Mitospore formation on pure cultures of Tuber japonicum (Tuberaceae, Pezizales) in vitro.

The members of the genus Tuber are Ascomycota that form ectomycorrhizal associations with various coniferous and broadleaf tree species. In the teleomorphic stage, the species of the genus produce fruit bodies known as true truffles. Recent studies have discovered mitosporic structures, including spore mats, of several Tuber species on forest soils, indicating the presence of a cryptic anamorphic stage or an unknown reproductive strategy. Here, we report in vitro mitospore formation on the mycelium of T. japonicum, which belongs to the Japonicum clade, collected in several regions in Japan. Twenty of the 25 strains formed mitospores on modified Melin-Norkrans agar medium, indicating that mitospore formation is likely a common trait among strains of T. japonicum. The fungus forms repeatedly branched conidiophores on aerial hyphae on colonies and generates holoblastic mitospores sympodially on the terminal and near apical parts and/or occasionally on the middle and basal parts of the conidiogenous cells. Mitospores are hyaline and elliptical, obovate, oblong, or occasionally bacilliform, with a vacuole and often distinct hilar appendices. Formation of mitospores by T. japonicum in vitro is useful in understanding the functions of mitospores in the genus Tuber under controlled environmental conditions.

In-vitro symbiotic germination of seeds of five mycoheterotrophic Gastrodia orchids with Mycena and Marasmiaceae fungi.

We performed in-vitro germination tests on seeds from five Gastrodia orchids (G. confusa, G. elata var. elata, G. elata var. pallens, G. nipponica, and G. pubilabiata) using one Marasmiaceae and two Mycena isolates. Mycena sp. 1 promoted germination of all five Gastrodia orchids, with root and/or tuber formation observed in G. confusa, G. nipponica, and G. pubilabiata. No additional growth was observed in the other two orchids. Mycena sp. 2 induced G. confusa, G. elata var. elata, and G. nipponica germination, whereas Marasmiaceae sp. 1 induced G. nipponica and G. pubilabiata germination. Phylogenetic analyses indicated that the two Mycena isolates represent distinct lineages within the Mycenaceae. Mycena sp. 1 and Marasmiaceae sp. 1 are closely related to Mycena abramsii and Marasmiellus rhizomorphogenus, respectively. Our results imply that Mycena and marasmioid fungi play important roles in early development in Gastrodia species, and that Mycena fungi in particular may be common mycobionts of Gastrodia species. Root and/or tuber development was observed with four plant-fungus combinations, implying that these associations persist throughout the life cycle, whereas G. elata var. elata may require different associates over time. Our findings will contribute to elucidating the mycorrhizal associations of mycoheterotrophic orchids throughout their life cycle.

Tuber torulosum: A new truffle species decorated with moniliform cystidia from Japan.

We describe a new truffle species, Tuber torulosum, based on molecular and morphological analyses. This species forms a single globose ascospore per ascus, pale yellow in color, as do Japanese T. flavidosporum and Chinese T. turmericum and T. xanthomonosporum in the Japonicum clade of the Tuber phylogeny. However, it can be distinguished from them microscopically by its whitish tomentose mycelium that partially covers the ascoma surface and the mesh size of its spore ornamentation. Cystidia are moniliform and yellowish to reddish. Molecular phylogenetic analysis using the internal transcribed spacer and partial large subunit regions of ribosomal DNA also supports T. torulosum as a distinct species. On the basis of our results, we provide a key to species in the Japonicum clade.

Physiological characteristics of pure cultures of a white-colored truffle Tuber japonicum.

A white-colored truffle Tuber japonicum, indigenous to Japan, is an ascomycetous ectomycorrhizal fungus. To clarify the physiological characteristics of this fungus, we investigated the influence of culture medium, temperature, and sources of nitrogen (N) and carbon (C) on the growth of five strains. Tuber japonicum strains grew better on malt extract and modified Melin-Norkrans medium, and showed peak growth at 20 °C or 25 °C. This fungus utilized inorganic (NH4 + and NO3 -) and organic N sources (casamino acids, glutamine, peptone, urea, and yeast extract). Additionally, this fungus utilized various C sources, such as monosaccharide (arabinose, fructose, galactose, glucose, and mannose), disaccharide (maltose, sucrose, and trehalose), polysaccharide (dextrin and soluble starch), and sugar alcohol (mannitol). However, nutrient sources that promote growth and their effects on growth promotion widely varied among strains. This can result from the strain difference in enzyme activities involved in the assimilation and metabolism of these sources.

Tuber iryudaense and T. tomentosum: Two new truffles encased in tomentose mycelium from Japan.

We describe two new Japanese truffle species, Tuber iryudaense and Tuber tomentosum, based on molecular and morphological analyses. Both species are clearly distinguishable from other Tuber species by the ocher tomentose mycelium covering the ascoma surfaces. Tuber iryudaense has one-spored asci that each contain a large (68-97 × 51-80 µm), reddish-brown ascospore; these microscopic characters are similar to those of closely related Chinese species, T. calosporum, T. gigantosporum, T. glabrum, T. monosporum, and T. sinomonosporum. Tuber tomentosum forms one to four ascospores per ascus with a reddish-brown color similar to that of ascospores of T. macrosporum and T. canaliculatum, although their spores are much larger than those of T. tomentosum (27‒64 × 26‒55 µm). Molecular phylogenetic analyses based on ribosomal internal transcribed spacer and partial 28S nuc rDNA sequences support that both species are distinct within the Macrosporum group.

Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi.

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant-fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

Phylogenetic placements and cultural characteristics of Tuber species isolated from ectomycorrhizas.

Pure cultures of Tuber were isolated from ectomycorrhizal root tips in Abies sachalinensis plantations in Hokkaido, Japan. Their phylogenetic relationships as well as vegetative hyphal characteristics on culture media were reported. Phylogenetic analysis based on the internal transcribed spacer within ribosomal DNA settled well-supported eight lineages within Puberulum, Latisporum, and Maculatum clades in Tuber. Three and one lineages were grouped with undescribed species of Puberulum clade in Japan and that of the Latisporum group in China, respectively. Two lineages were closely associated to but distinct from an undescribed species of Puberulum clade in Japan. One lineage did not group with any sequences in the International Nucleotide Sequence Database (INSD), proposing a new taxon in the Latisporum group. One lineage was grouped with T. foetidum in Maculatum clade. All strains in each lineage displayed yellowish white, thin, filamentous colonies on Melin-Norkrans agar medium. Various differences in morphological characteristics of hyphae on pure cultures of various strains were noted, but they were frequently uncommon among strains of the same taxa. Isolation from ectomycorrhizal root tips can be among the effective ways to acquire pure cultures of Tuber strains.

Component features, odor-active volatiles, and acute oral toxicity of novel white-colored truffle Tuber japonicum native to Japan.

Component analysis of a novel white-colored truffle native to Japan, Tuber japonicum, was performed to determine its characteristic features. The analysis of odor-active volatile compound showed a high contribution of 1-octen-3-ol and 3-methyl-2,4-dithiapentane to the odor of T. japonicum. Although 2,4-dithiapentane is a key odorant of well-known white truffle T. magnatum, 3-methyl-2,4-dithiapentane was detected from the ripe T. japonicum. The chemical components of T. japonicum showed no clear difference with those of edible truffles T. magnatum and T. melanosporum. It was rich in crude protein, crude fiber, and minerals (especially potassium), and low in crude fat. Glutamine and glutamic acid were detected in T. japonicum as free amino acids, while T. magnatum contained a large amount of alanine. Acute oral toxicity tests showed no abnormality, with an LD50 value of over 2000 mg/kg under the test conditions. The present study may support future market distribution of T. japonicum as a high-class foodstuff.

Inside Cover Image, Volume 8, Issue 1.

The cover image is based on the ORIGINAL RESEARCH Component features, odor-active volatiles, and acute oral toxicity of novel white-colored truffle Tuber japonicum native to Japan by Tomoko Shimokawa et al., https://doi.org/10.1002/fsn3.1325. Cover Credit: Cover image ©Tomoko Shimokawa Images.

Idiopathic anterior dislocation of the radial head: symptoms, radiographic findings, and management of 8 patients.

BACKGROUND: Radial head dislocation may occur during trauma or in association with congenital diseases, or it may be developmental or idiopathic. Reports of idiopathic dislocation of the radial head have been scarce. The symptoms, radiographic findings, and management of idiopathic dislocation of the radial head have not been well described in the literature. METHODS: During the past 28 years, we have encountered 8 cases of idiopathic anterior dislocation of the radial head (mean patient age, 12.5 years). In only 1 case did the patient and/or the patient's parents recall any preceding trauma or injury to the affected limb. Patients' complaints included a bulging mass, pain, and limited elbow flexion. Radiographically, the shape of the radial head was flat or slightly convex. Seven of the patients were treated with open reduction of the radial head and angulation osteotomy of the ulna. The other patient's radial head was stabilized without osteotomy. RESULTS: The mean postoperative follow-up period was 4.5 years. In patients whose elbow flexion was limited before surgery, improvement to more than 125° occurred. The bulging mass in the cubital fossa disappeared. None of the patients complained of disability during activities of daily living or sports participation. Radiographically, the radial head remained in the reduced position in all patients in whom open reduction of the radial head with angulation osteotomy of the ulna was performed. CONCLUSIONS: We have described the symptoms, radiographic characteristics, and treatment of idiopathic anterior dislocation of the radial head. Open reduction of the radial head combined with angulation osteotomy of the ulna yielded favorable results both clinically and radiographically.

Irreducible Dislocation of the Radial Head Associated With Pediatric Monteggia Lesions.

BACKGROUND: In pediatric patients with Monteggia lesions, the radial head can be reduced manually when displacement of the fractured ulna is corrected. Occasionally, however, a dislocated radial head could not be reduced manually even when the length and/or angulation of the fractured ulna had been corrected. We can find such cases in the literature, but those are single case reports. We encountered 17 cases of irreducible dislocation of the radial head in pediatric Monteggia lesions during the past 43 years. The purposes of this study were to identify the characteristics of our cases and to discuss the factors that inhibited reduction of the radial head. METHODS: Of 109 children treated for Monteggia lesions between 1972 and 2015, we encountered 17 cases of irreducible dislocation of the radial head. The patients' ages averaged 7.1 years, ranging from 2.6 to 12.1 years. Directions of the radial head dislocation were anterior in five cases, anteromedial in four, lateral in one, and anterolateral in seven. Most of the patients were referred to us from local orthopaedic clinics because of irreducibility of the radial head. We reduced the radial head surgically and confirmed the causes of irreducibility. RESULTS: In 10 of the 17 cases, the problem was identified as pseudoreduction. In those cases, the radial head was reduced in a supination position but redisplaced in a pronation position. Causes of irreducibility were traced to the annular ligament in 15 cases, biceps tendon in 1, and posterior interosseous nerve in 1. CONCLUSIONS: In cases of pediatric Monteggia lesions, we should pay attention to patients in whom the dislocated radial head is not reduced after closed reduction. The most frequent cause of hindered reduction was interposition of the annular ligament in the radiocapitellar joint. Here, the radial head seems to be reduced in the supination position but becomes redisplaced in the pronation position. After closed reduction, it is important to confirm whether the radial head is stable in both pronation and supination positions. LEVEL OF EVIDENCE: Diagnostic level IV.

Ectomycorrhizae formed by three Japanese truffle species (Tuber japonicum, T. longispinosum, and T. himalayense) on indigenous oak and pine species.

Modern truffle cultivation systems started in Europe in the early 1970s and are now successfully used for several European truffles throughout the world. However, systems for indigenous novel truffle species need to be developed in several regions, especially where truffle cultivation has not been attempted so far, such as in Japan. Recently, two new and one known truffle species that are expected to be edible were reported from Japan: Tuber japonicum, T. longispinosum, and T. himalayense. Here, we conducted mycorrhization trials between these three truffle species and four native tree species in Japan (Quercus acutissima, Q. phillyraeoides, Q. serrata, and Pinus densiflora) using spore suspension and trap-plant seedling techniques under axenic pot culture conditions to understand their potential host ranges and mycorrhizal morphologies and to determine whether these inoculation methods are applicable for mycorrhization of Japanese truffles with native host plants. Of the 12 combinations, nine were successful for mycorrhization, including both oak and pine trees. The T. japonicum mycorrhiza was characterized by short, needle-shaped cystidia without septa, whereas the two black truffles, T. longispinosum and T. himalayense, were indistinguishable from each other because they shared the same morphological and anatomical characters such as brownish, long cystidia with right angle ramification. These features were similar to related black truffle species. The results of the present study indicate that the inoculation method used for European truffles can also be applied for mycorrhization between Japanese truffle species and compatible native pine and/or oak hosts in Japan.

Using mating-type loci to improve taxonomy of the Tuber indicum complex, and discovery of a new species, T. longispinosum.

Black truffles that morphologically resemble Tuber indicum have been known to occur in Japan since 1979. Our previous studies showed that there are two phylotypes of these truffles, both of which fell into the T. indicum complex (hereinafter "Tuber sp. 6" and "Tuber sp. 7"). However, their taxonomic treatment is still unclear. In this study, we conducted morphological and phylogenetic analyses for a total of 52 specimens from Japan (16 Tuber sp. 6 and 13 Tuber sp. 7), China (10 T. himalayense and 8 T. indicum), and Taiwan (5 T. formosanum). We compared ascospore ornamentation, size, distribution of asci with average number of spores per ascus, spine size and shape of the Japanese specimens with their allied taxa. For phylogenetic analysis, we sequenced two mating loci (MAT1-1-1 and MAT1-2-1) and three commonly used loci (ITS, ß-tubulin, and TEF1-α). Three distinct lineages were recognized by phylogenetic analyses based on the sequences of the two mating-related loci and three independent loci. The Tuber sp. 6 sequences clustered with those of T. himalayense and T. formosanum, and there was no clear difference in morphology among them. Tuber sp. 7 formed a distinct lineage in each phylogram. The specimens tended to have five-spored asci more frequently than other allied species and could be characterized as having ascospore ornamentation with longer spines and narrower spine bases. We therefore described Tuber sp. 7 as a new species (T. longispinosum), and treat Tuber sp. 6 and T. formosanum as synonyms of T. himalayense.

Fungal partner shifts during the evolution of mycoheterotrophy in Neottia.

PREMISE OF THE STUDY: Few previous studies have examined how mycobionts change during the evolution from autotrophy to mycoheterotrophy based on phylogenetic hypotheses. Neottia (Orchidaceae) comprises leafy species that are autotrophic and related leafless mycoheterotrophic species, and the phylogenetic relationships among them have been clarified. Accordingly, Neottia is a suitable taxon for investigating the question above. Here we clarified the diversity of mycobionts in Neottia plants and elucidated changes in the character of symbiotic associations during the evolution of mycoheterotrophy. METHODS: We sequenced the internal transcribed spacer (ITS) regions of nuclear ribosomal (nr) DNA for mycobionts of Neottia plants. Furthermore, we selected one representative DNA sample from each fungal operational taxonomic unit (OTU) and used it to amplify the large subunit (LSU) nrDNA sequences. Phylogenetic analyses of Sebacinales (basidiomycetes), the dominant mycobiont of Neottia, were conducted and sample-based rarefaction curves generated for the observed mycobiont richness on each OTU. KEY RESULTS: Leafy and leafless species in Neottia were associated with Sebacinales Group B and Sebacinales Group A, respectively. The composition and specificity level of fungal partners varied among Neottia species. CONCLUSIONS: Fungal partner composition and specificity level changed with speciation in both leafy and leafless Neottia species. In particular, mycorrhizal associations likely shifted from Sebacinales Group B to Group A during the evolution from autotrophy to mycoheterotrophy. Partner shifts to Sebacinales Group A have also been reported in the evolution of mycoheterotrophy of other plant groups, suggesting that convergence to this fungal group occurs in association with the evolution of mycoheterotrophy.

How do fungal partners affect the evolution and habitat preferences of mycoheterotrophic plants? A case study in Gastrodia.

PREMISE OF THE STUDY: Since mycoheterotrophic plants (MHPs) completely depend on their mycorrhizal fungi for carbon, selection of fungal partners has an important role in the speciation of MHPs. However, the causes and mechanisms of mycobiont changes during speciation are not clear. We tested fungal partner shifts and changes in mycorrhizal specificity during speciation of three closely related MHPs-Gastrodia confusa (Gc), G. pubilabiata (Gp), and G. nipponica (Gn) (Orchidaceae)-and correlations between these changes and the vegetation types where each species grows. METHODS: We investigated the diversity of mycobionts of the three species by sequencing nrDNA ITS, and the sequence data were subjected to test changes in fungal specificity and fungal partner shifts among the three species. Furthermore, we conducted multivariate analysis to test for differences in mycobiont communities of vegetation types where each species grows. KEY RESULTS: Two saprobic Basidiomycota, Marasmiaceae and Mycenaceae, were dominant fungal partners of the three species, and Gn was simultaneously associated with the ectomycorrhizal Russulaceae and Sebacinaceae. Although mycobiont composition differed among the three species, they also sometimes shared identical fungal species. Multivariate analysis revealed that mycobiont communities of the three species in bamboo thickets differed significantly from those in other vegetation types. CONCLUSIONS: Fungal partner shifts are not necessarily associated with the evolution of MHPs, and fungal specificity of Gc and Gp was significantly higher than that of Gn, implying that the specificity fluctuates during speciation. Further, Gc exclusively inhabits bamboo thickets, which suggests that adaptation to particular fungi specific to bamboo thickets triggered speciation of this species.

Polysomnographic Assessment of Sleep Comorbidities in Drug-Naïve Narcolepsy-Spectrum Disorders--A Japanese Cross-Sectional Study.

This is a large cross-sectional study which aimed to investigate comorbidity rate, degree of sleep-related breathing disorder, polysomnigraphically diagnosible rapid eye movement sleep behavior disorder/rapid eye movement sleep without atonia and periodic limb movements during sleep in Japanese drug-naïve patients with narcolepsy-spectrum disorders. A total of 158 consecutive drug naïve patients with narcolepsy with cataplexy, 295 patients with narcolepsy without cataplexy and 395 patients with idiopathic hypersomnia without long sleep time were enrolled. From retrospectively analyzed data of nocturnal polysomnography and multiple sleep latency test, higher rates of periodic limb movements during sleep (> = 15 h(-1)) (10.2%) and polysomnographically diagnosable rapid eye movement sleep behavior disorder (1.9%) were found in patients with narcolepsy with cataplexy. They had more severe periodic limb movements during sleep especially during rapid eye movement sleep and higher percentages of rapid eye movement sleep without atonia than the other two patient groups. In the present large sample study, Japanese drug naïve patients with narcolepsy with cataplexy showed the highest comorbidity rates of periodic limb movements during sleep, polysomnographically diagnosable rapid eye movement sleep behavior disorder and rapid eye movement sleep without atonia among those with the other narcolepsy-spectrum disorders; the rates were lower than those for Western patients.

Revisiting the host effect on ectomycorrhizal fungal communities: implications from host-fungal associations in relict Pseudotsuga japonica forests.

Host identity is among the most important factors in structuring ectomycorrhizal (ECM) fungal communities. Both host-fungal coevolution and host shifts can account for the observed host effect, but their relative significance in ECM fungal communities is not well understood. To investigate these two host-related mechanisms, we used relict forests of Pseudotsuga japonica, which is an endangered endemic species in Japan. As with other Asian Pseudotsuga species, P. japonica has been isolated from North American Pseudotsuga spp. since the Oligocene and has evolved independently as a warm-temperate species. We collected 100 soil samples from four major localities in which P. japonica was mixed with other conifers and broadleaf trees. ECM tips in the soil samples were subjected to molecular analyses to identify both ECM fungi and host species. While 136 ECM fungal species were identified in total, their communities were significantly different between host groups, confirming the existence of the host effect on ECM fungal communities. None of the 68 ECM fungal species found on P. japonica belonged to Pseudotsuga-specific lineages (e.g., Rhizopogon and Suillus subgroups) that are common in North America. Most of ECM fungi on P. japonica were shared with other host fungi or phylogenetically close to known ECM fungi on other hosts in Asia. These results suggest that after migrating, Pseudotsuga-specific fungal lineages may have become extinct in small isolated populations in Japan. Instead, most of the ECM fungal symbionts on P. japonica likely originated from host shifts in the region.

Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage.

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae--the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (~156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.

Multiple origins of sequestrate basidiomes within Entoloma inferred from molecular phylogenetic analyses.

The genus Entoloma comprises diverse trophic modes and basidiome morphologies. Although Entoloma includes some sequestrate species, their origins are not clearly understood in relation to phylogenetic position and trophic status. In this study, we collected 34 sequestrate Entoloma specimens in Japan over a 9-y period. Their identities and phylogenetic positions were determined by molecular analyses using three nuclear loci [internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA and RNA polymerase II second LSU (rpb2) gene]. Based on species delimitation of 97 % sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified four sequestrate Entoloma species. Molecular phylogenetic analyses that included all related sequences in the International Nucleotide Sequence Database revealed that the four sequestrate Entoloma species belonged to two major phylogroups. One of the phylogroups was Inocephalus-Cyanula, which is composed only of saprotrophic species. Three of the Japanese sequestrate species, as well as three previously known sequestrate species from other regions, fell into at least two independent clades in this phylogroup, indicating multiple origins of sequestrate forms within this saprotrophic lineage. Another phylogroup, Rhodopolioid, was also shown to include a sequestrate species for the first time. Because the Rhodopolioid phylogroup is composed exclusively of mycorrhizal species (ectomycorrhizal and tuberculate mycorrhizal species), the sequestrate form may also have evolved from a mycorrhizal ancestor. Our results suggest that sequestrate basidiomes have evolved multiple times, irrespective of their trophic status in Entoloma. Finally, based on molecular and morphological characteristics, here we describe two new sequestrate Entoloma species, i.e., Entoloma prismaticum Sasaki, Kinoshita et Nara, sp. nov. and Entoloma hypogaeum Sasaki, Kinoshita et Nara, sp. nov.

Phylogeny and diversity of Japanese truffles (Tuber spp.) inferred from sequences of four nuclear loci.

The genus Tuber, which includes some highly valued truffles, comprises ascomycetous ectomycorrhizal fungi associated with ecologically important tree species. Although the genus is distributed over northern temperate regions, we know little about the phylogeny and diversity of Tuber species in Japan. We have collected 186 new Tuber ascoma samples in Japan over a 10 y period. The identities and phylogenies of the samples were analyzed with sequences of four nuclear loci (i.e. internal transcribed spacer [ITS] and large subunit [LSU] regions of rDNA, elongation factor 1 alpha [EF1-α], and RNA polymerase II large subunit [rpb2] genes). Based on the species delimitation of 95% sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified 20 Tuber species. The number of observed species did not reach an asymptote with our maximum sampling localities in a species accumulation curve. The Chao2 species richness estimator indicated that at least 40 Tuber species should be present in Japan. Molecular phylogenetic analyses revealed that Japanese Tuber species belong to five major phylogroups, including Macrosporum, which had not been reported previously in Asia. Two Japanese species were morphologically and phylogenetically distinct from other known phylogroups, and here we propose a new Tuber phylogroup, Japonicum. In addition most of the other Japanese species formed separate clades within individual major phylogroups and deserve to be proposed as new species. Detailed molecular phylogeny within individual phylogroups revealed the existence of phylogeographic structures at both continental and within-Asia scales, indicating that migration and allopatric speciation have occurred even between the mainland and islands in Asia. Although our findings substantially advance current understanding of Tuber diversity and phylogeny, comparable richness estimation and multilocus phylogeny in other geographic regions are necessary to unequivocally address global patterns of Tuber diversity and biogeography.