Changes

:::: - 1. The "historical contingency" hypothesis depicted in Figure 3 is contradicted by the abstract and by the data presented in the Third Experiment in Table 1 of his paper. Specifically, Figure 3 states the hypothesis as consisting of an historical contingency at about the 31,000th generation, while the abstract states that "some potentiating mutation arose by 20,000 generations." Alternatively, if Figure 3 is not changed, then the paper should clearly state that the "historical contingency" hypothesis as depicted was disproved by the Third Experiment.:::: - 2. Lenski's two alternative hypotheses suggest a fixed mutation rate, but the failure of the mutations in his experiments to increase based on scale tends to disprove both hypotheses. It has been suggested that the second and third replay experiments were so dissimilar to the first replay experiment that they cannot be compared for scale, but if that were true then it was an error to treat all three experiments as similar for the statistical analysis. :::: - 3. Lenski incorrectly included generations of the E. coli already known to contain Cit+ variants in his experiments, and he had no means to exclude their presence in the samples. The same reason that the paper admits an inability to "exclude an earlier origin" (p. 7901) for the Cit+ variants also results in an inability to exclude Cit+ variants from the samples taken after generation 31,000. There is no scientific basis for including these Cit+ populations in this study, and it only serves to distort the results. Indeed, the statistically improbable occurrence of four Cit+ variants from the 32,000 generation in the Second Experiment is strongly suggestive of pre-existing Cit+ variants.:::: - 4. Lenski's Third Experiment failed to support his hypothesis with statistical significance, even with the incorrect inclusion of the Cit+ variant generations. The paper incorrectly combined the Third Experiment with the other two based on outcome rather than sample size, thereby yielding a false claim of overall statistical significance.:::: - 5. The lag time between the potentiating mutation in the largest experiment disproves Lenski's implicit assumption that it likely occurred in proximity with the occurrence of the Cit+ variant.:::: - 6. Lenski has not disclosed key portions of his data, such as how thoroughly the samples were screened for pre-existing Cit⁺ variants at what specific generations these variants were actually observed in the replay experiments. In fact, while the paper states the generation periods for the First (replay) Experiment, it does not disclose the generation periods for the Second and Third Experiments.