Fitness of incompatible relative to compatible strains following competition.

<p>Independent cultures of compatible (<i>kMLH1-kPMS1</i>, EAY3242) and incompatible (<i>cMLH1-kPMS1</i>, EAY3236) strains were grown for the indicated number of transfers in YPD + 1.2 M NaCl. Fitness values were separately determined after competition experiments in which evolved cultures were randomly mixed at a 1:1 ratio and grown for an additional 24 hours in YPD or YPD + 1.2 M NaCl (see examples of the raw data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#pgen.1005407.s002" target="_blank">S2 Fig</a>). Fitness (<i>w</i>) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#pgen.1005407.ref034" target="_blank">34</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#pgen.1005407.ref035" target="_blank">35</a>] was calculated as <i>w</i> = ((p_t/q_t)/(p_o/q_o))^1/t, where t equals the number of generations after 24 hrs of competition (7 generations), p_o and q_o are the number of incompatible and compatible cells, respectively at 0 hrs, and p_t and q_t are the number of incompatible and compatible cells, respectively, at 24 hrs. n is the number of unique competitions performed for each data set. One-way ANOVA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#pgen.1005407.ref036" target="_blank">36</a>] was used to test whether mean fitness values are different in YPD + 1.2M NaCl vs. YPD.</p><p>Fitness of incompatible relative to compatible strains following competition.</p

<i>pmr1</i> mutations identified in this study.

<p>Clones from the indicated compatible (C) and incompatible (I) lines at Transfer 10 or 16 were analyzed by DNA sequencing. WGS indicates whole genome sequencing that was confirmed by Sanger sequencing the PCR-amplified <i>PMR1</i> locus. “Sanger, <i>PMR1</i> PCR” indicates that the <i>PMR1</i> locus was amplified by PCR and sequenced by the Sanger method. <i>pmr1</i> mutations are indicated by the wild-type sequence, followed by base pair or amino acid position, and then the mutant sequence. n/a, not applicable. *T to A substitution 220 bp upstream of the <i>PMR1</i> start codon.</p><p><i>pmr1</i> mutations identified in this study.</p

Incompatible strains display a fitness advantage in high salt media.

<p>Independent cultures of compatible (<i>kMLH1-kPMS1</i>, EAY3242) and incompatible (<i>cMLH1-kPMS1</i>, EAY3236) strains were grown for up to Transfer 16 (~ 2 x 10⁷ cells per transfer) in YPD (unevolved) or YPD + 1.2 M NaCl (evolved). Cultures at the indicated transfers were diluted to an OD₆₀₀ of 0.1 (~ 2 x 10⁷ cells per transfer) in YPD + 1.2 M NaCl and monitored for growth for 12 hrs. A representative experiment involving three replicates for each genotype is shown. Mean OD₆₀₀, +/- standard deviation, is presented for each time point. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#sec010" target="_blank">Materials and Methods</a> for details. </p

A model for how MMR incompatible populations arise in nature [22,27].

<p>In this cartoon, a common ancestor bearing the Mlh1 Gly 761 and Pms1 Arg 818/822 alleles can sustain mutations, neutral or beneficial, that give rise to the derived S288c (purple, Asp 761, Arg 818/822) and SK1 (green, Gly 761, Lys 818/822) group strains. Mating between the derived strains can yield an allele combination (Mlh1 Asp 761, Pms1 Lys 818/822) that had not been selected for function, leading to a negative epistatic interaction and a mutator phenotype. Sequencing analysis of a 32-kb region in the derived groups provided evidence for recombination between the two, supporting the idea that these two groups can meet in nature, exchange genetic information, and form a hybrid mutator [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005407#pgen.1005407.ref022" target="_blank">22</a>].</p

A single locus is likely responsible for high salt adaptation in compatible and incompatible strains.

<p>A, High salt resistant clones (red) isolated from evolved compatible and incompatible cultures were crossed to an unevolved strain (blue). Diploids were selected and streaked onto YPD + 1.2 M NaCl plates to determine if NaCl^r in the evolved strain was dominant or recessive. Subsequently, diploids were sporulated and tetrad dissected, and spore clones were analyzed for resistance to NaCl. B. Left panel. An evolved NaCl resistant clone showing a dominant phenotype. Growth of the indicated haploid and diploid strains on YPD + 1.2 M NaCl plates is shown. Right panel, example of 2:2 NaCl^r:NaCl^ssegregation. Growth on YPD + 1.2 M NaCl plates is shown for the spore clones of a single tetrad obtained by mating an evolved NaCl^r clone to an unevolved strain. C. The indicated incompatible and compatible evolved NaCl^r clones were each mated to an unevolved haploid strain and analyzed for segregation of NaCl resistance in tetrad analysis. For each mating the vast majority displayed 2:2 segregation of resistance to sensitivity. In total nine tetrads deviated from this pattern (“other” category) with eight showing 3:1 or 1:3 segregation and one showing 4:0 segregation.</p

Whole genome sequencing indicates <i>PMR1</i> linkage to NaCl resistance.

<p>The indicated evolved clones obtained from Transfer 10 were each mated to an unevolved compatible strain (EAY3241 for mating to incompatible evolved, EAY3191 for mating to compatible evolved). The resulting diploids were sporulated and tetrad dissected and germinated spore clones were analyzed for growth on YPD containing 1.2 M NaCl. For each mating at least 18 NaCl^r and 18 NaCl^s spore clones were separately pooled. The resistant and sensitive pools were then analyzed by whole genome sequencing (Materials and Methods). The sequence for the indicated position is shown for the unevolved reference (WT) and the evolved strains (SNP), followed by the number of WT and SNP reads detected in each pool. Using the Fisher Exact test, all SNPs we found that were defined as linkage differ from random segregation with a p value <0.0001.</p><p>Whole genome sequencing indicates <i>PMR1</i> linkage to NaCl resistance.</p

<i>pmr1</i> mutations identified in evolved lines are causative for resistance to 1.2 M NaCl and 0.4 M LiCl.

<p>In both A and B, wild type and <i>pmr1Δ</i> unevolved strains were plated in 10-fold serial dilutions onto YPD, YPD + 1.2 M NaCl, and YPD + 0.4 M LiCl plates. In panel A, representative NaCl-evolved strains (I, incompatible, C, compatible, with the Transfer indicated) bearing <i>pmr1</i> mutations are shown. In panel B, unevolved strains transformed to contain the indicated <i>pmr1</i> alleles (Replacement) are shown, with the corresponding evolved strain plated side by side.</p

cohort SNP file

variant file of all strains, contains all single nucleotide polymorphic sites that are bi-allelic only

Variants for all S. paradoxus strains listed in Table 1 of the publication

This .vcf file includes only biallelic loci, with no missing data, after filters were applied as described in the publication