Two-speed genomes of Epichloe fungal pathogens show contrasting signatures of selection between species and across populations.

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.

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.

Genetic Diversity of Epichloë Endophytes Associated with Brachypodium and Calamagrostis Host Grass Genera including Two New Species.

Fungi of genus Epichloë (Ascomycota, Clavicipitaceae) are common endophytic symbionts of Poaceae, including wild and agronomically important cool-season grass species (subfam. Poöideae). Here, we examined the genetic diversity of Epichloë from three European species of Brachypodium (B. sylvaticum, B. pinnatum and B. phoenicoides) and three species of Calamagrostis (C. arundinacea, C. purpurea and C. villosa), using DNA sequences of tubB and tefA genes. In addition, microsatellite markers were obtained from a larger set of isolates from B. sylvaticum sampled across Europe. Based on phylogenetic analyses the isolates from Brachypodium hosts were placed in three different subclades within the Epichloë typhina complex (ETC) but did not strictly group according to host grass species, suggesting that the host does not always select for particular endophyte genotypes. Analysis of microsatellite markers confirmed the presence of genetically distinct lineages of Epichloësylvatica on B. sylvaticum, which appeared to be tied to different modes of reproduction (sexual or asexual). Among isolates from Calamagrostis hosts, two subclades were detected which were placed outside ETC. These endophyte lineages are recognized as distinct species for which we propose the names E. calamagrostidis Leuchtm. & Schardl, sp. nov. and E. ftanensis Leuchtm. & A.D. Treindl, sp. nov. This study extends knowledge of the phylogeny and evolutionary diversification of Epichloë endophytes that are symbionts of wild Brachypodium and Calamagrostis host grasses.

Telomere-to-Telomere Genome Sequences across a Single Genus Reveal Highly Variable Chromosome Rearrangement Rates but Absolute Stasis of Chromosome Number.

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.

Cross-species transcriptomics identifies core regulatory changes differentiating the asymptomatic asexual and virulent sexual life cycles of grass-symbiotic Epichloë fungi.

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.

Chromosome-level genomes provide insights into genome evolution, organization and size in Epichloe fungi.

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.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Assortative mating in sympatric ascomycete fungi revealed by experimental fertilizations.

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.

Large Scale Screening of Epichloë Endophytes Infecting Schedonorus pratensis and Other Forage Grasses Reveals a Relation Between Microsatellite-Based Haplotypes and Loline Alkaloid Levels.

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.

Botanophila flies, vectors of Epichloë fungal spores, are infected by Wolbachia.

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.

Genomewide signatures of selection in Epichloë reveal candidate genes for host specialization.

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 FST and DXY 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.

Assessing effects of the entomopathogenic fungus Metarhizium brunneum on soil microbial communities in Agriotes spp. biological pest control.

The release of large quantities of microorganisms to soil for purposes such as pest control or plant growth promotion may affect the indigenous soil microbial communities. In our study, we investigated potential effects of Metarhizium brunneum ART2825 on soil fungi and prokaryota in bulk soil using high-throughput sequencing of ribosomal markers. Different formulations of this strain, and combinations of the fungus with garlic as efficacy-enhancing agent, were tested over 4 months in a pot and a field experiment carried out for biological control of Agriotes spp. in potatoes. A biocontrol effect was observed only in the pot experiment, i.e. the application of FCBK resulted in 77% efficacy. Colony counts combined with genotyping and marker sequence abundance confirmed the successful establishment of the applied strain. Only the formulated applied strain caused small shifts in fungal communities in the pot experiment. Treatment effects were in the same range as the effects caused by barley kernels, the carrier of the FCBK formulation and temporal effects. Garlic treatments and time affected prokaryotic communities. In the field experiment, only spatial differences affected fungal and prokaryotic communities. Our findings suggest that M. brunneum may not adversely affect soil microbial communities.

The effect of Piriformospora indica on the root development of maize (Zea mays L.) and remediation of petroleum contaminated soil.

As the depth of soil petroleum contamination can vary substantially under field conditions, a rhizotron experiment was performed to investigate the influence of endophyte, P. indica, on maize growth and degradation of petroleum components in a shallow and a deep-reaching subsurface layer of a soil. For control, a treatment without soil contamination was also included. The degree in contamination and the depth to which it extended had a strong effect on the growth of the plant roots. Contaminated soil layers severely inhibited root growth thus many roots preferred to bypass the shallow contaminated layer and grow in the uncontaminated soil. While the length and branching pattern of these roots were similar to those of uncontaminated treatment. Inoculation of maize with P. indica could improve root distribution and root and shoot growth in all three contamination treatments. This inoculation also enhanced petroleum degradation in soil, especially in the treatment with deep-reaching contamination, consequently the accumulation of petroleum hydrocarbons (PAHs) in the plant tissues were increased.

Multiplexed microsatellite markers for seven Metarhizium species.

Cross-species transferability of 41 previously published simple sequence repeat (SSR) markers was assessed for 11 species of the entomopathogenic fungus Metarhizium. A collection of 65 Metarhizium strains including all 54 used in a recent phylogenetic revision of the genus were characterized. Between 15 and 34 polymorphic SSR markers produced scorable PCR amplicons in seven species, including M. anisopliae, M. brunneum, M. guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii. To provide genotyping tools for concurrent analysis of these seven species fifteen markers grouped in five multiplex pools were selected based on high allelic diversity and easy scorability of SSR chromatograms.

Two distinct Epichloë species symbiotic with Achnatherum inebrians, drunken horse grass.

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.

Genetic evidence for reproductive isolation among sympatric Epichloë endophytes as inferred from newly developed microsatellite markers.

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.

Nomenclatural realignment of Neotyphodium species with genus Epicholë.

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.

Effects of natural hybrid and non-hybrid Epichloë endophytes on the response of Hordelymus europaeus to drought stress.

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.

The Epichloe endophytes associated with the woodland grass Hordelymus europaeus including four new taxa.

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.

Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae.

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.