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Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë-host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Epichloe fungi are endophytes of cool season grasses, both wild species and commercial cultivars, where they may exhibit mutualistic or pathogenic lifestyles. The Epichloe-grass symbiosis is of great interest to agricultural research for the fungal bioprotective properties conferred to host grasses but also serves as an ideal system to study the evolution of fungal plant-pathogens in natural environments. Here, we assembled and annotated gapless chromosome-level genomes of two pathogenic Epichloe sibling species. Both genomes have a bipartite genome organization, with blocks of highly syntenic gene-rich regions separated by blocks of AT-rich DNA. The AT-rich regions show an extensive signature of RIP (repeat-induced point mutation) and the expansion of this compartment accounts for the large difference in genome size between the two species. This study reveals how the rapid evolution of repeat structure can drive divergence between closely related taxa and highlights the evolutionary role of dynamic compartments in fungal genomes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mate recognition mechanisms resulting in assortative mating constitute an effective reproductive barrier that may promote sexual isolation and speciation. While such mechanisms are widely documented for animals and plants, they remain poorly studied in fungi. We used two interfertile species of Epichloë (Clavicipitaceae, Ascomycota), E. typhina and E. clarkii, which are host-specific endophytes of two sympatrically occurring grasses. The life cycle of these obligatory outcrossing fungi entails dispersal of gametes by a fly vector among external fungal structures (stromata). To test for assortative mating, we mimicked the natural fertilization process by applying mixtures of spermatia from both species and examined their reproductive success. Our trials revealed that fertilization is non-random and preferentially takes place between conspecific mating partners, which is indicative of assortative mating. Additionally, the viability of hybrid and non-hybrid ascospore offspring was assessed. Germination rates were lower in E. clarkii than in E. typhina and were reduced in ascospore progeny from treatments with high proportions of heterospecific spermatia. The preferential mating between conspecific genotypes and reduced hybrid viability represent important reproductive barriers that have not been documented before in Epichloë. Insights from fungal systems will deepen our understanding of the evolutionary mechanisms leading to reproductive isolation and speciation., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Host specialization is a key process in ecological divergence and speciation of plant-associated fungi. The underlying determinants of host specialization are generally poorly understood, especially in endophytes, which constitute one of the most abundant components of the plant microbiome. We addressed the genetic basis of host specialization in two sympatric subspecies of grass-endophytic fungi from the Epichloë typhina complex: subsp. typhina and clarkii. The life cycle of these fungi entails unrestricted dispersal of gametes and sexual reproduction before infection of a new host, implying that the host imposes a selective barrier on viability of the progeny. We aimed to detect genes under divergent selection between subspecies, experiencing restricted gene flow due to adaptation to different hosts. Using pooled whole-genome sequencing data, we combined F ST and D XY population statistics in genome scans and detected 57 outlier genes showing strong differentiation between the two subspecies. Genomewide analyses of nucleotide diversity (π), Tajima's D and dN/dS ratios indicated that these genes have evolved under positive selection. Genes encoding secreted proteins were enriched among the genes showing evidence of positive selection, suggesting that molecular plant-fungus interactions are strong drivers of endophyte divergence. We focused on five genes encoding secreted proteins, which were further sequenced in 28 additional isolates collected across Europe to assess genetic variation in a larger sample size. Signature of positive selection in these isolates and putative identification of pathogenic function supports our findings that these genes represent strong candidates for host specialization determinants in Epichloë endophytes. Our results highlight the role of secreted proteins as key determinants of host specialization., (© 2018 John Wiley & Sons Ltd.)

Reproductive isolation is central to the maintenance of species, and especially in sympatry, effective barriers to prevent interspecific crosses are expected. Host specificity is thought to constitute an effective mechanism for the formation of barriers in different genera of Fungi, but evidence for endophytes is so far lacking. Sexual Epichloë species (Ascomycota, Clavicipitaceae) represent an ideal study system to investigate the mechanisms underlying speciation as mediated by host specificity because they include species complexes with several host-specific taxa. Here, we studied genetic differentiation of three host-specific Epichloë species using microsatellite markers that were newly in silico identified on the genome of Epichloë poae. Among these, 15 were experimentally tested and applied to study an extensive sampling of isolates representing Epichloë typhina infecting Dactylis glomerata and Epichloë clarkii infecting Holcus lanatus from a site with sympatric populations in Switzerland, as well as a reduced sampling of E. poae infecting Poa nemoralis to create a three-taxon dataset. Both principal coordinate analysis and Bayesian clustering algorithm showed three genetically distinct groups representing the three host-specific species. High pairwise F ST values among the three species, as well as sequencing data of the tefA gene revealing diagnostic single nucleotide polymorphisms (SNPs), further support the hypothesis of genetic discontinuities among the taxa. These results provide genotypic evidence of the maintenance of reproductive isolation of the species in a context of sympatry. In silico testing of 885 discovered microsatellites on the genome of Epichloë festucae extend their applicability to a wider taxonomic range of Epichloë.

Achnatherum inebrians, colloquially known as drunken horse grass, is associated with livestock toxicity in northern China. Epichloë gansuensis (Eg) was described from endophyte isolates from A. inebrians in Sunan County, Gansu Province, whereas a morphologically distinct variety, E. gansuensis var. inebrians (Ei), was described based on two isolates from A. inebrians seeds collected in Urumqi County, Xinjiang Province. Genome sequencing and alkaloid analyses also distinguish these taxa; the Ei isolates produce neurotropic lysergic acid amides (ergot alkaloids), and an Eg isolate produces paxilline (an indole-diterpene alkaloid). To better elucidate the taxonomic diversity of Epichloë spp. symbiotic with A. inebrians, we surveyed eight populations in Xinjiang, Gansu and Inner Mongolia provinces of China and analyzed their genotypes by multiplex PCR for alkaloid biosynthesis genes and mating-type genes. Genotypes consistent with Ei were present in all eight populations, of which they dominated seven. The Ei isolates were all mating type A and tested positive for the ergot alkaloid gene, dmaW. In contrast Eg isolates were all mating type B and had the indole-diterpene gene, idtG. The genome was sequenced from an Ei isolate from seeds collected in Xiahe County, Gansu, and compared to that of the varietal ex type isolate from Urumqi. Alkaloid genes and four different housekeeping genes were nearly identical between the two sequenced Ei isolates and were distinct from a sequenced Eg isolate. Phylogenetic analysis placed Ei, Eg and Epichloë sibirica into respective subclades of a clade that emanated from the base of the Epichloë phylogeny. Given its chemotypic, genotypic, morphological and phylogenetic distinctiveness, its widespread occurrence in rangelands of northern China, and its importance in livestock toxicity, we propose raising Ei to species rank as Epichloë inebrians., (© 2015 by The Mycological Society of America.)

Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloë species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloë descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloë. We have reexamined classification of several described Epichloë and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloë, including distinct species, subspecies, and varieties. We exclude from Epichloë the two taxa Neotyphodium starrii, as nomen dubium, and Neotyphodium chilense, as an unrelated taxon.

Interspecific hybrid endophytes of the genus Epichloë (Ascomycota, Clavicipitaceae) are prevalent in wild grass populations, possibly because of their larger gene variation, resulting in increased fitness benefits for host plants; however, the reasons are not yet known. We tested hypotheses regarding niche expansion mediated by hybrid endophytes, population-dependent interactions and local co-adaptation in the woodland grass Hordelymus europaeus, which naturally hosts both hybrid and non-hybrid endophyte taxa. Seedlings derived from seeds of four grass populations made endophyte free were re-inoculated with hybrid or non-hybrid endophyte strains, or left endophyte free. Plants were grown in the glasshouse with or without drought treatment. Endophyte infection increased plant biomass and tiller production by 10-15% in both treatments. Endophyte types had similar effects on growth, but opposite effects on reproduction: non-hybrid endophytes increased seed production, whereas hybrid endophytes reduced or prevented it completely. The results are consistent with the observation that non-hybrid endophytes in H. europaeus prevail at dry sites, but cannot explain the prevalence of hybrid endophytes. Thus, our results do not support the hypothesis of niche expansion of hybrid-infected plants. Moreover, plants inoculated with native relative to foreign endophytes yielded higher infections, but both showed similar growth and survival, suggesting weak co-adaptation., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Epichloë endophytes (Clavicipitaceae, Ascomycota), including asexual forms placed in Neotyphodium, are common in cool-season grasses. Here we characterize the endophytes of the European woodland grass Hordelymus europaeus based on growth characteristics, morphology of conidiophores and conidia and phylogenetic relationships. Of the six different taxa found on H. europaeus, four are new, for which we propose the species names E. hordelymi, E. disjuncta, E. danica and subspecies E. sylvatica subsp. pollinensis. The other two are assigned to previously described E. bromicola and E. sylvatica. E. hordelymi, E. disjuncta and E. danica are asexual interspecific hybrids, while the other taxa are haploid. Only E. sylvatica subsp. pollinensis was found to reproduce sexually on H. europaeus. The high diversity of endophytes may be explained by repeated host jumps to H. europaeus with and without subsequent interspecific hybridizations.

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

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., Competing Interests: The authors have declared that no competing interests exist.

Epichloid fungal endophytes (Epichloë and Neotyphodium spp.) are excellent model systems for studying speciation processes because of their variable life history traits that are linked to host grass fitness. Presumed jumps to new hosts and subsequent somatic hybridizations appear to be common among epichloid endophytes resulting in increased genetic variation upon which selection can act and speciation be initiated. In this study, we explored the endophyte diversity of a rare European native woodland grass species, Hordelymus europaeus, along a latitudinal transect covering the entire distribution range of H. europaeus. From 28 populations in six countries, isolates were sampled and molecularly characterized. Based on the sequences of tubB and tefA, six distinct epichloid taxa (interspecific hybrid or cryptic haploid species) were found, of which four were novel and two have been previously reported from this host. Of the novel endophytes, two were presumed to be interspecific hybrids and two of nonhybrid origin. While previously known endophytes of H. europaeus are seed-born and strictly asexual, one of the novel nonhybrid endophytes found in the glacial refugium of the Apennine peninsula reproduced sexually in cultured plants. This is the first case of a seed-borne, but sexually reproducing endophyte of this host. We discuss the origin, and possible ancestral species, of the six epichloid taxa using phylogenetic analyses. Repeated host jumps and somatic hybridizations characterize the diversity of the endophytes. To date, no other grass species is known to host a larger diversity of endophytes than H. europaeus., (© 2012 Blackwell Publishing Ltd.)

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., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Genome rearrangements in filamentous fungi are prevalent but little is known about the modalities of their evolution, in part because few complete genomes are available within a single genus. To address this, we have generated and compared 15 complete telomere-to-telomere genomes across the phylogeny of a single genus of filamentous fungi, Epichloë. We find that the striking distinction between gene-rich and repeat-rich regions previously reported for isolated species is ubiquitous across the Epichloë genus. We built a species phylogeny from single-copy gene orthologs to provide a comparative framing to study chromosome composition and structural change through evolutionary time. All Epichloë genomes have exactly seven nuclear chromosomes, but despite this conserved ploidy, analyses reveal low synteny and substantial rearrangement of gene content across the genus. These rearrangements are highly lineage-dependent, with most occurring over short evolutionary distances, with long periods of structural stasis. Quantification of chromosomal rearrangements shows they are uncorrelated with numbers of substitutions and evolutionary distances, suggesting that different modes of evolution are acting to create nucleotide and chromosome-scale changes. [ABSTRACT FROM AUTHOR]

Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloë. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus , S. giganteus , and Lolium perenne. All plants were screened for the presence of Epichloë endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of loline alkaloids were measured in 218 infected meadow fescue plants. Average amount of total loline and the proportions of the single loline alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and alkaloid variation observed in E. uncinata. [ABSTRACT FROM AUTHOR]

Epichloë fungi are endophytes within grasses that can form stromata on culms of their hosts. Botanophila flies visit the stromata for egg laying and in the process can vector spermatial spores, thereby cross fertilising the fungus. Following egg hatch, larval flies consume fungal tissue and spores. Thus, Epichloë individuals with traits that limit larval consumption could be at a selective advantage. We assessed Botanophila fly larvae from sites within the United States and Europe for infection by the bacterial sexual parasite Wolbachia through amplification of the Wolbachia surface protein gene (wsp). Nearly 70% of fly larvae in our samples were infected by Wolbachia. This is the first record of infection by Wolbachia within Botanophila and could have far reaching effects on not only the fly host, but also the Epichloë fungi upon which Botanophila feeds as well as the grass host within which the fungi live. For example, infection by Wolbachia could limit consumption of Epichloë spores by Botanophila larvae if the bacteria promoted premature larval death. [ABSTRACT FROM AUTHOR]

Achnatherum inebrians, colloquially known as drunken horse grass, is associated with livestock toxicity in northern China. Epichloe? gansuensis (Eg) was described from endophyte isolates from A. inebrians in Sunan County, Gansu Province, whereas a morphologically distinct variety, E. gansuensis var. inebrians (Ei), was described based on two isolates from A. inebrians seeds collected in Urumqi County, Xinjiang Province. Genome sequencing and alkaloid analyses also distinguish these taxa; the Ei isolates produce neurotropic lysergic acid amides (ergot alkaloids), and an Eg isolate produces paxilline (an indole-diterpene alkaloid). To better elucidate the taxonomic diversity of Epichloe? spp. symbiotic with A. inebrians, we surveyed eight populations in Xinjiang, Gansu and Inner Mongolia provinces of China and analyzed their genotypes by multiplex PCR for alkaloid biosynthesis genes and mating-type genes. Genotypes consistent with Ei were present in all eight populations, of which they dominated seven. The Ei isolates were all mating type A and tested positive for the ergot alkaloid gene, dmaW. In contrast Eg isolates were all mating type B and had the indole-diterpene gene, idtG. The genome was sequenced from an Ei isolate from seeds collected in Xiahe County, Gansu, and compared to that of the varietal ex type isolate from Urumqi. Alkaloid genes and four different housekeeping genes were nearly identical between the two sequenced Ei isolates and were distinct from a sequenced Eg isolate. Phylogenetic analysis placed Ei, Eg and Epichloe? sibirica into respective subclades of a clade that emanated from the base of the Epichloe? phylogeny. Given its chemotypic, genotypic, morphological and phylogenetic distinctiveness, its widespread occurrence in rangelands of northern China, and its importance in livestock toxicity, we propose raising Ei to species rank as Epichloe? inebrians. [ABSTRACT FROM AUTHOR]

Grass-infecting Epichloё fungi depend on flies of the genus Botanophila (Diptera: Anthomyiidae) for the transfer of spermatia (gametes) and fertilization. Flies lay eggs on fungal structures and their larvae use this as a source of food, hence, this interaction is generally considered to be obligatory and mutualistic. In the present study, we monitored the occurrence of Botanophila flies in a population of Epichloë-infected wild orchardgrass (Dactylis glomerata) in central Poland. The number of eggs per stroma ranged from 0 to 14. Overall, 24.83% of all examined stromata had no eggs or brood chambers, whereas the incidence of fertilized stromata ranged from 89.66% to 100% depending on the year. Nearly all stromata that exhibited no signs of fly visitation were fertilized. These results suggest that in the Polish population of D. glomerata, sexual reproduction of Epichloё fungi depends on spermatia-carrying vectors other than Botanophila. [ABSTRACT FROM AUTHOR]

Abstract: Epichloë fungi (Ascomycota: Clavicipitaceae) are endophytes of grasses that can produce epiphytic stromata on the culms of their hosts. The fungal stromata are visited by Botanophila flies for feeding and egg laying. We review research that has documented the heterothallic mating system of Epichloë, the mutualistic service of spermatization flies provide for the fungus, and host selection by flies. Flies display an active, stereotypical behavior immediately following oviposition by which spermatia are transferred endozoochorously to stromata. After eggs hatch larvae feed on developing perithecia. Several studies have focused on the cost (consumption of ascospores) to the fungus of engaging in the mutualism with its insect visitors. Generally, researchers have found benefits outweigh costs suggesting the mutualism is stable, however exceptions have been reported for Epichloë infecting some commercially cultivated grasses. Because Epichloë fungi are obligate outcrossers and flies are the major (perhaps only) vector of spermatia, the possibility exists that flies could promote reproductive isolation among Epichloë species through specific behavior. This idea has been tested using different approaches including observations of flies within screened cages containing Epichloë species and an analysis of fly gut content, which revealed no or very moderate selectivity by flies. Volatile fungal compounds are responsible for fly attraction and differ among Epichloë species. However, in a field bioassay using species-specific blends of the two predominant volatiles – a sesquiterpene alcohol (chokol K) and a methyl ester – flies showed no preference for specific blends. That is, flies do not appear able to distinguish between different fungal species based on their odor profiles. Thus, it appears that the flies'' role in maintaining reproductive isolation among Epichloë species may be minor and that mechanisms of post-zygotic reproductive isolation are more important in keeping compatible species distinct. However, ethological mechanisms such as ‘stroma constancy’ favoring intraspecific mating may operate at a local scale and need to be investigated further. [Copyright &y& Elsevier]

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 attraction. [ABSTRACT FROM AUTHOR]

• Grass-infecting Epichloëendophytes (Ascomycota, Calvicipitaceae) depend on Botanophila flies for gamete transfer, while fly larvae feed and develop on the fertilized fungal fruiting structures. Flies are known to be attracted by volatile signals, but the exact mechanisms of chemical communication and the degree of specialization are unknown. • Headspace samples collected from five different Epichloëspecies were analysed with respect to physiologically active substances using Botanophila flies. In field bioassays using synthetic compounds, their attractiveness and the specificity of the Epichloë–Botanophila attraction were investigated. • The identification of a new natural product, methyl ( Z)-3-methyldodec-2-enoate, attracting Botanophila flies is reported here, and chokol K is confirmed as an attractive compound. Different blends of the two compounds attracted Botanophila flies under field conditions, but the three fly taxa present at the study site showed no preference for specific blends of volatiles. • Chemical communication in the Epichloë– Botanophila system relies on a few specific compounds, known as a communication system with ‘private channels’. Although ratios of emitted compounds vary in different Epichloë species, this seems not to lead to specialized attraction of Botanophila flies. Low selective pressure for specialization may have maintained a more generalist interaction between fungi and flies. [ABSTRACT FROM AUTHOR]

Grasses (family Poaceae) and fungi of the family Clavicipitaceae have a long history of symbiosis ranging in a continuum from mutualisms to antagonisms. This continuum is particularly evident among symbioses involving the fungal genus Epichloë (asexual forms Neotyphodiurn spp.). In the more mutualistic symbiota, the epichloë endophytes are vertically transmitted via host seeds, and in the more antagonistic symbiota they spread contagiously and suppress host seed set. The endophytes gain shelter, nutrition, and dissemination via host propagules, and can contribute an array of host fitness enhancements including protection against insect and vertebrate herbivores and root nematodes, enhancements of drought tolerance and nutrient status, and improved growth particularly of the root. In some systems, such as the tall fescue N. coenophialum symbioses, the plant may depend on the endophyte under many natural conditions. Recent advances in endophyte molecular biology promise to shed light on the mechanisms of the symbioses and host benefits. [ABSTRACT FROM AUTHOR]

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) are systemic symbionts of cool-season grasses. Their interactions with grass hosts may vary between mutualistic and pathogenic depending on the mode of endophyte reproduction. Sexual strains prevent flowering and seed set (choke disease) of the host and can be horizontally transmitted by ascospores, while asexual strains remain asymptomatic and are vertically transmitted through seeds. In Switzerland nearly all plants of Brachypodium sylvaticum (Huds.) P.B. are infected by Epichloeë sylvatica Leuchtmann & Schardl, but choke symptoms arc formed very rarely, and are restricted to particular locations and to a minority of plants at those locations. Earlier research has revealed that F. sylvatica is genetically differentiated into sexual and asexual subpopulations. Given the high level of infection and assuming horizontal transmission of sexual strains, multiple host infections have been predicted. In this study, 25 plants out of 63 examined by isozyme analysis were found to he infected by two or three different endophyte genotypes. In most cases endophyte genotypes appeared to he correlated with the symptom type of a particular tiller, suggesting that the fungal genome controls choke formation and that the sexual and asexual subpopulations are separated at the ramet (tiller) level rather than at the genet (plant) level. These conclusions were further supported by analyses with log-linear models of the population structure of E. sylvatica at four locations where choke symptoms were present. These analyses also revealed a geographic structure in the asexual subpopulation hut not in the sexual subpopulation which could be caused by the different dispersal ranges of their propagules. The rare occurrence of sexually reproducing strains and the dominance of a single genotype in asymptomatic plant populations may be explained by the colonization history of B. sylvaticum and its endophyte in Switzerland. [ABSTRACT FROM AUTHOR]