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Fungi shape the diversity of life. Characterizing the evolution of fungi is critical to understanding symbiotic associations across kingdoms. In this study, we investigate the genomic and metabolomic diversity of the genus Escovopsis , a specialized parasite of fungus-growing ant gardens. Based on 25 high-quality draft genomes, we show that Escovopsis forms a monophyletic group arising from a mycoparasitic fungal ancestor 61.82 million years ago (Mya). Across the evolutionary history of fungus-growing ants, the dates of origin of most clades of Escovopsis correspond to the dates of origin of the fungus-growing ants whose gardens they parasitize. We reveal that genome reduction, determined by both genomic sequencing and flow cytometry, is a consistent feature across the genus Escovopsis, largely occurring in coding regions, specifically in the form of gene loss and reductions in copy numbers of genes. All functional gene categories have reduced copy numbers, but resistance and virulence genes maintain functional diversity. Biosynthetic gene clusters (BGCs) contribute to phylogenetic differences among Escovopsis spp., and sister taxa in the Hypocreaceae. The phylogenetic patterns of co-diversification among BGCs are similarly exhibited across mass spectrometry analyses of the metabolomes of Escovopsis and their sister taxa. Taken together, our results indicate that Escovopsis spp. evolved unique genomic repertoires to specialize on the fungus-growing ant-microbe symbiosis.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis . Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia and Paraburkholderia from 50% of the 52 colonies sampled, indicating that members of the family Burkholderiaceae are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. IMPORTANCE Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis . The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia spp., bacteria that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia , is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates' ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis .

Herbivores must overcome a variety of plant defenses, including coping with plant secondary compounds (PSCs). To help detoxify these defensive chemicals, several insect herbivores are known to harbor gut microbiota with the metabolic capacity to degrade PSCs. Leaf-cutter ants are generalist herbivores, obtaining sustenance from specialized fungus gardens that act as external digestive systems and which degrade the diverse collection of plants foraged by the ants. There is in vitro evidence that certain PSCs harm Leucoagaricus gongylophorus , the fungal cultivar of leaf-cutter ants, suggesting a role for the Proteobacteria -dominant bacterial community present within fungus gardens. In this study, we investigated the ability of symbiotic bacteria present within fungus gardens of leaf-cutter ants to degrade PSCs. We cultured fungus garden bacteria, sequenced the genomes of 42 isolates, and identified genes involved in PSC degradation, including genes encoding cytochrome P450 enzymes and genes in geraniol, cumate, cinnamate, and α-pinene/limonene degradation pathways. Using metatranscriptomic analysis, we showed that some of these degradation genes are expressed in situ Most of the bacterial isolates grew unhindered in the presence of PSCs and, using gas chromatography-mass spectrometry (GC-MS), we determined that isolates from the genera Bacillus , Burkholderia , Enterobacter , Klebsiella , and Pseudomonas degrade α-pinene, β-caryophyllene, or linalool. Using a headspace sampler, we show that subcolonies of fungus gardens reduced α-pinene and linalool over a 36-h period, while L. gongylophorus strains alone reduced only linalool. Overall, our results reveal that the bacterial communities in fungus gardens play a pivotal role in alleviating the effect of PSCs on the leaf-cutter ant system. IMPORTANCE Leaf-cutter ants are dominant neotropical herbivores capable of deriving energy from a wide range of plant substrates. The success of leaf-cutter ants is largely due to their external gut, composed of key microbial symbionts, specifically, the fungal mutualist L. gongylophorus and a consistent bacterial community. Both symbionts are known to have critical roles in extracting energy from plant material, yet comparatively little is known about their roles in the detoxification of plant secondary compounds. In this study, we assessed if the bacterial communities associated with leaf-cutter ant fungus gardens can degrade harmful plant chemicals. We identify plant secondary compound detoxification in leaf-cutter ant gardens as a process that depends on the degradative potential of both the bacterial community and L. gongylophorus Our findings suggest that the fungus garden and its associated microbial community influence the generalist foraging abilities of the ants, underscoring the importance of microbial symbionts in plant substrate suitability for herbivores., (Copyright © 2020 Francoeur et al.)