Hyphal Mantle Research Articles

Description, identification, and growth of Tuber borchii Vittad. mycorrhized Pinus sylvestris L. seedlings on different lime contents.

Tuber borchii forms ectomycorrhiza with oaks, hazel, and pines, including Pinus sylvestris. However, its ectomycorrhiza morphotype with P. sylvestris was not comprehensively described so far, and molecular analyses are missing despite a high danger of misidentification of T. borchii ectomycorrhiza with other closely related and less valuable truffle species. We described for the first time the morphology and anatomy of T. borchii-P. sylvestris ectomycorrhiza using differential interference contrast technique and semi-thin sections in combination with molecular confirmation of identity. Color of ectomycorrhiza is reddish to dark brown, and morphotypes are unevenly but densely covered by warts-bearing pin-like cystidia. All layers of the hyphal mantle are pseudoparenchymatous with outer mantle layer formed of epidermoid cells. T. borchii ectomycorrhiza was identified by a molecular comparison with fruitbodies used for inoculation and its respective ectomycorrhizae. T. borchii has a wide ecological amplitude. To get a better insight in mycorrhization requirements, we investigated growth of P. sylvestris and its ectomycorrhiza infection rate with T. borchii in substrate with different lime content. The mycorrhization of P. sylvestris with T. borchii in the mycorrhization substrate and cultivation in greenhouse conditions was successful, with colonization of P. sylvestris varying between 36.5 and 48.1%. There was no significant correlation of mycorrhization to applied lime contents, and consequently to pH in substrate, while the increased levels of lime improved growth of the P. sylvestris seedlings.

An unusual specimen of the enigmatic fungal reproductive unit Windipila spinifera from the Lower Devonian Rhynie cherts of Scotland

Windipila spinifera from the Rhynie cherts is a spheroidal microfossil enveloped in a hyphal mantle from which extend prominent spines and otherwise shaped projections. It is believed to be a reproductive unit of a fungus in the Glomeromycota or zygomycetes, but features to determine the systematic affinities have not hitherto been documented. This study describes a new specimen of W. spinifera that contains a single spherical structure from which a hypha arises that extends outside and terminates in what appears to be a sporangium. The specimen is reminiscent of germinated zygospores of the germ-sporangial type, and thus may suggest affinities of W. spinifera to the zygomycetes. However, the interior sphere and its outgrowth could also be a part of another organism that had invaded W. spinifera.

Sieverdingia gen. nov., S. tortuosa comb. nov., and Diversispora peloponnesiaca sp. nov. in the Diversisporaceae (Glomeromycota)

Phylogenetic analyses of 18S–ITS–28S nuc rDNA sequences indicated that the arbuscular mycorrhizal fungus originally described as Glomus tortuosum and later transferred to the genus Corymbiglomus represents a separate, previously unrecognized clade at the rank of genus in the family Diversisporaceae (order Diversisporales, phylum Glomeromycota). The analyses located the clade between clades representing the genera Desertispora and Redeckera. Consequently, a new genus, Sieverdingia, was erected, with S. tortuosa comb. nov. The unique morphological feature of S. tortuosa is the formation of glomoid-like spores with a single-layered spore wall covered with a hyphal mantle. Importantly, the erection of Sieverdingia clarified the definition of Corymbiglomus, which currently consists of three species producing glomoid-like spores with one, three- to four-layered spore wall. The features of the innermost layer, which is hyaline, laminate, flexible to semi-flexible, indicate that it is a synapomorphy of Corymbiglomus. The definitions of Corymbiglomus and its species were emended. Moreover, the distribution of S. tortuosa and the three species of Corymbiglomus was discussed based on own studies, literature data, and molecular sequences deposited in public databases. We concluded that the distribution of S. tortuosa and C. globiferum known in environmental studies based on their partial 28S nuc rDNA sequences only may be understated because the main molecular characteristics distinguishing these species reside outside the 28S region. Finally, we described a new species in the genus Diversispora originating from Mediterranean dunes of the Peloponnese peninsula, Greece. The same phylogenetic analyses mentioned above indicated that the closest relative of the new species, producing dark-coloured spores, is D. clara, whose spores are creamy white at most.

Multigene phylogeny of Endogonales, an early diverging lineage offungi associated with plants

Endogonales is a lineage of early diverging fungi within Mucoromycota. Many species in this order produce small sporophores (“sporocarps”) containing a large number of zygospores, and many species form symbioses with plants. However, due to limited collections, subtle morphological differentiation, difficulties in growing these organisms in vitro, and idiosyncrasies in their rDNA that make PCR amplification difficult, the systematics and character evolution of these fungi have been challenging to resolve. To overcome these challenges we generated a multigene phylogeny of Endogonales using sporophores collected over the past three decades from four continents. Our results show that Endogonales harbour significant undescribed diversity and form two deeply divergent and well-supported phylogenetic clades, which we delimit as the families Endogonaceae and Densosporaceae fam. nov. The family Densosporaceae consists of the genus Densospora, Sphaerocreas pubescens, and many diverse lineages known only from environmental DNA sequences of plant-endosymbiotic fungi. Within Endogonaceae there are two clades. One corresponds to Endogone and includes the type species, E. pisiformis. Species of Endogone are characterized by above- and below-ground sporophores, a hollow and infolded sporophore form, a loose zygosporangial hyphal mantle, homogeneous gametangia, and an enigmatic trophic mode with no evidence of ectomycorrhizal association for most species. For the other clade we introduce a new generic name, Jimgerdemannia gen. nov. Members of that genus (J. flammicorona and J. lactiflua species complexes, and an undescribed species) are characterized by hypogeous sporophores with a solid gleba, a well-developed zygosporangial hyphal mantle, heterogeneous gametangia, and an ectomycorrhizal trophic mode. Future studies on Densosporaceae and Endogonaceae will be important for understanding fungal innovations including evolution of macroscopic sporophores and symbioses with plants.

Sacculospora felinovii, a novel arbuscular mycorrhizal fungal species (Glomeromycota) from dunes on the west coast of India

During an arbuscular mycorrhiza fungal spore survey on a primary coastal sand-dune system in Goa on the west coast of India, entrophosporoid spores tightly covered with a dense hyphal mantle were recovered. When intact, the spores, at first sight, seemed to be identical in morphology to those of Sacculospora baltica (originally described as Entrophospora baltica) extracted from Polish maritime sand dunes and, to date, the sole member of the recently described genus Sacculospora in the new family Sacculosporaceae, phylum Glomeromycota. Later detailed morphological studies indicated that both fungi produce two-walled spores but the structure and phenotypic features of components of the outer spore wall in the novel fungus differ considerably from those of S. baltica. Differences between the fungi were subsequently confirmed in the phylogenetic analysis of SSU–ITS–LSU nrDNA sequences. Consequently, we describe the novel species as Sacculospora felinoviisp. nov.

Morphology and ontogenetic development of Zwergimyces vestitus, a fungal reproductive unit enveloped in a hyphal mantle from the Lower Devonian Rhynie chert

Zwergimyces vestitus, a fungal reproductive unit enveloped in a mantle of interlaced circumferential hyphae from the Lower Devonian Rhynie chert, was formally described based on limited information obtained from six specimens. In this paper, we provide additional insights into the organization of Z. vestitus, together with specific developmental details, from >100 new specimens. A marked expansion of the hyphal mantle may occur on one side of this reproductive unit. Moreover, the units appear to have been embedded in a loose meshwork of delicate hyphae. Variations in the organization of the mantle among the specimens suggest mantle formation took place by repeated branching of hyphae on the surface of the developing unit and by additional hyphae extending between the pre-existing mantle hyphae. What was initially described as one non-hyphal wall lining the inner surface of the mantle is now demonstrated as an outer sporangium wall, which continues into the subtending hypha, and an inner spore wall. The diagnoses for Zwergimyces and Z. vestitus are emended. Small spherules in several Z. vestitus specimens are interpreted as intrusive organisms. The new material of Z. vestitus provides the first insights into the ontogenetic development of one of the Rhynie chert mantled fungal reproductive units.

Hartig' net formation of Tricholoma vaccinum-spruce ectomycorrhiza in hydroponic cultures.

For re-forestation of metal-contaminated land, ectomycorrhizal trees may provide a solution. Hence, the study of the interaction is necessary to allow for comprehensive understanding of the mutually symbiotic features. On a structural level, hyphal mantle and the Hartig' net formed in the root apoplast are essential for plant protection and mycorrhizal functioning. As a model, we used the basidiomycete Tricholoma vaccinum and its host spruce (Picea abies). Using an optimized hydroponic cultivation system, both features could be visualized and lower stress response of the tree was obtained in non-challenged cultivation. Larger spaces in the apoplasts could be shown with high statistical significance. The easy accessibility will allow to address metal stress or molecular responses in both partners. Additionally, the proposed cultivation system will enable for other experimental applications like addressing flooding, biological interactions with helper bacteria, chemical signaling, or other biotic or abiotic challenges relevant in the natural habitat.

Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza

Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown.

Fungal reproductive units enveloped in a hyphal mantle from the Lower Pennsylvanian of Great Britain, and their relevance to our understanding of Carboniferous fungal “sporocarps”

An assemblage of distinctive fungal reproductive units occurs in a fragment of permineralized xylem from the Lower Coal Measures (Carboniferous) of Great Britain. Specimens are spherical to irregular in outline, 70–95μm in diameter, and consist of a central cavity surrounded by an investment of tightly interlaced hyphae. Clusters of specimens may additionally be enveloped in a confluent hyphal meshwork. This complement of features is quite similar to the morphology seen in mantled zygosporangia/zygospores of certain extant zygomycetous fungi. Closely associated with several of the reproductive units are single or paired smaller spherical to elongate structures, which we interpret as apposed gametangia. This discovery represents only the third record of zygomycetous fungi from the Carboniferous. Moreover, the reproductive units resemble certain Carboniferous microfossils termed fungal sporocarps in the genus Mycocarpon, and thus indirectly support the hypothesis that the systematic affinities of these sporocarps may lie with the zygomycetous fungi.

Morphology, anatomy, and molecular studies of the ectomycorrhiza formed axenically by the fungus Sistotrema sp. (Basidiomycota)

Several species of the corticioid (resupinate) genus Sistotrema of the cantharelloid clade (Basidiomycota) were recently found to be ectomycorrhizal. This changed the traditional assertion that all Sistotrema species are strictly wood rotting and suggests that the genus may be polyphyletic. In the present investigation, a still unknown root tip-associated fungal specimen (EW63) was isolated and found to be associated with an above-ground fruiting body. Sequencing of the ITS and the nucLSU DNA regions and phylogenetic analyses verified that the root-associated fungus and the fruiting body represented the same species, which was found to belong to the genus Sistotrema. To prove the ectomycorrhizal status of this strain, axenic Pinus sylvestris resyntheses in flask cultures were conducted. Growth parameters of the seedlings were determined and the morphology and anatomy of the synthesized mycorrhizas were described. Length and dry mass of the Pinus shoot as well as those of the total root tips were found to be enhanced as a result of the mycorrhizal association. Mycorrhizal frequency was high (51.5%) in these cultures. Mycorrhizal root tips were cottony light ochre with a thin plectenchymatic hyphal mantle. The clamps of the fruiting body hyphae as well as the mycorrhiza were ampullately inflated. This is the first report proving in axenic culture that a fungus belonging to the genus Sistotrema forms true ectomycorrhiza.

Asexual reproduction of Hygrophorus olivaceoalbus by intracellular microsclerotia in root cells of Picea abies – A winner of ozone stress?

Hygrophorus olivaceoalbus has long been known as an ectomycorrhizal fungus, formerly designated with the artificial binomen Piceirhiza gelatinosa. Recently, it has been found to be abundant and very frequent under double ambient ozone free air fumigation of mature Norway spruce trees. As it has already been reported that this fungus can form intercellular hyphae within the root meristem, a more detailed study was performed to clarify its type of root colonization. The present study not only revealed intercellular hyphae within the meristematic zone but also intracellular hyphae within root cortex cells which grow towards and into large tannin droplets—phenolic compounds usually deposited as defensive aids—to finally fill them completely. The hyphal assemblages become globular which at first sit as separated hyphal balls within cells still containing cytoplasm. Later on, they are apparently released from the root cells, presumably as microsclerotia for dispersal of the species. Old ectomycorrhizae (ECM) show an apical pore, and later a large orifice of a tube-like cylinder formed by the thick, persistent hyphal mantle. The root tissue is progressively degrading towards proximal parts. Disintegrating root cells apparently liberate the microsclerotia through the orifice. Further studies have to find out the mechanism by which the microsclerotia are liberated and whether they operate as asexual propagules and lastly how and by whom they are propagated. As these ECM are found under ozone stress, and with identical features at higher altitudes, stress impact on trees might be the causative agent for the high frequency and abundance of Hygrophorus olivaceoalbus ECM.

The ectomycorrhizal morphotype Pinirhiza sclerotia is formed by Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii

Relatively few ectomycorrhizal fungal species are known to form sclerotia. Usually, sclerotia are initiated at the extraradical mycelium. In this study, we present anatomical and ultrastructural evidence for the formation of sclerotia directly in the hyphal mantle of the mycorrhizal morphotype Pinirhiza sclerotia. A dark-pigmented fungal strain was isolated from Pinirhiza sclerotia and identified by molecular tools as Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii s.l. As dark septate fungi are known to be mostly endophytic, resyntheses with Pinus sylvestris and A. macrosclerotiorum as well as Populus tremula x Populus tremuloides and A. macrosclerotiorum or P. fortinii s.l. were performed under axenic conditions. No mycorrhizas were found when hybrid aspen was inoculated with A. macrosclerotiorum or P. fortinii. However, A. macrosclerotiorum formed true ectomycorrhizas in vitro with P. sylvestris. Anatomical and ultrastructural features of this ectomycorrhiza are presented. The natural and synthesized ectomycorrhizal morphotypes were identical and characterized by a thin hyphal mantle that bore sclerotia in a later ontogenetic stage. The Hartig net was well-developed and grew up to the endodermis. To our knowledge, this is the first evidence at the anatomical and ultrastructural level that a close relative of P. fortinii s.l. forms true ectomycorrhizas with a coniferous host.

The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress.

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (−50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.

Hydraulic redistribution of water from Pinus ponderosa trees to seedlings: evidence for an ectomycorrhizal pathway

While there is strong evidence for hydraulic redistribution (HR) of soil water by trees, it is not known if common mycorrhizal networks (CMN) can facilitate HR from mature trees to seedlings under field conditions. Ponderosa pine (Pinus ponderosa) seedlings were planted into root-excluding 61-microm mesh barrier chambers buried in an old-growth pine forest. After 2 yr, several mature trees were cut and water enriched in D(2)O and acid fuchsin dye was applied to the stumps. Fine roots and mycorrhizal root tips of source trees became heavily dyed, indicating reverse sap flow in root xylem transported water from stems throughout root systems to the root hyphal mantle that interfaces with CMN. Within 3 d, D(2)O was found in mesh-chamber seedling foliage > 1 m from source trees; after 3 wk, eight of 10 mesh-chamber seedling stem samples were significantly enriched above background levels. Average mesh-chamber enrichment was 1.8 x greater than that for two seedlings for which the connections to CMN were broken by trenching before D(2)O application. Even small amounts of water provided to mycorrhizas by HR may maintain hyphal viability and facilitate nutrient uptake under drying conditions, which may provide an advantage to seedlings hydraulically linked by CMN to large trees.

Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populus×canescens

In order to characterise the effect of ectomycorrhiza on Na+-responses of the salt-sensitive poplar hybrid Populus x canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na+ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na+ and Cl- in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na+ and Cl- concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H2O2 in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H2O2 formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na+ and increased supply with K+.

Formation of Mycorrhiza-like Structures in Cultured Root/Callus of Cathaya argyrophylla Chun et Kuang Infected with the Ectomycorrhizal Fungus Cenococcum geophilum Fr.

An in vitro system was used for ectomycorrhizal synthesis of Cenococcum geophilum Fr. with Cathaya argyrophylla Chun et Kuang, an endangered species. Calli initiated from stem segments and adventitious roots differentiated from young seedlings were removed and cocultured with Cenococcum geophilum on a modified Murashige-Skoog medium. Fungal hyphae were visible within intercellular spaces of the callus 4 weeks after inoculation, but definite and well-developed Hartig net structures did not form in the calli 8 weeks after inoculation. The typical ectomycorrhizal structures (i.e. hyphal mantle and intracortical Hartig net) were observed in root segments 8 weeks after inoculation. This is the first report of aseptic ectomycorrhizal-like formation/infection between root organ/callus of Cathaya argyrophylla and the ectomycorrhizal fungus Cenococcum geophilum. This culture system is useful for further investigation of mycorrhizal synthesis in Cathaya trees. (Managing editor: Ya-Qin Han)

Changes in polyamine content and localization of Pinus sylvestris ADC and Suillus variegatus ODC mRNA transcripts during the formation of mycorrhizal interaction in an in vitro cultivation system

The involvement of polyamines (PAs) in the interaction between Pinus sylvestris L. seedlings and an ectomycorrhizal fungus Suillus variegatus (Swatz: Fr.) O. Kunze was studied in an in vitro cultivation system. PA concentrations in seedlings were analysed after 1, 3, and 5 weeks in dual culture with S. variegatus, and changes in PA pools were compared with the growth of the seedlings. Pinus sylvestris arginine decarboxylase (ADC) and S. variegatus ornithine decarboxylase (ODC) mRNA transcripts were localized during the formation of mycorrhizas. During mycorrhiza formation, Suillus variegatus ODC transcripts were found in developing hyphal mantle and Hartig net, and P. sylvestris ADC transcripts in specific root parenchyma cells adjacent to tracheids and in mitotic cells of the root apical meristem. However, no unambiguous difference in ADC transcript localization between inoculated and non-inoculated roots was observed. Regardless of the unchanged distribution of ADC transcripts, inoculation with S. variegatus increased free putrescine, spermidine, and spermine concentrations in roots within the first week in dual culture. The concentration of free and conjugated putrescine and conjugated spermidine also increased in the needles due to the fungus. The fungus-induced lateral root formation and main root elongation were greatest between the first and third week in dual culture, coinciding with retarded accumulation or a decrease of free PAs. These results show that accumulation of PAs in the host plant is one of the first indicators of the establishment of ectomycorrhizal interaction between P. sylvestris and S. variegatus in the in vitro system.

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.

Russulaceae and Thelephoraceae form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical mountain rain forest of southern Ecuador

* Three members of the Nyctaginaceae, two Neea species and one Guapira species, occurred scattered within a very species-rich neotropical mountain rain forest. The three species were found to form ectomycorrhizas of very distinctive characters, while all other tree species examined formed arbuscular mycorrhizas. * The ectomycorrhizas were structurally typified according to light and transmission electron microscope investigations. The internal transcribed spacer (ITS) rDNA and part of the nuclear large subunit (LSU, 28S) rDNA of the mycorrhiza forming fungi were amplified and sequenced. Phylogenetic analyses were carried out. * Neea species 1 was found to form typical ectomycorrhizas with five different fungal species, Russula puiggarii, Lactarius sp., two Tomentella or Thelephora species, and one ascomycete. Neea species 2 and the Guapira species were associated with only one fungus each, a Tomentella/Thelephora species clustering closely together in an ITS-neighbour-joining tree. The long and fine rootlets of the Guapira species showed proximally a hyphal mantle and a Hartig net, but distally intracellular fungal colonization of the epidermis and root hair development. The ectomycorrhizal segments of the long roots of Neea species 2 displayed a hyphal mantle and a Hartig net around alive root-hair-like outgrowths of the epidermal cells. * The distribution and the evolution of ectomycorrhizas in the predominantly neotropic Nyctaginaceae are discussed.

Graffenrieda emarginata (Melastomataceae) forms mycorrhizas with Glomeromycota and with a member of the Hymenoscyphus ericae aggregate in the organic soil of a neotropical mountain rain forest

Graffenrieda emarginata (Ruiz & Pav.) Triana (Melastomataceae) was found to be the most frequent tree on the nutrient-poor organic soil in a species-rich tropical mountain rain forest of southern Ecuador. Our studies revealed that the rootlets are symbiotically associated with arbuscular mycorrhizal fungi and simultaneously form ectomy corrhizas displaying a superficial Hartig net but no hyphal mantle. Molecular phylogeny revealed a multitude of previously unknown glomeromycete sequence types. Ultrastructure of the ectomycorrhiza-forming fungus indicated an ascomycete, and molecular phylogenetic studies identified the fungal symbiont as a new taxon within the Hymenoscy phus ericae (Read) Korf & Kernan aggregate (Ascomycota). The mycorrhizal state of G. emarginata is considered crucial for its competitiveness and abundance in these sites.Key words: arbuscular mycorrhiza, ectomycorrhiza, Glomeromycota, Hymenoscyphus ericae aggregate, neotropical mountain rain forest, Ecuador.