Determinants of divergent adaptation and Dobzhansky-Muller interaction in experimental yeast populations.

Divergent adaptation can be associated with reproductive isolation in speciation [1]. We recently demonstrated the link between divergent adaptation and the onset of reproductive isolation in experimental populations of the yeast Saccharomyces cerevisiae evolved from a single progenitor in either a high-salt or a low-glucose environment [2]. Here, whole-genome resequencing and comparative genome hybridization of representatives of three populations revealed 17 mutations, six of which explained the adaptive increases in mitotic fitness. In two populations evolved in high salt, two different mutations occurred in the proton efflux pump gene PMA1 and the global transcriptional repressor gene CYC8; the ENA genes encoding sodium efflux pumps were overexpressed once through expansion of this gene cluster and once because of mutation in the regulator CYC8. In the population from low glucose, one mutation occurred in MDS3, which modulates growth at high pH, and one in MKT1, a global regulator of mRNAs encoding mitochondrial proteins, the latter recapitulating a naturally occurring variant. A Dobzhansky-Muller (DM) incompatibility between the evolved alleles of PMA1 and MKT1 strongly depressed fitness in the low-glucose environment. This DM interaction is the first reported between experimentally evolved alleles of known genes and shows how reproductive isolation can arise rapidly when divergent selection is strong.

Gene expression and evolution of antifungal drug resistance.

Permanent changes in gene expression result from certain forms of antifungal resistance. In this study, we asked whether any changes in gene expression are required for the evolution of a drug-resistant phenotype in populations. We examined the changes in gene expression resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae with two antifungal drugs, fluconazole (FLC) in a previous study and amphotericin B (AmB) in this study, in which five populations were subjected to increasing concentrations of AmB, from 0.25 to 128 microg/ml in twofold increments. Six genes, YGR035C, YOR1, ICT1, GRE2, PDR16, and YPLO88W, were consistently overexpressed with resistance to AmB reported here and with resistance to FLC involving a mechanism of increased efflux reported previously. We then asked if the deletion of these genes impaired the ability of populations to evolve resistance to FLC over 108 generations of asexual reproduction in 32 and 128 microg/ml FLC, the same conditions under which FLC-resistant types evolved originally. For each of three deletion strains, YOR1, ICT1, and PDR16 strains, extinctions occurred in one of two replicate populations growing in 128 microg/ml FLC. Each of these three deletion strains was mixed 1:1 with a marked version of the wild type to measure the relative ability of the deletion strain to adapt over 108 generations. In these assays, only the PDR16 deletion strain consistently became extinct both at 32 and at 128 microg/ml FLC. The deletion of PDR16 reduces the capacity of a population to evolve to resistance to FLC.

Incipient speciation by divergent adaptation and antagonistic epistasis in yeast.

Establishing the conditions that promote the evolution of reproductive isolation and speciation has long been a goal in evolutionary biology. In ecological speciation, reproductive isolation between populations evolves as a by-product of divergent selection and the resulting environment-specific adaptations. The leading genetic model of reproductive isolation predicts that hybrid inferiority is caused by antagonistic epistasis between incompatible alleles at interacting loci. The fundamental link between divergent adaptation and reproductive isolation through genetic incompatibilities has been predicted, but has not been directly demonstrated experimentally. Here we empirically tested key predictions of speciation theory by evolving the initial stages of speciation in experimental populations of the yeast Saccharomyces cerevisiae. After replicate populations adapted to two divergent environments, we consistently observed the evolution of two forms of postzygotic isolation in hybrids: reduced rate of mitotic reproduction and reduced efficiency of meiotic reproduction. This divergent selection resulted in greater reproductive isolation than parallel selection, as predicted by the ecological speciation theory. Our experimental system allowed controlled comparison of the relative importance of ecological and genetic isolation, and we demonstrated that hybrid inferiority can be ecological and/or genetic in basis. Overall, our results show that adaptation to divergent environments promotes the evolution of reproductive isolation through antagonistic epistasis, providing evidence of a plausible common avenue to speciation and adaptive radiation in nature.

Antagonism between two mechanisms of antifungal drug resistance.

This study tested for interaction between two independently evolved mechanisms of fluconazole resistance in Saccharomyces cerevisiae. One set of strains was from a 400-generation evolution experiment, during which the concentration of fluconazole was increased from 16 to 256 microg/ml in four increments. At 100 generations, populations became fixed for resistance mutations in either of two transcriptional regulators, PDR1 or PDR3. At 400 generations, replicate populations became fixed for another resistance mutation in UNK1, an unmapped gene further increasing resistance. Another genotype used in this study came from a population placed initially in 128 microg/ml of fluconazole; this environment selects for resistance through loss of function at ERG3, resulting in altered sterol metabolism. Mutant strains carrying PDR1(r) or PDR3(r) were crossed with the erg3(r) mutant strain, and the doubly mutant, haploid offspring were identified. The double-mutant strains grew less well than the parent strains at all concentrations of fluconazole tested. In genome-wide assays of gene expression, several ABC transporter genes that were overexpressed in one parent and several ERG genes that were overexpressed in the other parent were also overexpressed in the double mutants. Of the 43 genes that were consistently overexpressed in the PDR1(r) parents at generation 100, however, 31 were not consistently overexpressed in the double mutants. Of these 31 genes, 30 were also not consistently overexpressed after a further 300 generations of evolution in the PDR1(r) parent populations. The two independently evolved mechanisms of fluconazole resistance are strongly antagonistic to one another.

Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae.

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.

Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae.

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.