A new species of the genus Aleurodiscus sensu lato (Russulales, Basidiomycota) from Hachijo Island, Japan.

Aleurodiscus sagittisporus sp. nov. is described and illustrated. This species is characterized by producing basidiomata with a monomitic hyphal system, clampless-septate hyphae, arrowhead-shaped, amyloid, finely verrucose basidiospores, gloeocystidia, dendrohyphidium-like branched paraphysoid hyphae, and variously shaped swelling cells in the hymenium. Phylogenetic analyses based on nuclear rDNA LSU and ITS sequences revealed that the species is distinct from the lineage of Aleurodiscus s. str. and related genera in the Aleurodiscus s. lat. clade. Basidiomata of A. sagittisporus have been collected only from dead petioles attached to living trees of Livistona chinensis var. subglobosa on Hachijo Island, Japan.

Identification of Cyclocybe erebia metabolites that affect the circadian rhythm of Eluc expression under control of Bmal1 promoter in mouse fibroblast cells.

Pharmacological intervention of circadian rhythms is a potentially useful approach for ameliorating various health problems caused by disturbed circadian rhythms including sleep disorder and metabolic diseases. To find compounds that affect circadian rhythms, we screened mushroom extracts using mouse cells expressing the luciferase gene under the control of the mouse Bmal1 promoter. The culture filtrate extract from the basidiomycete Cyclocybe erebia enhanced the oscillation of bioluminescence caused by the expression of the luciferase gene and prolonged the period of bioluminescence. Bioassay-guided fractionation of the extract resulted in purification of compounds 1 and 2. Spectroscopic analyses along with single-crystal X-ray diffraction analysis, revealed that these compounds were diterpenoids with a unique skeleton and a fused ring system comprising 3-, 7-, and 5-membered rings. Compounds 1 and 2 were named cyclocircadins A and B, respectively. These findings suggested that natural diterpenoids could be a source of compounds with the activity affecting circadian rhythms.

Hypochnicium sensu lato (Polyporales, Basidiomycota) from Japan, with descriptions of a new genus and three new species.

Species of Hypochnicium (Polyporales, Basidiomycota) collected from Japan were studied on their taxonomy by morphological and phylogenetic approaches. Phylogenetic analyses based on a nrDNA LSU and ITS dataset including the Japanese specimens and other publicly available ones show that Hypochnicium is polyphyletic. Since the clade containing the type species H. bombycinum was well-supported, we defined this clade as Hypochnicium s. str., and emended Hypochnicium to include restricted taxa with only smooth basidiospores. The new genus Neohypochnicium is proposed to accommodate the remaining taxa excluded from the genus Hypochnicium s. str., which includes both species with smooth basidiospores and ornamented ones. Three new species, Gyrophanopsis japonica, N. asiaticum and N. perlongicystidiosum are described and illustrated based on morphological and phylogenetic analyses using an ITS region dataset. In addition, the following 15 new combinations are proposed: N. albostramineum, N. aotearoae, N. capitulateum, N. cremicolor, N. cystidiatum, N. geogenium, N. guineense, N. huinayense, N. michelii, N. microsporum, N. patagonicum, N. pini, N. punctulatum, N. subrigescens and N. wakefieldiae. An identification key to Japanese species of Bulbillomyces, Gyrophanopsis, Hypochnicium and Neohypochnicium is provided.

Taxonomy and ecology of Physisporinus forming synnema-like structures in freshwater environments.

Since 2011, we have collected fungi that form synnema-like structures (SSs) bearing many acanthophyses at the apex on water-splashed wood in streams in various regions of Japan. A provisional phylogenetic analysis of strains isolated from SSs based on their nrDNA sequences implied affinity with Physisporinus (Polyporales, Basidiomycota). However, it has not been reported that this genus forms SSs in freshwater habitats. We found a fungus forming not only SSs on the water-boundary part of wood but also resupinate basidiocarps with poroid hymenophores on nonsubmerged parts, and the morphological characteristics of the basidiocarps matched those of Physisporinus. Therefore, we investigated the relationship between SS-forming fungi and their sexual states by taxonomic approaches. Phylogenetic analyses based on nrDNA internal transcribed spacer (ITS) and large subunit (LSU) sequences indicated that SS-forming fungi diverged into five clades in Physisporinus. Each clade was discriminated by the color of SSs, morphology of acanthophyses, and cultural characteristics. Of the five clades, Clade 1, which consisted only of sequences of strains isolated from SSs and basidiocarps produced on rhizomorphs, was closely related to P. eminens and P. undatus, but the morphology of basidiocarps and the manner of basidiocarp development differed. Clade 5 was closely related to P. castanopsidis, P. crocatus, P. pouzarii, P. sanguinolentus, P. subcrocatus, P. tibeticus, and P. vitreus, but the basidiocarp morphology differed. Therefore, Clades 1 and 5 were described as two new species. Regarding Clades 2, 3, and 4, further taxonomic studies with additional specimens are required. SS and acanthophysis formation in wet habitats in streams and in culture could be recognized as new taxonomic and ecological characters of Physisporinus.

Systematic revision of Hydnum species in Japan.

Hydnum (Hydnaceae, Basidiomycota) exhibits endemic species diversity in East Asia; however, few comprehensive systematic studies have been conducted to date. Here, we performed morphological, ecological, phylogenetic, and biological evaluations of the taxonomy of Hydnum species in Japan. In total, 186 Japanese Hydnum specimens were used for morphological observations. Phylogenetic trees were constructed using sequence data of nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) region and a portion of translation elongation factor 1-α (tef1). Intra- and interspecific mating tests using 78 monokaryotic strains of 13 species did not conflict with species delimitation inferred from their ITS and tef1 phylogenetic relationships. This study provides detailed morphological descriptions of 15 rigorously identified species from Japan, nine of which are described as new: H. alboluteum, H. albopallidum, H. pinicola, H. itachiharitake, H. minospororufescens, H. orientalbidum, H. subberkeleyanum, H. tomaense, and H. tottoriense. Three species documented in this work are new to Japan: H. boreorepandum, H. mulsicolor, and H. umbilicatum. The remaining three species (H. cremeoalbum, H. minus, and H. repando-orientale), previously reported from Japan, are redescribed using data from newly collected materials. We also transferred two old species (Hericium fimbrillatum and Sarcodon nauseofoetidus) from East Asian Hydnum into other genera.

Two new species of Sistotrema s.l. (Cantharellales) from Japan with descriptions of their ectomycorrhizae.

We describe two new species of resupinate Sistotrema sensu lato (Cantharellales) collected in Japan: S. flavorhizomorphae and S. chloroporum. Both species have urniform basidia with more than four sterigmata and monomitic hyphal system, oil-rich hyphae in subiculum, which is typical for this genus. Sistotrema chloroporum is characterized by poroid hymenophore partly yellowish-green, basidia 4-6-spored, medium-sized basidiospores (4.5-6.5 × 3.5-6 µm), and broadleaf forest habitat. Sistotrema flavorhizomorphae is characterized by hydnoid-irpicoid hymenophore, bright yellowish rhizomorphs, basidia 6-8-spored, small basidiospores (3-3.5 × 2.5-3 µm), and pine forest habitat. Phylogenetic trees inferred from the fungal nrDNA ITS and LSU and the rpb2 sequences supported that both species were distinct and grouped with other ectomycorrhizal Sistotrema and Hydnum species, but their generic boundary was unclear. Mycorrhizae underneath basidiomes of both species were identified and described via molecular techniques. Mycorrhizae of S. chloroporum have similar characteristics to those of other Sistotrema s.l. and Hydnum species, i.e., S. confluens and H. repandum, whereas S. flavorhizomorphae has a distinct morpho-anatomy, for example, a distinct pseudoparenchymatous mantle. Comprehensive characterizations of basidiomes and mycorrhizae improve the taxonomic analysis of mycorrhizal species of Sistotrema s.l.

Description of a new species of Gerhardtia (Lyophyllaceae, Agaricales) from Japan based on morphological and molecular phylogenetic analyses and live culture characteristics.

We describe a new species of Gerhardtia from Japan based on basidiomata morphology, live culture characteristics, and molecular phylogenetic analyses. Gerhardtia venosolamellata is found on broad-leaf litter, and is characterized by tricholomatoid to marasmioid basidiomata, an off-white to pale salmon-pink pileus surface with faint marginal striae, subdistant lamellae with lateral veins, a tomentose to strigose stipe base with hyphal strands generating arthroconidia measuring 4-7 × 2-3 µm, cyanophilic, elongate-ellipsoid to cylindrical, slightly verrucose or undulate basidiospores measuring 4.5-6 × 2.5-3 µm, and cyanophilic basidia measuring 25-35 × 5-6 µm and containing siderophilous granules. Phylogenetic analyses based on the internal transcribed spacer and large subunit regions of the fungal nrDNA indicates that G. venosolamellata is related to G. sinensis and G. highlandensis, but differs from the former with respect to basidiomata color, basidiospore shape, and habitat. An isotype specimen of G. highlandensis exhibited relatively close lamellae without veins, and slightly larger basidiospores (4.5-6.5 × 2.5-3 µm). Cultured mycelia of G. venosolamellata produced arthroconidia measuring 4.5-8.5 × 2.5-3 µm with both schizolytic and rhexolytic secession on MA and PDA media, and chlamydospores occasionally covered with crystals on MA and MYG media.

Mycorrhizal synthesis, morpho-anatomical characterization of mycorrhizae, and evaluation of mycorrhiza-forming ability of Hydnum albidum-like species using monokaryotic and dikaryotic cultures.

Despite the economic and ecological importance of Hydnum species, in vitro synthesis of ectomycorrhizae of this genus has not been reported due to difficulties in establishing pure cultures. We inoculated pure cultures of 12 monokaryotic and 3 dikaryotic mycelial strains of an undescribed Hydnum albidum-like species on roots of axenic Pinus densiflora seedlings to synthesize ectomycorrhizae and to evaluate their mycorrhiza-forming ability. Six months after inoculation, both monokaryotic and dikaryotic strains formed ectomycorrhizae with Hartig net hyphae at the root cortex. Monokaryotic and dikaryotic strains exhibited similar morpho-anatomical characteristics of ectomycorrhizae, with the exception for clamped septa of emanating and outer mantle hyphae in the latter. Between monokaryotic and descendant dikaryotic strains, there were no significant differences in number of mycorrhizae in pine seedlings, whereas monokaryotic strains showed a greater total number of root tips and lower colonization rates than the descendant dikaryotic strains. These results indicate that both monokaryotic and dikaryotic mycelia of the H. albidum-like species can form mycorrhizae under axenic condition, and that can be applied toward the cultivation of hedgehog mushrooms.

Four mycelial strains of Entoloma clypeatum species complex form ectomycorrhiza-like roots with Pyrus betulifolia seedlings in vitro, and one develops fruiting bodies 2 months after inoculation.

Entoloma clypeatum species complex (ECSC) forms ectomycorrhiza-like roots (EMLR) with host plant species of Rosaceae or Ulmaceae. The EMLR colonized with ECSC are characterized by a thick fungal mantle, absence of a Hartig net structure, and collapse of the apical meristem caused by hyphal invasion. Some researchers have suggested parasitism of ECSC because of this unique mode of colonization; however, the nature of the interaction between ECSC and host plants has not been investigated in co-culture because of the difficulty of culturing this group of fungi. We established a procedure to synthesize EMLR of ECSC on pear seedlings using fungal cultures. Three conspecific strains of ECSC isolated from basidiospores and one strain isolated from EMLR were tested. Cultured mycelia were inoculated onto a modified Norkrans' C (MNC) or Hyponex-yeast-glucose (HYG) medium slant on the bottom of a polycarbonate jar and covered with autoclaved andosol or a vermiculite/sphagnum moss mixture (VSM); an axenically cultivated Pyrus betulifolia seedling was then planted in the jar. Five months after inoculation, the formation of EMLR with Hartig net-like hyphae was confirmed in all of the experimental plots. However, the rate of root colonization was significantly higher in experimental plots using andosol than in those using VSM. The growth of pear seedlings was similar irrespective of the level of root colonization, suggesting commensalism rather than parasitism of ECSC. One experimental plot using strain A3, an MNC slant, and andosol as a substrate produced ECSC fruiting bodies with mature basidia and basidiospores. The results suggested that our procedure enables the synthesis of EMLR of ECSC and cultivation of their fruiting bodies.

Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka's perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a -80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.

Isolation of isolactarane sesquiterpenes from a Phlebia tremellosa culture filtrate and their growth promotion effects on lettuce roots.

The ethyl acetate extract of the culture filtrate of Phlebia tremellosa promoted elongation of the lateral roots of lettuce seedlings at 250 µg/mL. We purified two compounds that promote root elongation by using activity-guided chromatographic fractionation. On the basis of spectroscopic analyses, these compounds were identified to be isolactarane sesquiterpenes derived from the dehydrogenation of merulactone, which was previously isolated from the same species. We named the purified compounds phlelactones A and B. Phlelactones A and B promoted primary root elongation at 100-300 and 10-30 µg/mL and the elongation and formation of lateral roots at 300-1000 and 30-100 µg/mL, respectively.

A modified perlite protocol with a mixed dimethyl sulfoxide and trehalose cryoprotectant improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

Homolka's perlite protocol (HPP) was evaluated for cryopreservation of a wide range of ectomycorrhizal basidiomycete cultures, then a modified perlite protocol (MPP), in which cryoprotectant was added just before freezing rather than during the culturing process, was applied to cryosensitive strains that failed to survive when HPP was used. Further modifications of MPP with various cryoprotectants were explored to improve the cryopreservation of hard-to-preserve strains. The efficacy of HPP was assessed in 111 strains of 38 species of basidiomycetes of various cryosensitivities. After freezing strains using HPP, the viability and colony diameter of the strains were examined after 2 wk, 6 mo, and 1 y of storage at -80 C. Of the 111 strains tested, 91 survived after 1 y of storage with high viability of 80% or more, whereas the remaining 20 strains exhibited low and unstable viability. For those selected cryosensitive strains that did not survive well when HPP was used, MPP was applied with a mixture of cryoprotectants, dimethyl sulfoxide (DMSO), glycerol, and trehalose, at different concentrations and combinations. Toxicity testing of the cryoprotectants in the nonfrozen state revealed that 12% (v/v) glycerol was highly toxic for six strains (four species), whereas DMSO (5% and 10% [v/v]) was less toxic than glycerol. The viability of the cryosensitive strains after freezing demonstrated that DMSO was more efficient than glycerol, and trehalose enhanced the cryoprotective effects of both glycerol and DMSO when MPP was used for cryopreservation. Our comparative analysis of MPP with various combinations and concentrations of cryoprotectants revealed that a mixture of 5% DMSO and 10% trehalose was the most effective cryoprotectant, and that using MPP with this cryoprotectant was applicable to many cryosensitive strains.

Novel tyrosinase inhibitors from liquid culture of Neolentinus lepideus.

Tyrosinase is the key enzyme that controls melanin formation in the human skin. We performed a screening of 96 extracts of mushroom cultures and fruiting bodies for examining their inhibitory activity against mushroom tyrosinase. The ethyl acetate extracts of culture filtrate of Neolentinus lepideus exhibited the strongest inhibitory activity. The active compounds 1 and 2 were purified by repeated chromatographic separations from the extract. On the basis of spectroscopic analyses, 1 and 2 were identified to be 1,3-dihydroisobenzofuran-4,5,7-triol and 5-methoxy-1,3-dihydroisobenzofuran-4,7-diol, respectively. Lineweaver-Burk plot of the enzyme reaction in the presence of 1 indicated that 1 was a potent competitive inhibitor. The respective IC50 values of 1 and 2 were 173 and 263 µg/mL. Compound 1 at 15 µg/mL suppressed melanin accumulation stimulated by α-MSH in the murine melanoma B16 cells, as well as the induced accumulation of both tyrosinase transcript and protein without inhibiting cell proliferation.

Isaria takamizusanensis is the anamorph of Cordyceps ryogamimontana, warranting a new combination, Purpureocillium takamizusanense comb. nov.

The entomogenous anamorphic fungus Isaria takamizusanensis has not been resolved clearly in its teleomorphic state. We succeeded in inducing ascostroma formation by incubating conidiomata of I. takamizusanensis on cicada adults in a moist chamber. We observed the ascostroma and conducted a phylogenetic analysis based on ITS rDNA and EF-1α genes. The morphology of the ascostroma was identical to that of Cordyceps ryogamimontana. In the phylogenetic tree inferred from EF-1α, the isolate from the partspores grouped with nine strains derived from conidia of I. takamizusanensis, which was distinct from a clade including Purpureocillium lilacinum. Moreover, a conidial structure identical to that of I. takamizusanensis was rediscovered on the holotype specimen of C. ryogamimontana. As a result, we propose a new name, Purpureocillium takamizusanense, which is a combination of the teleomorph-anamorph connection of C. ryogamimontana-I. takamizusanensis, in accordance with the 'one fungus, one name' concept of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN).

The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: an application of One Fungus One Name.

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.

Trichoderma mienum sp. nov., isolated from mushroom farms in Japan.

During an investigation of Hypocrea/Trichoderma species inhabiting mushroom bedlogs, we found five strains of an undescribed species from a culture collection. These were analyzed using a combined approach, including morphology of holomorph, cultural studies, and phylogenetic analyses of the rRNA gene cluster of the internal transcribed spacer region, translation elongation factor 1-α, and RNA polymerase subunit II gene sequences. Distinctive morphological characters include stromata with green ascospores produced on potato dextrose agar medium, and Gliocladium-like to irregularly Verticillium-like conidiophores. In phylogenetic analyses, this species belongs to the Semiorbis clade, but its morphological characteristics do not match the other members of this clade. Based on morphological observations and phylogenetic analyses, we describe this as a new species, Trichoderma mienum, representing its Hypocrea teleomorph and Trichoderma anamorph.

Cryopreservation of cryosensitive basidiomycete cultures by application and modification of perlite protocol.

Homolka's perlite protocol (HPP) for cryopreservation of fungal cultures was evaluated in 12 strains (7 species) of cryosensitive basidiomycete cultures maintained in NBRC culture collection by investigating viability, time to recover, and basic morphological study after freezing and storing at -80 degree C for 6 months. The viability of the fungal strains was 60 percent in Phallus hadriani and 100 percent in remaining 11 strains, indicating the efficacy of HPP method for cryopreservation of some cryosensitive basidiomycetes. The HPP method was modified by changing the addition of cryoprotectant (glycerol) from prior precultivation to post precultivation, limiting the cryoprotectant exposure time to 48 hours, and increasing the glycerol concentration from 5 percent to 12 percent. The viability of P. hadriani strain increased from 60 percent to 100 percent with the modified perlite protocol after storage at -80 degree C for 6 months.

Trichoderma matsushimae and T. aeroaquaticum: two aero-aquatic species with Pseudaegerita-like propagules.

Four isolates tentatively identified as Pseudaegerita matsushimae on the basis of the morphology of bulbil-like propagules were collected from substrates submerged in water in Thailand and Japan. In culture studies the two Thai isolates were found to produce phialoconidia on conidiogenous cells and phialoconidiophores whose morphology was similar to that of Trichoderma. Phylogenetic analysis based on D1/D2 regions of LSU rDNA sequences showed that the four isolates were nested in Hypocrea/Trichoderma (Hypocreales) while P. corticalis, the type species of Pseudaegerita, belongs to Hyaloscypha (Helotiales). Preliminary analysis by ISTH Web tools based on 5.8S-ITS rDNA and phylogenetic analysis based on rpb2 and tef1-int4 genes showed that the isolates have specific sequences of Trichoderma (anchors 1-5) and belong to the Hamatum clade but they grouped apart from any known species of Trichoderma. The sequences of the tef1-int4 gene, which were amplified from the authentic specimen of P. matsushimae (IMI 266915), also showed that it belongs to the Hamatum clade closely clustering with T. yunnanense but separate from our four isolates. The morphology of P. matsushimae (IMI 266915), especially the sizes of phialides and phialoconidia, were different from T. yunnanense. Thus, we conclude that IMI 266915 and our isolates are to be assigned to two different species in the Hamatum clade of Trichoderma, although both species have similar morphology of bulbils and phialoconidia. Morphology and molecular data revealed that P. matsushimae should be assigned to the genus Trichoderma as T. matsushimae and the Thai and Japanese isolates are placed in T. aeroaquaticum sp. nov. This finding supports the interpretation that aero-aquatic fungi have evolved from terrestrial fungi. We assume that these fungi probably were derived from typically soil-inhabiting species of Trichoderma; an adaptation to aquatic environments is shown by formation of bulbil-like propagules floating on water.

Pichia porticicola sp. nov., a novel ascomycetous yeast related to Pichia acaciae isolated from galleries of ambrosia beetles in Japan.

Eleven strains of yeasts, isolated from galleries of ambrosia beetles in Japan, formerly identified as Pichia acaciae were found to have different sequences in the D1/D2 domain of the large subunit (LSU) rRNA gene. After detailed taxonomic studies including a DNA-DNA reassociation experiment, 11 strains were found to represent a novel species of the genus Pichia. It is described as Pichia porticicola sp. nov. (type strain NBRC 100302(T) = CBS 11715(T)). The eleven strains were isolated from various samples associated with different kinds of insects and trees which were collected in 11 prefectures, from the north to the south of Japan. This species might be a common species in galleries of ambrosia beetles in Japan.

Two new ascomycetous anamorphic yeast species related to Candida friedrichii--Candida jaroonii sp. nov., and Candida songkhlaensis sp. nov.--isolated in Thailand.

In a study of yeast diversity in Thailand, eight strains of hitherto undescribed anamorphic yeasts were isolated: four from insect frass, two from Marasmius sp. fruiting bodies, one from a flower, and one from jackfruit exudates. Phylogenetic analysis of the D1/D2 domain of 26S ribosomal DNA nucleotide sequences indicated that the eight strains represented two new species related to Candida friedrichii. Genetic separation of the two new species was further supported by DNA-DNA hybridization analysis, which resulted in between-species similarity values of less than 48%, and by electrophoretic karyotyping. The two new species are C. jaroonii sp. nov. (type strain, ST-300(T) = NBRC 103209(T) = BCC 11783(T) = CBS 10790(T)) and C. songkhlaensis sp. nov. (type strain, ST-328(T) = NBRC 103214(T) = BCC 11804(T) = CBS 10791(T)).