Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects.

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host's growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23-28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control.

Rearing zombie flies: Laboratory culturing of the behaviourally manipulating fungal pathogen Entomophthora muscae.

Insect pathogenic fungi (IPF) and insects have ubiquitous interactions in nature. The extent of these interkingdom host-pathogen interactions are both complex and diverse. Some IPF, notably of the order Entomophthorales, manipulate their species-specific host before death. The fungus-induced altered insect behaviours are sequential and can accurately be repeatedly characterised temporally, making them a valuable model for understanding the molecular and chemical underpinnings of behaviour and host-pathogen co-evolutionary biology. Here, we present methods for the isolation and laboratory culturing of the emerging behaviourally manipulating model IPF Entomophthora muscae for experimentation.•E. muscae isolation and culturing in vitro.•Establishing and maintaining an E. muscae culture in vivo in houseflies (Musca domestica).•Controlled E. muscae infections for virulence experiments and quantification of conidia discharge per cadaver.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. This is in part due to their large genome sizes and difficulty in obtaining sequenceable material. In this manuscript, we leveraged a recently-isolated, laboratory-tractable Entomophthora muscae isolate and improved long-read sequencing to obtain a largely-complete entomophthoralean genome. Our E. muscae assembly is 1.03 Gb, consists of 7,810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with other available (transcriptomic and genomic) datasets from entomophthoralean fungi, we provide new insight into the biology of these understudied pathogens. We offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex. Our findings suggest that substantial taxonomic revision is needed to define species within this group and we provide recommendations for differentiating strains and species in the context of the existing body of E. muscae scientific literature. We show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity, despite the presence of machinery to defend against transposable elements(RNAi). In addition, we find that E. muscae and its closest allies are enriched for M16A peptidases and possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We find that E. muscae has an expanded group of acid-trehalases, consistent with trehalose being the primary sugar component of fly (and insect) hemolymph. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Phenotypic variation and genomic variation in insect virulence traits reveal patterns of intraspecific diversity in a locust-specific fungal pathogen.

Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host-pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens.

A Rapid Method for Measuring In Vitro Growth in Entomopathogenic Fungi.

Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.

Blastospores from Metarhizium anisopliae and Metarhizium rileyi Are Not Always as Virulent as Conidia Are towards Spodoptera frugiperda Caterpillars and Use Different Infection Mechanisms.

Infective conidia from entomopathogenic fungi are widely used to control insect pests. Many entomopathogenic fungi also produce yeast-like cells called blastospores under specific liquid culture conditions that can directly infect insects. However, little is known about the biological and genetic factors that allow blastospores to infect insects and make them potentially effective for biological control in the field. Here, we show that while the generalist Metarhizium anisopliae produces a higher number of and smaller blastospores, the Lepidoptera specialist M. rileyi produces fewer propagules with a higher cell volume under high-osmolarity conditions. We compared the virulence of blastospores and conidia of these two Metarhizium species towards the economically important caterpillar pest Spodoptera frugiperda. Conidia and blastospores from M. anisopliae were equally infectious, but acted slower, and killed fewer insects than M. rileyi conidia and blastospores did, where M. rielyi conidia had the highest virulence. Using comparative transcriptomics during propagule penetration of insect cuticles, we show that M. rileyi blastospores express more virulence-related genes towards S. frugiperda than do M. anisopliae blastospores. In contrast, conidia of both fungi express more virulence-related oxidative stress factors than blastospores. Our results highlight that blastospores use a different virulence mechanism than conidia use, which may be explored in new biological control strategies.

Comparative transcriptomics of growth metabolism and virulence reveal distinct morphogenic profiles of yeast-like cells and hyphae of the fungus Metarhizium rileyi.

Metarhizium rileyiis an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhiziumspecies with broad host ranges. This species is also unique because the initial yeast-like growth on solid media is only observed in liquid culture in other Metharizium species. A lack of knowledge about the metabolism and genetic signatures of M. rileyiduring this yeast-like phase on solid and in liquid media is a bottleneck for its large-scale production as a commercial biocontrol agent.In this study wefound that M. rileyiyeast-like cells produced on solid medium infected and killed the important insect pest Spodoptera frugiperda with comparable efficiency as yeast-like cells grown in liquid medium. Secondly, we used comparative transcriptomic analysis to investigate theactive genes and genomic signatures of the M. rileyi yeast-like morphotypes produced on solid and in liquid media. Yeast-like cells grown in liquid medium had upregulated genes relating specifically to signal transduction andparticular membrane transporters. Thirdly, we compared the transcriptomic profiles of yeast-like phases of M. rileyi with those of M. anisopliae. The yeast-like phase of M. rileyi grown on solid medium upregulated unique genes not found in otherMetarhiziumspecies including specific membrane proteins and several virulence factors. Orthologous genes associated with heat shock protein, iron permease, membrane proteins and key virulence traits (e.g. collagen-like protein Mcl1) were upregulated in both species. Comparative transcriptome analyses of gene expression showed more differences than similarities between M. anisopliae and M. rileyi yeast-like cells.

Pathogenic fungus uses volatiles to entice male flies into fatal matings with infected female cadavers.

To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the profile of natural host cuticular hydrocarbons in infected female housefly (Musca domestica) cadavers. Healthy male houseflies respond to the fungal compounds and are enticed into mating with female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus exploits the willingness of male flies to mate and benefits from altering the behaviour of uninfected male host flies. The altered cuticular hydrocarbons and emitted volatiles thus underlie the evolution of an extended phenotypic trait.

Phylogenetic Revision and Patterns of Host Specificity in the Fungal Subphylum Entomophthoromycotina.

The Entomophthoromycotina, a subphylum close to the root of terrestrial fungi with a bias toward insects as their primary hosts, has been notoriously difficult to categorize taxonomically for decades. Here, we reassess the phylogeny of this group based on conserved genes encoding ribosomal RNA and RNA polymerase II subunits, confirming their general monophyly, but challenging previously assumed taxonomic relationships within and between particular clades. Furthermore, for the prominent, partially human-pathogenic taxon Conidiobolus, a new type species C. coronatus is proposed in order to compensate for the unclear, presumably lost previous type species C. utriculosus Brefeld 1884. We also performed an exhaustive survey of the broad host spectrum of the Entomophthoromycotina, which is not restricted to insects alone, and investigated potential patterns of co-evolution across their megadiverse host range. Our results suggest multiple independent origins of parasitism within this subphylum and no apparent co-evolutionary events with any particular host lineage. However, Pterygota (i.e., winged insects) clearly constitute the most dominantly parasitized superordinate host group. This appears to be in accordance with an increased dispersal capacity mediated by the radiation of the Pterygota during insect evolution, which has likely greatly facilitated the spread, infection opportunities, and evolutionary divergence of the Entomophthoromycotina as well.

The genus Entomophthora: bringing the insect destroyers into the twenty-first century.

The fungal genus Entomophthora consists of highly host-specific pathogens that cause deadly epizootics in their various insect hosts. The most well-known among these is the "zombie fly" fungus E. muscae, which, like other Entomophthora species, elicits a series of dramatic behaviors in infected hosts to promote optimal spore dispersal. Despite having been first described more than 160 years ago, there are still many open questions about Entomophthora biology, including the molecular underpinnings of host behavior manipulation and host specificity. This review provides a comprehensive overview of our current understanding of the biology of Entomophthora fungi and enumerates the most pressing outstanding questions that should be addressed in the field. We briefly review the discovery of Entomophthora and provide a summary of the 21 recognized Entomophthora species, including their type hosts, methods of transmission (ejection of spores after or before host death), and for which molecular data are available. Further, we argue that this genus is globally distributed, based on a compilation of Entomophthora records in the literature and in online naturalist databases, and likely to contain additional species. Evidence for strain-level specificity of hosts is summarized and directly compared to phylogenies of Entomophthora and the class Insecta. A detailed description of Entomophthora's life-cycle and observed manipulated behaviors is provided and used to summarize a consensus for ideal growth conditions. We discuss evidence for Entomophthora's adaptation to growth exclusively inside insects, such as producing wall-less hyphal bodies and a unique set of subtilisin-like proteases to penetrate the insect cuticle. However, we are only starting to understand the functions of unusual molecular and genomic characteristics, such as having large > 1 Gb genomes full of repetitive elements and potential functional diploidy. We argue that the high host-specificity and obligate life-style of most Entomophthora species provides ample scope for having been shaped by close coevolution with insects despite the current general lack of such evidence. Finally, we propose six major directions for future Entomophthora research and in doing so hope to provide a foundation for future studies of these fungi and their interaction with insects.

Diploidy within a Haploid Genus of Entomopathogenic Fungi.

Fungi in the genus Metarhizium are soil-borne plant-root endophytes and rhizosphere colonizers, but also potent insect pathogens with highly variable host ranges. These ascomycete fungi are predominantly asexually reproducing and ancestrally haploid, but two independent origins of persistent diploidy within the Coleoptera-infecting Metarhizium majus species complex are known and has been attributed to incomplete chromosomal segregation following meiosis during the sexual cycle. There is also evidence for infrequent sexual cycles in the locust-specific pathogenic fungus Metarhizium acridum (Hypocreales: Clavicipitaceae), which is an important entomopathogenic biocontrol agent used for the control of grasshoppers in agricultural systems as an alternative to chemical control. Here, we show that the genome of the M. acridum isolate ARSEF 324, which is formulated and commercially utilized is functionally diploid. We used single-molecule real-time sequencing technology to complete a high-quality assembly of ARSEF 324. K-mer frequencies, intragenomic collinearity between contigs and single nucleotide variant read depths across the genome revealed the first incidence of diploidy described within the species M. acridum. The haploid assembly of 44.7 Mb consisted of 20.8% repetitive elements, which is the highest proportion described of any Metarhizium species. The long-read diploid genome assembly sheds light on past research on this strain, such as unusual high UVB tolerance. The data presented here could fuel future investigation into the fitness landscape of fungi with infrequent sexual reproduction and aberrant ploidy levels, not least in the context of biocontrol agents.

Grandmother's pesticide exposure revealed bi-generational effects in Daphnia magna.

Man-made chemicals are a significant contributor to the ongoing deterioration of numerous ecosystems. Currently, risk assessment of these chemicals is based on observations in a single generation of animals, despite potential adverse intergenerational effects. Here, we investigate the effect of the fungicide prochloraz across three generations of Daphnia magna. We studied both the effects of continuous exposure over all generations and the effects of first-generation (F0) exposure on two subsequent generations. Effects at different levels of biological organization from genome-wide gene expression, whole organism metabolite levels, CYP enzyme activity and key phenotypic effects, such as reproduction, were monitored. Acclimation to prochloraz was found after continuous exposure. Following F0-exposure, embryonically exposed F1-offspring showed no significant effects. However, in the potentially germline exposed F2 animals, several parameters differed significantly from controls. A direct association between these F2 effects and the toxic mode of action of prochloraz was found, showing that chemicals can be harmful not only to the directly exposed generation, but also to prenatally exposed generations and in that way effects may even appear to skip a generation. This implies that current risk assessment practices are neglecting an important aspect of toxicity, such as delayed effects across generations due to a time gap between chemical exposure and emergence of effects.

Nutritional quality of Drosophila melanogaster as factitious prey for rearing the predatory bug Orius majusculus.

The predatory bug, Orius majusculus (Reuter), is an important predator of thrips commercially produced for augmentative releases using the eggs of the Mediterranean flour moth Ephestia kuehniella (Zeller). In this study, we assessed the potential for using frozen adults of fruit flies, Drosophila melanogaster (Meigen), either as nymphal rearing diet or as diet throughout the entire life-cycle. We compared life-history traits and reproduction of predators when fed D. melanogaster with high lipid body content (lipid-rich) and with high protein body content (protein-rich), using a diet of 100% E. kuehniella eggs as control. We also analyzed the biochemical composition of both prey and predator in order to assess the nutritional quality of each diet, which partially explained the adequacy of the different diets for O. majusculus. There were significant differences between predators fed the two types of D. melanogaster, with the protein-rich flies as diet providing the best results in terms of mortality and fecundity. Furthermore, we show that while feeding O. majusculus throughout their development with D. melanogaster increases mortality and reduces reproduction, protein-rich D. melanogaster can be used as nymphal diet with minimal reduction in reproductivity and minimal increase in mortality.

Going gentle into that pathogen-induced goodnight.

A diverse set of pathogens have evolved extended phenotypes that manipulate the moribund behavior of their various insect hosts. By elevating host positioning at death, a phenomenon called "summit disease", these pathogens have been shown to have higher fitness. Though a few summit disease systems have been intensively characterized, in particular the Ophiocordyceps-ant system, summit diseases lack an overarching theory for the underlying mechanisms of this complex behavioral manipulation. In this article, we combine the gamut of summiting systems into a cohesive framework: we propose two types of summit disease (juvenile and adult), which both exploit natural insect behaviors during periods of quiescence. We place this framework in the context of available literature and propose investigations that follow from this comprehensive understanding of summit disease in insects.

Comparative RNAseq Analysis of the Insect-Pathogenic Fungus Metarhizium anisopliae Reveals Specific Transcriptome Signatures of Filamentous and Yeast-Like Development.

The fungus Metarhizium anisopliae is a facultative insect pathogen used as biological control agent of several agricultural pests worldwide. It is a dimorphic fungus that is able to display two growth morphologies, a filamentous phase with formation of hyphae and a yeast-like phase with formation of single-celled blastospores. Blastospores play an important role for M. anisopliae pathogenicity during disease development. They are formed solely in the hemolymph of infected insects as a fungal strategy to quickly multiply and colonize the insect's body. Here, we use comparative genome-wide transcriptome analyses to determine changes in gene expression between the filamentous and blastospore growth phases in vitro to characterize physiological changes and metabolic signatures associated with M. anisopliae dimorphism. Our results show a clear molecular distinction between the blastospore and mycelial phases. In total 6.4% (n = 696) out of 10,981 predicted genes in M. anisopliae were differentially expressed between the two phases with a fold-change > 4. The main physiological processes associated with up-regulated gene content in the single-celled yeast-like blastospores during liquid fermentation were oxidative stress, amino acid metabolism (catabolism and anabolism), respiration processes, transmembrane transport and production of secondary metabolites. In contrast, the up-regulated gene content in hyphae were associated with increased growth, metabolism and cell wall re-organization, which underlines the specific functions and altered growth morphology of M. anisopliae blastospores and hyphae, respectively. Our study revealed significant transcriptomic differences between the metabolism of blastospores and hyphae. These findings illustrate important aspects of fungal morphogenesis in M. anisopliae and highlight the main metabolic activities of each propagule under in vitro growth conditions.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Fungal artillery of zombie flies: infectious spore dispersal using a soft water cannon.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic 'soft cannon' that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.