fifth and sixth mutations (wspF and wssA), but after that was observed again for the seventh and eighth mutations, which occurred in mwsR (Tab. 5.1). Although the modular organisation was interrupted by mutations in wspF followed by wssA, they still represent two out of four common mutational pathways involved in WS and SM evolution (Tab. 5.1).
The switcher genotype was detected during the ninth selection round and had a mutation in carB, which was not involved in any of the phenotypic changes that occurred previously. Other studies found a correlation between the fitness of a
carB* mutation and the proportion of WS types (Gallie, 2009). For example the fitness benefit of carB* in SBW25 decreased when competition between SBW25 and SBW25carB* took place over 72 hours instead of 48 hours, due to the occurrence of novel WS types. This fitness decrease was not observed in 1s4 after 72 hours because evolution was much slower. It was suggested that many of the common mutational routes that can lead to a WS type (wsp, wss, aws and mws) are available in genotypes that evolved during earlier selection rounds (e.g. SBW25, 1s1, 1s2). With proceeding evolution these common pathways are gradually
removed due to mutations and are therefore not available in 1s4 for WS evolution (Gallie, 2009). The depletion of common mutational pathways over time may explain the increased duration of evolution, the reduced proportion of novel types, the decreased diversity that was observed during the SREE in 1s3 and 1s4 in this study, and an elevated likelihood of switcher emergence (Arjan et al., 1999). Recently, most of the mutational pathways of all 12 replicate P. fluorescens lineages have been identified. The data suggest that the overlap in genetic changes is higher in the beginning of the evolutionary pathways and that lineages tend to have mutations in more diverse loci the longer they evolve (data not published). This suggests that after common mutational pathways have been used (McDonald et al., 2009), less likely and more diverse mutational pathways can arise that increase the chance of survival in that particular environment. It has been suggested that common mutational targets, because of genetic architecture and functionalities, have a higher rate of being translated into adaptive phenotypic change (McDonald et al., 2009). This may explain the high number and the fast evolution of new types in SBW25, 1s1 and 1s2, where more of these common mutational pathways are still available, however it remains curious that some pathways such mwsR can be used more than once in the evolution of a WS (Tab. 5.1).
5.4.4 Comparison of theoretical capacity of switcher evolution and