Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi.

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

A Review of Coccidioides Research, Outstanding Questions in the Field, and Contributions by Women Scientists.

PURPOSE OF REVIEW: Coccidioidomycosis is an infectious disease that gained clinical significance in the early 20th century. Many of the foundational contributions to coccidioidomycosis research, including the discovery of the fungal disease agent, Coccidioides spp., were made by women. We review recent progress in Coccidioides research and big questions remaining in the field, while highlighting some of the contributions from women. RECENT FINDINGS: New molecular-based techniques provide a promising method for detecting Coccidioides, which can help determine the dominate reservoir host and ideal environmental conditions for growth. Genetic and genomic analyses have allowed an understanding of population structure, species level diversity, and evolutionary histories. We present a current, comprehensive genome list, where women contributed many of these entries. Several efforts to develop a coccidioidomycosis vaccine are underway. SUMMARY: Women continue to pioneer research on Coccidioides, including the relationships between the fungi and the environment, genetics, and clinical observations. Significant questions remain in the field of Coccidioides, including the main host reservoir, the relationships between genotypic and phenotypic variation, and the underlying cause for chronic clinical coccidioidomycosis cases.

Microfluidics and Metabolomics Reveal Symbiotic Bacterial-Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

Increasing access to microfluidics for studying fungi and other branched biological structures.

BACKGROUND: Microfluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve "ready-to-use microfluidics." RESULTS: We present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi. CONCLUSIONS: This pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Genome-based estimates of fungal rDNA copy number variation across phylogenetic scales and ecological lifestyles.

Ribosomal DNA (rDNA) copy number variation (CNV) has major physiological implications for all organisms, but how it varies for fungi, an ecologically ubiquitous and important group of microorganisms, has yet to be systemically investigated. Here, we examine rDNA CNV using an in silico read depth approach for 91 fungal taxa with sequenced genomes and assess copy number conservation across phylogenetic scales and ecological lifestyles. rDNA copy number varied considerably across fungi, ranging from an estimated 14 to 1,442 copies (mean = 113, median = 82), and copy number similarity was inversely correlated with phylogenetic distance. No correlations were found between rDNA CNV and fungal trophic mode, ecological guild or genome size. Taken together, these results show that like other microorganisms, fungi exhibit substantial variation in rDNA copy number, which is linked to their phylogeny in a scale-dependent manner.

Emission Factors of Microbial Volatile Organic Compounds from Environmental Bacteria and Fungi.

Knowledge of the factors controlling the diverse chemical emissions of common environmental bacteria and fungi is crucial because they are important signal molecules for these microbes that also could influence humans. We show here not only a high diversity of mVOCs but that their abundance can differ greatly in different environmental contexts. Microbial volatiles exhibit dynamic changes across microbial growth phases, resulting in variance of composition and emission rate of species-specific and generic mVOCs. In vitro experiments documented emissions of a wide range of mVOCs (>400 different chemicals) at high time resolution from diverse microbial species grown under different controlled conditions on nutrient media, or residential structural materials ( N = 54, Ncontrol = 23). Emissions of mVOCs varied not only between microbial taxa at a given condition but also as a function of life stage and substrate type. We quantify emission factors for total and specific mVOCs normalized for respiration rates to account for the microbial activity during their stationary phase. Our VOC measurements of different microbial taxa indicate that a variety of factors beyond temperature and water activity, such as substrate type, microbial symbiosis, growth phase, and lifecycle affect the magnitude and composition of mVOC emission.

Bacterial-fungal interactions: ecology, mechanisms and challenges.

Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families engage in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to antagonism. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regard of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for research in order to stimulate synergy within the BFI research area and to resolve outstanding questions.

Integrated proteomics and metabolomics suggests symbiotic metabolism and multimodal regulation in a fungal-endobacterial system.

Many plant-associated fungi host endosymbiotic endobacteria with reduced genomes. While endobacteria play important roles in these tri-partite plant-fungal-endobacterial systems, the active physiology of fungal endobacteria has not been characterized extensively by systems biology approaches. Here, we use integrated proteomics and metabolomics to characterize the relationship between the endobacterium Mycoavidus sp. and the root-associated fungus Mortierella elongata. In nitrogen-poor media, M. elongata had decreased growth but hosted a large and growing endobacterial population. The active endobacterium likely extracted malate from the fungal host as the primary carbon substrate for energy production and biosynthesis of phospho-sugars, nucleobases, peptidoglycan and some amino acids. The endobacterium obtained nitrogen by importing a variety of nitrogen-containing compounds. Further, nitrogen limitation significantly perturbed the carbon and nitrogen flows in the fungal metabolic network. M. elongata regulated many pathways by concordant changes on enzyme abundances, post-translational modifications, reactant concentrations and allosteric effectors. Such multimodal regulations may be a general mechanism for metabolic modulation.

A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data.

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

New Boletaceae taxa from Guyana: Binderoboletus segoi gen. and sp. nov., Guyanaporus albipodus gen. and sp. nov., Singerocomus rubriflavus gen. and sp. nov., and a new combination for Xerocomus inundabilis.

Binderoboletus segoi gen. and sp. nov., Guyanaporus albipodus gen. and sp. nov. and Singerocomus rubriflavus gen. and sp. nov. (Boletaceae, Boletales, Basidiomycota) are described from the Pakaraima Mountains and adjacent lowlands of Guyana. Xerocomus inundabilis, originally described from the central Brazilian Amazon and based solely on the type collection, is redescribed from numerous collections from Guyana and transferred into Singerocomus. These boletes occur in Neotropical forests dominated by ectomycorrhizal trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae), Aldina (Fabaceae subfam. Papilionoideae) and Pakaraimaea (Dipterocarpaceae). Three of the species were repeatedly found in a multiyear sporocarp survey in Dicymbe corymbosa-monodominant forest. Macromorphological, micromorphological, habitat and multilocus DNA sequence data are provided for each species. A molecular phylogenetic analysis based on a large taxon set across the Boletaceae justifies erection of the new genera.

Sarcodon in the Neotropics I: new species from Guyana, Puerto Rico and Belize.

Four species of the ectomycorrhizal (ECM) genus Sarcodon (Bankeraceae, Thelephorales, Basidiomycota) are described as new to science. Sarcodon pakaraimensis sp. nov. is described from forests dominated by the ECM trees Pakaraimaea dipterocarpacea (Dipterocarpaceae) and Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae) in the Pakaraima Mountains of Guyana. Sarcodon portoricensis sp. nov. is described from lower montane wet forest within the El Yunque National Forest of Puerto Rico. Sarcodon quercophilus sp. nov. and Sarcodon umbilicatus sp. nov. are described from Quercus (Fagaceae) cloud forests within the Maya Mountains of Belize. The discovery of these species is significant given that the majority of the approximately 87 described Sarcodon species are north temperate or boreal in distribution and frequently associate with coniferous host plants; these constitute the most recent records for Sarcodon from the greater Neotropics. Each of the new species is morphologically consistent with accepted diagnostic characters for Sarcodon: pileate-stipitate stature, a dentate hymenophore, determinate basidiomatal development, fleshy, non-zonate context and brown, tuberculate basidiospores. DNA (ITS) sequence analysis corroborated the generic placement of S. pakaraimensis, S. portoricensis, S. quercophilus and S. umbilicatus and, along with morphological differences, supported their recognition as distinct species. Macromorphological, micromorphological, habitat and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species. A key to Neotropical Sarcodon species and similar extralimital taxa is provided.

Cantharellaceae of Guyana II: new species of Craterellus, new South American distribution records for Cantharellus guyanensis and Craterellus excelsus, and a key to the Neotropical taxa.

Craterellus olivaceoluteus sp. nov. and Craterellus cinereofimbriatus sp. nov. are described as new to science. These fungi were collected from Guyana in association with ectomycorrhizal host trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae) and Pakaraimaea (Dipterocarpaceae). Cantharellus guyanensis Mont., originally described from French Guiana, is redescribed from recent collections from Guyana, with additional range extensions for the species provided based on material examined from French Guiana, Venezuela, and north central, northeastern and southern Brazil, circumscribing nearly the entire Guiana Shield region and beyond. A new distribution record from French Guiana is provided for Craterellus excelsus T.W. Henkel & Aime. Macromorphological, micromorphological and habitat data are provided for the new species and C. guyanensis as well as DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU); additional sequence data is provided for C. guyanensis and C. excelsus specimens collected outside Guyana. The relationships of these taxa within the Cantharellaceae were evaluated with phylogenetic analyses of ITS and LSU sequence data. This work brings the total number of Cantharellaceae species known from Guyana to eight. A key to the Cantharellus and Craterellus species known from the lowland Neotropics and extralimital montane Central and South America is provided.

The ectomycorrhizal fungal community in a neotropical forest dominated by the endemic dipterocarp Pakaraimaea dipterocarpacea.

Ectomycorrhizal (ECM) plants and fungi can be diverse and abundant in certain tropical ecosystems. For example, the primarily paleotropical ECM plant family Dipterocarpaceae is one of the most speciose and ecologically important tree families in Southeast Asia. Pakaraimaea dipterocarpacea is one of two species of dipterocarp known from the Neotropics, and is also the only known member of the monotypic Dipterocarpaceae subfamily Pakaraimoideae. This Guiana Shield endemic is only known from the sandstone highlands of Guyana and Venezuela. Despite its unique phylogenetic position and unusual geographical distribution, the ECM fungal associations of P. dipterocarpacea are understudied throughout the tree's range. In December 2010 we sampled ECM fungi on roots of P. dipterocarpacea and the co-occurring ECM tree Dicymbe jenmanii (Fabaceae subfamily Caesalpinioideae) in the Upper Mazaruni River Basin of Guyana. Based on ITS rDNA sequencing we documented 52 ECM species from 11 independent fungal lineages. Due to the phylogenetic distance between the two host tree species, we hypothesized that P. dipterocarpacea would harbor unique ECM fungi not found on the roots of D. jenmanii. Although statistical tests suggested that several ECM fungal species did exhibit host preferences for either P. dipterocarpacea or D. jenmanii, most of the ECM fungi were multi-host generalists. We also detected several ECM fungi that have never been found in long-term studies of nearby rainforests dominated by other Dicymbe species. One particular mushroom-forming fungus appears to be unique and may represent a new ECM lineage of Agaricales that is endemic to the Neotropics.

New species and distribution records for Clavulina (Cantharellales, Basidiomycota) from the Guiana Shield, with a key to the lowland neotropical taxa.

Three new and one previously described species of Clavulina (Clavulinaceae, Cantharellales, Basidiomycota) are reported from the central Guiana Shield region from tropical rainforests dominated by ectomycorrhizal trees of the leguminous genus Dicymbe (Fabaceae subfam. Caesalpinioideae). We provide morphological, DNA sequence, habitat, and fruiting occurrence data for each species. The new species conform to a generic concept of Clavulina that includes coralloid, branched basidiomata with amphigenous hymenia, basidia with two or 2-4 incurved sterigmata and postpartal septa present or absent, and smooth, hyaline, guttulate basidiospores. Placements of the new species in Clavulina were corroborated with DNA sequence data from the internal transcribed spacer and large subunit of the nuclear ribosomal repeat, and their infrageneric relationships were examined with phylogenetic analyses based on DNA from the region coding for the second largest subunit of DNA-dependent RNA polymerase II (rpb2). To facilitate future studies of the genus in the neotropics, a key is provided for all Clavulina species described from the lowland neotropics.

Membranomyces species are common ectomycorrhizal symbionts in Northern Hemisphere forests.

Membranomyces (Clavulinaceae, Cantharellales) Jülich consists of two described species of resupinate (crust-like) basidiomycetes. Previous studies indicated that Membranomyces falls within the Clavulinaceae, but the phylogenetic position of the genus has not been fully resolved. Membranomyces species were thought to be saprotrophic until 2003 when Tedersoo et al. detected Membranomyces delectabilis on ectomycorrhizal roots of Populus and Picea. Membranomyces was previously known only from collections made in eastern Canada and Europe. We recently sequenced the ITS rDNA barcode region from Scandinavian herbarium specimens identified as M. delectabilis and Membranomyces spurius. Phylogenetic analyses of these sporocarp sequences and similar environmental sequences indicated that Membranomyces is more diverse than previously thought and forms ectomycorrhizas with hosts from a diverse range of plant families in many north temperate ecosystems.

Scaling up: examining the macroecology of ectomycorrhizal fungi.

Ectomycorrhizal (ECM) fungi play major ecological roles in temperate and tropical ecosystems. Although the richness of ECM fungal communities and the factors controlling their structure have been documented at local spatial scales, how they vary at larger spatial scales remains unclear. In this issue of Molecular Ecology, Tedersoo et al. (2012) present the results of a meta-analysis of ECM fungal community structure that sheds important new light on global-scale patterns. Using data from 69 study systems and 6021 fungal species, the researchers found that ECM fungal richness does not fit the classic latitudinal diversity gradient in which species richness peaks at lower latitudes. Instead, richness of ECM fungal communities has a unimodal relationship with latitude that peaks in temperate zones. Intriguingly, this conclusion suggests the mechanisms driving ECM fungal community richness may differ from those of many other organisms, including their plant hosts. Future research will be key to determine the robustness of this pattern and to examine the processes that generate and maintain global-scale gradients of ECM fungal richness.