Linda M. Kohn

A method for designing primer sets for speciation studies in filamentous ascomycetes

A simple method is described for designing primer sets that can amplify specific protein-encoding sequences in a wide variety of filamentous ascomycetes. Using this technique, we successfully desig...

A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea

Magnaporthe oryzae is described as a new species distinct from M. grisea. Gene trees were inferred for Magnaporthe species using portions of three genes: actin, beta-tubulin, and calmodulin. These gene trees were found to be concordant and distinguished two distinct clades within M. grisea. One clade is associated with the grass genus Digitaria and is therefore nomenclaturally tied to M. grisea. The other clade is associated with Oryza sativa and other cultivated grasses and is described as a new species, M. oryzae. While no morphological characters as yet distinguish them, M. oryzae is distinguished from M. grisea by several base substitutions in each of three loci as well as results from laboratory matings; M.oryzae and M. grisea are not interfertile. Given that M. oryzae is the scientifically correct name for isolates associated with rice blast and grey leaf spot, continued use of M. grisea for such isolates would require formal nomenclatural conservation.

Evolution of Drug Resistance in Experimental Populations of Candida albicans

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.

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.

Population genomics of drug resistance in Candida albicans

We followed adaptation in experimental microbial populations to inhibitory concentrations of an antimicrobial drug. The evolution of drug resistance was accompanied in all cases by changes in gene expression that persisted in the absence of the drug; the new patterns of gene expression were constitutive. The changes in gene expression occurred in four replicate populations of the pathogenic fungus Candida albicans during 330 generations of evolution in the presence of the antifungal drug fluconazole. Genome-wide expression profiling of over 5,000 ORFs identified 301 whose expression was significantly modulated. Cluster analysis identified three distinct patterns of gene expression underlying adaptation to the drug. One pattern was unique to one population and included up-regulation of the multidrug ATP-binding cassette transporter gene, CDR2. A second pattern occurred at a late stage of adaptation in three populations; for two of these populations profiled earlier in their evolution, a different pattern was observed at an early stage of adaptation. The succession of early- and late-stage patterns of gene expression, both of which include up-regulation of the multidrug major facilitator transporter gene, MDR1, must represent a common program of adaptation to this antifungal drug. The three patterns of gene expression were also identified in fluconazole-resistant clinical isolates, providing further evidence that these patterns represent common programs of adaptation to fluconazole.

Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotinia sclerotiorum

Two thousand seven hundred and forty‐seven isolates of Sclerotinia sclerotiorum were sampled from four field populations of canola in western Canada. Each field was sampled in a grid of 128 50‐m 50‐m quadrats plus four intensive quadrats each sampled in a diagonal transect. Sampling was done at two phases of the disease cycle: (1) from ascospore inoculum on petals and (2) from disease lesions in stems. A total of 594 unique genotypes was identified by DNA fingerprinting. In each field, a small group of clones represented the majority of the sample, with a large group of clones or genotypes sampled once or twice. Clone frequencies were compared by χ2 tests. The difference in profiles of clone frequencies for the two fields sampled in 1991 was not significant, but in 1992 the difference in profiles was marginally significant, indicating some local population substructure. The difference in profiles of clone frequencies for petals and lesions was not significant in each of the two fields sampled in 1991. In each of the two fields sampled in 1992, however, the difference was highly significant, consistent either with selection for some clones or with waves of immigration during the disease cycle. Nine of the 30 most frequently sampled clones from this study were previously recovered in a macrogeographical sample from western Canada in 1990. For spatial analyses, randomization tests indicated no significant spatial aggregation of either clones on petals or clones from lesions. Also, isolates of a clone on petals were not closer to isolates of the same clone from lesions than could be predicted by chance. Both observations suggest spatial mixing of ascospore inoculum from resident or immigrant sources.

PATTERNS OF DESCENT IN CLONAL LINEAGES AND THEIR MULTILOCUS FINGERPRINTS ARE RESOLVED WITH COMBINED GENE GENEALOGIES

Clonal lineages in the filamentous ascomycete (fungi) Sclerotinia sclerotiorum were determined by analysis of genealogies of four loci: the intergenic spacer of the nuclear ribosomal repeat (IGS; approximately 4 kb), the translation elongation factor (EF‐1α; approximately 300 bp), an anonymous region (44.11; approximately 700 bp), and the calmodulin gene (CAL; approximately 400 bp). Three of the four loci are physically unlinked. The combined analysis of the four loci provided the best estimate of phylogeny, which is consistent with a pattern of some recombination among clonal lineages against a background of predominant clonality. Comparison of gene genealogies with a phylogeny inferred from DNA fingerprints and a combined phylogeny of the entire dataset identified convergent or parallel changes in fingerprints. Analysis of the entire data matrix allowed us to resolve patterns of descent among clonal lineages that could not be inferred from fingerprints alone and to discern recent episodes of divergence that were not detected in gene genealogies. Prerequisites for applying this approach to other systems are a haploid context for inferring multiple gene genealogies (such as the mitochondrial genome) that indicate limited recombination and another data matrix that identifies recently evolved genotypes.

The mycorrhizal status of plants at Alexandra Fiord, Ellesmere Island, Canada, a high arctic site

To determine whether or not mycorrhizae were present at Alexandra Fiord, as well as which types were present, twenty-four species of plants were sampled and their roots examined for fungal colonization

A microbial population - species interface: nested cladistic and coalescent inference with multilocus data

Using sequence data from seven nuclear loci in 385 isolates of the haploid, plant parasitic, ascomycete fungus, Sclerotinia, divergence times of populations and of species were distinguished. The evolutionary history of haplotypes on both population and species scales was reconstructed using a combination of parsimony, maximum likelihood and coalescent methods, implemented in a specific order. Analysis of site compatibility revealed recombination blocks from which alternative (marginal) networks were inferred, reducing uncertainty in the network due to recombination. Our own modifications of Templeton and co‐workers’ cladistic inference method and a coalescent approach detected the same phylogeographic processes. Assuming neutrality and a molecular clock, the boundary between divergent populations and species is an interval of time between coalescence (to a common ancestor) of populations and coalescence of species.

Mycelial interactions inSclerotinia sclerotiorum

Abstract Mycelial interactions were examined among 35 isolates of Sclerotinia sclerotiorum and two Asian species, Sclerotinia asari and an unnamed, Japanese species. Pairings were scored as compatible when strains merged to form one colony and incompatible when strains grew to form two distinct colonies. Incompatible mycelial pairings resulted in an interaction zone in which a distinct reaction line and abundant aerial mycelium or thin mycelium were observed with some variation among replicates. All pairings of a strain with itself were compatible. Of the 31 strains of S. sclerotiorum tested, 21 were mycelially incompatible with all others. Among the remaining 10 strains of S. sclerotiorum , there were four mycelial compatibility groups consisting of two or three strains each. Pairings of S. asari with all other strains resulted in a unique incompatible reaction, a mycelium-free interaction zone. Two of three strains of the Japanese species were intercompatible, but pairings of each of the three strains with all other strains were incompatible. Microscopically, mycelial interactions in pairings of strains were complex. Anastomosis between paired strains was not always observed. This may be due in part to the conversion of many hyphal tips, in both compatible and incompatible interactions, to sites of microconidiogenesis no longer capable of hyphal fusion. Incompatible pairings were followed by hyphal deterioration in one or both strains; hyphal deterioration was not observed in compatible interactions. Of the 31 strains tested, 4 strains of S. sclerotiorum produced apothecia. Pairings between single ascospore isolates within each strain were compatible, as were pairings with the parent isolate. Mycelial interactions of single ascospore isolates with other strains were identical to those of the parent isolate, indicating that the parent fruitbody was homozygous for any determinant(s) of mycelial incompatibility. The data from this study suggest that a high level of mycelial incompatibility exists among strains of S. sclerotiorum , comparable to levels of vegetative incompatibility reported in other ascomycetes, that the extent of mycelial incompatibility indicates that genetic heterogeneity exists within the species, and that mycelial compatibility/incompatibility reactions may be an effective way of categorizing intraspecific heterogeneity.