Environmental complexity is more important than mutation in driving the evolution of latent novel traits in E. coli.

Recent experiments show that adaptive Darwinian evolution in one environment can lead to the emergence of multiple new traits that provide no immediate benefit in this environment. Such latent non-adaptive traits, however, can become adaptive in future environments. We do not know whether mutation or environment-driven selection is more important for the emergence of such traits. To find out, we evolve multiple wild-type and mutator E. coli populations under two mutation rates in simple (single antibiotic) environments and in complex (multi-antibiotic) environments. We then assay the viability of evolved populations in dozens of new environments and show that all populations become viable in multiple new environments different from those they had evolved in. The number of these new environments increases with environmental complexity but not with the mutation rate. Genome sequencing demonstrates the reason: Different environments affect pleiotropic mutations differently. Our experiments show that the selection pressure provided by an environment can be more important for the evolution of novel traits than the mutational supply experienced by a wild-type and a mutator strain of E. coli.

Low protein expression enhances phenotypic evolvability by intensifying selection on folding stability.

Protein abundance affects the evolution of protein genotypes, but we do not know how it affects the evolution of protein phenotypes. Here we investigate the role of protein abundance in the evolvability of green fluorescent protein (GFP) towards the novel phenotype of cyan fluorescence. We evolve GFP in E. coli through multiple cycles of mutation and selection and show that low GFP expression facilitates the evolution of cyan fluorescence. A computational model whose predictions we test experimentally helps explain why: lowly expressed proteins are under stronger selection for proper folding, which facilitates their evolvability on short evolutionary time scales. The reason is that high fluorescence can be achieved by either few proteins that fold well or by many proteins that fold less well. In other words, we observe a synergy between a protein's scarcity and its stability. Because many proteins meet the essential requirements for this scarcity-stability synergy, it may be a widespread mechanism by which low expression helps proteins evolve new phenotypes and functions.

Multiple Novel Traits without Immediate Benefits Originate in Bacteria Evolving on Single Antibiotics.

How new traits originate in evolution is a fundamental question of evolutionary biology. When such traits arise, they can either be immediately beneficial in their environment of origin, or they may become beneficial only in a future environment. Compared to immediately beneficial novel traits, novel traits without immediate benefits remain poorly studied. Here we use experimental evolution to study novel traits that are not immediately beneficial but that allow bacteria to survive in new environments. Specifically, we evolved multiple E. coli populations in five antibiotics with different mechanisms of action, and then determined their ability to grow in more than 200 environments that are different from the environment in which they evolved. Our populations evolved viability in multiple environments that contain not just clinically relevant antibiotics, but a broad range of antimicrobial molecules, such as surfactants, organic and inorganic salts, nucleotide analogues and pyridine derivatives. Genome sequencing of multiple evolved clones shows that pleiotropic mutations are important for the origin of these novel traits. Our experiments, which lasted fewer than 250 generations, demonstrate that evolution can readily create an enormous reservoir of latent traits in microbial populations. These traits can facilitate adaptive evolution in a changing world.

Adapting in larger numbers can increase the vulnerability of Escherichia coli populations to environmental changes.

Larger populations generally adapt faster to their existing environment. However, it is unknown if the population size experienced during evolution influences the ability to face sudden environmental changes. To investigate this issue, we subjected replicate Escherichia coli populations of different sizes to experimental evolution in an environment containing a cocktail of three antibiotics. In this environment, the ability to actively efflux molecules outside the cell is expected to be a major fitness-affecting trait. We found that all the populations eventually reached similar fitness in the antibiotic cocktail despite adapting at different speeds, with the larger populations adapting faster. Surprisingly, although efflux activity (EA) enhanced in the smaller populations, it decayed in the larger ones. The evolution of EA was largely shaped by pleiotropic responses to selection and not by drift. This demonstrates that quantitative differences in population size can lead to qualitative differences (decay/enhancement) in the fate of a character during adaptation to identical environments. Furthermore, the larger populations showed inferior fitness upon sudden exposure to several alternative stressful environments. These observations provide a novel link between population size and vulnerability to environmental changes. Counterintuitively, adapting in larger numbers can render bacterial populations more vulnerable to abrupt environmental changes.

Extent of adaptation is not limited by unpredictability of the environment in laboratory populations of Escherichia coli.

Environmental variability is on the rise in different parts of the earth, and the survival of many species depends on how well they cope with these fluctuations. Our current understanding of how organisms adapt to unpredictably fluctuating environments is almost entirely based on studies that investigate fluctuations among different values of a single environmental stressor such as temperature or pH. How would unpredictability affect adaptation when the environment fluctuates between qualitatively very different kinds of stresses? To answer this question, we subjected laboratory populations of Escherichia coli to selection over ~ 260 generations. The populations faced predictable and unpredictable environmental fluctuations across qualitatively different selection environments, namely, salt and acidic pH. We show that predictability of environmental fluctuations does not play a role in determining the extent of adaptation, although the extent of ancestral adaptation to the chosen selection environments is of key importance.

Environmental fluctuations do not select for increased variation or population-based resistance in Escherichia coli.

Little is known about the mechanisms that enable organisms to cope with unpredictable environments. To address this issue, we used replicate populations of Escherichia coli selected under complex, randomly changing environments. Under four novel stresses that had no known correlation with the selection environments, individual cells of the selected populations had significantly lower lag and greater yield compared to the controls. More importantly, there were no outliers in terms of growth, thus ruling out the evolution of population-based resistance. We also assayed the standing phenotypic variation of the selected populations, in terms of their growth on 94 different substrates. Contrary to expectations, there was no increase in the standing variation of the selected populations, nor was there any significant divergence from the ancestors. This suggested that the greater fitness in novel environments is brought about by selection at the level of the individuals, which restricts the suite of traits that can potentially evolve through this mechanism. Given that day-to-day climatic variability of the world is rising, these results have potential public health implications. Our results also underline the need for a very different kind of theoretical approach to study the effects of fluctuating environments.

ANIMAL COGNITION. Comment on "Number-space mapping in the newborn chick resembles humans' mental number line".

Rugani et al. (Reports, 30 January 3015, p. 534) tested 3-day-old domestic chicks using an innovative experimental setup and demonstrate the presence of the mental number line. We raise concerns regarding this conclusion by highlighting the possible loopholes in the experimental design and the data analysis procedures. We further suggest auxiliary experiments that can substantiate the authors' claim.