Introduction
Fluctuating environments pose several challenges to evolving
populations. While some potential adaptations might confer benefits
across all relevant selection regimes (Bennett and Lenski 1999; Buckling
et al. 2000; Buckling et al. 2007; Kassen and Bell 1998; Satterwhite and
Cooper 2015), others will confer benefits in some and costs in others
(Bailey and Kassen 2012; Jasmin and Kassen 2007; Lee and Marx 2012;
McGee et al. 2015; Roemhild et al. 2015). Indeed, even if
unconditionally beneficial mutations are initially available, they are
likely to become less common over time (Martin and Lenormand 2015;
Satterwhite and Cooper 2015; Schick et al. 2015). When a mutation that
confers a benefit in one environment, and a cost in another, fixes in a
population, it creates selection for subsequent mutations that
compensate for that cost (Maisnier-Patin et al. 2002; Moore et al. 2000;
Moura de Sousa et al. 2017; Wood et al. 2013).
Compensatory mutations have long been used in molecular genetic studies
as a tool to identify physical and genetic changes that can suppress the
effects of a focal mutation, thereby identifying interacting components
(Blank et al. 2014; Jarvik and Botstein 1975; Kacar et al. 2017;
Manson 2000; Ponmani and Munavar 2014; van Leeuwen et al. 2016).
Increasingly, they are also recognized as being important in broadening
the scope of evolutionary trajectories a population can follow (Szamecz
et al. 2014; Zee et al. 2014), allowing adaptations to be selected that
might otherwise prove to be evolutionary dead-ends (Covert et al. 2013;
Harrison et al. 2015), and influencing the ability of populations to
simultaneously adapt to multiple environments (Melnyk et al. 2017).
Mutation interactions arising following selection in fluctuating
environments have been extensively studied in the context of the
evolution of antibiotic resistance. Antibiotic resistance mutations
often confer a cost to bacteria in antibiotic free environments (Moura
de Sousa et al. 2017; Nilsson et al. 2003; Rozen et al. 2007). This cost
generates selection for subsequent compensatory mutations that relieve
the cost (Levin et al. 2000; Björkman et al. 1998; Björkman et al. 2000;
Nagaev et al. 2001). Compensation allows resistance mutations to be
maintained when they would normally be selected against, influencing
short-term evolutionary outcomes and perhaps longer-term potential.
Similar patterns of compensatory mutations depending on earlier
resistance mutations for their benefit have been seen in studies of
bacterial resistance to bacteriophage (Lenski 1988; Wielgloss et al.
2016). Compensation during evolution has also been found to occur to
overcome the loss of essential genes (Blank et al. 2014), the negative
effects of synonymous (Knöppel et al. 2016) and gene deletion (Szamecz
et al. 2014) mutations, and to restore a social trait (Zee et al. 2014).
In contrast to studies that have focused on mutations that compensate
for a specific genetic perturbation, few studies have examined
compensation during more general adaptation to an environment,
especially when this adaptation involves repeating rounds of selection
in contrasting environments. This distinction might be important.
Compensation to a specific genetic perturbation, as to an antibiotic
resistance mutation or a deletion of a focal gene, is thought to
generally act locally to reverse the costly effect (Brandis et al. 2012;
Filteau et al. 2015; Szamecz et al. 2014), although it can also arise in
pathways unrelated to the perturbation (Blank et al. 2014). At least in
experimentally evolving populations, adaptation often involves mutations
in regulatory genes that are likely to have highly pleiotropic
consequences (Cooper et al. 2003; Kurlandzka et al. 1991; MacLean et al.
2004; Rosenzweig et al. 1994). If costs of such pleiotropic mutations
are revealed following an environmental change, it is not clear how
subsequent compensatory mutations might affect fitness in the original
environment. Indeed, it is easy to imagine that compensation causing a
reduction in the cost of a focal mutation in a new environment might be
associated with a reduction of the original benefit. In that case,
reversion to the original environment might select for reversal of the
effects of the compensatory mutation. This could occur through its
direct reversion or through a second compensatory mutation, creating
potentially complex patterns of environmentally dependent epistatic
interactions between selected mutations.
The particular nature of the environmental fluctuations a population is
exposed to is expected to play a major role in the selection of
compensatory mutations. In a rapidly changing environment, mutations
that increase in frequency are likely to confer a net benefit across the
different environments (Buckling et al. 2007; Melnyk et al. 2017; Turner
and Elena 2000). With this limitation, such mutations can only confer at
most relatively small costs in any one environmental component so that
the strength of selection for compensation may be small (Poon and Chao
2005a; Poon and Chao 2006). In a more slowly fluctuating environment,
mutations selected in one component might fix before the population
experiences a second component in which they might confer substantial
costs (Bennett and Lenski 2007; Kassen and Bell 1998; Phillips et al.
2016). Such differential costs are consistent with the generally higher
between-environment trade-offs seen in populations selected in slowly
compared to quickly fluctuating environments (Bono et al. 2017;
Satterwhite and Cooper 2015; Schick et al. 2015).
We examine compensation to an adaptive mutation selected in a series of
experimentally evolved populations selected in environments that
contained either lactose or glucose alone or a combination of lactose
and glucose fluctuating daily or every 2000 generations (Cooper and
Lenski 2010). Mutational inactivation of the LacI repressor was rapidly
selected in many of the replicate bacterial populations that were
selected in the presence of lactose (Quan et al. 2012). Loss of LacI
causes the lac operon, a set of genes required for utilization of
lactose, to be constitutively expressed (Markiewicz et al. 1994; Quan et
al. 2012). When engineered into the ancestor of the evolution
experiment, constitutive expression of the lac operon provided a
benefit of ~9% during growth in lactose by shortening
the lag time before resumption of growth following transfer into fresh
medium (Quan et al. 2012). It also conferred a cost of
~3% in an environment containing glucose as the sole
resource, probably due to some combination of the energetic cost of
expressing unnecessary genes and toxicity of the LacY permease (Dekel
and Alon 2005; Stoebel et al. 2008; Quan et al. 2012; Eames and Kortemme
2012). In populations selected in environments containing both lactose
and glucose, this trade-off in the effect of lacI- mutations
creates potential for subsequent mutations to provide a fitness benefit
by compensating for the cost of the mutation in glucose.
We test whether evolved populations that fixed the lacI-mutation, and therefore constitutively express the lacoperon, evolved mechanisms that alleviate the cost of this expression
during growth in glucose and, if so, whether this compensation comes at
a cost of the benefit conferred by lacI- in lactose. To do this,
we isolated strains from populations evolved for 8,000 generations in
lactose, glucose, and combinations of both fluctuating daily and every
2,000 generations. The lacI- mutation was reverted in those
strains that had substituted it, and its effect on fitness determined.
We found that the fitness cost of lacI - in glucose was variable,
including, in one strain, becoming beneficial, but did not differ
consistently between populations evolved in lactose only, where
compensation is not expected to be selected, and in environments
containing glucose, where it is. Similarly, strains varied in their
relationship between the fitness benefit conferred by the lacI -
mutation in lactose and costs in glucose, but this variation did not
depend on their selection environment. Together, these results
demonstrate the potential for the action of compensatory mutations to
influence costs of adaptation but indicate that their effects may either
be idiosyncratic or be overwhelmed by the effects of additionally
selected mutations.