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.

Taxonomic study of polymorphic basidiomycetous fungi Sirobasidium and Sirotrema: Sirobasidium apiculatum sp. nov., Phaeotremella translucens comb. nov. and rediscovery of Sirobasidium japonicum in Japan.

Species in the genera Sirobasidium and Sirotrema (Tremellales, Tremellomycetes, Agaricomycotina, Basidiomycota) have been described based solely on the morphology of teleomorph, and many of them lack both isolates of anamorphic yeast state and nucleotide sequence data. Strains of Sirotrema translucens and Sirobasidium japonicum were established for the first time from basidiocarps collected in Japan. Also, an undescribed species in the genus Sirobasidium was isolated. Sirobasidium sp. was characterized by its apiculate epibasidia and 2-celled basidia divided by a longitudinal septum, which is a unique combination of characteristics in the genus. Although the phylogenetic placement of Sb. japonicum within the Tremellales was not resolved in our analysis, Sirobasidium sp. formed a well-supported monophyletic clade with Sb. magnum and Fibulobasidium spp., and Sirotrema translucens was located in the genus Phaeotremella. Mating experiments using single-basidiospore strains showed that Sb. japonicum produced basidia, epibasidia, and basidiospores on a nutrient-poor medium, and the life cycle was successfully completed in controlled conditions. In conclusion, we propose Sirobasidium apiculatum sp. nov. and Phaeotremella translucens comb. nov.

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.

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.

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.

Fungus symbionts colonizing the galleries of the ambrosia beetle Platypus quercivorus.

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.

D-Fructose Assimilation and Fermentation by Yeasts Belonging to Saccharomycetes: Rediscovery of Universal Phenotypes and Elucidation of Fructophilic Behaviors in Ambrosiozyma platypodis and Cyberlindnera americana.

The purpose of this study was to investigate the ability of ascomycetous yeasts to assimilate/ferment d-fructose. This ability of the vast majority of yeasts has long been neglected since the standardization of the methodology around 1950, wherein fructose was excluded from the standard set of physiological properties for characterizing yeast species, despite the ubiquitous presence of fructose in the natural environment. In this study, we examined 388 strains of yeast, mainly belonging to the Saccharomycetes (Saccharomycotina, Ascomycota), to determine whether they can assimilate/ferment d-fructose. Conventional methods, using liquid medium containing yeast nitrogen base +0.5% (w/v) of d-fructose solution for assimilation and yeast extract-peptone +2% (w/v) fructose solution with an inverted Durham tube for fermentation, were used. All strains examined (n = 388, 100%) assimilated d-fructose, whereas 302 (77.8%) of them fermented d-fructose. In addition, almost all strains capable of fermenting d-glucose could also ferment d-fructose. These results strongly suggest that the ability to assimilate/ferment d-fructose is a universal phenotype among yeasts in the Saccharomycetes. Furthermore, the fructophilic behavior of Ambrosiozyma platypodis JCM 1843 and Cyberlindnera americana JCM 3592 was characterized by sugar consumption profiles during fermentation.

Revision of Xylonaceae (Xylonales, Xylonomycetes) to include Sarea and Tromera.

The resinicolous fungi Sarea difformis and S. resinae (Sareomycetes) were taxonomically revised on the basis of morphological observations and phylogenetic analyses of the nucleotide sequences of the nSSU-LSU-rpb1-rpb2-mtSSU genes. The results of phylogenetic analyses show that S. difformis and S. resinae are grouped with members of Xylonomycetes. According to the results of phylogenetic analyses and their sexual and asexual morphs resemblance, Sareomycetes is synonymized with Xylonomycetes. Although Tromera has been considered a synonym of Sarea based on the superficial resemblance of the sexual morph, we show that they are distinct genera and Tromera should be resurrected to accommodate T. resinae (= S. resinae). Xylonomycetes was morphologically re-circumscribed to comprise a single family (Xylonaceae) with four genera (Sarea, Trinosporium, Tromera, and Xylona) sharing an endophytic or plant saprobic stage in their lifecycle, ascostroma-type ascomata with paraphysoid, Lecanora-type bitunicate asci, and pycnidial asexual morphs. Phylogenetic analyses based on ITS sequences and environmental DNA (eDNA) implied a worldwide distribution of the species. Although Symbiotaphrinales has been treated as a member of Xylonomycetes in previous studies, it was shown to be phylogenetically, morphologically, and ecologically distinct. We, therefore, treated Symbiotaphrinales as Pezizomycotina incertae sedis.

Gut bacterial and fungal communities in ground-dwelling beetles are associated with host food habit and habitat.

Beetles (Coleoptera) have the highest species diversity among all orders, and they have diverse food habits. Gut microbes may have contributed to this diversification of food habits. Here, we identified the pattern of the relationship between ground-dwelling beetles and their gut microbial communities (bacteria and fungi) in the field. We collected 46 beetle species of five families from secondary deciduous forests and grasslands in Japan and extracted microbial DNA from whole guts for amplicon sequencing. The gut bacterial and fungal communities differed among all habitats and all food habits of their hosts (carnivores, herbivores, omnivores, and scavengers) except for the fungal communities between carnivores and scavengers. Specifically, the abundant bacterial group varied among food habits: Xanthomonadaceae were abundant in scavengers, whereas Enterobacteriaceae were abundant in carnivores and herbivores. Phylogenetically closely related beetles had phylogenetically similar communities of Enterobacteriaceae, suggesting that the community structure of this family is related to the evolutionary change in beetle ecology. One of the fungal groups, Yarrowia species, which has been reported to have a symbiotic relationship with silphid beetles, was also detected from various carnivorous beetles. Our results suggest that the symbiotic relationships between ground-dwelling beetles and these microbes are widespread.

Draft Genome Sequence of Novel Metschnikowia sp. Strain JCM 33374, a Nectar Yeast Isolated from a Bumblebee.

Here, we report the draft genome sequence of Metschnikowia sp. strain JCM 33374, a nectar yeast isolated from a bumblebee (Bombus diversus). The genome of 20.1 Mb is a naturally heterozygous diploid. Phylogenetic analysis with related taxa demonstrated that the strain is very likely a novel species.

Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca.

Autotrophic eukaryotes have evolved by the endosymbiotic uptake of photosynthetic organisms. Interestingly, many algae and plants have secondarily lost the photosynthetic activity despite its great advantages. Prototheca and Helicosporidium are non-photosynthetic green algae possessing colourless plastids. The plastid genomes of Prototheca wickerhamii and Helicosporidium sp. are highly reduced owing to the elimination of genes related to photosynthesis. To gain further insight into the reductive genome evolution during the shift from a photosynthetic to a heterotrophic lifestyle, we sequenced the plastid and nuclear genomes of two Prototheca species, P. cutis JCM 15793 and P. stagnora JCM 9641, and performed comparative genome analyses among trebouxiophytes. Our phylogenetic analyses using plastid- and nucleus-encoded proteins strongly suggest that independent losses of photosynthesis have occurred at least three times in the clade of Prototheca and Helicosporidium. Conserved gene content among these non-photosynthetic lineages suggests that the plastid and nuclear genomes have convergently eliminated a similar set of photosynthesis-related genes. Other than the photosynthetic genes, significant gene loss and gain were not observed in Prototheca compared to its closest photosynthetic relative Auxenochlorella. Although it remains unclear why loss of photosynthesis occurred in Prototheca, the mixotrophic capability of trebouxiophytes likely made it possible to eliminate photosynthesis.

Lipid production via simultaneous conversion of glucose and xylose by a novel yeast, Cystobasidium iriomotense.

The yeast strains IPM32-16, ISM28-8sT, and IPM46-17, isolated from plant and soil samples from Iriomote Island, Japan, were explored in terms of lipid production during growth in a mixture of glucose and xylose. Phylogenetically, the strains were most closely related to Cystobasidium slooffiae, based on the sequences of the ITS regions and the D1/D2 domain of the LSU rRNA gene. The strains were oleaginous, accumulating lipids to levels > 20% dry cell weight. Moreover, kinetic analysis of the sugar-to-lipid conversion of a 1:1 glucose/xylose mixture showed that the strains consumed the two sugars simultaneously. IPM46-17 attained the highest lipid content (33%), mostly C16 and C18 fatty acids. Thus, the yeasts efficiently converted lignocellulosic sugars to lipids, aiding in biofuel production (which benefits the environment, promotes rural jobs, and strengthens fuel security). The strains constituted a novel species of Cystobasidium, for which we propose the name Cystobasidium iriomotense (type strain ISM28-8sT = JCM 24594T = CBS 15015T).

Draft Genome Sequences of the Oomycete Pilasporangium apinafurcum Strains JCM 30513 and JCM 30514, Formerly Classified as Pythium apinafurcum.

Pilasporangium apinafurcum, formerly classified as Pythium apinafurcum, is a unique oomycete that infects plants asymptomatically. Here, we present the draft genome sequences of two variants of P. apinafurcum, JCM 30513 and JCM 30514, isolated from uncultivated field soil in Wakayama Prefecture, Japan.

Draft Genome Sequence of Raffaelea quercivora JCM 11526, a Japanese Oak Wilt Pathogen Associated with the Platypodid Beetle, Platypus quercivorus.

The Japanese oak wilt pathogen Raffaelea quercivora and the platypodid beetle, Platypus quercivorus, cause serious mass mortality of Quercus spp. in Japan. Here, we present the first draft genome sequence of R. quercivora JCM 11526 to increase our understanding of the mechanism of pathogenicity and symbiosis with the ambrosia beetle.

Lipid production through simultaneous utilization of glucose, xylose, and L-arabinose by Pseudozyma hubeiensis: a comparative screening study.

Co-fermentation of glucose, xylose and L-arabinose from lignocellulosic biomass by an oleaginous yeast is anticipated as a method for biodiesel production. However, most yeasts ferment glucose first before consuming pentoses, due to glucose repression. This preferential utilization results in delayed fermentation time and lower productivity. Therefore, co-fermentation of lignocellulosic sugars could achieve cost-effective conversion of lignocellulosic biomass to microbial lipid. Comprehensive screening of oleaginous yeasts capable of simultaneously utilizing glucose, xylose, and L-arabinose was performed by measuring the concentration of sugars remaining in the medium and of lipids accumulated in the cells. We found that of 1189 strains tested, 12 had the ability to co-ferment the sugars. The basidiomycete yeast Pseudozyma hubeiensis IPM1-10, which had the highest sugars consumption rate of 94.1 %, was selected by culturing in a batch culture with the mixed-sugar medium. The strain showed (1) simultaneous utilization of all three sugars, and (2) high lipid-accumulating ability. This study suggests that P. hubeiensis IPM1-10 is a promising candidate for second-generation biodiesel production from hydrolysate of lignocellulosic biomass.

Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production.

The lipid-accumulating ability of 500 yeast strains isolated in Japan was evaluated. Primary screening revealed that 31 strains were identified as potential lipid producers, from which 12 strains were cultivated in a medium containing 3% glucose. It was found that JCM 24511 accumulated the highest lipid content, up to 61.53%, while JCM 24512 grew the fastest. They were tentatively identified as Cryptococcus sp. and Cryptococcus musci, respectively. The maximum lipid concentration of 1.49g/L was achieved by JCM 24512. Similarly, JCM 24511 also achieved a high lipid production of 1.37g/L. High lipid productivity is the most important characteristic of oleaginous yeasts from the viewpoint of practical production. Among the strains tested here, JCM 24512 had the best lipid productivity, 0.37g/L/day. The results show that the isolated yeasts could be promising candidates for biodiesel production.

Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus terricola could be used for fuel production through consolidated bioprocessing. C. terricola JCM 24523 exhibited the highest lipid content of 61.96% on medium with 5% starch at 10 days. Fatty acid methyl ester analysis showed that this strain produced high proportions of C16:0 and C18 fatty acids when grown on starch, which are ideal for use in biodiesel. Considering the yield and cost, lipids derived from starch using C. terricola would be a promising alternative source for biodiesel production.

Comparative genomics of Taphrina fungi causing varying degrees of tumorous deformity in plants.

Taphrina fungi are biotrophic plant pathogens that cause plant deformity diseases. We sequenced the genomes of four Taphrina species-Taphrina wiesneri, T. deformans, T. flavorubra, and T. populina-which parasitize Prunus, Cerasus, and Populus hosts with varying severity of disease symptoms. High levels of gene synteny within Taphrina species were observed, and our comparative analysis further revealed that these fungi may utilize multiple strategies in coping with the host environment that are also found in some specialized dimorphic species. These include species-specific aneuploidy and clusters of highly diverged secreted proteins located at subtelomeres. We also identified species differences in plant hormone biosynthesis pathways, which may contribute to varying degree of disease symptoms. The genomes provide a rich resource for investigation into Taphrina biology and evolutionary studies across the basal ascomycetes clade.