Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale

In most ectomycorrhizal (EM) community studies involving molecular identification methods there is a poor correspondence between fungi that appear dominant as sporocarps and those that appear dominant on EM roots and the species richness belowground is higher than that above ground. As a consequence, many fungi from root tip samples remain unidentified. In most studies, genetic data from multiple samples of an EM morphotype collected from various sampling locations are compared to genetic data from one to a few sporocarps of each species for identification purposes. The mismatch between above- and belowground species richness may be influenced by these different sampling efforts. To address this, intra-specific variation in the ITS region first investigated in Kårén et al. (1997) is revisited here, but at a spatial scale in which variation is expected to be low. Sporocarps were collected across a 7 km region of the Oregon Dunes National Recreation Area in western North America. ITS–RFLP data are presented for 3-18 sporocarps from each of 44 EM species in 18 genera. A total of 311 sporocarps were analyzed. Fifty-three ITS–RFLP types were observed. Of the 44 species, 38 (86% of total) yielded a single, species specific, RFLP type. No 2 species had the same RFLP type. Polymorphic ITS–RFLP types were observed in six species (14%). The following three species had two ITS-RFLP types with one type dominating: Inocybe lacera, Laccaria proxima, and Rhizopogon subcaerulescens. The following three species had three RFLP types with no type dominating: Laccaria laccata, Lactarius deliciosus, and Tricholoma flavovirens. A phylogenetic analysis of ITS sequences in Tricholoma revealed that two of the RFLP types in T. flavovirenswere apparently the result of intra-specific variation, while the third RFLP type was likely a cryptic species. All the other RFLP types observed in Tricholoma represented unique phylogenetic species. These results suggest that ITS–RFLP data from single samples (sporocarp or EM) are robust for characterizing most of the species at this scale. However, restriction endonucleases detect a limited amount of existing nucleotide variation and thus have limited value to detect cryptic species. Therefore, additional analyses of sequence data should be added to the RFLP matching technique to identify unknown RFLP types. These data also suggest that polymorphic RFLP types within species do not adequately explain the mismatch between above- and belowground views of EM species richness.     

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.     

Ectomycorrhizae between <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. and <Emphasis Type="Italic">Naucoria escharoides</Emphasis> (Fr.:Fr.) Kummer from Argentina

Field ectomycorrhizae of Naucoria escharoides on Alnus acuminata ("andean alder", "aliso del cerro") are described in detail for the first time. Naturally occurring ectomycorrhizal roots were sampled beneath sporocarps of N. escharoides. The samples were taken from four natural forest plots at two homogeneous A. acuminata sites (Tucumán and Catamarca Provinces, Argentina). The ectomycorrhizae were characterized morphologically and compared by means of PCR/RFLP analysis of the internal transcribed spacer region of the nuclear rDNA. The most important morphological features of the ectomycorrhizae are a white to pale yellow mantle, simple to monopodial branches, hyaline emanating hyphae, abundant hyphal bundles emerging more or less perpendicularly from a plectenchymatous mantle, and an acute or rounded apex with or without a mantle. N. escharoides fruitbodies have white basal mycelium with emanating hyphae similar to those of andean alder ectomycorrhizae. The RFLP profiles of sporocarps and mycorrhizae were the same.     

RAMARIA SUBGENERA RAMARIA AND LAETICOLORA IN YUNNAN

Three taxa of Ramaria subgenus Ramaria, and 18 of subg. Laeticolora are reported from Yunnan Province. Of these 13 are considered new to science. Only one taxon (R. formosa) is considered of widespread distribution, the others being known olay from the Indo-Asian subcontinent. Wide distribution patterns, based on phenetic similarity, seem to be at the infraspecific to species complex level, but only rarely contaxie. New taxa are as follows: subg. Ramaria: R. eryuanensis; subg. Laeticolora: R. brunneipes, R. distinctissima, R. ephemeroderma, R. hilaris, R. lacviformosoides, R. linearioides, R. linearis, R. nanispora, R. rubri-attenuipcs, R. sino-conjunctipes, R. rubricarnata var. laeta, R. botrytoides var. microspora, and R. neoformosa var. sinensis. Keys to Yunnan taxa of Ramaria subgenera Ramarla and Laeticolora are furnished.     

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fungal sporocarps house diverse and host-specific communities of fungicolous fungi

Sporocarps (fruit bodies) are the sexual reproductive stage in the life cycle of many fungi. They are highly nutritious and consequently vulnerable to grazing by birds and small mammals, and invertebrates, and can be infected by microbial and fungal parasites and pathogens. The complexity of communities thriving inside sporocarps is largely unknown. In this study, we revealed the diversity, taxonomic composition and host preference of fungicolous fungi (i.e., fungi that feed on other fungi) in sporocarps. We carried out DNA metabarcoding of the ITS2 region from 176 sporocarps of 11 wood-decay fungal host species, all collected within a forest in northeast Finland. We assessed the influence of sporocarp traits, such as lifespan, morphology and size, on the fungicolous fungal community. The level of colonisation by fungicolous fungi, measured as the proportion of non-host ITS2 reads, varied between 2.8–39.8% across the 11 host species and was largely dominated by Ascomycota. Host species was the major determinant of the community composition and diversity of fungicolous fungi, suggesting that host adaptation is important for many fungicolous fungi. Furthermore, the alpha diversity was consistently higher in short-lived and resupinate sporocarps compared to long-lived and pileate ones, perhaps due to a more hostile environment for fungal growth in the latter too. The fungicolous fungi represented numerous lineages in the fungal tree of life, among which a significant portion was poorly represented with reference sequences in databases.Subject terms: Microbial ecology, Molecular evolution     

Molecular identification of Tuber magnatum ectomycorrhizae in the field

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).     

Four ectomycorrhizae of Pyronemataceae (Pezizomycetes) on Chinese Pine (<Emphasis Type="Italic">Pinus tabulaeformis</Emphasis>): morpho-anatomical and molecular-phylogenetic analyses

Morphological and anatomical characters of four ectomycorrhizae with affinities to the genera Humaria, Geopora, and Trichophaea of Pyronemataceae (Pezizomycetes, Ascomycota) on Chinese Pine (Pinus tabulaeformis) are described. The ectomycorrhizae are yellowish brown to brown, and have pseudoparenchymatous outer mantle layers and partially warty emanating hyphae with thick walls and without clamps. Intrahyphal hyphae are present, and no rhizomorphs are formed. The four ectomycorrhizae are distinguishable by differences in cell shape of outer mantle layers and the presence of cystidia. Ectomycorrhizae of a possible Humaria species (Pinirhiza humarioides) lack cystidia and have irregularly inflated cells on the outer mantle layer that are connected with thin septa. The two ectomycorrhizae showing probable affinities to Geopora species (“P. daqingensis” and “P. geoporoides”) possess row-like arranged cells in the outer mantle layer and cell heaps, and differ by the presence or absence of cystidia as well as by the structure of the inner mantle layers. Ectomycorrhizae likely having been formed by a Trichophaea species (“P. trichophaeoides”) have oval to polygonal cells and no cystidia. The possible taxa affiliations were assessed by molecular-phylogenetic analyses of the internal transcribed spacer (ITS) and partial large subunit (LSU) nrDNA. Morphological and anatomical characters are discussed against the background of the LSU phylogeny.     

Tuberculate ectomycorrhizae of angiosperms: The interaction between Boletus rubropunctus (Boletaceae) and Quercus species (Fagaceae) in the United States and Mexico

Tuberculate ectomycorrhizae (TECM) are unique structures in which aggregates of ectomycorrhizal roots are encased in a covering of fungal hyphae. The function of TECM is unknown, but they probably enhance the nitrogen nutrition and disease resistance of host plants. Trees in the Pinaceae form TECM with species of Rhizopogon and Suillus (Suillineae, Boletales). Similar tubercules are found with diverse angiosperms, but their mycobionts have not been phylogenetically characterized. We collected TECM in Mexico and the USA that were similar to TECM in previous reports. We describe these TECM and identify both the plant and fungal symbionts. Plant DNA confirms that TECM hosts are Quercus species. ITS sequences from tubercules and sclerotia (hyphal aggregations that serve as survival structures) matched sporocarps of Boletus rubropunctus. Phylogenetic analyses confirm that this fungus belongs to the suborder Boletineae (Boletales). This is the first published report of TECM formation in the Boletineae and of sclerotia formation by a Boletus species. Our data suggest that the TECM morphology is an adaptive feature that has evolved separately in two suborders of Boletales (Suillineae and Boletineae) and that TECM formation is controlled by the mycobiont because TECM are found on distantly related angiosperm and gymnosperm host plants.     

Ectomycorrhizal fungi are shared between seedlings and adults in a monodominant Gilbertiodendron dewevrei rain forest in Cameroon

Ectomycorrhizal networks may facilitate the establishment and survival of seedlings regenerating under the canopies of tropical forests and are often invoked as a potential contributor to monodominance. We identified ectomycorrhizal fungi in a monodominant Gilbertiodendron dewevrei (Fabaceae) rain forest in Cameroon, using sporocarps and ectomycorrhizae of three age categories (seedlings, intermediate trees, and large trees) and tentatively revealed nutrient transfer through ectomycorrhizal networks by measuring spontaneous isotopic (¹³C and ¹⁵N) abundances in seedlings. Sporocarp surveys revealed fewer ectomycorrhizal fungal taxa (59 species from 1030 sporocarps) than molecular barcoding of ectomycorrhizal roots (75 operational taxonomic units from 828 ectomycorrhizae). Our observations suggest that ectomycorrhizal fungal diversity is similar to that in other mixed tropical forests and provide the first report of the Tuber‐Helvella lineage in a tropical forest. Despite some differences, all age categories of G. dewevrei had overlapping ectomycorrhizal fungal communities, with families belonging to Thelephoraceae, Russulaceae, Sebacinaceae, Boletaceae, and Clavulinaceae. Of the 49 operational taxonomic units shared by the three age categories (65.3% of the ectomycorrhizal fungal community), 19 were the most abundant on root tips of all categories (38.7% of the shared taxa), supporting the likelihood of ectomycorrhizal networks. However, we obtained no evidence for nutrient transfer from trees to seedlings. We discuss the composition of the ectomycorrhizal fungal community among the G. dewevrei age categories and the possible role of common ectomycorrhizal networks in this rain forest.     

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.     

Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M. Rich. and neighbouring tree ectomycorrhizae

Several achlorophyllous orchids associate with ectomycorrhizal hymenomycetes deriving carbon from surrounding trees for the plant. However, this has not been shown for achlorophyllous orchids associating with species of Rhizoctonia, a complex of basal lineages of hymenomycetes that are the most common orchid partners. We analysed Neottia nidus-avis, an achlorophyllous orchid symbiotic with a Rhizoctonia, to identify its symbionts by internal transcribed spacer (ITS) sequencing. Analysis of 61 root systems from 23 French populations showed that N. nidus-avis associates highly specifically with a group of species of Sebacinaceae. Their diversity emphasizes the need for further investigations in the Sebacinaceae systematics. Sebacinoid ITS sequences were often identical within orchid populations and a trend to regional variation in symbionts was observed. Using ITS and intergenic spacer (IGS) polymorphism, we showed that each root system harboured a single species, but that several genets colonized it. However, no polymorphism of these markers was found among portions of each root: this is consistent with the putative mode of entry of the fungus, i.e. from the rhizome into roots but not repeatedly from the soil. In addition, ectomycorrhizae were always found within the N. nidus-avis root systems: 120 of the 144 ectomycorrhizae typed by ITS sequencing were colonized by a sebacinoid fungus identical in ITS sequence to the respective orchid symbiont (even for the IGS polymorphism in some cases). Because sebacinoids were demonstrated recently to be ectomycorrhizal, the orchid is likely to derive its resources from surrounding trees, a mycorrhizal cheating strategy similar to other myco-heterotrophic plants studied to date.     

Occurrence and importance of mycorrhizae in aquatic trees of New South Wales,Australia

Vesicular arbuscular mycorrhizal (VAM) infection was found in KOH-cleared and lactophenolblue-stained roots of Salix babylonica, Melaleuca quinquenervia and Casuarina cunninghamiana. These are all trees growing on creeks and river banks, in stationary or slowly flowing fresh or brackish waters in swamps, creeks, drains and channels, and in seepage areas of New South Wales, Australia. Larger and older roots lacked VAM infection in the inner cortex, probably due to suberisation of cells, and the endophyte was restricted to the epidermal layers. Spores and sporocarps of the VAM fungi Glomus fasciculatus, G. mosseae, Sclerocystis rubiformis, Gigaspora margarita and an unidentified Scutellospora sp. were wet sieved and decanted from aquatic sediments and soils. The presence of similar VAM fungal spores in the aquatic sediments and terrestrial soil suggests that they probably enter the aquatic sediments through run off from the land ecosystem. All three plants formed vesicular arbuscular (VA) mycorrhizae almost exclusively in the marshy, periodically inundated soils, but the same plant species formed endo-/ ectomycorrhizae when growing in soil with higher redox potentials (E _h). Salix and Melaleuca tree roots possessed both VAmycorrhizae and ectomycorrhizae. VAM roots of Casuarina were equipped with both N-fixing Frankia nodules and proteoid roots. VAM endophytes did not invade nodular cortical tissues, suggesting the presence of an exclusion mechanism which needs further study. The highest VAM infection was found in nodulated specimens. Free-floating roots growing in water close to the banks were non-mycorrhizal but were mycorrhizal in the bottom-rooting state. VAM spore number and mycorrhizal infection seem to be associated with redox-potential, i.e. lower at sites such as swamps, water or sediments with lower E _h values than in terrestrial soils with higher E _h values. A relationship between soil moisture gradient and VAM infection pattern became apparent from the study of a C. cunninghamiana transect on a creek embankment, i.e. typical vesicles and arbuscules were found in roots from drier soils, there was a lack of arbuscules in relatively wet soils but large lipid-filled intracellular vesicles were present, and typical vesicles and arbuscules were absent in flooded creek beds where roots were associated with coenocytic intercellular hyphae with abundant lipid droplets. The importance of VA mycorrhiza, ectomycorrhizae, N-fixing root nodules and proteoid roots at the land-water interface is discussed with reference to the use of these trees as pioneering species for stabilising river and stream banks, reducing erosion, windbreaking, and as a long-term and inexpensive means of achieving biological control of aquatic weeds by shading waterways.     

云南松幼苗上红菇类菌根真菌的物种多样性及其菌根形态

以滇中1～2年生云南松幼苗为研究对象,观察鉴定与其共生的红菇属真菌外生菌根。形态观察发现了6种形态型（morphotypes）。本文对这6种形态型的外观和显微特征进行了详细描述,尤其强调了菌套形态特征。对rDNAITS片段比对分析表明,6种形态型对应6种红菇属真菌,它们分别是与Russula li-vescens、R.violeipes、R.densifolia、R.nigricans、R.sanguinea及R.nauseosa相近的红菇种类。本研究表明,形成的菌根及其菌套和囊状体的特征在红菇属真菌的系统分支间表现出较为稳定的差异。这一研究与前人对该属真菌的菌根形态及分类学研究基本吻合。红菇属真菌是云南松1～2年幼苗期的主要共生真菌类群。     

蛋白和酯酶电泳在根霉鉴定上的应用

对根霉(Rhizopus)属的十个种或变种共二十四株菌的菌体可溶性蛋白和酯酶同工酶进行了电泳的研究得到对这个属分类上更多的依据.在严格控制培养,提取和电泳条件的情况下,同一株菌不同批次所得菌体蛋白电泳图谱有较好的重复性.在相同的条件下,每个种的根霉有各自特征性蛋白图谱,种内不同菌株的蛋白图谱和酯酶酶谱基本相同.特别是形态特征明显、分类地位明确的种,种内各株的图谱也较一致,如R. stolonifer;与R.circinans.在确定新变种R. delemar var. latoapicalis时,电泳图谱与R.delemar var.delema:有明显不同,起到了佐证作用.因此认为,蛋白图谱与酯酶酶谱相辅相成,在根霉种的分类中是一有效的辅助手段.     

Establishment of ectomycorrhizae on the roots of two species of Eucalyptus from fungal spores contained in the faeces of the long-nosed potoroo (Potorous tridactylus)

Abstract The long-nosed potoroo (Potorous tridactylus), a medium-sized, ground-dwelling marsupial, inhabits the foothills and coastal forests of southeastern mainland Australia. Faecal analysis confirmed the mycophagous habit of P. tridactylus at a eucalypt forest site in East Gippsland, Victoria; in one month, the spores of 33 different fungi were identified. The majority of species (27) were hypogeal or subhypogeal gasteromycetes that form ectomycorrhizae with forest trees and shrubs, and included the widespread and common Mesophellia pachytrix. The possible role of mycophagy in establishment of eucalypt-fungal mycorrhizae was investigated in several ways. Fungal spores were viable after passage through the gut of P. tridactylus; application of spore-bearing faeces to seedlings of Eucalyptus sieberi and Eucalyptus globoidea grown in sterile soil produced ectomycorrhizae. Spores of M. pachythrix were among the most common spores in these faeces and some of the resulting ectomycorrhizae were of the type expected from M. pachythrix. In contrast, direct application of M. pachythrix spores from sporocarp tissue to seedlings produced no ectomycorrhizae. Ectomycorrhizae also were established on seedlings grown in unsterilized forest soil, indicating that soil-borne spores had been conditioned, either by passage through the gut of a mammal or some other means, for reaction with the roots of eucalypts. Addition of faeces improved root and shoot growth of seedlings, although we could not determine whether this was primarily due to the formation of ectomycorrhizae, the addition of nutrients in the faeces, or a combination of both. Potorous tridactylus may enhance the re-establishment of mycorrhizal colonies in logged and/or burned forest sites by dispersing in its faeces, the spores of mycorrhizal fungi from sporocarps consumed within the disturbed area, as well as spores from sporocarps eaten in adjacent undisturbed habitat. In performing this function, P. tridactylus may also aid in the recovery of regenerating silvertop ash-stringybark forests.     

Species Diversity of Russuloid Mycorrhizae forming Fungi on Pinus yunnanensis Seedlings and the Mycorrhizal Morphology

Russuloid ectomycorrhizae on 1-2 years old seedlings of Pinus yunnanensis collected from the central Yunnan, China were investigated. Six morphotypes were recognized by macro and anatomical morphological approaches as well as molecular analyses. The six morphotypes were confirmed to represent six phylotypes by matching rDNA ITS sequences. The fungal partners in the six morpho phylotypes are those closely related with Russula livescens, Rvioleipes ,Rdensifolia ,Rnigricans ,Rsanguinea and Rnauseosa respectively. The correlation between the morphology of mantles and cystidia and the phylogenetic clades is further supported in our work. The framework, in which this report is included, shows that russuloid mycorrhizae are one of the most dominant representative ectomycorrhizae formed on 1-2 years seedlings of Pinus yunnanensis.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

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.