Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum.

Budding yeasts (subphylum Saccharomycotina) are found in every biome and are as genetically diverse as plants or animals. To understand budding yeast evolution, we analyzed the genomes of 332 yeast species, including 220 newly sequenced ones, which represent nearly one-third of all known budding yeast diversity. Here, we establish a robust genus-level phylogeny comprising 12 major clades, infer the timescale of diversification from the Devonian period to the present, quantify horizontal gene transfer (HGT), and reconstruct the evolution of 45 metabolic traits and the metabolic toolkit of the budding yeast common ancestor (BYCA). We infer that BYCA was metabolically complex and chronicle the tempo and mode of genomic and phenotypic evolution across the subphylum, which is characterized by very low HGT levels and widespread losses of traits and the genes that control them. More generally, our results argue that reductive evolution is a major mode of evolutionary diversification.

The Ground State and Evolution of Promoter Region Directionality.

Eukaryotic promoter regions are frequently divergently transcribed in vivo, but it is unknown whether the resultant antisense RNAs are a mechanistic by-product of RNA polymerase II (Pol II) transcription or biologically meaningful. Here, we use a functional evolutionary approach that involves nascent transcript mapping in S. cerevisiae strains containing foreign yeast DNA. Promoter regions in foreign environments lose the directionality they have in their native species. Strikingly, fortuitous promoter regions arising in foreign DNA produce equal transcription in both directions, indicating that divergent transcription is a mechanistic feature that does not imply a function for these transcripts. Fortuitous promoter regions arising during evolution promote bidirectional transcription and over time are purged through mutation or retained to enable new functionality. Similarly, human transcription is more bidirectional at newly evolved enhancers and promoter regions. Thus, promoter regions are intrinsically bidirectional and are shaped by evolution to bias transcription toward coding versus non-coding RNAs.

Spathaspora marinasilvae sp. nov., a xylose-fermenting yeast isolated from galleries of passalid beetles and rotting wood in the Amazonian rainforest biome.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

Signatures of optimal codon usage in metabolic genes inform budding yeast ecology.

Reverse ecology is the inference of ecological information from patterns of genomic variation. One rich, heretofore underutilized, source of ecologically relevant genomic information is codon optimality or adaptation. Bias toward codons that match the tRNA pool is robustly associated with high gene expression in diverse organisms, suggesting that codon optimization could be used in a reverse ecology framework to identify highly expressed, ecologically relevant genes. To test this hypothesis, we examined the relationship between optimal codon usage in the classic galactose metabolism (GAL) pathway and known ecological niches for 329 species of budding yeasts, a diverse subphylum of fungi. We find that optimal codon usage in the GAL pathway is positively correlated with quantitative growth on galactose, suggesting that GAL codon optimization reflects increased capacity to grow on galactose. Optimal codon usage in the GAL pathway is also positively correlated with human-associated ecological niches in yeasts of the CUG-Ser1 clade and with dairy-associated ecological niches in the family Saccharomycetaceae. For example, optimal codon usage of GAL genes is greater than 85% of all genes in the genome of the major human pathogen Candida albicans (CUG-Ser1 clade) and greater than 75% of genes in the genome of the dairy yeast Kluyveromyces lactis (family Saccharomycetaceae). We further find a correlation between optimization in the GALactose pathway genes and several genes associated with nutrient sensing and metabolism. This work suggests that codon optimization harbors information about the metabolic ecology of microbial eukaryotes. This information may be particularly useful for studying fungal dark matter-species that have yet to be cultured in the lab or have only been identified by genomic material.

Kondoa tianchiensis f.a., sp. nov., an anamorphic yeast species isolated from plant leaves.

Two yeast strains, NYNU 236122 and NYNU 236180, were isolated from plant leaves collected in Tianchi Mountain, Henan Province, central China. Molecular phylogenetic analyses revealed the closest relatives of the strains are three described Kondoa species, Kondoa chamaenerii, Kondoa miscanthi, and Kondoa subrosea. Genetically, the isolated strains differed from the type strains of their three related species by 2-11(0.2-1.8%) base substitutions in the D1/D2 domain, 16-40 (2.6-5.6%) base mismatches in the internal transcribed spacer region, and more than 10.1% base substitutions in the partial RPB2 gene. Furthermore, the two strains differ physiologically from their closest related species, K. chamaenerii, in their ability to assimilate dl-lactate, nitrite, and l-lysine and their inability to assimilate nitrate. Additionally, they differ from K. miscanthi and K. subrosea in their ability to assimilate inulin, d-gluconate, and l-lysine. The species name of Kondoa tianchiensis f.a., sp. nov. is proposed with holotype CICC 33616T (Mycobank MB 853544).

Spencermartinsiella nicolii sp. nov., a potential opportunistic pathogenic yeast species isolated from rotting wood in Brazil.

Nineteen isolates representing a candidate for a novel yeast species belonging to the genus Spencermartinsiella were recovered from rotting wood samples collected at different sites in Atlantic Rainforest and Amazonian Forest ecosystems in Brazil. Similarity search of the nucleotide sequence of the intergenic spacer (ITS)-5.8S and large subunit D1/D2 regions of the ribosomal gene cluster showed that this novel yeast is closely related to Spencermartinsiella cellulosicola. The isolates differ by four nucleotide substitutions in the D1/D2 domain and six substitutions and 31 indels in the ITS region from the holotype of S. cellulosicola. Phylogenomic analysis based on 1474 single-copy orthologues for a set of Spencermartinsiella species whose whole genome sequences are available confirmed that the novel species is phylogenetically close to S. cellulosicola. The low average nucleotide identity value of 83% observed between S. cellulosicola and the candidate species confirms that they are distinct. The novel species produced asci with hemispherical ascospores. The name Spencermartinsiella nicolii sp. nov. is proposed. The holotype is CBS 14238T. The MycoBank number is MB855027. Interestingly, the D1/D2 sequence of the S. nicolii was identical to that of an uncultured strain of Spencermartinsiella causing systemic infection in a male adult crocodile (Crocodylus niloticus). The characterization of some virulence factors and antifungal susceptibility of S. nicolii isolates suggest that this yeast may be an opportunistic pathogen for animals, including humans; the isolates grow at 37 °C.

Cyberlindnera qingyuanensis f.a., sp. nov., a yeast species isolated from rotting wood.

Two yeast strains were isolated from rotting wood samples collected on Qingyuan Mountain, Fujian Province, PR China. Phylogenetic analysis, based on the concatenated sequences of the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit rRNA gene, revealed that these two strains represent a novel species of the genus Cyberlindnera. The proposed name for this new species is Cyberlindnera qingyuanensis f.a., sp. nov. (holotype: GDMCC 2.300; ex-type: PYCC 9925) although the formation of ascospores was not observed. The novel species differs from its close relative Cyberlindnera galapagoensis by 7.7% sequence divergence (37 substitutions and seven indels) in the D1/D2 domain and 9.7% sequence divergence (42 substitutions and 34 indels) in the ITS region, respectively. Additionally, Cyb. qingyuanensis differs from its close relative Cyb. galapagoensis by its ability to grow in cellobiose, l-rhamnose, ribitol, galactitol, and dl-lactate, its growth at 37 °C, and its inability to ferment raffinose. The Mycobank number is MB 854693.

Yamadazyma thunbergiae sp. nov., a novel yeast species associated with Bengal clock vines and soil in Okinawa, Japan.

Two strains, designated JCM 36746T and JCM 36749, were isolated from Bengal clock vine (Thunbergia grandiflora) and soil, respectively, in Okinawa, Japan. Analysis of the internal transcribed spacer (ITS) regions and D1/D2 domains of the large subunit rRNA gene sequences revealed identical sequences in both strains, indicating that they belong to the same species. Sequence analysis and physiological characterization identified these strains as representing a novel yeast species in the genus Yamadazyma. The sequence similarities of the concatenated ITS regions and D1/D2 domains indicated that JCM 36746T and JCM 36749 formed a well-supported distinct from closely related species belonging to the Yamadazyma clade, including Candida dendronema, C. diddensiae, C. germanica, C. kanchanaburiensis, C. naeodendra, C. vaughaniae, Y. akitaensis, Y. koratensis, Y. nakazawae, Y. philogaea, Y. phyllophila, Y. siamensis, Y. ubonensis, and three undescribed species, comprising Candida aff. naeodendra/diddensiae Y151, Candida sp. GE19S08, and Yamadazyma sp. strain NYNU 22830. The sequences of the D1/D2 domains and ITS regions differed in nucleotide substitutions by 1.51% and 2.57% or greater, respectively, from those of the previously described and undescribed related species. In addition, the physiological characteristics of the novel species were distinct from those of the closely related described species. On the basis of these findings, we propose the name Yamadazyma thunbergiae sp. nov. to classify this species within the genus Yamadazyma. The holotype used is JCM 36746T (ex-type strains CBS 18614 and NBRC 116657). The MycoBank accession number is MB 853823.

Mrakia polaris sp. nov. and Mrakia amundsenii sp. nov. (Mrakiaceae, Cystofilobasidiales), two novel psychrophilic yeast species isolated from Arctic habitats.

Four yeast strains belonging to the basidiomycetous yeast genus Mrakia were isolated from diverse habitats in the Ny-Ålesund region (Svalbard, High Arctic): two from vascular plants, one from seawater and one from freshwater. Phylogenetic analysis, based on the ITS region and the D1/D2 domain of the 28S rRNA gene, identified these four strains as representing two novel species within the genus Mrakia. The names Mrakia polaris sp. nov. (MycoBank number: MB 852063) and Mrakia amundsenii sp. nov. (MycoBank number: MB 852064) are proposed. These two new species show distinct psychrophilic adaptations, as they exhibit optimal growth at temperatures between 10 and 15°C, while being unable to grow at 25°C. The holotype of M. polaris sp. nov. is CPCC 300345T, and the holotype of M. amundsenii sp. nov. is CPCC 300572T.

Wickerhamiella lachancei f.a. sp. nov., a novel ascomycetous yeast species isolated from flowers of Lantana camara in India.

Two isolates representing a novel species of the genus Wickerhamiella were obtained in India from nectar of flowers of Lantana camara, an ornamental exotic species native to Central and South America. Phylogenetic analyses of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene, internal transcribed spacer (ITS) region, and physiological characteristics, supported the recognition of the novel species, that we designate Wickerhamiella lachancei sp. nov (MycoBank no. MB851709), with MCC 9929T as the holotype and PYCC 10003T as the isotype. Considering pairwise sequence similarity, the type strain of the novel species differs from the type strain of the most closely related species, Wickerhamiella drosophilae CBS 8459T, by 16 nucleotide substitutions and two gaps (3.9 % sequence variation) in the D1/D2 region (560 bp compared) and 28 nucleotide substitutions and five gaps (7.22 % sequence variation) in the ITS region (444 bp compared).

A Cluster of Diutina catenulata Funguria in Patients with Coronavirus Disease 2019 (COVID-19) Hospitalized in a Tertiary Reference Hospital from Rio de Janeiro, Brazil.

During the COVID-19 pandemic, fungal infections, especially pulmonary aspergillosis, mucormycosis, and invasive candidiasis, have emerged as a significant health concern. Beyond Candida albicans, the most common cause of invasive candidiasis, other rare ascomycetous yeast species have been described in tertiary care units, potentially posing a broader health threat. We have isolated, from September 2020 to June 2021, nine Diutina catenulata strains from urine samples of six patients. This was intriguing as this fungus had not been previously identified in our institution, nor after June 2021. Therefore, we decided to outline the clinical features of the patients with this rare pathogen, to describe phenotypic characteristics, including antifungal susceptibility profiles, of this yeast species and to identify the genetic makeup through whole-genome sequencing analysis to evaluate if this was a cluster of genetically similar D. catenulata isolates in our institution. The strains were identified through MALDI-TOF MS analyses and Sanger sequencing of two rDNA regions. All patients yielding D. catenulata from urine samples needed ventilator support and used urinary catheters during hospitalization for treatment of COVID-19. None of them had received COVID-19 vaccines. Morphological and biochemical profiles of the nine strains were largely consistent, although fluconazole susceptibility varied, ranging from 4 to 32 µg/mL. Phylogenomic analysis revealed minimal genetic variation among the isolates, with low intrapopulation variation, supported by the identification of only 84 SNPs across all strains. Therefore, we propose that the yeast strains isolated were part of a cluster of D. catenulata funguria in the context of COVID-19.

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

Evolutionary biology through the lens of budding yeast comparative genomics.

The budding yeast Saccharomyces cerevisiae is a highly advanced model system for studying genetics, cell biology and systems biology. Over the past decade, the application of high-throughput sequencing technologies to this species has contributed to this yeast also becoming an important model for evolutionary genomics. Indeed, comparative genomic analyses of laboratory, wild and domesticated yeast populations are providing unprecedented detail about many of the processes that govern evolution, including long-term processes, such as reproductive isolation and speciation, and short-term processes, such as adaptation to natural and domestication-related environments.

Metahyphopichia suwanaadthiae sp. nov., an anamorphic yeast species in the order Saccharomycetales and reassignment of Candida silvanorum to the genus Metahyphopichia.

Seven yeast strains, representing a single novel anamorphic species, were isolated in Thailand. They consisted of five strains (DMKU-MRY16T, DMKU-SK18, DMKU-SK25, DMKU-SK30 and DMKU-SK32) obtained from five different mushrooms, and two strains (ST-224 and 11-14.2) derived from insect frass and soil, respectively. The pairwise sequence analysis indicated that all seven strains had identical sequences in the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region. Metahyphopichia silvanorum was the most closely related species, but with 11.9-12.4% nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 13.1-13.3% nucleotide substitutions in the ITS region. The phylogenetic analyses based on the concatenated sequences of the ITS region and the D1/D2 domains of the LSU rRNA gene showed that the seven strains form a well-separated subclade in a clade containing M. silvanorum and Metahyphopichia laotica with high bootstrap support. A phylogenetic analysis of a multilocus dataset including the small subunit (SSU) rRNA gene, the ITS region, the D1/D2 domains of the LSU rRNA gene, translation elongation factor 1-alpha gene, actin gene and the RNA polymerase II subunit 2 gene, confirmed the presence of the monophyletic clade that also includes M. silvanorum and M. laotica, and strongly supported the phylogenetic isolation of the seven strains from its neighbouring species. Therefore, the seven strains were assigned as a single novel species of the genus Metahyphopichia, according to their phylogenetic relationships. The name Metahyphopichia suwanaadthiae sp. nov. is proposed to accommodate the seven strains. The holotype is DMKU-MRY16T (TBRC 11775T=NBRC 114386T=PYCC 8655T). The MycoBank number of the novel species is MB 841280. In addition, Candida silvanorum is reassigned to the genus Metahyphopichia. The MycoBank number of M. silvanorum comb. nov. is MB 841279.

Wickerhamiella nakhonpathomensis f.a. sp. nov., a novel ascomycetous yeast species isolated from a mushroom and a flower in Thailand.

Strains SU22T (TBRC 14875T) and FLA11.5, representing a novel anamorphic yeast species, were respectively isolated from a fruiting body of a Coprinus species and an inflorescence of a Coffea species collected in Thailand. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed that the two strains differed by two nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and were identical in the ITS regions. Wickerhamiella drosophilae CBS 8459T was the most closely related species, but with 24-26 nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 24 nucleotide substitutions in the ITS regions. A phylogenetic analysis, based on the sequences of the D1/D2 domains, indicated that the two strains represented a species in the genus Wickerhamiella which was distinct from other recognized species of the genus. Therefore, the two strains were assigned as a novel species, for which we propose the name Wickerhamiella nakhonpathomensis f.a. sp. nov. The holotype is TBRC 14875T (isotype PYCC 8914T). The MycoBank number of the novel species is MB 840833.

Analysis of glucose and xylose metabolism in new indigenous Meyerozyma caribbica strains isolated from corn residues.

Aiming to broaden the base of knowledge about wild yeasts, four new indigenous strains were isolated from corn residues, and phylogenetic-tree assemblings on ITS and LSU regions indicated they belong to Meyerozyma caribbica. Yeasts were cultivated under full- and micro-aerobiosis, starting with low or high cell-density inoculum, in synthetic medium or corn hydrolysate containing glucose and/or xylose. Cells were able to assimilate both monosaccharides, albeit by different metabolic routes (fermentative or respiratory). They grew faster in glucose, with lag phases ~ 10 h shorter than in xylose. The hexose exhaustion occurred between 24 and 34 h, while xylose was entirely consumed in the last few hours of cultivation (44-48 h). In batch fermentation in synthetic medium with high cell density, under full-aerobiosis, 18-20 g glucose l-1 were exhausted in 4-6 h, with a production of 6.5-7.0 g ethanol l-1. In the xylose medium, cells needed > 12 h to consume the carbohydrate, and instead of ethanol, cells released 4.4-6.4 g l-1 xylitol. Under micro-aerobiosis, yeasts were unable to assimilate xylose, and glucose was more slowly consumed, although the ethanol yield was the theoretical maximum. When inoculated into the hydrolysate, cells needed 4-6 h to deplete glucose, and xylose had a maximum consumption of 57%. Considering that the hydrolysate contained ~ 3 g l-1 acetic acid, it probably has impaired sugar metabolism. Thus, this study increases the fund of knowledge regarding indigenous yeasts and reveals the biotechnological potential of these strains.

The budding yeast life cycle: More complex than anticipated?

The budding yeast, Saccharomyces cerevisiae, has served as a model for nearly a century to understand the principles of the eukaryotic life cycle. The canonical life cycle of S. cerevisiae comprises a regular alternation between haploid and diploid phases. Haploid gametes generated by sporulation are expected to quickly restore the diploid phase mainly through inbreeding via intratetrad mating or haploselfing, thereby promoting genome homozygotization. However, recent large population genomics data unveiled that heterozygosity and polyploidy are unexpectedly common. This raises the interesting paradox of a haplo-diplobiontic species being well-adapted to inbreeding and able to maintain high levels of heterozygosity and polyploidy, thereby suggesting an unanticipated complexity of the yeast life cycle. Here, we propose that unprogrammed mating type switching, heterothallism, reduced spore formation and viability, cell-cell fusion and dioecy could play key and uncharted contributions to generate and maintain heterozygosity through polyploidization.

Cyberlindnera dasilvae sp. nov., a xylitol-producing yeast species isolated from rotting wood and frass of cerambycid larva.

Six yeast isolates were obtained from rotting wood samples in Brazil and frass of a cerambycid beetle larva in French Guiana. Sequence analysis of the ITS-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of Cyberlindnera. This novel species is related to Cyberlindnera japonica, Cyberlindnera xylosilytica, Candida easanensis and Candida maesa. It is heterothallic and produces asci with two or four hat-shaped ascospores. The name Cyberlindnera dasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Cy. dasilvae is CBS 16129T and the designated paratype is CBS 16584. The MycoBank number is 838252. All isolates of Cy. dasilvae were able to convert xylose into xylitol with maximum xylitol production within 60 and 72 h. The isolates produced xylitol with values ranging from 12.61 to 31.79 g l-1 in yeast extract-peptone-xylose medium with 5% xylose. When the isolates were tested in sugarcane bagasse hydrolysate containing around 35-38 g l-1d-xylose, isolate UFMG-CM-Y519 showed maximum xylitol production.

Zygotorulaspora dagestanica sp. nov., a novel ascomycetous yeast species associated with the Georgian honeysuckle (Lonicera iberica M. Bieb.).

During an investigation of the yeast communities associated with wild fruit shrubs in Dagestan (Caucasus, Russia), four fermenting ascospore-producing yeast strains were isolated from leaves of the Georgian honeysuckle (Lonicera iberica M. Bieb.) and from soil underneath this plant. Phylogenetic analyses based on concatenated sequences of the ITS region and D1/D2 domains of the large subunit rRNA gene and concatenated sequences of the ribosomal DNA cystron, RPB2 and TEF1 genes showed that the isolated strains represented a new species of the genus Zygotorulaspora. The new species was placed in the basal position to other species of the clade and close to Zygotorulaspora mrakii. Based on the results of phylogenetic analyses and the phenotypic characteristics of the four studied strains, a novel species is described, for which the name Zygotorulaspora dagestanica sp. nov. is proposed. The holotype is KBP Y-4591T, three metabolically inactive cryopreserved isotype cultures are DSM 100088, VKM Y-3060 and VKPM Y-4318. The MycoBank number is MB 838285.

Cyberlindnera sylvatica sp. nov., a yeast species isolated from forest habitats.

Five yeast strains isolated from forest habitats in Hungary and Germany were characterized phenotypically and by sequencing of the D1/D2 domain of the large subunit rRNA gene and the ITS1-5.8S-ITS2 (ITS) region of the rRNA gene. The strains have identical D1/D2 domain and ITS region sequences. By sequence comparisons, Candida mycetangii and Candida maritima were identified as the closest relatives among the currently recognized yeast species. The DNA sequences of the investigated strains differ by 1.2 % (six substitutions) in the D1/D2 domain and by 3.5 % (12 substitutions and eight indels) in the ITS region from the type strain of C. mycetangii (CBS 8675T) while by 1.2 % (six substitutions and one indel) in the D1/D2 domain and by 7 % (32 substitutions and seven indels) in the ITS region from the type strain of C. maritima (CBS 5107T). Because the intraspecies heterogeneity seems to be very low and the distance to the most closely related species is above the commonly expected level for intraspecies variability Cyberlindnera sylvatica sp. nov. (holotype, CBS 16335T; isotype, NCAIM Y.02233T; MycoBank no., MB 835268) is proposed to accommodate the above-noted five yeast strains. Phenotypically the novel species can be distinguished from C. mycetangii and C. maritima by the formation of ascospores. Cyberlindnera sylvatica forms one or two hat-shaped ascospores per ascus on many different media as well as well-developed pseudohyphae and true hyphae. Additionally, we propose the transfer of three anamorphic members of the Cyberlindnera americana sub-clade to the genus Cyberlindnera as the following new taxonomic combinations Cyberlindnera maritima f.a., comb. nov., Cyberlindnera mycetangii f.a., comb. nov. and Cyberlindnera nakhonratchasimensis f.a., comb. nov.