Genetic Covariation Between the Vertically Transmitted Endophyte Epichloë canadensis and Its Host Canada Wildrye.

Symbiotic mutualisms are thought to be stabilized by correlations between the interacting genotypes which may be strengthened via vertical transmission and/or reduced genetic variability within each species. Vertical transmission, however, may weaken interactions over time as the endosymbionts would acquire mutations that could not be purged. Additionally, temporal variation in a conditional mutualism could create genetic variation and increased variation in the interaction outcome. In this study, we assessed genetic variation in both members of a symbiosis, the endosymbiotic fungal endophyte Epichloë canadensis and its grass host Canada wildrye (Elymus canadensis). Both species exhibited comparable levels of diversity, mostly within populations rather than between. There were significant differences between populations, although not in the same pattern for the two species, and the differences were not correlated with geographic distance for either species. Interindividual genetic distance matrices for the two species were significantly correlated, although all combinations of discriminant analysis of principle components (DAPC) defined multilocus genotype groups were found suggesting that strict genotype matching is not necessary. Variation in interaction outcome is common in grass/endophyte interactions, and our results suggest that the accumulation of mutations overtime combined with temporal variation in selection pressures increasing genetic variation in the symbiosis may be the cause.

Does the Degree of Mutualism between Epichloë Fungi and Botanophila Flies Depend upon the Reproductive Mode of the Fungi?

Epichloë (Ascomycota: Clavicipitaceae) fungi can form an intriguing interaction with Botanophila flies. The fungi live within above-ground shoots of grasses. Some species (type I) only reproduce sexually by forming stromata on all host culms (choke disease). Stromata produce haploid spores (spermatia) that fertilize stromata of opposite mating type to form dikaryotic cells. A second category of Epichloë species (type II) produces stromata on only some of the host culms; culms without choke produce flowers and seeds. These Epichloë can reproduce asexually by invading host seed, as well as sexually. Female Botanophila flies visit stromata for feeding and oviposition. Spermatia pass through the gut of Botanophila intact and viable. Flies can cross-fertilize the fungus during defecation after egg laying. Hence, we described the interaction as a mutualism similar to pollination. Yet, subsequent work by others and ourselves showed that visitation by Botanophila flies was not necessary for cross fertilization of Epichloë. We believe these contradictory results can be reconciled from an evolutionary perspective, if one takes into account the reproductive mode of the fungus. We explore a novel hypothesis to reconcile this contradiction, its predictions and discuss ways in which to test them.

Complex Interactions among Sheep, Insects, Grass, and Fungi in a Simple New Zealand Grazing System.

Epichloë fungi (Ascomycota) live within aboveground tissues of grasses and can have important implications for natural and managed ecosystems through production of alkaloids. Nonetheless, vertebrate herbivores may possess traits, like oral secretions, that mitigate effects of alkaloids. We tested if sheep saliva mitigates effects of Epichloë alkaloids on a beetle pest of perennial ryegrass (Lolium perenne L.) in a New Zealand pasture setting. Plants with one of several fungal isolates were clipped with scissors, grazed by sheep, or clipped with sheep saliva applied to cut ends of stems. We then assessed feeding damage by Argentine stem weevils on blade segments collected from experimental plants. We found that clipping plants induced synthesis of an alkaloid that reduces feeding by beetles and that sheep saliva mitigates this effect. Unexpectedly, the alkaloid (perloline) that explains variation in beetle feeding is one produced not by the endophyte, but rather by the plant. Yet, these effects depended upon fungal isolate. Such indirect, complex interactions may be much more common in both managed and natural grassland systems than typically thought and could have implications for managing grazing systems.

Primers to amplify SNP markers in Epichloë canadensis (Clavicipitaceae).

PREMISE OF THE STUDY: Primers were designed to produce short amplicons containing single-nucleotide polymorphisms (SNPs) in ß-tubulin (tubB) and translation elongation factor 1-α (tefA) in Epichloë canadensis (Clavicipitaceae), an endophytic fungus of Elymus canadensis (Poaceae). METHODS AND RESULTS: Primers to amplify regions of tubB and tefA containing suspected SNPs were designed and tested on individuals from six populations. Two tubB alleles were identified that differed by a single SNP, and three tefA alleles were identified that differed by a combination of two SNPs. All six populations tested were polymorphic for the tefA marker, and three of the populations were also polymorphic for the tubB marker. These primers are also predicted to amplify these regions in 11 additional epichloid species. CONCLUSIONS: Primers for short amplicons within tubB and tefA genes can be used to successfully genotype E. canadensis, making them useful markers for population genetic or landscape genomic studies.

Do Botanophila flies provide reproductive isolation between two species of Epichloë fungi? A field test.

Epichloë spp., fungal endophytes of cool season grasses, produce collars of mycelium (stromata) on host stems that Botanophila flies visit for egg laying. Flies transfer fungal gametes among stromata and thereby serve to cross-fertilize fungi. Hence, the interaction is analogous to insect pollination in angiosperms. While most Epichloë species are not interfertile, Epichloë typhina and Epichloë clarkii can hybridize. We investigated whether Botanophila flies play a role in the reproductive isolation of the two Epichloë species at a field site in southwestern Switzerland. We estimated the density of stromata and collected fly larvae and stromata occurring on plants. While most ascospores collected from both species indicated intraspecific mating, 9.3% of fungal fruiting bodies contained spores of hybrid origin. Two species of Botanophila larvae occurred on stromata and both preferred E. typhina. Yet, both fly species laid eggs on both fungal species. While preferences by Botanophila flies should influence reproductive isolation between the fungi, other mechanisms are likely more important. Our data, which show hybrid ascospores are produced, suggest postzygotic isolating mechanisms are an important means of reproductive isolation.

Effects of methyl jasmonate and an endophytic fungus on plant resistance to insect herbivores.

Tall fescue (Lolium arundinaceum) forms a mutualistic relationship with the fungal endophyte Neotyphodium coenophialum. The endophyte provides constitutive resistance to herbivores through its production of alkaloid compounds. Moreover, herbivore attack induces elevated synthesis of loline alkaloids, that is, the fungus also provides wound-inducible resistance for its host. Jasmonic acid and its conjugates are key signaling compounds in many plant species and play a role systemically in the upregulation of defensive compounds within plants following attack by herbivores. The purpose of our study was to determine if and how the plant and fungus respond to methyl jasmonate (MJ) exposure and if these responses interact in antagonistic or synergistic ways. Plants were exposed to MJ via gaseous diffusion within a controlled environment chamber. Response to MJ was assessed with an herbivore bioassay, gas chromatography-mass spectrometry to quantify alkaloids, and real-time reverse transcriptase-polymerase chain reaction to quantify mRNA from a loline alkaloid biosynthesis gene. We found that MJ hindered endophyte-infected tall fescue's resistance against aphids by downregulating transcription of the LolC gene. The opposite pattern was observed for endophyte-free tall fescue; its exposure to MJ resulted in a significant increase in resistance to aphids, apparently through stimulating defense compounds produced by the plant. These results indicate that, when tall fescue lacks fungal infection, MJ induces the plant to produce its own defensive compounds. In contrast, while endophyte-infected plants are defended from herbivores by fungally produced lolines, this defense is compromised by MJ.

Symbiont-mediated changes in Lolium arundinaceum inducible defenses: evidence from changes in gene expression and leaf composition.

Plants have multiple strategies to deal with herbivory, ranging from chemical or physical defenses to tolerating damage and allocating resources for regrowth. Grasses usually tolerate herbivory, but for some cool-season grasses, their strategy may depend upon their interactions with intracellular symbionts. Neotyphodium endophytes are common symbionts in pooid grasses, and, for some host species, they provide chemical defenses against both vertebrate and invertebrate herbivores. Here, it was tested whether defenses provided by Neotyphodium coenophialum in Lolium arundinaceum (tall fescue) are inducible by both mechanical damage and herbivory from an invertebrate herbivore, Spodoptera frugiperda (fall armyworm), via a bioassay and by quantifying mRNA expression for lolC, a gene required for loline biosysnthesis. Both mechanical and herbivore damage had a negative effect on the reproduction of a subsequent herbivore, Rhopalosiphum padi (bird cherry-oat aphid), and herbivore damage caused an up-regulation of lolC. Uninfected grass hosts also had significantly higher foliar N% and lower C:N ratio compared with infected hosts, suggesting greater allocation to growth rather than defense. For L. arundinaceum, N. coenophialum appears to switch its host's defensive strategy from tolerance via compensation to resistance.

"Pollination" of a fungus by a fly.

The mechanism resulting in fertilization of Epichloë typhina, a heterothallic ascomycete that is an endophytic pathogen of grasses, has now been discovered. Conidia of one mating type are produced in stromata and are then transferred by insects to individuals of the opposite mating type. One insect, Phorbia phrenione, is a particularly important vector of conidia. Once conidia of the opposite mating type have been transferred to a stroma, the life cycle continues with the formation of perithecia.

Selective oviposition by a leaf miner in response to temporal variation in abscission.

We studied within-tree variaton in density and mortality of the leaf miner, Cameraria sp. nov., on Emory oak. Miner densities were lower in the peripheral than the central parts of trees. Following tagged mined and unmined leaves revealed that peripheral leaves fall sooner than central leaves and that more Cameraria mining peripheral leaves die due to abscission than those mining central leaves. We discuss factors that might influence interactions between leaf miners and abscission of the host-leaves they attack.

Abundance and community structure of forest floor spiders following litter manipulation.

We conducted a field manipulation of forest litter to determine effects of litter structure (flat vs curly leaves) on forest floor spiders in natural and artificial leaf litter. Artificial litter made of vinyl, was nondecomposable and non-nutritive. In this way, we separated interactions of effects of litter as a nutritional base and as a spatially heterogeneous environment on litter-dwelling spiders.Structural complexity of litter significantly affected abundances of some forest floor spiders. In particular, abundances of web-building spiders were lower in treatments of flat leaves. Litter nutritional content and structural complexity only slightly affected spider species composition and richness. Results suggest that litter depth is more influential than are litter structural complexity or nutritional content, in organizing forest floor spider communities.