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Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely results in asci with fewer than eight ascospores. This study provides new insights into the poorly understood life cycle of Morchella species and more broadly improves knowledge of conidia formation and reproductive strategies in Pezizomycetes.

Summary: Fungi are among the most biodiverse organisms in the world. Accurate species identification is imperative for studies on fungal ecology and evolution. The internal transcribed spacer (ITS) rDNA region has been widely accepted as the universal barcode for fungi. However, several recent studies have uncovered intragenomic sequence variation within the ITS in multiple fungal species. Here, we mined the genome of 2414 fungal species to determine the prevalence of intragenomic variation and found that the genomes of 641 species, about one-quarter of the 2414 species examined, contained multiple ITS copies. Of those 641 species, 419 (∼65%) contained variation among copies revealing that intragenomic variation is common in fungi. We proceeded to show how these copies could result in the erroneous description of hundreds of fungal species and skew studies evaluating environmental DNA (eDNA) especially when making diversity estimates. Additionally, many genomes were found to be contaminated, especially those of unculturable fungi.

Laboulbeniales (Ascomycota, Laboulbeniomycetes) are biotrophic microfungi always attached to the exoskeleton of their arthropod hosts. They do not form hyphae or a mycelium; instead, they undergo determinate growth, developing from a two-celled ascospore to form a multicellular thallus. Hesperomyces virescens has been reported on over 30 species of ladybirds (Coleoptera, Coccinellidae); in reality, it represents a complex of species, presumably segregated by host genus association. In this study, we report on Hesperomyces thalli on Hyperaspis vinciguerrae from the Canary Islands and compare them with the Hesperomyces hyperaspidis described on Hyperaspis sp. from Trinidad. We generated the sequences of the internal transcribed spacer (ITS) region, the large subunit (LSU) nuclear ribosomal RNA gene, and the minichromosome maintenance complex component 7 (MCM7) protein-coding gene. Our phylogenetic reconstruction of Hesperomyces based on a concatenated ITS–LSU–MCM7 dataset revealed Hesperomyces sp. ex Hy. vinciguerrae as a member of the He. virescens species complex distinct from He. virescens sensu stricto (s.s.). It also revealed that the Hesperomyces sp. ex Chilocorus bipustulatus from Algeria is different from He. virescens s.s., which is associated with Chilocorus stigma from the USA. This suggests that the species of Hesperomyces are not solely segregated by host association, but that there is also a biogeographical component involved. Based on these data, we refrained from referring our material from Hy. vinciguerrae to He. hyperaspidis. Finally, we discuss the usefulness of MCM7 as a useful marker for species delimitation in Hesperomyces.

Botanical gardens have extensive spatial databases of their plant specimens; however, the fungi occurring in them are generally unstudied. Botanical gardens, with their great plant diversity, undoubtedly harbor a wide range of symbiotic fungi, including those that are plant-pathogenic. One such group of fungi is powdery mildews (Erysiphaceae). The powdery mildews are among the most prevalent and economically important plant pathogens in the world, with an estimated 906 species in 19 genera. They are known to infect more than 10,000 species of flowering plants and although some species occur across a range of hosts, many are associated with specific plants. Powdery mildews have undergone a long and dynamic coevolution with their host plants, resulting in co-speciation. Botanical gardens provide a living laboratory in which to study these fungi, leading to a wealth of undiscovered fungal diversity. Furthermore, monitoring pathogens in botanical gardens has led to important ecological findings related to the plant sciences and plant protection. Between 2018 and 2022, a collaborative citizen science project was established with 10 botanical gardens in the United States and Mexico. A total of more than 300 powdery mildew specimens were collected on 220 different host taxa. We sequenced the entire internal transcribed spacer (ITS) and large subunit (LSU) rDNA loci and phylogenetically and morphologically analyzed these collections revealing 130 species, of which 31 are likely unknown to science. This research highlights the importance of botanical gardens as a reservoir of fungal diversity. Future research will further elucidate the coevolutionary relationship between powdery mildews and their hosts and extend the current study to evaluate other plant pathogens and fungi in botanical gardens.

Abstract Since its resurrection, the resinicolous discomycete genus Sarea has been accepted as containing two species, one with black apothecia and pycnidia, and one with orange. We investigate this hypothesis using three ribosomal (nuITS, nuLSU, mtSSU) regions from and morphological examination of 70 specimens collected primarily in Europe and North America. The results of our analyses support separation of the traditional Sarea difformis s.lat. and Sarea resinae s.lat. into two distinct genera, Sarea and Zythia. Sarea as circumscribed is shown to conservatively comprise three phylospecies, with one corresponding to Sarea difformis s.str. and two, morphologically indistinguishable, corresponding to the newly combined Sarea coeloplata. Zythia is provisionally maintained as monotypic, containing only a genetically and morphologically variable Z. resinae. The new genus Atrozythia is erected for the new species A. klamathica. Arthrographis lignicola is placed in this genus on molecular grounds, expanding the concept of Sareomycetes by inclusion of a previously unknown type of asexual morph. Dating analyses using additional marker regions indicate the emergence of the Sareomycetes was roughly concurrent with the diversification of the genus Pinus, suggesting that this group of fungi emerged to exploit the newly-available resinous ecological niche supplied by Pinus or another, extinct group of conifers. Our phylogeographic studies also permitted us to study the introductions of these fungi to areas where they are not native, including Antarctica, Cape Verde, and New Zealand and are consistent with historical hypotheses of introduction.

Studies of Trochila (Leotiomycetes, Helotiales, Cenangiaceae) are scarce. Here, we describe two new species based on molecular phylogenetic data and morphology. Trochila bostonensis was collected at the Boston Harbor Islands National Recreation Area, Massachusetts. It was found on the stem of Asclepias syriaca, representing the first report of any Trochila species from a plant host in the family Apocynaceae. Trochila urediniophila is associated with the uredinia of the rust fungus Cerotelium fici. It was discovered during a survey for rust hyperparasites conducted at the Arthur Fungarium, in a single sample from 1912 collected in Trinidad. Macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses are presented. The two new species are placed in Trochila with high support in both our six-locus (SSU, ITS, LSU, rpb1, rpb2, tef1) and two-locus (ITS, LSU) phylogenetic reconstructions. In addition, two species are combined in Trochila: Trochila colensoi (formerly placed in Pseudopeziza) and T. xishuangbanna (originally described as the only species in Calycellinopsis). This study reveals new host plant families, a new ecological strategy, and a new country record for the genus Trochila. Finally, our work emphasizes the importance of specimens deposited in biological collections such as fungaria.

Powdery mildews are a group of economically and ecologically important plant pathogens. In the past 25 years the use of ribosomal DNA (rDNA) in the powdery mildews has led to major taxonomic revisions. However, the broad scale use of rDNA has also revealed multiple species complexes that cannot be differentiated based on ITS + LSU data alone. Currently, there are only two powdery mildew taxonomic studies that took a multi-locus approach to resolve a species complex. In the present study, we introduce primers to sequence four additional regions (CAM, GAPDH, GS, and RPB2) that have the potential to improve support values in both broad and fine scale phylogenetic analyses. The primers were applied to a broad set of powdery mildew genera in China and the United States, and phylogenetic analyses included some of the common complexes. In taxa with nearly identical ITS sequences the analyses revealed a great amount of diversity. In total 154 non-rDNA sequences from 11 different powdery mildew genera were deposited in NCBI’s GenBank, laying the foundation for secondary barcode databases for powdery mildews. The combined and single loci phylogenetic trees constructed generally followed the previously defined species/genus concepts for the powdery mildews. Future research can use these primers to conduct in depth phylogenetic, and taxonomic studies to elucidate the evolutionary relationships of species and genera within the powdery mildews.

During fungal surveys between 2012 and 2014 in pine-dominated forests of the western Himalayas in Pakistan, several collections of Pseudosperma (Agaricales, Inocybaceae) were made. These were documented, based on morphological and molecular data. During this work, three new species came to light, which are here formally described as Pseudosperma brunneoumbonatum, P. pinophilum and P. triacicularis. These species belong in the genus Pseudosperma fide Matheny et al. (2019) = Pseudosperma clade fide Matheny (2005) = Inocybe sect. Rimosae s.s. fide Larsson et al. (2009). Macro- and micro-morphological descriptions, illustrations and molecular phylogenetic reconstructions of the studied taxa are provided. The new species are differentiated from their close relatives by basidiospore size and colouration of basidiomata. Molecular phylogenetic relationships are inferred using ITS (ITS1–5.8S–ITS2), nrLSU and mtSSU sequence data. All three newly-described taxa likely share an ectomycorrhizal association with trees in the genus Pinus. In addition, five names are recombined in Inosperma, Mallocybe and Pseudosperma. These are Inosperma vinaceobrunneum, Mallocybe erratum, Pseudosperma alboflavellum, Pseudosperma friabile and Pseudosperma neglectum.

Tympanis species (Leotiales) are plant pathogens distributed mostly in northern temperate ecosystems. The diversity and identity of some species remains unclear. Tympanis vagabunda, found in Sicilia (Italy) on dry twigs of Rosa, Rubus, and Pistacia, is one example of an obscure and poorly known species. During the study of its type specimen in S, which contained one twig with a wood anatomy fitting neither of the three mentioned hosts, the microanatomic structures indicated that it belongs to the genus Rutstroemia (Helotiales). To investigate its identity, the types of R. fruticeti, R. juniperi, R. urceolus, and R. longiasca were studied for comparison. The species for which molecular data were available were included in a dataset that contained identified species of Rutstroemia, along with other select species from the families Rutstroemiaceae and Sclerotiniaceae. R. fruticeti, a saprobe frequently reported from Rubus fruticosus in Europe, is found to be a later synonym of T. vagabunda, and the combination Rutstroemia vagabunda is proposed. R. juniperi is an infrequently reported European species on twigs of Juniperus and is morphologically hard to distinguish from R. vagabunda; available molecular data support its recognition as a distinct species. R. longiasca differs from R. vagabunda in its black apothecia, smaller asci, and narrower ascospores. R. urceolus differs from R. vagabunda in having black apothecia and smaller inamyloid asci, and excipulum at the flanks and margin is composed of dark-walled hyphae.

Triblidiaceae is a family of uncommonly encountered, non-lichenized discomycetes. A recent classification circumscribed the family to include Triblidium (4 spp. and 1 subsp.), Huangshania (2 spp.) and Pseudographis (2 spp. and 1 var.). The apothecia of these fungi are persistent and drought-tolerant; they possess stromatic, highly melanized covering layers that open and close with fluctuations of humidity. Triblidialean fungi occur primarily on the bark of Quercus, Pinaceae and Ericaceae, presumably as saprobes. Though the type species of Huangshania is from China, these fungi are mostly known from collections originating from Western Hemisphere temperate and boreal forests. The higher-rank classification of triblidialean fungi has been in flux due in part to an overemphasis on ascospore morphology. Muriform ascospores are observed in species of Triblidium and in Pseudographis elatina. An intense, dark blue/purple ascospore wall reaction in iodine-based reagents is observed in species of Pseudographis. These morphologies have led, in part, to these genera being shuffled among unrelated taxa in Hysteriaceae (Dothideomycetes, Hysteriales) and Graphidaceae (Lecanoromycetes, Ostropales). Triblidiaceae has been placed within the monofamilial order Triblidiales (affinity Lecanoromycetes). Here, we demonstrate with a three-gene phylogenetic approach that triblidialean fungi are related to taxa in Rhytismatales (Leotiomycetes). We synonymize Triblidiales under Rhytismatales and emend Triblidiaceae to include Triblidium and Huangshania, with Pseudographis placed within Rhytismataceae. A history of Triblidiaceae is provided along with a description of the emended family. We discuss how the inclusion of triblidialean fungi in Rhytismatales brings some rarely observed or even unique ascospore morphologies to the order and to Leotiomycetes.

Abstract The Russula globispora lineage is a morphologically and phylogenetically well-defined group of ectomycorrhizal fungi occurring in various climatic areas. In this study we performed a multi-locus phylogenetic study based on collections from boreal, alpine and arctic habitats of Europe and Western North America, subalpine collections from the southeast Himalayas and collections from subtropical coniferous forests of Pakistan. European and North American collections are nearly identical and probably represent a single species named R. dryadicola distributed from the Alps to the Rocky Mountains. Collections from the southeast Himalayas belong to two distinct species: R. abbottabadensis sp. nov. from subtropical monodominant forests of Pinus roxburghii and R. tengii sp. nov. from subalpine mixed forests of Abies and Betula. The results suggest that speciation in this group is driven by a climate disjunction and adaptation rather than a host switch and geographical distance.

Closed cleistothecia-like ascomata have repeatedly evolved in non-related perithecioid and apothecioid lineages of lichenized and non-lichenized Ascomycota. The evolution of a closed, darkly pigmented ascoma that protects asci and ascospores is conceived as either an adaptation to harsh environmental conditions or a specialized dispersal strategy. Species with closed ascomata have mostly lost sterile hymenial elements (paraphyses) and the capacity to actively discharge ascospores. The class Leotiomycetes, one of the most speciose classes of Ascomycota, is mainly apothecioid, paraphysate, and possesses active ascospore discharge. Lineages with closed ascomata, and their morphological variants, have evolved independently in several families, such as Erysiphaceae, Myxotrichaceae, Rutstroemiaceae, etc. Thelebolales is a distinctive order in the Leotiomycetes class. It has two widespread families (Thelebolaceae, Pseudeurotiaceae) with mostly closed ascomata, evanescent asci, and thus passively dispersed ascospores. Within the order, closed ascomata dominate and a great diversity of peridia have evolved as adaptations to different dispersal strategies. The type genus, Thelebolus, is an exceptional case of ascomatal evolution within the order. Its species are the most diverse in functional traits, encompassing species with closed ascomata and evanescent asci, and species with open ascomata, active ascospore discharge, and paraphyses. Open ascomata were previously suggested as the ancestral state in the genus, these ascomata depend on mammals and birds as dispersal agents. In this scheme, species with closed ascomata, a lack of paraphyses, and passive ascospore discharge exhibit derived traits that evolved in adaptation to cold ecosystems. Here, we used morphological and phylogenetic methods, as well as the reconstruction of ancestral traits for ascomatal type, asci dehiscence, the presence or absence of paraphyses, and ascospore features to explore evolution within Thelebolales. We demonstrate the apothecial ancestry in Thelebolales and propose a new hypothesis about the evolution of the open ascomata in Thelebolus, involving a process of re-evolution where the active dispersal of ascospores appears independently twice within the order. We propose a new family, Holwayaceae, within Thelebolales, that retains the phenotypic features exhibited by species of Thelebolus, i.e., pigmented capitate paraphyses and active asci discharge with an opening limitation ring.

Harmonia axyridis is an invasive alien ladybird in North America and Europe. Studies show that multiple natural enemies are using Ha. axyridis as a new host. However, thus far, no research has been undertaken to study the effects of simultaneous infection by multiple natural enemies on Ha. axyridis. We hypothesized that high thallus densities of the ectoparasitic fungus Hesperomyces virescens on a ladybird weaken the host’s defenses, thereby making it more susceptible to infection by other natural enemies. We examined mortality of the North American-native Olla v-nigrum and Ha. axyridis co-infected with He. virescens and an entomopathogenic fungus—either Beauveria bassiana or Metarhizium brunneum. Laboratory assays revealed that He. virescens-infected O. v-nigrum individuals are more susceptible to entomopathogenic fungi, but Ha. axyridis does not suffer the same effects. This is in line with the enemy release hypothesis, which predicts that invasive alien species in new geographic areas experience reduced regulatory effects from natural enemies compared to native species. Considering our results, we can ask how He. virescens affects survival when confronted by other pathogens that previously had little impact on Ha. axyridis.

Abstract As public policies and conservation requirements for biodiversity evolve there is a need for a term for the kingdom Fungi equivalent to Fauna and Flora. This need is considered to be urgent in order to simplify projects oriented toward implemention of educational and conservation goals. In an informal meeting held during the IX Congreso Latinoamericano de Micología by the authors, the idea of clarifying this matter initiated an extensive search of pertinent terminologies. As a result of these discussions and reviews, we propose that the word Funga be employed as an accurate and encompassing term for these purposes. This supports the proposal of the three Fs, Fauna, Flora and Funga, to highlight parallel terminology referring to treatments of these macrorganism of particular geographical areas. Alternative terms and proposals are acknowledged and discussed.

Abstract The new genus Aotearoamyces is proposed to accommodate a single species that was repeatedly collected on fallen wood in Nothofagaceae forests of New Zealand and was previously misidentified as a Claussenomyces species. This monotypic genus belongs to Tympanidaceae, a recently erected family in Phacidiales. Aotearoamyces is differentiated from other Tympanidaceae by phragmospores that do not form conidia either in or outside the asci, an exciple of textura intricata with hyphae widely spaced and strongly gelatinized (plectenchyma), and apically flexuous. partly helicoid paraphyses. The asexual morph was studied in pure culture. Phylogenetic analyses of combined SSU, ITS and LSU sequences strongly support a sister relationship between the sexually typified Aotearoamyces and the asexually typified “Collophorina” paarla characterized morphologically by forming endoconidia, a feature not found in the genetically distinct type species of Collophorina. Based on our molecular results, we place the genus Epithamnolia in the Mniaecia lineage within Phacidiales.

Abstract The aim of this study was to explore the diversity of ectoparasitic fungi (Ascomycota, Laboulbeniales) that use bat flies (Diptera, Hippoboscoidea) as hosts. Bat flies themselves live as ectoparasites on the fur and wing membranes of bats (Mammalia, Chiroptera); hence this is a tripartite parasite system. Here, we collected bats, bat flies, and Laboulbeniales, and conducted phylogenetic analyses of Laboulbeniales to contrast morphology with ribosomal sequence data. Parasitism of bat flies by Laboulbeniales arose at least three times independently, once in the Eastern Hemisphere (Arthrorhynchus) and twice in the Western Hemisphere (Gloeandromyces, Nycteromyces). We hypothesize that the genera Arthrorhynchus and Nycteromyces evolved independently from lineages of ectoparasites of true bugs (Hemiptera). We assessed phylogenetic diversity of the genus Gloeandromyces by considering the LSU rDNA region. Phenotypic plasticity and position‐induced morphological adaptations go hand in hand. Different morphotypes belong to the same phylogenetic species. Two species, G. pageanus and G. streblae, show divergence by host utilization. In our assessment of coevolution, we only observe congruence between the Old World clades of bat flies and Laboulbeniales. The other associations are the result of the roosting ecology of the bat hosts. This study has considerably increased our knowledge about bats and their associated ectoparasites and shown the necessity of including molecular data in Laboulbeniales taxonomy.

Parasola is a genus of small, veil-less coprinoid mushrooms in the family Psathyrellaceae (Agaricales). The genus is not well documented in Asia, specifically in Pakistan. In this study we describe two new species Parasola glabra and P. pseudolactea from Pakistan, based on morphological and molecular data. Phylogeny based on three DNA regions: nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with the 5.8S rDNA (ITS), nuc 28S rDNA D1-D2 domains (28S) and translation elongation factor 1α gene (TEF1α) show that the new taxa are clustered in a clade formed by the members of section Parasola of genus Parasola. Parasola glabra with grayish pileus, slightly depressed pileal disc, lamellae separated from the stipe by pseudocollarium, basidiospores 14.5–16.5 × 9.5–11.5 × 8.0–10.5 µm, in front view broadly ovoid to oblong, some with rhomboidal outline, in side view ellipsoid, with eccentric germ-pore of 1.5 µm diameter. Parasola pseudolactea with yellowish brown to dull brown pileus, disc indistinctly umbonate, lamellae free, pseudocollarium absent, basidiospores 13.5–14.5 × 10.5–12.0 × 9.5–10.5 µm, in face view rounded triangular to heart shaped, rarely ovoid to subglobose, in side view ellipsoid to oblong, with eccentric germ-pore of 1.5 µm diam. In addition to these new species, P. auricoma and P. lilatincta were also studied. Morphological descriptions for the new species and comparison with known Parasola species are provided. Our observations highlight the diversity of Parasola in northern Pakistan and further document the need for additional systematic focus on the region’s fungi.

Harmonia axyridis is an invasive ladybird (Coleoptera, Coccinellidae) with the potential to outcompete native ladybird species in its invasive distribution area. It was introduced as a biological control agent in many countries but has also spread unintentionally in many others. Hesperomyces virescens (Ascomycota, Laboulbeniales) is a minute (200–400 µm in size) biotrophic fungus that infects over 30 species of ladybirds. The aim of this study was to evaluate whether the elytral color pattern, size, and sex of Ha. axyridis affect infection by H. virescens. Coloration in Ha. axyridis has been linked to the presence of an antimicrobial alkaloid (harmonine). In fall 2016, we collected 763 Ha. axyridis individuals in Cambridge, Massaschusetts, of which 119 (16%) bore H. virescens fruiting bodies. We analyzed 160 individuals (80 infected, 80 uninfected) concerning the intensity of infection by H. virescens. Elytral sizes and coloration patterns were quantified using digital photography and analytical methods. Smaller ladybirds had a higher prevalence and higher intensity of parasitism. Additionally, male ladybirds bore more thalli compared to female ladybirds. Elytral color patterns had an effect on neither prevalence nor intensity of infection by Laboulbeniales in our dataset, although we found a slight trend to higher intensity of parasitism in more melanic males. This suggests that the development of Laboulbeniales might be affected by certain insect alkaloids.