CLOCI: unveiling cryptic fungal gene clusters with generalized detection.

Gene clusters are genomic loci that contain multiple genes that are functionally and genetically linked. Gene clusters collectively encode diverse functions, including small molecule biosynthesis, nutrient assimilation, metabolite degradation, and production of proteins essential for growth and development. Identifying gene clusters is a powerful tool for small molecule discovery and provides insight into the ecology and evolution of organisms. Current detection algorithms focus on canonical 'core' biosynthetic functions many gene clusters encode, while overlooking uncommon or unknown cluster classes. These overlooked clusters are a potential source of novel natural products and comprise an untold portion of overall gene cluster repertoires. Unbiased, function-agnostic detection algorithms therefore provide an opportunity to reveal novel classes of gene clusters and more precisely define genome organization. We present CLOCI (Co-occurrence Locus and Orthologous Cluster Identifier), an algorithm that identifies gene clusters using multiple proxies of selection for coordinated gene evolution. Our approach generalizes gene cluster detection and gene cluster family circumscription, improves detection of multiple known functional classes, and unveils non-canonical gene clusters. CLOCI is suitable for genome-enabled small molecule mining, and presents an easily tunable approach for delineating gene cluster families and homologous loci.

A global phylogenomic analysis of the shiitake genus Lentinula.

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Genome sequencing progenies of magic mushrooms (Psilocybe subaeruginosa) identifies tetrapolar mating and gene duplications in the psilocybin pathway.

Knowledge of breeding systems and genetic diversity is critical to select and combine desired traits that advance new cultivars in agriculture and horticulture. Mushrooms that produce psilocybin, magic mushrooms, may potentially be used in therapeutic and wellness industries, and stand to benefit from genetic improvement. We studied haploid siblings of Psilocybe subaeruginosa to resolve the genetics behind mating compatibility and advance knowledge of breeding. Our results show that mating in P. subaeruginosa is tetrapolar, with compatibility controlled at a homeodomain locus with one copy each of HD1 and HD2, and a pheromone/receptor locus with four homologs of the receptor gene STE3. An additional two pheromone/receptor loci homologous to STE3 do not appear to regulate mating compatibility. Alleles in the psilocybin gene cluster did not vary among the five siblings and were likely homozygous in the parent. Psilocybe subaeruginosa and its relatives have three copies of PsiH genes but their impact on production of psilocybin and its analogues is unknown. Genetic improvement in Psilocybe will require access to genetic diversity from the centre of origin of different species, identification of genes behind traits, and strategies to avoid inbreeding depression.

Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum.

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the "C" clade are not resolved as distinct from the "A" clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the "B" clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

The Architecture of Metabolism Maximizes Biosynthetic Diversity in the Largest Class of Fungi.

Ecological diversity in fungi is largely defined by metabolic traits, including the ability to produce secondary or "specialized" metabolites (SMs) that mediate interactions with other organisms. Fungal SM pathways are frequently encoded in biosynthetic gene clusters (BGCs), which facilitate the identification and characterization of metabolic pathways. Variation in BGC composition reflects the diversity of their SM products. Recent studies have documented surprising diversity of BGC repertoires among isolates of the same fungal species, yet little is known about how this population-level variation is inherited across macroevolutionary timescales. Here, we applied a novel linkage-based algorithm to reveal previously unexplored dimensions of diversity in BGC composition, distribution, and repertoire across 101 species of Dothideomycetes, which are considered the most phylogenetically diverse class of fungi and known to produce many SMs. We predicted both complementary and overlapping sets of clustered genes compared with existing methods and identified novel gene pairs that associate with known secondary metabolite genes. We found that variation among sets of BGCs in individual genomes is due to nonoverlapping BGC combinations and that several BGCs have biased ecological distributions, consistent with niche-specific selection. We observed that total BGC diversity scales linearly with increasing repertoire size, suggesting that secondary metabolites have little structural redundancy in individual fungi. We project that there is substantial unsampled BGC diversity across specific families of Dothideomycetes, which will provide a roadmap for future sampling efforts. Our approach and findings lend new insight into how BGC diversity is generated and maintained across an entire fungal taxonomic class.

Acurin A, a novel hybrid compound, biosynthesized by individually translated PKS- and NRPS-encoding genes in Aspergillus aculeatus.

This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.

Phlox Species Show Quantitative and Qualitative Resistance to a Population of Powdery Mildew Isolates from the Eastern United States.

Ornamental plants in the genus Phlox are extensively planted in landscapes and home gardens around the world. A major limitation to a more widespread use of these plants is their susceptibility to powdery mildew (PM). In this study, we used multilocus sequence typing (MLST) analysis to gain insights into the population diversity of 32 Phlox PM pathogen (Golovinomyces magnicellulatus and Podosphaera sp.) isolates collected from the eastern United States and relate it to the ability to overcome host resistance. Low genetic diversity and a lack of structure were found within our population. Whole genome comparison of two isolates was used to support low genetic diversity evidence found with the MLST analysis. Recombination was suggested by the incongruences observed in the six phylogenetic trees generated from the housekeeping genes TEF-1α, CSI, ITS, IGS, H3, and TUB. Contrasting with low genetic diversity, we found high phenotypic diversity when using 10 of the 32 isolates to evaluate host resistance in four different Phlox species (P. paniculata 'Dunbar Creek', P. amoena OPGC 3598, P. glaberrima OPGC 3594, and P. subulata OPGC 4185) using in vitro bioassays. We observed quantitative and qualitative resistance in all Phlox species and a consistent low disease severity in our control, P. paniculata 'Dunbar Creek'. Taken together, the results generated in this study constitute a robust screening of popular Phlox germplasm that can be incorporated into breeding programs for PM resistance and provides significant information on the evolution of PM pathogens.

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

BACKGROUND: Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood. RESULTS: In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects. CONCLUSIONS: This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Comparative genomics of biotechnologically important yeasts.

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.

Differential Retention of Gene Functions in a Secondary Metabolite Cluster.

In fungi, distribution of secondary metabolite (SM) gene clusters is often associated with host- or environment-specific benefits provided by SMs. In the plant pathogen Alternaria brassicicola (Dothideomycetes), the DEP cluster confers an ability to synthesize the SM depudecin, a histone deacetylase inhibitor that contributes weakly to virulence. The DEP cluster includes genes encoding enzymes, a transporter, and a transcription regulator. We investigated the distribution and evolution of the DEP cluster in 585 fungal genomes and found a wide but sporadic distribution among Dothideomycetes, Sordariomycetes, and Eurotiomycetes. We confirmed DEP gene expression and depudecin production in one fungus, Fusarium langsethiae. Phylogenetic analyses suggested 6-10 horizontal gene transfers (HGTs) of the cluster, including a transfer that led to the presence of closely related cluster homologs in Alternaria and Fusarium. The analyses also indicated that HGTs were frequently followed by loss/pseudogenization of one or more DEP genes. Independent cluster inactivation was inferred in at least four fungal classes. Analyses of transitions among functional, pseudogenized, and absent states of DEP genes among Fusarium species suggest enzyme-encoding genes are lost at higher rates than the transporter (DEP3) and regulatory (DEP6) genes. The phenotype of an experimentally-induced DEP3 mutant of Fusarium did not support the hypothesis that selective retention of DEP3 and DEP6 protects fungi from exogenous depudecin. Together, the results suggest that HGT and gene loss have contributed significantly to DEP cluster distribution, and that some DEP genes provide a greater fitness benefit possibly due to a differential tendency to form network connections.

Iterative l-Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication.

Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the "magic" mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.

Fungal adaptation to plant defences through convergent assembly of metabolic modules.

The ongoing diversification of plant defence compounds exerts dynamic selection pressures on the microorganisms that colonize plant tissues. Evolutionary processes that generate resistance towards these compounds increase microbial fitness by giving access to plant resources and increasing pathogen virulence. These processes entail sequence-based mechanisms that result in adaptive gene functions, and combinatorial mechanisms that result in novel syntheses of existing gene functions. However, the priority and interactions among these processes in adaptive resistance remain poorly understood. Using a combination of molecular genetic and computational approaches, we investigated the contributions of sequence-based and combinatorial processes to the evolution of fungal metabolic gene clusters encoding stilbene cleavage oxygenases (SCOs), which catalyse the degradation of biphenolic plant defence compounds known as stilbenes into monophenolic molecules. We present phylogenetic evidence of convergent assembly among three distinct types of SCO gene clusters containing alternate combinations of phenolic catabolism. Multiple evolutionary transitions between different cluster types suggest recurrent selection for distinct gene assemblages. By comparison, we found that the substrate specificities of heterologously expressed SCO enzymes encoded in different clusters types were all limited to stilbenes and related molecules with a 4'-OH group, and differed modestly in substrate range and activity under the experimental conditions. Together, this work suggests a primary role for genome structural rearrangement, and the importance of enzyme modularity, in promoting fungal metabolic adaptation to plant defence chemistry.

The evolution of fungal metabolic pathways.

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.

Physical linkage of metabolic genes in fungi is an adaptation against the accumulation of toxic intermediate compounds.

Genomic analyses have proliferated without being tied to tangible phenotypes. For example, although coordination of both gene expression and genetic linkage have been offered as genetic mechanisms for the frequently observed clustering of genes participating in fungal metabolic pathways, elucidation of the phenotype(s) favored by selection, resulting in cluster formation and maintenance, has not been forthcoming. We noted that the cause of certain well-studied human metabolic disorders is the accumulation of toxic intermediate compounds (ICs), which occurs when the product of an enzyme is not used as a substrate by a downstream neighbor in the metabolic network. This raises the hypothesis that the phenotype favored by selection to drive gene clustering is the mitigation of IC toxicity. To test this, we examined 100 diverse fungal genomes for the simplest type of cluster, gene pairs that are both metabolic neighbors and chromosomal neighbors immediately adjacent to each other, which we refer to as "double neighbor gene pairs" (DNGPs). Examination of the toxicity of their corresponding ICs shows that, compared with chromosomally nonadjacent metabolic neighbors, DNGPs are enriched for ICs that have acutely toxic LD50 doses or reactive functional groups. Furthermore, DNGPs are significantly more likely to be divergently oriented on the chromosome; remarkably, ∼40% of these DNGPs have ICs known to be toxic. We submit that the structure of synteny in metabolic pathways of fungi is a signature of selection for protection against the accumulation of toxic metabolic intermediates.

In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins.

Most fungal plant pathogens secrete effector proteins during pathogenesis to manipulate their host's defense and promote disease. These are so highly diverse in sequence and distribution, they are essentially considered as species-specific. However, we have recently shown the presence of homologous effectors in fungal species of the Dothideomycetes class. One such example is Ecp2, an effector originally described in the tomato pathogen Cladosporium fulvum but later detected in the plant pathogenic fungi Mycosphaerella fijiensis and Mycosphaerella graminicola as well. Here, using in silico sequence-similarity searches against a database of 135 fungal genomes and GenBank, we extend our queries for homologs of Ecp2 to the fungal kingdom and beyond, and further study their history of diversification. Our analyses show that Ecp2 homologs are members of an ancient and widely distributed superfamily of putative fungal effectors, which we term Hce2 for Homologs of C. fulvum Ecp2. Molecular evolutionary analyses show that the superfamily originated and diversified within the fungal kingdom, experiencing multiple lineage-specific expansions and losses that are consistent with the birth-and-death model of gene family evolution. Newly formed paralogs appear to be subject to diversification early after gene duplication events, whereas at later stages purifying selection acts to preserve diversity and the newly evolved putative functions. Some members of the Hce2 superfamily are fused to fungal Glycoside Hydrolase family 18 chitinases that show high similarity to the Zymocin killer toxin from the dairy yeast Kluyveromyces lactis, suggesting an analogous role in antagonistic interactions. The observed high rates of gene duplication and loss in the Hce2 superfamily, combined with diversification in both sequence and possibly functions within and between species, suggest that Hce2s are involved in adaptation to stresses and new ecological niches. Such findings address the need to rationalize effector biology and evolution beyond the perspective of solely host-microbe interactions.

Fragmentation of an aflatoxin-like gene cluster in a forest pathogen.

Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D. septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D. septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.

Multiple GAL pathway gene clusters evolved independently and by different mechanisms in fungi.

A notable characteristic of fungal genomes is that genes involved in successive steps of a metabolic pathway are often physically linked or clustered. To investigate how such clusters of functionally related genes are assembled and maintained, we examined the evolution of gene sequences and order in the galactose utilization (GAL) pathway in whole-genome data from 80 diverse fungi. We found that GAL gene clusters originated independently and by different mechanisms in three unrelated yeast lineages. Specifically, the GAL cluster found in Saccharomyces and Candida yeasts originated through the relocation of native unclustered genes, whereas the GAL cluster of Schizosaccharomyces yeasts was acquired through horizontal gene transfer from a Candida yeast. In contrast, the GAL cluster of Cryptococcus yeasts was assembled independently from the Saccharomyces/Candida and Schizosaccharomyces GAL clusters and coexists in the Cryptococcus genome with unclustered GAL paralogs. These independently evolved GAL clusters represent a striking example of analogy at the genomic level. We also found that species with GAL clusters exhibited significantly higher rates of GAL pathway loss than species with unclustered GAL genes. These results suggest that clustering of metabolic genes might facilitate fungal adaptation to changing environments both through the acquisition and loss of metabolic capacities.