the minimal glucose medium used for the competition assays (Lenski et al., 1991). Competitions are always performed between reciprocally marked ancestral and derived lines, such that the colony color serves only as a marker to distinguish evolutionary derivation. In these experiments, each determination of W was done with six replicate measurements, and the mean and 95% confidence limits are reported.

Change in fitness (ΔW) is measured by comparing W of a 20°C line relative to Anc with that of its immediate historical progenitor, also assessed relative to Anc. (Direct competitions are not possible because both competitors share the same genetic marker state.) For example, the extent of adaptation of the 32/20 − 1 line to 20°C is determined by W of 32/20 − 1 at 20°C minus W of 32 − 1 at 20°C. Values of ΔW significantly >0 in the selective environment (20°C) indicate evolutionary adaptation, whereas values significantly <0 in the nonselective environment (40°C) indicate a trade-off.

Adaptation by each derived line to 20°C was analyzed by a one-tailed t test on the six replicate measurements of ΔW at 20°C. Mean ΔW of each of the 24 derived lines at 20°C was used to determine the generality of the adaptive response (one-tailed t test on 24 lines). Trade-off by each derived line at 40°C was analyzed with a one-tailed t test on the six experimental replicate measurements of ΔW at 40°C, except as noted, when we explored, independently tested, and confirmed the finding that one lineage in fact showed a correlated improvement at this temperature. Mean ΔW of each of the 24 derived lines at 40°C was used to determine the generality of the trade-off response (one-tailed t test on 24 lines). The quantitative nature of the trade-off was analyzed by determining the correlation coefficient between ΔW at 20°C and ΔW at 40°C for each of the 24 lineages. The effects of historical thermal environment on adaptation to 20°C and trade-off at 40°C were analyzed with one-way ANOVAs on historical temperature (32°C, 37°C, 42°C, or 32–42°C).

We thank Pamela McDonald for assistance with these experiments and Catherine Loudon for statistical assistance. We appreciate the comments of two anonymous reviewers, which greatly improved the manuscript. This research was supported by the National Aeronautics and Space Administration (NASA) Astrobiology Institute (NASA Grant 632731, “Center for Genomic and Evolutionary Studies on Microbial Life at Low Temperatures”).