Kluyveromyces osmophilus is not a synonym of Zygosaccharomyces mellis; reinstatement as Zygosaccharomyces osmophilus comb. nov.

Kluyveromyces osmophilus, a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis. Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis. During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata, as well as ripe honey of Tetragonisca angustula. The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they represent the same species. Phylogenomic analyses using 4038 orthologous genes support the reinstatement of K. osmophilus as a member of the genus Zygosaccharomyces. We, therefore, propose the name Zygosaccharomyces osmophilus comb. nov. (lectotype ATCC 22027; MycoBank no. MB 833739).

Takashi Nakase's last tweet: what is the current direction of microbial taxonomy research?

During the last few decades, type strains of most yeast species have been barcoded using the D1/D2 domain of their LSU rRNA gene and internal transcribed spacer (ITS) region. Species identification using DNA sequences regarding conspecificity in yeasts has also been studied. Most yeast species can be identified according to the sequence divergence of their ITS region or a combination of the D1/D2 and ITS regions. Studies that have examined intraspecific diversity have used multilocus sequence analyses, whereas the marker regions used in this analysis vary depending upon taxa. D1/D2 domain and ITS region sequences have been used as barcodes to develop primers suitable for the detection of the biological diversity of environmental DNA and the microbiome. Using these barcode sequences, it is possible to identify relative lineages and infer their gene products and function, and how they adapt to their environment. If barcode sequence was not variable enough to identify a described species, one could investigate the other biological traits of these yeasts, considering geological distance, environmental circumstances and isolation of reproduction. This article is dedicated to late Dr Takashi Nakase (1939-2018).

The Suhomyces clade: from single isolate to multiple species to disintegrating sex loci.

Candida tanzawaensis clade members are now placed in Suhomyces. The group was virtually unknown until the early 2000s. Here, we review progress made on Suhomyces over the last two decades and provide data from reports of new members of the group from distant localities worldwide, their habitats and a new study of mating loci that helps explain earlier failed compatibility tests. Phylogenetic studies indicate early diverging members are mostly associated with plants, but later diverging species are usually fungus-feeding insect associates. The genome of S. tanzawaensis was known to have a heterothallic mating allele arrangement with a single MAT α idiomorph. For this review, we generate sequence data and compare the MAT gene arrangement of 30 strains from nine Suhomyces species. These varied from MAT α loci containing mating genes α1 and α2, hypothetical MAT a loci without detectable mating genes a1 and a2 to truncated, possibly completely dissociated MAT loci with intraspecific variation. The absence of a second MAT in a genome locus precludes the possibility of mating type switching. Sympatric speciation likely occurred after MAT locus deterioration began in isolated habitats. Although asexual reproduction may be an effective short-term strategy, theory predicts it will not endure over the extreme long term.

Multilocus phylogeny of the Trichophyton mentagrophytes species complex and the application of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry for the rapid identification of dermatophytes.

Despite intensive studies of the Trichophyton mentagrophytes species complex, its taxonomy still causes confusion. In this study, more than 70 dermatophytes were analyzed based on nuc rDNA ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (D1D2), and ß-tubulin gene (TUBB) sequences to clarify phylogenetic relationships in the complex. This demonstrated that strains of the complex were divided into three major lineages with high statistical support: (i) T. benhamiae and related species; (ii) T. simii and two related species, T. quinckeanum and T. schoenleinii; and (iii) T. mentagrophytes, T. interdigitale, and related species. The major lineages could be further divided into 18 phylogroups, representing either individual species or phylogenetically distinct groups within species. Among strains of T. benhamiae, African isolates American Type Culture Collection (ATCC) 28064 and 28065 formed a phylogenetically distinct phylogroup from their type strain and were considered a distinct species. Strains of T. mentagrophytes were divided into at least four phylogroups based on combined sequence analysis, but some phylogroups showed closer relationships to T. interdigitale, T. equinum, and T. tonsurans when compared by individual genes. This indicates that identifying those species with one gene could lead to incorrect results. For rapid identification of those dermatophytes, each phylogroup was tested by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry using a database with customized reference spectra of each phylogroup. This system was able to identify all the tested strains to species level with higher than 91% accuracy, except for strains of T. interdigitale. The three phylogroups of T. benhamiae were well distinguished from one another with high identification accuracy, whereas phylogroups of T. mentagrophytes were often cross-identified to one another or to T. interdigitale. Further research should improve identification accuracy for some species, but the results suggested that MALDI-TOF MS could be a rapid and efficient identification tool for closely related dermatophytes in the T. mentagrophytes species complex.

Taxonomic annotation of public fungal ITS sequences from the built environment - a report from an April 10-11, 2017 workshop (Aberdeen, UK).

Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS barcode sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.

Characterization of the medically important yeast Trichosporon mucoides and its close sister Trichosporon dermatis by traditional and advanced technologies.

Trichosporon dermatis is a causative agent of several mycoses in immunocompromised patients but is often misidentified as Trichosporon mucoides due to their phenotypic resemblance. In order to evaluate the current identification keys for these species and to develop a rapid and reliable identification method, 11 strains of these yeasts were fully characterized in this study by traditional and advanced technologies. DNA sequences of the internal transcribed spacer (ITS), IGS1, and D1/D2 regions identified six of the yeasts as T. dermatis that were previously known as T. mucoides, including ATCC 204094 that has been used as the quality-control strain of T. mucoides for the VITEK 2 system and other commercial yeast identification kits. These two species could not be differentiated reliably by any previously known phenotypic keys for the species, such as growth patterns on ethylamine, phloroglucinol and tyramine, or by the VITEK 2 system. On the other hand, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) proved to be a rapid and reliable identification tool for the two closely related yeasts. With newly added superspectra from fully authenticated reference strains, the VITEK MS system using MALDI-TOF MS successfully separated strains of T. dermatis and T. mucoides at a similarity level of approximately 67 % for the mass spectra data, and could identify these strains at the species level with 100 % accuracy in repeated tests. Furthermore, the in vitro susceptibility results indicated that itraconazole, posaconazole and voriconazole were more effective against both T. mucoides and T. dermatis than the other antifungal agents tested in this study.

Scheffersomyces parashehatae f.a., sp. nov., Scheffersomyces xylosifermentans f.a., sp. nov., Candida broadrunensis sp. nov. and Candida manassasensis sp. nov., novel yeasts associated with wood-ingesting insects, and their ecological and biofuel implications.

During a survey of yeasts associated with wood-ingesting insects, 69 strains in the Scheffersomyces clade and related taxa were isolated from passalid and tenebrionid beetles and the decayed wood inhabited by them. The majority of these yeasts was found to be capable of fermenting xylose, and was recognized as Scheffersomyces stipitis or its close relative Scheffersomyces illinoinensis, which are known to be associated with wood-decaying beetles and rotten wood. Yeasts in 'Scheffersomyces' ( = Candida) ergatensis and 'Scheffersomyces' ( = Candida) coipomoensis were also frequently isolated. The remaining six strains were identified as representing four novel species in the genera Scheffersomyces and Candida based on multilocus sequence analyses of nuclear rRNA genes and four protein-coding genes, as well as other taxonomic characteristics. Two xylose-fermenting species, Scheffersomyces parashehatae f.a., sp. nov. (type strain ATCC MYA-4653(T) = CBS 12535(T) = EH045(T); MycoBank MB805440) and Scheffersomyces xylosifermentans f.a., sp. nov. (type strain ATCC MYA-4859(T) = CBS 12540(T) = MY10-052(T); MycoBank MB805441), formed a clade with Scheffersomyces shehatae and related Scheffersomyces species. Interestingly, S. xylosifermentans can survive at 40 °C, which is a rare property among xylose-fermenting yeasts. Candida broadrunensis sp. nov. (type strain ATCC MYA-4650(T) = CBS 11838(T) = EH019(T); MycoBank MB805442) is a sister taxon of C. ergatensis, while Candida manassasensis sp. nov. (type strain ATCC MYA-4652(T) = CBS 12534(T) = EH030(T); MycoBank MB805443) is closely related to Candida palmioleophila in the Candida glaebosa clade. The multilocus DNA sequence comparisons in this study suggest that the genus Scheffersomyces needs to be circumscribed to the species near S. stipitis (type species) and S. shehatae that can be characterized by the ability to ferment xylose.

Proposal of Zygosaccharomyces parabailii sp. nov. and Zygosaccharomyces pseudobailii sp. nov., novel species closely related to Zygosaccharomyces bailii.

Twenty-three yeast strains traditionally identified as Zygosaccharomyces bailii were studied in order to clarify their taxonomy and phylogenetic relationships. The molecular phylogeny from rRNA gene sequences showed that these yeasts were well divided into three major groups, and two of the groups could be clearly distinguished from the type strain of Z. bailii at the species level. Therefore, we propose Zygosaccharomyces parabailii sp. nov. (type strain ATCC 56075(T)  = NBRC 1047(T)  = NCYC 128(T)  = CBS 12809(T)) and Zygosaccharomyces pseudobailii sp. nov. (type strain ATCC 56074(T)  = NBRC 0488(T)  = CBS 2856(T)) to accommodate the yeasts belonging to the two groups. By conventional physiological tests, Z. bailii and the two novel species are not clearly distinguished from one another, as variations exist more frequently between individual strains and are not species-specific. However, the conclusions from rRNA gene sequence analyses are well supported by genome fingerprinting patterns as well as other protein-coding gene sequence comparisons.

Trichosporon vanderwaltii sp. nov., an asexual basidiomycetous yeast isolated from soil and beetles.

During a survey of unidentified yeast isolates deposited in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection housed at the Department of Microbial, Biochemical and Food Biotechnology of the University of the Free State, one isolate obtained from soil in South Africa showed 100 % identity in D1/D2 rDNA sequence with undescribed basidiomycetous yeasts isolated from the gut of beetles from the United States of America and forest soil from Taiwan in the NCBI sequence database. Phylogenetic analyses using sequences of the D1/D2 rDNA and ITS regions indicated that all these isolates form a well-supported sub-clade that is the sister clade to the Brassicae plus Porosum clades of Trichosporon in the order Trichosporonales. Subsequent phenotypic tests revealed that asexual reproduction by budding is rare but dominated by arthroconidia resulting from segmentation of hyphae and that fusiform giant cells are characterized by budding from a broad base. These findings further suggest that these isolates belong to a single tremellomycetous yeast species for which the name Trichosporon vanderwaltii CBS 12124(T) (=NRRL Y-48732(T), =UOFS Y-1920(T)) is proposed.

Bandoniozyma gen. nov., a genus of fermentative and non-fermentative tremellaceous yeast species.

BACKGROUND: Independent surveys across the globe led to the proposal of a new basidiomycetous yeast genus within the Bulleromyces clade of the Tremellales, Bandoniozyma gen. nov., with seven new species. METHODOLOGY/PRINCIPAL FINDINGS: The species were characterized by multiple methods, including the analysis of D1/D2 and ITS nucleotide sequences, and morphological and physiological/biochemical traits. Most species can ferment glucose, which is an unusual trait among basidiomycetous yeasts. CONCLUSIONS/SIGNIFICANCE: In this study we propose the new yeast genus Bandoniozyma, with seven species Bandoniozyma noutii sp. nov. (type species of genus; CBS 8364(T)  =  DBVPG 4489(T)), Bandoniozyma aquatica sp. nov. (UFMG-DH4.20(T)  =  CBS 12527(T)  =  ATCC MYA-4876(T)), Bandoniozyma complexa sp. nov. (CBS 11570(T)  =  ATCC MYA-4603(T)  =  MA28a(T)), Bandoniozyma fermentans sp. nov. (CBS 12399(T)  =  NU7M71(T)  =  BCRC 23267(T)), Bandoniozyma glucofermentans sp. nov. (CBS 10381(T)  =  NRRL Y-48076(T)  =  ATCC MYA-4760(T)  =  BG 02-7-15-015A-1-1(T)), Bandoniozyma tunnelae sp. nov. (CBS 8024(T)  =  DBVPG 7000(T)), and Bandoniozyma visegradensis sp. nov. (CBS 12505(T)  =  NRRL Y-48783(T)  =  NCAIM Y.01952(T)).

Viability of fastidious Phytophthora following different cryopreservation treatments.

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the -1 °C min(-1) freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae).

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.

Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginosus sp. nov.

Two new species in the anamorphic basidiomycetous genus Trichosporon (Tremellomycetes, Agaricomycotina) were uncovered in a DNA sequence-based molecular analysis of oleaginous yeasts maintained in the ATCC Mycology Collection. One yeast is named as Trichosporon cacaoliposimilis sp. nov. for its capability of synthesizing and accumulating a large amount of lipids having a composition equivalent to that of natural cacao butter. The type strain is ATCC 20505(T), originally deposited as Trichosporon sp. The other can use food industry wastes and agricultural byproducts as the substrate for growth and accumulation of a high level of oil and accordingly is named Trichosporon oleaginosus sp. nov. The type strain is ATCC 20509(T), previously identified as Cryptococcus curvatus. Molecular phylogenetic analyses indicate that T. cacaoliposimilis is a novel taxon in the Gracile clade of the genus, close to T. gracile and T. dulcitum, and that T. oleaginosus belongs to the Cutaneum clade, with T. jirovecii as the closest sister taxon. Other oleaginous yeasts were identified as new strains of known taxa, T. insectorum, Candida orthopsilosis and C. palmioleophila.

Kazachstania intestinalis sp. nov., an ascosporogenous yeast from the gut of passalid beetle Odontotaenius disjunctus.

Three ascosporogenous yeast strains were isolated from the gut of the passalid beetle, Odontotaenius disjunctus, inhabiting on rotten oak trees. DNA sequence comparison and other taxonomic characteristics identified the strains as a novel species in the genus Kazachstania. The name Kazachstania intestinalis sp. nov. (type strain EH085(T) = ATCC MYA-4658(T) = CBS 11839(T)) is proposed for the strains. The yeast is homothallic, producing persistent asci with 1-4 spheroidal ascospores. Molecular phylogeny from ribosomal RNA gene sequences placed this novel species on the basal lineage of a clade including Kazachstania lodderae, Kazachstania exigua, Kazachstania martiniae, and other related Kazachstania spp., but none of those species was a close sister to K. intestinalis.

Yeasts in the Sugiyamaella clade associated with wood-ingesting beetles and the proposal of Candida bullrunensis sp. nov.

During a survey of yeasts associated with wood-ingesting insects, six strains of the Sugiyamaella clade were isolated from the gut of passalid and tenebrionid beetles and the decayed wood inhabited by them. Phylogeny based on rRNA gene sequences placed these yeasts as members of Sugiyamaella smithiae, Sugiyamaella americana, Candida lignohabitans and a novel species closely related to Su. americana. The only strain of the novel species, EH008(T), could be unquestionably distinguished from its relatives by DNA sequences and other taxonomic characteristics. Ascospore production was not observed under the laboratory conditions tested. Therefore, this novel species is proposed as Candida bullrunensis sp. nov. (type strain EH008(T) = ATCC MYA-4660(T) = CBS 11840(T)).

Trichosporon xylopini sp. nov., a hemicellulose-degrading yeast isolated from the wood-inhabiting beetle Xylopinus saperdioides.

Four arthroconidium-producing yeasts were isolated from the gut of wood-inhabiting tenebrionid and passalid beetles. The rRNA genes of these yeast strains were sequenced, compared and analysed. The sequence results and other taxonomic characterizations placed two of the strains into Trichosporon porosum, and the remaining strains, EH024(T) and EH026 which were isolated from Xylopinus saperdioides (Coleoptera: Tenebrionidae), into a novel species of the genus Trichosporon in the Porosum clade. Strain EN6S23 was independently isolated from forest soil in Taiwan and was identified as the same novel species based on identical sequences in the internal transcribed spacers (ITS) and the D1/D2 region of the LSU rRNA gene and similar physiological characteristics to those of strains EH024(T) and EH026. The three strains can assimilate cellulose and xylan as sole carbon source, and are clearly distinguished from their closest taxon, T. porosum, by 14 nt differences in the ITS and D1/D2 region. These strains did not reproduce sexually under the laboratory conditions tested. The novel species is proposed as Trichosporon xylopini sp. nov. (type strain EH024(T)  = ATCC MYA-4670(T)  = CBS 11841(T)).

Methylotrophic yeasts near Ogataea (Hansenula) polymorpha: a proposal of Ogataea angusta comb. nov. and Candida parapolymorpha sp. nov.

Ogataea (Hansenula) polymorpha and related yeasts were studied to clarify their taxonomy and phylogenetic relationships. The molecular analyses based on ribosomal DNA sequences revealed that (1) ATCC 14755, type strain of Pichia angusta, is phylogenetically distinguished from the majority of O. polymorpha strains including ATCC 34438(T) (=NRRL Y-5445(T)=CBS 4732(T)), type of the species; (2) Ogataea thermophila is conspecific to O. polymorpha; and (3) two of the strains, ATCC 26012 and ATCC 58401, are a novel Candida species closely related to O. polymorpha. The conclusions were also supported by physiological characteristics and other taxonomic features of these strains. Therefore, we propose here two novel species, Ogataea angusta comb. nov. (ATCC 14755(T)=CBS 7073(T)=NRRL Y-2214(T)) and Candida parapolymorpha sp. nov. (ATCC 26012(T)=NRRL Y-7560(T)), and conclude that O. thermophila is a synonym of O. polymorpha.

Diatrypasimilis australiensis, a novel xylarialean fungus from mangrove.

A marine xylarialean fungus, isolated from roots of Rhizophora (mangrove) in Australia, displays morphology of eight ellipsoidal dark brown ascospores in a cylindrical ascus having a refractive apical apparatus. Each ascospore has a longitudinal germ slit. The fungus grew very slowly and produced dark brown water-soluble pigment(s) on various media. It developed unique, column-shaped, indeterminate synnemata on which needle-shaped conidia were produced. The sexual stage of this fungus was not observed under the laboratory conditions tested. Molecular phylogenetic analyses of the combined sequences of nuclear ribosomal RNA genes and their internal transcribed spacers placed it at a basal position in the clade of Diatrypaceae of the Xylariales with comparatively high statistical support. However the morphological features and phylogenetic position of this organism do not closely resemble any known fungal taxa. Therefore this fungus is proposed to be a representative of a novel taxon and described as Diatrypasimilis australiensis gen. et sp. nov.

Yeasts associated with the curculionid beetle Xyloterinus politus: Candida xyloterini sp. nov., Candida palmyrensis sp. nov. and three common ambrosia yeasts.

Seven yeast strains were isolated from the body surface and galleries of Xyloterinus politus, the ambrosia beetle that attacks black oak trees. Based on rDNA sequence comparisons and other taxonomic characteristics, five of the strains were identified as members of the species Saccharomycopsis microspora, Wickerhamomyces hampshirensis and Candida mycetangii, which have been reported previously as being associated with insects. The remaining two yeast strains were proposed as representatives of two novel species, Candida xyloterini sp. nov. (type strain ATCC 62898(T)=CBS 11547(T)) and Candida palmyrensis sp. nov. (type strain ATCC 62899(T)=CBS 11546(T)). C. xyloterini sp. nov. is a close sister taxon to Ogataea dorogensis and assimilates methanol as a sole carbon source but lacks ascospores. On the other hand, C. palmyrensis sp. nov. is phylogenetically distinct from any other ambrosia yeast reported so far. The species was placed near Candida sophiae-reginae and Candida beechii based on DNA sequence analyses, but neither of these were close sister taxa to C. palmyrensis sp. nov.

The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.