Aerial warfare: An inducible production of volatile bioactive metabolites in a novel species of Scytinostroma sp.

Antimicrobial volatile organic compounds (VOCs) may provide fungi an advantage over other competing microorganisms. As these defensive metabolites are often produced in response to microbial competitors, they are easily overlooked in axenic cultures. We used media supplemented with spent medium from Candida albicans to induce the expression of a broad-spectrum antimicrobial response in a previously uncharacterised white-rot fungus, Scytinostroma sp. Crude extractions of Scytinostroma sp. metabolites were found to be cytotoxic to fibroblast cells and antimicrobial to filamentous fungi, yeasts and Gram-positive bacteria. Volatile antimicrobial activity was observed for Scytinostroma sp. cultures and metabolite extracts using antimicrobial assays in bi-compartmentalised plates. Culture headspace analysis using solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) revealed a pronounced shift in Scytinostroma sp. VOCs when cultured on media supplemented with C. albicans spent medium. We observed a significant increase in the levels of 45 identified VOCs, including 7 metabolites with reported antimicrobial activity. Using preparative HPLC combined with GC-MS, we determined that isovelleral is likely to be the main broad-spectrum antimicrobial metabolite produced by Scytinostroma sp. Isovelleral is a sesquiterpene dialdehyde with both antibiotic and antifeedant properties, previously detected in fruit bodies of other Basidiomycetes.

Comparative genomics reveals a dynamic genome evolution in the ectomycorrhizal milk-cap (Lactarius) mushrooms.

Ectomycorrhizal fungi play a key role in forests by establishing mutualistic symbioses with woody plants. Genome analyses have identified conserved symbiosis-related traits among ectomycorrhizal fungal species, but the molecular mechanisms underlying host specificity remain poorly known. We sequenced and compared the genomes of seven species of milk-cap fungi (Lactarius, Russulales) with contrasting host specificity. We also compared these genomes with those of symbiotic and saprotrophic Russulales species, aiming to identify genes involved in their ecology and host specificity. The size of Lactarius genomes is significantly larger than other Russulales species, owing to a massive accumulation of transposable elements and duplication of dispensable genes. As expected, their repertoire of genes coding for plant cell wall-degrading enzymes is restricted, but they retained a substantial set of genes involved in microbial cell wall degradation. Notably, Lactarius species showed a striking expansion of genes encoding proteases, such as secreted ectomycorrhiza-induced sedolisins. A high copy number of genes coding for small secreted LysM proteins and Lactarius-specific lectins were detected, which may be linked to host specificity. This study revealed a large diversity in the genome landscapes and gene repertoires within Russulaceae. The known host specificity of Lactarius symbionts may be related to mycorrhiza-induced species-specific genes, including secreted sedolisins.

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi.

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE 'nests', or dense aggregations of TEs. Some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

Lactifluus (Russulaceae) diversity in Central America and the Caribbean: melting pot between realms.

Species of the ectomycorrhizal genus Lactifluus, and often entire sections, are typically unique to a single continent. Given these biogeographic patterns, an interesting region to study their diversity is Central America and the Caribbean, since the region is closely connected to and often considered a part of the North American continent, but biogeographically belong to the Neotropical realm, and comprises several regions with different geologic histories. Based on a multi-gene phylogeny and morphological study, this study shows that Central America, Mexico and the Caribbean harbour at least 35 Lactifluus species, of which 33 were never reported outside of this region. It was found that species from the Caribbean generally show affinities to South American taxa, while species from the Central American mainland generally show affinities to Northern hemispheric taxa. We hypothesise that host specificity and/or climate play a crucial role in these different origins of diversity. Because of these different affinities, Caribbean islands harbour a completely different Lactifluus diversity than the Central American mainland. The majority of species occurring on the islands can be considered endemic to certain islands or island groups. In this paper, detailed morphological descriptions are given, with a focus on the unique diversity of the islands, and identification keys to all hitherto described Lactifluus species occurring in Central America and the Caribbean are provided. One new section, Lactifluus sect. Nebulosi, and three new species, Lactifluus guadeloupensis, Lactifluus lepus and Lactifluus marmoratus are described.

Biochemical and genetic basis of orsellinic acid biosynthesis and prenylation in a stereaceous basidiomycete.

The prenylphenols are a class of natural products that have been frequently isolated from basidiomycetes, e.g., from the genus Stereum (false turkey tail fungi) and other Russulales as well as from ascomycetes. Biosynthetically, these compounds are considered hybrids, as the orsellinic acid moiety is a polyketide and the prenyl side chain originates from the terpene metabolism, although no literature on the genetic and biochemical background of the biosynthesis is available. In a stereaceous basidiomycete, referred to as BY1, a new prenylphenol, now termed cloquetin, was identified and its structure elucidated by mass spectrometry and nuclear magnetic resonance spectroscopy. Genes for two non-reducing polyketide synthases (PKS1 and PKS2) were identified in the BY1 genome, and heterologously expressed in Aspergillus niger. Product formation identified both PKSs as orsellinic acid synthases. A putative prenyltransferase gene (BYPB) found in the BY1 genome was expressed in Escherichia coli. In vitro characterization showed that BYPB activity depends on bivalent cations and that it uses orsellinic acid as acceptor substrate for the transfer of a prenyl group. The two orsellinic acid synthases support the emerging notion that fungi secure individual metabolic steps or entire pathways by redundant enzymes.

Two new Lactarius species from a subtropical cloud forest in eastern Mexico.

Two species of Lactarius from the subtropical montane cloud forest of central Veracruz are described as new. Both species are placed in subgenus Lactarius based on an accurate study of macro- and micromorphological features and supported by molecular phylogenetic analyses of a single nuc rDNA ITS1-5.8S-ITS2 (ITS), a concatenated ITS, D1, and D2 domains of nuc 28S rDNA (28S), and part of the second largest subunit of the RNA polymerase II (rpb2) (6-7 region) sequence datasets. In the phylogenetic reconstruction presented, the two species appear nested in two clearly distinct, well supported clades that reveal their phylogenetic position among members of subgenus Lactarius (= subg. Piperites s. auct.), a complex group of which a worldwide phylogeny requires further study. According to field observations, both taxa are possible ectomycorrhizal associates of Quercus spp. and one of Fagus grandifolia var. mexicana Taxonomic descriptions based on the study of specimens gathered during fieldwork from 2012 to 2014 are presented. Descriptions are accompanied with illustrations of macro and micromorphological characters and a discussion of related species is provided.

Sequestrate fungi from Patagonian Nothofagus forests: Cystangium (Russulaceae, Basidiomycota).

Six species of Cystangium, a genus of sequestrate taxa related to Russula, were collected in Patagonia (Argentina and Chile) during autumn 2001. Two species, C. depauperatum Singer & A.H. Sm. and C. nothofagi (E. Horak) Trappe, Castellano & T. Lebel, were already known from this region, while four new species, C. domingueziae, C. gamundiae, C. grandihyphatum and C. longisterigmatum, are described, illustrated and a key to the species is provided. In addition, sequences of the ITS (rDNA) region were obtained to explore the phylogenetic relationships of our South American Cystangium species.

Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.

Russula brunneoaurantiaca, a novel taxon of Russula subg. Crassotunicata from West Bengal, India, with morpho-molecular analysis and scanning electron microscopy.

This paper describes a new Russula species, R. brunneoaurantiaca, from India with morphological and molecular sequence (nrITS) data, field pictures of basidiocarps, and comparisons with close relatives. Russula brunneoaurantiaca has a brownish orange pileus with a mucilaginous surface, sub-decurrent lamellae that are white to pale orange, a white stipe that turns yellowish brown to brown when bruised, a strong, unpleasant smell, globose to subglobose basidiospores (5.0-9.0 5.0-7.8 m) with an inamyloid suprahilar spot and ornamentation of small isolated conical warts, fusiform hymenial cystidia on gill sides (62.5-82 × 7.5-12.5 µm) and lageniform to sub-lageniform cystidia with filiform apex near the gill edge (80-113 × 7.5-10 µm), fusiform to spindle-shaped pileocystidia, and habitat in association with Castanopsis sp. A complete morphological description, photographs, and molecular sequence-based phylogenetic trees demarcating the position of the novel taxon are provided. RESEARCH HIGHLIGHTS: Scanning electron microscopy (SEM) and subsequent DNA analysis revealed a new species of the genus Russula. SEM analysis is an additional technique to describe the size and shape of its basidiospores as well as their ornamentation. The diagnostic characteristics, habit, habitat, and similarities to related species are given.

Four new species of Russula from the Xizang Autonomous Region and other provinces of China.

Russula is the largest genus in the Russulales and is widespread throughout the world. Almost all Russula species are known to be ectomycorrhizal with high ecological and edible values, and some are lethal poisonous. In this study, four new species belonging to the subgenus Russula crown clade are identified based on morphological and phylogenetic evidence from the Xizang Autonomous Region and other provinces of China. Morphologically, Russula paragraveolens (sect. Polychromae, subsect. Xerampelinae) is mainly characterised by a cherry red to blood red pileus centre, a reddish orange pileus margin; R. pseudograveolens (sect. Polychromae, subsect. Xerampelinae) is characterised by a violet brown to brownish red pileus centre, a pale red to pastel red pileus margin and short basidia; R. shigatseensis (sect. Flavisiccantes, subsect. Lepidinae) is characterised by a brownish orange to madder red pileus centre, pinkish red pileus margin, and having lateral branches or branches of hyphal terminations in pileipellis; R. yadongensis (sect. Tenellae, subsect. Laricinae) is characterised by a dark purplish red pileus centre with brownish purple tints and having isolated to clustered spines of spore ornamentations. Their distinct taxonomic status is confirmed by the positions of the four new species in both the ITS and 4-locus (nucLSU, mtSSU, rpb2, tef1) phylogenetic trees.

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

A new and threatened species of Bondarzewia from the Brazilian cloud forests.

A new and threatened polypore species, Bondarzewia loguerciae, is described from the cloud forests of southern Brazil. It is characterized by single-pileate basidiomata that grow on dead branches and along living stems of standing trunks and present a context with dark lines and resinous tubes. When growing in axenic culture, this species also develops chlamydospores. We provide an illustrated morphological description and molecular analysis. Our specimens from Brazil form a monophyletic group among other species of the Southern Hemisphere. The conservation status of B. loguerciae is assessed and published as "Critically Endangered" based on the International Union for Conservation of Nature  (IUCN) criteria. Additionally, a key to the species is provided.

Hericium ophelieae sp. nov., a novel species of Hericium (Basidiomycota: Russulales, Hericiaceae) from the Southern Afrotemperate forests of South Africa.

A novel species of Hericium was recently collected in the Afrotemperate forests (Knysna - Amatole region) of Southern Africa. The novel species shares many similar, dentate features common to other species in Hericium, and its basidiome first appears stark white and yellows with age. However, the substrate choice and gloeocystidia and basidiospore sizes of the specimens collected were distinct from other Hericium species. This was confirmed by sequencing the ITS and 28S genetic markers, respectively. The novel species is described as Hericium ophelieae sp. nov. and appears unique as it grows on hardwoods indigenous to Southern Africa. The species has larger basidiospores and wider gloeocystidia compared to its closest relative. H. ophelieae sp. nov. is the first endemic species of the medicinal mushroom genus Hericium to be described from Southern Africa, and the second to be described from Africa, after its closest relative, H. bembedjaense, which was isolated in Cameroon. Although this is the first Hericium to be described from the Southern African region, there are likely others to be discovered, and this study highlights the need for further research into the fungal diversity of Afrotemperate environments.

The genome sequence of the Fruity Milkcap, Lactarius evosmus (Kühner & Romagn., 1954).

We present a genome assembly from an individual Lactarius evosmus (Fruity Milkcap; Basidiomycota; Agaricomycetes; Russulales; Russulaceae). The genome sequence is 57.2 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 61.51 kilobases in length.

Russulaceae of the Pakaraima Mountains of Guyana. IV. New species forming a distinct lineage of Lactarius subg. Plinthogalus.

The Neotropics have recently emerged as an important region for studies of tropical ectomycorrhizal (ECM) fungi. Specific neotropical areas with high ECM host tree densities have ECM fungal diversities rivaling those of higher-latitude forests. Some forests of the Guiana Shield are dominated by endemic ECM trees of the Fabaceae, including species of Dicymbe (subfam. Detarioideae), Aldina (subfam. Papilionoideae), and Pakaraimaea (Cistaceae). One of the most species-rich ECM fungal families present in each of these systems is Russulaceae. Long-term sampling in forests in Guyana's Pakaraima Mountains has revealed a number of species of the Russulaceae genera Lactarius, Lactifluus, and Russula. In this study, we document a previously unknown, distinct lineage of Lactarius subg. Plinthogalus containing eight species from the Guiana Shield. Here, we describe five of these species from Guyana as new to science: Lactarius humiphilus, Lactarius mycenoides, Lactarius guyanensis, Lactarius dicymbophilus, and Lactarius aurantiolamellatus. Morphological descriptions, habit, habitat, and known distribution are provided for each new species. Sequence data for the barcode internal transcribed spacer (ITS) locus are provided for types and most other collections of the new taxa, and a molecular phylogenetic analysis based on the ITS, 28S, and RPB2 (second-largest subunit of the RNA polymerase II) loci across the genus Lactarius corroborates their morphology-based infrageneric placement. The discovery of this lineage changes our insights into the biogeography and evolutionary history of Lactarius subg. Plinthogalus.

Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora.

Symbiosis between insects and fungi arose multiple times during the evolution of both groups, and some of the most biologically diverse and economically important are mutualisms in which the insects cultivate and feed on fungi. Among these are bark beetles, whose ascomycetous cultivars are better known and studied than their frequently-overlooked and poorly understood basidiomycetous partners. In this study, we propose five new species of Entomocorticium, fungal mutualists in the Russulales (Basidiomycota) that are mutualistic symbionts of scolytine beetles. We have isolated these fungi from the beetle mycangia, which are structures adapted for the selective storage and transportation of fungal mutualists. Herein, we present the most complete phylogeny of the closely related genera Entomocorticium and Peniophora and provide insights into how an insect-associated taxon (Entomocorticium) evolved from within a wood-decaying, wind-dispersed lineage (Peniophora). Our results indicate that following a transition from angiosperms to gymnosperms, fungal domestication by beetles facilitated the evolution and diversification of Entomocorticium. We additionally propose four new species: Entomocorticium fibulatum Araújo, Li & Hulcr, sp. nov.; E. belizense Araújo, Li & Hulcr, sp. nov.; E. perryae Araújo, Li & Hulcr, sp. nov.; and E. macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov. Our findings highlight the fact that insect-fungi associations remain an understudied field and that these associations harbor a large reservoir of novel fungal species.

A short story of nearly everything in Lactifluus (Russulaceae).

Fungi are a large and hyper-diverse group with major taxa present in every ecosystem on earth. However, compared to other eukaryotic organisms, their diversity is largely understudied. Since the rise of molecular techniques, new lineages are being discovered at an increasing rate, but many are not accurately characterised. Access to comprehensive and reliable taxonomic information of organisms is fundamental for research in different disciplines exploring a variety of questions. A globally dominant ectomycorrhizal (ECM) fungal family in terrestrial ecosystems is the Russulaceae (Russulales, Basidiomycota) family. Amongst the mainly agaricoid Russulaceae genera, the ectomycorrhizal genus Lactifluus was historically least studied due to its largely tropical distribution in many underexplored areas and the apparent occurrence of several species complexes. Due to increased studies in the tropics, with a focus on this genus, knowledge on Lactifluus grew. We demonstrate here that Lactifluus is now one of the best-known ECM genera. This paper aims to provide a thorough overview of the current knowledge of Lactifluus, with information on diversity, distribution, ecology, phylogeny, taxonomy, morphology, and ethnomycological uses of species in this genus. This is a result of our larger study, aimed at building a comprehensive and complete dataset or taxonomic framework for Lactifluus, based on molecular, morphological, biogeographical, and taxonomical data as a tool and reference for other researchers. Citation: De Crop E, Delgat L, Nuytinck J, Halling RE, Verbeken A (2021). A short story of nearly everything in Lactifluus (Russulaceae). Fungal Systematics and Evolution 7: 133-164. doi: 10.3114/fuse.2021.07.07.

Enlightening the black and white: species delimitation and UNITE species hypothesis testing in the Russula albonigra species complex.

Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R. albonigra s. lat. represents a species complex consisting of at least five European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit R. albonigra are not always reliable. Therefore, a new delimitation of the R. albonigra complex is proposed and a key to the described European species of R. subgen. Compactae is presented. A lectotype and an epitype are designated for R. albonigra and three new European species are described: R. ambusta, R. nigrifacta, and R. ustulata. Different thresholds of UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality sequences.

Morphological and genetic diversification of Russula floriformis, sp. nov., along the Isthmus of Panama.

Species of Russula are ubiquitous members of ectomycorrhizal fungal communities in tropical ecosystems. However, an important part of the total tropical diversity of this genus and its biogeographic patterns is unknown due to the lack of studies on Russula in tropical ecosystems. We combined molecular, morphological, ecological, and biogeographic data to elaborate concepts for two new subspecies of R. floriformis (subsection Substriatinae). Russula floriformis subsp. floriformis and R. floriformis subsp. symphoniae are described as new from montane forest dominated by Quercus and/or Oreomunnea (Fagales) from Colombia and Panama, respectively. Phylogenies were constructed using nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S), and partial regions of the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-alpha (tef1). Similar environmental conditions, similar morphology, and an ITS sequence similarity higher than 99% with only three different positions indicate that these two subspecies are closely related. Detailed observations of microscopic structures and analyses of further DNA loci, however, revealed morphological and molecular characteristics that allow distinguishing the two subspecies of R. floriformis. Spatial distribution and phylogenetic proximity of the two Russula subspecies and their ectomycorrhizal hosts, i.e., species of Quercus, suggest that their diversification is a result of comigration, adaptation, and geographic isolation along the Isthmus of Panama during the Pliocene and Pleistocene.

Looks can be deceiving: the deceptive milkcaps (Lactifluus, Russulaceae) exhibit low morphological variance but harbour high genetic diversity.

The ectomycorrhizal genus Lactifluus is known to contain many species complexes, consisting of morphologically very similar species, which can be considered cryptic or pseudocryptic. In this paper, a thorough molecular study is performed of the clade around Lactifluus deceptivus (originally described by Peck from North America) or the deceptive milkcaps. Even though most collections were identified as L. deceptivus, the clade is shown to contain at least 15 species, distributed across Asia and America, indicating that the L. deceptivus clade represents a species complex. These species are morphologically very similar and are characterized by a tomentose pileus with thin-walled hyphae and a velvety stipe with thick-walled hyphae. An ITS1 sequence was obtained through Illumina sequencing for the lectotype of L. deceptivus, dating from 1885, revealing which clade represents the true L. deceptivus. In addition, it is shown that three other described species also belong to the L. deceptivus clade: L. arcuatus, L. caeruleitinctus and L. mordax, and molecularly confirmed that L. tomentoso-marginatus represents a synonym of L. deceptivus. Furthermore, two new Neotropical species are described: Lactifluus hallingii and L. domingensis.