To estimate the number and diversity of beneficial mutations, we experimentally evolved 115 populations of Escherichia coli to 42.2°C for 2000 generations and sequenced one genome from each population. We identified 1331 total mutations, affecting more than 600 different sites. Few mutations were shared among replicates, but a strong pattern of convergence emerged at the level of genes, operons, and functional complexes. Our experiment uncovered a set of primary functional targets of high temperature, but we estimate that many other beneficial mutations could contribute to similar adaptive outcomes. We inferred the pervasive presence of epistasis among beneficial mutations, which shaped adaptive trajectories into at least two distinct pathways involving mutations either in the RNA polymerase complex or the termination factor rho.

SUMMARY Over the past two decades, comparative biological analyses have undergone profound changes with the incorporation of rigorous evolutionary perspectives and phylogenetic information. This change followed in large part from the realization that traditional methods of statistical analysis tacitly assumed independence of all observations, when in fact biological groups such as species are differentially related to each other according to their evolutionary history. New phylogenetically based analytical methods were then rapidly developed, incorporated into `the comparative method', and applied to many physiological, biochemical, morphological and behavioral investigations. We now review the rationale for including phylogenetic information in comparative studies and briefly discuss three methods for doing this(independent contrasts, generalized least-squares models, and Monte Carlo computer simulations). We discuss when and how to use phylogenetic information in comparative studies and provide several examples in which it has been helpful, or even crucial, to a comparative analysis. We also consider some difficulties with phylogenetically based statistical methods, and of comparative approaches in general, both practical and theoretical. It is our personal opinion that the incorporation of phylogeny information into comparative studies has been highly beneficial, not only because it can improve the reliability of statistical inferences, but also because it continually emphasizes the potential importance of past evolutionary history in determining current form and function.

Resting and maximal levels of oxygen consumption of endothermic vertebrates exceed those of ectotherms by an average of five- to tenfold. Endotherms have a much broader range of activity that can be sustained by this augmented aerobic metabolism. Ectotherms are more reliant upon, and limited by, anaerobic metabolism during activity. A principal factor in the evolution of endothermy was the increase in aerobic capacities to support sustained activity.

The contributions of adaptation, chance, and history to the evolution of fitness and cell size were measured in two separate experiments using bacteria. In both experiments, populations propagated in identical environments achieved similar fitnesses, regardless of prior history or subsequent chance events. In contrast, the evolution of cell size, a trait weakly correlated with fitness, was more strongly influenced by history and chance.

Phenotypic acclimation is generally assumed to confer an advantage in the environment that stimulates the response. To test this beneficial aclimation assumption explicitly, we investigated the consequences of temperature acclimation for the fitness ofEscherichia coliat two temperatures, 32?C and 41.50C. Both temperatures permit growth and long-term persistence of the genotypes in serial culture. We found that prior acclimation to 3rC, relative to acclimation to 41.5'C, enhanced fitness at 32C, consistent with the assumption. But prior acclimation to 41.5C actually reduced fitness at 41.50C, relative to acclimation to 32rC. Hence, the assumption that acclimation always confers an advantage is demonstrated to be false. Acclimation to 41.50C did, however, improve survival at