The genomic landscape of compensatory evolution.

Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes

Molecular Modeling and Simulation: Force Field Development, Evaporation Processes and Thermophysical Properties of Mixtures

To gain physical insight into the behavior of fluids on a microscopic level as well as to broaden the data base for thermophysical properties especially for mixtures, molecular modeling and simulation is utilized in this work. Various methods and applications are discussed, including a procedure for the development of new force field models. The evaporation of liquid nitrogen into a supercritical hydrogen atmosphere is presented as an example for large scale molecular dynamics simulation. System-size dependence and scaling behavior are discussed in the context of Kirkwood-Buff integration. Further, results for thermophysical mixture properties are presented, i.e. the Henry’s law constant of aqueous systems and diffusion coefficients of a ternary mixture

Comparisons of the transcriptome profiles of wild-type, ancestor, and evolved lines.

<p>(A) Heatmaps of transcriptome profiles of deletion mutants <i>Δrpl43a</i>, <i>Δpop2</i>, <i>Δmdm34</i>, <i>Δrsc2</i>, <i>Δifm1</i>, <i>Δrpb9</i>, and <i>Δbud20</i> and their corresponding evolved lines. For each deletion mutant, the fold-changes (FC) are shown for the ancestor strain versus the wild type, the evolved strain versus the wild type and the evolved strain versus the ancestor strain (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s008" target="_blank">Table S3</a>). Color scales as indicated. Individual transcripts are depicted if they change significantly (FC>1.7, <i>p</i><0.05) at least once in one of these comparisons. (B) The Euclidean distances of microarray profiles of the evolved evolutionary line from its ancestor and from wild type (WT) were calculated and normalized to the ancestor–wild type distance for each genotype. The distances of the points in the figure are proportional to the calculated profile distances. For each genotype triplet, distances were calculated on the basis of those genes that are differentially expressed in at least one of the pairwise comparisons. For each deletion strain, the edges of the triangle represent Euclidean distances of log₂ mRNA expression fold-changes between the wild-type (WT), ancestor (anc), and evolved (evo) lines. To calculate these distances we used the average of four replicate expression measurements (two biological and two technical replicates). Circles around average values represent the Euclidean distance between the two biological replicates (calculated as the average based on the two technical replicates). For each genotype triplet, distances were calculated on the basis of those genes that are differentially expressed (FC>1.7, <i>p</i><0.05) in at least one of the pairwise comparisons (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s011" target="_blank">Table S6</a>). (C) Within the subset of genes that showed expression change upon gene deletion, the barplot shows the fraction of these genes that changed expression during evolution in the opposite direction (i.e., evolution towards restoration of wild-type expression level; see inset). With one major exception (lines disrupted in <i>mdm34</i>), only a small fraction of the expression changes were restored in the evolved lines (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s011" target="_blank">Table S6</a>). The threshold for expression change was 1.7-fold-change and <i>p</i><0.05, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935-vanWageningen1" target="_blank">[62]</a>.</p

Compensation of the <i>MDM34</i> gene deletion.

<p>(A) The cardiolipin synthesis pathway with an emphasis on the ERMES complex. The complex tethers the endoplasmatic reticulum to the mitochondria, and is central for the transfer of phospholipids between the two compartments. <i>De novo</i> mutations in the independent evolutionary lines affected different, but related cellular subsystems, including upregulation of the unsaturated fatty acid synthesis (<i>MGA2</i>), another step of the cardiolipin synthesis pathway downstream of the ERMES complex (<i>MDM35</i>), and another mitochondrial transport process (<i>CRC1</i>), which most likely affects respiration by modulating the interaction between carnitine and cardiolipin. For further details on the underlying mechanisms see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s018" target="_blank">Text S1</a>. The green arrow represents transcriptional upregulation; the dashed arrow indicates indirect positive effect. The mutations in <i>MGA2</i>, <i>MDM35</i>, and <i>CRC1</i> genes were found in Δ<i>mdm34</i> evolved lines 1, 3, and 4, respectively. (B) The cumulative fitness effects of the compensatory mutations in Δ<i>mdm34</i> and “wild type” (Δ<i>mdm34</i>+<i>MDM34</i> reintroduced) backgrounds (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s012" target="_blank">Table S7</a>). (C) Epistatic interactions between mutations in two environments (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s012" target="_blank">Table S7</a>). The bars in (B) and (C) indicate means ± standard error. Arrows indicate fitness costs and the extent of compensation.</p

Large-scale phenotypic screen of evolved lines.

<p>(A) Fitness trade-offs in evolved lines carrying a deletion across 14 environments (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>). Lines are ranked according to the number of environments in which they display improved fitness (brown). Grey and black dots indicate conditions where the fitness of the line is statistically equal or lower, respectively, than that of the corresponding ancestor. (B) Fitness variation in independently evolving lines carrying the same gene deletion. The figure shows the coefficient of variation in the in the medium of selection (YPD) versus all other media (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>). The difference is highly significant (Wilcoxon rank sum test <i>p</i>-value<10⁻⁷). The bars indicate mean of the coefficients of variations ± standard error. (C) Gene deletions showing larger fitness gains have higher variance of fitness between replicate lines across other environments (Spearman rank correlation, rho = 0.36, <i>p</i> = 0.0001). Each point represents a gene deletion genotype. The x-axis shows the mean of the fitness gains of the parallel evolving replicates of a given gene deletion, while the y-axis shows the mean of the coefficient of variations measured in each alternative media between the parallel evolving replicates after 104 days of lab evolution (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>). The gray line indicates fit by linear regression.</p

Environment-dependent compensation by a loss-of-function mutation.

<p>(A) <i>Δrpb9</i> and <i>Δwhi2</i> mutations were crossed by SGA using haploid parental strains as shown. To compare the double mutant <i>Δrpb9 Δwhi2</i> with the wild-type control and corresponding single mutants, the resistance cassettes required by the SGA method were introduced into wild-type and single mutants by crossing them with parental strains where the corresponding resistance cassettes reside at a non-functional locus (<i>Δhis3</i>::KanMX4 and <i>Δho</i>::NatMX4). (B) Relative fitness was measured as colony sizes on YPD and YPD supplemented with cycloheximide (CYC), values were normalized to WT. The arrow shows the extent of compensation of <i>Δrpb9</i> by <i>Δwhi2</i> on glucose medium (Wilcoxon rank sum test <i>p</i> = 0.005, error bars show standard error) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s013" target="_blank">Table S8</a>). (C) Relative fitness of <i>Δrpb9</i> replicate evolving line 2 and <i>Δrpb9 Δwhi2</i> double mutant were measured as colony sizes grown on different media. Genotypes are indicated on the left, the growth media are indicated above the heat map. For media composition and abbreviations, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s009" target="_blank">Table S4</a>. Values are normalized to <i>Δrpb9</i> ancestor. Log₂ values are shown according to the color coding (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s013" target="_blank">Table S8</a>).</p

Compensation of fitness loss during laboratory evolution.

<p>(A) Experimental scheme to estimate evolutionary compensation of gene defects. See text for details. (B) Distribution of relative fitness improvement (RFI) of the knock-out mutant strains and the evolving control lineages (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>), where RFI = (evolved fitness/initial fitness)−1. (C) Relative compensation (RC) of the compensated knock-out mutant strains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>), where RC is the fraction of the initial fitness defect that was compensated for during laboratory evolution (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#s4" target="_blank">Materials and Methods</a>). (D) Compensation does not depend on pleiotropy (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>). The bars indicate mean ± standard error, Wilcoxon rank sum test <i>p</i>-values for the three comparisons are: 0.71, 0.44, and 0.36, respectively. (E) Genotypes with lower initial fitness were more likely to be compensated for during laboratory evolution (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001935#pbio.1001935.s006" target="_blank">Table S1</a>). Lines were divided into groups by initial fitness, the fraction of compensated lines among all the lines in the group is shown as bars (chi-squared test for trend in proportions, <i>p</i><10⁻¹³, number of lines in the groups from left to right: 38, 56, 201, 337).</p