observation excluded the possibility that the clones were contaminants, rather than spontaneous mutants. In a series of experiments that "replayed" the tape of Ara-3 evolution from Cit− clones isolated from samples frozen at various time points in the population's history, they demonstrated that the ability to grow aerobically on citrate was more likely to re-evolve in a subset of genetically pure, evolved clones. In these experiments, they observed 19 new, independent instances of Cit+ re-evolution, but only when starting from clones isolated from after generation 20,000. Fluctuation tests showed that clones from this generation and later displayed a rate of mutation to the Cit+ trait which was significantly higher than the ancestral rate. Even in these later clones, the rate of mutation to Cit+ was on the order of one occurrence per trillion cell divisions. Lenski and his colleagues concluded that the evolution of the Cit+ function in this one population arose due to one or more earlier, possibly nonadaptive, "potentiating" mutations that increased the rate of mutation to an accessible level. The data suggested that citrate usage involved at least two mutations subsequent to these "potentiating" mutations. More generally, the authors suggest these results indicate, following the argument of Stephen Jay Gould, "that historical contingency can have a profound and lasting impact" on the course of evolution. These findings have come to be considered a significant instance of the impact of historical contingency on evolution. Genomic analysis of the Cit+ trait and implications for evolutionary innovation In 2012, Lenski and his team reported the results of a genomic analysis of the Cit+ trait that shed light on the genetic basis and evolutionary history of the trait. The researchers had sequenced the entire genomes of twenty-nine clones isolated from various time points in the Ara-3 population's history. They used these sequences to reconstruct the phylogenetic history of the population; this reconstruction showed that the population had diversified into three clades by 20,000 generations. The Cit+ variants had evolved in one of these, which they called Clade 3. Clones that had been found to be potentiated in earlier research were distributed among all three clades, but were over-represented in Clade 3. This led the researchers to conclude that there had been at least two potentiating mutations involved in Cit+ evolution. The researchers also found that all Cit+ clones had mutations in which a 2933-base-pair segment of DNA was duplicated or amplified.