Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea.

Clonostachys rosea is a necrotrophic mycoparasitic fungus, used for biological control of plant pathogenic fungi. A better understanding of the underlying mechanisms resulting in successful biocontrol is important for knowledge‐based improvements of the application and use of biocontrol in agricultural production systems. Transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the fungal prey species Botrytis cinerea and Fusarium graminearum. Genes predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate‐active enzymes were induced during the mycoparasitic attack. Predicted major facilitator superfamily (MFS) transporters constituted 54% of the induced genes, and detailed phylogenetic and evolutionary analyses showed that a majority of these genes belonged to MFS gene families evolving under selection for increased paralog numbers, with predicted functions in drug resistance and transport of carbohydrates and small organic compounds. Sequence analysis of MFS transporters from family 2.A.1.3.65 identified rapidly evolving loop regions forming the entry to the transport tunnel, indicating changes in substrate specificity as a target for selection. Deletion of the MFS transporter gene mfs464 resulted in mutants with increased growth inhibitory activity against F. graminearum, providing evidence for a function in interspecific fungal interactions. In summary, we show that the mycoparasite C. rosea can distinguish between fungal prey species and modulate its transcriptomic responses accordingly. Gene expression data emphasize the importance of secondary metabolites in mycoparasitic interactions.

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1. The effect of nutrient enrichment on structural (invertebrate indices) and functional (leaf-litter breakdown rates) characteristics of stream integrity was studied in nine boreal streams. 2. The results showed predicted changes in biotic indices and leaf-litter breakdown along a complex (principal component) nutrient gradient. Biotic indices were better correlated with nutrient effects than leaf-litter breakdown. 3. Fungal biomass and invertebrate densities in the litter bags were positively correlated with leaf-litter breakdown, and both were also positively related to the nutrient gradient. 4. Invertebrate community composition influenced breakdown rate. High breakdown rates at one site were associated with the high abundance of the detritivore Asellus aquaticus. 5. This study lends support to the importance of invertebrate and fungi as mediators of leaf-litter decomposition. However, our study also shows that study design (length of incubation) can confound the interpretation of nutrient-induced effects on decomposition.

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Comparative sequencing studies among a wide range of taxonomic groups, including fungi, provide the overall pattern that reproductive genes evolve more rapidly than other genes, and this divergence is believed to be important in the establishment of reproductive barriers between species. In this study, we investigated the molecular evolution of the pheromone receptor genes pre-1 and pre-2 of strains belonging to 12 and 13 heterothallic taxa, respectively, of the model genus Neurospora. Furthermore, we examined the regulatory pattern of both pheromone precursor and receptor genes during sexual crosses of Neurospora crassa and Neurospora intermedia, for which reinforcement of interspecific reproductive barriers in sympatry previously has been documented. We conclude that the part encoding the C-terminal intracellular domain of pre-1 and pre-2 genes evolves rapidly. Both stochastic and directional processes drive this divergence; both genes contain neutrally evolving codons, and in addition, pre-1 contains codons evolving under positive selection, whereas in pre-2 we found highly variable regions with numerous repeats encoding glycine, threonine, or aspartic acid. In addition, we found regulatory changes of the pheromone and receptor genes during crosses between N. crassa and N. intermedia with different reproductive success. Gene expression levels are higher in the interspecific sympatric crosses with low reproductive success than in their intraspecific and/or allopatric equivalents, both at the stage of initial communication and contact and later at postfertilization stages. Taken together, our data indicate that pheromones and receptors are important key players during reproductive isolation between Neurospora species, and this study provides a general framework for future studies on the role of reproductive proteins for reproductive isolation.

In the vast majority of sexual life cycles, fusion between single-celled gametes is directly followed by nuclear fusion, leading to a diploid zygote and a lifelong commitment between two haploid genomes. Mushroom-forming basidiomycetes differ in two key respects. First, the multicellular haploid mating partners are fertilized in their entirety, each cell being a gamete that simultaneously can behave as a female, i.e. contributing the cytoplasm to a zygote by accepting nuclei, and a male gamete, i.e. only donating nuclei to the zygote. Second, after gamete union, the two haploid genomes remain separate so that the main vegetative stage, the dikaryon, has two haploid nuclei per cell. Only when the dikaryon produces mushrooms, do the nuclei fuse to enter a short diploid stage, immediately followed by meiosis and haploid spore formation. So in basidiomycetes, gamete fusion and genome mixing (sex) are separated in time. The ‘living apart together’ of nuclei in the dikaryon maintains some autonomy for nuclei to engage in a relationship with a different nucleus. We show that competition among the two nuclei of the dikaryon for such ‘extramarital affairs’ may lead to genomic conflict by favouring genes beneficial at the level of the nucleus, but deleterious at that of the dikaryon.This article is part of the themed issue ‘Weird sex: the underappreciated diversity of sexual reproduction’.

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