Otidea subterranea sp. nov.: Otidea goes below ground

Evidence suggests that truffle-like sporocarp forms have evolved many times in the Pezizales, but primarily from epigeous ancestors within ectomycorrhizal clades. There are several ectomycorrhizal clades, however, that contain no known hypogeous species. We collected specimens of an unusual unidentified truffle from mixed oak woodlands in Iowa. Although clearly a member of the Pezizales (Ascomycota), this hypogeous species did not belong to any of the described truffle genera. Based on a combination of ecological, phylogenetic, and morphological evidence we determined that this new truffle is a hypogeous member of the genus Otidea (Pyronemataceae), a lineage with no described truffle species. We describe it here as a new species, Otidea subterranea.     

Report on a New Truffle Species,Tuber koreanum sp. nov., from Korea

The truffle and ectomycorrhizal roots formed by Tuber sp. were collected from the rhizosphere of Quercus aliena in Korea. The morphological characteristics of the ascoma, and molecular phylogenetic analysis using sequences from the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA, translation elongation factor 1-alpha (TEF), and RNA polymerase second largest subunit (RPB2) regions confirmed the distinct morphology of the truffle. This truffle belongs to a monophyletic clade among the other Tuber species in the phylogeny. This study describes the truffle, Tuber koreanum, as a new species reported from Korea.     

Early evolution in the Pinaceae

Nineteen species of structurally preserved ovulate cones of the Pinaceae are known from the Cretaceous. One of these belongs to Pinus, while the others contain anatomical features necessitating their classification in the organ genera Pityostrobus and Pseudoaraucaria. The six species of the latter group are very similar to one another and probably represent a natural, thought extinct, genus. By contrast, there is so much variety in the twelve Cretaceous species of Pityostrobus that when they are considered with respect to the uniformity of cone structure within each of the modern genera, each of the species of this organ genus may well reperesent an natural genus by itself. All expect one of these fossil forms contain features that are today characteristic of Pinus. This, combined with the Early Cretaceous occurrence of a structurally preserved Pinus cone, suggests that Pinus or something very close to it represents the phylogenetic centrum of the ancestral complex. Lack of cones showing distinct affinity with other modern genera supports this idea and further suggests that, while Pinus was in existence in the Early Cretaceous, other Recent genera of the Pinaceae may not have diverged from the complex until the Late Cretaceous or Early Tertiary.     

Discovery of Rhizopogon associated with Larix from northeastern Siberia: Insights into host shift of ectomycorrhizal fungi

Rhizopogon (Boletales) is an ectomycorrhizal fungal genus that exhibits a strong specificity to Pinaceae. This strict association occurs almost exclusively with Pinus and Pseudotsuga, while associations with other genera in Pinaceae are inconclusive. Here, we describe Rhizopogon laricinus sp. nov. associated with Larix cajanderi distributed in northeastern Siberia, where forest fires are frequent. We confirmed the host identity by comparing rDNA internal transcribed spacer (ITS) sequences obtained from basidiomata and ectomycorrhizal root tips collected at the same sites. Morphological characteristics and molecular identification revealed that R. laricinus is a new species associated with Larix unequivocally. The molecular phylogeny based on ITS sequences placed this species sister to the subgenus Roseoli, which is specific to Pinus, and not to the Pseudotsuga-specific subgenus Villosuli. Thus, R. laricinus evolution does not correspond to host phylogeny as Larix and Pseudotsuga form a monophyletic clade. Instead, ecological traits of Rhizopogon for adapting to disturbed habitats may have driven the host shift to Larix under high-frequency fire ecosystems.     

Two new deer truffle species,Elaphomyces marmoratus and Elaphomyces fuscus spp. nov., from a secondary forest in Japan

We describe two new species of deer truffle, Elaphomyces marmoratus and E. fuscus spp. nov., collected from a secondary forest dominated by Quercus serrata, in Tokyo, Japan. Both species have morphological similarities to E. muricatus and E. granulatus; however, the former has a gleba without a purplish color and the latter has reticulated ascospores more closely resembling E. asperulus. Molecular phylogenetic analyses also support the assignment of these specimens as new species. ITS rDNA homologies with known species were low (<91% and <97%). In addition, phylogenetic trees using neighbor-joining and maximum likelihood methods showed that the sequences of the two new species each formed a monophyletic group within section Elaphomyces with bootstrap support of 99%. Analyses of ectomycorrhizal roots collected concurrently with the ascomata revealed that E. marmoratus is associated with at least Pinus densiflora, while E. fuscus is associated with at least Q. serrata.     

Ectomycorrhizae and vascular plants growing in <Emphasis Type="Italic">brûlés</Emphasis> as indicators of below and above ground microecology of black truffle production areas in Navarra (Northern Spain)

The diversity below (ectomycorrhizae) and above (vascular flora) ground in brûlés of black truffle production areas have been studied together for the first time, both in plantations and in natural areas, as possible indicators of the microecology of these zones. Studies on the ectomycorrhizal community of mature plantations are scarce. However, monitoring the dynamics of such systems is important to understand the conditions that promote truffle fructification. In the study described here the most frequent ectomycorrhizae are Tuber melanosporum and Quercirrhiza quadratum. In the plantations, Q. quadratum is the most abundant morphotype and in the natural area it is Cenococcum geophilum. The development of truffle ecosystems involves the appearance of competitor species with wide networks of hyphae and rhizomorphs. On the other hand, there are few studies concerning the special composition of the vascular flora growing in brûlés. We identified 199 taxa, most of them Mediterranean or Eurosiberian xerophiles and therophytes. This is consistent with the ecology of truffle production areas (dry, sunny and stony). These plants are heavily influenced by the inhibiting substances produced by the truffle and, as a result, they suffer from inhibited growth and in some cases cannot complete their life cycle.     

A new Rhizopogon species associated with Pinus amamiana in Japan

Pinus amamiana is an endangered Pinus species found only on Yakushima Island and Tanegashima Island, Japan. We surveyed remaining P. amamiana forests and found some sporocarps of Rhizopogon (Boletales), many species of which exhibit strict host specificity to a narrow range of Pinaceae trees and play critical roles in host establishment. Based on morphological characteristics and molecular phylogeny, here we describe Rhizopogon yakushimensis sp. nov. This new species belongs to a new clade, phylogenetically related to the subgenera Versicolores and Rhizopogon. We also confirmed its ectomycorrhizal association with P. amamiana by comparing rDNA ITS sequences between the sporocarps and ectomycorrhizal root tips.     

New evidence for the symbiosis between Tuber aestivum and Picea abies

The Burgundy truffle (Tuber aestivum Vittad.), an ectomycorrhizal fungus living in association with host plants, is one of the most exclusive delicacies. The symbiosis with deciduous oak, beech, and hazel dominates our concept of truffle ecophysiology, whereas potential conifer hosts have rarely been reported. Here, we present morphological and molecular evidence of a wildlife T. aestivum symbiosis with Norway spruce (Picea abies Karst.) and an independent greenhouse inoculation experiment, to confirm our field observation in southwest Germany. A total of 27 out of 50 P. abies seedlings developed T. aestivum ectomycorrhizae with a mean mycorrhization rate of 19.6 %. These findings not only suggest P. abies to be a productive host species under suitable biogeographic conditions but also emphasize the broad ecological amplitude and great symbiotic range of T. aestivum. While challenging common knowledge, this study demonstrates a significant expansion of the species' cultivation potential to the central European regions, where P. abies forests occur on calcareous soils.     

<Emphasis Type="Italic">Luteoamylascus aculeatus</Emphasis> (Pezizomycetes,Pezizaceae): a new genus and species near the <Emphasis Type="Italic">Pachyphlodes</Emphasis>–<Emphasis Type="Italic">Amylascus</Emphasis> lineage

The new taxon Luteoamylascus aculeatus described in this article is proposed to accommodate two collections of a hypogeous ascomycete from central Spain, characterized by a tomentose yellowish peridium, labyrinth-like gleba filled with whitish hyphae, and intensely reacting amyloid asci. ITS, 28S, and RPB2 data suggest that this new taxon is an independent lineage proposed here as the new genus Luteoamylascus. Until now, this lineage was only known from ectomycorrhizal root tips and mitotic spore mats. In phylogenetic analyses, the Luteoamylascus lineage is placed close to the genera Amylascus, Pachyphlodes, and Scabropezia. Morphological data suggest an affinity with Amylascus.     

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.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

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.     

Phylogenetic relationship,biogeography, and conservation genetics of endangered Fraxinus chiisanensis (Oleaceae), endemic to South Korea

Endemic plants are important for understanding phylogenetic relationships, biogeographical history, and genetic variation because of their restricted distribution and their role in conserving biodiversity. Here, we investigated the phylogenetic relationships of the Korean endemic Fraxinus chiisanensis by reconstructing the molecular phylogeny of Fraxinus based on two nuclear DNA (nrITS and phantastica) and two chloroplast DNA (psbA-trnH and rpl32-trnL) regions. Within our fossil-calibrated phylogenetic framework, we also inferred the biogeographical history of F. chiisanensis. To provide a scientific basis for the conservation of F. chiisanensis, we determined the levels of genetic diversity and genetic differentiation in this species. Combining information from nuclear and chloroplast DNA sequence data, our molecular phylogenetic analyses identified F. chiisanensis as a genetically distinct unit from its sister group, Fraxinus platypoda from Japan. Our molecular dating analyses using nuclear and chloroplast DNA data sets show F. chiisanensis diverged from its sister F. platypoda in the Early or Middle Miocene and differentiated in the Late Miocene on the Korean Peninsula. Our results suggest that the divergence of F. chiisanensis was associated with the submergence of the East China Sea land bridge and enhanced monsoons in East Asia. When compared to F. platypoda, F. chiisanensis exhibits low genetic diversity within populations and high genetic differentiation among populations. These results help us to understand the evolutionary history of F. chiisanensis and to develop a conservation strategy for this species.     

Stable isotope analysis,field observations and synthesis experiments suggest that Odontia is a non-mycorrhizal sister genus of Tomentella and Thelephora

Ectomycorrhizal symbiosis has evolved multiple times in plants and fungi, but the trophic status of certain fungal groups remains poorly understood due to their unculturability or ambiguous interpretation of biotrophic associations. Combining field observations, molecular identification of root tips, synthesis experiments and analysis of stable isotopes, we address the lifestyle of Tomentella crinalis and another species closely related to T. fibrosa that represents a sister group to the ectomycorrhizal genera Tomentella and Thelephora. Based on molecular analyses these two and other related species are moved to the genus Odontia. In Odontia species, ectomycorrhizal associations were not observed in nature or in various synthesis experiments. Although Odontia species normally fruit in old forests, Odontia ferruginea has also been identified from a deep belowground mine. Unlike saprotrophs, Odontia spp. and ectomycorrhizal fungi were not enriched in ¹³C compared with their woody fruiting substratum, suggesting that wood is not their major energy source. In contrast to ectomycorrhizal fungi, Odontia species and saprotrophs were not enriched in ¹⁵N relative to their substratum. Taken together, we suggest that Odontia spp. are non-mycorrhizal, but their nutrition differs from typical wood-rotting Basidiomycota.     

Diversity of ectomycorrhizal Thelephoraceae in <Emphasis Type="Italic">Tuber melanosporum</Emphasis>-cultivated orchards of Northern Spain

Truffles are edible hypogeous ascomycetes highly appreciated worldwide, especially the black truffle (Tuber melanosporum Vittad.). In recent decades, the cultivation of the black truffle has expanded across the Mediterranean climate regions in and outside its native range. Members of the Thelephoraceae (Thelephorales, Agaricomycetes, Basidiomycota) are commonly found in truffle plantations, but their co-occurrence with Tuber species and other members of the fungal community has been scarcely reported. Thelephoraceae is one of the most represented families of the ectomycorrhizal fungal community in boreal and Mediterranean forests. To reveal the diversity of these fungi in T. melanosporum-cultivated plantations, ten orchards located in the Navarra region (Northern Spain) were surveyed for 2 years. Morphological and molecular approaches were used to detect and identify the Thelephoraceae ectomycorrhizas present in those plantations. Ten different mycorrhizal types were detected and described. Four of them were morphologically identified as Tomentella galzinii, Quercirhiza cumulosa, Q. squamosa, and T39 Thelephoraceae type. Molecular analyses revealed 4–6 operational taxonomic units (OTUs), depending on the nucleotide database used, but similarities remained under 95 % and no clear species assignments could be done. The results confirm the diversity and abundance of this fungal family in the ectomycorrhizal community of black truffle plantations, generally established in Mediterranean areas. The occurrence and relative abundance of Thelephoraceae ectomycorrhizas is discussed in relation to their possible influence on truffle production.     

Mycorrhizal detection of native and non-native truffles in a historic arboretum and the discovery of a new North American species, <Emphasis Type="Italic">Tuber arnoldianum</Emphasis> sp. nov.

During a study comparing the ectomycorrhizal root communities in a native forest with those at the Arnold Arboretum in Massachusetts (USA), the European species Tuber borchii was detected on the roots of a native red oak in the arboretum over two successive years. Since T. borchii is an economically important edible truffle native to Europe, we conducted a search of other roots in the arboretum to determine the extent of colonization. We also wanted to determine whether other non-native Tuber species had been inadvertently introduced into this 140-year-old Arboretum because many trees were imported into the site with intact soil and roots prior to the 1921 USDA ban on these horticultural practices in the USA. While T. borchii was not found on other trees, seven other native and exotic Tuber species were detected. Among the North American Tuber species detected from ectomycorrhizae, we also collected ascomata of a previously unknown species described here as Tuber arnoldianum. This new species was found colonizing both native and non-native tree roots. Other ectomycorrhizal taxa that were detected included basidiomycetes in the genera Amanita, Russula, Tomentella, and ascomycetes belonging to Pachyphlodes, Helvella, Genea, and Trichophaea. We clarify the phylogenetic relationships of each of the Tuber species detected in this study, and we discuss their distribution on both native and non-native host trees.     

Soil spore banks of ectomycorrhizal fungi in endangered Japanese Douglas-fir forests

Interactions between trees and ectomycorrhizal fungi are critical to the growth and survival of both partners. However, ectomycorrhizal symbiosis has barely been explored in endangered trees, and no information is available regarding soil spore banks of ectomycorrhizal fungi from forests of threatened trees. Here, we evaluated soil spore banks of ectomycorrhizal fungi from endangered Japanese Douglas-fir (Pseudotsuga japonica) forests using bioassay approaches with congeneric P. menziesii and Pinus densiflora seedlings in combination with molecular identification techniques. Rhizopogon togasawariana was predominant in soil propagule banks and was found in all remaining P. japonica forests when assayed with P. menziesii, while no colonization of this fungus was observed on Pinus seedlings. Given the observed specificity of R. togasawariana for P. menziesii and its phylogenetic position within the Pseudotsuga-specific Rhizopogon lineage, its geographical distribution is likely restricted to the remaining Japanese Douglas-fir forests, indicating a high extinction risk for this fungus as well as its endangered host. Spore banks of R. togasawariana remained highly infective after preservation for 1 year or heat treatment at 70 °C, suggesting an ecological strategy of establishing ectomycorrhizal associations on regenerating Japanese Douglas-fir seedlings after disturbance, as observed in other Rhizopogon–Pinaceae combinations. Therefore, the regeneration of Japanese Douglas-fir seedlings may depend largely on the soil spore banks dominated by R. togasawariana, which has co-evolved with the Japanese Douglas-fir for over 30 million years. More attention must be paid to underground ectomycorrhizal fungi for the conservation of endangered tree species, especially in the era of human-induced mass extinction.     

Truffle Br?lés Have an Impact on the Diversity of Soil Bacterial Communities

Background

The development of Tuber melanosporum mycorrhizal symbiosis is associated with the production of an area devoid of vegetation (commonly referred to by the French word ‘brûlé’) around the symbiotic plants and where the fruiting bodies of T. melanosporum are usually collected. The extent of the ecological impact of such an area is still being discovered. While the relationship between T. melanosporum and the other fungi present in the brûlé has been assessed, no data are available on the relationship between this fungus and the bacteria inhabiting the brûlé.

Methodology/Principal Findings

We used DGGE and DNA microarrays of 16S rRNA gene fragments to compare the bacterial and archaeal communities inside and outside of truffle brûlés. Soil samples were collected in 2008 from four productive T. melanosporum/Quercus pubescens truffle-grounds located in Cahors, France, showing characteristic truffle brûlé. All the samples were analyzed by DGGE and one truffle-ground was analyzed also using phylogenetic microarrays. DGGE profiles showed differences in the bacterial community composition, and the microarrays revealed a few differences in relative richness between the brûlé interior and exterior zones, as well as differences in the relative abundance of several taxa.

Conclusions/Significance

The different signal intensities we have measured for members of bacteria and archaea inside versus outside the brûlé are the first demonstration, to our knowledge, that not only fungal communities, but also other microorganisms are affected by T. melanosporum. Firmicutes (e.g., Bacillus), several genera of Actinobacteria, and a few Cyanobacteria had greater representation inside the brûlé compared with outside, whereas Pseudomonas and several genera within the class Flavobacteriaceae had higher relative abundances outside the brûlé. The findings from this study may contribute to future searches for microbial bio-indicators of brûlés.