Diversity and habitat relationships of hypogeous fungi. I. Study design, sampling techniques and general survey results

Hypogeous fungi are a large yet unknown component of biodiversity in forests of south-eastern mainland Australia. To better define their diversity and habitat relationships, we identified and counted fruit-bodies at 136 study sites sampling the climatic, geological and topographic features of the region. In one year 7451 fruit-bodies representing 209 species were collected in an autumn and spring sampling period. Only 57 of these species were previously described. Within genera, the number of species ranged from 1 to 21. Sites sampled in autumn averaged higher diversity of species and greater number of fruit-bodies than the same sites sampled in spring. Most major taxa occurred at more sites in autumn than in spring, whereas a few occurred more frequently in spring than in autumn. These patterns are consistent with those identified in previous smaller studies and likely reflect seasonal changes in soil moisture and temperature levels. Subsequent papers will explore factors influencing the occurrence, relative abundance and numbers of species of hypogeous fungi at the study sites and their community structure and possible host–plant relationships.     

Exotic Mammals Disperse Exotic Fungi That Promote Invasion by Exotic Trees

Biological invasions are often complex phenomena because many factors influence their outcome. One key aspect is how non-natives interact with the local biota. Interaction with local species may be especially important for exotic species that require an obligatory mutualist, such as Pinaceae species that need ectomycorrhizal (EM) fungi. EM fungi and seeds of Pinaceae disperse independently, so they may use different vectors. We studied the role of exotic mammals as dispersal agents of EM fungi on Isla Victoria, Argentina, where many Pinaceae species have been introduced. Only a few of these tree species have become invasive, and they are found in high densities only near plantations, partly because these Pinaceae trees lack proper EM fungi when their seeds land far from plantations. Native mammals (a dwarf deer and rodents) are rare around plantations and do not appear to play a role in these invasions. With greenhouse experiments using animal feces as inoculum, plus observational and molecular studies, we found that wild boar and deer, both non-native, are dispersing EM fungi. Approximately 30% of the Pinaceae seedlings growing with feces of wild boar and 15% of the seedlings growing with deer feces were colonized by non-native EM fungi. Seedlings growing in control pots were not colonized by EM fungi. We found a low diversity of fungi colonizing the seedlings, with the hypogeous Rhizopogon as the most abundant genus. Wild boar, a recent introduction to the island, appear to be the main animal dispersing the fungi and may be playing a key role in facilitating the invasion of pine trees and even triggering their spread. These results show that interactions among non-natives help explain pine invasions in our study area.     

Hypogeous sequestrate fungi in South America – how well do we know them?

Collecting and studying hypogeous sequestrate fungi and their particular fruiting biology has always been challenging and intriguing for scientists. However, knowledge of hypogeous taxa has for a long time been limited mainly to the Northern Hemisphere, and more recently, Australia. Nevertheless, cumulative information on sequestrate fungi for South America (SA) has increased considerably over the years, and constitutes by itself, the aim of this review. We have reviewed the available published literature, from 1880 until recent times, to extract information on records, ecology, and morphological characteristics of hypogeous sequestrate fungi from SA. Based on the 172 taxa cited in the available literature, a trend of increasing interest in the study of these fungi in the region is apparent, yet with an uneven distribution among countries, climate belts, and nature of forest habitats. Hypogeous truffle-like species in SA play a key role in regulating nutrient and carbon cycles and in all ecosystem multifunctionality. The symbiotic status is provided for most species listed, and mutualism, especially ectomycorrhizal, is predominant (82 %). The hypogeous sequestrate fungi in SA are an understudied group of fungi, with exceptional anatomical and biological features as well as in many cases intriguing phylogenetic relationships, requiring more attention and analysis from mycologists.     

Differential hypogeous sporocarp production from Nothofagus dombeyi and N. pumilio forests in southern Argentina

Mycorrhizal fungi that form hypogeous sporocarps are an important component of the temperate forest soil community. In many regions, such as the Nothofagus forest in the Patagonian Andes, this group of fungi has been poorly studied. Here we examined the spring and autumn community composition of "sequestrate fungi", based on sporocarp production in pure forests of Nothofagus dombeyi (evergreen) and N. pumilio (deciduous). We investigated the possible relationships between these communities and environmental factors over 2 y. The rarefaction curves and the minimal richness estimates converged at nearly the same level for each forest type, and the asymptotes suggested that the sampling effort was sufficient to capture most of the hypogeous sporocarp richness in these forest stands. In total 27 species were recovered. Basidiomycota, Ascomycota and Glomeromycota respectively accounted for nine, two and one genera. Species richness of hypogeous sporocarps varied in relation to forest type but not to season (fall and spring), whereas sporocarp biomass varied according to an interaction between season and forest type. Species richness and sporocarp biomass were positively correlated with rainfall and negatively correlated with altitude. In addition sporocarp species richness was positively related to number of trees per transect. We found that two different forest stands, each dominated by different species of Nothofagus, exhibited different hypogeous sporocarp communities.     

Interrelationships among some ectomycorrhizal trees,hypogeous fungi and small mammals: Western Australian and northwestern American parallels

Species of the hypogeous fungus genus. Mesophellia have been shown to form ectomycorrhiza with Eucalyptus diversicolor and improve seedling growth. Since the morphology of these fungi increases their dependence on animal mycophagy for spore dispersal, these results imply a tripartite interrelationship between eucalypts, hypogeous fungi and marsupials which parallels the North American interrelationship between ectomycorrhizal pine, hypogeous fungi and rodents. An understanding of the independent but analogous interrelationships on the two continents has implications for forest management, especially in regard to the effects of forest conversion on other members of the ecosystem.     

Allocasuarina tree hosts determine the spatial distribution of hypogeous fungal sporocarps in three tropical Australian sclerophyll forests

Across three tropical Australian sclerophyll forest types, site-specific environmental variables could explain the distribution of both quantity (abundance and biomass) and richness (genus and species) of hypogeous fungi sporocarps. Quantity was significantly higher in the Allocasuarina forest sites that had high soil nitrogen but low phosphorous. Three genera of hypogeous fungi were found exclusively in Allocasuarina forest sites including Gummiglobus, Labyrinthomyces and Octaviania, as were some species of Castoreum, Chondrogaster, Endogone, Hysterangium and Russula. However, the forest types did not all group according to site-scale variables and subsequently the taxonomic assemblages were not significantly different between the three forest types. At site scale, significant negative relationships were found between phosphorous concentration and the quantity of hypogeous fungi sporocarps. Using a multivariate information theoretic approach, there were other more plausible models to explain the patterns of sporocarp richness. Both the mean number of fungal genera and species increased with the number of Allocasuarina stems, at the same time decreasing with the number of Eucalyptus stems. The optimal conditions for promoting hypogeous fungi sporocarp quantity and sporocarp richness appear to be related to the presence and abundance of Allocasuarina (Casuarinaceae) host trees. Allocasuarina tree species may have a higher host receptivity for ectomycorrhizal hypogeous fungi species that provide an important food resource for Australian mycophagous animals.     

Interactions between mammals and ectomycorrhizal fungi

Many ectomycorrhizal (ECM) fungi produce fruit-bodies below ground and rely on animals, especially mammals, for dispersal of spores. Mammals may therefore play an important role in the maintenance of mycorrhizal symbiosis and biodiversity of ECM fungi in many forest ecosystems. Given the pivotal role played by mycorrhizal fungi In the nutrition of their plant hosts and, possibly, in the determination of plant community structure, the ecological significance of mycophagous mammals may extend to the productivity and diversity of plant communities. Mycologists and mammalogists have been aware of the interaction between their study organisms for many years, but recent research has produced new insights Into the evolution of mammal-vectored spore dispersal among ECM fungi, the ecological importance of mycophagy to small mammals, and the effectiveness of mammals as spore-dispersal agents.     

How many fungi make sclerotia?

Most fungi produce some type of durable microscopic structure such as a spore that is important for dispersal and/or survival under adverse conditions, but many species also produce dense aggregations of tissue called sclerotia. These structures help fungi to survive challenging conditions such as freezing, desiccation, microbial attack, or the absence of a host. During studies of hypogeous fungi we encountered morphologically distinct sclerotia in nature that were not linked with a known fungus. These observations suggested that many unrelated fungi with diverse trophic modes may form sclerotia, but that these structures have been overlooked. To identify the phylogenetic affiliations and trophic modes of sclerotium-forming fungi, we conducted a literature review and sequenced DNA from fresh sclerotium collections. We found that sclerotium-forming fungi are ecologically diverse and phylogenetically dispersed among 85 genera in 20 orders of Dikarya, suggesting that the ability to form sclerotia probably evolved ≥14 different times in fungi.     

Does secotioid inertia drive the evolution of false-truffles?

Secotioid inertia is a model implemented to explain the prevalence of highly derived false-truffles with no obvious connection to the Homobasidiomycetes. The model accommodates the apparent lack of epigeous sister taxa for some highly derived hypogeous lineages by assuming that gasteromycetation in some fungi leads to the extinction of their epigeous sister population. The derived state of some hypogeous lineages suggests that they arose early in the evolution of Homobasidiomycetes and that those groups were subject to conditions that favoured hypogeous lineages such that the hypogeous fruit body form became the predominant form for some lineages. The directional selection component of secotioid inertia, termed secotioid drive, led to the extinction of their epigeous sister taxon. Morphological and molecular data from Russulaceae are used to model the evolutionary stages of secotioid inertia. The resulting phylogenetic results are compared with data from the order Leucogastrales, and the genus Destuntzia. The implications of secotioid drive are discussed with reference to gasteromycete phylogenetics, evolution, and conservation. Specifically, secotioid inertia can be used to account for reversals in fruit body morphology and instability in mycorrhizal formation.     

Role of fungi in marine ecosystems

Marine fungi are an ecological rather than a taxonomic group and comprise an estimated 1500 species, excluding those that form lichens. They occur in most marine habitats and generally have a pantropical or pantemperate distribution. Marine fungi are major decomposers of woody and herbaceous substrates in marine ecosystems. Their importance lies in their ability to aggressively degrade lignocellulose. They may be important in the degradation of dead animals and animal parts. Marine fungi are important pathogens of plants and animals and also form symbiotic relationships with other organisms. The effect of disturbances on marine fungi is poorly investigated. Keystone marine species may exist, especially in mutualistic symbioses. However, as many saprophytes appear to carry out the same function simultaneously, they may be functionally redundant. The need for a concerted effort to investigate the biodiversity and role of marine fungi globally and on as many substrata as possible is presented.     

The interrelationship of mycophagous small mammals and ectomycorrhizal fungi in primeval, disturbed and managed Central European mountainous forests

Small forest dwelling mammals are considered to be major consumers and vectors of hypogeous ectomycorrhizal (ECM) fungi, which have lost the ability of active spore discharge. Fungal spore dispersal by mycophagy is deemed an important process involved in forest regeneration, resilience and vitality, primarily based on evidence from Australia and the Pacific Northwestern USA, but is poorly known for Central European mountainous forests thus far. Small mammal mycophagy was investigated by live trapping and microscopical analysis of faecal samples. All small mammal species recorded (Myodes glareolus, Microtus agrestis, Pitymys subterraneus, Apodemus spp., Glis glis, Sorex spp.) had ingested spores of ECM fungi, albeit in varying amounts. My. glareolus was found to be the most important vector of ECM fungal spores, both in quantity and diversity. Species of the genus Sorex seem to play a hitherto underestimated role as dispersers of fungal spores. Glis glis is likely to be an important vector owing to its large home range. Hypogeous ECM basidiomycetes accounted for most spores found in the faecal samples. The frequency of various genera of hypogeous ECM ascomycetes and ECM epigeous fungi was much lower. Comparison with null models indicated a non-random structure of the mycophagy network similar to other mutualistic bipartite networks. Mycophagy can be considered (1) to contribute to nutrition of small forest mammals, (2) to play a pivotal role for forest regeneration and functioning by providing mycorrhizal inoculum to tree seedlings and (3) to be vital for reproduction and diversity of the still poorly known hypogeous fungi.     

Interactions between fire,mycophagous mammals,and dispersal of ectromycorrhizal fungi in Eucalyptus forests

Several species of marsupials in Eucalyptus forests in Australia feed predominantly on the sporocarps of hypogeous fungi. This feeding is apparently beneficial to the fungi as it results in dispersal of spores. As these fungi are in almost all cases ectomycorrhiza-forming species, mycophagy by mammals may play an important role in the maintenance of mycorrhizal symbiosis in Eucalyptus forests. Fire is frequent and a dominant ecological factor in these forests, and this study tested the hypothesis that fire triggers both increased sporocarp production by some hypogeous ectomycorrhizal fungi associated with eucalypts, and increased mycophagy by mammals. Three experimental burns were set in E. tenuiramus forest in southeastern Tasmania. Digging activity (which reflects feeding on hypogeous fungi) by a mycophagous marsupial, the Tasmanian bettong Bettongia gaimardi, increased up to ten-fold after fire, with a peak about 1 month post-fire. This was associated with a similar pattern of increase in sporocarp production, which was due to species in the family Mesophelliaceae (especially Castoreum tasmanicum and Mesophellia spp.). This family appears to have radiated in association with eucalypts and has an exclusively Australasian distribution, unlike many of the other ectomycorrhizal fungi collected in this study which are cosmopolitan and have broad host ranges. No B. gaimardi were killed by fire, and there was no increase in mortality following fire. Population density increased after fire as a result of immigration of adult males. However, body condition and fecundity of individual B. gaimardi were maintained at pre-fire levels. This suggests that the availability of energy to B. gaimardi increased as a result of fire, and the fact that the contribution of fungus to the diet of B. gaimardi was high on burnt relative to control sites suggests further that this increase in energy availability was provided by hypogeous fungi. Effects of fire on hypogeous fungi and B. gaimardi were short-lived; all measured variables returned to control values about 4 months after fire. The capacity of B. gaimardi to survive fire and to harvest the increased sporocarp production triggered by fire provides a mechanism for the rapid dispersal of spores after fire. This should result in the establishment of ectomycorrhizae very early in post-fire succession. Because only some species of ectomycorrhizal fungi fruited in response to burning, fire probably has a strong influence on community structure among ectomycorrhizal fungi.     

Diversity and habitat relationships of hypogeous fungi. III. Factors influencing the occurrence of fire-adapted species

Among the huge array of hypogeous ectomycorrhizal fungi so far documented from Australia, six genera and more than 30 species occur within the family Mesophelliaceae, all of which show various adaptations for surviving in fire-prone landscapes. These mostly endemic fungi are critical to postfire reestablishment of regenerating vegetation, and their fruit-bodies provide essential food resources for diverse ground-dwelling fauna. We developed habitat models for five common representatives of the Mesophelliaceae based on repeat collections of their fruit-bodies from 136 study plots situated along a series of environmental gradients across the south-eastern mainland of Australia. At a meso- or landscape scale, temperature influenced the occurrence of Castoreum radicatum, Mesophellia clelandii and Nothocastoreum cretaceum, with the type of response varying. Below a threshold, C. radicatum preferred sites with cooler mean annual temperatures. In contrast, M. clelandii and N. cretaceum had optimal ranges of temperature, above and below which the probability of detecting them dropped. Also at a landscape scale, C. radicatum was more likely to be detected at sites with lower levels of precipitation during the driest quarter of the year. At a micro-site scale, M. clelandii and N. cretaceum were more likely to occur in stands with an intermediate number of host eucalypt stems, likely relating to successional age of the stand. Sites with a higher number of large fallen trees were more likely to have N. cretaceum, while sites with intermediate litter depths were more likely to have C. radicatum and M. clelandii. Mesophellia glauca and M. trabalis showed no consistent patterns. They are apparently the most broadly adaptable in terms of the independent variables tested. Although fire has been previously suggested to be heavily implicated in the life cycle of several members of the Mesophelliaceae, we found no relationship between time since disturbance by fire and other factors and likelihood of occurrence. Instead, other habitat attributes appeared to be more important in explaining their distribution. The complex and differing responses of the species of Mesophelliaceae studied here, to features of their environment, reinforce the need to manage multiple-use forest landscapes across the region for a diversity of attributes.     

Fungus diversity in revegetated paddocks compared with remnant woodland in a south‐eastern Australian agricultural landscape

Summary Despite the importance of fungi for restoration, their presence in revegetated sites has received little attention. We compared the diversity and composition of macrofungi (i.e. those that form fleshy mushrooms and truffles) in 12 sites where 3‐to‐6‐year‐old native trees and shrubs had been planted (woodland restoration sites), with that in six woodland remnants. All sites were within an agricultural landscape near Holbrook in New South Wales. Of 58 fungal genera recorded, 57% occurred in woodland restoration sites and 83% in nearby patches of remnant woodland. Of the genera found in restoration sites, 70% were also found in the woodland remnants. The dominance of early successional genera such as Lacceria and Scleroderma in restoration sites suggests windblown colonisation by fungi. The reduced proportion of hypogeous genera (truffles) that rely on mammal vectors, which are less likely to occur in the restoration sites, also supports the view that most fungi occurred in restoration through colonisation rather than being generated from soil spores. Greatest overall fungal diversity occurred in large remnants that had greater structural complexity. Across all sites, epigeous genera (mushrooms) were most common (78% of all taxa collected across 46 genera) and of the nutritional modes, mycorrhizal genera (forming symbiotic associations with plants) were the most common (206 collections, 71%, 25 genera). Both hypogeous and mycorrhizal fungi were positively associated with the diversity of native forb species (wildflowers), suggesting that lower fungal diversity in restoration sites is likely to be a consequence of long‐term agricultural practices, particularly fertilizer use.     

Molecular phylogeny of the mycorrhizal desert truffles (Terfezia and Tirmania), host specificity and edaphic tolerance

Terfezia and Tirmania, so called desert truffles, are mycorrhizal fungi mostly endemic to arid and semi-arid areas of the Mediterranean Region, where they are associated with Helianthemum species. The aim of this work was to study the phylogenetic relationships in these pezizalean hypogeous fungi. The restriction fragment length polymorphism (RFLP) and DNA sequences of internal transcribed spacers (ITS) of the nuclear rDNA were studied for several morphological species, Terfezia arenaria, T. boudieri, T. claveryi, T. leptoderma, T. terfezioides (=Mattirolomyces terfezioides), Tirmania nivea and T. pinoyi. The sequences were analyzed with distance and parsimony methods. Phylogenetic analyses indicated a close genetic relationship between Tirmania and Terfezia. They may have arisen from a single evolutionary lineage of pezizalean fungi that developed the hypogeous habit as an adaptation to heat and drought in Mediterranean ecosystems. This analysis also supports the re-establishment of the genus Mattirolomyces. The genera Tirmania and Terfezia were monophyletic, and morphological species corresponded to phylogenetic species. The Tirmania clade comprises desert truffles with smooth spores and amyloid asci, which were found in deserts. The Terfezia clade grouped species found in semi-arid habitats having ornamented and spherical spores. These species are adapted to exploit different types of soil (either acid or basic soils) in association with specific hosts (either basophilous or acidophilous species). Although other factors might also play a role, host specialization and edaphic tolerances (fungus and/or host tolerances) might be the key in the species diversity of these genera.     

Diets of native and introduced tree squirrels in Washington

Competition for food resources can be a primary mechanism for displacement of native species by introduced species. Investigation of dietary partitioning between potential competitors and spatiotemporal variability in food resources can reveal contested food items and intensity of competition. Introduced eastern gray squirrels (Sciurus carolinensis) have been implicated as competitors with western gray squirrels (S. griseus) and Douglas’ squirrels (Tamiasciurus douglasii), but little is known about interactions among these species. We collected foraging observations and fecal pellets of sympatric gray and Douglas’ squirrels trapped and radio-tracked in western Washington, USA, from 2007 to 2012. We compared food resources consumed across species, seasons, and years to evaluate dietary overlap and the potential for competitive interactions. All squirrel species ate hypogeous fungi throughout all seasons; spores of several genera were present in 272 of 275 fecal samples and observed in all months. Rhizopogon, Geopora, and Melanogaster spp. occurred in most fecal pellets for all squirrels, but the diet of the Douglas’ squirrels had slightly lower richness of hypogeous fungi than gray squirrels. Although foraging observations suggest that eastern and western gray squirrels shared important dietary items such as acorns, strong differences in consumption of seeds of a common conifer may facilitate their coexistence. Our results suggest that dietary overlap among arboreal squirrels could lead to competitive interactions during periods of food scarcity, but subtle differences may be sufficient to permit long-term coexistence. Tree squirrels in Washington would likely benefit from forest management practices that promote or sustain robust crops of hypogeous fungi. © 2019 The Wildlife Society.     

Mycophagy by invasive wild boar (Sus scrofa) facilitates dispersal of native and introduced mycorrhizal fungi in Patagonia,Argentina

Fleshy hypogeous fungi produce scents that enable mycophagous mammals and invertebrates to locate them and disperse their spores. The European wild boar (Sus scrofa) was introduced in central Argentina in 1900s and later expanded into Patagonia. Here, we determined the diversity and abundance of fungal taxa, and the frequency of hypogeous fungal spores in wild boar feces in Patagonia. We collected fecal samples on Isla Victoria, Nahuel Huapi National Park, and identified fungi using microscope and DNA metabarcoding of ITS2 rDNA. Hypogeous fungal spores occurred in almost all fecal samples. The most abundant species belonged to the genera Hysterangium, Melanogaster, Radiigera and Gautieria. In addition to the symbiotrophic hypogeous taxa, we also identified numerous pathotrophic and saprotrophic taxa. Not only diverse native hypogeous fungi, but also introduced ones are part of the diet of the wild boar in forests of Patagonia. If viable, introduced fungi are being dispersed as far as 2.5 km from the nearest plantation, highlighting how the introduced wild boar might alter the local distribution and composition of fungal communities.     

Nitrogen content, amino acid composition and digestibility of fungi from a nutritional perspective in animal mycophagy

Fungi comprise a major part of the diet of many animals. Even so, the nutritional value of fungi has been much debated, with some arguing that fungi are nutritionally poor. However, the chemical composition of fungi and of the biology of the animals that eat them are not well understood, particularly in reference to amino acid (AA) composition of fungi and digestibility of fungal protein. We analysed fibre, total nitrogen (N), available N, and AA contents and measured in vitro digestibility of a wide range of epigeous and hypogeous fungi collected in Australia and the USA to test three hypotheses: (i) fungi are nutritionally poor because they contain few nutrients or are otherwise of low digestibility, (ii) fungi vary substantially in their nutritional composition; and (iii) animals can counter this variable quality by eating diverse taxa. Resultant data indicate many fungi are a reasonable source of AAs and digestible nitrogen. However, they vary highly between species in AA content, and the protein has a poor balance of digestible AAs. This helps explain why many mycophagous animals eat a wide array of fungi and often have digestive strategies to cope with fungi, such as foregut fermentation. Another common strategy is to supplement the diet with high quality protein, such as insect protein. Accordingly, evaluating nutritional value of fungi requires consideration of physiology of the animal species and their whole diet.     

What determines species composition and diversity of hypogeous fungi in the diet of small mammals? A comparison across mammal species,habitat types and seasons in Central European mountains

Interactions between diverse groups of organisms influence the functioning and diversity of ecosystems. Salient examples of such relationships are those among hypogeous fungi, trees and mycophagous mammals. To investigate the role of small mammals in transporting fungal spores within and outside forests as well as the influence of seasons, habitats and species on small mammal mycophagy, we set up a study in the Pieniny Mts, Western Carpathians (Southern Poland). The droppings of small mammals were collected during live trapping in July and September 2016 and 2017, to analyze richness, composition and frequency of fungal spores present in faeces. The yellow-necked mouse Apodemus flavicollis, the bank vole Myodes glareolus and the common vole Microtus arvalis were the most frequently trapped. Spores of 27 fungal taxa from 16 genera were retrieved from nearly 70% of faecal samples of rodents and shrews, with up to 9 spore taxa recorded per sample. Spore diversity in samples was higher in September than in July, although seasonal variation was year and animal dependent. The highest mean number of fungal taxa per sample was recorded for the bank vole and the yellow-necked mouse, with the former species showing a higher degree of mycophagy. The two rodents differed in the average frequencies of consumed fungi in samples, which could result from some degree of specialization in the choice of particular fungal species, as shown by the laboratory-based experiment. Within particular animal species, differences in the fungal diet were found between seasons. The spores of hypogeous fungi were transported from forests to meadows mostly by the yellow-necked mouse and, to a lesser extent, by the common vole. However, both, the diversity and the number of transported spores diminished with distance from the forest edge.     

Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis.

Extensive morphological convergence or divergence, a common occurrence in fungi, tends to obscure recognition of phylogenetic relationships among Pezizales, widespread filamentous Ascomycetes with either enclosed underground (hypogeous) or exposed (epigeous) fruit bodies, that often establish mutualistic interactions with arboreous plants. Focusing on hypogeous Pezizales commonly known as truffles, we sequenced the 18S rDNA from nine species belonging to three different families (Tuberaceae, Terfeziaceae, and Balsamiaceae). A data set consisting of 1700 secondary structure-aligned sites, including 24 homologous sequences from the GenBank DNA database and using three reconstruction methods, was employed to infer phylogenies in an interval ranging from the subordinal to the subgeneric level. As revealed by the 18S phylogenetic scheme, Balsamiaceae represent a monophyletic clade, comprising the hypogeous taxa Balsamia and Barssia, nested within Helvellaceae. Similarly, the terfeziacean genera Pachyphloeus and Terfezia constitute together with Cazia a distinct hypogeous clade nested within Pezizaceae. The lack of clustering between Terfezia arenaria and Terfezia terfezioides strongly supports the reassignment of the latter taxon to the original monotypic genus Mattirolomyces. Within Tuberaceae, which are sister to the highly evolved Helvellaceae, the genus Tuber cannot be considered monophyletic if Choiromyces is recognized. The paraphyly of Tuber and other relationships that were not supported by high bootstrap values, nor corroborated by morphological evidence, were supported by a parallel analysis of the faster evolving internal transcribed spacer (ITS) rDNA. Distinct episodes of fruit body morphology shifts are discernable in the 18S rDNA phylogenetic tree. In all cases, the shift from an epigeous to a hypogeous form is the most parsimonious interpretation of character transformation, without any instance of character reversal.