Different selective pressures lead to different genomic outcomes as newly-formed hybrid yeasts evolve
© Piotrowski et al; licensee BioMed Central Ltd. 2012
Received: 13 November 2011
Accepted: 2 April 2012
Published: 2 April 2012
Interspecific hybridization occurs in every eukaryotic kingdom. While hybrid progeny are frequently at a selective disadvantage, in some instances their increased genome size and complexity may result in greater stress resistance than their ancestors, which can be adaptively advantageous at the edges of their ancestors' ranges. While this phenomenon has been repeatedly documented in the field, the response of hybrid populations to long-term selection has not often been explored in the lab. To fill this knowledge gap we crossed the two most distantly related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum, and established a mixed population of homoploid and aneuploid hybrids to study how different types of selection impact hybrid genome structure.
As temperature was raised incrementally from 31°C to 46.5°C over 500 generations of continuous culture, selection favored loss of the S. uvarum genome, although the kinetics of genome loss differed among independent replicates. Temperature-selected isolates exhibited greater inherent and induced thermal tolerance than parental species and founding hybrids, and also exhibited ethanol resistance. In contrast, as exogenous ethanol was increased from 0% to 14% over 500 generations of continuous culture, selection favored euploid S. cerevisiae x S. uvarum hybrids. Ethanol-selected isolates were more ethanol tolerant than S. uvarum and one of the founding hybrids, but did not exhibit resistance to temperature stress. Relative to parental and founding hybrids, temperature-selected strains showed heritable differences in cell wall structure in the forms of increased resistance to zymolyase digestion and Micafungin, which targets cell wall biosynthesis.
This is the first study to show experimentally that the genomic fate of newly-formed interspecific hybrids depends on the type of selection they encounter during the course of evolution, underscoring the importance of the ecological theatre in determining the outcome of the evolutionary play.
Interspecific hybridization occurs in every eukaryotic kingdom and can lead to reticulated rather than branching phylogenies [1, 2]. Hybrid progeny are often at a strong selective disadvantage (e.g., they may be sterile or have reduced viability). However, in some instances the increased genome size and complexity of interspecific hybrids may result in greater fecundity and/or adaptive flexibility than either ancestral species , particularly at the edges of the ancestral species' range, where they are more likely to encounter stress . This phenomenon is amply documented in the agricultural literature as well as in field-based evolutionary studies [1, 2, 5–10]. Laboratory studies of interspecific hybridization have largely been confined to the Drosophila species complex, where foundational studies have shaped our understanding of the genetic basis for pre-zygotic and post-zygotic reproductive isolation [11–13]. Long-term experimental studies aimed at discerning the evolutionary trajectories open to newly-formed hybrids under different types of selection are lacking, a knowledge gap due in part to the scarcity of hybrid eukaryotes that have the short generation time and ease of preservation needed to undertake experiments lasting hundreds of generations.
Experimental evolution studies using microbes have enlarged our understanding of the tempo of adaptive change [14–16], the shape of the adaptive landscape , and the manner in which genotypes navigate that landscape . Increasingly, the budding yeast Saccharomyces cerevisiae has become a favored model for such studies because of its genetic tractability, arsenal of post-genomic tools, and homology of many of its genes to those in higher eukaryotes. Yeast's short generation time, simple, heterogonic life cycle and ease of preservation ideally suit it for studying evolution in the laboratory [16, 19–21], where it has yielded insights into the physiology of adaptive traits  and how genome structure evolves under selection [23, 24].
The 7 members of the Saccharomyces sensu stricto group, though closely related, have long been recognized as biological species by virtue of their post-zygotic reproductive isolation [25, 26]. While bona fide representatives of each species are easily recovered from nature , retrospective comparative genomics studies [28, 29] suggest that interspecific hybridization has occurred repeatedly during the group's evolutionary history. Indeed, the lager yeast S. pastorianus, which likely arose ~500-600 years ago , is a natural hybrid of two species, S. cerevisiae and the newly discovered S. eubayanus . Saccharomyces hybrids have most often been studied with the aim of developing industrially useful traits [31, 32], typically by focusing on the physiology of single clones. However, Grieg et al. (2002) showed that the sensu stricto group could also be used to investigate hybrid speciation in the lab . Currently lacking, however, are prospective studies of interspecific hybrid evolution under different types of selection, using either single clones or, more realistically, populations of hybrid clones, such as one might expect to arise in natural hybrid zones.
The two most distantly-related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum (formerly S. bayanus var. uvarum ), are largely syntenic, exhibit 80% and 62% nucleotide identity in coding regions and intergenic regions, respectively, and are thought to have diverged ~20 million years ago . The two species differ in their stress tolerances, with S. cerevisiae being much more thermal tolerant [35–37] and slightly more ethanol tolerant ; S. cerevisiae x S. uvarum hybrids can exhibit greater ethanol tolerance than either parental species . These genetic and phenotypic differences, coupled with the availability of genomic resources for both species, make S. cerevisiae x S. uvarum hybrids an attractive system in which to investigate how newly-formed hybrid genomes evolve under different types of stress. We therefore sporulated a S. cerevisiae x S. uvarum hybrid and mass-mated the progeny to create a pool of S. cerevisiae x S. uvarum homoploid and aneuploid hybrids that was used to found six replicate populations. Because fungi in nature are chronically nitrogen limited [40, 41], experimental populations were evolved in a glucose-sufficient, nitrogen-limited 'common garden;' three were subjected to incremental increases in temperature, and three were subjected to incremental increases in ambient ethanol. Because the two ancestral species and their F1 hybrid exhibit differential sensitivity to temperature and ethanol, and because stress has been shown to increase mitiotic recombination, including chromosome missegregation , we hypothesized that the two selective pressures would lead to different genomic outcomes. As prior experiments in S. cerevisiae had shown that thermal tolerance confers cross-protection against other types of stress [37, 43], we further hypothesized that hybrids evolving under temperature selection would not only become more thermal tolerant than their ancestors but also exhibit greater ethanol tolerance, and vice versa.
We tested these hypotheses using an integrated approach that combined physiological assays with analysis of genome structure by Clamped Homogeneous Electric Field (CHEF) electrophoresis and array-Comparative Genomic Hybridization (a-CGH). Consistent with our primary hypothesis, temperature selection resulted in loss of the S. uvarum genome from interspecific hybrids, while ethanol selection resulted in yeast that retained essentially both S. cerevisiae and S. uvarum genomes. Consistent with our secondary hypothesis, cross-protection to ethanol was evident in the temperature selected isolates; however, cross-protection to thermal stress was not observed among ethanol-selected clones. Thus, as hybrid populations evolve under different selection pressures their fate may depend not only on the outcome of competition between individual variants, but also on the outcome of competition between the ancestral genomes themselves.
Results and Discussion
Creation of the founder hybrid population and experimental design
Hybrid cells with complete or almost-complete chromosomal complements tended to grow faster than those cells that were multiply aneuploid (data not shown). Consequently, our starting inoculum was a complex mixture of F1 and F2 hybrids having different levels of aneuploidy and specific growth rates. A mixture of F1, haploid and aneuploid F2 hybrids is an evolutionarily relevant starting point for our experiments, inasmuch as it mimics the genetic complexity one might expect to see within a hybrid zone . We refer to this pool as the founder hybrid population. For purposes of comparison with clones subsequently isolated after many generations of selective growth, three isolates were randomly chosen from the founder hybrid population and denoted H1, H2, and H3. All strains used in this study are listed in Additional file 1: Table S1.
Steady-state population size declines as evolving hybrid populations respond to increases in either temperature or ethanol
Yeasts recovered from temperature selection are more thermotolerant than members of the founder population
Yeasts recovered from ethanol selection do not appear to be more ethanol tolerant than members of the founder population
The manner and extent to which interspecific hybrid populations responded to ethanol selection was less clear-cut. When cultured until stationary phase (48 h) in liquid, low-nitrogen minimal medium amended with 8% ethanol, culture densities among ethanol-selected isolates were statistically indistinguishable from those of S. cerevisiae, the F1 interspecific hybrid, founder population isolates H1 and H2, and the temperature-selected isolates (Additional file 2: Figure S1). Only the S. uvarum parent and founder isolate H3 proved to be ethanol sensitive by this assay (P < 0.05, Tukey's HSD). Furthermore, temperature selected isolates were no less ethanol tolerant than ethanol-selected isolates.
Temperature and ethanol selection can lead to changes in cell wall integrity
We also observed that temperature-selected isolates were more resistant to Micafungin, a drug that targets β-1,3 glucan synthesis  (Additional file 3: Figure S2). Isolates from vessels A and C exhibited significantly greater resistance to this drug than S. cerevisiae, S. uvarum, the F1 and founding hybrids (P < 0.05, Tukey's HSD). This suggests that changes in cell wall composition or deposition contribute to the thermal tolerant phenotype observed in isolates from vessels A and C, whereas isolates from vessel B may have evolved a different resistance mechanism. This suggestion is supported by the fact that vessel B isolates, while not Micafungin-resistant, did exhibit resistance to zymolyase digestion.
Although interspecific hybrid populations responded weakly, if at all, to ethanol selection, we nevertheless assayed for heritable changes in cell wall integrity. Interestingly, similar to members of the temperature-selected populations, ethanol-selected isolates were more zymolyase resistance than parental species, the F1 hybrid and founder isolates (Figure 5B), though only isolates from vessel E showed significantly greater zymolyase resistance than founding hybrids after 1 h of treatment (P < 0.05, T-test). Strains isolated from ethanol-selected populations showed no difference in Micafungin sensitivity relative to the parental species, the S. cerevisiae x S. uvarum F1 hybrid, or isolates drawn from the founder population (Figure 5B).
Temperature and ethanol selection lead to different genomic outcomes
Temperature selection on the founding hybrid population favors loss of the S. Uvarum genome
We speculate that haploids arose when diploid hybrids sporulated under aerobic nitrogen limitation. Alternatively, a rare haploid, or nearly-haploid, F1 gamete may have been present in the initial founder hybrid population. If such a gamete contained only S. cerevisiae chromosomes and no S. uvarum chromosomes, it would have been HO- (and separated from potential opposite-mating-type spores by turbulence in the liquid medium), and could therefore have remained haploid. Whatever their origin, multiple temperature-selected clones exhibited chromosome rearrangements at 500 generations (Figure 6B). S. cerevisiae-like haploids likely acquired a competitive advantage at temperatures exceeding 35°C, a speculation supported by reports that the cardinal growth temperature of S. cerevisiae is significantly greater than S. uvarum [35, 36] and (Figure 3). Given that temperature tolerance is a multi-locus, quantitative trait , haploidization resulting in nearly-complete loss of the S. uvarum genome complement provides the shortest path to thermal tolerance for S. cerevisiae X S. uvarum hybrids. Further, haploid S. cerevisae are known to exhibit greater induced thermotolerance than isogenic diploids , and haploids outcompete diploids at elevated temperature (37°C) when serially propagated in either YPD or low-nitrogen, minimal medium . Consistent with these observations, after 48 h of growth at 40°C the haploid form of our S. cerevisiae parent attained higher cell density than the diploid (A600 = 0.28 ± 0.01 vs. 0.19 ± 0.01).
Two species array comparative genomic hybridization supports the inference that genome evolution in interspecific hybrid populations is selection-specific
Using a-CGH we examined the same 9 temperature-selected isolates described above (A1 through A3, B1 through B3, and C1 through C3, Figure 1). For all 9 clones, the genome complement appears to be comprised solely of that of the S. cerevisiae parent (Figure 8). The a-CGH results for the three vessel A clones thus closely agree with their CHEF gel results (Figure 6B); furthermore, a-CGH reveals that all three vessel A clones contain a small deletion in the S. cerevisiae genome; because its length is only ~1-3 kb it was undetectable on CHEF gels. This deletion is located on Chromosome IV and corresponds to the ARS in the region between HXT6 and HXT7 (Figure 8, indicated by an arrow). As the deletion appears identical among the three clones within the detection limits of a-CGH, it likely arose from the same member of the founder hybrid population within population A and does not represent multiple independent evolutionary events. Additional experiments will be required to ascertain both its prevalence in the vessel A population (i.e., whether it is "fixed"), as well as its possible adaptive value.
For the three 500-generation clones from population B and C, our a-CGH results do not intuitively agree with the CHEF results. CHEF gel karyotypes for all B clones appear to show the presence of an additional chromosome; although the band is similar (but not identical) to mobility of S. uvarum Chromosome XI, a-CGH results from all assayed 500-generation vessel B clones show the presence of only the parental S. cerevisiae genome (Figure 8) indicating that the band must derive from a copy-neutral event within the S. cerevisiae genome. Similarly, the CHEF electrokaryotypes of the clones from population C show increase in size of at least two of the smaller chromosomes, but again, only the parental S. cerevisiae genome is present (Figure 8), suggesting the occurrence of copy-neutral rearrangements.
We also performed a-CGH on 3 ethanol-selected isolates from each of Vessels D, E, and F (Figure 7). We investigated clones from the 400 generation time-point rather than at 500 generations, as we had observed greater karyotypic diversity in the 400 generation populations (Figure 6C vs. Additional file 5: Figure S4). An ethanol-selected clone from vessel D (Figure 7, EtOH 400 gen D2) appeared to be an essentially euploid F1 hybrid with a large deletion in the S. cerevisiae genome (but not deleted in the corresponding S. uvarum region): 100 kb of the right end of Chromosome XII, starting just proximal to (and including) the TUS1 gene and extending to the right telomere, is deleted in this isolate. Interestingly, Tus1 is involved in cell integrity signalling  and its deletion results in increased chitin deposition ; ECM30, which is also included in the deleted region, is a gene possibly involved in cell wall biosynthesis. This deletion may explain the increased zymolyase resistance observed in Vessel D isolates (see Figure 5B). The Vessel E isolate examined by a- CGH again appeared to be an essentially euploid F1 (Figure 8, EtOH 400 gen E3), but exhibited a 15 kb deletion on its S. cerevisiae Chromosome XIV starting between MEP2 and AAH1 and extending to a tRNA gene just beyond FYV6. The deleted region contains genes involved in a variety of functions including nitrogen utilization (AAH1), recombination (THO2 and FYV6) and chromatin modifications (EAF7 and FPR1). The ethanol-selected isolate from Vessel F lost its S. cerevisiae mitochondrial genome, yet otherwise appeared as an intact euploid F1 hybrid (Figure 8, EtOH 400 gen F4).
This study is the first to report how populations of interspecific hybrid organisms evolving in the laboratory follow dramatically different evolutionary trajectories depending on the selective pressures applied. Indeed, to the best of our knowledge this is the first long-term study in experimental microbial evolution initiated with a heterogenous population of genetically diverse variants rather than with a homogenous population derived from a single clone. This collection of variants was used to found six replicate populations that evolved under glucose-sufficient, nitrogen-limiting conditions: three under temperature selection and three under ethanol selection.
In clones that arose under temperature selection, thermal tolerance was significantly greater than that of isolates in the founder population and of the parental F1 strain, a result likely due in part to segregational loss of the temperature-sensitive S. uvarum genome, resulting in S. cerevisiae haploids. This event occurred independently in all populations under temperature selection, although the haploid variants arose at different times and swept their respective populations over different time intervals. Interestingly, loss of heterozygosity under thermal and oxidative stress has recently been documented in Candida albicans, where increased stress appears to elevate rates of recombination, including chromosome missegration . In diploid S. cerevisiae, haploidization has now been shown to provide an escape for persistent DNA rearrangement stress due to the presence of mutator alleles . Thus, strong selection to augment thermal tolerance as well as to diminish DNA stress may help to explain the outcome of temperature selection on a population of newly-formed interspecific hybrids.
Heritable thermotolerance among these strains was evident in their ability to grow at temperatures restrictive to founding hybrid isolates on solid (data not shown) and in liquid media, as well as in their intrinsic and acquired resistance to heat shock. Temperature-selected strains were also ethanol resistant, most likely due to loss of the ethanol-sensitive S. uvarum genome . Based on CHEF analysis, two of three temperature-selected populations came to be dominated by a unique karyotype, while one remained polymorphic, indicating multiple pathways to the evolution of thermotolerance. Evolution of multiple heat stress tolerance mechanisms was also manifest as differential, strain-specific resistance to zymolyase digestion as well as to Micafungin, a drug that targets cell wall biosynthesis. Because increased zymolyase resistance correlates with increased ethanol resistance [61, 62], adaptive changes in cell wall integrity in temperature-selected clones may underlie "cross-protection" against ethanol stress.
Under temperature selection, adaptive changes in zymolyase and Micafungin resistance could be expected inasmuch as cell wall integrity has been shown to play a key role in thermal stress response . Acquisition of thermotolerance in diauxic and stationary phase yeast has also been linked to accumulation of the disaccharide trehalose [64, 65], however, we uncovered no evidence that trehalose hyper-accumulates in heat-shock resistant clones that arose under temperature selection (data not shown).
We uncovered little evidence for adaptive evolution of ethanol tolerance. Populations evolved under ethanol selection became dominated by euploid S. cerevisiae x S. uvarum hybrids. These hybrids showed improved growth on ethanol-amended media relative to some (e.g., founder H3), but not all founding hybrids, nor to the parental F1 strain. Evidence suggesting heritable changes in cell wall composition in these isolates was limited to higher (but not significantly higher) zymolyase resistance. We therefore conclude that a subset of euploid hybrids in the original founder population was at or near a fitness peak for ethanol tolerance , providing limited scope for selection.
It is important to bear in mind that temperature and ethanol tolerance assays were performed on only a few clones that together represent a small fraction of the genetic variation latent in our terminal populations. Other clones may exist that exhibit even greater thermal tolerance and, perhaps also greater ethanol tolerance. Indeed, in follow-up experiments we found that the temperature-selected clones we had randomly chosen for analysis grew poorly in chemostat monoculture at > 45°C, suggesting that other more highly tolerant variants exist in the terminal populations. Also, it may be that a slow ramp-up in temperature may be required even for thermotolerant clones to achieve their true performance maxima. We acknowledge the possibility that highly stress tolerant variants may have been present in the hybrid founder population used for these experiments. Such clones may have persisted at low frequency under slow growth conditions (D = 0.15 h-1) until selection favored them over more abundant, stress-sensitive clones. This possibility highlights an outstanding unresolved issue in experimental evolution, namely the extent to which adaptation results from accumulation of de novo mutations as opposed to selection of rare adaptive mutants that may exist at the onset of selection. In this regard, it would be interesting to perform a high-throughput phenotypic screen of hundreds of variants in the founder and terminal populations, as well as to perform population sequencing at the beginning and end of these experiments. Still, given that selection was applied over the course of 500 generations it is virtually certain that multiple de novo mutations distinguish members of the terminal populations from their common ancestors.
The industrial applications of hybrid variability have already been recognized , and our approach of selecting on a diverse hybrid population may be used to enhance routine industrial strain development. However, our findings highlight the need to carefully choose appropriate parental strains as the selection process cannot rely solely on hybrid vigor. Genome plasticity under strong selection may lead to unexpected results, such as the shedding of one or another parental genome. In our experiments this remarkable occurrence seems to provide the most direct route to thermal tolerance, a trait whose many genetic determinants are widely distributed across the S. uvarum and S. cerevisiae genomes. Selection on a genetically diverse population of S. cerevisiae alone might produce comparable gains in fitness at high temperatures. Indeed, wild isolates of that species have been isolated which can grow at temperatures exceeding 45°C , and genome shuffling experiments  involving recursive protoplast fusions have produced S. cerevisiae strains that aggressively ferment at temperatures up to 48°C . Most importantly, our findings highlight the importance of the ecological theatre in determining the outcome of the evolutionary play. Euploid interspecific S. cerevisiae x S. uvarum hybrids are genetically stable and highly fit as ambient levels of ethanol increase, but poorly fit under rising temperature. Thus, the evolutionary fate of hybrids in nature likely depends as much on their environmental context as on their genetic potential.
Strains and hybrid creation
All yeast strains used in this work are derivatives of the prototrophic diploid strains CEN.PK (Saccharomyces cerevisiae)  and CBS7001 (S. uvarum)  the former obtained from D. Botstein, the latter from E. Louis. To obtain F1 hybrids, S. cerevisiae CEN.PK and S. uvarum CBS7001 were transformed to G418 and hygromycin antibiotic resistance, respectively, using 2 μ-based YEp352-KanMX and YEp352-hph plasmids. After verifying plasmid segregation, transformants were sporulated for three days on sporulation medium (1% potassium acetate, 0.1% yeast extract and 0.05% glucose) and then mixed and plated on rich medium supplied with G418 and hygromycin at 200 μg mL-1 and 300 μg mL-1, respectively. F1 progeny were selected as clones resistant to both antibiotics. After confirming segregational loss of both plasmids, a single F1 clone was sporulated for 3 days in liquid sporulation medium (1% potassium acetate); unsporulated cells were then digested by a combination of Zymolyase T100 and a detergent, as described in , leaving F2 hybrid spores, which represent rare viable spore progeny of the F1 clone. These spores were left to germinate and mate overnight, then after verifying cell titer, spread on 48 large plates so that every cell could grow into a colony, unencumbered by others. Approximately 10,000 colonies were washed with 5 mL of sterile ddH2O per plate and combined to make the initial hybrid pool.
Media and growth conditions
Unless otherwise indicated, all media used was the inorganic nitrogen-limiting (0.15 g L-1 (NH4)2SO4) medium used for batch and chemostat cultures described by Verduyn et al. . For chemostat experiments 10 L of basal medium were prepared in 13 L glass carboys. To each liter a post-sterile addition was made of: 1.0 mL 1000× vitamins, 1.0 mL 1000× trace metals, and 45.0 mL 20% glucose (final conc. 9 g L-1) , a formulation hereafter referred to as low-nitrogen, minimal medium. Populations were cultured in water-jacketed chemostats (200 mL working volume), which were mixed and aerated using sterile, humidified house air at a flow rate of 10 L h-1 (0.8 vvm). For temperature selections, triplicate experimental populations were founded by adding cells from an inoculum prepared as described to a final density of ~108 cells per mL, with the initial target dilution rate set at D = 0.15 h-1 and the initial temperature at 31°C. Every 25 generations (about every week), culture temperature was increased by 1°C; as cell yield declined steeply above 37°C, later adjustments were made on a bi-weekly basis. To avoid wash-out, dilution rate at higher temperatures was lowered to D = 0.05 h-1 at 41°C and remained at this level until 500 generations. The final vessel temperature at 500 generations was 46.5°C. For ethanol selections, the same founding population was used to inoculate three identical 200 mL chemostat vessels, which were kept at room temperature (25-28°C). The basal medium was identical to that used for temperature selection; ethanol content of this medium was increased by 1% approximately every 10 d. Evaporation was impeded by layering sterile mineral oil atop ethanol-amended, low-nitrogen minimal medium. Because cell growth was strongly inhibited at ethanol amendments > 12%, at these concentrations chemostat dilution rate was reduced to D = 0.05 h-1 to prevent wash-out.
Sampling and assay of growth parameters
Optical density at λ = 600 nm was measured daily using a Biomate3 spectrophotometer (Thermo Electron Corp, Waltham, MA, USA.) by sterile removal of 1 mL of culture from each vessel, and measuring absorbance of 1:10 dilutions. Approximately every 15 generations 5 mL were removed from each vessel (i) to archive samples as 15% glycerol stocks at -80°C, (ii) to estimate viable cell counts by plating serial dilutions on YPD, and (iii) to determine concentrations of glucose and ethanol, as described below. Three-mL aliquots archived for analysis of residual growth substrate and ethanol were filtered using a 0.2 μm nylon filter and stored at -20°C until assayed.
Temperature and ethanol tolerance assays
For follow-up experiments we used the diploid parental strains S. cerevisiae CEN.PK and S. uvarum CBS7001, the F1 hybrid, three isolates from the founder hybrid population, and isolates from each of six experimental populations at the final 500-generation time-point. An overview of how the hybrid population was generated and the naming scheme for the isolates is shown in Figure 1, and a detailed list of all tested evolved isolates is presented in Additional file 1: Table S1. Representatives of the founder hybrid population are the same isolates shown in Figure 6A, lanes 1, 2, 3, and are referred to as H1, H2, and H3. Temperature selected isolates tested are shown in Figure 6B Lanes 1, 2, 3 (Temperature selected A1, A2, A3); Lanes 8, 9, 10 (Temperature selected B1, B2, B3); and Lanes 15, 16, 17 (Temperature selected C1, C2, C3). Ethanol evolved isolates tested are shown in Figure 6C Lanes 1, 2, 3 (Ethanol selected D1, D2, D3); Lanes 9, 10, 11 (Ethanol selected E1, E2, E3); and Lanes 16, 17, 18 (Ethanol selected F1, F2, F3). In certain instances, to test the performance of the ancestral haploid form we included a S. cerevisiae CEN.PK haploid. For each experimental parameter tested, individual isolates were run in triplicate.
Growth in liquid media
For all experiments we used parental isolates, F1, 3 founding hybrid isolates, and three isolates from each terminal population (Figure 1). Pre-cultures were grown overnight in 200 Lof low-nitrogen, minimal medium at 25°C and used to inoculate test cultures to a starting A600 of 0.01. All test cultures were similarly grown in 200 μL of low-nitrogen, minimal medium in 96-well microtiter plates. To assay temperature tolerance, cultures were grown at 40°C; to test ethanol tolerance cultures were grown at 25°C in the same medium, amended with 8% ethanol. All cultures were grown in triplicate to stationary phase (48 h). Optical density was measured spectrophotometrically at λ = 600 nm every 6 h to confirm that all cultures were in stationary phase before the final measure (Spectramax 340PC, Molecular Devices, Sunnyvale, CA, USA). Replicate estimates of growth parameters for temperature or ethanol isolates (A1-F3) were pooled by vessel for statistical comparison to the parent, F1 and founding strains.
Induced versus inherent thermotolerance
Each parental stain, the F1, the three representative founder stains and two isolates from each temperature selection (isolates 1 and 2 from each vessel in Additional file 1: Table S1) were grown in triplicate overnight at 25°C in 50 mL low-nitrogen, minimal medium to A600 0.4-0.6. To test strain-specific differences in induced versus inherent thermotolerance, each culture was apportioned into two vessels: one was placed in a 37°C water bath for 5 min and then incubated at 37°C on a shaker (induced thermotolerance), while the other was shaken at room temperature (inherent thermotolerance). After 50 min, cultures were diluted into fresh pre-warmed media, then placed in a 48°C water bath, whereafter samples were removed every hour for 5 h and diluted before plating onto YPD agar. Survivorship was reported as the percentage of viable cells remaining at each time-point, relative to viable cell counts at T = 0 hours.
Analysis of cell wall phenotypes
To determine whether observed changes in thermal tolerance were correlated with changes in cell wall composition we performed a spheroplast assay, as described in . Cultures were diluted to an A600 = 0.8 in spheroplast medium (1.2 M sorbitol), whereupon zymolyase was added to achieve a final assay concentration of 250 μg mL-1. Decrease in A600, resulting from cell wall digestion, was measured over the course of 1 h at 37°C. All strains were tested in triplicate, and the selected isolates used were the "1" isolates (e.g., A1, B1, etc.) from each evolved population (Additional file 1: Table S1). Strain-specific resistance to Micafungin (Astellas Pharma, Tokyo), a compound that targets fungal cell wall biosynthesis was tested by culturing cells at 25°C in 200 μL of low-nitrogen, with 150 nM Micafungin or a solvent control (DMSO). Cells were inoculated to an A600 = 0.01 and incubated until all cultures were in stationary phase and measured spectrophotometrically. Growth was calculated relative to the solvent-only control. All isolates were run in triplicate.
CHEF analysis was conducted as previously described [70, 71]. To assay ploidy flow cytometry was performed using SYTOX green as described in . Microarray-based Comparative Genome Hybridization (array-CGH) was performed as described in . Microarray data have been deposited in the GEO repository under accession GSE24479.
We used one-way ANOVA to compare differences in response to temperature and ethanol tolerance, zymolyase, and Micafungin resistance, using Tukey's HSD. For individual comparisons we used a T-test. We used Sigma Plot 11 (Dundas software LTD, Germany) for all statistical analyses.
The authors wish to thank Carla Boulianne-Larsen and Jeff Good for critically reading the manuscript. Gregory Koniges performed heat shock experiments to distinguish between induced and inherent thermal resistance. Flow cytometry was performed with the assistance of Pamela Shaw. We thank Minoru Yoshida and Astellas Pharma for the use of Micafungin. This work was supported by grants to GS from Global Climate and Energy Project (Grant #33450) and to FR from NASA NNX07AJ28G, as well as by NIH grant P20RR017670 to the University of Montana Fluorescence Cytometry Core Facility.
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