Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

<div><p>The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of <i>Escherichia coli</i> were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.</p></div

Growth rate changes in the evolution for thermal adaptation.

<p>The duplicated Line1 and Line2 from the final population at generation 7580 were passaged daily. The daily exponential growth rates at various temperatures were calculated based on the absorbance at 600 nm as described in the Materials and Methods section. a) Each color indicates the growth rates of the bacterial cells at 44.8°C (black), 45.0°C (pink), 45.2°C (blue), 45.4°C (orange), 45.6°C (green), 45.8°C (purple), and 46.0°C (red). The cell populations that were finally acquired at 46.0°C were subjected to genome sequencing analysis. b) The populations at generation 7580 (white) and the final populations of Line1 (gray) and Line2 (dark gray) were cultured to observe the growth rates at 20.0°C, 37.0°C, 45.0°C, 46.0°C, 46.5°C and 47.0°C. The average and standard errors were obtained by repeated cultures (n = 5–6).</p

The linear accumulation of mutations over generations.

<p>The number of non-synonymous (top), synonymous (middle) and non-coding (bottom) mutations were plotted at the time point when the frequency of the mutated genome first became greater than 10%. The mutated genomes that appeared at the latter stage in Line1 and declined at the end, in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005392#pgen.1005392.g003" target="_blank">Fig 3</a>, which correspond to clusters R4 and R5 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005392#pgen.1005392.s003" target="_blank">S2 Table</a>, were omitted to focus on only the lineages dominating at the ends of the two lines. The dashed and solid lines represent the regression lines for non-synonymous and synonymous mutations, respectively (n = 21, including no mutation at generation 5212, P < 10⁻¹⁶ for non-synonymous and P < 10⁻¹⁰ for synonymous).</p

Temporal frequency change for each mutation.

<p>The frequency estimated by Sanger sequencing is plotted for each substituted base for Line1 (upper) and Line2 (bottom). The mutations that were inherited in Line1 were colored red, while those that were suppressed at the end of Line1 are colored in purple. The blue lines correspond to the mutations that were maintained at the end of Line2. The gray lines represent mutations that were inherited in both Line1 and Line2.</p

The fixation processes of the genomes.

<p>The frequency of each mutated genome at each generation was calculated as the average over the mutations in the same clusters. The bars are the standard errors. The standard error for the genomes only with single mutations was replaced with the standard deviation from Sanger sequencing (6.2%, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005392#pgen.1005392.s001" target="_blank">S1 Fig</a>). The spread for each genome began with an arbitrary height at one sampling point before the frequency went beyond the detection limit of 5%. A frequency higher than 95% was regarded as 100%. The gray scaled spreads correspond to the genome mutations that were inherited at the ends of Line1 and Line2. The red spreads correspond to the mutations that were inherited in Line1, while the blue spreads indicate the mutations that survived until the end of Line2.</p

The effects of the five non-synonymous mutations in the first cluster on the growth rate.

<p>CloneA was isolated from the population at generation 5076, while CloneB was isolated from the population at generation 5504. Sanger sequencing confirmed that CloneB had all the mutations in the first cluster, while CloneA did not. The average and standard error for the growth rate at 44.8°C were determined by repeated growth experiments for each clone (n = 6–27). The five recombinant genotypes from Clone1 on one of the five mutations on <i>mutH</i>, <i>helD</i>, <i>cyaA</i>, <i>nadR</i> and <i>phoU/bglG</i> and the five recombinant genotypes back-mutated from CloneB on one of the five mutations are indicated by the names of the corresponding five genes. Clone_1/94, the clone found among randomly isolating 94 clones from the population at generation 5358 exhibited the same growth rate as the clone back-mutated on <i>cyaA</i> from CloneB.</p