BACKGROUND
most research in evolution pursues the comparative method, in which the present-day patterns of organismal diversity are examined in order to infer historical processes of change  <cit> .
research in paleontology allows a more direct examination of the past  <cit> , but fossil data are limited in certain respects, including the inability to measure the performance abilities of organisms.
a third approach for studying evolution is to perform long-term studies, either observational  <cit>  or experimental  <cit> , that allow one to observe evolution in action across many generations.
in recent years, bacteria and viruses have become especially popular for experimental evolution, owing to their rapid generations that allow studies to run for hundreds or even thousands of generations  <cit> .
in a long-term experiment, lenski and colleagues have propagated  <dig> populations of e. coli for more than  <dig>  generations at 37°c in a minimal-salts medium supplemented with glucose  <cit> .
the dynamics of both phenotypic  <cit>  and genomic  <cit>  evolution have been characterized in a variety of ways.
during the  <dig>  generations, the bacteria have genetically adapted to their selective environment, such that their mean fitness relative to the ancestor increased by about 70%, based on direct competitions  <cit> .
interestingly, four of the  <dig> populations evolved defects in their dna repair mechanisms, which caused them to become hypermutable  <cit> .
the evolving bacteria also increasingly became ecological specialists, in the sense that their performance in some, but not all, other test environments tended to decline  <cit> .
the parallel trajectories between increasing fitness in the selective environment and declining performance in other environments suggest that most of the decline in other environments is the result of pleiotropic side-effects of the same mutations that produce adaptation in the selective environment  <cit> .
the fact that the four populations that became mutators do not show much more specialization is also consistent with this interpretation  <cit> .
stressful environments, such as prolonged freezing or repeated freeze-thaw cycles, may reveal other performance tradeoffs in the evolved lines.
in fact, freezing and thawing impose several interconnected stresses including dehydration, hyperosmotic stress, ice formation, oxidative stress, and low temperature  <cit> .
the acute responses of bacterial cells to freezing and thawing, including the effects of prior exposure to cold and other stresses on survival  <cit> , are reasonably well understood.
many other variables also contribute to whether bacteria survive freezing and thawing, including their nutritional status and growth phase as well as the cooling rate employed  <cit> .
freezing and thawing e. coli cells without an exogenously supplied cryoprotective agent, such as glycerol, severely decreases their viability  <cit> .
loss of viability is proportional to the number of freeze-thaw cycles that cells experience  <cit> .
therefore, the elapsed time that cells are frozen generally influences viability less than the processes of freezing and thawing.
by contrast, much less is known about how and why different bacterial strains and species vary in their capacity to survive these stresses  <cit> .
in this study, we examine how evolutionary adaptation by populations of e. coli to serial propagation on a minimal glucose medium at a constant temperature of 37°c affected survival during prolonged freezing and repeated freeze-thaw cycles in the absence of cryoprotectant.
in particular, we test whether there was an evolutionary trade-off such that adaptation to this benign environment led to correlated losses in survival capacity under these stresses.
we also evaluate whether replicate lines that evolved under the same regime show heterogeneous changes in their stress responses.
one of our motivating interests in this research is to identify strains and conditions suitable for a future experiment that will investigate evolutionary adaptation to repeated freeze-thaw-growth cycles.
we want to identify conditions in which some survival is possible, but where there is sufficient mortality to impose strong selection.
also, evidence for heritable variation among lines in survival under these extreme conditions would indicate the potential, at least, for evolutionary adaptation in that future experiment.
RESULTS
effects of freeze-only and freeze-thaw regimes on survival of the ancestor
it is quite clear that most of this mortality was caused by the repeated bouts of freezing and thawing, as opposed to the time that cells spent frozen, because the cumulative mortality under the freeze-only regime was far less.
in fact, over the entire  <dig> days at -80°c, with one thaw, the viable population size declined by only  <dig> %.
this decline almost exactly matches the cell death observed after one day in the freeze-thaw regime , implying that no further death occurred during the other  <dig> days at constant -80°c.
in fact, the slope of the cell-survival trajectory from day  <dig> to day  <dig> under the freeze-only regime was not significantly different from zero .
thus, there was little or no mortality, even over several weeks, beyond that caused by the single freeze-thaw cycle that was a necessary part of the survival assay procedures for all samples, regardless of how long they had spent at -80°c.
therefore, the ancestral strain used for the long-term evolution experiment at 37°c is quite hardy with respect to prolonged freezing at -80°c.
however, it is much more sensitive to repeated cycles of freezing and thawing.
in the next section, we examine whether the lines that previously evolved for  <dig>  generations in a benign environment became less tolerant of either prolonged freezing or repeated freeze-thaw cycles.
effects of freeze-only and freeze-thaw regimes on survival of the evolved lines in comparison with the ancestor
we performed 10-day experiments under both freeze-only and freeze-thaw regimes using the  <dig> lines that evolved by serial propagation on a minimal glucose medium at constant 37°c for  <dig>  generations and their ancestor.
each evolved line had three replicates, while the ancestor was replicated six-fold .
figure  <dig> compares the average mortality rates of the evolved lines and their ancestor under the freeze-thaw regime.
the ancestor experienced a mortality rate of  <dig> % per day under the freeze-thaw regime, a value almost identical to our first experiment.
by contrast, all  <dig> evolved lines experienced greater mortality, with an average rate of  <dig> % per day.
the difference in freeze-thaw mortality rates between ancestral and evolved rates is highly significant .
however, we observed no significant difference between the evolved lines and their ancestor in mortality rates during prolonged freezing at -80°c .
mortality rates under this regime were calculated, as described in the materials and methods, such that they correct for the effect of one cycle of freezing and thawing.
the average mortality rates estimated for the ancestor and evolved lines under the freeze-only regime were  <dig> % and  <dig> % per day, respectively.
the ancestral value is somewhat higher than estimated in the first experiment, but it is still much lower than the mortality rates measured during repeated freeze-thaw cycles in either experiment.
thus, the 37°c-evolved lines as a group remained about as robust as their ancestor to the effects of prolonged freezing at -80°c.
however, the evolved lines are much more sensitive than their ancestor to the effects of repeated freeze-thaw cycles, with the average mortality rate increasing from  <dig> % to  <dig> % per daily cycle.
in the section that follows, we examine variation among the evolved lines in their freeze-thaw sensitivity.
heterogeneity among the evolved lines in freeze-thaw survival
mortality rates were estimated with three-fold replication for each of the  <dig> evolved lines.
the very low p-value indicates significant heritable variation among these lines in their survival capacity under the freeze-thaw regime.
the four lines that evolved defects in dna repair, and which have much higher mutation rates , do not have consistently higher mortality rates, as a group, than do the other evolved lines.
also, there is no significant correlation between competitive fitness in the benign environment and survival under the freeze-only treatment , consistent with the absence of any significant difference between the ancestor and evolved lines as a group in this stress-related capacity.
the variation among lines that evolved in the benign selective environment indicate that they acquired different sets of mutations that differentially affect their sensitivity to freeze-thaw cycles.
the differences between lines are not associated with variation in dna repair function and mutability, which implies that pleiotropic effects of the mutations that were selected for their beneficial effects during the evolution experiment in minimal glucose medium at 37°c are responsible for the increased susceptibility to freezing and thawing.
if, alternatively, mutations that harmed freeze-thaw survival had accumulated by neutral drift, then we would expect significantly higher susceptibility among the four lines with defective dna repair functions  <cit> .
CONCLUSIONS
e. coli b cells experience little mortality during prolonged freezing at -80°c, even in the absence of added cryoprotectant.
however, they are much more susceptible to repeated freezing and thawing, consistent with earlier studies  <cit> .
twelve lines that previously evolved for  <dig>  generations in a benign environment, consisting of serial transfer in a minimal glucose medium at constant 37°c, all became more susceptible to freeze-thaw mortality than was their ancestor.
moreover, those evolved lines with higher fitness gains in the benign selective environment also tended to have greater susceptibility to freeze-thaw cycles, further supporting the trade-off in performance between these environments.
however, the variation among the evolved lines was not associated with differences in dna repair function and mutability that arose during the evolution experiment  <cit> .
therefore, increased susceptibility to freeze-thaw cycles in the evolved lines probably reflects pleiotropic effects of mutations that were beneficial to the bacteria during evolution in the benign environment with minimal glucose medium and constant 37°c.
in any case, significant variation among the lines in their freeze-thaw survival implies that there is genetic variation for this trait, such that it can be selected, which will be a focus of our future research.
future research directions include evolving e. coli populations under freeze-thaw-growth cycles.
the growth phase will allow populations to recover from mortality caused by freezing and thawing; selection should favour a reduction in freeze-thaw mortality, a faster transition to growth after thawing, or both.
another future direction includes identifying individual mutations responsible for increased susceptibility to freezing and thawing in the lines evolved in the benign environment of constant 37°c, or increased resistance in lines evolved under the freeze-thaw-growth regime.
finally, phenotypic and genetic analyses of freeze-thaw resistance could be extended to natural isolates of e. coli.
comparisons between food-borne pathogens and commensals would be of particular interest because the ability to survive and recover from freezing and thawing might be an important adaptation of some food-borne pathogens.

 most research in evolution pursues the comparative method, in which the present-day patterns of organismal diversity are examined in order to infer historical processes of change. research in paleontology allows a more direct examination of the past, but fossil data are limited in certain respects, including the inability to measure the performance abilities of organisms. a third approach for studying evolution is to perform long-term studies, either observational or experimental, that allow one to observe evolution in action across many generations. in recent years, bacteria and viruses have become especially popular for experimental evolution, owing to their rapid generations that allow studies to run for hundreds or even thousands of generations. in a long-term experiment, lenski and colleagues have propagated populations of e. coli for more than generations at 37°c in a minimal-salts medium supplemented with glucose. the dynamics of both phenotypic and genomic evolution have been characterized in a variety of ways. during the generations, the bacteria have genetically adapted to their selective environment, such that their mean fitness relative to the ancestor increased by about 70%, based on direct competitions. interestingly, four of the populations evolved defects in their dna repair mechanisms, which caused them to become hypermutable. the evolving bacteria also increasingly became ecological specialists, in the sense that their performance in some, but not all, other test environments tended to decline. the parallel trajectories between increasing fitness in the selective environment and declining performance in other environments suggest that most of the decline in other environments is the result of pleiotropic side-effects of the same mutations that produce adaptation in the selective environment. the fact that the four populations that became mutators do not show much more specialization is also consistent with this interpretation. stressful environments, such as prolonged freezing or repeated freeze-thaw cycles, may reveal other performance tradeoffs in the evolved lines. in fact, freezing and thawing impose several interconnected stresses including dehydration, hyperosmotic stress, ice formation, oxidative stress, and low temperature. the acute responses of bacterial cells to freezing and thawing, including the effects of prior exposure to cold and other stresses on survival, are reasonably well understood. many other variables also contribute to whether bacteria survive freezing and thawing, including their nutritional status and growth phase as well as the cooling rate employed. freezing and thawing e. coli cells without an exogenously supplied cryoprotective agent, such as glycerol, severely decreases their viability. loss of viability is proportional to the number of freeze-thaw cycles that cells experience. therefore, the elapsed time that cells are frozen generally influences viability less than the processes of freezing and thawing. by contrast, much less is known about how and why different bacterial strains and species vary in their capacity to survive these stresses. in this study, we examine how evolutionary adaptation by populations of e. coli to serial propagation on a minimal glucose medium at a constant temperature of 37°c affected survival during prolonged freezing and repeated freeze-thaw cycles in the absence of cryoprotectant. in particular, we test whether there was an evolutionary trade-off such that adaptation to this benign environment led to correlated losses in survival capacity under these stresses. we also evaluate whether replicate lines that evolved under the same regime show heterogeneous changes in their stress responses. one of our motivating interests in this research is to identify strains and conditions suitable for a future experiment that will investigate evolutionary adaptation to repeated freeze-thaw-growth cycles. we want to identify conditions in which some survival is possible, but where there is sufficient mortality to impose strong selection. also, evidence for heritable variation among lines in survival under these extreme conditions would indicate the potential, at least, for evolutionary adaptation in that future experiment.  effects of freeze-only and freeze-thaw regimes on survival of the ancestor it is quite clear that most of this mortality was caused by the repeated bouts of freezing and thawing, as opposed to the time that cells spent frozen, because the cumulative mortality under the freeze-only regime was far less. in fact, over the entire days at -80°c, with one thaw, the viable population size declined by only %. this decline almost exactly matches the cell death observed after one day in the freeze-thaw regime, implying that no further death occurred during the other days at constant -80°c. in fact, the slope of the cell-survival trajectory from day to day under the freeze-only regime was not significantly different from zero. thus, there was little or no mortality, even over several weeks, beyond that caused by the single freeze-thaw cycle that was a necessary part of the survival assay procedures for all samples, regardless of how long they had spent at -80°c. therefore, the ancestral strain used for the long-term evolution experiment at 37°c is quite hardy with respect to prolonged freezing at -80°c. however, it is much more sensitive to repeated cycles of freezing and thawing. in the next section, we examine whether the lines that previously evolved for generations in a benign environment became less tolerant of either prolonged freezing or repeated freeze-thaw cycles. effects of freeze-only and freeze-thaw regimes on survival of the evolved lines in comparison with the ancestor we performed 10-day experiments under both freeze-only and freeze-thaw regimes using the lines that evolved by serial propagation on a minimal glucose medium at constant 37°c for generations and their ancestor. each evolved line had three replicates, while the ancestor was replicated six-fold. figure compares the average mortality rates of the evolved lines and their ancestor under the freeze-thaw regime. the ancestor experienced a mortality rate of % per day under the freeze-thaw regime, a value almost identical to our first experiment. by contrast, all evolved lines experienced greater mortality, with an average rate of % per day. the difference in freeze-thaw mortality rates between ancestral and evolved rates is highly significant. however, we observed no significant difference between the evolved lines and their ancestor in mortality rates during prolonged freezing at -80°c. mortality rates under this regime were calculated, as described in the materials and methods, such that they correct for the effect of one cycle of freezing and thawing. the average mortality rates estimated for the ancestor and evolved lines under the freeze-only regime were % and % per day, respectively. the ancestral value is somewhat higher than estimated in the first experiment, but it is still much lower than the mortality rates measured during repeated freeze-thaw cycles in either experiment. thus, the 37°c-evolved lines as a group remained about as robust as their ancestor to the effects of prolonged freezing at -80°c. however, the evolved lines are much more sensitive than their ancestor to the effects of repeated freeze-thaw cycles, with the average mortality rate increasing from % to % per daily cycle. in the section that follows, we examine variation among the evolved lines in their freeze-thaw sensitivity. heterogeneity among the evolved lines in freeze-thaw survival mortality rates were estimated with three-fold replication for each of the evolved lines. the very low p-value indicates significant heritable variation among these lines in their survival capacity under the freeze-thaw regime. the four lines that evolved defects in dna repair, and which have much higher mutation rates, do not have consistently higher mortality rates, as a group, than do the other evolved lines. also, there is no significant correlation between competitive fitness in the benign environment and survival under the freeze-only treatment, consistent with the absence of any significant difference between the ancestor and evolved lines as a group in this stress-related capacity. the variation among lines that evolved in the benign selective environment indicate that they acquired different sets of mutations that differentially affect their sensitivity to freeze-thaw cycles. the differences between lines are not associated with variation in dna repair function and mutability, which implies that pleiotropic effects of the mutations that were selected for their beneficial effects during the evolution experiment in minimal glucose medium at 37°c are responsible for the increased susceptibility to freezing and thawing. if, alternatively, mutations that harmed freeze-thaw survival had accumulated by neutral drift, then we would expect significantly higher susceptibility among the four lines with defective dna repair functions.  e. coli b cells experience little mortality during prolonged freezing at -80°c, even in the absence of added cryoprotectant. however, they are much more susceptible to repeated freezing and thawing, consistent with earlier studies. twelve lines that previously evolved for generations in a benign environment, consisting of serial transfer in a minimal glucose medium at constant 37°c, all became more susceptible to freeze-thaw mortality than was their ancestor. moreover, those evolved lines with higher fitness gains in the benign selective environment also tended to have greater susceptibility to freeze-thaw cycles, further supporting the trade-off in performance between these environments. however, the variation among the evolved lines was not associated with differences in dna repair function and mutability that arose during the evolution experiment. therefore, increased susceptibility to freeze-thaw cycles in the evolved lines probably reflects pleiotropic effects of mutations that were beneficial to the bacteria during evolution in the benign environment with minimal glucose medium and constant 37°c. in any case, significant variation among the lines in their freeze-thaw survival implies that there is genetic variation for this trait, such that it can be selected, which will be a focus of our future research. future research directions include evolving e. coli populations under freeze-thaw-growth cycles. the growth phase will allow populations to recover from mortality caused by freezing and thawing; selection should favour a reduction in freeze-thaw mortality, a faster transition to growth after thawing, or both. another future direction includes identifying individual mutations responsible for increased susceptibility to freezing and thawing in the lines evolved in the benign environment of constant 37°c, or increased resistance in lines evolved under the freeze-thaw-growth regime. finally, phenotypic and genetic analyses of freeze-thaw resistance could be extended to natural isolates of e. coli. comparisons between food-borne pathogens and commensals would be of particular interest because the ability to survive and recover from freezing and thawing might be an important adaptation of some food-borne pathogens. 
by contrast, there was no significant difference between the evolved lines and their ancestor in mortality during prolonged freezing. in order to study the dynamics of evolutionary change, populations of e. coli b were serially propagated for generations in minimal glucose medium at constant 37°c. correlated changes in various other traits have been previously associated with the improvement in competitive fitness in the selective environment. those lines that had evolved higher competitive fitness in the minimal glucose medium at 37°c also had higher mortality during freeze-thaw cycles. this study examines whether these evolved lines changed in their ability to tolerate the stresses of prolonged freezing and repeated freeze-thaw cycles during adaptation to a benign environment. all lines that evolved in the benign environment for generations are more sensitive to freeze-thaw cycles than their ancestor. the evolved lines have an average mortality rate of 54% per daily cycle, compared to the ancestral rate of 34%. the consistency of the evolutionary declines in freeze-thaw tolerance, the correlation between fitness in glucose medium at 37°c and mortality during freeze-thaw cycles, and the absence of greater declines in freeze-thaw survival among the hypermutable lines all indicate a trade-off between performance in minimal glucose medium at 37°c and the capacity to tolerate this stress. there was also some variability among the evolved lines in susceptibility to repeated freeze-thaw cycles.
 in order to study the dynamics of evolutionary change, populations of e. coli b were serially propagated for generations in minimal glucose medium at constant 37°c. correlated changes in various other traits have been previously associated with the improvement in competitive fitness in the selective environment. this study examines whether these evolved lines changed in their ability to tolerate the stresses of prolonged freezing and repeated freeze-thaw cycles during adaptation to a benign environment.  all lines that evolved in the benign environment for generations are more sensitive to freeze-thaw cycles than their ancestor. the evolved lines have an average mortality rate of 54% per daily cycle, compared to the ancestral rate of 34%. by contrast, there was no significant difference between the evolved lines and their ancestor in mortality during prolonged freezing. there was also some variability among the evolved lines in susceptibility to repeated freeze-thaw cycles. those lines that had evolved higher competitive fitness in the minimal glucose medium at 37°c also had higher mortality during freeze-thaw cycles. this variability was not associated, however, with differences among lines in dna repair functionality and mutability.  the consistency of the evolutionary declines in freeze-thaw tolerance, the correlation between fitness in glucose medium at 37°c and mortality during freeze-thaw cycles, and the absence of greater declines in freeze-thaw survival among the hypermutable lines all indicate a trade-off between performance in minimal glucose medium at 37°c and the capacity to tolerate this stress. analyses of the mutations that enhance fitness at 37°c may shed light on the physiological basis of this trade-off. 