Are dark septate endophytes an ancestral ecological state in the evolutionary history of the order Chaetothyriales?

Dark septate endophytes (DSE) are pigmented fungi that colonize plant roots. They represent a morpho-functional status composed of many species belonging to the phylum Ascomycota, distributed in different orders. The order Chaetothyriales has representatives with diverse lifestyles, among which the rock-inhabiting one has been proposed to be the ancestral ecological character state. However, all taxa have the phenotypic characteristic of being highly melanized. This trait has been considered relevant in most Chaetothyriales because it allows them to tolerate extreme or toxic environmental conditions. In the present study, aiming to reconstruct the evolutionary history of this order, we analyzed the contribution of the DSE habit to the diversification of the Chaetothyriales. We also report the distribution of the DSE habit among the main families and/or clades within the order. Our results suggest that DSE had a key position in the evolution of the order Chaetothyriales, both as an ancestral ecological character and as a character from which other specialized forms such as Domatium probably derived.

Sooty molds from the Jurassic of Patagonia, Argentina.

PREMISE: The sooty molds are a globally distributed ecological group of ascomycetes with epiphyllous, saprotrophic habit, comprising several phylogenetically distant taxa (i.e., members of the classes Dothideomycetes and Eurotiomycetes). Their fossil record extends almost continuously back to the early Cretaceous; however, they are hypothesized to have originated in the early Mesozoic. Here, we describe new specimens of sooty molds associated with conifer leaves from Jurassic hot spring deposits of Patagonia, Argentina. METHODS: Thin sections of chert samples from the La Matilde Formation, Deseado Massif (Santa Cruz, Argentina) were observed using light microscopy. RESULTS: The fungi occur on the surface and axils of leafy twigs with podocarpaceous affinities, forming dense subicula comprised by opaque moniliform hyphae. Additionally, several asexual and sexual reproductive structures are observed. On the basis of vegetative (i.e., dense subicula composed of moniliform hyphae; hyphae composed of opaque cells deeply constricted at the septa) and reproductive characters (i.e., poroconidial and sympodioconidial asexual stages and diverse spores), two morphotypes were identified with affinities within lineages of the subphylum Pezizomycotina that encompass the ecological group of sooty molds, and a third morphotype was within the phylum Ascomycota. CONCLUSIONS: This finding extends the fossil record of sooty molds to the Jurassic and their geographic fossil range to the South American continent. In particular, their association with podocarpaceous conifers is shown to be ancient, dating back to the Jurassic. This new record provides an additional reference point on the diversity of interactions that characterized Jurassic forests in Patagonia.

Mating type (MAT) locus and possible sexuality of the opportunistic pathogen Exophiala dermatitidis.

Sexual reproduction among the black yeasts is generally limited to environmental saprobic species and is rarely observed among opportunists in humans. To date, a complete sexual cycle has not been observed in Exophiala dermatitidis. In this study, we aimed to gain insight into the reproductive mode of E. dermatitidis by characterizing its mating type (MAT) locus, conducting MAT screening of environmental and clinical isolates, examining the expression of the MAT genes and analyzing the virulence of the isolates of different mating types. Similar to other members of the Pezizomycotina, the E. dermatitidis genome harbors a high mobility group (HMG) domain gene (MAT1-2-1) in the vicinity of the SLA2 and APN2 genes. The MAT loci of 74 E. dermatitidis isolates (11 clinical and 63 environmental) were screened by PCR, and the surrounding region was amplified using long-range PCR. Sequencing of the ∼ 12-kb PCR product of a MAT1-1 isolate revealed an α-box gene (MAT1-1-1). The MAT1-1 idiomorph was 3544-bp long and harbored the MAT1-1-1 and MAT1-1-4 genes. The MAT1-2 idiomorph was longer, 3771-bp, and harbored only the MAT1-2-1 gene. This structure suggests a heterothallic reproduction mode. The distribution of MAT among 74 isolates was ∼ 1:1 with a MAT1-1:MAT1-2 ratio of 35:39. RT-PCR analysis indicated that the MAT genes are transcribed. No significant difference was detected in the virulence of isolates representing different mating types using a Galleria mellonella model (P > 0.05). Collectively, E. dermatitidis is the first opportunistic black yeast in which both MAT idiomorphs have been characterized. The occurrence of isolates bearing both idiomorphs, their approximately equal distribution, and the expression of the MAT genes suggest that E. dermatitidis might reproduce sexually.

A Comparison of Isolation Methods for Black Fungi Degrading Aromatic Toxins.

The prevalence of black fungi in the order Chaetothyriales has often been underestimated due to the difficulty of their isolation. In this study, three methods which are often used to isolate black fungi are compared. Enrichment on aromatic hydrocarbon appears effective in inhibiting growth of cosmopolitan microbial species and allows appearance of black fungi. We miniaturized the method for high-throughput purposes. The new procedure saves time, consumes less space and can process multiple samples simultaneously.

A phylogenetic perspective on the association between ants (Hymenoptera: Formicidae) and black yeasts (Ascomycota: Chaetothyriales).

The frequency and the geographical extent of symbiotic associations between ants and fungi of the order Chaetothyriales have been highlighted only recently. Using a phylogenetic approach based on seven molecular markers, we showed that ant-associated Chaetothyriales are scattered through the phylogeny of this order. There was no clustering according to geographical origin or to the taxonomy of the ant host. However, strains tended to be clustered according to the type of association with ants: strains from ant-made carton and strains from plant cavities occupied by ants ('domatia') rarely clustered together. Defining molecular operational taxonomic units (MOTUs) with an internal transcribed spacer sequence similarity cut-off of 99% revealed that a single MOTU could be composed of strains collected from various ant species and from several continents. Some ant-associated MOTUs also contained strains isolated from habitats other than ant-associated structures. Altogether, our results suggest that the degree of specialization of the interactions between ants and their fungal partners is highly variable. A better knowledge of the ecology of these interactions and a more comprehensive sampling of the fungal order are needed to elucidate the evolutionary history of mutualistic symbioses between ants and Chaetothyriales.

ITS1 metabarcoding highlights low specificity of lichen mycobiomes at a local scale.

As self-supporting and long-living symbiotic structures, lichens provide a habitat for many other organisms beside the traditionally considered lichen symbionts-the myco- and the photobionts. The lichen-inhabiting fungi either develop diagnostic phenotypes or occur asymptomatically. Because the degree of specificity towards the lichen host is poorly known, we studied the diversity of these fungi among neighbouring lichens on rocks in an alpine habitat. Using a sequencing metabarcoding approach, we show that lichen mycobiomes clearly reflect the overlap of multiple ecological sets of taxa, which differ in their trophic association with lichen thalli. The lack of specificity to the lichen mycobiome is further supported by the lack of community structure observed using clustering and ordination methods. The communities encountered across samples largely result from the subsampling of a shared species pool, in which we identify three major ecological components: (i) a generalist environmental pool, (ii) a lichenicolous/endolichenic pool and (iii) a pool of transient species. These taxa majorly belong to the fungal classes Dothideomycetes, Eurotiomycetes and Tremellomycetes with close relatives in adjacent ecological niches. We found no significant evidence that the phenotypically recognized lichenicolous fungi influence the occurrence of the other asymptomatic fungi in the host thalli. We claim that lichens work as suboptimal habitats or as a complex spore and mycelium bank, which modulate and allow the regeneration of local fungal communities. By performing an approach that minimizes ambiguities in the taxonomic assignments of fungi, we present how lichen mycobiomes are also suitable targets for improving bioinformatic analyses of fungal metabarcoding.

Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota).

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

The genus Anthopsis and its phylogenetic position in Chaetothyriales.

The genus Anthopsis was introduced for a black fungus with peculiar, inverted phialides and triangular conidia. The genus accommodates, in addition to the type species Anthopsis deltoidea, which once was reported as a cause of human phaeohyphomycosis, two further taxa: A. catenata and A. microspora. Current taxonomy is mainly based on microscopic structures of phialides. To assess the phylogenetic position of the genus, sequences of the internal transcribed spacer region and partial LSU rDNA were obtained for Anthopsis spp. and compared with sequences from public databases. Phylogenetic analyses based on both loci were used to assess the evolutionary relationships of Anthopsis spp. at the family and ordinal levels. Anthopsis s.str. was found to cluster in Chaetothyriales, while A. catenata proved to be of helotialean affinity. Thermotolerance and morphology of each species were recorded.

Convergent structure and function of mycelial galleries in two unrelated Neotropical plant-ants.

The construction process and use of galleries by Azteca brevis (Myrmicinae: Dolichoderinae) inhabiting Tetrathylacium macrophyllum (Salicaceae) were compared with Allomerus decemarticulatus (Myrmicinae: Solenopsidini) galleries on Hirtella physophora (Chrysobalanaceae). Though the two ant species are phylogenetically distant, the gallery structure seems to be surprisingly similar and structurally convergent: both are pierced with numerous holes and both ant species use Chaetothyrialean fungi to strengthen the gallery walls. Al. decemarticulatus is known to use the galleries for prey capture and whether this is also the case for Az. brevis was tested in field experiments. We placed Atta workers as potential prey/threat on the galleries and recorded the behaviour of both ant species. We found considerable behavioural differences between them: Al. decemarticulatus was quicker and more efficient at capture than was Az. brevis. While most Atta workers were captured after the first 5 min by Al. decemarticulatus, significantly fewer were captured by Az. brevis even after 20 min. Moreover, the captured Atta were sometimes simply discarded and not taken to the nest by Az. brevis. As a consequence, the major function of the galleries built by Az. brevis may, therefore, be defense against intruders in contrast to Al. decemarticulatus which uses them mainly for prey capture. This may be due to a higher need for protein in Al. decemarticulatus compared to coccid-raising Az. brevis.

Biodiversity and human-pathogenicity of Phialophora verrucosa and relatives in Chaetothyriales.

Phialophora as defined by its type species P. verrucosa is a genus of Chaetothyriales, and a member of the group known as 'black yeasts and relatives'. Phialophora verrucosa has been reported from mutilating human infections such as chromoblastomycosis, disseminated phaeohyphomycosis and mycetoma, while morphologically similar fungi are rather commonly isolated from the environment. Phenotypes are insufficient for correct species identification, and molecular data have revealed significant genetic variation within the complex of species currently identified as P. verrucosa or P. americana. Multilocus analysis of 118 strains revealed the existence of five reproductively isolated species apparently having different infectious potentials. Strains of the sexual morph Capronia semiimmersa cluster within P. americana. The newly defined taxa differ markedly in their predilection for the human host.

Three new drimane-type sesquiterpenoids, chaetothyrins A-C, from an endolichenic fungus Chaetothyriales sp.

Three new drimane-type sesquiterpenoids chaetothyrins A-C (1-3), were isolated from an endolichenic fungus Chaetothyriales sp. (4341B). Their structures were determined by analysis of MS and NMR data as well as single-crystal X-ray diffraction analysis.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility.

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

The Metallotolerance and Biosorption of As(V) and Cr(VI) by Black Fungi.

A collection of 34 melanized fungi isolated previously from anthropogenic contaminated sites were assessed for their tolerance to toxic concentrations of As(V) and Cr(VI) anions. Three strains of the species Cyphellophora olivacea, Rhinocladiella similis, and Exophiala mesophila (Chaetothyriales) were identified as hyper-metallotolerant, with estimated IC50 values that ranged from 11.2 to 16.9 g L-1 for As(V) and from 2.0 to 3.4 g L-1 for Cr(VI). E. mesophila and R. similis were selected for subsequent assays on their biosorption capacity and kinetics under different pH values (4.0 and 6.5) and types of biomass (active and dead cells and melanin extracts). The fungal biosorption of As(V) was relatively ineffective, but significant removal of Cr(VI) was observed from liquid cultures. The Langmuir model with second-order kinetics showed maximum sorption capacities of 39.81 mg Cr6+ g-1 for R. similis and 95.26 mg Cr6+ g-1 for E. mesophila on a dry matter basis, respectively, while the kinetic constant for these two fungi was 1.32 × 10-6 and 1.39 × 10-7 g (mg Cr6+ min)-1. Similar experiments with melanin extracts of E. mesophila showed maximum sorption capacities of 544.84 mg Cr6+ g-1 and a kinetic constant of 1.67 × 10-6 g (mg Cr6+ min)-1. These results were compared to bibliographic data, suggesting that metallotolerance in black fungi might be the result of an outer cell-wall barrier to reduce the diffusion of toxic metals into the cytoplasm, as well as the inner cell wall biosorption of leaked metals by melanin.

Fungi as mutualistic partners in ant-plant interactions.

Associations between fungi and ants living in mutualistic relationship with plants ("plant-ants") have been known for a long time. However, only in recent years has the mutualistic nature, frequency, and geographical extent of associations between tropical arboreal ants with fungi of the ascomycete order Chaetothyriales and Capnodiales (belonging to the so-called "Black Fungi") become clear. Two groups of arboreal ants displaying different nesting strategies are associated with ascomycete fungi: carton-building ants that construct nest walls and galleries on stems, branches or below leaves which are overgrown by fungal hyphae, and plant-ants that make their nests inside living plants (myrmecophytes) in plant provided cavities (domatia) where ants cultivate fungi in small delimited "patches". In this review we summarize the current knowledge about these unsuspected plant-ant-fungus interactions. The data suggest, that at least some of these ant-associated fungi seem to have coevolved with ants over a long period of time and have developed specific adaptations to this lifestyle.

Discovery of Formicomyces microglobosus gen. et sp. nov. strengthens the hypothesis of independent evolution of ant-associated fungi in Trichomeriaceae.

Different groups of fungi have been reported to interact with ants. Recent studies have shown that fungi of the order Chaetothyriales are important components of ant-fungus networks, including members of the family Trichomeriaceae, which is particularly rich in fungi isolated from carton ants nests. One of the still understudied ant-related environments are ants' infrabuccal pockets and pellets, which often contain fungal matter. The aim of this work was to determine the systematic and phylogenetic position of two slow growing strains of Trichomeriaceae isolated from infrabuccal pellets of Formica polyctena ants. Molecular analyses based on maximum likelihood and bayesian inference, using sequences of two ribosomal DNA markers: ITS and LSU have shown that the isolated strains form a monophyletic clade within the family Trichomeriaceae, sister to a clade formed by representatives of the genus Trichomerium. Morphological analyses additionally justified distinctiveness of the isolated strains, which have different morphology of conidia and conidiophores than Trichomerium representatives. Therefore, our results show that the isolated strains represent a new species within a not yet described fungal genus. Due to the strains' isolation source and their close relatedness to a fungal strain isolated from a carton nest of Lasius fuliginosus, we propose a name Formicomyces microglobosus Siedlecki & Piatek for this fungus. While our discovery strengthens a hypothesis of the multiple, independent evolution of ant-associated fungi in the family Trichomeriaceae, the ecology of F. microglobosus still remains to be characterized.

Localization of the Swainsonine-Producing Chaetothyriales Symbiont in the Seed and Shoot Apical Meristem in Its Host Ipomoea carnea.

Several species of fungi from the orders Chaetothyriales and Pleosporales have been reported to produce swainsonine and be associated as symbionts with plants of the Convolvulaceae and Fabaceae, respectively. An endosymbiont belonging to the Chaetothyriales produces swainsonine and grows as an epibiont on the adaxial leaf surfaces of Ipomoea carnea, but how the symbiont passes through plant growth and development is unknown. Herein, different types of microscopy were used to localize the symbiont in seeds and in cross sections of plant parts. The symbiont was found in several tissues including the hilum, the sclereids, and the hypocotyl of seeds. In five-day old seedlings and mature plants, the symbiont was found in the shoot apical meristem (SAM) and the adaxial surface of immature folded leaves. The mycelia generally formed a close association with peltate glandular trichomes. This report provides further data explaining the relationship between the seed transmitted Chaetothyriales symbiont and Ipomoea carnea. These results provide a possible explanation for how this symbiont, and others like Periglandula may persist and are transmitted over time.

Black fungi and ants: a genomic comparison of species inhabiting carton nests versus domatia.

Some members of Chaetothyriales, an order containing potential agents of opportunistic infections in humans, have a natural habitat in nests of tropical arboreal ants. In these black fungi, two types of ant symbiosis are known, i.e. occurrence in domatia inside living plants, or as components of carton constructions made of ant-chewed plant tissue. In order to explain differences between strains from these types of association, we sequenced and annotated genomes of two newly described carton species, Incumbomyces lentus and Incumbomyces delicatus, and compared these with genomes of four domatia species and related Chaetothyriales. General genomic characteristics, CYP genes, carbohydrate-active enzymes (CAZymes), secondary metabolism, and sex-related genes were included in the study.

Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi.

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.

Novel black yeast-like species in chaetothyriales with ant-associated life styles.

Among ancestral fungi in Chaetothyriales, several groups have a life style in association with tropical ants, either in domatia or in carton-nests. In the present study, two strains collected from ant carton in Thailand and Malaysia were found to represent hitherto undescribed species. Morphological, physiological, phylogenetic data and basic genome information are provided for their classification. Because of the relatively large phylogenetic distances with known species confirmed by overall genome data, large subunit (LSU) and Internal Transcribed Spacer (ITS) ribosomal DNA sequences were sufficient for taxonomic circumscription of the species. The analyzed strains clustered with high statistical support as a clade in the family Trichomeriaceae. Morphologically they were rather similar, lacking sporulation in vitro. In conclusion, Incumbomyces delicatus and Incumbomyces lentus were described as new species based on morphological, physiological and phylogenetic analysis.

Slow-growing fungi belonging to the unnamed lineage in Chaetothyriomycetidae form hyphal coils in vital ericaceous rhizodermal cells in vitro.

The diversity and functionality of ericoid mycorrhizal (ErM) fungi are still being understudied. Members of Chaetothyriomycetidae evolved a specific lifestyle of inhabiting extreme, poor, or toxic environments. Some taxa in this subclass, especially in Chaetothyriales, are also putative ErM taxa, but their mycorrhizal ability is mostly unknown because the members are generally hard to isolate from roots. This study herein focused on eight root isolates and provided their phylogeny and morphology of root colonization. Phylogenetic analysis based on rRNA sequences clarified that the isolated strains were not classified into Chaetothyriales, but in an unnamed lineage in Chaetothyriomycetidae. This lineage also contains rock isolates, bryosymbionts, and a resinicolous species as well as various environmental sequences obtained from soil/root samples. All strains grew extremely slow by mycelia on cornmeal or malt extract agar (2.9-8.5 mm/month) and formed hyphal coils in vital rhizodermal cells of sterile blueberry seedlings in vitro. This study illustrated the presence of a novel putative ErM lineage in Chaetothyriomycetidae.