Phylogenetics of the rust fungi (Pucciniales) of South Africa, with notes on their life histories and possible origins.

South Africa has an indigenous rust (Pucciniales) funga of approximately 460 species. This funga was sampled with species from as many genera as possible. The nuclear ribosomal large subunit (28S) region was amplified from samples representing 110 indigenous species, as well as the small subunit (18S) region and the cytochrome c oxidase subunit 3 (CO3) in some cases, and these were used in phylogenetic analyses. One new species is described, 12 new combinations made, six names reinstated, and two life history connections made. The life histories of this funga were summarized; it is dominated by species with contracted life histories. The majority of species are autoecious, with a small proportion being heteroecious. Of the autoecious species, many will likely be homothallic with no spermagonia. A shortened life history with homothallism allows for a single basidiospore infection to initiate a local population buildup under the prevailing unpredictable climatic conditions. Suggestions are made as to the possible origin of this funga based on the development of the modern South African flora. It is postulated that the rusts of South Africa are of relatively recent origin, consisting of three groups. Firstly, there is an African tropical element with members of the Mikronegerineae (Hemileia), the Sphaerophragmiaceae (Puccorchidium, Sphaerophragmium), and certain Uredinineae (Stomatisora). Their immediate ancestors likely occurred in the tropical forests of Africa during the Paleogene. Secondly, there is a pantropical element including the Raveneliaceae (e.g., Diorchidium, Maravalia, Ravenelia sensu lato, Uropyxis). This likely diversified during the Neogene, when the mimosoids became the dominant trees of the developing savannas. Thirdly, the Pucciniaceae invaded Africa as this continent pushed northward closing the Tethys Sea. They diversified with the development of the savannas as these become the dominant habitat in most of Africa, and are by far the largest component of the South African rust funga.

A revised phylogeny of Boletaceae using whole genome sequences.

The porcini mushroom family Boletaceae is a diverse, widespread group of ectomycorrhizal (ECM) mushroom-forming fungi that so far has eluded intrafamilial phylogenetic resolution based on morphology and multilocus data sets. In this study, we present a genome-wide molecular data set of 1764 single-copy gene families from a global sampling of 418 Boletaceae specimens. The resulting phylogenetic analysis has strong statistical support for most branches of the tree, including the first statistically robust backbone. The enigmatic Phylloboletellus chloephorus from non-ECM Argentinian subtropical forests was recovered as a new subfamily sister to the core Boletaceae. Time-calibrated branch lengths estimate that the family first arose in the early to mid-Cretaceous and underwent a rapid radiation in the Eocene, possibly when the ECM nutritional mode arose with the emergence and diversification of ECM angiosperms. Biogeographic reconstructions reveal a complex history of vicariance and episodic long-distance dispersal correlated with historical geologic events, including Gondwanan origins and inferred vicariance associated with its disarticulation. Together, this study represents the most comprehensively sampled, data-rich molecular phylogeny of the Boletaceae to date, establishing a foundation for future robust inferences of biogeography in the group.

Rust HUBB: DNA barcode-based identification of Pucciniales.

Rust fungi (Pucciniales, Basidiomycota) are a species-rich (ca. 8000 species), globally distributed order of obligate plant pathogens. Rust species are host-specific, and as a group they cause disease on many of our most economically and/or ecologically significant plants. As such, the ability to accurately and rapidly identify these fungi is of particular interest to mycologists, botanists, agricultural scientists, farmers, quarantine officials, and associated stakeholders. However, the complexities of the rust life cycle, which may include production of up to five different spore types and alternation between two unrelated host species, have made standard identifications, especially of less-documented spore states or alternate hosts, extremely difficult. The Arthur Fungarium (PUR) at Purdue University is home to one of the most comprehensive collections of rust fungi in the world. Using material vouchered in PUR supplemented with fresh collections we generated DNA barcodes of the 28S ribosomal repeat from > 3700 rust fungal specimens. Barcoded material spans 120 genera and > 1100 species, most represented by several replicate sequences. Barcodes and associated metadata are hosted in a publicly accessible, BLAST searchable database called Rust HUBB (Herbarium-based Universal Barcode Blast) and will be continuously updated.

The surface of leaves and fruits of Peruvian cacao is home for several Hannaella yeast species, including the new species Hannaella theobromatis sp. nov.

As part of a long-term study aiming to isolate and identify yeast species that inhabit the surface of leaves and fruits of native fine-aroma cacao in the department of Amazonas, Peru, we obtained multiple isolates of Hannaella species. Yeasts of the genus Hannaella are common inhabitants of the phyllosphere of natural and crop plants. On the basis of morphological, and physiological characteristics, and sequence analysis of the D1/D2 domains of the large subunit rRNA gene (LSU) and the internal transcribed spacer region (ITS), we identified five species of Hannaella from the phyllosphere of Peruvian cacao. Four have been previously described: H. phyllophila (isolates KLG-073, KLG-091), H. pagnoccae (KLG-076), H. sinensis (KLG-121), and H. taiwanensis (KLG-021). A fifth, represented by eight isolates (KLG-034, KLG-063, KLG-074, KLG-078, KLG-79, KLG-082, KLG-084, KLG-085), is not conspecific with any previously described Hannaella species, and forms the sister clade to H. surugaensis in the phylogenetic analysis. It has 2.6-3.9% (18-27 substitutions, 2-4 deletions, and 1-3 insertions in 610-938 bp-long alignments), and 9.8-10.0% nucleotide differences (37 substitutions and 14 insertions in 511-520 bp-long alignments) in the LSU and ITS regions, respectively, to H. surugaensis type strain, CBS 9426. Herein, the new species Hannaella theobromatis sp. nov. is described and characterised. The species epithet refers to its epiphytic ecology on its host Theobroma cacao.

Extensive intragenomic variation in the internal transcribed spacer region of fungi.

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.

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.

Molecular phylogenetic analyses and micromorphology reveal placement of the enigmatic tropical discomycete Polydiscidium in Sclerococcum (Sclerococcales, Eurotiomycetes).

Polydiscidium is an enigmatic, monotypic, and rarely reported genus of Ascomycota of uncertain placement. The morphologically unique Polydiscidium martynii grows on dead wood and forms compound ascomata composed of thick, black, gelatinous somatic tissue that branches out from a common base. Multiple apothecia are located on the branches, mostly toward the tips, and are composed of 8-spored asci and paraphyses embedded in a gelatinous matrix that turns blue in Melzer's reagent. The species was previously known from only three collections from Guyana (holotype), Trinidad, and the Democratic Republic of the Congo and no sequences exist. Due to its peculiar morphology, taxonomic affinities of Polydiscidium have been debated, with different authors having placed it in Helotiaceae, Leotiaceae, or Leotiomycetes incertae sedis. Recent collections of this species resulting from long-term field work in Guyana and Cameroon led us to revisit the morphology and phylogenetic position of this fungus. Newly generated sequences of P. martynii were added to an Ascomycota-wide six-locus data set. The resulting phylogeny showed Polydiscidium to be a member of order Sclerococcales (Eurotiomycetes). Next, a four-locus (18S, ITS, 28S, mtSSU) phylogenetic reconstruction revealed that Polydiscidium is congeneric with Sclerococcum. A new combination is proposed for this species, Sclerococcum martynii. Micromorphological features, including the gelatinous hymenium composed of asci with amyloid gel cap and septate brown ascospores, are in agreement with Sclerococcum. New combinations are proposed for two additional species: Sclerococcum chiangraiensis and S. fusiformis. Finally, Dactylosporales is considered a later synonym of Sclerococcales.

Sexual reproduction is the null hypothesis for life cycles of rust fungi.

Sexual reproduction, mutation, and reassortment of nuclei increase genotypic diversity in rust fungi. Sexual reproduction is inherent to rust fungi, coupled with their coevolved plant hosts in native pathosystems. Rust fungi are hypothesised to exchange nuclei by somatic hybridisation with an outcome of increased genotypic diversity, independent of sexual reproduction. We provide criteria to demonstrate whether somatic exchange has occurred, including knowledge of parental haplotypes and rejection of fertilisation in normal rust life cycles.

The life cycle of Puccinia digitariae on Digitaria eriantha and Solanum species in South Africa.

Rust fungi are important plant pathogens and have been extensively studied on crops and other host plants worldwide. This study describes the heterecious life cycle of a rust fungus on Digitaria eriantha (finger grass) and the Solanum species S. lichtensteinii (large yellow bitter apple), S. campylacanthum (bitter apple), and S. melongena (eggplant) in South Africa. Following field observations, inoculation studies involving telial isolates collected from Digitaria plants produced spermogonia and aecia on S. lichtensteinii, S. campylacanthum, and S. melongena. Likewise, inoculation of finger grass with aeciospores collected from the aforementioned Solanum species produced uredinia on D. eriantha. Pennisetum glaucum (pearl millet varieties Milkstar and Okashana, as well as 17 experimental lines) and S. elaeagnifolium (silverleaf nightshade or bitter apple) were resistant to the rust isolates. Morphological descriptions and molecular phylogenetic data confirmed the identity of the rust on Digitaria as P. digitariae, herein reinstated as a species and closely related to P. penicillariae the pearl millet rust, also reinstated. Puccinia digitariae has a macrocyclic, heterecious life cycle in which teliospores overwinter on dormant D. eriantha plants. Aecia sporulate on species of Solanum during spring and early summer to provide inocula that infect new growth of Digitaria.

Sporobolomyces lactucae sp. nov. (Pucciniomycotina, Microbotryomycetes, Sporidiobolales): An Abundant Component of Romaine Lettuce Phylloplanes.

Shifts in food microbiomes may impact the establishment of human pathogens, such as virulent lineages of Escherichia coli, and thus are important to investigate. Foods that are often consumed raw, such as lettuce, are particularly susceptible to such outbreaks. We have previously found that an undescribed Sporobolomyces yeast is an abundant component of the mycobiome of commercial romaine lettuce (Lactuca sativa). Here, we formally describe this species as Sporobolomyces lactucae sp. nov. (Pucciniomycotina, Microbotryomycetes, and Sporidiobolales). We isolated multiple strains of this yeast from commercial romaine lettuce purchased from supermarkets in Illinois and Indiana; additional isolates were obtained from various plant phylloplanes in California. S. lactucae is a red-pigmented species that is similar in appearance to other members of the genus Sporobolomyces. However, it can be differentiated by its ability to assimilate glucuronate and D-glucosamine. Gene genealogical concordance supports S. lactucae as a new species. The phylogenetic reconstruction of a four-locus dataset, comprising the internal transcribed spacer and large ribosomal subunit D1/D2 domain of the ribosomal RNA gene, translation elongation factor 1-α, and cytochrome B, places S. lactucae as a sister to the S. roseus clade. Sporobolomyces lactucae is one of the most common fungi in the lettuce microbiome.

Draft Genome Sequence of an Unusual Ectomycorrhizal Fungus, Pseudotulostoma volvatum.

Here, we report the draft genome sequence of Pseudotulostoma volvatum, an unusual ectomycorrhizal fungus in the "mold" order Eurotiales (Ascomycota, Pezizomycotina). The assembled genome is 60.4 Mbp and contains an estimated 5,492 genes. Compared with closely related species, the P. volvatum genome is depauperate in secondary metabolite gene clusters.

Coffee Leaf Rust (Hemileia vastatrix) from the Recent Invasion into Hawaii Shares a Genotypic Relationship with Latin American Populations.

Hawaii has long been one of the last coffee-producing regions of the world free of coffee leaf rust (CLR) disease, which is caused by the biotrophic fungus Hemileia vastatrix. However, CLR was detected in coffee farms and feral coffee on the island of Maui in February 2020 and subsequently on other islands of the Hawaiian archipelago. The source of the outbreak in Hawaii is not known, and CLR could have entered Hawaii from more than 50 coffee-producing nations that harbor the pathogen. To determine the source(s) of the Hawaii inoculum, we analyzed a set of eleven simple sequence repeat markers (SSRs) generated from Hawaii isolates within a dataset of 434 CLR isolates collected from 17 countries spanning both old and new world populations, and then conducted a minimum spanning network (MSN) analysis to trace the most likely pathway that H. vastatrix could have taken to Hawaii. Forty-two multilocus genotypes (MLGs) of H. vastatrix were found in the global dataset, with all isolates from Hawaii assignable to MLG 10 or derived from it. MLG 10 is widespread in Central America and Jamaica, making this region the most probable source of inoculum for the outbreak in Hawaii. An examination of global weather patterns during the months preceding the introduction of CLR makes it unlikely that the pathogen was windborne to the islands. Likely scenarios for the introduction of CLR to Hawaii are the accidental introduction of spores or infected plant material by travelers or seasonal workers, or improperly fumigated coffee shipments originating from Central America or the Caribbean islands.

Global Analysis of Hemileia vastatrix Populations Shows Clonal Reproduction for the Coffee Leaf Rust Pathogen Throughout Most of Its Range.

Hemileia vastatrix is the most important fungal pathogen of coffee and the causal agent of recurrent disease epidemics that have invaded nearly every coffee growing region in the world. The development of coffee varieties resistant to H. vastatrix requires fundamental understanding of the biology of the fungus. However, the complete life cycle of H. vastatrix remains unknown, and conflicting studies and interpretations exist as to whether the fungus is undergoing sexual reproduction. Here we used population genetics of H. vastatrix to infer the reproductive mode of the fungus across most of its geographic range, including Central Africa, Southeast Asia, the Caribbean, and South and Central America. The population structure of H. vastatrix was determined via eight simple sequence repeat markers developed for this study. The analyses of the standardized index of association, Hardy-Weinberg equilibrium, and clonal richness all strongly support asexual reproduction of H. vastatrix in all sampled areas. Similarly, a minimum spanning network tree reinforces the interpretation of clonal reproduction in the sampled H. vastatrix populations. These findings may have profound implications for resistance breeding and management programs against H. vastatrix.

The evolving species concepts used for yeasts: from phenotypes and genomes to speciation networks.

Here we review how evolving species concepts have been applied to understand yeast diversity. Initially, a phenotypic species concept was utilized taking into consideration morphological aspects of colonies and cells, and growth profiles. Later the biological species concept was added, which applied data from mating experiments. Biophysical measurements of DNA similarity between isolates were an early measure that became more broadly applied with the advent of sequencing technology, leading to a sequence-based species concept using comparisons of parts of the ribosomal DNA. At present phylogenetic species concepts that employ sequence data of rDNA and other genes are universally applied in fungal taxonomy, including yeasts, because various studies revealed a relatively good correlation between the biological species concept and sequence divergence. The application of genome information is becoming increasingly common, and we strongly recommend the use of complete, rather than draft genomes to improve our understanding of species and their genome and genetic dynamics. Complete genomes allow in-depth comparisons on the evolvability of genomes and, consequently, of the species to which they belong. Hybridization seems a relatively common phenomenon and has been observed in all major fungal lineages that contain yeasts. Note that hybrids may greatly differ in their post-hybridization development. Future in-depth studies, initially using some model species or complexes may shift the traditional species concept as isolated clusters of genetically compatible isolates to a cohesive speciation network in which such clusters are interconnected by genetic processes, such as hybridization.

Symbiotic nitrogen fixation in the reproductive structures of a basidiomycete fungus.

Nitrogen (N) fixation is a driving force for the formation of symbiotic associations between N2-fixing bacteria and eukaryotes.1 Limited examples of these associations are known in fungi, and none with sexual structures of non-lichenized species.2-6 The basidiomycete Guyanagaster necrorhizus is a sequestrate fungus endemic to the Guiana Shield.7 Like the root rot-causing species in its sister genera Armillaria and Desarmillaria, G. necrorhizus sporocarps fruit from roots of decaying trees (Figures 1A-1C),8 and genome sequencing is consistent with observations that G. necrorhizus is a white-rotting decomposer. This species also represents the first documentation of an arthropod-dispersed sequestrate fungus. Numerous species of distantly related wood-feeding termites, which scavenge for N-rich food, feed on the mature spore-bearing tissue, or gleba, of G. necrorhizus. During feeding, mature spores adhere to termites for subsequent dispersal.9 Using chemical assays, isotope analysis, and high-throughput sequencing, we show that the sporocarps harbor actively N2-fixing Enterobacteriaceae species and that the N content within fungal tissue increases with maturation. Untargeted proteomic profiling suggests that ATP generation in the gleba is accomplished via fermentation. The use of fermentation-an anaerobic process-indicates that the sporocarp environment is anoxic, likely an adaptation to protect the oxygen-sensitive nitrogenase enzyme. Sporocarps also have a thick outer covering, possibly to limit oxygen diffusion. The enriched N content within mature sporocarps may offer a dietary inducement for termites in exchange for spore dispersal. These results show that the flexible metabolic capacity of fungi may facilitate N2-fixing associations, as well as higher-level organismal associations.

Inopinatum lactosum gen. & comb. nov., the first yeast-like fungus in Leotiomycetes.

Sporobolomyces lactosus is a pink yeast-like fungus that is not congeneric with other members of Sporobolomyces (Basidiomycota, Microbotryomycetes, Sporidiobolales). During our ongoing studies of pink yeasts we determined that S. lactosus was most closely related to Pseudeurotium zonatum (Ascomycota, Leotiomycetes, Thelebolales). A molecular phylogenetic analysis using sequences of the ITS region and the small and large subunit (SSU, LSU) rRNA genes, indicated that four isolates of S. lactosus, including three ex-type isolates, were placed in Thelebolales with maximum support. A new genus is proposed to accommodate S. lactosus, Inopinatum. This is the first pink yeast reported in Leotiomycetes.

Epidemics and the future of coffee production.

In this perspective, we draw on recent scientific research on the coffee leaf rust (CLR) epidemic that severely impacted several countries across Latin America and the Caribbean over the last decade, to explore how the socioeconomic impacts from COVID-19 could lead to the reemergence of another rust epidemic. We describe how past CLR outbreaks have been linked to reduced crop care and investment in coffee farms, as evidenced in the years following the 2008 global financial crisis. We discuss relationships between CLR incidence, farmer-scale agricultural practices, and economic signals transferred through global and local effects. We contextualize how current COVID-19 impacts on labor, unemployment, stay-at-home orders, and international border policies could affect farmer investments in coffee plants and in turn create conditions favorable for future shocks. We conclude by arguing that COVID-19's socioeconomic disruptions are likely to drive the coffee industry into another severe production crisis. While this argument illustrates the vulnerabilities that come from a globalized coffee system, it also highlights the necessity of ensuring the well-being of all. By increasing investments in coffee institutions and paying smallholders more, we can create a fairer and healthier system that is more resilient to future social-ecological shocks.

Host Adaptation and Virulence in Heteroecious Rust Fungi.

Rust fungi (Pucciniales, Basidiomycota) are obligate biotrophic pathogens that cause rust diseases in plants, inflicting severe damage to agricultural crops. Pucciniales possess the most complex life cycles known in fungi. These include an alternation of generations, the development of up to five different sporulating stages, and, for many species, the requirement of infecting two unrelated host plants during different parts of their life cycle, termed heteroecism. These fungi have been extensively studied in the past century through microscopy and inoculation studies, providing precise descriptions of their infection processes, although the molecular mechanisms underlying their unique biology are poorly understood. In this review, we cover recent genomic and life cycle transcriptomic studies in several heteroecious rust species, which provide insights into the genetic tool kits associated with host adaptation and virulence, opening new avenues for unraveling their unique evolution.

How to publish a new fungal species, or name, version 3.0.

It is now a decade since The International Commission on the Taxonomy of Fungi (ICTF) produced an overview of requirements and best practices for describing a new fungal species. In the meantime the International Code of Nomenclature for algae, fungi, and plants (ICNafp) has changed from its former name (the International Code of Botanical Nomenclature) and introduced new formal requirements for valid publication of species scientific names, including the separation of provisions specific to Fungi and organisms treated as fungi in a new Chapter F. Equally transformative have been changes in the data collection, data dissemination, and analytical tools available to mycologists. This paper provides an updated and expanded discussion of current publication requirements along with best practices for the description of new fungal species and publication of new names and for improving accessibility of their associated metadata that have developed over the last 10 years. Additionally, we provide: (1) model papers for different fungal groups and circumstances; (2) a checklist to simplify meeting (i) the requirements of the ICNafp to ensure the effective, valid and legitimate publication of names of new taxa, and (ii) minimally accepted standards for description; and, (3) templates for preparing standardized species descriptions.