Contrasting selective signatures of gene expression plasticity in response to antifungal clotrimazole and common environmental stresses in yeast

Domain: biology
Published: 2026-03-25
Editorial outcome: major_revision

Abstract
Phenotypic plasticity plays a key role in adaptation to fluctuating environments. However, its evolutionary significance remains debated, with conflicting views on whether it is actively maintained by natural selection or a neutral consequence of molecular constraints. In this study, we examine the evolutionary role of gene expression plasticity in yeast populations exposed to both common, ancestrally experienced stressors (NaCl and heat at 37°C) and a novel stressor (the antifungal drug clotrimazole). Using mutation accumulation lines to separate the effects of natural selection from genetic drift, we compare gene expression responses (referred to as plastic change) between 22 natural strains, 15 mutation accumulation lines, and their progenitor under non-stressed and stressed conditions. Our results show that, in the antifungal environment, gene expression plasticity is selectively maintained, as indicated by its reduction in magnititude, reversal in directionality, and loss of stress-responsive pathways in mutation accumulation lines. In contrast, plasticity under the common stress conditions appears largely neutral, with random variation across mutation accumulation lines. This study provides direct evidence of the adaptive role of gene expression plasticity in response to novel stressors and offers insights into the molecular mechanisms driving these adaptive responses.

Introduction
Phenotypic plasticity, the ability of a genotype to produce different phenotypes in response to environmental changes, is a fundamental feature of biological systems1. While it is widely recognized as an important biological property, its evolutionary origins, maintenance, and role in adaptation remain controversial. A central debate in evolutionary biology concerns whether plasticity is an adaptive mechanism actively maintained by natural selection or a byproduct of molecular constraints and regulatory networks2–7. Specifically, the extended evolutionary synthesis challenges the traditional modern synthesis by arguing that plasticity plays a foundational role in adaptation and that its evolutionary importance has been underappreciated8–12. According to the "plasticity-first" hypothesis12, when an environment shifts, the reaction norm of an organism allows it to produce a novel phenotype that may be partially or fully adapted to the new conditions. In other words, plastic phenotypic changes are often necessary for survival in novel environments, providing a crucial buffer that allows populations to persist while genetic adaptation occurs13. For example, plasticity facilitates adaptation by pre-aligning phenotypic traits with their optimal states, thereby serving as a stepping stone for genetic adaptation14. However, an alternative view posits that plastic changes are often reversed rather than reinforced during evolution, suggesting that they may not always be beneficial or necessary for adaptation15.

Gene expression plasticity, a key aspect of phenotypic plasticity, is particularly relevant in microbial populations, where rapid transcriptional responses to environmental stress can directly impact survival and fitness16. By using gene expression levels as focal traits, studying plasticity at the transcriptional level provides an effective and unbiased framework for assessing its evolutionary significance across a group of traits15,17. However, it remains uncertain whether gene expression plasticity is selectively maintained as an adaptive feature or simply represents a transient, non-adaptive response to environmental fluctuations. Moreover, the evolutionary outcomes of plasticity may vary depending on the type of environment encountered, particularly between conditions that have been common throughout an organism’s evolutionary history and those that are novel or rarely experienced. When a stressor has been repeatedly encountered over evolutionary time, such as high salinity (NaCl exposure) or heat stress, organisms are likely to evolve specific adaptive responses to mitigate its effects. Notwithstanding, if such stressors persist over long timescales, plastic responses may eventually become genetically assimilated, resulting in the fixation of adaptive traits and a consequent loss of plasticity5,6,18. In contrast, exposure to novel stressors that were absent from an organism’s evolutionary history poses different challenges. In these cases, organisms may lack specialized adaptive responses but might still cope with stress through mechanisms that overlap with responses to familiar stressors or through broad, general stress-response pathways19.

To address the role of natural selection in the evolution of plasticity under both common and novel stressors, experimental evolution provides a powerful approach by directly assessing the role of selection in shaping gene expression plasticity. Mutation accumulation (MA) experiments, in which populations undergo repeated single-cell bottlenecks to minimize the effects of natural selection, provide a rigorous approach to determine whether plasticity is selectively maintained or subject to genetic drift. If gene expression plasticity is adaptive, selection in natural populations would typically remove mutations that reduce plasticity, preserving an optimal level of gene expression responsiveness. However, in MA lines, where random mutations accumulate primarily due to genetic drift and selection is largely absent, deleterious mutations affecting plasticity would not be purged. As a result, plasticity should systematically decline in MA lines due to the accumulation of mutations that impair its regulatory mechanisms; In contrast, if gene expression plasticity is neutral, meaning it does not provide a selective advantage or disadvantage, its presence or absence in a given strain would be governed by stochastic processes rather than selective pressures. In this case, MA lines would show random deviations from the progenitor in plasticity, with some lines exhibiting higher or lower plasticity compared to the progenitor strain, but without any consistent directional trend. Thus, comparing the magnitude and direction of gene expression plasticity between the progenitor strain and MA lines provides a way to infer whether plasticity is actively maintained by selection.

In this study, we investigate the adaptive significance of gene expression plasticity using yeast MA lines exposed to common and novel stressors. We compare gene expression responses in natural strains, where natural selection remains intact, with MA lines, where selection is largely absent. Our results reveal that gene expression plasticity in the antifungal environment is predominantly adaptive, as indicated by its reduced magnitude in MA lines and the loss of stress-responsive pathways. Conversely, expression plasticity in the common stress conditions (NaCl and heat) appears largely neutral, with no clear pattern of selective maintenance. These findings provide empirical evidence supporting the adaptive role of gene expression plasticity in response to clotrimazole, offering insights into how microbial populations rapidly respond to environmental stressors and how selection shapes these responses over evolutionary timescales.

Results
1. Study design and transcriptome sequencing of yeast MA lines and natural strains
To investigate the selective pressures acting on expression plasticity, we utilized yeast mutation accumulation (MA) lines generated in a previous study20. These lines were established using BY4741 as the ancestral strain and underwent approximately 1500 generations of MA in the solid nutrient-rich (YPD) medium at 30 ℃, which lacked the stressors employed in the current study. To accelerate mutation accumulation, the mismatch repair gene MSH2 was deleted before MA and reinserted afterward. Consequently, each MA line accumulated an average of ~900 mutations.

To ensure comparability between the common and novel stress conditions, we selected MA lines that were capable of stable growth in both the novel stress and at least one of the common stress environments. Initially, 21 MA lines were chosen. We then conducted whole-genome sequencing to verify the identity of the MA lines. This analysis revealed that 6 lines were contaminated by the progenitor strain, as they carried ≤3 mutations, and were therefore excluded from the study (Supplementary Data 1 and Methods for details). As a result, a total of 15 MA lines, their progenitor BY4741, and 22 natural strains21 were collected for analysis (Figure 1a and Supplementary Data 2). Given the relatively short evolutionary timeframe of the MA experiment, the genetic diversity among the MA lines averaged 1.8×10⁻⁵, significantly lower than the 3.0×10⁻³ observed in the natural strains (Figures 1b and 1c). To test if the plastic response differs between common and novel stressors, transcriptional profiles for each strain were obtained under four conditions: a non-stressful medium (YPD), two common stressors (YPD with 0.5 M NaCl and YPD at 37℃), and a novel stressor inflicted by a synthetic antifungal drug (YPD with 1 μM Clotrimazole (Clo)). NaCl induces osmotic stress22 and distorts intracellular Na+ concentration23, 37℃ induces heat stress, while clotrimazole, an antifungal drug developed in the 1960s24, inhibits ergosterol synthesis in fungal cell membranes25. As expected, growth rates were significantly reduced in stress environments, reaching only 50%-70% of those in YPD (Figure 1d). For each strain, transcriptome sequencing was performed in three biological replicates, yielding an average of 6.9 million reads per sample (Supplementary Data 3 and Supplementary Figure 1). The high consistency among biological replicates (Pearson’s r = 0.98, P < 10 -10; Supplementary Figure 2) confirmed the robustness of the sequencing data.

Dimensionality reduction analysis of the transcriptome profiles revealed two major transcriptional clusters corresponding to MA and natural strains, with further subgrouping based on environmental conditions (Figure 1e). These transcriptomic profiles were used to quantify gene expression plasticity by comparing expression levels across YPD and the three stress conditions (Figure 1a).

2. Reduced expression plasticity in the antifungal environment after MA
To evaluate whether ancestral expression plasticity was selectively maintained, suppressed, or neutral, we analyzed changes in plasticity across MA lines. If ancestral plasticity was maintained by selection, MA lines would exhibit reduced plasticity due to gradual erosion of its genetic basis during mutation accumulation. Conversely, if plasticity was suppressed, MA lines would show increased plasticity, while a neutral model would predict random variation in plasticity levels relative to the progenitor.

Gene expression levels were quantified by normalizing expression levels using log (FPKM+1) (Supplementary Data 4 and 5). This transformation is commonly employed to stabilize variance and prevent issues associated with zero counts, ensuring more robust downstream analysis26. Plasticity for each gene was then calculated as the absolute difference in expression levels between stressed and YPD conditions27 (Supplementary Data 6-8). To assess overall plasticity, we summed the absolute plasticity across all genes for each strain (SumAcrossAllGenes(| Expression - Expression |). For NaCl/YPD comparisons, 7 of 15 MA lines exhibited lower plasticity compared to the progenitor, a distribution that did not significantly different from randomness (Figure 2a; two-sided binomial test, proportion = 0.47 (7/15), P = 1.00, 95% confidence interval = 0.21-0.73). A similar pattern was observed for 37℃/30℃ comparisons (Figure 2b; two-sided binomial test, proportion = 0.33 (3/9), P = 0.51, 95% confidence interval = 0.07-0.70). However, for Clo/YPD comparisons, All 15 MA lines showed reduced plasticity (Figure 2c; two-sided binomial test, proportion = 1.00 (15/15), P = 6.1×10-5, 95% confidence interval = 0.78-1.00).

To further verify these findings, we conducted an additional analysis that weighted all genes equally. Specifically, for each MA line, we compared the expression plasticity of each gene to that of the progenitor and calculated the percentage of genes exhibiting increased plasticity. For NaCl/YPD, 6 of 15 MA lines had <50% of genes with increased plasticity, while the remaining 9 had >50% of genes with increased plasticity, consistent with neutrality (Figure 2d; two-sided exact binomial test, proportion = 0.40 (6/15), P = 0.61, 95% confidence interval = 0.16–0.68). Again, the pattern is similar for the plasticity between 37℃ and 30℃ (Figure 2e; two-sided binomial test, proportion = 0.56 (5/9), P = 1.00, 95% confidence interval = 0.21–0.86). In contrast, for Clo/YPD, all 15 MA lines had reduced plasticity across the majority of genes, demonstrated a reduced expression plasticity compared with their progenitor (Figure 2f; two-sided binomial test, proportion = 1.0 (15/15), P = 6.1×10-5, 95% confidence interval = 0.78-1.00), suggesting that the overall expression plasticity in the progenitor was selectively maintained.

This pattern held true when analyzing individual gene plasticity across the yeast genome. For each of the 6263 genes, we calculated the proportion of MA lines with increased plasticity compared to the progenitor. No significant bias was detected for NaCl/YPD or 37℃/30℃ plasticity (Figures 2g; two-sided binomial test, proportion = 0.50 (3127/6263), P = 0.92, 95% confidence interval = 0.49–0.51; Figure 2h; two-sided binomial test, proportion = 0.49 (3087/6263), P = 0.27, 95% confidence interval = 0.48–0.51). However, for Clo/YPD, a significant majority of genes in the MA lines showed reduced plasticity, providing strong evidence that plasticity was selectively maintained in the progenitor for the antifungal environment (Figure 2i; two-sided binomial test, proportion = 0.73 (4572/6263), P = 4.5×10-301, 95% confidence interval = 0.72–0.74).

A consistent pattern emerged when comparing MA lines with natural strains, with the largest differences observed in the Clo/YPD plasticity. In all forms of analysis (Figures 2c, 2f, and 2i), the magnitude of Clo/YPD plasticity was significantly lower in MA lines compared to natural strains.

3. Reduced number of significantly plastic genes in the antifungal environment after MA
To confirm that the above findings were not driven by stochastic variation in gene expression, we repeated the analyses using only genes with significant plasticity (SPGs) between YPD and stressed environments (SPGs were identified using DESeq2 with FDR < 0.05 and |log FoldChange| > 1). In the progenitor, 516 SPGs were identified between NaCl and YPD, with 346–1466 SPGs across MA lines and natural strains (Figure 3a). Among the 15 MA lines, 4 exhibited fewer SPGs than the progenitor, while 11 showed more, consistent with random expectations (Figure 3b; two-sided binomial test, proportion = 0.27 (4/15), P = 0.12, 95% confidence interval = 0.08–0.55). Between 37℃ and 30℃, 676 SPGs were found in the progenitor, and all the 9 MA lines exhibited more SPGs than the progenitor (Figures 3c and 3d; two-sided binomial test, proportion = 0.00 (0/9), P=0.0039, 95% confidence interval = 0.00–0.34). If anything, it suggest that the plastic expression under heat condition is selectively suppressed in the progenitor.

Between Clo and YPD conditions, 1876 SPGs were found in the progenitor, while MA lines and natural strains exhibited 234–2237 SPGs (Figure 3e). Importantly, all 15 MA lines showed reduced numbers of SPGs compared to the progenitor (Figure 3f; two-sided binomial test, proportion = 1.0 (15/15), P = 6.1×10-5, 95% confidence interval = 0.78-1.00), with an average of 605 SPGs in MA lines—less than half of the progenitor’s count. This significant reduction in transcriptional responsiveness highlights the role of selection in maintaining expression plasticity under clotrimazole stress.

4. Directional reversal of plasticity in the antifungal environment after MA
While the above analysis focused on changes in the magnitude of expression plasticity, we further investigated the evolutionary trajectory of plasticity by examining its directionality. To this end, we adapted a modified method from prior studies6,15 to categorize plastic expression changes into two distinct categories: codirectional and reversed. Plastic changes were classified as codirectional if the MA line retained the same directional response as the progenitor, regardless of magnitude (e.g., the expression level of a gene was higher in stressed than non-stressed conditions in both the MA line and the progenitor). Conversely, changes were classified as reversed if the direction of plasticity shifted in the opposite direction relative to the progenitor (e.g., the expression level of a gene was higher in stressed versus non-stressed conditions in the progenitor but lower under the same conditions in the MA line) (Figure 4a).

We hypothesized that if the directionality of ancestral plasticity was selectively maintained, reversed plasticity would occur more frequently than codirectional plasticity. This is because, in the absence of selective constraints, random mutations accumulating in MA lines are more likely to disrupt the regulatory mechanisms that sustain the ancestral plastic response. To test this, we generated a null distribution of genes between the two categories by treating the 22 natural strains as controls and comparing their plastic expression profiles with the progenitor of the MA lines. We classified all genes in the MA lines and natural strains based on plastic expression profiles in the three stressful environments (Figures 4b, 4c, and 4d) and compared the proportions of genes in each category between MA and natural strains.

For NaCl/YPD and 37℃/30℃ plasticity, no significant differences were found across the two categories between MA and natural strains (Figure 4e, two sided t-test, P = 0.10; Figure 4f, two sided t-test, P = 0.09), consistent with neutral expectation. However, significant differences were observed for Clo/YPD plasticity. The MA lines exhibited significantly fewer genes with codirectional plasticity and significantly more genes with reversed plasticity compared to natural strains (Figure 4g; two-sided t-test, P =3.0 × 10-7). The increased levels of reversed plasticity in MA lines further supports the hypothesis that expression plasticity in the progenitor was selectively maintained.

To further dissect these patterns, we refined our classification into six categories, separating ancestral inductions and suppressions under stress into three subtypes each: strengthened, weakened, and reversed (Supplementary Figure 3a). Strengthened plasticity indicates that the direction of the response is the same in the MA line and progenitor but with a greater magnitude in the MA line, suggesting that ancestral plasticity was selectively suppressed. Conversely, weakened and reversed plasticity imply that ancestral plasticity was selectively maintained. Consistent with our previous findings, the proportion of strengthened plasticity was significantly lower under clotrimazole treatment, whereas the proportions of weakened and reversed plasticity were significantly higher (Supplementary Figure 3b). Similar trends were observed for ancestral suppressions, although the differences among the three stressful environments were less pronounced (Supplementary Figure 3c). Collectively, these results indicate that expression plasticity in the clotrimazole environment was selectively maintained.

5. Shared pathway loss of stress response in the antifungal environment after MA
Our results indicate that gene expression plasticity in the antifungal environment is largely adaptive, suggesting that it provides functional benefits to yeast cells under stress. To identify the specific biological functions associated with this plasticity, we first identified genes with significant plasticity (SPGs) in the progenitor, MA lines, and natural strains. Gene Ontology (GO) enrichment analysis was then performed to identify overrepresented biological processes. If a biological process was functionally advantageous in the antifungal environment, it would likely be present in most natural strains but lost in some MA lines. To test this, we compared the presence of progenitor-enriched biological processes between natural strains and MA lines, analyzing up-regulated and down-regulated genes separately. This distinction is important because activators and repressors involved in the same biological process often exhibit opposite patterns of expression change under stress.

For genes up-regulated under clotrimazole environment, we identified 11 biological processes that were significantly enriched in the progenitor and natural strains but largely absent in MA lines (Figure 5; Fisher’s exact test, P-adjust < 0.05). These processes were frequently detected in natural strains (present in at least 12 of 22 strains) but rarely observed in MA lines (at most in 1 of 15 lines). Notably, processes associated with general stress response, such as response to oxidative stress and response to chemical stimulus, were among the most enriched. Oxidative stress response is commonly induced when yeast cells face diverse environmental stresses19, and azole antifungal drugs, including clotrimazole, are known to cause reactive oxygen species (ROS) accumulation in fungi28,29. Similarly, cellular response to chemical stimulus is frequently activated when cells encounter a wide range of toxic chemicals30,31. These findings suggest that even stressors absent from yeast’s evolutionary history can still be mitigated through general stress-response mechanisms. Other enriched biological processes included pathways involved in carbohydrate metabolism and purine and pyrimidine metabolism. Clotrimazole inhibits glucose metabolism by targeting the key glycolytic enzyme 6-phosphofructo-1-kinase32. This aligns with findings that clotrimazole is more effective against fungal species like C. albicans that rely on glucose rather than alternative carbon sources such as lactic acid33. Furthermore, elevated ROS levels can lead to DNA damage34, necessitating enhanced nucleotide synthesis for DNA repair.

In contrast, for genes up-regulated under NaCl stress, enriched pathways were mainly related to amino acid metabolism and showed no direct connection to canonical osmotic stress responses such as the HOG pathway22,35 (Supplementary Figure 4). No significant enrichment was observed for genes up-regulated under heat stress, nor for genes down-regulated in any of the stressed conditions. In summary, the up-regulated pathways in the progenitor and natural strains under antifungal stress likely conferred functional advantages by activating general stress responses, while their loss in MA lines underscores the selective maintenance of these pathways in natural populations.

Because our transcriptomic analyses revealed that genes associated with the oxidative stress response were among the most frequently lost pathways in MA lines under clotrimazole treatment, we hypothesized that this loss might contribute directly to reduced stress tolerance. To test this, we measured the competitive fitness of 15 MA lines in media containing 1 μM clotrimazole and 0.1 mM hydrogen peroxide (H₂O₂), the latter serving as a well-established inducer of oxidative stress. If the oxidative stress response pathway underlies the adaptive plasticity to clotrimazole, then MA lines impaired in this pathway should show parallel fitness reductions in both environments. Consistent with this prediction, we observed a significant positive correlation between fitness reductions (relative to YPD) across the two conditions (Pearson’s r = 0.62, P = 0.014; Supplementary Figure 5). These results suggest that the loss of oxidative stress response plasticity in MA lines contributes to decreased fitness under both clotrimazole and oxidative stress, supporting the conclusion that adaptive expression plasticity in the progenitor involves activation of a general oxidative stress response pathway.

Methods
Strains and media
This study utilized the commonly used laboratory yeast strain BY4741, 15 MA lines derived from BY474120, and 22 diverse natural strains21 (Supplementary Data 2). To accumulate mutations more efficiently, the MA lines were generated in a mutator genetic background, with each line accumulating approximately 900 random mutations on average. Following mutation accumulation, the non-mutator genotype was restored for each MA line20. The natural strains were subjected to sporulation, polyploid selection, and genetic modifications as described by Maclean et al.21. Specifically, the HO and URA3 genes in the natural strains were replaced with antibiotic markers, and all strains were maintained in haploid form.

Yeast strains were cultured in four media: standard YPD, YPD supplemented with 0.5 M NaCl (osmotic stress), YPD at 37°C (heat stress), and YPD supplemented with 1 µM clotrimazole (antifungal stress). The YPD medium was prepared by dissolving 20 g glucose, 20 g peptone, and 10 g yeast extract in 1 L of distilled deionized water (ddH O), followed by autoclaving at 121°C for 30 minutes. For osmotic stress conditions, 29.25 g of sodium chloride was added to 1 L of YPD medium to achieve a final concentration of 0.5 M NaCl. For antifungal stress conditions, 1.72 g of clotrimazole was dissolved in 50 mL of anhydrous ethanol, filter-sterilized, and added to 1 L of YPD medium to achieve a final concentration of 1 µM.

Sample preparation, transcriptome, and genome sequencing
To prepare samples for transcriptome sequencing, all strains were pre-adapted in the four media (YPD, YPD + NaCl, YPD at 37°C, and YPD + clotrimazole) for a full growth cycle, from initial inoculation to saturation. A 15 µL aliquot of each culture was inoculated into 985 µL of fresh medium to achieve a final volume of 1 mL. Cultures were incubated at 30°C, and optical density (OD) was measured every one hour. Cells were harvested during the logarithmic growth phase (OD 0.4–0.6) by centrifugation at 13,800 × g for 1 minute. The supernatant was discarded, and the cell pellets were flash-frozen in liquid nitrogen. Each strain was cultured in triplicate for each medium condition.

RNA extraction, reverse transcription, library preparation, and high-throughput sequencing were performed by Genewiz (https://climsprod.genewiz.com.cn). Sequencing was conducted on the Illumina NovaSeq 6000 platform using a PE150 configuration, generating an average of 6.9 million paired-end reads per sample (Supplementary Data 3).

Genome sequencing was performed on the 15 MA lines initially selected, using the Illumina HiSeq platform with a target output of 1 Gb per sample. Mutation calling was conducted as described previously20. Briefly, sequencing reads were first aligned using BWA39, followed by sorting and duplicate marking with Picard (https://broadinstitute.github.io/picard/). Finally, variants were called using GATK40.

Data analysis
Raw sequencing data were preprocessed using fastp (v0.23.2) 41 to remove low-quality reads with default parameters. The Saccharomyces cerevisiae reference genome (R64) and corresponding annotation files were obtained from the Yeast Genome Database (https://www.yeastgenome.org/). Filtered reads were aligned to the reference genome using HISAT2 (v2.2.1) 42 and sorted using SAMtools (v1.20) 43. Gene-level read counts were quantified using featureCounts44 implemented in the Subread package (v2.0.1). Normalized expression levels, expressed as fragments per kilobase per million (FPKM), and significant plastic genes (SPGs) were defined as genes with expression level differ significantly between two environments and were identified using the DESeq2 package (v1.42.1) 45 in R. Gene Ontology (GO) enrichment analysis was performed using clusterProfiler (v4.10.1) 46.

Expression plasticity was quantified using two methods. First, FPKM values for each gene were normalized using log (FPKM + 1) to represent gene expression levels (Supplementary Data 4 and 5). Expression plasticity was calculated as the difference between expression levels under stress and non-stress conditions (Supplementary Data 6-8). To ensure robustness, a second method was employed, where expression plasticity was calculated as the normalized difference, which is the difference between stress and non-stress expression levels divided by the sum of the two expression levels.

Growth rate measurement and competition assay
The maximum growth rates of yeast strains in the three media were measured as described previously47. Briefly, strains were pre-adapted in the respective medium for a full growth cycle. Saturated cultures were diluted 10-fold, and 3 µL of the diluted culture was added to 197 µL of fresh medium in a 96-well plate (final volume: 200 µL per well). Plates were incubated at 30°C in a microplate reader, and optical density (OD) at 660 nm was recorded every 20 minutes for 48-72 hours (Supplementary Figures 8 and 9). OD measurements were converted to cell densities using a pre-determined OD–cell density calibration curve. Slopes were calculated for every four measurements spaced 60 minutes apart along the growth curve by Δln(cell density)/60. To avoid potential artifacts, the two highest slopes were discarded, and the mean of the third to eighth highest slopes was used as the growth rate per minute38,47.

Competition assay was performed following a previous study48. Specifically, YPD and clotrimazole-containing YPD media were prepared as described above. Hydrogen peroxide (H O ) solution was prepared by adding 0.4 µL of 30% H O to 40 mL of YPD, yielding a final concentration of 0.003‰, which is approximately 0.1 mM. The ADE2 gene was knocked out in BY4741 to generate the Ade2-marked strain, which form pink colonies on low-adenine media. SC medium was used to visualize the pink colonies of the progenitor strain. Competitions were performed in liquid media between each MA strain and the Ade2-marked progenitor, with wildtype BY4741 versus Ade2-marked BY4741 competitions included as controls. Prior to competition, both progenitor and MA strains were pre-cultured in the corresponding environment until reaching an OD600 of approximately 0.3. For each competition, 10 µL of MA culture and 5 µL of Ade2 progenitor culture were inoculated into 1 mL of the corresponding medium. This initial time point was designated as t . Cultures were incubated at 30°C with shaking at 250 rpm for 12 hours, corresponding to t . At t , cells were diluted and plated on SC medium. Plates were incubated at 30°C for > 2 days and then stored at room temperature for an additional day to allow clear visualization of colonies.

Relative fitness of each MA strain was calculated with respect to the Ade2 progenitor. The relative growth rate, s, was calculated using the formula:
ln(T )−ln(T )
s = 1 0 ⑴
t
where T represents the ratio of MA (white colonies) to Ade2 (pink colonies) at t , and t is the duration of the competition experiment, here 12 hours. The relative fitness, f, was subsequently calculated as:
f = exp(s×generation time) ⑵
Generation times for each environment were determined by monitoring cell number changes of BY4741 over the 12-hour period. The estimation of MA strains’ relative fitness finally normalized to the Ade2-marked progenitor versus progenitor across different environmental conditions.

Data availability
Source data are provided as a Source Data file. All sequencing reads generated in this study have been deposited in the China National Genomics Data Center under the accession number CRA033816 (https://ngdc.cncb.ac.cn/gsa/browse/CRA033816).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper explores whether gene expression plasticity, the ability of genes to change expression level in response to different environments, is a trait that is selected for or an inherent property of molecular networks. The authors address this question by comparing the transcriptome response to two different environmental stressors: osmotic stress and “antibiotic” stress between 22 natural isolates of Saccharomyces cerevisiae yeast to 21 mutation accumulation (MA) lines and their progenitor. [Note: “antibiotic” should be changed to “antifungal” throughout the paper given that antibiotics specifically refer to drugs impacting growth of bacteria.] The authors reason that the MA lines capture the effects of genetic drift in the absence of selection. The authors find that gene expression plasticity is reduced in the MA lines as compared to the progenitor in response to antifungal stress. In osmotic stress, in contrast, fewer genes exhibit expression plasticity in the progenitor, and the MA lines do not show a significant reduction in gene expression plasticity. Furthermore, the direction of gene expression plasticity (increasing or decreasing) is more likely to match the progenitor’s direction in the natural strains in both stress environments and the MA lines in osmotic stress. In the MA lines experiencing antifungal stress, in contrast, the direction of gene expression plasticity is near 50% matching the progenitor’s direction and 50% opposing the progenitor’s direction. Finally, the authors examine Gene Ontology enrichment among genes with expression plasticity. In antifungal stress, they find significant enrichment of categories with known relation to clotrimazole exposure in the progenitor and the natural strains but not in the MA lines. In osmotic stress, in contrast, the authors state that the GO categories they found enrichment for have “no direct relevance to osmotic stress”. The authors conclude there is evidence that gene expression plasticity is being selectively maintained in response to antifungal stress but not osmotic stress. This paper is clearly written and the findings are for the most part presented in an elegant and interpretable manner. The author’s findings represent a significant advancement in our understanding of how gene expression plasticity evolves. We have a few minor suggestions organized into 3 primary categories plus some general line edits:\n1. Clarify figures\n1.a. In Fig 2 a) and b), the authors use a metric called “overall expression plasticity”, which is the sum of expression differences of all genes. A large value of “overall expression plasticity” could therefore be driven by a single, highly plastic gene. While the authors clarify in later plots that this is not the case, it would still be helpful for readers to understand what “overall expression plasticity” is measuring by providing an equation or visual representation alongside Fig2 a) and b). Something as simple as SumAcrossGenes( |expression_O - expression_P| ). As Supplementary Fig3 also has a metric called “overall expression plasticity”, but it is calculated differently, it would additionally help to include a similar equation showing how this version is normalized by mean expression level.\n1.b. In Fig 2 c)-f), the authors use the “Percentage” to mean two different things. The “Percentage” in c) and d) is “Percent of genes with increased plasticity”, n = total number of samples. In e) and f), the “Percentage” is “Percent of MA line samples where a certain gene has higher expression plasticity than the progenitor”, n = total number of genes. While fully explaining the difference between these two percentages may not fit under an x-axis, having some visual representation next to the plot would make it clear to readers that the percentages are different.\n1.c. Fig 1a: It is unclear what the second scatter plot with black and grey points is representing, possibly natural strains vs MA lines?\n2.a. The information about which MA lines were selected and why is not presented until the end of the manuscript, in lines 326-332. This information should be shared with the reader much closer to the start of the MS when the experiential design is introduced. Justification of which MA lines were selected for this analysis is critical to evaluating and interpreting the work.\n2.b. Lines 177-178: The authors identify genes with significant plasticity (SPGs) using DESeq2, but they methods and tests used to identify these genes are only described in the Methods. Please also provide a description in the main text making it clear how p-values were calculated and assessed for significance using DESeq2.\n2.c. At the beginning of results section 3, the authors transition from using an absolute difference in expression to measure plasticity to a DESeq2 test to identify significant plasticity genes (SPGs). The authors should more clearly explain and justify the use of the two different metrics at different points in the paper.\n3. Gene Ontology\n3.a. Line 257: The authors state that the Gene Ontology category they found enrichment in in their osmotic stress condition, amino acid synthesis, isn’t “directly relevant to osmotic stress”. This appears to be based on the author’s judgment and could be strengthened by including a clear statement of the author’s expectation. For example, other papers have looked at GO enrichment in response to osmotic stress (MacGillveray et al. 2018, as an example) that could be used to inform your expectations.\n3.b. Line 236: The authors perform separate gene ontology analyses on genes that had an increasing versus decreasing plastic response. As activators and repressors of same biological process will change expression in opposite directions, the authors should better defend why they analyzed the gene sets separately rather than analyzing all SPGs together regardless of direction.\n4. Discussion Points\n4.a. How does use of the MSH2 mutation during mutational accumulation change the spectrum of mutations introduced? How might this affect the comparison to natural strains?\n4.b. A core claim made in the paper is that it provides evidence for expression plasticity in response to Clotrimazole that has been maintained by natural selection. But there is no discussion of whether the natural strains used in this study have been exposed to Clotrimazole during their evolutionary history. Assuming this is not the case, do you think Clotrimazole exposure is a proxy for other conditions the strains are more likely to have experienced during their evolution. **This is a critical issue that must be addressed in a revised version.** The text on lines 291-292 seems to start to get at this point, but it should be expanded, more explicit, and apparent throughout the manuscript.\n4.c. In figures 4c and 5, MA21 and MA97 don’t show the same pattern as the other MA lines. Rather, they appear to more closely resemble results in the natural strains. Is there a reason the authors think these strains are so different from the others? It would be helpful to readers for the authors to recognize these outliers and discuss possible reasons why these MA lines in particular show a different pattern.\n4.d. Duveau et al. 2017 PNAS also suggested that plasticity was adaptive in some environments but not others by comparing the effects of new mutations and polymorphisms from natural strains in S. cerevisiae in a particular gene’s promoter. While this paper and the authors’ paper look at this question from different scales (single gene vs whole genome), mentioning this paper in the discussion (perhaps at the end of the paragraph ending on line 306) would make the reader aware that there is additional support to the conclusions presented. [There might be other published work that would be good to cite here as well.]\n5. Line edits\n5.a. Line 71: “To address this” has an unclear subject. Please consider something like “To address the role of selection in plasticity evolution,”\n5.b. Line 113: “each MA lines”, change to “each MA line”\n5.c. Lines 130-131: “two major transcriptional corresponding to…” appears to be missing a word. Perhaps the authors meant something like “two major axes of transcriptional variation”\n5.d. Line 145: change “Gene expression levels was quantified” to “Gene expression levels were quantified” FPKM and explain to the reader why log2(FPKM+1) was used.\n5.e. Line 148: “To access”, change to “to assess”\n5.f. Line 159: There is something missing after >50%. Perhaps it should say “plasticity, while the remaining 13 had >50% of genes with increased plasticity, consistent with neutrality”?.\n5g. Line 300: “fluctuation” implies expression plasticity is changing in one direction then changing back again over evolutionary time, but the MA lines are not sequential isolates, so the word fluctuation is misleading. “Random deviations from the progenitor” or something similar would be more appropriate.\n5.h. Reminder: throughout paper, replace “antibiotic” with “antifungal”, as Clotrimazole specifically targets fungi not bacteria.", "reviewer_2": "Qian et al use a very interesting and largely novel approach to address whether phenotypes in extant lineages have been established by selection or neutral drift. They argue that if a phenotype has been established through a selective process, then evolved changes to that phenotype, under strictly neutral evolution, would tend to more often reverse than reinforce it. The logic is neat: random changes to an already optimized trait would be expected to more often impair than further improve it, whereas changes to a neutral trait would be random in direction.\nThey employ this logic to a yeast model strain, S388c, and test whether its capacity to respond to stressful environments, exemplified by exposure to moderate amounts of either NaCl or the antifungal clotrimazole, through changes to transcript abundance, is systematically lost or not when the strain is evolved neutrally.\nThey find that, somewhat surprisingly, there are no directional evolutionary changes to the transcriptional response to NaCl, implying that it is neutral, but that evolutionary changes to clotrimazole transcriptional plasticity are directional and tend to reverse it, implying that it has been selectively favoured.\nI very much appreciate the fundamental thinking in this paper, which I find refreshing, sound and employable on a broad scale, to a range of microbial phenotypes. As such, I think it has the potential to be well cited, and I can see contexts in which I would cite it. The data are mostly analysed in a correct and careful way, and the clotrimazole results seems clear.\nUnfortunately, I am not entirely convinced about the conclusions, because of various design issues, and would like to see additional follow-up analysis before I am ready to accept the claim of selection.\nMajor concerns:\n1. Looking for natural selection for clotrimazole plasticity in a S288c derivative doesn’t make much evolutionary sense. First, S288c is an artificial hybrid, whose genetic variants never jointly have been exposed to any selection outside the laboratory. S288c also tends to be a phenotypic outlier (see e.g. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1002111). This can also be seen in Fig 1E for YPD and NaCl, which calls for caution in interpretations of YPD and NaCl responses. Second, clotrimazole, as other azoles of its class, is a synthetic compound. Although some azoles have very recently begun to be used as fungicides in agriculture, this short period could hardly have selected for an optimal clotrimazole transcriptional response even in natural yeast. With both these points in mind, I find it hard to understand how natural selection could have worked to achieve optimal S288c transcriptional responses to clotrimazole. This would presumably require many genetic variants being driven to high frequencies - and their beneficial combinations not having been disrupted by the process creating S288. It would have been far easier to argue for natural selection for plasticity under NaCl exposure (see below). And ideally, experiments would have been performed with a wild yeast strain and exposure omnipresent natural selection pressures, such as nitrogen limitation, a respiratory carbon source, or temperature. As is, I am left with strong doubts. The reduced clotrimazole expression plasticity in MA lines seems robust. But, either this plasticity was established by selection pressures other than azoles. Or, they are not reflective of selection at all. This needs to be resolved and discussed. Could the plastic changes observed simply be a consequence of MA strains growing at a different rate? Or - of MA strains not being in the same mid-log phase when harvested as the progenitor, but one of the two approaching the end of log-phase growth which has entirely different expression patterns? If cells are not in comparable physiological states, we would be looking at a technical artefact, not selection? At a minimum, we should be shown log-scale growth curves with points of harvest indicated, ideally with 1st derivatives included, to demonstrate that cells are in comparable, mid-log states. A comparison of observed plasticities to that of previous studies looking at how BY transcript abundance changes as a function of growth phase could also help reject the explanation of cultures being in different growth states. If technical explanations to the observed difference can be rejected, I would be more ready to accept the claim of selection – but what would be the selection pressure, given that it is highly unlikely to have been azoles? Are we looking at a general stress response? The yeast “general stress response” is typically defined by distinct transcription changes in the initial shock phase after shift to a new environment, but there may be remnants of that also in the subsequent growth phase. Are we looking at a response to reduced/increased membrane stability/permeability? What would the driving selection pressure have been? There are extensive datasets on yeast transcriptional responses, and a very rich yeast literature, that could be used to illuminate this. At this stage, given that the authors have just one positive signal for an environment of dubious selective importance, and a negative signal for the environment that I would readily accept having been of selective importance, and this using a strain never exposed to natural selection, I feel the obligation to be cautious.\n2. If the observed plasticities lost in MA lines are indeed beneficial in the progenitor - then fitness should be reduced in the neutrally evolved MA lines, relative the progenitor, in the relevant environments? In the absence of such a fitness loss, the arguments for selection appear circumstantial to me. Relevant environments would seem to be clotrimazole, even though it doesn’t make much evolutionary sense, or other commonly encountered environments that could induce very similar plastic changes. For example, the authors argue for the oxidative stress response component of the observed plasticity being selectively important. This could be tested, e.g. using hydrogenperoxide or paraquat. In the absence of data demonstrating that the loss of plasticity is accompanied by a loss of fitness, I find it hard to accept the selection argument. A competition experiment that competes several evolved MA lines individually against the progenitor in clotrimazole and e.g. paraquat, and that estimates the selection coefficient from frequency change, would at least associate plasticity loss to fitness loss, and growth data could potentially be used as well (see below). The really convincing experiment would be to identify and then engineer the causative plasticity loss mutations, only, in a WT and compete that against the WT to establish true causality between plasticity loss and fitness loss. As is, the inference of selection from MA line transcriptional change is too circumstantial for me, and would need confirmation by supporting data.\n3. Growth data, such as reported in Fig 1D, could convince us that MA lines are less fit, in e.g. clotrimazole, than the BY progenitor – but this does not appear to be the case? However, we should be shown growth curves for the BY and the MA lines. It may be that a fitness defect is reflected in growth properties other than growth rates? I also have question marks around how the growth was estimated. Optical density and cell density are not linearly related, something that is normally accounted for either by dilution down to the near-linear range, or a post-experimental mathematical correction for the non-linearity (see e.g. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-1134-2). Unless this was done, the estimated growth will be incorrect, which as outlined in point 1 and 2 could have serious implications. Fig 1D gives some cause for concern in that growth has been correctly analyzed. If what is shown here is the maximum specific growth rate, umax - and even more so if it is the average growth rate across the entire growth interval - values are unrealistically high. A umax of 0.35-0.45 in YPD is standard, a value of 0.8, as shown, would imply doubling times well below an hour, which is outside the realistic range.\n4. In paragraph 4, I feel that the division of evolved changes to plasticity into co-directional and reversed gives a too low degree of resolution. “Repressions turning into inductions” are in this classification grouped together with “inductions turning into repressions”, which does not make much biological sense. Moreover, potential trends of inductions and repressions being reduced - but not changing sign - which to me would be signs of selection and their absence of would be signs of neutrality - are overlooked. Increasing the resolution would also reveal to what degree selection has acted on clotrimazole inductions and repressions respectively.\n5. I struggle to accept that gene expression changes induced by NaCl would be selectively neutral, which is implied by the data and alluded to in the text. The NaCl transcriptional response in yeast is well studied, with HOG pathway dependent transcriptional inductions of glycerol production, e.g. via GPD1, and the transcriptional regulation of salt-extrusion via the ENA proteins, being widely accepted as beneficial, selectively favoured responses. A more detailed analysis of what aspects of the NaCl plasticity that are and are not reversed could shed light on what is going on, and form the basis of a more nuanced discussion. The absence of directional changes to NaCl plasticity is almost as surprising as the presence of directional changes to clotrimazole plasticity, and contributes to making it difficult to accept the stringency of the selection test used. Given that only two environments are included, this deserves to be more thoroughly analysed and discussed.\nMinor issues:\n6. The title inappropriately alludes to antibiotics, and is an overreach. Azoles are not antibiotics but antifungals, and this should be corrected throughout the text, and it is an overreach to generalize to “the antibiotic environment” at large, from a single environment. Moreover, I am not sure the authors have shown the majority of individual plastic changes to be beneficial, even in clotrimazole, so predominant is probably not the right word.\n7. Some additional details on the conditions of the MA line evolution would be welcome. The statement that mutations accumulate primarily due to drift comes with an important caveat, as strongly negative mutations are selected against and these constitute a very substantial fraction of the total, in many DFE estimates. The MA conditions would dictate what these strongly negative variants are – for example, if MA conditions contains substantial amounts of Na+, e.g. through setting the pH with NaOH, this would probably influence the NaCl data.\n8. NaCl is not only on osmolyte acting externally, as implied in the text, but induces Na+ saline stress intracellularly, e.g. by distorting the Na+/K+ ratio.", "reviewer_3": "This is a review of “Gene expression plasticity in the antibiotic environment is predominantly adaptive in the budding yeast”.\nQian et al explore the evolutionary dynamics of phenotypic plasticity in the form of gene expression. MA lines and natural lineages are used to determine if plasticity is adaptive or neutral. They test two stressful conditions - osmotic and antibiotic - and report that gene expression plasticity is adaptive in environments with antifungal but is neutral in osmotic conditions. This conclusion is based on the observation that MA lines, but not natural isolates, lose the ability to adjust their gene expression to environmental change. Thus, the authors conclude that natural selection prevents accumulation of mutations that destroy this ability, while MA experiments do not. We have several major concerns with the way the results are interpreted, and we do not feel that the broad and sweeping conclusions are justified by these data.\nMajor concern 1: While the experiments address an important question of interest to scientists in many fields, this is not the question discussed in the introduction. The focus of the paper as described in the introduction is the question of whether plasticity allows organisms to persist in a novel environment until a new genetic mutation arises that helps them in that environment. That is an important question, but these results do not pertain to that question. At no point do the authors look for novel genetic mutations occuring and being selected in the stressful environments. They seem to define “adaptive” in the introduction as allowing an organism to persist and survive until it gets a new mutation. In that sense, the authors have not shown plasticity is adaptive at all. This definition of adaptive relates to evolvability. Does plasticity increase evolvability? The experiments have nothing to do with this question and the introduction should be rewritten to focus on the narrower question below.\nMajor concern 2: Even when using a more narrow definition of “adaptive”, we still are not sure the results justify the broad sweeping conclusions. When an organism can change its gene expression to match the environment, that likely suggests the organism has previously encountered that environment. That is why gene regulation evolved, to deal with environmental change. So of course, plasticity is adaptive. If plasticity is absent, and an organism cannot change its gene expression to survive an environment, it likely means it never encountered that environment. This ecological perspective, the idea that gene expression regulation reflects an organism's history, is entirely absent from the introduction.\nMajor concern 3: The conclusions appear to relate to all possible drugs and all possible salt environments, while only one of each was studied. Relatedly, the authors only look at two conditions and do not make clear why either was chosen and whether these environments are likely to have been encountered in that past history. The manuscript would benefit from the inclusion of additional environments, particularly more drug conditions. For example, do MA lines consistently show reduced phenotypic plasticity across a range of drug stresses? Without testing other drugs, it’s difficult to support the claim that gene expression plasticity in response to antifungal stress is broadly adaptive, as suggested in the discussion. The same goes for the identified pathways. Perhaps the conclusions could be reframed to emphasize their relevance to clotrimazole specifically. Additionally, the manuscript repeatedly refers to clotrimazole as an \"antibiotic,\" but it is an antifungal which needs to be corrected. Finally, do you think if you tested higher levels of osmotic stress that you would still see a neutral response? Can you please address this in the discussion? Again, I’m not sure these broad generalizations are appropriate based on the tested conditions.\nMajor concern 4: Another concern is that the MA lines used in this study reportedly carry around 900 mutations each which is substantially more than other yeast MA lines, which typically accumulate only a few mutations (see work by Hall lab). Nine hundred is a very high number, and it’s difficult to imagine that all of these mutations truly accumulated in the absence of any selection. It’s well established that lethal mutations do not accumulate in MA experiments. If the salt stress response regulon is essential, it likely would not be mutated, and as a result, you would not observe degradation of plasticity in response to salt. This doesn’t mean plasticity isn’t present—it may simply mean the relevant regulatory pathways were protected from mutation, especially if all media used contained some level of salt. In contrast, the drug condition may have activated a non-essential regulon, which could have been more susceptible to the accumulated mutations. In this way, the experiment may be unintentionally testing for essentiality rather than plasticity.\nMajor concern 5: The figures, as currently presented, are not effective in highlighting the key findings and in many cases feel redundant.\nFigure 1:\na-c) The expression level and plasticity plots do not clearly convey a central point, and this plotting style is not used in any subsequent figures, which makes it feel disconnected from the rest of the manuscript. The phylogenetic trees might be better placed in the supplement. Additionally, the natural strain names used here are inconsistent with those used elsewhere in the paper. If phylogenetic trees are included, the best-fit model and root should be reported.\nd) Consider combining these panels into a single plot for clarity.\ne) It’s curious that BY4741 in clo clusters with the natural isolates rather than the MA lines—this is unexpected and warrants an explanation.\nFigure 2:\nThe main point of this figure is unclear.\nFigure 3:\nPanels b and d appear to duplicate information shown in a and c. If so, they may not be necessary.\nGeneral:\nWhy are the natural isolates excluded from Figures 1, 2, and 3? Including them could help contextualize the results and support broader comparisons.\nSupplemental Figures:\nSupplemental Figure 4 appears to be identical to panels b–e from the main text, including the figure legend. If this is the case, it should either be removed or clearly justified.\nMinor concern 1: The use of bulk RNAseq in this study feels somewhat outdated, especially given the availability of single-cell RNAseq methods. Bulk approaches average expression across thousands of cells, potentially masking meaningful cell-to-cell variation that could be critical for understanding plasticity. Gene expression plasticity may not be uniform across a population—some subpopulations may respond strongly while others do not, and this heterogeneity can be biologically important. Incorporating single-cell methods could provide deeper insight into the dynamics and distribution of plastic responses, particularly under stress conditions.\nMinor concern 2: There were several typos throughout the manuscript. A thorough re-read and careful editing are strongly recommended. Additionally, the inclusion of line numbers would have made it easier to provide specific, targeted feedback.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the three anonymous reviewers for their thoughtful and constructive feedback. In response, we have conducted additional experiments and analyses to address their concerns. The new results are consistent with our previous findings and further strengthen the conclusions of the manuscript. Detailed point-by-point responses to each comment are provided below, with revisions highlighted in blue for clarity.\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nThis paper explores whether gene expression plasticity, the ability of genes to change expression level in response to different environments, is a trait that is selected for or an inherent property of molecular networks. The authors address this question by comparing the transcriptome response to two different environmental stressors: osmotic stress and “antibiotic” stress between 22 natural isolates of Saccharomyces cerevisiae yeast to 21 mutation accumulation (MA) lines and their progenitor. [Note: “antibiotic” should be changed to “antifungal” throughout the paper given that antibiotics specifically refer to drugs impacting growth of bacteria.] The authors reason that the MA lines capture the effects of genetic drift in the absence of selection. The authors find that gene expression plasticity is reduced in the MA lines as compared to the progenitor in response to antifungal stress. In osmotic stress, in contrast, fewer genes exhibit expression plasticity in the progenitor, and the MA lines do not show a significant reduction in gene expression plasticity. Furthermore, the direction of gene expression plasticity (increasing or decreasing) is more likely to match the progenitor’s direction in the natural strains in both stress environments and the MA lines in osmotic stress. In the MA lines experiencing antifungal stress, in contrast, the direction of gene expression plasticity is near 50% matching the progenitor’s direction and 50% opposing the progenitor’s direction. Finally, the authors examine Gene Ontology enrichment among genes with expression plasticity. In antifungal stress, they find significant enrichment of categories with known relation to clotrimazole exposure in the progenitor and the natural strains but not in the MA lines. In osmotic stress, in contrast, the authors state that the GO categories they found enrichment for have “no direct relevance to osmotic stress”. The authors conclude there is evidence that gene expression plasticity is being selectively maintained in response to antifungal stress but not osmotic stress. This paper is clearly written and the findings are for the most part presented in an elegant and interpretable manner. The author’s findings represent a significant advancement in our understanding of how gene expression plasticity evolves. We have a few minor suggestions organized into 3 primary categories plus some general line edits:\nResponse: We sincerely thank the reviewer for the thoughtful and constructive evaluation of our manuscript. We appreciate the clear summary of our study’s objectives and findings, and we are pleased that the reviewer found the paper clearly written and the results meaningful in advancing understanding of how gene expression plasticity evolves.\nWe have carefully addressed all of the reviewer’s specific comments and suggestions.\nBelow we provide detailed, point-by-point responses. As recommended, we have replaced the term “antibiotic” with “antifungal” throughout the manuscript.\n1. Clarify figures\n1.a. In Fig 2 a) and b), the authors use a metric called “overall expression plasticity”, which is the sum of expression differences of all genes. A large value of “overall expression plasticity” could therefore be driven by a single, highly plastic gene. While the authors clarify in later plots that this is not the case, it would still be helpful for readers to understand what “overall expression plasticity” is measuring by providing an equation or visual representation alongside Fig2 a) and b).\nSomething as simple as SumAcrossGenes( |expression_O - expression_P| ). As Supplementary Fig3 also has a metric called “overall expression plasticity”, but it is calculated differently, it would additionally help to include a similar equation showing how this version is normalized by mean expression level.\nResponse: Following the recommendation, we have clarified the definition of “overall expression plasticity” in both the main text and the corresponding figure legends. Specifically, we now include explicit equations to illustrate how this metric is calculated in each case. In Figure 2, “overall expression plasticity” is defined as:\nSumAcrossAllGenes(| Expression - Expression |)\nYPD stress\nand in Supplementary Figure 3 is defined as:\nSumAcrossAllGenes(| Expression - Expression |)/(Expression + Expression )\nYPD stress YPD stress\n1.b. In Fig 2 c)-f), the authors use the “Percentage” to mean two different things. The “Percentage” in c) and d) is “Percent of genes with increased plasticity”, n = total number of samples. In e) and f), the “Percentage” is “Percent of MA line samples where a certain gene has higher expression plasticity than the progenitor”, n = total number of genes. While fully explaining the difference between these two percentages may not fit under an x-axis, having some visual representation next to the plot would make it clear to readers that the percentages are different.\nResponse: We have now changed the corresponding x-axis labels to “% of genes with increased plasticity” and “% of MA lines with increased plasticity”, respectively. The same changes have also been applied to Supplementary Figure 3.\n1.c. Fig 1a: It is unclear what the second scatter plot with black and grey points is representing, possibly natural strains vs MA lines?\nResponse: The black and grey dots in the scatter plot indeed represent natural strains and MA lines, respectively. To clarify this, we have updated the figure legend with the following sentence:\n“The four colors represent four different environments and the black and grey dots in the scatter plot correspond to natural strains and MA lines.”\n2.a. The information about which MA lines were selected and why is not presented until the end of the manuscript, in lines 326-332. This information should be shared with the reader much closer to the start of the MS when the experiential design is introduced. Justification of which MA lines were selected for this analysis is critical to evaluating and interpreting the work.\nResponse: In the revised manuscript, we conducted an additional experiment to measure gene expression under a new condition, heat stress at 37 °C. With this addition, the study now includes three environmental stressors, which we categorized into two groups: common stresses (NaCl and heat) that yeast is likely to have encountered throughout their evolutionary history, and a novel stress (the antifungal drug clotrimazole), which represents a recently introduced, synthetic challenge. To ensure meaningful comparisons between common and novel stresses, we selected MA lines that were able to maintain stable growth under clotrimazole and at least one of the common stress conditions. This clarification has been added to the beginning of the Results section, where the experimental design is first introduced.\n2.b. Lines 177-178: The authors identify genes with significant plasticity (SPGs) using DESeq2, but they methods and tests used to identify these genes are only described in the Methods. Please also provide a description in the main text making it clear how p-values were calculated and assessed for significance using DESeq2.\nResponse: We have now mentioned in the main text that the SPGs were identified using DESeq2 with FDR < 0.05 and |log FoldChange| > 1.\n2\n2.c. At the beginning of results section 3, the authors transition from using an absolute difference in expression to measure plasticity to a DESeq2 test to identify significant plasticity genes (SPGs). The authors should more clearly explain and justify the use of the two different metrics at different points in the paper.\nResponse: The reviewer is correct that we employed two metrics to quantify gene expression plasticity. At the beginning of the Results section, we used the absolute difference in expression between stressed and non-stressed conditions as a quantitative measure of plasticity magnitude at the whole-transcriptome level. This metric allowed us to compare the overall extent and direction of gene expression changes among natural strains, mutation accumulation (MA) lines, and the progenitor, independent of statistical significance. Subsequently, we employed DESeq2 differential expression analysis to identify significant plasticity genes (SPGs)—genes showing statistically supported expression changes between conditions. This step enabled us to focus on biologically meaningful and reproducible plastic responses, rather than stochastic fluctuations. Both approaches yielded consistent results, supporting the robustness and reliability of our findings.\nTo enhance clarity, the following sentence has been added to the beginning of Results section 3:\n“To confirm that the above findings were not driven by stochastic variation in gene expression, we repeated the analyses using only genes with significant plasticity (SPGs) between YPD and stressed environments (SPGs were identified using DESeq2 with FDR < 0.05 and |log FoldChange| > 1)”\n2\n3. Gene Ontology\n3.a. Line 257: The authors state that the Gene Ontology category they found enrichment in in their osmotic stress condition, amino acid synthesis, isn’t “directly relevant to osmotic stress”. This appears to be based on the author’s judgment and could be strengthened by including a clear statement of the author’s expectation. For example, other papers have looked at GO enrichment in response to osmotic stress (MacGillveray et al. 2018, as an example) that could be used to inform your expectations.\nResponse: We thank the reviewer for this helpful comment. We have now cited two studies (references 22 and 35) that characterized the osmotic stress response in yeast and identified the HOG pathway as the primary regulator of this process. However, genes associated with the HOG pathway were not significantly enriched in our GO analysis. This information is now provided in the main text.\n3.b. Line 236: The authors perform separate gene ontology analyses on genes that had an increasing versus decreasing plastic response. As activators and repressors of same biological process will change expression in opposite directions, the authors should better defend why they analyzed the gene sets separately rather than analyzing all SPGs together regardless of direction.\nResponse: We agree that separating upregulated and downregulated genes provides a more biologically meaningful interpretation, as these gene sets often participate in distinct functional processes. For this reason, we performed the GO enrichment analysis separately for upregulated and downregulated SPGs, allowing us to better capture the functional differences between genes activated and repressed under each stress condition. For clarity, the following sentence is added to the main text:\n“To test this, we compared the presence of progenitor-enriched biological processes between natural strains and MA lines, analyzing up-regulated and down-regulated genes separately. This distinction is important because activators and repressors involved in the same biological process often exhibit opposite patterns of expression change under stress.”\n4. Discussion Points\n4.a. How does use of the MSH2 mutation during mutational accumulation change the spectrum of mutations introduced? How might this affect the comparison to natural strains?\nResponse: We thank the reviewer for this important question. The following paragraph has been added to the Discussion to address the potential effects of MSH2 deletion on the mutational spectrum:\n“In our MA experiment, we used an MSH2 deletion strain to increase the mutation rate, which also alters the mutational spectrum by elevating the frequency of base substitutions and small insertion–deletion events, particularly in repetitive regions. While this spectrum differs somewhat from that of natural yeast populations, the primary purpose of MA experiments is to allow random mutations to accumulate under minimal selection, thereby gradually eroding the genome. In this sense, even though the precise mutation spectrum may differ, it serves the same functional role as naturally occurring mutations in revealing how random genetic changes affect gene expression and plasticity. Because our analyses focus on the selective patterns acting on these phenotypic outcomes rather than the specific mutation mechanisms, the conclusions regarding the selective maintenance or neutrality of expression plasticity should remain valid.”\n4.b. A core claim made in the paper is that it provides evidence for expression plasticity in response to Clotrimazole that has been maintained by natural selection. But there is no discussion of whether the natural strains used in this study have been exposed to Clotrimazole during their evolutionary history. Assuming this is not the case, do you think Clotrimazole exposure is a proxy for other conditions the strains are more likely to have experienced during their evolution. **This is a critical issue that must be addressed in a revised version.** The text on lines 291-292 seems to start to get at this point, but it should be expanded, more explicit, and apparent throughout the manuscript.\nResponse: We thank the reviewer for raising this critical issue. We have expanded on lines 291-292 (now lines 368-370) and incorporated the concept of the general stress response to explain the adaptive plasticity observed under Clotrimazole exposure.\nSpecifically, the following sentences have been added to the Discussion:\n“However, since Clotrimazole is a synthetic drug developed in the 1960s and has not been part of the long evolutionary history of budding yeasts, it raises the question of how yeast cells can still maintain adaptive expression plasticity when exposed to Clotrimazole. Our analysis of GO categories for genes induced by Clotrimazole, which are lost in the MA lines but preserved in the natural strains, identified two key categories: response to oxidative stress and response to chemical stimulus. These categories are integral to the yeast’s general stress response pathway and are often activated under a wide range of stressors (Gasch et al. 2000. Mol. Biol. Cell). These findings suggest that, even when exposed to novel stressors not previously encountered by yeast cells, they can still utilize these general stress response pathways to manage such challenges.”\n4.c. In figures 4c and 5, MA21 and MA97 don’t show the same pattern as the other MA lines. Rather, they appear to more closely resemble results in the natural strains. Is there a reason the authors think these strains are so different from the others? It would be helpful to readers for the authors to recognize these outliers and discuss possible reasons why these MA lines in particular show a different pattern.\nResponse: We thank the reviewer for this thoughtful comment. We also noticed that several MA lines exhibited expression patterns that differed from the others. To investigate the potential cause of these discrepancies, we performed whole-genome sequencing on all 21 MA lines and compared the results with the progenitor strain. Our analysis revealed that for 15 MA lines, we were able to accurately identify the mutations as reported in the original study that generated these lines. However, for six of the MA lines (21, 28, 45, 51, 69, and 97), we could not detect the expected mutations. In fact, compared to the progenitor strain, these lines showed very few mutations, suggesting possible sample contamination. As a result, we have removed these six MA lines from our analysis. Importantly, after their removal, the results and conclusions of our study remained unchanged.\n4.d. Duveau et al. 2017 PNAS also suggested that plasticity was adaptive in some environments but not others by comparing the effects of new mutations and polymorphisms from natural strains in S. cerevisiae in a particular gene’s promoter. While this paper and the authors’ paper look at this question from different scales (single gene vs whole genome), mentioning this paper in the discussion (perhaps at the end of the paragraph ending on line 306) would make the reader aware that there is additional support to the conclusions presented. [There might be other published work that would be good to cite here as well.]\nResponse: The following sentence has been added to the end of the paragraph on line 306 (now line 408):\n“This finding aligns with a previous study on the expression plasticity of the TDH3 gene in yeast, where the authors observed evidence of adaptive plasticity in some environments but not in others (Duveau et al. 2017 PNAS).”\n5. Line edits\n5.a. Line 71: “To address this” has an unclear subject. Please consider something like “To address the role of selection in plasticity evolution,”\nResponse: Corrected as suggested.\n5.b. Line 113: “each MA lines”, change to “each MA line”\nResponse: Corrected.\n5.c. Lines 130-131: “two major transcriptional corresponding to…” appears to be missing a word. Perhaps the authors meant something like “two major axes of transcriptional variation”\nResponse: The word “clusters” was missing. Now corrected as:\n“Dimensionality reduction analysis of the transcriptome profiles revealed two major transcriptional clusters corresponding to MA and natural strains, with further subgrouping based on environmental conditions.”\n5.d. Line 145: change “Gene expression levels was quantified” to “Gene expression levels were quantified” FPKM and explain to the reader why log2(FPKM+1) was used.\nResponse: “was” is corrected to “were” and the following sentence is added to explain why log2(FPKM+1) was used:\n“This transformation is commonly employed to stabilize variance and prevent issues associated with zero counts, ensuring more robust downstream analysis.”\n5.e. Line 148: “To access”, change to “to assess”\nResponse: Corrected.\n5.f. Line 159: There is something missing after >50%. Perhaps it should say “plasticity, while the remaining 13 had >50% of genes with increased plasticity, consistent with neutrality”?.\nResponse: The reviewer is correct that it should say “while the remaining 13 had >50% of genes with increased plasticity”.\n5g. Line 300: “fluctuation” implies expression plasticity is changing in one direction then changing back again over evolutionary time, but the MA lines are not sequential isolates, so the word fluctuation is misleading. “Random deviations from the progenitor” or something similar would be more appropriate.\nResponse: “random fluctuation” is now changed to “random deviations from the progenitor”.\n5.h. Reminder: throughout paper, replace “antibiotic” with “antifungal”, as Clotrimazole specifically targets fungi not bacteria.\nResponse: Corrected.\nReviewer #2 (Remarks to the Author):\nQian et al use a very interesting and largely novel approach to address whether phenotypes in extant lineages have been established by selection or neutral drift. They argue that if a phenotype has been established through a selective process, then evolved changes to that phenotype, under strictly neutral evolution, would tend to more often reverse than reinforce it. The logic is neat: random changes to an already optimized trait would be expected to more often impair than further improve it, whereas changes to a neutral trait would be random in direction.\nThey employ this logic to a yeast model strain, S388c, and test whether its capacity to respond to stressful environments, exemplified by exposure to moderate amounts of either NaCl or the antifungal clotrimazole, through changes to transcript abundance, is systematically lost or not when the strain is evolved neutrally.\nThey find that, somewhat surprisingly, there are no directional evolutionary changes to the transcriptional response to NaCl, implying that it is neutral, but that evolutionary changes to clotrimazole transcriptional plasticity are directional and tend to reverse it, implying that it has been selectively favoured.\nI very much appreciate the fundamental thinking in this paper, which I find refreshing, sound and employable on a broad scale, to a range of microbial phenotypes. As such, I think it has the potential to be well cited, and I can see contexts in which I would cite it. The data are mostly analysed in a correct and careful way, and the clotrimazole results seems clear.\nUnfortunately, I am not entirely convinced about the conclusions, because of various design issues, and would like to see additional follow-up analysis before I am ready to accept the claim of selection.\nMajor concerns:\n1. Looking for natural selection for clotrimazole plasticity in a S288c derivative doesn’t make much evolutionary sense. First, S288c is an artificial hybrid, whose genetic variants never jointly have been exposed to any selection outside the laboratory. S288c also tends to be a phenotypic outlier (see e.g.https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1002111). This can also be seen in Fig 1E for YPD and NaCl, which calls for caution in interpretations of YPD and NaCl responses.\nResponse: The reviewer is correct that S288c is a hybrid strain that created in the lab.\nHowever, its parental strains are all natural strains that have evolved under natural condition. As such, the alleles present in S288c have indeed undergone natural selection prior to the hybridization, which provides valuable evolutionary context.\nThe fact that S288c may appear as a genotypic outlier compared to other strains is likely a result of its frequent cultivation in laboratory environments like YPD and SC, which could have led to it acquiring higher fitness under these specific conditions. Finally, we selected the BY4741 background because it is one of the most widely used model strains in yeast genetics and genomics, with extensive genomic, transcriptomic, and functional resources available. This choice ensures experimental reproducibility and facilitates comparisons with prior studies investigating gene expression plasticity and mutation accumulation in yeast.\nSecond, clotrimazole, as other azoles of its class, is a synthetic compound. Although some azoles have very recently begun to be used as fungicides in agriculture, this short period could hardly have selected for an optimal clotrimazole transcriptional response even in natural yeast. With both these points in mind, I find it hard to understand how natural selection could have worked to achieve optimal S288c transcriptional responses to clotrimazole. This would presumably require many genetic variants being driven to high frequencies - and their beneficial combinations not having been disrupted by the process creating S288. It would have been far easier to argue for natural selection for plasticity under NaCl exposure (see below). And ideally, experiments would have been performed with a wild yeast strain and exposure omnipresent natural selection pressures, such as nitrogen limitation, a respiratory carbon source, or temperature. As is, I am left with strong doubts. The reduced clotrimazole expression plasticity in MA lines seems robust. But, either this plasticity was established by selection pressures other than azoles. Or, they are not reflective of selection at all. This needs to be resolved and discussed. Could the plastic changes observed simply be a consequence of MA strains growing at a different rate? Or - of MA strains not being in the same mid-log phase when harvested as the progenitor, but one of the two approaching the end of log-phase growth which has entirely different expression patterns? If cells are not in comparable physiological states, we would be looking at a technical artefact, not selection? At a minimum, we should be shown log-scale growth curves with points of harvest indicated, ideally with 1st derivatives included, to demonstrate that cells are in comparable, mid-log states. A comparison of observed plasticities to that of previous studies looking at how BY transcript abundance changes as a function of growth phase could also help reject the explanation of cultures being in different growth states. If technical explanations to the observed difference can be rejected, I would be more ready to accept the claim of selection – but what would be the selection pressure, given that it is highly unlikely to have been azoles? Are we looking at a general stress response? The yeast “general stress response” is typically defined by distinct transcription changes in the initial shock phase after shift to a new environment, but there may be remnants of that also in the subsequent growth phase. Are we looking at a response to reduced/increased membrane stability/permeability? What would the driving selection pressure have been? There are extensive datasets on yeast transcriptional responses, and a very rich yeast literature, that could be used to illuminate this. At this stage, given that the authors have just one positive signal for an environment of dubious selective importance, and a negative signal for the environment that I would readily accept having been of selective importance, and this using a strain never exposed to natural selection, I feel the obligation to be cautious.\nResponse: We sincerely thank the reviewer for this very thoughtful and comprehensive comment. We have carefully considered each point and made several additions and clarifications in response.\n1. Growth phase\nWe fully agree that differences in growth state could confound transcriptomic comparisons. To address this, we have now included a new Supplementary Figure 8 showing the complete growth curves for all strains under each experimental condition. As shown below, both the natural strains and MA lines were harvested at comparable mid-logarithmic growth phases (OD ≈ 0.4–0.6 after subtracting media OD), well\n660\nbefore the onset of stationary phase. Therefore, the transcriptional differences observed between conditions are unlikely to result from differences in growth phase..\nSupplementary Figure 8. Growth curves and sampling windows for MA lines, natural strains, and the progenitor in the four environments. a–d, Growth curves of MA lines and the progenitor; e–h, growth curves of natural strains. The gray shaded areas indicate the sampling windows.\n2. Evolutionary relevance of clotrimazole stress\nWe agree with the reviewer that clotrimazole, as a synthetic azole developed in the 1960s, does not represent a stressor that yeast would have encountered in its evolutionary history. We therefore consider clotrimazole to be a novel stressor, providing an opportunity to examine how yeast respond transcriptionally to completely unfamiliar chemical challenges.\nTo explore why yeast can still mount an apparently adaptive transcriptional response to clotrimazole, we analyzed DEGs between YPD and clotrimazole conditions. Among the genes whose expression was induced under clotrimazole, Gene Ontology (GO) enrichment analysis revealed significant overrepresentation of categories such as response to oxidative stress and response to chemical stimulus (Fisher’s exact test, P < 0.05 after BH correction). These categories were consistently enriched in natural strains but largely absent in MA lines. Previous studies have shown that oxidative stress response is broadly activated when yeast encounter diverse environmental challenges, while response to chemical stimulus encompasses reactions to a wide range of toxic or xenobiotic compounds, not limited to azoles.\nThese results strongly suggest that the transcriptional plasticity observed under clotrimazole treatment reflects activation of the general stress response rather than a clotrimazole-specific adaptation. Thus, although yeast have never experienced clotrimazole during their evolutionary history, they can still employ general stress-response pathways to mitigate the effects of such novel stressors. We have clarified this interpretation in the revised Discussion section, emphasizing that the observed adaptive plasticity likely stems from selection for general stress responsiveness.\n3. Neutral plasticity under NaCl\nTo test whether the neutral pattern observed under NaCl stress was specific to that condition, we performed an additional experiment exposing yeast to heat stress (37 °C), a ubiquitous environmental challenge. The results were consistent with those under NaCl stress, showing a neutral pattern of expression plasticity.\nWe have added a new paragraph in the Discussion introducing the concept of genetic assimilation to explain this pattern. Genetic assimilation describes the evolutionary process by which an environmentally induced phenotype becomes genetically fixed, leading to a loss of plasticity over time. Under this model, stressors that are frequently encountered, such as NaCl or temperature fluctuations, are more likely to drive fixation of adaptive expression patterns, resulting in the neutral plasticity observed in our study.\nIn contrast, novel stressors like clotrimazole can still reveal adaptive plasticity, as their responses remain under active selection through general stress pathways.\nFinally, we note that our analysis focuses on genome-wide patterns of plasticity; we cannot exclude the possibility that the plastic expression of specific genes under NaCl or heat stress remains adaptive. This clarification has been added to the Discussion section.\n2. If the observed plasticities lost in MA lines are indeed beneficial in the progenitor - then fitness should be reduced in the neutrally evolved MA lines, relative the progenitor, in the relevant environments? In the absence of such a fitness loss, the arguments for selection appear circumstantial to me. Relevant environments would seem to be clotrimazole, even though it doesn’t make much evolutionary sense, or other commonly encountered environments that could induce very similar plastic changes. For example, the authors argue for the oxidative stress response component of the observed plasticity being selectively important. This could be tested, e.g. using hydrogenperoxide or paraquat. In the absence of data demonstrating that the loss of plasticity is accompanied by a loss of fitness, I find it hard to accept the selection argument. A competition experiment that competes several evolved MA lines individually against the progenitor in clotrimazole and e.g. paraquat, and that estimates the selection coefficient from frequency change, would at least associate plasticity loss to fitness loss, and growth data could potentially be used as well (see below). The really convincing experiment would be to identify and then engineer the causative plasticity loss mutations, only, in a WT and compete that against the WT to establish true causality between plasticity loss and fitness loss. As is, the inference of selection from MA line transcriptional change is too circumstantial for me, and would need confirmation by supporting data.\nResponse: We agree that demonstrating an association between the loss of expression plasticity and a corresponding fitness reduction would provide stronger support for selection acting on plasticity.\nFollowing the reviewer’s recommendation, we conducted additional experiments to test whether the loss of plasticity observed in the MA lines is linked to decreased fitness in relevant environments. Specifically, because our results indicate that the oxidative stress response is a major component of the gene expression plasticity induced by clotrimazole, we reasoned that if this pathway is compromised by mutations accumulated during the MA process, then MA lines with lower fitness under clotrimazole should also show reduced fitness under oxidative stress.\nTo test this hypothesis, we measured the competitive fitness of all 15 MA lines in YPD media containing 1 μM clotrimazole and YPD media containing 0.1 mM hydrogen peroxide (H₂O₂). The results show a significant positive correlation between the fitness reductions (relative to YPD) across the two conditions (Pearson’s r = 0.62, P = 0.014; see new Supplementary Figure 5). This relationship suggests that the same biological mechanisms underlie the reduced fitness of MA lines in both environments.\nSupplementary Figure 5. The correlation of fitness reduction of MA lines in hydrogen peroxide and clotrimazole. Fitness was measured by competitive growth assay. Each gray point represents a MA line. The black line shows the fitted linear regression, with the shaded area indicating the 95% confidence interval.\n3. Growth data, such as reported in Fig 1D, could convince us that MA lines are less fit, in e.g. clotrimazole, than the BY progenitor – but this does not appear to be the case? However, we should be shown growth curves for the BY and the MA lines. It may be that a fitness defect is reflected in growth properties other than growth rates? I also have question marks around how the growth was estimated. Optical density and cell density are not linearly related, something that is normally accounted for either by dilution down to the near-linear range, or a post-experimental mathematical correction for the non-linearity (see e.g. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-1134-2). Unless this was done, the estimated growth will be incorrect, which as outlined in point 1 and 2 could have serious implications. Fig 1D gives some cause for concern in that growth has been correctly analyzed. If what is shown here is the maximum specific growth rate, umax - and even more so if it is the average growth rate across the entire growth interval - values are unrealistically high. A umax of 0.35-0.45 in YPD is standard, a value of 0.8, as shown, would imply doubling times well below an hour, which is outside the realistic range.\nResponse: We thank the reviewer for these detailed and thoughtful comments regarding the growth-rate analysis and fitness interpretation. We confirm that, as shown in Figure 1D, the MA lines indeed exhibit lower fitness than the progenitor strain in the clotrimazole environment. We agree that growth rate is only one aspect of fitness and that other growth properties such as lag time, maximum growth rate during the logarithmic phase, and final yield can all contribute to overall fitness. In this study, we used maximum growth rate as a proxy for fitness, as it provides a robust and comparable measure of physiological performance across strains and conditions.\nWe agree that optical density (OD) and cell density are not linearly correlated. To address this issue, we established an OD–cell density calibration curve based on manual cell counts and applied this calibration to convert OD measurements to actual cell densities prior to growth-rate estimation. This step minimizes potential non-linearity artifacts.\nFinally, we confirm that the reported growth-rate values represent maximum specific growth rates (μ ). To measure growth rate, OD was recorded every 20 minutes and\nmax\nconverted to cell density using the calibration curve. The slopes of ln (cell density) over time were computed for all overlapping 60-minute windows. To reduce the influence of transient fluctuations, the two highest slopes were discarded, and the mean of the third to eighth highest slopes was used to represent μ . We note that this value is\nmax\nexpressed per minute, and therefore a μ of 0.8 corresponds to approximately 0.8 ×\nmax\n60 = 48% increase per hour, which falls within the biologically reasonable range for yeast growing in rich medium. This clarification has been added to the revised Methods section.\n4. In paragraph 4, I feel that the division of evolved changes to plasticity into co-directional and reversed gives a too low degree of resolution. “Repressions turning into inductions” are in this classification grouped together with “inductions turning into repressions”, which does not make much biological sense. Moreover, potential trends of inductions and repressions being reduced - but not changing sign - which to me would be signs of selection and their absence of would be signs of neutrality - are overlooked. Increasing the resolution would also reveal to what degree selection has acted on clotrimazole inductions and repressions respectively.\nResponse: Following the reviewer’s recommendation, we have now refined our analysis by dividing the evolved plasticity changes into six categories. Specifically, we first separated genes based on whether their ancestral plasticity in the progenitor represented an induction or a repression under stress. Within each of these two major categories, we further classified the changes observed in the MA lines into three subtypes: strengthened, weakened, and reversed. Here, “strengthened” indicates that the MA line exhibited the same directional response as the progenitor but with a greater magnitude (representing potential non-adaptive plasticity), while “weakened” and “reversed” indicate a loss or reversal of the ancestral response (representing adaptive maintenance of plasticity in the progenitor).\nOur results show that for ancestral inductions, expression plasticity in MA lines under clotrimazole exhibited a significantly lower proportion of strengthened changes but significantly higher proportions of weakened and reversed changes compared to the other two stress conditions (Figure below and new Supplementary Figure 3). This pattern suggests that the expression plasticity in clotrimazole is under stronger selective maintenance than plasticity in NaCl or heat stress. A consistent, though less pronounced, trend was observed for ancestral repressions, where clotrimazole again showed a lower proportion of strengthened response and higher proportions of weakened and reversed responses.\nSupplementary Figure 3. The proportions of strengthened, weakened, and reversed plasticity for ancestral inductions and suppressions in the MA lines. a, Classification of expression plasticity based on progenitor plasticity (ancestral inductions or suppressions) then classified to strengthened, weakened, and reversed. On the x-axis, ‘O’ represents the expression level in the original environment and ‘P’ represents the expression level in the new environment. Black dots represent the progenitor and orange dots represent MA lines. Pink, purple, and green denote strengthened, weakened, and reversed plasticity, respectively. b, The proportions of strengthened, weakened, and reversed plasticity for ancestral inductions in MA lines across three environments. c, The proportions of strengthened, weakened, and reversed plasticity for ancestral suppressions in MA lines across three environments. In panels b and c, Wilcoxon-test is performed between every two bins with ‘NS.’ represents not significant, ‘*’ represents P < 0.05, and ‘***’ represents P < 0.001.\n5. I struggle to accept that gene expression changes induced by NaCl would be selectively neutral, which is implied by the data and alluded to in the text. The NaCl transcriptional response in yeast is well studied, with HOG pathway dependent transcriptional inductions of glycerol production, e.g. via GPD1, and the transcriptional regulation of salt-extrusion via the ENA proteins, being widely accepted as beneficial, selectively favoured responses. A more detailed analysis of what aspects of the NaCl plasticity that are and are not reversed could shed light on what is going on, and form the basis of a more nuanced discussion. The absence of directional changes to NaCl plasticity is almost as surprising as the presence of directional changes to clotrimazole plasticity, and contributes to making it difficult to accept the stringency of the selection test used. Given that only two environments are included, this deserves to be more thoroughly analysed and discussed.\nResponse: We thank the reviewer for this insightful comment and fully understand the concern regarding the apparent neutrality of gene expression plasticity under NaCl stress.\nFirst, to determine whether the neutral pattern observed under NaCl stress is a unique case or represents a general trend for long-standing stressors, we performed an additional experiment examining expression plasticity under heat stress (37 °C), which is another ubiquitous environmental challenge in yeast’s evolutionary history. The results under heat stress closely mirrored those for NaCl. Plastic expression changes showed no consistent directionality across MA lines relative to the progenitor, suggesting that the neutral pattern is not specific to NaCl but may instead represent a general feature of common stressors that yeast have frequently encountered over evolutionary time.\nSecond, to explain why gene expression plasticity under such common stressors appears neutral at the genome-wide level, we introduced the concept of genetic assimilation in the revised Discussion section. Genetic assimilation, first described by Waddington, refers to the process by which an environmentally induced phenotype becomes genetically fixed, leading to a loss of plasticity over time. This evolutionary mechanism can explain why the initially plastic response has likely been genetically stabilized through recurrent exposure over millions of years.\nThe following paragraph has been added to the Discussion section to address this point in detail:\n“While this conclusion might initially seem counterintuitive, it may be explained by recurrent genetic assimilation. Genetic assimilation, first described by Waddington, occurs when environmentally induced phenotypes (i.e., phenotypic plasticity) become fixed in a population over a few generations, eliminating the need for plasticity18. Later, Lande developed a quantitative-genetic model that demonstrated how a transient increase in phenotypic plasticity, followed by genetic assimilation, can drive adaptation to a novel environment5. The strongest experimental evidence for genetic assimilation comes from a recent study on Silene uniflora36, which has repeatedly adapted to zinc-contaminated soils over the last 250 years. By sequencing the transcriptomes of both zinc-sensitive and zinc-tolerant lineages of Silene uniflora in both zinc-containing and zinc-free environments, researchers identified 183 genes whose plastic expression was fixed in the adapted lineages, providing direct evidence for genetic assimilation. In contrast to the recent 250-year exposure to zinc in Silene uniflora, osmotic and heat stressors in yeast have likely occurred intermittently for millions of years during their evolutionary history. Therefore, much of the adaptive ancestral plastic expression to these stressors may have already been fixed through genetic assimilation. This could explain the neutral pattern of plasticity we observed under NaCl and heat stress.”\nMinor issues:\n6. The title inappropriately alludes to antibiotics, and is an overreach. Azoles are not antibiotics but antifungals, and this should be corrected throughout the text, and it is an overreach to generalize to “the antibiotic environment” at large, from a single environment. Moreover, I am not sure the authors have shown the majority of individual plastic changes to be beneficial, even in clotrimazole, so predominant is probably not the right word.\nResponse: The title is now changed to “Contrasting selective signatures of gene expression plasticity in response to antifungal clotrimazole and common environmental stresses in yeast”.\n7. Some additional details on the conditions of the MA line evolution would be welcome. The statement that mutations accumulate primarily due to drift comes with an important caveat, as strongly negative mutations are selected against and these constitute a very substantial fraction of the total, in many DFE estimates. The MA conditions would dictate what these strongly negative variants are – for example, if MA conditions contains substantial amounts of Na+, e.g. through setting the pH with NaOH, this would probably influence the NaCl data.\nResponse: For clarity, the following information have been added to the main text:\n“These lines were established using BY4741 as the ancestral strain and underwent approximately 1500 generations of MA in the solid nutrient-rich (YPD) medium at 30 ℃, which lacked the stressors employed in the current study.”\n8. NaCl is not only on osmolyte acting externally, as implied in the text, but induces Na+ saline stress intracellularly, e.g. by distorting the Na+/K+ ratio.\nResponse: For clarity, the following sentence have been added to the main text:\n“NaCl induces osmotic stress and distorts intracellular Na+ concentration.”\nReviewer #3 (Remarks to the Author):\nThis is a review of “Gene expression plasticity in the antibiotic environment is predominantly adaptive in the budding yeast”. Qian et al explore the evolutionary dynamics of phenotypic plasticity in the form of gene expression. MA lines and natural lineages are used to determine if plasticity is adaptive or neutral. They test two stressful conditions - osmotic and antibiotic - and report that gene expression plasticity is adaptive in environments with antifungal but is neutral in osmotic conditions. This conclusion is based on the observation that MA lines, but not natural isolates, lose the ability to adjust their gene expression to environmental change. Thus, the authors conclude that natural selection prevents accumulation of mutations that destroy this ability, while MA experiments do not. We have several major concerns with the way the results are interpreted, and we do not feel that the broad and sweeping conclusions are justified by these data.\nMajor concern 1: While the experiments address an important question of interest to scientists in many fields, this is not the question discussed in the introduction. The focus of the paper as described in the introduction is the question of whether plasticity allows organisms to persist in a novel environment until a new genetic mutation arises that helps them in that environment. That is an important question, but these results do not pertain to that question. At no point do the authors look for novel genetic mutations occuring and being selected in the stressful environments. They seem to define “adaptive” in the introduction as allowing an organism to persist and survive until it gets a new mutation. In that sense, the authors have not shown plasticity is adaptive at all. This definition of adaptive relates to evolvability. Does plasticity increase evolvability? The experiments have nothing to do with this question and the introduction should be rewritten to focus on the narrower question below.\nResponse: We thank the reviewer for this comment. We agree that our experiments do not directly address whether plasticity facilitates persistence until beneficial mutations arise, which concerns evolvability. Instead, our study focuses on whether gene expression plasticity itself is adaptive, i.e., whether it is selectively maintained by natural selection. To avoid confusion, we have revised the Introduction to clarify this narrower focus.\nSpecifically, we modified the framing of the research question and added the following sentences to highlight that our study examines the adaptive maintenance of plasticity, not its role in promoting evolvability:\n“However, it remains uncertain whether gene expression plasticity is selectively maintained as an adaptive feature or simply represents a transient, non-adaptive response to environmental fluctuations.”\n“To address the role of natural selection in the evolution of plasticity under both common and novel stressors…”\nMajor concern 2: Even when using a more narrow definition of “adaptive”, we still are not sure the results justify the broad sweeping conclusions. When an organism can change its gene expression to match the environment, that likely suggests the organism has previously encountered that environment. That is why gene regulation evolved, to deal with environmental change. So of course, plasticity is adaptive. If plasticity is absent, and an organism cannot change its gene expression to survive an environment, it likely means it never encountered that environment. This ecological perspective, the idea that gene expression regulation reflects an organism's history, is entirely absent from the introduction.\nResponse: We appreciate the reviewer’s thoughtful comment and agree that incorporating an ecological and historical perspective on gene regulation is essential for contextualizing the adaptive significance of plasticity. We have revised the Introduction to explicitly discuss how the evolutionary history of environmental exposure shapes the adaptiveness of gene expression plasticity.\nSpecifically, we added the following new paragraph to the Introduction:\n“Moreover, the evolutionary outcomes of plasticity may vary depending on the type of environment encountered, particularly between conditions that have been common throughout an organism’s evolutionary history and those that are novel or rarely experienced. When a stressor has been repeatedly encountered over evolutionary time, such as high salinity (NaCl exposure) or heat stress, organisms are likely to evolve specific adaptive responses to mitigate its effects. Notwithstanding, if such stressors persist over long timescales, plastic responses may eventually become genetically assimilated, resulting in the fixation of adaptive traits and a consequent loss of plasticity5,6,18. In contrast, exposure to novel stressors that were absent from an organism’s evolutionary history poses different challenges. In these cases, organisms may lack specialized adaptive responses but might still cope with stress through mechanisms that overlap with responses to familiar stressors or through broad, general stress-response pathways19.”\nMajor concern 3: The conclusions appear to relate to all possible drugs and all possible salt environments, while only one of each was studied. Relatedly, the authors only look at two conditions and do not make clear why either was chosen and whether these environments are likely to have been encountered in that past history. The manuscript would benefit from the inclusion of additional environments, particularly more drug conditions. For example, do MA lines consistently show reduced phenotypic plasticity across a range of drug stresses? Without testing other drugs, it’s difficult to support the claim that gene expression plasticity in response to antifungal stress is broadly adaptive, as suggested in the discussion. The same goes for the identified pathways. Perhaps the conclusions could be reframed to emphasize their relevance to clotrimazole specifically. Additionally, the manuscript repeatedly refers to clotrimazole as an \"antibiotic,\" but it is an antifungal which needs to be corrected. Finally, do you think if you tested higher levels of osmotic stress that you would still see a neutral response? Can you please address this in the discussion? Again, I’m not sure these broad generalizations are appropriate based on the tested conditions.\nResponse: We thank the reviewer for this insightful and constructive comment. We agree that our original framing was too broad and have revised the manuscript accordingly to clarify the scope and interpretation of our findings.\nFirst, we have reframed the study design and discussion around the comparison between common and novel stressors rather than generalizing across all drugs or all stress conditions. Specifically, NaCl and heat (37 °C) are categorized as common stressors that yeast is likely to have encountered repeatedly throughout their evolutionary history, while clotrimazole is treated as a novel stressor because it is a synthetic antifungal compound developed in the 1960s. We have replaced all instances of “antibiotic” with “antifungal” throughout the manuscript.\nTo strengthen this comparison, we have now included heat stress as an additional common stressor to test whether the neutral pattern of plasticity under NaCl stress generalizes to another familiar condition. We found that both NaCl and heat stress yielded consistent, largely neutral patterns of expression plasticity, consistent with the hypothesis of genetic assimilation, that ancestral adaptive plasticity to long-standing stressors has been gradually fixed as a stable phenotype (as discussed in the revised Discussion section).\nIn contrast, expression plasticity under clotrimazole stress showed a clear adaptive pattern, with reduced plasticity and loss of stress-responsive pathways in MA lines relative to the progenitor and natural strains. We have revised the text to explicitly emphasize that this adaptive plasticity pertains specifically to clotrimazole rather than to antifungal or drug stresses in general. Our discussion now focuses on how yeast cells can utilize general stress-response pathways (e.g., oxidative stress response and chemical stimulus response) to cope with novel stressors not encountered in their evolutionary past.\nRegarding the environmental relevance of our experimental conditions, we have clarified the rationale for choosing stress intensities that approximate natural conditions for yeast. Specifically, the NaCl concentration used (0.5 M) is comparable to seawater salinity (~0.6 M), and the applied heat stress (37 °C) represents a temperature yeast frequently encounters in natural environments. These choices ensure that the “common stressors” reflect ecologically relevant conditions. However, as the reviewer suggests, applying higher NaCl concentrations or more extreme temperatures would create conditions rarely experienced in nature and might elicit adaptive patterns similar to those observed under clotrimazole stress. We have added a paragraph in the Discussion addressing this possibility and acknowledging that stronger stress levels could alter the observed selective patterns.\nMajor concern 4: Another concern is that the MA lines used in this study reportedly carry around 900 mutations each which is substantially more than other yeast MA lines, which typically accumulate only a few mutations (see work by Hall lab). Nine hundred is a very high number, and it’s difficult to imagine that all of these mutations truly accumulated in the absence of any selection. It’s well established that lethal mutations do not accumulate in MA experiments. If the salt stress response regulon is essential, it likely would not be mutated, and as a result, you would not observe degradation of plasticity in response to salt. This doesn’t mean plasticity isn’t present—it may simply mean the relevant regulatory pathways were protected from mutation, especially if all media used contained some level of salt. In contrast, the drug condition may have activated a non-essential regulon, which could have been more susceptible to the accumulated mutations. In this way, the experiment may be unintentionally testing for essentiality rather than plasticity.\nResponse: We appreciate the reviewer’s thoughtful comment and agree that the relatively high number of accumulated mutations in our MA lines warrants clarification.\nIn our study, each MA line carries approximately 900 mutations, which is indeed higher than the number typically reported in standard yeast MA experiments. This difference arises because our MA lines were generated in a mutator background (with MSH2 deleted) to accelerate mutation accumulation, followed by restoration of the wild-type MSH2 allele after the MA phase. This approach was adopted deliberately to ensure that sufficient genetic variation accumulated across the genome, thereby increasing the likelihood that mutations would occur in genes or regulatory elements influencing gene expression plasticity. As the reviewer notes, if the MA lines contained too few mutations, the genetic networks underlying plasticity might remain largely untouched, making it difficult to detect any signal of selection. The following paragraph has been added to the Discussion to address the potential influence of MSH2 deletion on the results of our experiment:\n“In our MA experiment, we used an MSH2 deletion strain to increase the mutation rate, which also alters the mutational spectrum by elevating the frequency of base substitutions and small insertion–deletion events, particularly in repetitive regions. While this spectrum differs somewhat from that of natural yeast populations, the primary purpose of MA experiments is to allow random mutations to accumulate under minimal selection, thereby gradually eroding the genome. In this sense, even though the precise mutation spectrum may differ, it serves the same functional role as naturally occurring mutations in revealing how random genetic changes affect gene expression and plasticity. Because our analyses focus on the selective patterns acting on these phenotypic outcomes rather than the specific mutation mechanisms, the conclusions regarding the selective maintenance or neutrality of expression plasticity should remain valid.”\nWe fully agree that lethal mutations cannot accumulate in MA experiments, as such mutations would prevent lineage propagation. However, non-lethal or mildly deleterious mutations can still occur within essential genes if they do not completely abolish gene function. These partial-loss-of-function mutations may still impair regulatory mechanisms related to plasticity without being lethal. To further address the reviewer’s concern that our observed patterns might reflect differences in essentiality rather than plasticity, we compared the proportions of essential genes among DEGs responding to NaCl, heat, and clotrimazole in the progenitor strain. We agree that essential stress-response regulons are less likely to accumulate disruptive mutations, and this may indeed explain the relative stability of NaCl/heat plasticity compared with clotrimazole. However, we found that the proportion of essential genes among clotrimazole-responsive DEGs was significantly higher than that in NaCl or heat responses (χ² test, P < 0.01). This result suggests that the antifungal stress response involves a larger number of essential or core cellular functions, rather than a more dispensable regulon. Therefore, our findings are unlikely to be explained solely by differences in essentiality between stress responses.\nMajor concern 5: The figures, as currently presented, are not effective in highlighting the key findings and in many cases feel redundant.\nFigure 1:\na-c) The expression level and plasticity plots do not clearly convey a central point, and this plotting style is not used in any subsequent figures, which makes it feel disconnected from the rest of the manuscript. The phylogenetic trees might be better placed in the supplement. Additionally, the natural strain names used here are inconsistent with those used elsewhere in the paper. If phylogenetic trees are included, the best-fit model and root should be reported.\nd) Consider combining these panels into a single plot for clarity.\ne) It’s curious that BY4741 in clo clusters with the natural isolates rather than the MA lines—this is unexpected and warrants an explanation.\nFigure 2:\nThe main point of this figure is unclear.\nFigure 3:\nPanels b and d appear to duplicate information shown in a and c. If so, they may not be necessary.\nGeneral:\nWhy are the natural isolates excluded from Figures 1, 2, and 3? Including them could help contextualize the results and support broader comparisons.\nSupplemental Figures:\nSupplemental Figure 4 appears to be identical to panels b–e from the main text, including the figure legend. If this is the case, it should either be removed or clearly justified.\nResponse: We thank the reviewer for the detailed and constructive feedback on the figures. We have carefully revised and reorganized the figures to improve clarity, consistency, and alignment with the main conclusions of the study. The major changes are summarized below.\nFigure 1:\nPanel (a) presents a schematic overview of the experimental design and analytical workflow, which helps readers quickly grasp the logic of the study. The phylogenetic trees (Panels b and c) remain included to give readers a direct visual impression of the genetic relationships among the MA lines and natural strains. We have added details on phylogenetic tree construction to the figure legend as follows: “Phylogenetic trees were reconstructed using the maximum likelihood method implemented in IQ-TREE (v3.0.1) based on all identified variant sites. The TVM substitution model was applied, as determined by model selection within IQ-TREE.”\nRegarding the reviewer’s point that BY4741 under clotrimazole clusters with natural strains rather than MA lines, we agree that this is an interesting and important pattern. In fact, this pattern is indeed consistent with our key conclusion: expression profiles under clotrimazole stress are adaptively maintained. The clustering indicates that the progenitor (BY4741) and natural strains share conserved adaptive transcriptional responses that were largely lost in the MA lines, reflecting the selective maintenance of plasticity in the antifungal environment. We have now explicitly discussed this interpretation in the revised manuscript (Discussion section, lines 364-368).\nFigure 2:\nThis figure has been clarified to emphasize its main purpose. Specifically, we added the formula that used to calculated overall plasticity (SumAcrossAllGenes(| Expression\nYPD\n- Expression |) and changed the x-axis labels to more specific meanings (percentage\nstress\nof genes with increased plasticity and percentage of MA lines with increased plasticity).\nThe figure shows that Clo/YPD plasticity is significantly lower in MA lines than in the progenitor, whereas NaCl/YPD and 37 °C/30 °C plasticities show no such difference.\nThis pattern suggests that Clo/YPD plasticity has been selectively maintained.\nFigure 3:\nWhile Panels (b) and (d) do share related information with (a) and (c), they serve distinct purposes: Panels (a) and (c) illustrate the numbers of up- and down-regulated SPGs under each condition, while Panels (b) and (d) show the distribution of SPG numbers among MA lines and the statistical test of significance relative to the progenitor. To make this distinction clearer, we have revised both the figure and legend accordingly. Natural strains have also been added to Figures 2 and 3 for better context.\nSupplementary Figure 4:\nWe clarified that Supplementary Figure 4 differs from the main text figures in that it uses an alternative normalization method to calculate the magnitude of plasticity. This methodological distinction is now clearly stated in both the main text and the figure legend to avoid confusion.\nMinor concern 1: The use of bulk RNAseq in this study feels somewhat outdated, especially given the availability of single-cell RNAseq methods. Bulk approaches average expression across thousands of cells, potentially masking meaningful cell-to-cell variation that could be critical for understanding plasticity. Gene expression plasticity may not be uniform across a population—some subpopulations may respond strongly while others do not, and this heterogeneity can be biologically important. Incorporating single-cell methods could provide deeper insight into the dynamics and distribution of plastic responses, particularly under stress conditions.\nResponse: We thank the reviewer for this insightful comment. We fully agree that single-cell RNAseq offers powerful opportunities to explore cell-to-cell variation in gene expression. However, our study focuses on population-level transcriptional plasticity and on comparisons among multiple strains and MA lines across several environments. In this context, bulk RNAseq provides an established and appropriate approach for quantifying average gene expression responses at the population level. We noted that many recent studies addressing gene expression plasticity and its evolution (PMID: 26331546 and PMID: 36702857) have successfully used bulk RNA-seq to characterize environmentally induced transcriptional changes. Following this well-established framework ensures comparability with previous work and allows robust inference of plasticity at the transcriptome scale.\nMinor concern 2: There were several typos throughout the manuscript. A thorough re-read and careful editing are strongly recommended. Additionally, the inclusion of line numbers would have made it easier to provide specific, targeted feedback.\nResponse: We have thoroughly proofread the manuscript and corrected typographical and grammatical errors throughout. In addition, line numbers have been added to the submitted manuscript.\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1:\nI appreciate the changes made by the authors to address the comments from all reviewers. The addition of the 37C environment strengthens the claim that specifically the Clotrimazole-induced plasticity is maintained by selection, whereas more common environments don't see the same pattern. It seems like a missed opportunity to have not also included a second antifungal drug when you collected the 37C results, as it would be nice to know how general that result is to Clotrimazole. But that's more of a nice-to-have than a need-to-have.\nI have one major and one minor comment about the revised manuscript:\nMajor: To address questions about the outlier MA lines, you repeated whole genome sequencing of the previously-published 21 MA lines. You found that 6/21 of the original MA lines did not have the expected mutations and concluded that these were likely contaminated with the common lab strain. Rather than simply omit these 6 lines from the paper, I think the sequencing data and subsequent exclusion should be mentioned in the manuscript when the MA lines are introduced. There should also be a supplementary figure or some text describing why you could not detect any mutations in these 6 lines and comparing them to the 15 lines they could detect mutations in. How many mutations did you find in each strain? still ~900? What software/methods did you use to verify these mutations? Comparing Fig 2 from the original manuscript to the revised Fig 2, it appears that the 6 strains that were removed were also the biggest outliers in the original manuscript and that omitting them made the results stronger. But, to be confident in this, it would be helpful to know that there was a clear difference in the whole genome sequencing results for these 6 strains versus the 15 that were kept. Currently, this sequencing and analysis of all strains are not mentioned in the material and methods.\nResponse: Thank you for raising this important point. To verify the identity of the MA lines, we re-sequenced all 21 lines used in our study. Six of these lines showed ≤3 mutations relative to the progenitor, whereas the remaining 15 lines carried an average of 668 mutations each. Of the 10,018 total mutations detected in the 15 verified lines, 96% matched those reported previously. These results clearly distinguish the 6 low-mutation lines from the rest, indicating that they were likely contaminated by the progenitor strain. Accordingly, we have excluded them from all analyses.\nWe have included a Supplementary Table detailing the mutation counts for all 21 lines and have revised the Methods section to provide a comprehensive description of the sequencing and variant-calling procedures. This includes the use of the Illumina HiSeq platform for sequencing, as well as the software tools bwa, samtools, picard, and GATK for variant calling. Furthermore, the introduction of the MA lines now briefly mentions the verification process and the exclusion of contaminated lines.\nSupplementary Table 1. Number of mutations identified in each MA line.\nStrain Mutation count Status\nMA14 653 Included\nMA22 797 Included\nMA25 937 Included\nMA29 770 Included\nMA33 620 Included\nMA47 814 Included\nMA54 134 Included\nMA63 943 Included\nMA66 624 Included\nMA79 120 Included\nMA82 770 Included\nMA90 645 Included\nMA93 821 Included\nMA95 624 Included\nMA98 746 Included\nMA21 0 Excluded\nMA28 0 Excluded\nMA45 0 Excluded\nMA51 3 Excluded\nMA69 1 Excluded\nMA97 0 Excluded\nMinor: I like the new histograms in Fig2 and 3 that include the natural strains and MA strains using two separate fill colors. However, the natural strain fill is a little faint and the MA strain fill is a bit too opaque to properly compare the two distributions. Making the natural strains stand out a bit more would help, just a darker grey should work (and a lighter purple for the MA strains).\nResponse: Following the reviewer’s advice, we changed the color of natural strains from light grey to dark grey and the color of MA lines from purple to a lighter purple. Please see the updated figures attached below.\nFigure 2.\nFigure 3.\nReviewer #2:\nI remain appreciative of this submission. The idea of using the directionality of changes to plastic responses during forced neutral evolution is original and fundamentally sound. I still I am still not convinced about some of the interpretations, as detailed below.\nThe main issue, to me, is that the authors have no unquestionable positive control to validate their method and ensure that it captures true instances of historical natural selection on phenotypic plasticity. To me, the ideal positive controls would have been the plasticity associated with the key life history transitions in yeast, from stationary phase to growth, the diauxic shift, sporulation, or germination – key aspects of these are certainly established by natural selection. The absence of an excellent positive control leaves the paper open to criticism.\nThat said, I think the authors have gone to substantial lengths to alleviate my concerns. Therefore, and given the refreshing originality of the idea, I would recommend publication given that the below is addressed.\nNecessary revisions:\n1. The growth curves supplied to ensure that there are no differences in physiological state between MA and progenitor must be on log-scale. Unless curves are on a log-scale, the existence and length of an exponential phase remain unclear. If MA lines e.g. in clotrimazole have begun experiencing energy restrictions at the time of sampling, while the progenitor has not, there will be a systematic difference in gene expression that could bias results and potentially generate the clotrimazole trends. The truly convincing way to exclude this would be to show the derivatives of growth curves with exact sampling times indicated. If MA and progenitor cells are not in the same phase, this needs to be highlighted and discussed as a caveat.\nResponse: Thank you for this valuable suggestion. Following your recommendation, we have added a new supplementary figure 9 showing the derivatives of the growth curves (dOD/dt) with sampling times indicated. To generate these, we subtracted the background OD (~0.1) from raw readings and calculated growth rates as time derivatives of OD based on 20-min intervals from the plate reader. The resulting dOD/dt values were plotted against OD, with the sampling window (OD = 0.4–0.6) highlighted in grey. Because growth follows a logistic trajectory, we applied quadratic smoothing to reduce noise in the derivative estimates. These analyses confirm that all samples were collected during the exponential phase and that MA lines and the progenitor were in comparable physiological states at the time of sampling.\nSupplementary Figure 9. Derivatives of the growth curves (dOD/dt) and sampling windows for MA lines, natural strains, and the progenitor in the four environments. a–d, Derivatives of the growth curves of MA lines and the progenitor; e–h, Derivatives of the growth curves of natural strains. The gray shaded areas indicate the sampling windows. To calculate the derivatives, the background OD (~0.1) was subtracted from the raw readings, and growth rates were computed as time derivatives of OD based on 20-minute intervals from the plate reader. Given the logistic nature of growth, quadratic smoothing was applied to reduce noise in the derivative estimates.\n2. I am not at all convinced about the genetic assimilation argument. The yeast literature on NaCl plastic responses is very rich, and gives us a very solid base for evaluating such claims. From this literature, it seems quite clear to me that S. cerevisiae is not innately salt tolerant and has not genetically assimilated an original plastic response that since then has become obsolete. In contrast, it seems quite clear that plastic inductions of ENA1 and/or GPD1 remain absolutely critical for negotiating a period of elevated NaCl exposure in extant yeasts. Non-redundant genes involved in the plastic induction of ENA1 and/or GPD1 are critical for salt tolerance, as are ENA1 and either of GPD1/GPD2. A few other regulations also appear somewhat important, e.g. of NHA1. So – the key aspects of the plastic response to salt have not been genetically assimilated. The authors could instead argue that ENA1 and GPD1 inductions represent a small fraction of the many gene expression changes that occur during salt exposure – and that the overall response, considering the total number of regulations, is neutral. To me, that would amount to saying that as long as ENA1 and GPD1 and maybe NHA1 is induced, it doesn’t matter much what other gene expression changes occur – and that selection hasn’t acted on optimizing the plastic responses to NaCl beyond ensuring that the key inductions are achieved and weeding out any too costly unnecessary inductions/repressions. From a simple counting perspective, the few beneficial changes would then drown in the hundreds of neutral expression variations. In fact, the lack of overall correlation between what genes are important and what genes are induced during NaCl exposure https://pubmed.ncbi.nlm.nih.gov/14676322/ could be interpreted in this way. A discussion along these lines might add nuance and depth to how we see gene expression plasticity, but the authors should refrain from giving the false impression that the entire salt response is neutral, or genetically assimilated. I am not sure whether the heat response is sufficiently well understood for the same reasoning to hold.\nResponse: We appreciate the reviewer’s insightful and detailed critique. We fully agree that S. cerevisiae’s salt tolerance depends critically on plastic induction of ENA1, GPD1/GPD2, and NHA1, and that these pathways have not been genetically assimilated. Our intention was not to imply that the entire NaCl response is genetically fixed. Rather, our data indicate that, at the transcriptome-wide level, gene expression plasticity under NaCl stress shows a random distribution across MA lines (consistent with neutrality in most genes) while specific components (such as ENA1 and GPD1) remain functionally indispensable and adaptively plastic.\nTo address, we have revised the manuscript to more clearly distinguish between the adaptive core and the broader neutral response. Specifically, we emphasize that the canonical osmoadaptive modules, including the HOG pathway, ENA1, and GPD1/GPD2, remain actively induced and are under strong selective constraint. The apparent neutrality we report pertains to the global pattern of transcriptional plasticity, where many expression changes appear to be selectively unconstrained or evolve in a neutral manner. We suggest that selection maintains only the essential subset of plastic responses required for salt tolerance, while many peripheral expression changes are effectively neutral or reflect relaxed optimization, as the reviewer points out.\nIn light of these revisions, we have added the following paragraph to the Discussion section:\nWhile our study primarily focused on the overall pattern of plastic expression, we acknowledge that certain genes may still exhibit adaptive plasticity under NaCl stress. For example, the core osmoadaptive modules, particularly the HOG pathway, ENA1, and GPD1/GPD2, remain essential and show strong, adaptive induction in response to NaCl stress (Gasch et al., 2000; Warringer et al., 2003). However, our data suggest that beyond this essential core, the broader pattern of transcriptome-wide plastic expression changes appears largely random across MA lines, which is consistent with neutral variation in many genes. This observation indicates that while selection continues to preserve the critical plastic responses needed for osmotic balance and ion homeostasis, many peripheral transcriptional responses may evolve under relaxed selection or drift. In this way, adaptive plasticity is maintained where it is functionally necessary, but the entire stress-responsive network is not globally optimized.\nMinor issues.\n1. It is not correct to say that YPD lacks Na+. There is no added NaCl, but the yeast extract and peptones do contain the ions, in varying amounts depending on batch. The batch variations is one reason to use SC instead of YPD.\nResponse: Thank you for your valuable feedback. We have reviewed the documentation for the chemicals, including peptone and yeast extract, used in the MA experiment. According to the Certificates of Analysis for these materials:\nhttps://documents.thermofisher.com/TFS-Assets/certificate/MN/COA/COA_LP0037B_11042025030433_6208751_1.pdf\nhttps://documents.thermofisher.com/TFS-Assets/BPD/certificate/Certificate-of-Analysis/212720%20-%203257238.pdf\nOn average, peptone and yeast extract contain about 0.3% sodium chloride, resulting in a final sodium chloride concentration of approximately 0.012% in YPD medium. In comparison, the sodium chloride concentration in 0.5 M NaCl is 2.92%, which is more than two hundred times higher. Given this substantial difference, we determined that the amount of sodium chloride from peptone and yeast extract would have a minimal impact on our experiment. However, we will remain mindful that YPD does contain trace amounts of ions, which could influence future experiments.\n2. The protocol for growth rate estimations appears not to be from ref 44 but from\nhttps://pubmed.ncbi.nlm.nih.gov/14676322/?\nResponse: Thank you for pointing this out. You are correct that our growth rate estimation protocol follows the method described by Warringer et al. (2003; PMID: 14676322). We have now cited both this paper and our previously listed reference (ref. 44) to accurately acknowledge the original source of the method.\nReviewer #3:\nIn this revised version, the authors have responded thoroughly and constructively to our earlier concerns. They substantially revised the Introduction to focus on the question their data directly address, namely whether gene expression plasticity is selectively maintained rather than its role in evolvability. They also added important ecological and historical context by explicitly distinguishing between common and novel stressors and by invoking genetic assimilation for long-standing stresses. The scope of the conclusions has been appropriately narrowed to clotrimazole and to general stress-response mechanisms, rather than to antibiotics or drugs broadly, and terminology has been corrected throughout. Several concerns about potential technical and experimental artefacts have been alleviated by the inclusion of new growth-curve data, additional heat-stress experiments, refined analyses of plasticity categories, and competitive fitness assays in clotrimazole and hydrogen peroxide that link loss of plasticity to reduced fitness in relevant environments. The authors have also clarified the consequences of using an MSH2 mutator background, addressed the issue of essentiality, and improved the clarity and organization of the figures, including better integration of the natural strains. While the interpretation of adaptive plasticity may still depend on perspective, the main conclusions are now well supported by the data, and we are satisfied that major concerns have been adequately addressed.\nResponse: We thank the reviewer for their positive and constructive feedback. We are pleased that the revisions have addressed the earlier concerns and that the main conclusions are now considered well supported."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the changes made by the authors to address the comments from all reviewers. The addition of the 37C environment strengthens the claim that specifically the Clotrimazole-induced plasticity is maintained by selection, whereas more common environments don't see the same pattern. It seems like a missed opportunity to have not also included a second antifungal drug when you collected the 37C results, as it would be nice to know how general that result is to Clotrimazole. But that's more of a nice-to-have than a need-to-have.\nI have one major and one minor comment about the revised manuscript:\nMajor: To address questions about the outlier MA lines, you repeated whole genome sequencing of the previously-published 21 MA lines. You found that 6/21 of the original MA lines did not have the expected mutations and concluded that these were likely contaminated with the common lab strain. Rather than simply omit these 6 lines from the paper, I think the sequencing data and subsequent exclusion should be mentioned in the manuscript when the MA lines are introduced. There should also be a supplementary figure or some text describing why you could not detect any mutations in these 6 lines and comparing them to the 15 lines they could detect mutations in. How many mutations did you find in each strain? still ~900? What software/methods did you use to verify these mutations? Comparing Fig 2 from the original manuscript to the revised Fig 2, it appears that the 6 strains that were removed were also the biggest outliers in the original manuscript and that omitting them made the results stronger. But, to be confident in this, it would be helpful to know that there was a clear difference in the whole genome sequencing results for these 6 strains versus the 15 that were kept. Currently, this sequencing and analysis of all strains are not mentioned in the material and methods.\nMinor: I like the new histograms in Fig2 and 3 that include the natural strains and MA strains using two separate fill colors. However, the natural strain fill is a little faint and the MA strain fill is a bit too opaque to properly compare the two distributions. Making the natural strains stand out a bit more would help, just a darker grey should work (and a lighter purple for the MA strains).", "reviewer_2": "I remain appreciative of this submission. The idea of using the directionality of changes to plastic responses during forced neutral evolution is original and fundamentally sound. I still I am still not convinced about some of the interpretations, as detailed below.\nThe main issue, to me, is that the authors have no unquestionable positive control to validate their method and ensure that it captures true instances of historical natural selection on phenotypic plasticity. To me, the ideal positive controls would have been the plasticity associated with the key life history transitions in yeast, from stationary phase to growth, the diauxic shift, sporulation, or germination – key aspects of these are certainly established by natural selection. The absence of an excellent positive control leaves the paper open to criticism.\nThat said, I think the authors have gone to substantial lengths to alleviate my concerns. Therefore, and given the refreshing originality of the idea, I would recommend publication given that the below is addressed.\nNecessary revisions:\n1. The growth curves supplied to ensure that there are no differences in physiological state between MA and progenitor must be on log-scale. Unless curves are on a log-scale, the existence and length of an exponential phase remain unclear. If MA lines e.g. in clotrimazole have begun experiencing energy restrictions at the time of sampling, while the progenitor has not, there will be a systematic difference in gene expression that could bias results and potentially generate the clotrimazole trends. The truly convincing way to exclude this would be to show the derivatives of growth curves with exact sampling times indicated. If MA and progenitor cells are not in the same phase, this needs to be highlighted and discussed as a caveat.\n2. I am not at all convinced about the genetic assimilation argument. The yeast literature on NaCl plastic responses is very rich, and gives us a very solid base for evaluating such claims. From this literature, it seems quite clear to me that S. cerevisiae is not innately salt tolerant and has not genetically assimilated an original plastic response that since then has become obsolete. In contrast, it seems quite clear that plastic inductions of ENA1 and/or GPD1 remain absolutely critical for negotiating a period of elevated NaCl exposure in extant yeasts. Non-redundant genes involved in the plastic induction of ENA1 and/or GPD1 are critical for salt tolerance, as are ENA1 and either of GPD1/GPD2. A few other regulations also appear somewhat important, e.g. of NHA1. So – the key aspects of the plastic response to salt have not been genetically assimilated. The authors could instead argue that ENA1 and GPD1 inductions represent a small fraction of the many gene expression changes that occur during salt exposure – and that the overall response, considering the total number of regulations, is neutral. To me, that would amount to saying that as long as ENA1 and GPD1 and maybe NHA1 is induced, it doesn’t matter much what other gene expression changes occur – and that selection hasn’t acted on optimizing the plastic responses to NaCl beyond ensuring that the key inductions are achieved and weeding out any too costly unnecessary inductions/repressions. From a simple counting perspective, the few beneficial changes would then drown in the hundreds of neutral expression variations. In fact, the lack of overall correlation between what genes are important and what genes are induced during NaCl exposure https://pubmed.ncbi.nlm.nih.gov/14676322/ could be interpreted in this way. A discussion along these lines might add nuance and depth to how we see gene expression plasticity, but the authors should refrain from giving the false impression that the entire salt response is neutral, or genetically assimilated. I am not sure whether the heat response is sufficiently well understood for the same reasoning to hold.\nMinor issues.\n1. It is not correct to say that YPD lacks Na+. There is no added NaCl, but the yeast extract and peptones do contain the ions, in varying amounts depending on batch. The batch variations is one reason to use SC instead of YPD.\n2. The protocol for growth rate estimations appears not to be from ref 44 but from\nhttps://pubmed.ncbi.nlm.nih.gov/14676322/?", "reviewer_3": "In this revised version, the authors have responded thoroughly and constructively to our earlier concerns. They substantially revised the Introduction to focus on the question their data directly address, namely whether gene expression plasticity is selectively maintained rather than its role in evolvability. They also added important ecological and historical context by explicitly distinguishing between common and novel stressors and by invoking genetic assimilation for long-standing stresses. The scope of the conclusions has been appropriately narrowed to clotrimazole and to general stress-response mechanisms, rather than to antibiotics or drugs broadly, and terminology has been corrected throughout.\nSeveral concerns about potential technical and experimental artefacts have been alleviated by the inclusion of new growth-curve data, additional heat-stress experiments, refined analyses of plasticity categories, and competitive fitness assays in clotrimazole and hydrogen peroxide that link loss of plasticity to reduced fitness in relevant environments. The authors have also clarified the consequences of using an MSH2 mutator background, addressed the issue of essentiality, and improved the clarity and organization of the figures, including better integration of the natural strains. While the interpretation of adaptive plasticity may still depend on perspective, the main conclusions are now well supported by the data, and we are satisfied that major concerns have been adequately addressed.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the three anonymous reviewers for their thoughtful and constructive feedback. In response, we have conducted additional experiments and analyses to address their concerns. The new results are consistent with our previous findings and further strengthen the conclusions of the manuscript. Detailed point-by-point responses to each comment are provided below, with revisions highlighted in blue for clarity.\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nThis paper explores whether gene expression plasticity, the ability of genes to change expression level in response to different environments, is a trait that is selected for or an inherent property of molecular networks. The authors address this question by comparing the transcriptome response to two different environmental stressors: osmotic stress and “antibiotic” stress between 22 natural isolates of Saccharomyces cerevisiae yeast to 21 mutation accumulation (MA) lines and their progenitor. [Note: “antibiotic” should be changed to “antifungal” throughout the paper given that antibiotics specifically refer to drugs impacting growth of bacteria.] The authors reason that the MA lines capture the effects of genetic drift in the absence of selection. The authors find that gene expression plasticity is reduced in the MA lines as compared to the progenitor in response to antifungal stress. In osmotic stress, in contrast, fewer genes exhibit expression plasticity in the progenitor, and the MA lines do not show a significant reduction in gene expression plasticity. Furthermore, the direction of gene expression plasticity (increasing or decreasing) is more likely to match the progenitor’s direction in the natural strains in both stress environments and the MA lines in osmotic stress. In the MA lines experiencing antifungal stress, in contrast, the direction of gene expression plasticity is near 50% matching the progenitor’s direction and 50% opposing the progenitor’s direction. Finally, the authors examine Gene Ontology enrichment among genes with expression plasticity. In antifungal stress, they find significant enrichment of categories with known relation to clotrimazole exposure in the progenitor and the natural strains but not in the MA lines. In osmotic stress, in contrast, the authors state that the GO categories they found enrichment for have “no direct relevance to osmotic stress”. The authors conclude there is evidence that gene expression plasticity is being selectively maintained in response to antifungal stress but not osmotic stress. This paper is clearly written and the findings are for the most part presented in an elegant and interpretable manner. The author’s findings represent a significant advancement in our understanding of how gene expression plasticity evolves. We have a few minor suggestions organized into 3 primary categories plus some general line edits:\nResponse: We sincerely thank the reviewer for the thoughtful and constructive evaluation of our manuscript. We appreciate the clear summary of our study’s objectives and findings, and we are pleased that the reviewer found the paper clearly written and the results meaningful in advancing understanding of how gene expression plasticity evolves.\nWe have carefully addressed all of the reviewer’s specific comments and suggestions.\nBelow we provide detailed, point-by-point responses. As recommended, we have replaced the term “antibiotic” with “antifungal” throughout the manuscript.\n1. Clarify figures\n1.a. In Fig 2 a) and b), the authors use a metric called “overall expression plasticity”, which is the sum of expression differences of all genes. A large value of “overall expression plasticity” could therefore be driven by a single, highly plastic gene. While the authors clarify in later plots that this is not the case, it would still be helpful for readers to understand what “overall expression plasticity” is measuring by providing an equation or visual representation alongside Fig2 a) and b).\nSomething as simple as SumAcrossGenes( |expression_O - expression_P| ). As Supplementary Fig3 also has a metric called “overall expression plasticity”, but it is calculated differently, it would additionally help to include a similar equation showing how this version is normalized by mean expression level.\nResponse: Following the recommendation, we have clarified the definition of “overall expression plasticity” in both the main text and the corresponding figure legends. Specifically, we now include explicit equations to illustrate how this metric is calculated in each case. In Figure 2, “overall expression plasticity” is defined as:\nSumAcrossAllGenes(| Expression - Expression |)\nYPD stress\nand in Supplementary Figure 3 is defined as:\nSumAcrossAllGenes(| Expression - Expression |)/(Expression + Expression )\nYPD stress YPD stress\n1.b. In Fig 2 c)-f), the authors use the “Percentage” to mean two different things. The “Percentage” in c) and d) is “Percent of genes with increased plasticity”, n = total number of samples. In e) and f), the “Percentage” is “Percent of MA line samples where a certain gene has higher expression plasticity than the progenitor”, n = total number of genes. While fully explaining the difference between these two percentages may not fit under an x-axis, having some visual representation next to the plot would make it clear to readers that the percentages are different.\nResponse: We have now changed the corresponding x-axis labels to “% of genes with increased plasticity” and “% of MA lines with increased plasticity”, respectively. The same changes have also been applied to Supplementary Figure 3.\n1.c. Fig 1a: It is unclear what the second scatter plot with black and grey points is representing, possibly natural strains vs MA lines?\nResponse: The black and grey dots in the scatter plot indeed represent natural strains and MA lines, respectively. To clarify this, we have updated the figure legend with the following sentence:\n“The four colors represent four different environments and the black and grey dots in the scatter plot correspond to natural strains and MA lines.”\n2.a. The information about which MA lines were selected and why is not presented until the end of the manuscript, in lines 326-332. This information should be shared with the reader much closer to the start of the MS when the experiential design is introduced. Justification of which MA lines were selected for this analysis is critical to evaluating and interpreting the work.\nResponse: In the revised manuscript, we conducted an additional experiment to measure gene expression under a new condition, heat stress at 37 °C. With this addition, the study now includes three environmental stressors, which we categorized into two groups: common stresses (NaCl and heat) that yeast is likely to have encountered throughout their evolutionary history, and a novel stress (the antifungal drug clotrimazole), which represents a recently introduced, synthetic challenge. To ensure meaningful comparisons between common and novel stresses, we selected MA lines that were able to maintain stable growth under clotrimazole and at least one of the common stress conditions. This clarification has been added to the beginning of the Results section, where the experimental design is first introduced.\n2.b. Lines 177-178: The authors identify genes with significant plasticity (SPGs) using DESeq2, but they methods and tests used to identify these genes are only described in the Methods. Please also provide a description in the main text making it clear how p-values were calculated and assessed for significance using DESeq2.\nResponse: We have now mentioned in the main text that the SPGs were identified using DESeq2 with FDR < 0.05 and |log FoldChange| > 1.\n2\n2.c. At the beginning of results section 3, the authors transition from using an absolute difference in expression to measure plasticity to a DESeq2 test to identify significant plasticity genes (SPGs). The authors should more clearly explain and justify the use of the two different metrics at different points in the paper.\nResponse: The reviewer is correct that we employed two metrics to quantify gene expression plasticity. At the beginning of the Results section, we used the absolute difference in expression between stressed and non-stressed conditions as a quantitative measure of plasticity magnitude at the whole-transcriptome level. This metric allowed us to compare the overall extent and direction of gene expression changes among natural strains, mutation accumulation (MA) lines, and the progenitor, independent of statistical significance. Subsequently, we employed DESeq2 differential expression analysis to identify significant plasticity genes (SPGs)—genes showing statistically supported expression changes between conditions. This step enabled us to focus on biologically meaningful and reproducible plastic responses, rather than stochastic fluctuations. Both approaches yielded consistent results, supporting the robustness and reliability of our findings.\nTo enhance clarity, the following sentence has been added to the beginning of Results section 3:\n“To confirm that the above findings were not driven by stochastic variation in gene expression, we repeated the analyses using only genes with significant plasticity (SPGs) between YPD and stressed environments (SPGs were identified using DESeq2 with FDR < 0.05 and |log FoldChange| > 1)”\n2\n3. Gene Ontology\n3.a. Line 257: The authors state that the Gene Ontology category they found enrichment in in their osmotic stress condition, amino acid synthesis, isn’t “directly relevant to osmotic stress”. This appears to be based on the author’s judgment and could be strengthened by including a clear statement of the author’s expectation. For example, other papers have looked at GO enrichment in response to osmotic stress (MacGillveray et al. 2018, as an example) that could be used to inform your expectations.\nResponse: We thank the reviewer for this helpful comment. We have now cited two studies (references 22 and 35) that characterized the osmotic stress response in yeast and identified the HOG pathway as the primary regulator of this process. However, genes associated with the HOG pathway were not significantly enriched in our GO analysis. This information is now provided in the main text.\n3.b. Line 236: The authors perform separate gene ontology analyses on genes that had an increasing versus decreasing plastic response. As activators and repressors of same biological process will change expression in opposite directions, the authors should better defend why they analyzed the gene sets separately rather than analyzing all SPGs together regardless of direction.\nResponse: We agree that separating upregulated and downregulated genes provides a more biologically meaningful interpretation, as these gene sets often participate in distinct functional processes. For this reason, we performed the GO enrichment analysis separately for upregulated and downregulated SPGs, allowing us to better capture the functional differences between genes activated and repressed under each stress condition. For clarity, the following sentence is added to the main text:\n“To test this, we compared the presence of progenitor-enriched biological processes between natural strains and MA lines, analyzing up-regulated and down-regulated genes separately. This distinction is important because activators and repressors involved in the same biological process often exhibit opposite patterns of expression change under stress.”\n4. Discussion Points\n4.a. How does use of the MSH2 mutation during mutational accumulation change the spectrum of mutations introduced? How might this affect the comparison to natural strains?\nResponse: We thank the reviewer for this important question. The following paragraph has been added to the Discussion to address the potential effects of MSH2 deletion on the mutational spectrum:\n“In our MA experiment, we used an MSH2 deletion strain to increase the mutation rate, which also alters the mutational spectrum by elevating the frequency of base substitutions and small insertion–deletion events, particularly in repetitive regions. While this spectrum differs somewhat from that of natural yeast populations, the primary purpose of MA experiments is to allow random mutations to accumulate under minimal selection, thereby gradually eroding the genome. In this sense, even though the precise mutation spectrum may differ, it serves the same functional role as naturally occurring mutations in revealing how random genetic changes affect gene expression and plasticity. Because our analyses focus on the selective patterns acting on these phenotypic outcomes rather than the specific mutation mechanisms, the conclusions regarding the selective maintenance or neutrality of expression plasticity should remain valid.”\n4.b. A core claim made in the paper is that it provides evidence for expression plasticity in response to Clotrimazole that has been maintained by natural selection. But there is no discussion of whether the natural strains used in this study have been exposed to Clotrimazole during their evolutionary history. Assuming this is not the case, do you think Clotrimazole exposure is a proxy for other conditions the strains are more likely to have experienced during their evolution. **This is a critical issue that must be addressed in a revised version.** The text on lines 291-292 seems to start to get at this point, but it should be expanded, more explicit, and apparent throughout the manuscript.\nResponse: We thank the reviewer for raising this critical issue. We have expanded on lines 291-292 (now lines 368-370) and incorporated the concept of the general stress response to explain the adaptive plasticity observed under Clotrimazole exposure.\nSpecifically, the following sentences have been added to the Discussion:\n“However, since Clotrimazole is a synthetic drug developed in the 1960s and has not been part of the long evolutionary history of budding yeasts, it raises the question of how yeast cells can still maintain adaptive expression plasticity when exposed to Clotrimazole. Our analysis of GO categories for genes induced by Clotrimazole, which are lost in the MA lines but preserved in the natural strains, identified two key categories: response to oxidative stress and response to chemical stimulus. These categories are integral to the yeast’s general stress response pathway and are often activated under a wide range of stressors (Gasch et al. 2000. Mol. Biol. Cell). These findings suggest that, even when exposed to novel stressors not previously encountered by yeast cells, they can still utilize these general stress response pathways to manage such challenges.”\n4.c. In figures 4c and 5, MA21 and MA97 don’t show the same pattern as the other MA lines. Rather, they appear to more closely resemble results in the natural strains. Is there a reason the authors think these strains are so different from the others? It would be helpful to readers for the authors to recognize these outliers and discuss possible reasons why these MA lines in particular show a different pattern.\nResponse: We thank the reviewer for this thoughtful comment. We also noticed that several MA lines exhibited expression patterns that differed from the others. To investigate the potential cause of these discrepancies, we performed whole-genome sequencing on all 21 MA lines and compared the results with the progenitor strain. Our analysis revealed that for 15 MA lines, we were able to accurately identify the mutations as reported in the original study that generated these lines. However, for six of the MA lines (21, 28, 45, 51, 69, and 97), we could not detect the expected mutations. In fact, compared to the progenitor strain, these lines showed very few mutations, suggesting possible sample contamination. As a result, we have removed these six MA lines from our analysis. Importantly, after their removal, the results and conclusions of our study remained unchanged.\n4.d. Duveau et al. 2017 PNAS also suggested that plasticity was adaptive in some environments but not others by comparing the effects of new mutations and polymorphisms from natural strains in S. cerevisiae in a particular gene’s promoter. While this paper and the authors’ paper look at this question from different scales (single gene vs whole genome), mentioning this paper in the discussion (perhaps at the end of the paragraph ending on line 306) would make the reader aware that there is additional support to the conclusions presented. [There might be other published work that would be good to cite here as well.]\nResponse: The following sentence has been added to the end of the paragraph on line 306 (now line 408):\n“This finding aligns with a previous study on the expression plasticity of the TDH3 gene in yeast, where the authors observed evidence of adaptive plasticity in some environments but not in others (Duveau et al. 2017 PNAS).”\n5. Line edits\n5.a. Line 71: “To address this” has an unclear subject. Please consider something like “To address the role of selection in plasticity evolution,”\nResponse: Corrected as suggested.\n5.b. Line 113: “each MA lines”, change to “each MA line”\nResponse: Corrected.\n5.c. Lines 130-131: “two major transcriptional corresponding to…” appears to be missing a word. Perhaps the authors meant something like “two major axes of transcriptional variation”\nResponse: The word “clusters” was missing. Now corrected as:\n“Dimensionality reduction analysis of the transcriptome profiles revealed two major transcriptional clusters corresponding to MA and natural strains, with further subgrouping based on environmental conditions.”\n5.d. Line 145: change “Gene expression levels was quantified” to “Gene expression levels were quantified” FPKM and explain to the reader why log2(FPKM+1) was used.\nResponse: “was” is corrected to “were” and the following sentence is added to explain why log2(FPKM+1) was used:\n“This transformation is commonly employed to stabilize variance and prevent issues associated with zero counts, ensuring more robust downstream analysis.”\n5.e. Line 148: “To access”, change to “to assess”\nResponse: Corrected.\n5.f. Line 159: There is something missing after >50%. Perhaps it should say “plasticity, while the remaining 13 had >50% of genes with increased plasticity, consistent with neutrality”?.\nResponse: The reviewer is correct that it should say “while the remaining 13 had >50% of genes with increased plasticity”.\n5g. Line 300: “fluctuation” implies expression plasticity is changing in one direction then changing back again over evolutionary time, but the MA lines are not sequential isolates, so the word fluctuation is misleading. “Random deviations from the progenitor” or something similar would be more appropriate.\nResponse: “random fluctuation” is now changed to “random deviations from the progenitor”.\n5.h. Reminder: throughout paper, replace “antibiotic” with “antifungal”, as Clotrimazole specifically targets fungi not bacteria.\nResponse: Corrected.\nReviewer #2 (Remarks to the Author):\nQian et al use a very interesting and largely novel approach to address whether phenotypes in extant lineages have been established by selection or neutral drift. They argue that if a phenotype has been established through a selective process, then evolved changes to that phenotype, under strictly neutral evolution, would tend to more often reverse than reinforce it. The logic is neat: random changes to an already optimized trait would be expected to more often impair than further improve it, whereas changes to a neutral trait would be random in direction.\nThey employ this logic to a yeast model strain, S388c, and test whether its capacity to respond to stressful environments, exemplified by exposure to moderate amounts of either NaCl or the antifungal clotrimazole, through changes to transcript abundance, is systematically lost or not when the strain is evolved neutrally.\nThey find that, somewhat surprisingly, there are no directional evolutionary changes to the transcriptional response to NaCl, implying that it is neutral, but that evolutionary changes to clotrimazole transcriptional plasticity are directional and tend to reverse it, implying that it has been selectively favoured.\nI very much appreciate the fundamental thinking in this paper, which I find refreshing, sound and employable on a broad scale, to a range of microbial phenotypes. As such, I think it has the potential to be well cited, and I can see contexts in which I would cite it. The data are mostly analysed in a correct and careful way, and the clotrimazole results seems clear.\nUnfortunately, I am not entirely convinced about the conclusions, because of various design issues, and would like to see additional follow-up analysis before I am ready to accept the claim of selection.\nMajor concerns:\n1. Looking for natural selection for clotrimazole plasticity in a S288c derivative doesn’t make much evolutionary sense. First, S288c is an artificial hybrid, whose genetic variants never jointly have been exposed to any selection outside the laboratory. S288c also tends to be a phenotypic outlier (see e.g.https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1002111). This can also be seen in Fig 1E for YPD and NaCl, which calls for caution in interpretations of YPD and NaCl responses.\nResponse: The reviewer is correct that S288c is a hybrid strain that created in the lab.\nHowever, its parental strains are all natural strains that have evolved under natural condition. As such, the alleles present in S288c have indeed undergone natural selection prior to the hybridization, which provides valuable evolutionary context.\nThe fact that S288c may appear as a genotypic outlier compared to other strains is likely a result of its frequent cultivation in laboratory environments like YPD and SC, which could have led to it acquiring higher fitness under these specific conditions. Finally, we selected the BY4741 background because it is one of the most widely used model strains in yeast genetics and genomics, with extensive genomic, transcriptomic, and functional resources available. This choice ensures experimental reproducibility and facilitates comparisons with prior studies investigating gene expression plasticity and mutation accumulation in yeast.\nSecond, clotrimazole, as other azoles of its class, is a synthetic compound. Although some azoles have very recently begun to be used as fungicides in agriculture, this short period could hardly have selected for an optimal clotrimazole transcriptional response even in natural yeast. With both these points in mind, I find it hard to understand how natural selection could have worked to achieve optimal S288c transcriptional responses to clotrimazole. This would presumably require many genetic variants being driven to high frequencies - and their beneficial combinations not having been disrupted by the process creating S288. It would have been far easier to argue for natural selection for plasticity under NaCl exposure (see below). And ideally, experiments would have been performed with a wild yeast strain and exposure omnipresent natural selection pressures, such as nitrogen limitation, a respiratory carbon source, or temperature. As is, I am left with strong doubts. The reduced clotrimazole expression plasticity in MA lines seems robust. But, either this plasticity was established by selection pressures other than azoles. Or, they are not reflective of selection at all. This needs to be resolved and discussed. Could the plastic changes observed simply be a consequence of MA strains growing at a different rate? Or - of MA strains not being in the same mid-log phase when harvested as the progenitor, but one of the two approaching the end of log-phase growth which has entirely different expression patterns? If cells are not in comparable physiological states, we would be looking at a technical artefact, not selection? At a minimum, we should be shown log-scale growth curves with points of harvest indicated, ideally with 1st derivatives included, to demonstrate that cells are in comparable, mid-log states. A comparison of observed plasticities to that of previous studies looking at how BY transcript abundance changes as a function of growth phase could also help reject the explanation of cultures being in different growth states. If technical explanations to the observed difference can be rejected, I would be more ready to accept the claim of selection – but what would be the selection pressure, given that it is highly unlikely to have been azoles? Are we looking at a general stress response? The yeast “general stress response” is typically defined by distinct transcription changes in the initial shock phase after shift to a new environment, but there may be remnants of that also in the subsequent growth phase. Are we looking at a response to reduced/increased membrane stability/permeability? What would the driving selection pressure have been? There are extensive datasets on yeast transcriptional responses, and a very rich yeast literature, that could be used to illuminate this. At this stage, given that the authors have just one positive signal for an environment of dubious selective importance, and a negative signal for the environment that I would readily accept having been of selective importance, and this using a strain never exposed to natural selection, I feel the obligation to be cautious.\nResponse: We sincerely thank the reviewer for this very thoughtful and comprehensive comment. We have carefully considered each point and made several additions and clarifications in response.\n1. Growth phase\nWe fully agree that differences in growth state could confound transcriptomic comparisons. To address this, we have now included a new Supplementary Figure 8 showing the complete growth curves for all strains under each experimental condition. As shown below, both the natural strains and MA lines were harvested at comparable mid-logarithmic growth phases (OD ≈ 0.4–0.6 after subtracting media OD), well\n660\nbefore the onset of stationary phase. Therefore, the transcriptional differences observed between conditions are unlikely to result from differences in growth phase..\nSupplementary Figure 8. Growth curves and sampling windows for MA lines, natural strains, and the progenitor in the four environments. a–d, Growth curves of MA lines and the progenitor; e–h, growth curves of natural strains. The gray shaded areas indicate the sampling windows.\n2. Evolutionary relevance of clotrimazole stress\nWe agree with the reviewer that clotrimazole, as a synthetic azole developed in the 1960s, does not represent a stressor that yeast would have encountered in its evolutionary history. We therefore consider clotrimazole to be a novel stressor, providing an opportunity to examine how yeast respond transcriptionally to completely unfamiliar chemical challenges.\nTo explore why yeast can still mount an apparently adaptive transcriptional response to clotrimazole, we analyzed DEGs between YPD and clotrimazole conditions. Among the genes whose expression was induced under clotrimazole, Gene Ontology (GO) enrichment analysis revealed significant overrepresentation of categories such as response to oxidative stress and response to chemical stimulus (Fisher’s exact test, P < 0.05 after BH correction). These categories were consistently enriched in natural strains but largely absent in MA lines. Previous studies have shown that oxidative stress response is broadly activated when yeast encounter diverse environmental challenges, while response to chemical stimulus encompasses reactions to a wide range of toxic or xenobiotic compounds, not limited to azoles.\nThese results strongly suggest that the transcriptional plasticity observed under clotrimazole treatment reflects activation of the general stress response rather than a clotrimazole-specific adaptation. Thus, although yeast have never experienced clotrimazole during their evolutionary history, they can still employ general stress-response pathways to mitigate the effects of such novel stressors. We have clarified this interpretation in the revised Discussion section, emphasizing that the observed adaptive plasticity likely stems from selection for general stress responsiveness.\n3. Neutral plasticity under NaCl\nTo test whether the neutral pattern observed under NaCl stress was specific to that condition, we performed an additional experiment exposing yeast to heat stress (37 °C), a ubiquitous environmental challenge. The results were consistent with those under NaCl stress, showing a neutral pattern of expression plasticity.\nWe have added a new paragraph in the Discussion introducing the concept of genetic assimilation to explain this pattern. Genetic assimilation describes the evolutionary process by which an environmentally induced phenotype becomes genetically fixed, leading to a loss of plasticity over time. Under this model, stressors that are frequently encountered, such as NaCl or temperature fluctuations, are more likely to drive fixation of adaptive expression patterns, resulting in the neutral plasticity observed in our study.\nIn contrast, novel stressors like clotrimazole can still reveal adaptive plasticity, as their responses remain under active selection through general stress pathways.\nFinally, we note that our analysis focuses on genome-wide patterns of plasticity; we cannot exclude the possibility that the plastic expression of specific genes under NaCl or heat stress remains adaptive. This clarification has been added to the Discussion section.\n2. If the observed plasticities lost in MA lines are indeed beneficial in the progenitor - then fitness should be reduced in the neutrally evolved MA lines, relative the progenitor, in the relevant environments? In the absence of such a fitness loss, the arguments for selection appear circumstantial to me. Relevant environments would seem to be clotrimazole, even though it doesn’t make much evolutionary sense, or other commonly encountered environments that could induce very similar plastic changes. For example, the authors argue for the oxidative stress response component of the observed plasticity being selectively important. This could be tested, e.g. using hydrogenperoxide or paraquat. In the absence of data demonstrating that the loss of plasticity is accompanied by a loss of fitness, I find it hard to accept the selection argument. A competition experiment that competes several evolved MA lines individually against the progenitor in clotrimazole and e.g. paraquat, and that estimates the selection coefficient from frequency change, would at least associate plasticity loss to fitness loss, and growth data could potentially be used as well (see below). The really convincing experiment would be to identify and then engineer the causative plasticity loss mutations, only, in a WT and compete that against the WT to establish true causality between plasticity loss and fitness loss. As is, the inference of selection from MA line transcriptional change is too circumstantial for me, and would need confirmation by supporting data.\nResponse: We agree that demonstrating an association between the loss of expression plasticity and a corresponding fitness reduction would provide stronger support for selection acting on plasticity.\nFollowing the reviewer’s recommendation, we conducted additional experiments to test whether the loss of plasticity observed in the MA lines is linked to decreased fitness in relevant environments. Specifically, because our results indicate that the oxidative stress response is a major component of the gene expression plasticity induced by clotrimazole, we reasoned that if this pathway is compromised by mutations accumulated during the MA process, then MA lines with lower fitness under clotrimazole should also show reduced fitness under oxidative stress.\nTo test this hypothesis, we measured the competitive fitness of all 15 MA lines in YPD media containing 1 μM clotrimazole and YPD media containing 0.1 mM hydrogen peroxide (H₂O₂). The results show a significant positive correlation between the fitness reductions (relative to YPD) across the two conditions (Pearson’s r = 0.62, P = 0.014; see new Supplementary Figure 5). This relationship suggests that the same biological mechanisms underlie the reduced fitness of MA lines in both environments.\nSupplementary Figure 5. The correlation of fitness reduction of MA lines in hydrogen peroxide and clotrimazole. Fitness was measured by competitive growth assay. Each gray point represents a MA line. The black line shows the fitted linear regression, with the shaded area indicating the 95% confidence interval.\n3. Growth data, such as reported in Fig 1D, could convince us that MA lines are less fit, in e.g. clotrimazole, than the BY progenitor – but this does not appear to be the case? However, we should be shown growth curves for the BY and the MA lines. It may be that a fitness defect is reflected in growth properties other than growth rates? I also have question marks around how the growth was estimated. Optical density and cell density are not linearly related, something that is normally accounted for either by dilution down to the near-linear range, or a post-experimental mathematical correction for the non-linearity (see e.g. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-1134-2). Unless this was done, the estimated growth will be incorrect, which as outlined in point 1 and 2 could have serious implications. Fig 1D gives some cause for concern in that growth has been correctly analyzed. If what is shown here is the maximum specific growth rate, umax - and even more so if it is the average growth rate across the entire growth interval - values are unrealistically high. A umax of 0.35-0.45 in YPD is standard, a value of 0.8, as shown, would imply doubling times well below an hour, which is outside the realistic range.\nResponse: We thank the reviewer for these detailed and thoughtful comments regarding the growth-rate analysis and fitness interpretation. We confirm that, as shown in Figure 1D, the MA lines indeed exhibit lower fitness than the progenitor strain in the clotrimazole environment. We agree that growth rate is only one aspect of fitness and that other growth properties such as lag time, maximum growth rate during the logarithmic phase, and final yield can all contribute to overall fitness. In this study, we used maximum growth rate as a proxy for fitness, as it provides a robust and comparable measure of physiological performance across strains and conditions.\nWe agree that optical density (OD) and cell density are not linearly correlated. To address this issue, we established an OD–cell density calibration curve based on manual cell counts and applied this calibration to convert OD measurements to actual cell densities prior to growth-rate estimation. This step minimizes potential non-linearity artifacts.\nFinally, we confirm that the reported growth-rate values represent maximum specific growth rates (μ ). To measure growth rate, OD was recorded every 20 minutes and\nmax\nconverted to cell density using the calibration curve. The slopes of ln (cell density) over time were computed for all overlapping 60-minute windows. To reduce the influence of transient fluctuations, the two highest slopes were discarded, and the mean of the third to eighth highest slopes was used to represent μ . We note that this value is\nmax\nexpressed per minute, and therefore a μ of 0.8 corresponds to approximately 0.8 ×\nmax\n60 = 48% increase per hour, which falls within the biologically reasonable range for yeast growing in rich medium. This clarification has been added to the revised Methods section.\n4. In paragraph 4, I feel that the division of evolved changes to plasticity into co-directional and reversed gives a too low degree of resolution. “Repressions turning into inductions” are in this classification grouped together with “inductions turning into repressions”, which does not make much biological sense. Moreover, potential trends of inductions and repressions being reduced - but not changing sign - which to me would be signs of selection and their absence of would be signs of neutrality - are overlooked. Increasing the resolution would also reveal to what degree selection has acted on clotrimazole inductions and repressions respectively.\nResponse: Following the reviewer’s recommendation, we have now refined our analysis by dividing the evolved plasticity changes into six categories. Specifically, we first separated genes based on whether their ancestral plasticity in the progenitor represented an induction or a repression under stress. Within each of these two major categories, we further classified the changes observed in the MA lines into three subtypes: strengthened, weakened, and reversed. Here, “strengthened” indicates that the MA line exhibited the same directional response as the progenitor but with a greater magnitude (representing potential non-adaptive plasticity), while “weakened” and “reversed” indicate a loss or reversal of the ancestral response (representing adaptive maintenance of plasticity in the progenitor).\nOur results show that for ancestral inductions, expression plasticity in MA lines under clotrimazole exhibited a significantly lower proportion of strengthened changes but significantly higher proportions of weakened and reversed changes compared to the other two stress conditions (Figure below and new Supplementary Figure 3). This pattern suggests that the expression plasticity in clotrimazole is under stronger selective maintenance than plasticity in NaCl or heat stress. A consistent, though less pronounced, trend was observed for ancestral repressions, where clotrimazole again showed a lower proportion of strengthened response and higher proportions of weakened and reversed responses.\nSupplementary Figure 3. The proportions of strengthened, weakened, and reversed plasticity for ancestral inductions and suppressions in the MA lines. a, Classification of expression plasticity based on progenitor plasticity (ancestral inductions or suppressions) then classified to strengthened, weakened, and reversed. On the x-axis, ‘O’ represents the expression level in the original environment and ‘P’ represents the expression level in the new environment. Black dots represent the progenitor and orange dots represent MA lines. Pink, purple, and green denote strengthened, weakened, and reversed plasticity, respectively. b, The proportions of strengthened, weakened, and reversed plasticity for ancestral inductions in MA lines across three environments. c, The proportions of strengthened, weakened, and reversed plasticity for ancestral suppressions in MA lines across three environments. In panels b and c, Wilcoxon-test is performed between every two bins with ‘NS.’ represents not significant, ‘*’ represents P < 0.05, and ‘***’ represents P < 0.001.\n5. I struggle to accept that gene expression changes induced by NaCl would be selectively neutral, which is implied by the data and alluded to in the text. The NaCl transcriptional response in yeast is well studied, with HOG pathway dependent transcriptional inductions of glycerol production, e.g. via GPD1, and the transcriptional regulation of salt-extrusion via the ENA proteins, being widely accepted as beneficial, selectively favoured responses. A more detailed analysis of what aspects of the NaCl plasticity that are and are not reversed could shed light on what is going on, and form the basis of a more nuanced discussion. The absence of directional changes to NaCl plasticity is almost as surprising as the presence of directional changes to clotrimazole plasticity, and contributes to making it difficult to accept the stringency of the selection test used. Given that only two environments are included, this deserves to be more thoroughly analysed and discussed.\nResponse: We thank the reviewer for this insightful comment and fully understand the concern regarding the apparent neutrality of gene expression plasticity under NaCl stress.\nFirst, to determine whether the neutral pattern observed under NaCl stress is a unique case or represents a general trend for long-standing stressors, we performed an additional experiment examining expression plasticity under heat stress (37 °C), which is another ubiquitous environmental challenge in yeast’s evolutionary history. The results under heat stress closely mirrored those for NaCl. Plastic expression changes showed no consistent directionality across MA lines relative to the progenitor, suggesting that the neutral pattern is not specific to NaCl but may instead represent a general feature of common stressors that yeast have frequently encountered over evolutionary time.\nSecond, to explain why gene expression plasticity under such common stressors appears neutral at the genome-wide level, we introduced the concept of genetic assimilation in the revised Discussion section. Genetic assimilation, first described by Waddington, refers to the process by which an environmentally induced phenotype becomes genetically fixed, leading to a loss of plasticity over time. This evolutionary mechanism can explain why the initially plastic response has likely been genetically stabilized through recurrent exposure over millions of years.\nThe following paragraph has been added to the Discussion section to address this point in detail:\n“While this conclusion might initially seem counterintuitive, it may be explained by recurrent genetic assimilation. Genetic assimilation, first described by Waddington, occurs when environmentally induced phenotypes (i.e., phenotypic plasticity) become fixed in a population over a few generations, eliminating the need for plasticity18. Later, Lande developed a quantitative-genetic model that demonstrated how a transient increase in phenotypic plasticity, followed by genetic assimilation, can drive adaptation to a novel environment5. The strongest experimental evidence for genetic assimilation comes from a recent study on Silene uniflora36, which has repeatedly adapted to zinc-contaminated soils over the last 250 years. By sequencing the transcriptomes of both zinc-sensitive and zinc-tolerant lineages of Silene uniflora in both zinc-containing and zinc-free environments, researchers identified 183 genes whose plastic expression was fixed in the adapted lineages, providing direct evidence for genetic assimilation. In contrast to the recent 250-year exposure to zinc in Silene uniflora, osmotic and heat stressors in yeast have likely occurred intermittently for millions of years during their evolutionary history. Therefore, much of the adaptive ancestral plastic expression to these stressors may have already been fixed through genetic assimilation. This could explain the neutral pattern of plasticity we observed under NaCl and heat stress.”\nMinor issues:\n6. The title inappropriately alludes to antibiotics, and is an overreach. Azoles are not antibiotics but antifungals, and this should be corrected throughout the text, and it is an overreach to generalize to “the antibiotic environment” at large, from a single environment. Moreover, I am not sure the authors have shown the majority of individual plastic changes to be beneficial, even in clotrimazole, so predominant is probably not the right word.\nResponse: The title is now changed to “Contrasting selective signatures of gene expression plasticity in response to antifungal clotrimazole and common environmental stresses in yeast”.\n7. Some additional details on the conditions of the MA line evolution would be welcome. The statement that mutations accumulate primarily due to drift comes with an important caveat, as strongly negative mutations are selected against and these constitute a very substantial fraction of the total, in many DFE estimates. The MA conditions would dictate what these strongly negative variants are – for example, if MA conditions contains substantial amounts of Na+, e.g. through setting the pH with NaOH, this would probably influence the NaCl data.\nResponse: For clarity, the following information have been added to the main text:\n“These lines were established using BY4741 as the ancestral strain and underwent approximately 1500 generations of MA in the solid nutrient-rich (YPD) medium at 30 ℃, which lacked the stressors employed in the current study.”\n8. NaCl is not only on osmolyte acting externally, as implied in the text, but induces Na+ saline stress intracellularly, e.g. by distorting the Na+/K+ ratio.\nResponse: For clarity, the following sentence have been added to the main text:\n“NaCl induces osmotic stress and distorts intracellular Na+ concentration.”\nReviewer #3 (Remarks to the Author):\nThis is a review of “Gene expression plasticity in the antibiotic environment is predominantly adaptive in the budding yeast”. Qian et al explore the evolutionary dynamics of phenotypic plasticity in the form of gene expression. MA lines and natural lineages are used to determine if plasticity is adaptive or neutral. They test two stressful conditions - osmotic and antibiotic - and report that gene expression plasticity is adaptive in environments with antifungal but is neutral in osmotic conditions. This conclusion is based on the observation that MA lines, but not natural isolates, lose the ability to adjust their gene expression to environmental change. Thus, the authors conclude that natural selection prevents accumulation of mutations that destroy this ability, while MA experiments do not. We have several major concerns with the way the results are interpreted, and we do not feel that the broad and sweeping conclusions are justified by these data.\nMajor concern 1: While the experiments address an important question of interest to scientists in many fields, this is not the question discussed in the introduction. The focus of the paper as described in the introduction is the question of whether plasticity allows organisms to persist in a novel environment until a new genetic mutation arises that helps them in that environment. That is an important question, but these results do not pertain to that question. At no point do the authors look for novel genetic mutations occuring and being selected in the stressful environments. They seem to define “adaptive” in the introduction as allowing an organism to persist and survive until it gets a new mutation. In that sense, the authors have not shown plasticity is adaptive at all. This definition of adaptive relates to evolvability. Does plasticity increase evolvability? The experiments have nothing to do with this question and the introduction should be rewritten to focus on the narrower question below.\nResponse: We thank the reviewer for this comment. We agree that our experiments do not directly address whether plasticity facilitates persistence until beneficial mutations arise, which concerns evolvability. Instead, our study focuses on whether gene expression plasticity itself is adaptive, i.e., whether it is selectively maintained by natural selection. To avoid confusion, we have revised the Introduction to clarify this narrower focus.\nSpecifically, we modified the framing of the research question and added the following sentences to highlight that our study examines the adaptive maintenance of plasticity, not its role in promoting evolvability:\n“However, it remains uncertain whether gene expression plasticity is selectively maintained as an adaptive feature or simply represents a transient, non-adaptive response to environmental fluctuations.”\n“To address the role of natural selection in the evolution of plasticity under both common and novel stressors…”\nMajor concern 2: Even when using a more narrow definition of “adaptive”, we still are not sure the results justify the broad sweeping conclusions. When an organism can change its gene expression to match the environment, that likely suggests the organism has previously encountered that environment. That is why gene regulation evolved, to deal with environmental change. So of course, plasticity is adaptive. If plasticity is absent, and an organism cannot change its gene expression to survive an environment, it likely means it never encountered that environment. This ecological perspective, the idea that gene expression regulation reflects an organism's history, is entirely absent from the introduction.\nResponse: We appreciate the reviewer’s thoughtful comment and agree that incorporating an ecological and historical perspective on gene regulation is essential for contextualizing the adaptive significance of plasticity. We have revised the Introduction to explicitly discuss how the evolutionary history of environmental exposure shapes the adaptiveness of gene expression plasticity.\nSpecifically, we added the following new paragraph to the Introduction:\n“Moreover, the evolutionary outcomes of plasticity may vary depending on the type of environment encountered, particularly between conditions that have been common throughout an organism’s evolutionary history and those that are novel or rarely experienced. When a stressor has been repeatedly encountered over evolutionary time, such as high salinity (NaCl exposure) or heat stress, organisms are likely to evolve specific adaptive responses to mitigate its effects. Notwithstanding, if such stressors persist over long timescales, plastic responses may eventually become genetically assimilated, resulting in the fixation of adaptive traits and a consequent loss of plasticity5,6,18. In contrast, exposure to novel stressors that were absent from an organism’s evolutionary history poses different challenges. In these cases, organisms may lack specialized adaptive responses but might still cope with stress through mechanisms that overlap with responses to familiar stressors or through broad, general stress-response pathways19.”\nMajor concern 3: The conclusions appear to relate to all possible drugs and all possible salt environments, while only one of each was studied. Relatedly, the authors only look at two conditions and do not make clear why either was chosen and whether these environments are likely to have been encountered in that past history. The manuscript would benefit from the inclusion of additional environments, particularly more drug conditions. For example, do MA lines consistently show reduced phenotypic plasticity across a range of drug stresses? Without testing other drugs, it’s difficult to support the claim that gene expression plasticity in response to antifungal stress is broadly adaptive, as suggested in the discussion. The same goes for the identified pathways. Perhaps the conclusions could be reframed to emphasize their relevance to clotrimazole specifically. Additionally, the manuscript repeatedly refers to clotrimazole as an \"antibiotic,\" but it is an antifungal which needs to be corrected. Finally, do you think if you tested higher levels of osmotic stress that you would still see a neutral response? Can you please address this in the discussion? Again, I’m not sure these broad generalizations are appropriate based on the tested conditions.\nResponse: We thank the reviewer for this insightful and constructive comment. We agree that our original framing was too broad and have revised the manuscript accordingly to clarify the scope and interpretation of our findings.\nFirst, we have reframed the study design and discussion around the comparison between common and novel stressors rather than generalizing across all drugs or all stress conditions. Specifically, NaCl and heat (37 °C) are categorized as common stressors that yeast is likely to have encountered repeatedly throughout their evolutionary history, while clotrimazole is treated as a novel stressor because it is a synthetic antifungal compound developed in the 1960s. We have replaced all instances of “antibiotic” with “antifungal” throughout the manuscript.\nTo strengthen this comparison, we have now included heat stress as an additional common stressor to test whether the neutral pattern of plasticity under NaCl stress generalizes to another familiar condition. We found that both NaCl and heat stress yielded consistent, largely neutral patterns of expression plasticity, consistent with the hypothesis of genetic assimilation, that ancestral adaptive plasticity to long-standing stressors has been gradually fixed as a stable phenotype (as discussed in the revised Discussion section).\nIn contrast, expression plasticity under clotrimazole stress showed a clear adaptive pattern, with reduced plasticity and loss of stress-responsive pathways in MA lines relative to the progenitor and natural strains. We have revised the text to explicitly emphasize that this adaptive plasticity pertains specifically to clotrimazole rather than to antifungal or drug stresses in general. Our discussion now focuses on how yeast cells can utilize general stress-response pathways (e.g., oxidative stress response and chemical stimulus response) to cope with novel stressors not encountered in their evolutionary past.\nRegarding the environmental relevance of our experimental conditions, we have clarified the rationale for choosing stress intensities that approximate natural conditions for yeast. Specifically, the NaCl concentration used (0.5 M) is comparable to seawater salinity (~0.6 M), and the applied heat stress (37 °C) represents a temperature yeast frequently encounters in natural environments. These choices ensure that the “common stressors” reflect ecologically relevant conditions. However, as the reviewer suggests, applying higher NaCl concentrations or more extreme temperatures would create conditions rarely experienced in nature and might elicit adaptive patterns similar to those observed under clotrimazole stress. We have added a paragraph in the Discussion addressing this possibility and acknowledging that stronger stress levels could alter the observed selective patterns.\nMajor concern 4: Another concern is that the MA lines used in this study reportedly carry around 900 mutations each which is substantially more than other yeast MA lines, which typically accumulate only a few mutations (see work by Hall lab). Nine hundred is a very high number, and it’s difficult to imagine that all of these mutations truly accumulated in the absence of any selection. It’s well established that lethal mutations do not accumulate in MA experiments. If the salt stress response regulon is essential, it likely would not be mutated, and as a result, you would not observe degradation of plasticity in response to salt. This doesn’t mean plasticity isn’t present—it may simply mean the relevant regulatory pathways were protected from mutation, especially if all media used contained some level of salt. In contrast, the drug condition may have activated a non-essential regulon, which could have been more susceptible to the accumulated mutations. In this way, the experiment may be unintentionally testing for essentiality rather than plasticity.\nResponse: We appreciate the reviewer’s thoughtful comment and agree that the relatively high number of accumulated mutations in our MA lines warrants clarification.\nIn our study, each MA line carries approximately 900 mutations, which is indeed higher than the number typically reported in standard yeast MA experiments. This difference arises because our MA lines were generated in a mutator background (with MSH2 deleted) to accelerate mutation accumulation, followed by restoration of the wild-type MSH2 allele after the MA phase. This approach was adopted deliberately to ensure that sufficient genetic variation accumulated across the genome, thereby increasing the likelihood that mutations would occur in genes or regulatory elements influencing gene expression plasticity. As the reviewer notes, if the MA lines contained too few mutations, the genetic networks underlying plasticity might remain largely untouched, making it difficult to detect any signal of selection. The following paragraph has been added to the Discussion to address the potential influence of MSH2 deletion on the results of our experiment:\n“In our MA experiment, we used an MSH2 deletion strain to increase the mutation rate, which also alters the mutational spectrum by elevating the frequency of base substitutions and small insertion–deletion events, particularly in repetitive regions. While this spectrum differs somewhat from that of natural yeast populations, the primary purpose of MA experiments is to allow random mutations to accumulate under minimal selection, thereby gradually eroding the genome. In this sense, even though the precise mutation spectrum may differ, it serves the same functional role as naturally occurring mutations in revealing how random genetic changes affect gene expression and plasticity. Because our analyses focus on the selective patterns acting on these phenotypic outcomes rather than the specific mutation mechanisms, the conclusions regarding the selective maintenance or neutrality of expression plasticity should remain valid.”\nWe fully agree that lethal mutations cannot accumulate in MA experiments, as such mutations would prevent lineage propagation. However, non-lethal or mildly deleterious mutations can still occur within essential genes if they do not completely abolish gene function. These partial-loss-of-function mutations may still impair regulatory mechanisms related to plasticity without being lethal. To further address the reviewer’s concern that our observed patterns might reflect differences in essentiality rather than plasticity, we compared the proportions of essential genes among DEGs responding to NaCl, heat, and clotrimazole in the progenitor strain. We agree that essential stress-response regulons are less likely to accumulate disruptive mutations, and this may indeed explain the relative stability of NaCl/heat plasticity compared with clotrimazole. However, we found that the proportion of essential genes among clotrimazole-responsive DEGs was significantly higher than that in NaCl or heat responses (χ² test, P < 0.01). This result suggests that the antifungal stress response involves a larger number of essential or core cellular functions, rather than a more dispensable regulon. Therefore, our findings are unlikely to be explained solely by differences in essentiality between stress responses.\nMajor concern 5: The figures, as currently presented, are not effective in highlighting the key findings and in many cases feel redundant.\nFigure 1:\na-c) The expression level and plasticity plots do not clearly convey a central point, and this plotting style is not used in any subsequent figures, which makes it feel disconnected from the rest of the manuscript. The phylogenetic trees might be better placed in the supplement. Additionally, the natural strain names used here are inconsistent with those used elsewhere in the paper. If phylogenetic trees are included, the best-fit model and root should be reported.\nd) Consider combining these panels into a single plot for clarity.\ne) It’s curious that BY4741 in clo clusters with the natural isolates rather than the MA lines—this is unexpected and warrants an explanation.\nFigure 2:\nThe main point of this figure is unclear.\nFigure 3:\nPanels b and d appear to duplicate information shown in a and c. If so, they may not be necessary.\nGeneral:\nWhy are the natural isolates excluded from Figures 1, 2, and 3? Including them could help contextualize the results and support broader comparisons.\nSupplemental Figures:\nSupplemental Figure 4 appears to be identical to panels b–e from the main text, including the figure legend. If this is the case, it should either be removed or clearly justified.\nResponse: We thank the reviewer for the detailed and constructive feedback on the figures. We have carefully revised and reorganized the figures to improve clarity, consistency, and alignment with the main conclusions of the study. The major changes are summarized below.\nFigure 1:\nPanel (a) presents a schematic overview of the experimental design and analytical workflow, which helps readers quickly grasp the logic of the study. The phylogenetic trees (Panels b and c) remain included to give readers a direct visual impression of the genetic relationships among the MA lines and natural strains. We have added details on phylogenetic tree construction to the figure legend as follows: “Phylogenetic trees were reconstructed using the maximum likelihood method implemented in IQ-TREE (v3.0.1) based on all identified variant sites. The TVM substitution model was applied, as determined by model selection within IQ-TREE.”\nRegarding the reviewer’s point that BY4741 under clotrimazole clusters with natural strains rather than MA lines, we agree that this is an interesting and important pattern. In fact, this pattern is indeed consistent with our key conclusion: expression profiles under clotrimazole stress are adaptively maintained. The clustering indicates that the progenitor (BY4741) and natural strains share conserved adaptive transcriptional responses that were largely lost in the MA lines, reflecting the selective maintenance of plasticity in the antifungal environment. We have now explicitly discussed this interpretation in the revised manuscript (Discussion section, lines 364-368).\nFigure 2:\nThis figure has been clarified to emphasize its main purpose. Specifically, we added the formula that used to calculated overall plasticity (SumAcrossAllGenes(| Expression\nYPD\n- Expression |) and changed the x-axis labels to more specific meanings (percentage\nstress\nof genes with increased plasticity and percentage of MA lines with increased plasticity).\nThe figure shows that Clo/YPD plasticity is significantly lower in MA lines than in the progenitor, whereas NaCl/YPD and 37 °C/30 °C plasticities show no such difference.\nThis pattern suggests that Clo/YPD plasticity has been selectively maintained.\nFigure 3:\nWhile Panels (b) and (d) do share related information with (a) and (c), they serve distinct purposes: Panels (a) and (c) illustrate the numbers of up- and down-regulated SPGs under each condition, while Panels (b) and (d) show the distribution of SPG numbers among MA lines and the statistical test of significance relative to the progenitor. To make this distinction clearer, we have revised both the figure and legend accordingly. Natural strains have also been added to Figures 2 and 3 for better context.\nSupplementary Figure 4:\nWe clarified that Supplementary Figure 4 differs from the main text figures in that it uses an alternative normalization method to calculate the magnitude of plasticity. This methodological distinction is now clearly stated in both the main text and the figure legend to avoid confusion.\nMinor concern 1: The use of bulk RNAseq in this study feels somewhat outdated, especially given the availability of single-cell RNAseq methods. Bulk approaches average expression across thousands of cells, potentially masking meaningful cell-to-cell variation that could be critical for understanding plasticity. Gene expression plasticity may not be uniform across a population—some subpopulations may respond strongly while others do not, and this heterogeneity can be biologically important. Incorporating single-cell methods could provide deeper insight into the dynamics and distribution of plastic responses, particularly under stress conditions.\nResponse: We thank the reviewer for this insightful comment. We fully agree that single-cell RNAseq offers powerful opportunities to explore cell-to-cell variation in gene expression. However, our study focuses on population-level transcriptional plasticity and on comparisons among multiple strains and MA lines across several environments. In this context, bulk RNAseq provides an established and appropriate approach for quantifying average gene expression responses at the population level. We noted that many recent studies addressing gene expression plasticity and its evolution (PMID: 26331546 and PMID: 36702857) have successfully used bulk RNA-seq to characterize environmentally induced transcriptional changes. Following this well-established framework ensures comparability with previous work and allows robust inference of plasticity at the transcriptome scale.\nMinor concern 2: There were several typos throughout the manuscript. A thorough re-read and careful editing are strongly recommended. Additionally, the inclusion of line numbers would have made it easier to provide specific, targeted feedback.\nResponse: We have thoroughly proofread the manuscript and corrected typographical and grammatical errors throughout. In addition, line numbers have been added to the submitted manuscript.\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1:\nI appreciate the changes made by the authors to address the comments from all reviewers. The addition of the 37C environment strengthens the claim that specifically the Clotrimazole-induced plasticity is maintained by selection, whereas more common environments don't see the same pattern. It seems like a missed opportunity to have not also included a second antifungal drug when you collected the 37C results, as it would be nice to know how general that result is to Clotrimazole. But that's more of a nice-to-have than a need-to-have.\nI have one major and one minor comment about the revised manuscript:\nMajor: To address questions about the outlier MA lines, you repeated whole genome sequencing of the previously-published 21 MA lines. You found that 6/21 of the original MA lines did not have the expected mutations and concluded that these were likely contaminated with the common lab strain. Rather than simply omit these 6 lines from the paper, I think the sequencing data and subsequent exclusion should be mentioned in the manuscript when the MA lines are introduced. There should also be a supplementary figure or some text describing why you could not detect any mutations in these 6 lines and comparing them to the 15 lines they could detect mutations in. How many mutations did you find in each strain? still ~900? What software/methods did you use to verify these mutations? Comparing Fig 2 from the original manuscript to the revised Fig 2, it appears that the 6 strains that were removed were also the biggest outliers in the original manuscript and that omitting them made the results stronger. But, to be confident in this, it would be helpful to know that there was a clear difference in the whole genome sequencing results for these 6 strains versus the 15 that were kept. Currently, this sequencing and analysis of all strains are not mentioned in the material and methods.\nResponse: Thank you for raising this important point. To verify the identity of the MA lines, we re-sequenced all 21 lines used in our study. Six of these lines showed ≤3 mutations relative to the progenitor, whereas the remaining 15 lines carried an average of 668 mutations each. Of the 10,018 total mutations detected in the 15 verified lines, 96% matched those reported previously. These results clearly distinguish the 6 low-mutation lines from the rest, indicating that they were likely contaminated by the progenitor strain. Accordingly, we have excluded them from all analyses.\nWe have included a Supplementary Table detailing the mutation counts for all 21 lines and have revised the Methods section to provide a comprehensive description of the sequencing and variant-calling procedures. This includes the use of the Illumina HiSeq platform for sequencing, as well as the software tools bwa, samtools, picard, and GATK for variant calling. Furthermore, the introduction of the MA lines now briefly mentions the verification process and the exclusion of contaminated lines.\nSupplementary Table 1. Number of mutations identified in each MA line.\nStrain Mutation count Status\nMA14 653 Included\nMA22 797 Included\nMA25 937 Included\nMA29 770 Included\nMA33 620 Included\nMA47 814 Included\nMA54 134 Included\nMA63 943 Included\nMA66 624 Included\nMA79 120 Included\nMA82 770 Included\nMA90 645 Included\nMA93 821 Included\nMA95 624 Included\nMA98 746 Included\nMA21 0 Excluded\nMA28 0 Excluded\nMA45 0 Excluded\nMA51 3 Excluded\nMA69 1 Excluded\nMA97 0 Excluded\nMinor: I like the new histograms in Fig2 and 3 that include the natural strains and MA strains using two separate fill colors. However, the natural strain fill is a little faint and the MA strain fill is a bit too opaque to properly compare the two distributions. Making the natural strains stand out a bit more would help, just a darker grey should work (and a lighter purple for the MA strains).\nResponse: Following the reviewer’s advice, we changed the color of natural strains from light grey to dark grey and the color of MA lines from purple to a lighter purple. Please see the updated figures attached below.\nFigure 2.\nFigure 3.\nReviewer #2:\nI remain appreciative of this submission. The idea of using the directionality of changes to plastic responses during forced neutral evolution is original and fundamentally sound. I still I am still not convinced about some of the interpretations, as detailed below.\nThe main issue, to me, is that the authors have no unquestionable positive control to validate their method and ensure that it captures true instances of historical natural selection on phenotypic plasticity. To me, the ideal positive controls would have been the plasticity associated with the key life history transitions in yeast, from stationary phase to growth, the diauxic shift, sporulation, or germination – key aspects of these are certainly established by natural selection. The absence of an excellent positive control leaves the paper open to criticism.\nThat said, I think the authors have gone to substantial lengths to alleviate my concerns. Therefore, and given the refreshing originality of the idea, I would recommend publication given that the below is addressed.\nNecessary revisions:\n1. The growth curves supplied to ensure that there are no differences in physiological state between MA and progenitor must be on log-scale. Unless curves are on a log-scale, the existence and length of an exponential phase remain unclear. If MA lines e.g. in clotrimazole have begun experiencing energy restrictions at the time of sampling, while the progenitor has not, there will be a systematic difference in gene expression that could bias results and potentially generate the clotrimazole trends. The truly convincing way to exclude this would be to show the derivatives of growth curves with exact sampling times indicated. If MA and progenitor cells are not in the same phase, this needs to be highlighted and discussed as a caveat.\nResponse: Thank you for this valuable suggestion. Following your recommendation, we have added a new supplementary figure 9 showing the derivatives of the growth curves (dOD/dt) with sampling times indicated. To generate these, we subtracted the background OD (~0.1) from raw readings and calculated growth rates as time derivatives of OD based on 20-min intervals from the plate reader. The resulting dOD/dt values were plotted against OD, with the sampling window (OD = 0.4–0.6) highlighted in grey. Because growth follows a logistic trajectory, we applied quadratic smoothing to reduce noise in the derivative estimates. These analyses confirm that all samples were collected during the exponential phase and that MA lines and the progenitor were in comparable physiological states at the time of sampling.\nSupplementary Figure 9. Derivatives of the growth curves (dOD/dt) and sampling windows for MA lines, natural strains, and the progenitor in the four environments. a–d, Derivatives of the growth curves of MA lines and the progenitor; e–h, Derivatives of the growth curves of natural strains. The gray shaded areas indicate the sampling windows. To calculate the derivatives, the background OD (~0.1) was subtracted from the raw readings, and growth rates were computed as time derivatives of OD based on 20-minute intervals from the plate reader. Given the logistic nature of growth, quadratic smoothing was applied to reduce noise in the derivative estimates.\n2. I am not at all convinced about the genetic assimilation argument. The yeast literature on NaCl plastic responses is very rich, and gives us a very solid base for evaluating such claims. From this literature, it seems quite clear to me that S. cerevisiae is not innately salt tolerant and has not genetically assimilated an original plastic response that since then has become obsolete. In contrast, it seems quite clear that plastic inductions of ENA1 and/or GPD1 remain absolutely critical for negotiating a period of elevated NaCl exposure in extant yeasts. Non-redundant genes involved in the plastic induction of ENA1 and/or GPD1 are critical for salt tolerance, as are ENA1 and either of GPD1/GPD2. A few other regulations also appear somewhat important, e.g. of NHA1. So – the key aspects of the plastic response to salt have not been genetically assimilated. The authors could instead argue that ENA1 and GPD1 inductions represent a small fraction of the many gene expression changes that occur during salt exposure – and that the overall response, considering the total number of regulations, is neutral. To me, that would amount to saying that as long as ENA1 and GPD1 and maybe NHA1 is induced, it doesn’t matter much what other gene expression changes occur – and that selection hasn’t acted on optimizing the plastic responses to NaCl beyond ensuring that the key inductions are achieved and weeding out any too costly unnecessary inductions/repressions. From a simple counting perspective, the few beneficial changes would then drown in the hundreds of neutral expression variations. In fact, the lack of overall correlation between what genes are important and what genes are induced during NaCl exposure https://pubmed.ncbi.nlm.nih.gov/14676322/ could be interpreted in this way. A discussion along these lines might add nuance and depth to how we see gene expression plasticity, but the authors should refrain from giving the false impression that the entire salt response is neutral, or genetically assimilated. I am not sure whether the heat response is sufficiently well understood for the same reasoning to hold.\nResponse: We appreciate the reviewer’s insightful and detailed critique. We fully agree that S. cerevisiae’s salt tolerance depends critically on plastic induction of ENA1, GPD1/GPD2, and NHA1, and that these pathways have not been genetically assimilated. Our intention was not to imply that the entire NaCl response is genetically fixed. Rather, our data indicate that, at the transcriptome-wide level, gene expression plasticity under NaCl stress shows a random distribution across MA lines (consistent with neutrality in most genes) while specific components (such as ENA1 and GPD1) remain functionally indispensable and adaptively plastic.\nTo address, we have revised the manuscript to more clearly distinguish between the adaptive core and the broader neutral response. Specifically, we emphasize that the canonical osmoadaptive modules, including the HOG pathway, ENA1, and GPD1/GPD2, remain actively induced and are under strong selective constraint. The apparent neutrality we report pertains to the global pattern of transcriptional plasticity, where many expression changes appear to be selectively unconstrained or evolve in a neutral manner. We suggest that selection maintains only the essential subset of plastic responses required for salt tolerance, while many peripheral expression changes are effectively neutral or reflect relaxed optimization, as the reviewer points out.\nIn light of these revisions, we have added the following paragraph to the Discussion section:\nWhile our study primarily focused on the overall pattern of plastic expression, we acknowledge that certain genes may still exhibit adaptive plasticity under NaCl stress. For example, the core osmoadaptive modules, particularly the HOG pathway, ENA1, and GPD1/GPD2, remain essential and show strong, adaptive induction in response to NaCl stress (Gasch et al., 2000; Warringer et al., 2003). However, our data suggest that beyond this essential core, the broader pattern of transcriptome-wide plastic expression changes appears largely random across MA lines, which is consistent with neutral variation in many genes. This observation indicates that while selection continues to preserve the critical plastic responses needed for osmotic balance and ion homeostasis, many peripheral transcriptional responses may evolve under relaxed selection or drift. In this way, adaptive plasticity is maintained where it is functionally necessary, but the entire stress-responsive network is not globally optimized.\nMinor issues.\n1. It is not correct to say that YPD lacks Na+. There is no added NaCl, but the yeast extract and peptones do contain the ions, in varying amounts depending on batch. The batch variations is one reason to use SC instead of YPD.\nResponse: Thank you for your valuable feedback. We have reviewed the documentation for the chemicals, including peptone and yeast extract, used in the MA experiment. According to the Certificates of Analysis for these materials:\nhttps://documents.thermofisher.com/TFS-Assets/certificate/MN/COA/COA_LP0037B_11042025030433_6208751_1.pdf\nhttps://documents.thermofisher.com/TFS-Assets/BPD/certificate/Certificate-of-Analysis/212720%20-%203257238.pdf\nOn average, peptone and yeast extract contain about 0.3% sodium chloride, resulting in a final sodium chloride concentration of approximately 0.012% in YPD medium. In comparison, the sodium chloride concentration in 0.5 M NaCl is 2.92%, which is more than two hundred times higher. Given this substantial difference, we determined that the amount of sodium chloride from peptone and yeast extract would have a minimal impact on our experiment. However, we will remain mindful that YPD does contain trace amounts of ions, which could influence future experiments.\n2. The protocol for growth rate estimations appears not to be from ref 44 but from\nhttps://pubmed.ncbi.nlm.nih.gov/14676322/?\nResponse: Thank you for pointing this out. You are correct that our growth rate estimation protocol follows the method described by Warringer et al. (2003; PMID: 14676322). We have now cited both this paper and our previously listed reference (ref. 44) to accurately acknowledge the original source of the method.\nReviewer #3:\nIn this revised version, the authors have responded thoroughly and constructively to our earlier concerns. They substantially revised the Introduction to focus on the question their data directly address, namely whether gene expression plasticity is selectively maintained rather than its role in evolvability. They also added important ecological and historical context by explicitly distinguishing between common and novel stressors and by invoking genetic assimilation for long-standing stresses. The scope of the conclusions has been appropriately narrowed to clotrimazole and to general stress-response mechanisms, rather than to antibiotics or drugs broadly, and terminology has been corrected throughout.\nSeveral concerns about potential technical and experimental artefacts have been alleviated by the inclusion of new growth-curve data, additional heat-stress experiments, refined analyses of plasticity categories, and competitive fitness assays in clotrimazole and hydrogen peroxide that link loss of plasticity to reduced fitness in relevant environments. The authors have also clarified the consequences of using an MSH2 mutator background, addressed the issue of essentiality, and improved the clarity and organization of the figures, including better integration of the natural strains. While the interpretation of adaptive plasticity may still depend on perspective, the main conclusions are now well supported by the data, and we are satisfied that major concerns have been adequately addressed.\nResponse: We thank the reviewer for their positive and constructive feedback. We are pleased that the revisions have addressed the earlier concerns and that the main conclusions are now considered well supported."}]

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