However, 14% of trajectories on the chlorcycloguanil landscape have I164L as a first step. This finding underscores the importance of the I164L mutation in leading to high-level resistance to chlorcycloguanil specifically. Our findings further demonstrate that,PD 0332991 based on the chlorcycloguanil landscape in yeast, I164L is not necessarily a late arising mutation. This likely reflects a fundamental difference between the adaptive landscapes for pyrimethamine and chlorcycloguanil and underscores the importance of drug specific patterns of evolution. Simultaneously we recognize that I164L may not be as readily found as a single mutation in nature due to some additional fitness deficit that is not related to selection by pyrimethamine use but which we cannot determine in this context. It has already been observed that early mutations may entail some loss of fitness under a regime where no drug pressure is present but fitness is later restored by compensatory mutations. Trajectories containing I164L as an initial step seem to follow such a pattern. An important observation is gained from the startling presence of I164L among the initial mutational steps found on among many trajectories. That is,PCI-32765 the probability of evolutionary trajectories and the order of the steps that make up those trajectories can be heavily influenced by differences in the underlying landscape based on the drug treatment used. The second most often accessed endpoint is the quintuple mutant 11121, an allele not generally observed in clinical settings. Conceivably this may simply be due to a smaller sample size for alleles associated with chlorcycloguanil resistance. Chlorcycloguanil is not and has not been used as widely as pyrimethamine, and there is less data available on resistance mutations in the field apart from clinical trials. However, there are also intrinsic explanations for this observation. As two more mutations are required to reach this allele, it may be expected to occur more infrequently than a triple mutant given that mutations at all positions are equally likely. Alternatively, the relative dearth of pathway realizations leading to the quintuple mutant 11121 may be a reflection of the negative interaction between the mutations found in this allele. This allele contains the mutation S108T, which is much more rarely observed than S108N and usually occurs in combination with A16V.