Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

The Quaternary cold periods in Europe are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in both animals and plants. The phylogeographies of 10 taxa, including mammals (Ursus arctos, Sorex spp., Crocidura suaveolens, Arvicola spp.), amphibians (Triturus spp.), arthropods (Chorthippus parallelus), and plants (Abies alba, Picea abies, Fagus sylvatica, Quercus spp.), were analysed to elucidate general trends across Europe. Only a small degree of congruence was found amongst the phylogeographies of the 10 taxa, but the likely postglacial colonization routes exhibit some similarities. A Brooks parsimony analysis produced an unrooted area phylogram, showing that: (i) the northern regions were colonized generally from the Iberic and Balkanic refugia; and (ii) the Italian lineages were often isolated due to the presence of the Alpine barrier. The comparison of colonization routes highlighted four main suture-zones where lineages from the different refugia meet. Some of the intraspecific genetic distances among lineages indicated a prequaternary divergence that cannot be connected to any particular cold period, but are probably related mainly to the date of arrival of each taxon in the European continent. As a consequence, molecular genetics so far appears to be of limited use in dating Quaternary events.

Comparative phylogeography and postglacial colonization routes in Europe

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 method for designing primer sets for speciation studies in filamentous ascomycetes

A simple random‐walk method for obtaining ab initio solutions of the Schrodinger equation is examined in its application to the case of the molecular ion H+3 in the equilateral triangle configuration with side length R=1.66 bohr. The method, which is based on the similarity of the Schrodinger equation and the diffusion equation, involves the random movement of imaginary particles (psips) in electron configuration space subject to a variable chance of multiplication or disappearance. The computation requirements for high accuracy in determining energies of H+3 are greater than those of existing LCAO–MO–SCF–CI methods. For more complex molecular systems the method may be competitive.

A random‐walk simulation of the Schrödinger equation: H+3

ASEXUALLY reproducing organisms occur in a variety of taxa in all biological kingdoms1 and distinguishing asexually propagated genotypes is essential for the understanding of their population biology. Among the higher fungi, however, the clonal 'individual' is especially difficult to define2 because most of the fungal thallus consists of a network of anastamosing hyphae embedded in the substratum. Whether fruit-bodies, the most recognizable part of a fungus, are produced by a single supporting mycelium can only be determined by establishing direct physiological continuity or genetic identity. We report a means by which individual fungi can be unambiguously identified within local populations and identify an individual of Armillaria bulbosa that occupies a minimum of 15 hectares, weighs in excess of 10,000kg, and has remained genetically stable for more than 1,500 years.

The fungus Armillaria bulbosa is among the largest and oldest living organisms

The random‐walk method of solving the Schrodinger equation for molecular wavefunctions is extended to incorporate the effects of electron spin in several one‐ to four‐electron systems. Improved calculation procedures reduce computation requirements for high accuracy by a factor of about 10. Results are given for the systems H 2P, H+3 D3h 1A′1, H2 3Σ+u, linear equidistant H4 1Σ+g, and Be 1S.

Quantum chemistry by random walk. H 2P, H+3D3h1A′1, H23Σ+u, H41Σ+g, Be 1S

Like other microorganisms, fungi exist in populations that are adaptable. Under the selection imposed by antifungal drugs, drug-sensitive fungal pathogens frequently evolve resistance. Although the molecular mechanisms of resistance are well-characterized, there are few measurements of the impact of these mechanisms on pathogen fitness in different environments. To predict resistance before a new drug is prescribed in the clinic, the full spectrum of potential resistance mutations and the interactions among combinations of divergent mechanisms can be determined in evolution experiments. In the search for new strategies to manage drug resistance, measuring the limits of adaptation might reveal methods for trapping fungal pathogens in evolutionary dead ends.

Evolution of antifungal-drug resistance: mechanisms and pathogen fitness

The evolution of drug resistance is an important process that affects clinical outcomes. Resistance to fluconazole, the most widely used antifungal, is often associated with acquired aneuploidy. Here we provide a longitudinal study of the prevalence and dynamics of gross chromosomal rearrangements, including aneuploidy, in the presence and absence of fluconazole during a well-controlled in vitro evolution experiment using Candida albicans, the most prevalent human fungal pathogen. While no aneuploidy was detected in any of the no-drug control populations, in all fluconazole-treated populations analyzed an isochromosome 5L [i(5L)] appeared soon after drug exposure. This isochromosome was associated with increased fitness in the presence of drug and, over time, became fixed in independent populations. In two separate cases, larger supernumerary chromosomes composed of i(5L) attached to an intact chromosome or chromosome fragment formed during exposure to the drug. Other aneuploidies, particularly trisomies of the smaller chromosomes (Chr3–7), appeared throughout the evolution experiment, and the accumulation of multiple aneuploid chromosomes per cell coincided with the highest resistance to fluconazole. Unlike the case in many other organisms, some isolates carrying i(5L) exhibited improved fitness in the presence, as well as in the absence, of fluconazole. The early appearance of aneuploidy is consistent with a model in which C. albicans becomes more permissive of chromosome rearrangements and segregation defects in the presence of fluconazole.

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James B. Anderson

Papers

A random‐walk simulation of the Schrödinger equation: H+3

The fungus Armillaria bulbosa is among the largest and oldest living organisms

Quantum chemistry by random walk. H 2P, H+3D3h1A′1, H23Σ+u, H41Σ+g, Be 1S

Evolution of antifungal-drug resistance: mechanisms and pathogen fitness

Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance.