Championing inclusive terminology in ecology and evolution.

Amid a growing disciplinary commitment to inclusion in ecology and evolutionary biology (EEB), it is critical to consider how the use of scientific language can harm members of our research community. Here, we outline a path for identifying and revising harmful terminology to foster inclusion in EEB.

Genomic diversification of the specialized parasite of the fungus-growing ant symbiosis.

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.

Prey identity affects fitness of a generalist consumer in a brown food web.

The use of ever-advancing sequencing technologies has revealed incredible biodiversity at the microbial scale, and yet we know little about the ecological interactions in these communities. For example, in the phytotelmic community found in the purple pitcher plant, Sarracenia purpurea, ecologists typically consider the bacteria as a functionally homogenous group. In this food web, bacteria decompose detritus and are consumed by protozoa that are considered generalist consumers. Here, we tested whether a generalist consumer benefits from all bacteria equally. We isolated and identified 22 strains of bacteria, belonging to six genera, from S. purpurea plants. We grew the protozoa, Tetrahymena sp. with single isolates and strain mixtures of bacteria and measured Tetrahymena fitness. We found that different bacterial strains had different effects on protozoan fitness, both in isolation and in mixture. Our results demonstrate that not accounting for the composition of prey communities may affect the predicted outcome of predator-prey interactions.

From Plants to Ants: Fungal Modification of Leaf Lipids for Nutrition and Communication in the Leaf-Cutter Ant Fungal Garden Ecosystem.

Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry, we evaluated differences between the molecular species of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens, we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression toward 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an interkingdom communication tool in leaf-cutter ant fungal gardens.IMPORTANCE In this work, we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an interkingdom communication tool, in this case, as an attractant rather than as a repellant, which is more often seen.

Metagenomics Reveals Diet-Specific Specialization of Bacterial Communities in Fungus Gardens of Grass- and Dicot-Cutter Ants.

Leaf-cutter ants in the genus Atta are dominant herbivores in the Neotropics. While most species of Atta cut dicots to incorporate into their fungus gardens, some species specialize on grasses. Here we examine the bacterial community associated with the fungus gardens of grass- and dicot-cutter ants to examine how changes in substrate input affect the bacterial community. We sequenced the metagenomes of 12 Atta fungus gardens, across four species of ants, with a total of 5.316 Gbp of sequence data. We show significant differences in the fungus garden bacterial community composition between dicot- and grass-cutter ants, with grass-cutter ants having lower diversity. Reflecting this difference in community composition, the bacterial functional profiles between the fungus gardens are significantly different. Specifically, grass-cutter ant fungus garden metagenomes are particularly enriched for genes responsible for amino acid, siderophore, and terpenoid biosynthesis while dicot-cutter ant fungus gardens metagenomes are enriched in genes involved in membrane transport. Differences between community composition and functional capacity of the bacteria in the two types of fungus gardens reflect differences in the substrates that the ants incorporated. These results show that different substrate inputs matter for fungus garden bacteria and shed light on the potential role of bacteria in mediating the ants' transition to the use of a novel substrate.

Bacteria Contribute to Plant Secondary Compound Degradation in a Generalist Herbivore System.

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, ß-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.

Stingless Bee Larvae Require Fungal Steroid to Pupate.

The larval stage of the stingless bee Scaptotrigona depilis must consume a specific brood cell fungus in order to continue development. Here we show that this fungus is a member of the genus Zygosaccharomyces and provides essential steroid precursors to the developing bee. Insect pupation requires ecdysteroid hormones, and as insects cannot synthesize sterols de novo, they must obtain steroids in their diet. Larval in vitro culturing assays demonstrated that consuming ergosterol recapitulates the developmental effects on S. depilis as ingestion of Zygosaccharomyces sp. cells. Thus, we determined the molecular underpinning of this intimate mutualistic symbiosis. Phylogenetic analyses showed that similar cases of bee-Zygosaccharomyces symbiosis may exist. This unprecedented case of bee-fungus symbiosis driven by steroid requirement brings new perspectives regarding pollinator-microbiota interaction and preservation.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates.

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and ß-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores.

Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants.

Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

The relative abundance of mountain pine beetle fungal associates through the beetle life cycle in pine trees.

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.

Target-specific PCR primers can detect and differentiate ophiostomatoid fungi from microbial communities associated with the mountain pine beetle Dendroctonus ponderosae.

The aim of this study was to develop DNA probes that could identify the major fungal species associated with mountain pine beetles (MPB). The beetles are closely associated with fungal species that include ophiostomatoid fungi that can be difficult to differentiate morphologically. The most frequently isolated associates are the pine pathogens Grosmannia clavigera and Leptographium longiclavatum, the less pathogenic Ophiostoma montium, and an undescribed Ceratocystiopsis species (Cop. sp.). Because growing, isolating and extracting DNA from fungi vectored by MPB can be time and labour intensive, we designed three rDNA primer sets that specifically amplify short rDNA amplicons from O. montium, Cop. sp. and the pine Leptographium clade. We also designed two primer sets on a gene of unknown function that can differentiate G. clavigera and L. longiclavatum. We tested the primers on 76 fungal isolates that included MPB associates. The primers reliably identified their targets from DNA obtained from pure fungal cultures, pulverized beetles, beetle galleries, and tree phloem inoculated with G. clavigera. The primers will facilitate large-scale work on the ecology of the MPB-fungal-lodgepole pine ecosystem, as well as phytosanitary/quarantine sample screening.

Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes.

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.