The taxonomic position of the genus Heydenia (Pyronemataceae, Pezizales) based on molecular and morphological data

Molecular and morphological data indicate that the genus Heydenia is closely related to the cleistothecial ascomycete Orbicula (Pyronemataceae, Pezizales). Observations on the disposition and the immediate surroundings of immature spores within the spore capsule suggest that the Heydenia fruiting bodies are teleomorphs producing early evanescent asci in stipitate cleistothecia. The once advocated identity of Heydenia with Onygena is refuted on molecular grounds. Onygena arietina E. Fischer is transferred to Heydeni

Taxon-specific PCR primers to detect two inconspicuous arbuscular mycorrhizal fungi from temperate agricultural grassland

Taxon-specific polymerase chain reaction (PCR) primers enable detection of arbuscular mycorrhizal fungi (AMF, Glomeromycota) in plant roots where the fungi lack discriminative morphological and biochemical characters. We designed and validated pairs of new PCR primers targeted to the flanking regions of the variable domain 1 of the nuclear ribosomal large subunit RNA gene to specifically detect Acaulospora paulinae and an undescribed member of the Diversisporaceae. These two fungal taxa, sporulating late in soil-trap cultures and showing small, faintly coloured spores and weakly staining intraradical structures, were frequently found in roots of Trifolium repens from a high-input agricultural grassland. The newly developed PCR primers may thus enable studies on two inconspicuous AMF taxa that appear to have been overlooked in previous molecular AMF community analyses and for which no specific PCR primers have been publishe

Variation of Insect Attracting Odor in Endophytic Epichloë Fungi: Phylogenetic Constrains Versus Host Influence

Odor is a key trait for pollinator attraction in flowering plants, and many studies have investigated odor evolution in the light of pollinator selection by emphasizing the importance of the plant phylogenetic history. By contrast, little is known on the evolution of odors in fungus-insect interactions. In this study, profiles of three volatile compounds that are emitted by grass-inhabiting Epichloë fungi (Clavicipitaceae, Ascomycota) and that have a confirmed or likely role in the attraction of gamete-transferring Botanophila flies were investigated. We collected headspace samples from stromata of six European Epichloë species (including various host races) that originated from different locations in Switzerland, France, Poland, and UK for conducting gas chromatography analyses. Odor profiles exhibited considerable variation, but profiles of most species overlapped and did not discriminate at the species level. The exception was Epichloë festucae, which had a profile dominated by methyl (Z)-3-methyldodec-2-enoate. Based on an Epichloë phylogeny, there was some hierarchical structuring regarding levels of chokol K, another confirmed Botanophila attractant. However, patterns of odor profiles appeared to be largely dependant on particular Epichloë-host associations. The observed variation may be the result of complex selective pressures imposed by Botanophila gametic vectors, local environment, and mycoparasite

Ecological role of volatiles produced by Epichloë: differences in antifungal toxicity

Species of Epichloë (Ascomycota, Clavicipitaceae) are endophytic symbionts of pooid grasses. Sexual reproduction of the fungus depends on gamete-transferring Botanophila flies, which in earlier studies were shown to be specifically attracted by the fungal volatiles chokol K and methyl (Z)-3-methyldodec-2-enoate. As several Epichloë volatiles are known to have antimicrobial properties, it was hypothesised that the original function of insect-attracting volatiles is microbial deterrence. However, the origin of volatile compounds and their toxicity within an ecological context has not yet been clarified. We examined the inhibitory effect of chokol K and methyl (Z)-3-methyldodec-2-enoate on mycoparasites, plant pathogenic fungi and on Epichloë itself at ecologically relevant concentrations, and assessed volatile production in pure cultures of Epichloë on complex and defined media supplemented with inorganic sources of carbon and nitrogen. Chokol K reduced the spore germination of all tested fungi, whereas methyl (Z)-3-methyldodec-2-enoate had no inhibitory effect. Moreover, only chokol K was produced in culture, confirming its fungal origin. Our findings are consistent with the proposed scenario that fungal volatile substances have followed an evolutionary pathway from defence to attractio

Botanophila-Epichloë Interaction in a Wild Grass, Puccinellia distans, Lacks Dependence on the Fly Vector

In grass-infecting Epichloë (Ascomycetes: Clavicipitaceae) fungi, the transfer of spermatia for fungal fertilization depends on an insect vector: flies of the genus Botanophila (Diptera: Anthomyiidae). The flies use the fungal stroma, a spore-producing fungal structure surrounding the grass inflorescence, for laying eggs and as a food source for both adults and larvae. This fly-fungus interaction is generally regarded as obligatory and mutualistic. Two Botanophila taxa were noted among four populations of the nonagricultural grass Puccinellia distans (L.) Parl. that were infected with the fungus Epichloë typhina (Pers.) Tul. However, during the 7 yr of field observations, Botanophila flies were present every year in only one population of P. distans. The number of eggs per stroma ranged from zero to four and differed with year and site. Overall, eggs (or larvae) were observed on only 132 (19.2%) of the 687 stromata examined during the survey, with one (13.8%), two (4.5%), or more than two (0.9%) per stroma. However, 90.8% of the examined stromata were fertilized and produced perithecia, suggesting that other mechanisms or vectors of spermatia were responsible for fertilizatio

Botanophila–Epichloe Interaction in a Wild Grass, Puccinellia distans, Lacks Dependence on the Fly Vector

In grass-infecting Epichloe (Ascomycetes: Clavicipitaceae) fungi, the transfer of spermatia for fungal fertilization depends on an insect vector: flies of the genus Botanophila (Diptera: Anthomyiidae). The flies use the fungal stroma, a spore-producing fungal structure surrounding the grass inßorescence, for laying eggs and as a food source for both adults and larvae. This fly-fungus interaction is generally regarded as obligatory and mutualistic. Two Botanophila taxa were noted among four populations of the nonagricultural grass Puccinellia distans (L.) Parl. that were infected with the fungus Epichloe typhina (Pers.) Tul. However, during the 7 yr of field observations, Botanophila flies were present every year in only one population of P. distans. The number of eggs per stroma ranged from zero to four and differed with year and site. Overall, eggs (or larvae) were observed on only 132 (19.2%) of the 687 stromata examined during the survey, with one (13.8%), two (4.5%), or more than two (0.9%) per stroma. However, 90.8% of the examined stromata were fertilized and produced perithecia, suggesting that other mechanisms or vectors of spermatia were responsible for fertilization

Genetic diversity and population structure of Epichloë fungal pathogens of plants in natural ecosystems

Understanding the population genetic processes driving the evolution of plant pathogens is of central interest to plant pathologists and evolutionary biologists alike. However, most studies focus on host-pathogen associations in agricultural systems of high genetic and environmental homogeneity and less is known about the genetic structure of pathogen populations infecting wild plants in natural ecosystems. We performed parallel population sampling of two pathogenic Epichloë species occurring sympatrically on different host grasses in natural and seminatural grasslands in Europe: E. typhina infecting Dactylis glomerata and E. clarkii infecting Holcus lanatus. We sequenced 422 haploid isolates and generated genome-wide SNP datasets to investigate genetic diversity and population structure. In both species geographically separated populations formed genetically distinct groups, however, population separation was less distinct in E. typhina compared to E. clarkii. The patterns of among population admixture also differed between species across the same geographic range: we found higher levels of population genetic differentiation and a stronger effect of isolation by distance in E. clarkii compared to E. typhina, consistent with lower levels of gene flow in the former. This pattern may be explained by the different dispersal abilities of the two pathogens and is expected to be influenced by the genetic structure of host populations. In addition, genetic diversity was higher in E. typhina populations compared to E. clarkii, indicative of higher effective population size in E. typhina. These results suggest that the effect of genetic drift and the efficacy of selection may differ in the two species. Our study provides evidence of how ecologically similar species occupying the same geographical space can experience different evolutionary contexts, which could influence local adaptation and co-evolutionary dynamics of these fungal pathogens

The occurrence and preference of Botanophila flies (Diptera: Anthomyiidae) for particular species of Epichloë fungi infecting wild grasses

Specific associations between species frequently occur in ecological interactions. The aim of this study was to determine the preferences of anthomyiid flies of the genus Botanophila for particular species of fungi as sites for laying eggs and as food for both larvae and adults. The associations of their eggs, larvae and flies with the stromata of different species of Epichloë fungi infecting 7 species of grass in Poland were analyzed. Scanning electron microscopy of the surface of their eggs and an analysis of the genetic sequences of their mitochondrial cytochrome oxidase (COII) were used to identify the taxa of the flies studied. Three types of eggs were distinguished based on their shape, colour and the presence of dorsal folds and sculpturing on the shells. Tentatively,these eggs were assigned to the following species: B. laterella, B. phrenione, B. dissecta and B. lobata. COII sequences obtained from larvae that hatched from two of the types of eggs formed three distinct clades associated with the reference sequences for Botanophila phrenione, B. lobata (new to the fauna of Poland) and a putative species, “Taxon 1”. Only one of these flies (B. lobata) was restricted to a single species of Epichloë (E. bromicola on Elymus repens); B. phrenione was recorded mainly from E. typhina infecting three different species of grass. The results of this study confirm that there is not a close species specific association between this fungus and this insect

Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses