Spore germination and ectomycorrhizae formation of Tricholoma matsutake on pine root systems with previously established ectomycorrhizae from a dikaryotic mycelial isolate of T. matsutake.

In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

Dual colonization of Mucoromycotina and Glomeromycotina fungi in the basal liverwort, Haplomitrium mnioides (Haplomitriopsida).

In general, Glomeromycotina was thought to be the earliest fungi forming mycorrhiza-like structure (MLS) in land plant evolution. In contrast, because the earliest divergent lineage of extant land plants, i.e. Haplomitriopsida liverworts, associates only with Mucoromycotina mycobionts, recent studies suggested that those fungi are novel candidates for the earliest mycobionts. Therefore, Mucoromycotina-Haplomitriopsida association currently attracts attention as an ancient mycorrhiza-like association. However, mycobionts were identified in only 7 of 16 Haplomitriopsida species and the mycobionts diversity of this lineage is largely unclarified. To clarify the taxonomic composition of mycobionts in Haplomitriopsida, we observed MLSs in the rhizome of Haplomitrium mnioides (Haplomitriopsida), the Asian representative Haplomitriopsida species, and conducted molecular identification of mycobionts. It was recorded for the first time that Glomeromycotina and Mucoromycotina co-occur in Haplomitriopsida as mycobionts. Significantly, the arbuscule-like branching (ALB) of Glomeromycotina was newly described. As the Mucoromycotina fungi forming MLSs in H. mnioides, Endogonaceae and Densosporaceae were detected, in which size differences of hyphal swelling (HS) were found between the fungal families. This study provides a novel evidence in the MLS of Haplomitriopsida, i.e. the existence of Glomeromycotina association as well as the dominant Mucoromycotina association. In addition, since hyphal characteristics of the HS-type MLS were quite similar to those of fine endophytes (FE) of Endogonales in other bryophytes and vascular plants previously described, this MLS is suggested to be included in FE. These results suggest that Glomeromycotina and Mucoromycotina were acquired concurrently as the mycobionts by the earliest land plants evolved into arbuscular mycorrhizae and FE. Therefore, dual association of Haplomitriopsida, with Endogonales and Glomeromycotina will provide us novel insight on how the earliest land plants adapted to terrestrial habitats with fungi.

Repeated fruiting of Japanese golden chanterelle in pot culture with host seedlings.

Yellow chanterelles are among the most popular wild edible ectomycorrhizal mushrooms worldwide. The representative European golden chanterelle, Cantharellus cibarius, has only once been reported to fruit under greenhouse conditions, due to the difficulty of establishing pure culture. Recently, we developed a new technique for establishing a pure culture of a Japanese golden chanterelle (Cantharellus anzutake), and conducted in vitro ectomycorrhizal synthesis using established strains and Pinus densiflora. Acclimated pine mycorrhizal seedlings colonized with C. anzutake in a pot system under laboratory conditions produced small but distinct basidiomata with developed basidiospores. C. anzutake mycorrhizae were established on Quercus serrata seedlings by inoculation of mycorrhizal root tips of the fungus synthesized on P. densiflora. A scaled-up C. anzutake-host system in larger pots (4 L soil volume) exhibited repeated fruiting at 20-24 °C under continuous light illumination at 150 µmol m-2 s-1 during a 2-year incubation period. Therefore, a C. anzutake cultivation trial is practical under controlled environmental conditions.

The (oxalato)aluminate complex as an antimicrobial substance protecting the "shiro" of Tricholoma matsutake from soil micro-organisms.

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, "matsutake" in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called "shiro" in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.

First detection of Endogone ectomycorrhizas in natural oak forests.

The order Endogonales in Mucoromycotina, an early divergent lineage of fungi, includes ectomycorrhizal (EM) fungi. This order is therefore considered a key taxon for elucidation of the evolution of EM associations. Recent studies have revealed high diversity of EM lineages of Basidiomycota and Ascomycota; however, EM associations of Endogonales and its relatives remain largely unknown. In this study, EM root tips with a unique fungal sheath, with aseptate and highly branched hyphae of variable widths, were identified in Quercus acutissima and Quercus crispula forests in the temperate zone of Japan. The mycobionts were confirmed as Endogone sp., which were placed as a sister clade of Endogone pisiformis, based on phylogenetic analyses of the small and large subunits of the nuclear ribosomal RNA and elongation factor-1α genes. This is the first report of EM of Endogone in natural forests of the Northern Hemisphere and the first finding on Quercus.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

Ectomycorrhizas in vitro between Tricholoma matsutake, a basidiomycete that associates with Pinaceae, and Betula platyphylla var. japonica, an early-successional birch species, in cool-temperate forests.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae in the Northern Hemisphere and produces prized "matsutake" mushrooms. We questioned whether the symbiont could associate with a birch that is an early-successional species in boreal, cool-temperate, or subalpine forests. In the present study, we demonstrated that T. matsutake can form typical ectomycorrhizas with Betula platyphylla var. japonica; the associations included a Hartig net and a thin but distinct fungal sheath, as well as the rhizospheric mycelial aggregate "shiro" that is required for fruiting in nature. The in vitro shiro also emitted a characteristic aroma. This is the first report of an ectomycorrhizal formation between T. matsutake and a deciduous broad-leaved tree in the boreal or cool-temperate zones that T. matsutake naturally inhabits.

The host ranges of conifer-associated Tricholoma matsutake, Fagaceae-associated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature.

Tricholoma matsutake is the most commercially important edible mushroom in pine forests in Japan. Tricholoma bakamatsutake and T. fulvocastaneum, species closely related to T. matsutake, occur in Fagaceae forests. We examined ectomycorrhizal (EM) formation by these Tricholoma species by in vitro synthesis among seven strains (two of T. matsutake, four of T. bakamatsutake, one of T. fulvocastaneum) and axenic plants of pine (Pinus densiflora) and oak (Quercus serrata, Q. phillyraeoides). All strains, except for one of T. matsutake, formed EM associations with both pine and oak. Plant growth and mycelial development were differently affected by EM formation depending on the plant-fungus combination.

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa.

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or "shiro". We questioned whether this phenomenon could have occurred because of plant-microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant-fungal symbiosis.

Detection of the edible ectomycorrhizal fungus Lyophyllum shimeji colonising seedlings of cultivated conifer species in New Zealand.

Lyophyllum shimeji is an edible ectomycorrhizal fungus that is widely distributed in East Asia and also present in the northern regions of Europe. In Japan, L. shimeji is a culinary delicacy, considered amongst all edible mushrooms to have the best taste and to be second only to Tricholoma matsutake in price. Traditionally, fruiting bodies of L. shimeji have been collected from the wild but fruiting of L. shimeji is now relatively uncommon and cannot keep up with increasing consumer demand. As a result, methods for its cultivation are being developed for commercial production in Japan and other countries. In this work, techniques were developed to cultivate L. shimeji on coniferous seedlings using a pure culture inoculum. They resulted in successful mycorrhization of Pinus pinaster and Picea abies in only 8 to 10 months. As ectomycorrhizae of L. shimeji are difficult to identify morphologically, mycorrhization was confirmed using an L. shimeji-specific PCR diagnostic, which was designed following a phylogenetic analysis of the Lyophyllum section Difformia using DNA sequences of the internal transcribed spacer (ITS), intergenic spacer (IGS) and elongation factor 1-α (EF1-α) gene. L. shimeji is a member of the Lyophyllum decastes complex in section Difformia, which also includes Lyophyllum fumosum and L. decastes. This analysis confirmed the separation of L. shimeji from closely related Lyophyllum spp. and enabled its unambiguous detection using an IGS-based PCR diagnostic. This is the first report of successful mycorrhization of L. shimeji on P. pinaster and P. abies and provides an opportunity for its commercial cultivation on conifers in New Zealand.

Quantum-classical correspondence and dissipative to dissipationless crossover in magnetotransport phenomena.

The three-dimensional magneto-conductivity tensor was derived in a gauge invariant form based on the Kubo formula considering quantum effects under a magnetic field, such as the Landau quantization and quantum oscillations. We analytically demonstrated that the quantum formula of the magneto-conductivity can be obtained by adding a quantum oscillation factor to the classical formula. This result establishes the quantum-classical correspondence, which has long been missing in magnetotransport phenomena. Moreover, we found dissipative-to-dissipationless crossover in the Hall conductivity by paying special attention to the analytic properties of the thermal Green's function. Finally, by calculating the magnetoresistance of semimetals, we identified a phase shift in quantum oscillation originating from the dissipationless transport predominant at high fields.

Inaoside A: New antioxidant phenolic compound from the edible mushroom Laetiporus cremeiporus.

Laetiporus cremeiporus is the edible mushroom distributed in East Asia. It has been reported that an extract of L. cremeiporus exhibited DPPH radical scavenging activity. The aim of this study is to identify the antioxidant compound from L. cremeiporus. Guided by DPPH radical scavenging activity, a new antioxidant phenolic compound inaoside A (1) and three well-known bioactive compounds 5'-S-methyl-5'-thioadenosine (MTA, 2), nicotinamide (3), and adenosine (4) were isolated from L. cremeiporus. An antioxidant compound was isolated from L. cremeiporus for the first time. This is the first report of the isolation of 1, 2, and 4 from L. cremeiporus. The structures were determined by one- and two-dimensional NMR spectroscopic analysis and chemical derivatization. The antioxidant activities of extracts, fractions, and isolated compounds were evaluated by a DPPH radical scavenging assay. Compound 1 exhibited significant DPPH radical scavenging activity (80 % inhibition at 100 µg/mL, IC50 79.9 µM, trolox equivalent antioxidant capacity (TEAC) = 0.36).

Visualizing the spatial distribution of ustalic acid in the fruiting body of Tricholoma kakishimeji.

Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross sections of the materials at a resolution of 120 µm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-IMS) revealed the localization of UA and its biogenically related metabolites. MALDI-IMS confirmed that UA was predominantly located on the entire surface of the fruiting body and accumulated in higher amounts in younger fruiting bodies than in mature ones. UA is the first toxic secondary metabolite in the genus Tricholoma locally identified using IMS in mushrooms.

In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora.

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar's mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar's mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar's mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar's mushrooms and their acclimatization under non-sterile conditions.

Mobile DNA distributions refine the phylogeny of "matsutake" mushrooms, Tricholoma sect. Caligata.

"Matsutake" mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric "shiro".

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.

Classification of patients who experience a higher distress level to transoral esophagogastroduodenoscopy than to transnasal esophagogastroduodenoscopy.

BACKGROUND: In Japanese routine clinical practice, endoscopy is generally carried out without sedation. The present study aimed to identify the factors essential for appropriate selection of transnasal esophagogastroduodenoscopy (TN-EGD) as an alternative to unsedated transoral esophagogastroduodenoscopy (TO-EGD). PATIENTS AND METHODS: Subjects in this prospective cohort study comprised consecutive outpatients who underwent EGD at a single center. Factors predicting TO-EGD-induced distress were evaluated on a visual analog scale (VAS) and analyzed. Patients were classified into a two-layered system on the basis of these predictive factors, and the severity of distress between the TN-EGD and TO-EGD groups was compared using VAS and the change in the rate-pressure product as subjective and objective indices, respectively. RESULTS: In total, 728 outpatients (390 male, 338 female; mean age, 63.1 ± 0.5 years; TO-EGD group, 630; TN-EGD group, 98)met the inclusion criteria. Multivariate logistic regression analysis confirmed that age <65 years (P < 0.01; odds ratio [OR], 1.69; 95% confidence interval [CI], 1.14-2.52), gender (female; P < 0.01; OR,1.97; 95% CI, 1.34-2.91), marital status (single; P < 0.01; OR, 1.96; 95% CI, 1.18-3.27), and anxiety towards TO-EGD (P < 0.001; OR, 3.62; 95% CI, 2.44-5.37) were independently associated with intolerance. Both indices were significantly higher in the TO-EGD subgroup than in the TN-EGD subgroup in the high predictive class, but not in the low predictive class. CONCLUSION: Predictive factors for detecting intolerance to unsedated TO-EGD may be useful to appropriately select patients who transpose unsedated TO-EGD to TN-EGD.

Near-complete de novo assembly of Tricholoma bakamatsutake chromosomes revealed the structural divergence and differentiation of Tricholoma genomes.

Tricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species in Tricholoma section Caligata. We generated a highly contiguous whole-genome sequence for T. bakamatsutake SF-Tf05 isolated in an Oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average guanine and cytosine (GC) content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison of T. bakamatsutake and Tricholoma matsutake (TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least 3 phylogenetic clades in Tricholoma section Caligata. Specifically, all T. bakamatsutake strains belonged to the "bakamatsutake" clade, which is most proximal to the "caligatum" clade consisting of Tricholoma caligatum and Tricholoma fulvocastaneum. The constructed highly contiguous nearly telomere-to-telomere genome sequence of a T. bakamatsutake isolate will serve as a fundamental resource for future research on the evolution and differentiation of Tricholoma species.

The genome of Lyophyllum shimeji provides insight into the initial evolution of ectomycorrhizal fungal genomes.

Mycorrhizae are one of the most fundamental symbioses between plants and fungi, with ectomycorrhizae being the most widespread in boreal forest ecosystems. Ectomycorrhizal fungi are hypothesized to have evolved convergently from saprotrophic ancestors in several fungal clades, especially members of the subdivision Agaricomycotina. Studies on fungal genomes have identified several typical characteristics of mycorrhizal fungi, such as genome size expansion and decreases in plant cell-wall degrading enzymes (PCWDEs). However, genomic changes concerning the evolutionary transition to the ectomycorrhizal lifestyle are largely unknown. In this study, we sequenced the genome of Lyophyllum shimeji, an ectomycorrhizal fungus that is phylogenetically related to saprotrophic species and retains some saprotroph-like traits. We found that the genome of Ly. shimeji strain AT787 lacks both incremental increases in genome size and reduced numbers of PCWDEs. Our findings suggest that the previously reported common genomic traits of mycorrhizal fungi are not essential for the ectomycorrhizal lifestyle, but are a result of abolishing saprotrophic activity. Since Ly. shimeji is commercially consumed as an edible mushroom, the newly available genomic information may also impact research designed to enhance the cultivation of this mushroom.

Phylogenetic relationship and species delimitation of matsutake and allied species based on multilocus phylogeny and haplotype analyses.

Tricholoma matsutake (S. Ito & S. Imai) Singer and its allied species are referred to as matsutake worldwide and are the most economically important edible mushrooms in Japan. They are widely distributed in the northern hemisphere and established an ectomycorrhizal relationship with conifer and broadleaf trees. To clarify relationships among T. matsutake and its allies, and to delimit phylogenetic species, we analyzed multilocus datasets (ITS, megB1, tef, gpd) with samples that were correctly identified based on morphological characteristics. Phylogenetic analyses clearly identified four major groups: matsutake, T. bakamatsutake, T. fulvocastaneum and T. caligatum; the latter three species were outside the matsutake group. The haplotype analyses and median-joining haplotype network analyses showed that the matsutake group included four closely related but clearly distinct taxa (T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare) from different geographical regions; these were considered to be distinct phylogenetic species.