Molecular systematics and biological diversification of Boletales

Historical patterns of morphological evolution and ecology in the Boletales are largely unresolved but appear to involve extensive convergence. We studied phylogenetic relationships of Boletales based on two datasets. The nuc-lsu dataset is broadly sampled and includes roughly 30% of the described species of Boletales and 51 outgroup taxa across the Hymenomycetes. The multigene dataset (nuc-ssu, nuc-lsu, 5.8S, mt-lsu, atp6) sampled 42 key species of Boletales in a framework of 14 representative Hymenomycetes. The Boletales are strongly supported as monophyletic in our analyses on both datasets with parsimony, maximum likelihood and Bayesian approaches. Six major lineages of Boletales that currently are recognized on subordinal level, Boletineae, Paxillineae, Sclerodermatineae, Suillineae, Tapinellineae, Coniophorineae, received varied support. The backbone of the Boletales was moderately resolved in the analyses with the nuc-lsu dataset, but support was strong for most major groups. Nevertheless, most brown-rot producing forms were placed as a paraphyletic grade at the base of the Boletales. Analyses on the multigene dataset confirm sister group relationships among Boletales, Agaricales and Atheliales. Boletineae and Suillineae received the highest support values; Paxillineae and Sclerodermatineae were not consistently resolved as monophyletic groups. The Coniophorineae were not monophyletic in any analyses. The Tapinellineae consisting of morphologically diverse brown-rotting fungi forms the basal group in the Boletales. We performed ancestral state reconstruction with BayesMultiState, which suggested that the ancestor of the Boletales was a resupinate or polyporoid saprotrophic fungus, producing a brown-rot.     

Molecular phylogenetics of porcini mushrooms (Boletus section Boletus)

Porcini (Boletus section Boletus: Boletaceae: Boletineae: Boletales) are a conspicuous group of wild, edible mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is "stuffed" with white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Northern Hemisphere. Little progress has been made on the systematics of this group using modern molecular phylogenetic tools because sampling has been limited primarily to European species and the genes employed were insufficient to resolve the phylogeny. We examined the evolutionary history of porcini by using a global geographic sampling of most known species, new discoveries from little explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal transcribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not porcini form a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six gene. Contrary to previous studies, our separate and combined phylogenetic analyses support the monophyly of porcini. We also report the discovery of two taxa that expand the known distribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incentive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal symbiosis.     

Comparative phylogenies and host specialization in the alder ectomycorrhizal fungi Alnicola, Alpova and Lactarius (Basidiomycota) in Europe

Background  

Mycorrhizal fungi form intimate associations with their host plants that constitute their carbon resource and habitat. Alnus spp. (Betulaceae) are known to host an exceptional species-poor and specialized ectomycorrhizal (ECM) fungal community compared to other tree species, but the host-specificity pattern and its significance in terms of fungal diversification and speciation remain poorly documented. The degree of parallel speciation, host switching, and patterns of biogeography were explored in the historical associations between alders and three ECM taxa of Basidiomycetes: Alnicola (Agaricales), Alpova (Boletales), and Lactarius (Russulales). The aim was to develop an evolutionary framework on host specificity and diversification of Basidiomycetes in this highly specialized plant-fungus symbiosis.     

Phylogenetic constrains on mycorrhizal specificity in eight Dendrobium(Orchidaceae) species

Plant phylogeny constrains orchid mycorrhizal(OrM) fungal community composition in some orchids. Here, we investigated the structures of the OrM fungal communities of eight Dendrobium species in one niche to determine whether similarities in the OrM fungal communities correlated with the phylogeny of the host plants and whether the Dendrobium-OrM fungal interactions are phylogenetically conserved. A phylogeny based on DNA data was constructed for the eight coexisting Dendrobium species,and the OrM fungal communities were characterized by their roots. There were 31 different fungal lineages associated with the eight Dendrobium species. In total, 82.98% of the identified associations belonging to Tulasnellaceae, and a smaller proportion involved members of the unknown Basidiomycota(9.67%). Community analyses revealed that phylogenetically related Dendrobium tended to interact with a similar set of Tulasnellaceae fungi. The interactions between Dendrobium and Tulasnellaceae fungi were significantly influenced by the phylogenetic relationships among the Dendrobium species. Our results provide evidence that the mycorrhizal specificity in the eight coexisting Dendrobium species was phylogenetically conserved.     

Cryptic species and host specificity in the ectomycorrhizal genus Strobilomyces (Strobilomycetaceae)

Taxonomical classification of higher fungi remains an important challenge and can benefit from the application of molecular analysis. We propose that the ectomycorrhizal (EM) fungal taxa might include a number of cryptic species because there are few morphological characteristics useful for distinguishing among these fungi. Previously, host specificity in most EM fungi was thought to be low, but we suspect that confusion of cryptic species has led to an underestimate of fungal host specificity. We analyzed both nuclear and mitochondrial DNA sequences from Strobilomyces fungi and obtained evidence that what were previously described as four species can be grouped into 14 distinct lineages, suggesting that these lineages might be distinct biological species. Moreover, we identified host plants for Strobilomyces via nucleotide sequencing of both fungal and plant DNA from EM samples. Most lineages of Strobilomyces tested in this study were associated only with Fagaceae trees, even though Strobilomyces species were previously thought to be generalists with regard to hosts. Thus, we present an approach useful for identifying cryptic species and detecting the true host range of a set of EM fungi in natural conditions.     

Derivation of a polymorphic lineage of Gasteromycetes from boletoid ancestors

The phylogeny of selected gasteromycetes and hymenomycetes was inferred from partial nuclear large subunit rDNA (nuc-lsu, 28S) sequences, delimited by primers LR0R and LR5. Taxon sampling with emphasis on relationships within the Boletales further included some gasteroid groups, which obviously have evolved convergent fruiting body morphology, and therefore remained controversial in taxonomy. This study confirms the close relationship of Geastrales, Gauteriales and Phallales and the presumable derivation of Nidulariales and Tulostomatales within the euagarics clade, as widely accepted. In addition, four Hymenogaster species investigated were found to be in the euagarics clade and a relationship to the Cortinariaceae was indicated. The gasteroid fungus Zelleromyces stephensii is an example for maintaining morphological linkage by a lactiferous hyphal system to the genus Lactarius in the Russulales, and this relationship was affirmed in the sequence analysis. Several previously suggested relationships of gasteromycetes and Boletales were reproducible by analyzing nuc-lsu sequences. As a new result, Astraeus hygrometricus, the barometer earth star, is an additional representative of the Boletales. Together with Boletinellus, Phlebopus, Pisolithus, Calostoma, Gyroporus, Scleroderma, and Veligaster, Astraeus forms an unusual group comprising pileate-stipitate hymenomycetes and polymorphic gasteromycetes. This group is a major lineage within the Boletales and we propose the new suborder Sclerodermatineae, including the six families Boletinellaceae fam. nov. (Boletinellus and Phlebopus), Gyroporaceae (Singer) fam. nov. (Gyroporus), Pisolithaceae (Pisolithus), Astraeaceae (Astraeus), Calostomataceae (Calostoma), and the typus subordinis Sclerodermataceae (Scleroderma and Veligaster). Morphological and ecological characters, and pigment synthesis support the delimitation of the Sclerodermatineae, and indicate the radiation of different lineages in the Boletales originating from fungi with primitive tubular hymenophores. We regard such boletes with gyroid-boletinoid hymenophores, like Boletinellus, Gyrodon, Gyroporus, Paragyrodon and Phlebopus as key taxa in the evolution of Paxillineae, Sclerodermatineae and Boletineae.     

Soil fungal communities in a Castanea sativa (chestnut) forest producing large quantities of Boletus edulis sensu lato (porcini): where is the mycelium of porcini?

A study was conducted in a Castanea sativa forest that produces large quantities of the edible mushroom porcini (Boletus edulis sensu lato). The primary aim was to study porcini mycelia in the soil, and to determine if there were any possible ecological and functional interactions with other dominant soil fungi. Three different approaches were used: collection and morphological identification of fruiting bodies, morphological and molecular identification of ectomycorrhizae by rDNA-ITS sequence analyses and molecular identification of the soil mycelia by ITS clone libraries. Soil samples were taken directly under basidiomes of Boletus edulis, Boletus aestivalis, Boletus aereus and Boletus pinophilus. Thirty-nine ectomycorrhizal fungi were identified on root tips whereas 40 fungal species were found in the soil using the cloning technique. The overlap between above- and below-ground fungal communities was very low. Boletus mycelia, compared with other soil fungi, were rare and with scattered distribution, whereas their fruiting bodies dominated the above-ground fungal community. Only B. aestivalis ectomycorrhizae were relatively abundant and detected as mycelia in the soil. No specific fungus-fungus association was found. Factors triggering formation of mycorrhizae and fructification of porcini appear to be too complex to be simply explained on the basis of the amount of fungal mycelia in the soil.     

Characterization and identification of field ectomycorrhizae of Boletus edulis and Cistus ladanifer

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.     

Correlation analysis between arbuscular mycorrhizal fungal community and host plant phylogeny

 为弄清丛枝菌根(arbuscular mycorrhiza, AM)真菌群落随宿主植物演化的变异规律,通过对MaarjAM数据库进行数据挖掘, 根据每个分子虚拟种(virtual taxa, VT)包含的DNA序列不少于5条的标准, 筛选出188种菌根植物。通过分析植物与其根内AM真菌的关系发现: AM真菌的物种丰富度随着寄主植物的分化而增加; 在不同的植物系统类群中, AM真菌的物种丰富度显著不同; 在起源时间较晚的被子植物和裸子植物中, AM真菌的物种丰富度显著高于起源较早的苔类、角苔类和蕨类植物类群, 而与寄生植物共生的AM真菌物种丰富度与早期植物无显著差异; 不同寄主植物进化类群间AM真菌组成差异显著。以上结果表明: AM真菌群落随着寄主植物进化而发生变化。在进化过程中, 寄主植物倾向于选择保留共生效率较高的AM真菌。     

Community composition and diversity of Neotropical root‐associated fungi in common and rare trees

Interactions between plants and root‐associated fungi can affect the assembly, diversity, and relative abundances of tropical plant species. Host–symbiont compatibility and some degree of host specificity are prerequisites for these processes to occur, and these prerequisites may vary with host abundance. However, direct assessments of whether specificity of root‐associated fungi varies with host abundance are lacking. Here, in a diverse tropical forest in Los Tuxtlas, Mexico, we couple DNA metabarcoding with a sampling design that controls for host phylogeny, host age, and habitat variation, to characterize fungal communities associated with the roots of three confamilial pairs of host species that exhibit contrasting (high and low) relative abundances. We uncovered a functionally and phylogenetically diverse fungal community composed of 1,038 OTUs (operational taxonomic units with 97% genetic similarity), only 14 of which exhibited host specificity. Host species was a significant predictor of fungal community composition only for the subset of OTUs composed of putatively pathogenic fungi. We found no significant difference in the number of specialists associating with common versus rare trees, but we found that host abundance was negatively correlated with the diversity of root fungal communities. This latter result was significant for symbiotrophs (mostly arbuscular mycorrhizal fungi) and, to a lesser extent, for pathotrophs (mostly plant pathogens). Thus, root fungal communities differ between common and rare trees, which may impact the strength of conspecific negative density dependence. Further studies from other tropical sites and host lineages are warranted, given the role of root‐associated fungi in biodiversity maintenance.     

Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographical structure

The Monotropoideae (Ericaceae) are nonphotosynthetic plants that obtain fixed carbon from their fungal mycorrhizal associates. To infer the evolutionary history of this symbiosis we identified both the plant and fungal lineages involved using a molecular phylogenetic approach to screen 331 plants, representing 10 of the 12 described species. For five species no prior molecular data were available; for three species we confirmed prior studies which used limited samples; for five species all previous reports are in conflict with our results, which are supported by sequence analysis of multiple samples and are consistent with the phylogenetic patterns of host plants. The phylogenetic patterns observed indicate that: (i) each of the 13 plant phylogenetic lineages identified is specialized to a different genus or species group within five families of ectomycorrhizal Basidiomycetes; (ii) mycorrhizal specificity is correlated with phylogeny; (iii) in sympatry, there is no overlap in mature plant fungal symbionts even if the fungi and the plants are closely related; and (iv) there are geographical patterns to specificity.     

Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)

Burkholderia endophytes were identified within the leaves of non-nodulated members of the genus Psychotria. In contrast to leaf-nodulated Psychotria species, which are known to accommodate their endosymbionts into specialized endosymbiont-housing structures, non-nodulated species lack bacterial leaf nodules and harbor endosymbionts intercellularly between mesophyll cells. Based on molecular data (rps16, trnG, and trnLF), the phylogenetic reconstruction of the host plants revealed a separate origin of leaf-nodulated and non-nodulated Psychotria species. Despite a distinct phylogenetic position of the two host clades, the endophytes of the non-nodulated plants were not placed into a single monophyletic group but were found to be closely related to the leaf-nodulated endosymbionts. The observation of genetically similar endophytes in both nodulated and non-nodulated Psychotria lineages suggests that the host plant is playing a crucial role in the induction of leaf nodule formation. Moreover, the concentration of endosymbionts into specialized leaf nodules may be considered as a more derived evolutionary adaptation of the host plant, serving as an interface structure to facilitate metabolic exchange between plant and endosymbiont.     

Unravelling evolutionary relationships between epifoliar Meliolaceae and angiosperms

Epifoliar fungi are a group of poorly studied fungal symbionts that coinhabit the surface of living plants. Meliolaceae is the largest group of epifoliar fungi and has been considered as obligate parasites. We investigated the taxonomy of Meliolaceae and the coevolutionary events with their host plants using time-calibrated cophylogeny based on large subunit, small subunit, and internal transcribed spacer (ITS) sequence data obtained from 17 different fungal taxa and rbcL, ITS, and trnH-psbA sequence data from their corresponding hosts. Nine new fungal species are introduced in this paper and Appendiculella is synonymized under Asteridiella. The dominant coevolutionary events during the Cretaceous and Cenozoic are cospeciation and host shift, respectively. We hypothesize that the evolutionary history of epifoliar fungi can be divided into three major periods: origins of families, formations of genera, and diversification of species. The rise of angiosperms prompted the evolution of modern epifoliar fungi and the diversification of orders of Angiospermae fostered the formation of epifoliar fungal genera. Phylogenetically, epifoliar fungal genera can be delimited according to their coevolutionary patterns and divergent periods.     

Endophytic fungi assemblages from 10 <Emphasis Type="Italic">Dendrobium</Emphasis> medicinal plants (Orchidaceae)

Dendrobium is the largest genus of tropical epiphytic orchid, some of which are traditional Chinese medicinal plants. The therapeutic components varied significantly among species. Endophytic microbes (fungi) hidden in medicinal plants may play an important effect on the overall quality of herb. Investigation of fungal composition in host plants is the first step toward elucidating the relationship endophyte-therapeutic content of herbal medicine. In this study, 401 culturable fungal endophytes were isolated and identified from 10 species of medicinal Dendrobium based on morphological and molecular techniques. The results showed that endophytic fungi from Dendrobium plants exhibited high biodiversity (37 genera, about 80 species). Acremonium, Alternaria, Ampelomyces, Bionectria, Cladosporium, Colletotrichum, Fusarium, Verticillium and Xylaria were the dominant fungal endophytes. Tropical epiphytic orchids appear to vary in degree of host specificity in their endophytic fungi.     

Mycorrhizal features and fungal partners of four mycoheterotrophic Monotropoideae (Ericaceae) species from Yunnan, China

We provide a preliminary report of the mycobionts found within four Monotropoideae (Ericaceae) species from China: Monotropa uniflora, Hypopitys monotropa, Monotropastrum humile and Monotropastrum sciaphilum (a rare endemic species never previously studied for mycorrhizae). Such achlorophyllous Monotropoideae plants obtain their carbohydrates from mycorrhizal fungi linking them to surrounding trees, on which these fungi form ectomycorrhizae. Since Monotropoideae were rarely studied in continental Asia, the root systems of the four species sampled in Yunnan were examined using morphological and molecular methods. All the roots of these four species exhibit a typical monotropoid mycorrhizal morphology, including a fungal mantle, a Hartig net and hyphal pegs. In M. uniflora and M. humile mycorrhizae, cystidia typical of Russula symbionts covered the fungal mantle. ITS barcoding revealed that Russulales were the most frequent colonizers in all species, but Hypopitys monotropa displayed various additional mycorrhizal taxa. Moreover, a few additional ectomycorrhizal and saprotrophic Basidiomycota taxa were identified in the three other species, challenging that these four Monotropoideae species are as strictly fungal specific as the other Monotropoideae species hitherto studied. Moreover, a comparison with accompanying fungus sporocarps revealed that the fruiting fungal community significantly differed from that associated with the Monotropoideae roots, so that a clear fungal preference was evident. Finally, four fungal species were found on more than one Monotropoideae species: this contrasted with previous reports of sympatrically growing mycoheterotrophic plants, which did not reveal any overlap. This again challenges the idea of strict fungal specificity.     

Links between tree species, symbiotic fungal diversity and ecosystem functioning in simplified tropical ecosystems

We studied the relationships among plant and arbuscular mycorrhizal (AM) fungal diversity, and their effects on ecosystem function, in a series of replicate tropical forestry plots in the La Selva Biological Station, Costa Rica. Forestry plots were 12 yr old and were either monocultures of three tree species, or polycultures of the tree species with two additional understory species. Relationships among the AM fungal spore community, host species, plant community diversity and ecosystem phosphorus-use efficiency (PUE) and net primary productivity (NPP) were assessed. Analysis of the relative abundance of AM fungal spores found that host tree species had a significant effect on the AM fungal community, as did host plant community diversity (monocultures vs polycultures). The Shannon diversity index of the AM fungal spore community differed significantly among the three host tree species, but was not significantly different between monoculture and polyculture plots. Over all the plots, significant positive relationships were found between AM fungal diversity and ecosystem NPP, and between AM fungal community evenness and PUE. Relative abundance of two of the dominant AM fungal species also showed significant correlations with NPP and PUE. We conclude that the AM fungal community composition in tropical forests is sensitive to host species, and provide evidence supporting the hypothesis that the diversity of AM fungi in tropical forests and ecosystem NPP covaries.     

中国牛肝菌目的研究和增补

在研究我国川西、滇、桂、海南岛的牛肝菌目标本中,发现二新种:网盖金牛肝菌(Aureoboletus reticuloceps)和黑牛肝菌(Boletus nigricans),后者属黑牛肝菌组,新组(Boletus sect.Nigres)。并报道了婆罗洲牛肝菌、美丽牛肝菌、怪形牛肝菌、焰色牛肝菌、黑紫牛肝菌、长柄条孢牛肝菌和西藏金牛肝菌的分布型和所呈现的印度——马来西亚热带成分的相适关联。     

Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups

The Monotropoideae (Ericaceae) are non-photosynthetic angiosperms that obtain fixed carbon from basidiomycete ectomycorrhizal fungi. In previous work, we showed that each plant species is associated with a single genus or a set of closely related genera of ectomycorrhizal fungi. Here we show that the level of specificity is much higher. We used a molecular phylogenetic approach to contrast specificity patterns among eight plant lineages and three fungal genera. We relied on fungal nuclear internal transcribed spacer (nrITS) sequence data obtained from 161 basidiocarps and 85 monotropoid roots representing 286 sampled plants screened using restriction length polymorphisms. From the phylogenetic placement of fungal symbionts in fungal phylograms, we found that three basal (Sarcodes, Pterospora, Pleuricospora) and one derived lineage (Allotropa) of plants target narrow clades of closely related species groups of fungi, and four derived lineages (Monotropa hypopithys species group, Pityopus) target more distant species groups. Within most plant lineages, geography and photobiont association constrain specificity. Specificity extended further in Pterospora andromedea, in which sequence haplotypes at the plastid trn L-F region of 73 plants were significantly associated with different fungal species groups even in sympatry. These results indicate that both the macro- and microevolution of the Monotropoideae are tightly coupled to their mycorrhizal symbionts.     

Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays)

The focus of many fungal endophyte studies has been how plants benefit from endophyte infection. Few studies have investigated the role of the host plant as an environment in shaping endophyte community diversity and composition. The effects that different attributes of the host plant, that is, host genetic variation, host variation in resistance to the fungal pathogen Ustilago maydis and U. maydis infection, have on the fungal endophyte communities in maize (Zea mays) was examined. The internal transcribed spacer (ITS) region of the rDNA was sequenced to identify fungi and the endophyte communities were compared in six maize lines that varied in their resistance to U. maydis. It was found that host genetic variation, as determined by maize line, had significant effects on species richness, while the interactions between line and U. maydis infection and line and field plot had significant effects on endophyte community composition. However, the effects of maize line were not dependent on whether lines were resistant or susceptible to U. maydis. Almost 3000 clones obtained from 58 plants were sequenced to characterize the maize endophyte community. These results suggest that the endophyte community is shaped by complex interactions and factors, such as inoculum pool and microclimate, may be important.