Endophytic fungi of the genus Epichloë and their asexual Neotyphodium forms are thought to interact mutualistically with their host grasses, providing protection for the host against herbivores and pathogens mediated by fungal alkaloids. Most previous research has concentrated on agronomically important grasses, such as tall fescue, and its interactions with livestock grazers or invertebrate herbivores. In this study we focus on the woodland grass Brachypodium sylvaticum which is infected by the strictly host-specific endophyte Epichloë sylvatica. This fungus has two alternative modes of reproduction: the predominant asexual strains are seed-transmitted, whereas the rare sexual strains are capable of contagious spread by ascospores produced on stromata. To assess potential host protection from herbivory, we tested to what extent development of Spodoptera frugiperda, a noctuid generalist herbivore, was affected when fed on different genotypes of naturally infected (E), artificially infected (F), or uninfected (E) leaf material of B. sylvaticum. In a feeding assay, insect larvae performed significantly better on a diet of uninfected leaves, even though previous studies have not detected alkaloid production by E. sylvatica. A possible explanation for this result may be the presence of an unknown compound in infected plants, acting as metabolic toxin against S. frugiperda larvae. The negative effect on insect larvae was increased when they were fed on a diet artificially infected by a particular genotype (F), suggesting that content of allelochemicals may depend on the fungal genotype. In a dual-choice test, neonate S. frugiperda larvae initially preferred uninfected seedlings over naturally infected seedlings, but only during the first 6 h of the experiment. This suggests that the unknown compounds can act as weak insect-feeding deterrents. To assess herbivory in natural stands of the host grass, nine infected populations of B. sylvaticum were examined for feeding damage. Damage due to microherbivores and macroherbivores was equal in most populations. However, microherbivores (mostly insects) showed a clear preference for tillers bearing fungal stromata, whereas asymptomatically infected tillers were less damaged. Thus, herbivore resistance of infected plants appears to be correlated with the mode of reproduction of fungal genotypes. This result is of evolutionary significance, since asexual, seed-transmitted endophytes depend on host fitness and seed production for their dispersal.

Grass endophytes (Clavicipitaceae, Ascomycota) are generally considered to be mutualists which increase the host's fitness. Infected plants are often more persistent and competitive than uninfected plants, influencing population dynamics and plant community diversity. However, most of this empirical evidence is based on studies focusing on agronomically important grass species such as tall fescue or perennial ryegrass and their implications for livestock and man‐made habitats. Recent studies indicate that endophyte‐plant associations may be more variable, ranging from parasitic to mutualistic. In the present study, we investigated the influence of endophyte infection on two wild woodland grasses, which are naturally infected with distinct fungal endophytes: Brachypodium sylvaticum with Epichloë sylvatica and Bromus benekenii with Epichloë bromicola. An intraspecific competition experiment was conducted over two growing seasons in the greenhouse and in an experimental garden. At first harvest (after 12 weeks growing), endophyte infection had a significant negative effect on above ground dry matter yield (DMY) of B. sylvaticum, but a significant positive effect on DMY of Br. benekenii under competition. The same differential effects on DMY and on total seed number were also observed at final harvest (after 62 weeks growing). Results from Br. benekenii were consistent with our hypothesis of increased competitive abilities of infected plants in nature which could explain the high infection rate observed in natural populations. In contrast, this explanation does not hold true for B. sylvaticum, and other factors such as increased herbivore and pathogen resistance together with frequent horizontal transmission may be responsible for the very high incidence of this association in nature. Our results confirm previous predictions that beneficial effects of endophyte infection in wild grasses can vary for different grass species, even in comparable habitats.

Host shifts of plant-feeding insects and parasites promote adaptational changes that may result in the formation of host races, an assumed intermediate stage in sympatric speciation. Here, we report on genetically differentiated and host-adapted races of the fungal endophyte Epichloë bromicola, which presumably emerged after a shift from the grass Bromus erectus to other Bromus hosts. Fungi of the genus Epichloë (Ascomycota) and related anamorphs of Neotyphodium are widespread endophytes of cool-season grasses. Sexually reproducing strains sterilize the host by formation of external fruiting structures (stromata), whereas asexual strains are asymptomatic and transmitted via seeds. In E. bromicola, strains infecting B. erectus are sexual, and strains from two woodland species, B. benekenii and B. ramosus, are asexual and seed transmitted. Analyses of amplified fragment length polymorphism fingerprinting and of intron sequences of the tub2 and tef1 genes of 26 isolates from the three Bromus hosts collected at natural sites in Switzerland and nearby France demonstrated that isolates are genetically differentiated according to their host, indicating that E. bromicola does not form a single, randomly mating population. Phylogenetic analyses of sequence data did not unambiguously resolve the exact origin of asexual E. bromicola strains, but it is likely they arose from within sexual populations on B. erectus.Incongruence of trees derived from different genes may have resulted from recombination at some time in the recent history of host strains. Reciprocal inoculations of host plant seedlings showed that asexual isolates from B. benekenii and B. ramosus were incapable of infecting B. erectus, whereas the sexual isolates from B. erectus retained the assumed ancestral trait of broad compatibility with Bromus host seedlings. Because all isolates were interfertile in experimental crosses, asexual strains may not be considered independent biological species. We suggest that isolates infecting B. benekenii and B. ramosus represent long-standing host races or incipient species that emerged after host shifts and that may evolve through host-mediated reproductive isolation toward independent species.

Abstract. Host shifts of plant-feeding insects and parasites promote adaptational changes that may result in the formation of host races, an assumed intermediate stage in sympatric speciation. Here, we report on genetically differentiated and host-adapted races of the fungal endophyte Epichloë bromicola, which presumably emerged after a shift from the grass Bromus erectus to other Bromus hosts. Fungi of the genus Epichloë (Ascomycota) and related anamorphs of Neotyphodium are widespread endophytes of cool-season grasses. Sexually reproducing strains sterilize the host by formation of external fruiting structures (stromata), whereas asexual strains are asymptomatic and transmitted via seeds. In E. bromicola, strains infecting B. erectus are sexual, and strains from two woodland species, B. benekenii and B. ramosus, are asexual and seed transmitted. Analyses of amplified fragment length polymorphism fingerprinting and of intron sequences of the tub2 and tef1 genes of 26 isolates from the three Bromus hosts collected at natural sites in Switzerland and nearby France demonstrated that isolates are genetically differentiated according to their host, indicating that E. bromicola does not form a single, randomly mating population. Phylogenetic analyses of sequence data did not unambiguously resolve the exact origin of asexual E. bromicola strains, but it is likely they arose from within sexual populations on B. erectus.Incongruence of trees derived from different genes may have resulted from recombination at some time in the recent history of host strains. Reciprocal inoculations of host plant seedlings showed that asexual isolates from B. benekenii and B. ramosus were incapable of infecting B. erectus, whereas the sexual isolates from B. erectus retained the assumed ancestral trait of broad compatibility with Bromus host seedlings. Because all isolates were interfertile in experimental crosses, asexual strains may not be considered independent biological species. We suggest that isolates infecting B. benekenii and B. ramosus represent long-standing host races or incipient species that emerged after host shifts and that may evolve through host-mediated reproductive isolation toward independent species.

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