Knowledge, Attitudes, and Practices of Artisanal and Small-Scale Miners regarding Tuberculosis, Human Immunodeficiency Virus, and Silicosis in Zimbabwe.

In Zimbabwe, artisanal and small-scale miners (ASMs) have a high prevalence of tuberculosis (TB), human immunodeficiency virus (HIV), and silicosis. Previous studies on ASMs utilised programme data, and it was not possible to understand reasons for the high prevalence of these comorbidities. We conducted a cross-sectional study to investigate the knowledge, attitudes, and practices of ASMs regarding TB, HIV, and silicosis. We enrolled a convenience sample of 652 ASMs. Their mean (standard deviation) age was 34.2 (10.8) years. There were 602 (92%) men and over 75% had attained secondary education. A total of 504 (80%) of the ASMs knew that TB is a curable disease, and 564 (87%) knew that they were at higher risk of TB than the general population. However, they were less likely to know that HIV increases the risk of TB disease, 340 (52%), with only 226 (35%) who perceived the risk of TB infection to be high among ASMs. Only 564 (59%) were aware that silica dust causes permanent and incurable lung diseases. Six hundred and twenty (97%) showed a positive attitude towards healthcare when they were sick, and 97% were willing to use special respirators to prevent dust inhalation. On practices, only 159 (30%) reported consistent use of either cloth or respirators to prevent dust inhalation. Three hundred and five (49%) ASMs reported consistent use of condoms outside their homes and 323 (50%) reported use of water to suppress dust. Only 480 (75%) of ASMs sought healthcare services when sick. ASMs cited challenges of accessing healthcare services due to lack of money to pay for healthcare (50%), long distances to clinics (17%), and the shortage of medicines at clinics (11%). Effective control of TB, silicosis, and HIV among ASMs requires addressing the identified knowledge gaps and barriers that are faced by ASMs in accessing personal protective equipment and healthcare services. This will require multisector collaboration and the involvement of ASMs in co-designing a package of healthcare services that are tailored for them.

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Divergent Evolution of Early Terrestrial Fungi Reveals the Evolution of Mucormycosis Pathogenicity Factors.

Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

Mycoparasites, Gut Dwellers, and Saprotrophs: Phylogenomic Reconstructions and Comparative Analyses of Kickxellomycotina Fungi.

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.

Evolution of zygomycete secretomes and the origins of terrestrial fungal ecologies.

Fungi survive in diverse ecological niches by secreting proteins and other molecules into the environment to acquire food and interact with various biotic and abiotic stressors. Fungal secretome content is, therefore, believed to be tightly linked to fungal ecologies. We sampled 132 genomes from the early-diverging terrestrial fungal lineage zygomycetes (Mucoromycota and Zoopagomycota) and characterized their secretome composition. Our analyses revealed that phylogeny played an important role in shaping the secretome composition of zygomycete fungi with trophic mode contributing a smaller amount. Reconstruction of the evolution of secreted digestive enzymes revealed lineage-specific expansions, indicating that Mucoromycota and Zoopagomycota followed different trajectories early in their evolutionary history. We identified the presence of multiple pathogenicity-related proteins in the lineages known as saprotrophs, suggesting that either the ecologies of these fungi are incompletely known, and/or that these pathogenicity-related proteins have important functions associated with saprotrophic ecologies, both of which invite further investigation.

Protocol for single-cell isolation and genome amplification of environmental microbial eukaryotes for genomic analysis.

We describe environmental microbial eukaryotes (EMEs) sample collection, single-cell isolation, lysis, and genome amplification, followed by the rDNA amplification and OTU screening for recovery of high-quality species-specific genomes for de novo assembly. These protocols are part of our pipeline that also includes bioinformatic methods. The pipeline and its application on a wide range of phyla of different sample complexity are described in our complementary paper. In addition, this protocol describes optimized lysis, genome amplification, and OTU screening steps of the pipeline. For complete details on the use and execution of this protocol, please refer to Ciobanu et al. (2021).

A single-cell genomics pipeline for environmental microbial eukaryotes.

Single-cell sequencing of environmental microorganisms is an essential component of the microbial ecology toolkit. However, large-scale targeted single-cell sequencing for the whole-genome recovery of uncultivated eukaryotes is lagging. The key challenges are low abundance in environmental communities, large complex genomes, and cell walls that are difficult to break. We describe a pipeline composed of state-of-the art single-cell genomics tools and protocols optimized for poorly studied and uncultivated eukaryotic microorganisms that are found at low abundance. This pipeline consists of seven distinct steps, beginning with sample collection and ending with genome annotation, each equipped with quality review steps to ensure high genome quality at low cost. We tested and evaluated each step on environmental samples and cultures of early-diverging lineages of fungi and Chromista/SAR. We show that genomes produced using this pipeline are almost as good as complete reference genomes for functional and comparative genomics for environmental microbial eukaryotes.

Taxonomic notes on eight species of obligate mycoparasites in the genus Syncephalis isolated from soil and dung.

Species of Syncephalis (Zoopagomycotina, Piptocephalidaceae) are obligate mycoparasites that grow on common saprobic species of Mortierellomycotina and Mucoromycotina in soil and dung. Despite their ubiquitous occurrence across the globe, fungi in the genus Syncephalis are understudied, and there are few modern taxonomic treatments of these fungi. In order to clarify species concepts in the genus, we provide morphological data and discuss seven classical Syncephalis species: S. basibulbosa, S. cordata, S. depressa, S. hypogena, S. intermedia, S. nodosa, and S. sphaerica. Three of these species are only known as herbarium specimens (S. basibulbosa, S. cordata, S. intermedia). We have isolated co-cultures of the remaining parasites (S. depressa, S. nodosa, and S. sphaerica) on their host fungi both from nature and from culture collections. The remaining taxon (S. hypogena) was revived from a lyophilized culture. We provide photos and updated descriptions for all of these species as well as new geographic data and references to documented herbarium specimens for each taxon. In addition, we also describe the new species S. latigena.

Phylogenetic and morphological analyses of the mycoparasitic genus Piptocephalis.

The Piptocephalidaceae (Zoopagales, Zoopagomycota) contains three genera of mycoparasitic, haustoria-forming fungi: Kuzuhaea, Piptocephalis, and Syncephalis. Although the species in this family are diverse and ubiquitous in soil and dung, they are among the least studied fungi. Co-cultures of Piptocephalis and their hosts are relatively easy to isolate from soil and dung samples across the globe, making them a good model taxon for the order Zoopagales. This study focuses on the systematics of the genus Piptocephalis. Despite the fact that there are approximately 40 described Piptocephalis species, there are no modern taxonomic or molecular phylogenetic treatments of this group. Minimal sequence data are available, and relatively little is known about the true diversity or biogeography of the genus. Our study addresses two aspects: Piptocephalis systematics and analyses of the length and inter- and infraspecific variation of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. First, we generated a large subunit (28S) nuc rDNA phylogeny and evaluated several morphological characters by testing their correlation with the phylogeny using Bayesian Tip-association Significance testing (BaTS). We found monophyly of Piptocephalis species identified based on morphological traits, but morphological character states were not conserved across clades, suggesting that there have been multiple gains and losses of morphological characters. We also found that Kuzhuaea is nested within Piptocephalis. Second, we amplified the ITS from many Piptocephalis isolates, created a sequence alignment, and measured the lengths using the software ITSx. Piptocephalis species had ITS regions that were longer than the average for most Dikarya but were similar in length to those of the related genus Syncephalis.

Genome-scale phylogenetics reveals a monophyletic Zoopagales (Zoopagomycota, Fungi).

Previous genome-scale phylogenetic analyses of Fungi have under sampled taxa from Zoopagales; this order contains many predacious or parasitic genera, and most have never been grown in pure culture. We sequenced the genomes of 4 zoopagalean taxa that are predators of amoebae, nematodes, or rotifers and the genome of one taxon that is a parasite of amoebae using single cell sequencing methods with whole genome amplification. Each genome was a metagenome, which was assembled and binned using multiple techniques to identify the target genomes. We inferred phylogenies with both super matrix and coalescent approaches using 192 conserved proteins mined from the target genomes and performed ancestral state reconstructions to determine the ancestral trophic lifestyle of the clade. Our results indicate that Zoopagales is monophyletic. Ancestral state reconstructions provide moderate support for mycoparasitism being the ancestral state of the clade.

Leveraging single-cell genomics to expand the fungal tree of life.

Environmental DNA surveys reveal that most fungal diversity represents uncultured species. We sequenced the genomes of eight uncultured species across the fungal tree of life using a new single-cell genomics pipeline. We show that, despite a large variation in genome and gene space recovery from each single amplified genome (SAG), ≥90% can be recovered by combining multiple SAGs. SAGs provide robust placement for early-diverging lineages and infer a diploid ancestor of fungi. Early-diverging fungi share metabolic deficiencies and show unique gene expansions correlated with parasitism and unculturability. Single-cell genomics holds great promise in exploring fungal diversity, life cycles and metabolic potential.

Notes on Syncephalis (Zoopgales, Zoopagomycota) from the Farlow Herbarium, with the description of a new species, Syncephalis aethiopica.

The Harvard mycologist Roland Thaxter collected and studied a wide variety of fungi. Although he described >1000 fungal species, he was unable to describe all the specimens he collected before his death in 1932. His collections and microscope slides remain an important resource for studying fungal diversity at the Farlow Herbarium at Harvard University. Thaxter described three species in the obligate mycoparasite genus Syncephalis: S. pycnosperma, S. tenuis, and S. wynneae. He retained slide mounts of several other species, including a new species from his collection that was not described. Here, we describe that new species as S. aethiopica based on morphological analysis of his preserved slides. We also discuss the morphology of S. reflexa, a species apparently only known from slides preserved at the Farlow Herbarium.

Phylogenetic systematics of Syncephalis (Zoopagales, Zoopagomycotina), a genus of ubiquitous mycoparasites.

We examined phylogenetic relationships among species of the mycoparasite genus Syncephalis using sequences from three nuclear ribsosomal DNA genes (18S, 5.8S, and 28S nuc rDNA) and a gene encoding the largest subunit of RNA polymerase II (RPB1). Our data set included 88 Syncephalis isolates comprising 23 named species and several unnamed taxa. We also revived a culturing technique using beef liver and cellophane to grow several Syncephalis isolates without their host fungi to obtain pure parasite DNA. Most isolates, however, were grown in co-cultures with their host fungi, so we designed Syncephalis-specific primers to obtain sequence data. Individual and combined data sets were analyzed by maximum likelihood (ML) and Bayesian methods. We recovered 20 well-supported lineages and 38 operational taxonomic units (OTUs). Most major clades contained isolates from distant localities on multiple continents. There were taxonomic and nomenclature issues within several clades, probably due to high phenotypic plasticity or species dimorphism. We also conducted an analysis of Syncephalis nuc rDNA internal transcribed spacer (ITS) sequences for 31 phylogenetically diverse isolates, and we determined that most Syncephalis species have long ITS sequences relative to other fungi. Although commonly employed eukaryotic and fungal primers are compatible with diverse Syncephalis species, the ITS sequences of Syncepahlis are nonetheless rarely recovered in environmental molecular diversity surveys.

Preliminary phylogeny of Coemansia (Kickxellales), with descriptions of four new species from Taiwan.

Four new species of Coemansia from Taiwan are described. Three produce spirally twisted sporangiophores, and these new taxa increase the number of species in the Coemansia spiralis complex from three to six. Each new taxon is morphologically unique. Coemansia biformis, sp. nov., has two different asexual reproductive types on the same thallus; one is straight and the other has a spiral fertile region. Coemansia helicoidea, sp. nov., has stoloniferous sporangiophores with a helicoid fertile region. Coemansia pennisetoides, sp. nov., has a sporangiophore with a fertile region that resembles the inflorescence of the plant genus Pennisetum. Coemansia umbellata, sp. nov., has an umbellate sporangiophore branching pattern and a spirally twisted fertile region on the lowest branches. A dichotomous key was provided to identify the 23 accepted Coemansia species. Phylogenetic analysis based on a combined data set of D1-D2 domains of nuc 28S ribosomal RNA (rDNA) and partial nuc 18S rDNA identifies several independent evolutionary lineages within Coemansia and suggests that Spirodactylon aureum and Kickxella alabastrina may be nested within the genus Coemansia. Sequences of nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) are also used to support the description of these new species of Coemansia.

Five new species of the obligate mycoparasite Syncephalis (Zoopagales, Zoopagomycotina) from soil.

More than 520 soil samples were surveyed for species of the mycoparasitic zygomycete genus Syncephalis using a culture-based approach. These fungi are relatively common in soil using the optimal conditions for growing both the host and parasite. Five species obtained in dual culture are unknown to science and are described here: (i) S. digitata with sporangiophores short, merosporangia separate at the apices, simple, 3-5 spored; (ii) S. floridana, which forms galls in the host and has sporangiophores up to 170 µm long with unbranched merosporangia that contain 2-4 spores; (iii) S. pseudoplumigaleta, with an abrupt apical bend in the sporophore; (iv) S. pyriformis with fertile vesicles that are long-pyriform; and (v) S. unispora with unispored merosporangia. To facilitate future molecular comparisons between species of Syncephalis and to allow identification of these fungi from environmental sampling datasets, we used Syncephalis-specific PCR primers to generate internal transcribed spacer (ITS) sequences for all five new species.

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.

An eight-gene molecular phylogeny of the Kickxellomycotina, including the first phylogenetic placement of Asellariales.

Kickxellomycotina is a recently described subphylum encompassing four zygomycete orders (Asellariales, Dimargaritales, Harpellales, Kickxellales). These fungi are united by the formation of disciform septal pores containing lenticular plugs. Morphological diversification and life history evolution has made the relationships within and among the four orders difficult to resolve on those grounds alone. Here we infer the phylogeny of the Kickxellomycotina based on an eight-gene supermatrix including both ribosomal rDNA (18S, 28S, 5.8S) and protein sequences (MCM7, TSR1, RPB1, RPB2, ß-tubulin). The results of this study demonstrate that Kickxellomycotina is monophyletic and related to members of the Zoopagomycotina. Eight unique clades are distinguished in the Kickxellomycotina, including the four defined orders (Asellariales, Dimargaritales, Harpellales, Kickxellales) as well as four genera previously placed within two of these orders (Barbatospora, Orphella, Ramicandelaber, Spiromyces). Dimargaritales and Ramicandelaber are the earliest diverging members of the subphylum, although the relationship between these taxa remains uncertain. The remaining six clades form a monophyletic group, with Barbatospora diverging first. The next split divides the remaining members of the subphylum into two subclades: (i) Asellariales and Harpellales and (ii) Kickxellales, Orphella and Spiromyces. Estimation of ancestral states for four potentially informative morphological and ecological characters reveals that arthropod endosymbiosis might have been an important factor in the early evolution of the Kickxellomycotina.

Phylogenetic analysis of the genus Modicella reveals an independent evolutionary origin of sporocarp-forming fungi in the Mortierellales.

Most studies of tissue differentiation and development have focused on animals and plants but many fungi form multi-cellular aggregations of spore-bearing tissue known as fruiting bodies or sporocarps. The ability to form sporocarps has arisen independently in several different evolutionary lineages of fungi. Evolutionary relationships of most sporocarp-forming fungi are well known, but the enigmatic zygomycete genus Modicella contains two species of sporocarp-forming fungi for which the phylogenetic affinities have not been explored based on molecular data. Species of Modicella have an uncertain trophic mode and have alternatively been considered members of the order Endogonales (which contains documented species of sporocarp-forming fungi) or the order Mortierellales (which contains no previously documented species of sporocarp-forming fungi). In this study we perform phylogenetic analyses based on ribosomal DNA of Modicella malleola from the Northern Hemisphere and Modicella reniformis from the Southern Hemisphere to determine the evolutionary affinities of the genus Modicella. Our analyses indicate that Modicella is a monophyletic genus of sporocarp-forming fungi nested within the Mortierellales, a group of microfungi with no previously documented sporocarp-forming species. Because Modicella is distantly related to all other known sporocarp-forming fungi, we infer that this lineage has independently evolved the ability form sporocarps. We conclude that the genus Modicella should be a high priority for comparative genomics studies to further elucidate the process of sporocarp formation in fungi.

Two new Ramicandelaber species from Taiwan.

Two new species of Ramicandelaber isolated from soil in Taiwan are described. Ramicandelaber fabisporus sp. nov. is characterized by having bean-shaped spores that are the smallest in the genus and also by the absence of lateral branches on the sporangiophores. Ramicandelaber taiwanensis sp. nov. is characterized by small, fusiform spores and it produces lateral branches that arise from the sporangiophores. Morphological characters and molecular phylogenetic analysis (D1/D2 domains of the LSU rRNA genes and ITS region) justify these new species of Ramicandelaber. A key is provided to the recognized species of Ramicandelaber.

Evidence implicating Thamnostylum lucknowense as an etiological agent of rhino-orbital mucormycosis.

In this report, we present a case of rhino-orbital mucormycosis in a 57-year-old female with poorly controlled diabetes mellitus. The only mold cultured at 25°C, 37°C, and 40°C from a specimen of the nasal crust was identified phenotypically and independently using nuclear ribosomal DNA sequence data as Thamnostylum lucknowense. To our knowledge, this report presents the first data implicating this mucoraceous fungus as a mycotic agent of human infection.