Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.

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.

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.T HE predicted advantages and disadvantages of hapotide site, and their level of dominance can be measured loidy and diploidy, like those of recombination (Kondirectly. drashov 1993), hinge on the ability of populations (a)Two factors are preeminent in determining the effect to cope with deleterious mutation (Kondrashov and of ploidy on the rate of adaptation (Orr and Otto 1994): Crow 1991) and (b) to adapt to new environments (Orr the waiting time for mutations to appear and the fixation and Otto 1994). Our goal in this study was to make a time required for mutations to spread to high frequency direct comparison of ploidy states by measuring the in a population in response to directional selection. Under relative abilities of isogenic haploids and diploids of the conditions of finite population size where the waiting yeast Saccharomyces cerevisiae to adapt to the presence of time for beneficial mutations is the rate-limiting step in the antifungal drug fluconazole (FLC). We followed the adaptation, diploids should, at first glance, have the faster initial, rather than the long-term, responses of haploid rate of adaptation. This is because diploids have twice the and diploid populations to pinpoint the relati...

Summary Divergent adaptation can be associated with reproductive isolation in the process of 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, we used whole-genome re-sequencing of representatives of three populations to identify 17 candidate mutations, six of which explained the adaptive increases in mitotic fitness in the two environments. 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 over-expressed once through expansion of this gene cluster and once due to 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.

Twenty‐five primers produced unambiguous amplification products of 23 microsatellite‐containing loci and two microsatellite‐like polymorphic loci, with 2–10 alleles at each locus in the plant pathogenic fungus, Sclerotinia sclerotiorum. Haplotypes are polymorphic among individuals sharing the same DNA fingerprint and DNA sequence haplotype, facilitating epidemiological monitoring worldwide. Fourteen of these primers also successfully amplified the closely related S. trifoliorum and S. minor.