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.

Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Description of four Apiotrichum and two Cutaneotrichosporon species isolated from guano samples from bat-inhabited caves in Japan.

Four new yeast species belonging to the genus Apiotrichum and two new yeast species belonging to Cutaneotrichosporon are described for strains isolated from guano samples from bat-inhabited caves in Japan. In 2005, we reported these isolates as Trichosporon species based on sequence analyses of the D1/D2 domain of large subunit (LSU) rRNA genes according to available basidiomycetous yeast classification criteria; however, to date, they have not been officially published as new species with descriptions. Their phylogenetic positions have been reanalysed based on comparison of internal transcribed spacer (ITS) region sequences (including the 5.8S rRNA gene) and the D1/D2 domain of the LSU rRNA gene with those of known species; we confirmed clear separation from previously described species. Physiological and biochemical properties of the isolates also suggest their distinctiveness. Therefore, we describe Apiotrichum akiyoshidainum (holotype JCM 12595T), Apiotrichum chiropterorum (JCM 12594T), Apiotrichum coprophilum (JCM 12596T), Apiotrichum otae (JCM 12593T), Cutaneotrichosporon cavernicola (JCM 12590T) and Cutaneotrichosporon middelhovenii (JCM 12592T) as new species. C. cavernicola showed particularly distinctive morphology including large inflated anomalous cells on the hyphae and germination from the cells, although clear clamp connections on the hyphae were not confirmed. Further study is needed to elucidate the morph of this species.

Global deceleration of gene evolution following recent genome hybridizations in fungi.

Polyploidization events such as whole-genome duplication and inter-species hybridization are major evolutionary forces that shape genomes. Although long-term effects of polyploidization have been well-characterized, early molecular evolutionary consequences of polyploidization remain largely unexplored. Here, we report the discovery of two recent and independent genome hybridizations within a single clade of a fungal genus, Trichosporon Comparative genomic analyses revealed that redundant genes are experiencing decelerations, not accelerations, of evolutionary rates. We identified a relationship between gene conversion and decelerated evolution suggesting that gene conversion may improve the genome stability of young hybrids by restricting gene functional divergences. Furthermore, we detected large-scale gene losses from transcriptional and translational machineries that indicate a global compensatory mechanism against increased gene dosages. Overall, our findings illustrate counteracting mechanisms during an early phase of post-genome hybridization and fill a critical gap in existing theories on genome evolution.

Recognition and delineation of yeast genera based on genomic data: Lessons from Trichosporonales.

Delineation and characterization of genera in Trichosporonales (Agaricomycotina, Basidiomycota) was performed using 24 haploid and 3 naturally occurring hybrid genomes, with 3 Tremellales genomes used as outgroups. Orthologous group analysis of those genomes showed presence-absence patterns of orthologs that were consistent with the genus classifications. Many shared unique orthologs were identified in the well-supported lineages (genera Apiotrichum and Trichosporon), supporting the definitions of the genera Apiotrichum and Trichosporon from a genomic perspective. Specifically, we obtained 24 and 285 genus-specific genes from eight Apiotrichum and five Trichosporon species, respectively, and propose that these genus-specific genes can be used for delineation of those genera. On the other hand, the genus Cutaneotrichosporon shared only one genus-specific gene among eight genomes, indicating that this genus definition might require re-examination based on genomic data. In addition, taxonomic revisions are presented in this study, including the proposal of two genera, Pascua and Prillingera. Because genomic data can be systematically obtained and analyzed to compare species from a comprehensive viewpoint, they can be used not only to reconstruct reliable phylogenetic trees, but also to re-examine the definitions of taxonomic classifications. To our knowledge, this is the first report to discuss the 'natural system' of genus level classification in fungi based on genomic data.

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).

Heterocephalacria mucosa sp. nov., a new basidiomycetous yeast species isolated from a mangrove forest in Thailand.

Strains of yeast were isolated under a nitrogen-depleted culture condition from decaying tree bark (strain N-12.1) and from mangrove forest water (strain 1-7W.1) sampled at different locations within a mangrove forest site in Ranong province, Thailand. They were found to be genetically and phenotypically different from any currently recognised yeast species. Phylogenetic analysis of nucleotide sequence of three genes, the internal transcribed spacer region 1 and 2 plus 5.8S ribosomal RNA gene (ITS), D1/D2 domain of the large subunit ribosomal RNA gene (LSU D1/D2) and the small subunit of the ribosomal RNA gene (SSU), revealed that these two strains were related to but distinguished from Heterocephalacriaarrabidensis. Several distinct physiological characteristics of these two strains were detected, namely inability to assimilate glycerol, dl-lactic acid, succinic acid, citric acid, d-gluconic acid, and ability to grow well at 25 °C, which were different from those of H. arrabidensis. Accordingly, the name Heterocephalacria mucosa sp. nov., is proposed to accommodate this novel species. The MycoBank number is MB 828624.

A Trichosporonales genome tree based on 27 haploid and three evolutionarily conserved 'natural' hybrid genomes.

To construct a backbone tree consisting of basidiomycetous yeasts, draft genome sequences from 25 species of Trichosporonales (Tremellomycetes, Basidiomycota) were generated. In addition to the hybrid genomes of Trichosporon coremiiforme and Trichosporon ovoides that we described previously, we identified an interspecies hybrid genome in Cutaneotrichosporon mucoides (formerly Trichosporon mucoides). This hybrid genome had a gene retention rate of ~55%, and its closest haploid relative was Cutaneotrichosporon dermatis. After constructing the C. mucoides subgenomes, we generated a phylogenetic tree using genome data from the 27 haploid species and the subgenome data from the three hybrid genome species. It was a high-quality tree with 100% bootstrap support for all of the branches. The genome-based tree provided superior resolution compared with previous multi-gene analyses. Although our backbone tree does not include all Trichosporonales genera (e.g. Cryptotrichosporon), it will be valuable for future analyses of genome data. Interest in interspecies hybrid fungal genomes has recently increased because they may provide a basis for new technologies. The three Trichosporonales hybrid genomes described in this study are different from well-characterized hybrid genomes (e.g. those of Saccharomyces pastorianus and Saccharomyces bayanus) because these hybridization events probably occurred in the distant evolutionary past. Hence, they will be useful for studying genome stability following hybridization and speciation events. Copyright © 2017 John Wiley & Sons, Ltd.

Allodekkera sacchari gen. nov., sp. nov., a yeast species in the Saccharomycetales isolated from a sugar factory.

Three yeast isolates, G5-5(5)T, G5-9(3) and G5-9(4), were obtained from the sugar cane juice and waste from sugar production plant (Korach Industry Co., Ltd) in Korach province, Thailand. They were found to belong to the same species based on DNA sequence identity of the small subunit ribosomal RNA gene (SSU) and the D1/D2 region of the large subunit rRNA gene (LSU D1/D2). A blastn search of the GenBank database revealed they had 93 % nucleotide sequence identity to Dekkera bruxellensis for the SSU (1742 bp), but their LSU D1/D2 sequence (572 bp) showed less than 90 % identity to all available sequences in the database. Phylogenetic analyses with neighbour-joining and maximum-parsimony methods using the aligned LSU D1/D2 and SSU sequences (a total of 2072 positions after removal of gaps) inferred that the three isolates were separated from all known taxa in the Saccharomycetales, and that the neighbouring taxa were species of Dekkera/Brettanomyces. Physiological and biochemical characters revealed distinct differences between the three isolates and Dekkera/Brettanomyces species, including the ability to assimilate several carbon sources and inability to ferment glucose. Thus, isolates G5-5(5)T, G5-9(3) and G5-9(4) should be assigned to a novel taxon, for which the name Allodekkera sacchari gen. nov., sp. nov. is proposed. The type strain of the type species is G5-5(5)T (=CBS 14167T=JCM 18455T=TISTR 5950T), with MycoBank number MB815477 (for the genus) and MB817751 (for the species). Two additional strains of the species are G5-9(3) (=JCM 18456) and G5-9(4) (=JCM 18457).

Occultifur plantarum f.a., sp. nov., a novel cystobasidiomycetous yeast species.

Nine strains representing a single anamorphic novel yeast species in dispersed tropical and subtropical habitats were isolated from sugarcane leaf tissue (DMKU-SE24, DMKU-SE45T, DMKU-SE129 and DMKU-SE134) and corn leaf tissue (DMKU-CE36) in Thailand, phylloplane and rhizoplane of sugarcane in Brazil (IMUFRJ 52018 and IMUFRJ 52019), bromeliad leaf tissue in Brazil (IMUFRJ 51954) and plant leaf in Japan (IPM31-24). These strains showed identical or only 1 nt substitution in the sequences of the D1/D2 region of the LSU rRNA gene and 0-5 nt substitutions in the internal transcribed spacer (ITS) region. Phylogenetic analysis based on the combined sequences of the ITS and the D1/D2 regions showed that the eight of these strains represented a single species in the genus Occultifur that was distinct from related species. Occultifur kilbournensis was the most closely related species, but with 0.9-1.2 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, and 2.4-2.6 % nucleotide substitutions in the ITS region. They are therefore considered to represent a novel species of the genus Occultifur although the formation of basidia was not observed. The name Occultifur plantarum f.a., sp. nov. is proposed. The type strain is DMKU-SE45T (=CBS 14554T=TBRC 6561T).

Trichosporon heliocopridis sp. nov., a urease-negative basidiomycetous yeast associated with dung beetles (Heliocopris bucephalus Fabricius).

Ninety-six yeast isolates associated with dung beetles (Heliocopris bucephalus Fabricius) were examined based on a culture-dependent method. A comparison of the colony morphology and PCR-fingerprints obtained by (GTG)5 microsatellite-primed PCR indicated that 84 of these isolates belonged to one group. Five strains (DD1-1T, DD2-33, DD4-11, DD5-15 and DD6-1) were selected as the representatives of this main group, where each of the five selected strains had been derived from a different dung beetle collected in northern Thailand. A comparison of the D1/D2 domain sequence of the large subunit rRNA gene (LSU D1/D2) and the internal transcribed spacer (ITS) sequences revealed that these five strains were the same and were related to the genus Trichosporon. Phylogenetic analysis based on the LSU D1/D2 plus ITS sequences placed this group within the Trichosporon brassicae clade, but it was clearly separated from any known species. In addition, physiological tests showed that this group had the unusual property of the inability to hydrolyse urea, which was distinctly different from the related taxon. Therefore a novel yeast species named Trichosporon heliocopridis sp. nov. (ex-type strain DD1-1T = TISTR 5946T = JCM 30786T = CBS 14168T) is proposed. The MycoBank number is MB812098.

Detection and characterization of plasminogen receptors on clinical isolates of Trichosporon asahii.

Trichosporon asahii is the major causative agent of deep-seated trichosporonosis. The virulence factors of this yeast pathogen remain uncharacterized. To investigate the pathogenicity of T. asahii, we focused on the interactions between surface molecules of the yeast and host biomolecules. We examined the ability of surface molecules to bind human plasminogen using clinical isolates of T. asahii. Living T. asahii cells accelerated the conversion of plasminogen to plasmin in a dose-dependent manner in the presence of tissue plasminogen activator. Extracts from cells using lithium chloride contained plasminogen-binding molecules based on surface plasmon resonance (SPR) analyses. In all strains tested, several of the fractions obtained using DEAE column chromatography bound and accelerated the conversion of plasminogen to plasmin. Based on far-Western blotting analyses, a common protein was identified within the four strains, which was identified as a hypothetical protein from genome analyses of T. asahii. blast searches suggested the protein might be heparinase, and heparinase activity was detected in the T. asahii extract. Furthermore, affinity chromatography using plasminogen as a ligand detected one protein band by SDS-PAGE, which was identified as thioredoxin-dependent peroxide reductase.SPR analyses suggested the presence of molecules on T. asahii cells that could bind plasminogen with differing affinities.

Hannaella pagnoccae sp. nov., a tremellaceous yeast species isolated from plants and soil.

Several independent surveys of yeasts associated with different plant materials and soil led to the proposal of a novel yeast species belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences of the D1/D2 domains and internal transcribed spacer region of the large subunit of the rRNA gene suggested affinity to a phylogenetic lineage that includes Hannaella coprosmaensis, Hannaella oryzae and Hannaella sinensis. Thirty-two isolates were obtained from different sources, including bromeliads, nectar of Heliconia psittacorum (Heliconiaceae), flowers of Pimenta dioica (Myrtaceae), roots and leaves of sugar cane (Saccharum spp.) in Brazil, leaves of Cratoxylum maingayi, Arundinaria pusilla and Vitis vinifera in Thailand, soil samples in Taiwan, and prairie soil in the USA. Sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene showed that the novel species differs from Hannaella coprosmaensis and Hannaella oryzae by 36 and 46 nt substitutions, respectively. A novel species is suggested to accommodate these isolates, for which the name Hannaella pagnoccae sp. nov. is proposed. The type strain is BI118(T) ( = CBS 11142(T) = ATCC MYA-4530(T)).

Three new basidiomycetous yeasts, Pseudozyma alboarmeniaca sp. nov., Pseudozyma crassa sp. nov. and Pseudozyma siamensis sp. nov. isolated from Thai patients.

We previously reported the first isolation of Pseudozyma species from the blood of Thai patients. In this study, three additional new Pseudozyma species were isolated from clinical specimens from Thai patients. The Pseudozyma species showed relatively low sensitivity to azole antifungal agents. The names proposed for these isolates are Pseudozyma alboarmeniaca (DMST 17135(T) = JCM 12454(T ) = CBS 9961(T)), Pseudozyma crassa (DMST 17136(T ) = JCM 12455(T) = CBS 9959(T)) and Pseudozyma siamensis (DMST 17137(T ) = JCM 12456(T) CBS 9960(T)), where DMST is Department of Medical Sciences Culture Collection, JCM is Japan Collection of Microorganisms and CBS is Centraalbureau voor Schimmelcultures.

Probing membrane deformation energy by KcsA potassium channel gating under varied membrane thickness and tension.

This study investigated how membrane thickness and tension modify the gating of KcsA potassium channels when simultaneously varied. The KcsA channel undergoes global conformational changes upon gating: expansion of the cross-sectional area and longitudinal shortening upon opening. Thus, membranes impose differential effects on the open and closed conformations, such as hydrophobic mismatches. Here, the single-channel open probability was recorded in the contact bubble bilayer, by which variable thickness membranes under a defined tension were applied. A fully open channel in thin membranes turned to sporadic openings in thick membranes, where the channel responded moderately to tension increase. Quantitative gating analysis prompted the hypothesis that tension augmented the membrane deformation energy when hydrophobic mismatch was enhanced in thick membranes.

Hyphal Growth in Trichosporon asahii Is Accelerated by the Addition of Magnesium.

Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

Bacteria can maintain rRNA operons solely on plasmids for hundreds of millions of years.

It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

Moniliella carnis sp. nov. and Moniliella dehoogii sp. nov., two novel species of black yeasts isolated from meat processing environments.

Thirteen strains of yeasts typical of the genus Moniliella were isolated from fermenting meat and meat processing tools in Vietnam. PCR fingerprints generated by primer (GAC)(5) subdivided the strains into two distinctive genetic groups. In a phylogenetic tree based on D1/D2 large subunit rRNA gene sequences, the strains formed a well-supported clade with Moniliella spathulata and Moniliella suaveolens but represented two new lineages. The names Moniliella carnis sp. nov. and Moniliella dehoogii sp. nov. are proposed. The two novel species can be distinguished from each other and from known species of Moniliella based on phenotypic characteristics. It is assumed that the yeasts were associated with fatty substances that contaminated the meat processing tools. The type strain of Moniliella carnis is KFP 246(T) ( = CBS 126447(T) = NRRL Y-48681(T)) and the type strain of Moniliella dehoogii is KFP 211(T) ( = CBS 126564(T) = NRRL Y-48682(T)).

Taxonomy of Pathogenic Yeasts Candida, Cryptococcus, Malassezia, and Trichosporon.

This review describes the changes in yeast species names in the previous decade. Several yeast species have been reclassified to accommodate the "One fungus=One name" (1F=1N) principle of the Code. As the names of medically important yeasts have also been reviewed and revised, details of the genera Candida, Cryptococcus, Malassezia, and Trichosporon are described in Section 3, along with the history of name changes. Since the phylogenetic positions of Candida species in several clades have not been clarified, revision of this species has not been completed. Among the species that remain unrevised despite their importance in the medical field, we propose the transfer of six Candida species to be reclassified in the Nakaseomyces clade, including Nakaseomyces glabratus and Nakaseomyces nivalensis.