Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella.

Delimitation of species and the search for a proper threshold for defining phylogenetic species in fungi are under discussion. In this study, morphological and molecular data are correlated to delimit species of Tulasnella, the most important mycobionts of Orchidaceae, which suffer from poor taxonomy. Resupinate basidiomata of Tulasnella species were collected in Ecuador and Germany, and 11 specimens (seven from Ecuador, four from Germany) were assigned to traditional species concepts by use of morphological keys. The specimens were compared by micro-anatomical examination with 75 specimens of Tulasnella borrowed from fungaria to obtain better insights on variation of characters. Sequences of the ITS region (127) were obtained after cloning from the fresh basidiomata and from pure cultures. Proportional variability of ITS sequences was analyzed within and among the cultures and the specimens designated to different morphospecies. Results suggested an intragenomic variation of less than 2%, an intraspecific variation of up to 4% and an interspecific divergence of more than 9% in Tulasnella. Cryptic species in Tulasnella, mostly from Ecuador, were revealed by phylogenetic analyses with 4% intraspecific divergence as a minimum threshold for delimiting species. Conventional diagnostic morphological characters appeared insufficient for species characterization. Arguments are presented for molecular delimitation of the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. cf. pinicola, T. tomaculum and T. violea.

The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids.

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co-evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.

Atractiellomycetes belonging to the 'rust' lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids.

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the 'rust' lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.

Structural characterization and molecular identification of arbuscular mycorrhiza morphotypes of Alzatea verticillata (Alzateaceae), a prominent tree in the tropical mountain rain forest of South Ecuador.

The vast majority of the highly diverse trees in the tropical mountain rain forest of South Ecuador form arbuscular mycorrhizas, and previous molecular investigations revealed a high diversity of fungi. In this study, we present a first trial to link fungal DNA-sequences with defined morphotypes characterized on the basis of partly new mycelial features obtained from field material of one tree species, Alzatea verticillata. Fine roots were halved lengthwise to study the mycelium anatomy on one half and to obtain fungal nuclear rDNA coding for the small subunit rRNA of Glomeromycota from the other half. Light microscopy revealed conspicuously large amounts of mycelium attaching to the surface of the rootlets. The mycelium formed fine- or large-branched appressoria-like plates, vesicles of regular or irregular shape, and very fine, multibranched structures ensheathed by septate hyphae. These previously undescribed features of the supraradical mycelia combined with intraradical mycelium structures were used for distinguishing of four main morphogroups and subordinate 14 morphotypes. DNA sequences of Glomus group A, Acaulospora and Gigaspora, were obtained and linked to three morphogroups. Two sequence types within Glomus group A could be tentatively associated to subordinate morphotypes.

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.

Phylogenetic analysis of nuclear small subunit rDNA sequences suggests that the endangered African Pencil Cedar, Juniperus procera, is associated with distinct members of Glomeraceae.

The endangered indigenous tree species Juniperus procera, commonly known as African Pencil Cedar, is an important component of the dry Afromontane vegetation of Ethiopia and was shown to be AM in earlier studies. Here we describe the composition of AM fungi in colonized roots of J. procera from two dry Afromontane forests of Ethiopia. The nuSSU rDNA gene was amplified from colonized roots, cloned and sequenced using AM fungal specific primers that were partly developed for this study. Molecular phylogenetic analysis revealed that all the glomeralean sequences obtained belonged exclusively to the genus Glomus (Glomeraceae). Seven distinct Glomus sequence types were identified that all are new to science. The composition of the AM fungal communities between the sampled trees, and between the two study sites in general, differed significantly. Isolation and utilization of the indigenous AM fungal taxa from the respective sites might be required for successful enrichment plantation of this threatened Juniperus species.

Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador.

Cavendishia nobilis var. capitata is an endemic member of the Ericaceae growing as a hemiepiphyte in the tropical mountain rain forest of southern Ecuador. Mycorrhizas were collected from 20 individuals along an altitudinal gradient between 1850 and 2300 m. Transmission electron microscopy was used to study the symbiotic association in detail, and phylogenetic analyses based on nuclear rDNA coding for the ribosomal large subunit (nucLSU) were carried out to identify the associated mycorrhizal fungi. Microscopic and ultrastructural investigations showed the formation of a hyphal sheath, intercellular penetration of fine hyphae and colonization of the cortical cells by swollen hyphae of the same fungus. These structures were formed by hymenomycetes and ascomycetes. Molecular phylogenetic analysis detected seven groups of mycorrhizal fungi belonging to the Sebacinales. This is the first study to obtain evidence of ectendomycorrhizas in the Vaccinioideae. The ascomycetous nucLSU sequences belonged to members of the Leotiomycetes. The ectendomycorrhiza of C. nobilis with Sebacinales is discussed as a specific, hitherto undescribed mycorrhizal subcategory of ectomycorrhizas. We propose the term 'cavendishioid mycorrhiza'. This subcategory is most likely specific for the Andean clade of Ericaceae.

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.

Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry.

The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver

Subcellular localization of copper in the root cells of Allium sativum by electron energy loss spectroscopy (EELS).

Ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cu (1, 10 and 100 microM) for 9 days was carried out using transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The results presented here indicate that excess Cu induces ultrastructural changes such as strong vacuolization, condensed nuclear chromatin, decreased endoplasmic reticulum (ER) and ribosome and serious plasmolysis. EELS analysis indicated that electron dense granules containing Cu appeared in the cells after Cu treatment. The vacuoles of the root tip cells were the main Cu-accumulation site. Small amounts of copper were also localized to cytoplasmic vesicles or cell walls of cortical cells. The results of the present investigation have significant importance in further understanding the mechanisms of absorption, transportation and accumulation of heavy metals in plants grown in polluted soil.

Molecular diversity of arbuscular mycorrhizal fungi in Prunus africana, an endangered medicinal tree species in dry Afromontane forests of Ethiopia.

• The molecular diversity of arbuscular mycorrhizal (AM) fungi colonizing roots of Prunus africana and of AM fungal spores obtained from baiting cultures of indigenous soils from two dry afromontane forests of Ethiopia was investigated. • The internal transcribed spacer (ITS) rDNA region from colonized roots and single spores of three AM fungal spore types was amplified, cloned and sequenced using AM fungal specific primers. • Phylogenetic analysis using the 5.8S rDNA data set revealed that 109 of the sequences obtained belong to members of the Glomeromycota. Subsequent 5.8S/ITS2 rDNA sequence analysis indicated high AM fungal diversity and dominance of Glomus types. Twenty sequence types belonged to the Glomeraceae and one each to the Diversisporaceae and Archaeosporaceae. Two of the three spore types were identified as Glomus etunicatum and Glomus mosseae. • Twenty of the AM fungal types identified are new to Ethiopia and to science. The AM fungal community differed between the two sites studied.

Heterobasidiomycetes form symbiotic associations with hepatics: Jungermanniales have sebacinoid mycobionts while Aneura pinguis (Metzgeriales) is associated with a Tulasnella species.

In order to evaluate substrate dependence of the symbiotic fungal associations in leafy liverworts (Jungermanniopsida), 28 species out of 12 families were investigated by transmission electron microscopy and molecular methods. Samples were obtained from the diverse substrates: from naked soil, from the forest floor on needle litter, from between peat moss, from rotten bark of standing trees, and from stumps and rotten wood. Associations with ascomycetes were found in most of the specimens independent from the substrate. Seven species sampled from soil were found to contain basidiomycete hyphae. Ultrastructure consistently showed dolipores with imperforate parenthesomes. Molecular phylogenetic studies revealed that three specimens belonging to the Jungermanniales were associated with members of Sebacinaceae, while Aneura pinguis (Metzgeriales) was associated with a Tulasnella species. These taxa are so far the only basidiomycetes known to be symbiotically associated with leafy liverworts. The probability that the associations with Sebacinaceae are evolutionary old, but the Tulasnella associations more derived is discussed. The sebacinoid mycobionts form a similar interaction type with the jungermannialian leafy liverworts as do the associated ascomycetes. The term 'jungermannioid mycorrhiza' is proposed for this distinctive symbiotic interaction type.

Subcellular localization of Cd in the root cells of Allium sativum by electron energy loss spectroscopy.

The ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cd (100 mM, 1 mM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.

The effect of drought on mycorrhizas of beech (Fagus sylvatica L.): changes in community structure, and the content of carbohydrates and nitrogen storage bodies of the fungi.

In a water-exclusion experiment, five different ecotypes of beech (Fagus sylvatica L.; representing regions of different environmental and climatic conditions in Baden-Württemberg, Germany) were subjected to drought conditions of different severity between July and September of two consecutive years. Drought stress as characterised by the water content and the pre-dawn water potential of the leaves was related to the degree of mycorrhization, the type of ectomycorrhiza, and the physiological properties of individual fungus/plant interactions at the fine roots of different beech ecotypes. Our data show that decreased soil water availability did not significantly change either the degree of fungal colonisation of beech roots (measured by the amount of ergosterol) or the number of ectomycorrhizal types per root system. Drought did, however, have an influence on the composition of the ectomycorrhizal community, and different mycorrhizal types responded to drought differently in terms of their patterns of occurrence/abundance. While the abundance of the dominant mycorrhizal types, formed with Byssocorticium atrovirens and Lactarius subdulcis, was not affected, drought increased the abundance of mycorrhiza formed between beech and Xerocomus chrysenteron. A detailed analysis of plant and fungal carbohydrates in mycorrhizas indicated that different drought intensities led to distinguishable responses. In plants exhibiting a pre-dawn water potential of down to -1.96 MPa, drought caused the accumulation of sucrose, glucose and fructose, and of fungus-specific compounds such as mannitol and arabitol in mycorrhizal roots at the expense of, e.g. trehalose. The accumulation of sugar alcohols, which constitute compatible solutes known to counteract drought stress, was species-specific. Mycorrhizas with X. chrysenteron formed large amounts of arabitol, while those with L. subdulcis accumulated mannitol. Sustained partitioning of carbon towards the mycorrhizal fungi under drought was also reflected by an increase of nitrogen storage in the fungal vacuoles. In treatments where the pre-dawn water potential reached values of as low as -2.4 MPa, such alterations were no longer found. In such plants, the starch and soluble sugars content was generally reduced, which also resulted in a lack of increase in protective, fungus-specific sugar alcohols. In summary, the data show that, within certain limits, an increase in drought causes a shift in plant/fungus communities. The shift in the pattern of fungus-specific compounds could possibly be used as a sensitive measure of physiological stress imposed on this symbiosis.

Studies on the root associations of the truffle Terfezia terfezioides.

The paper contains an overview of the results of the studies made on the truffle Terfezia terfezioides, particularly the investigations related to the associations of this fungus with plants. Twelve plant species originated from a natural habitat of the fungus were supposed to be connected with T. terfezioides based on the anatomy of the endogenous fungal structures in their roots. Aseptic experiments were carried out on modified MMN substrates with different phosphate concentrations to study the interaction of T. terfezioides with Robinia pseudoacacia and Helianthemum ovatum. The colonization of the roots of black locust was always weaker than that of Helianthemum. The main characteristics were the intracellular coiled, branched, frequently septated hyphae in dead root cells. The intercellular hyphae formed Hartig-net with finger like structures only in Helianthemum, the interactions could not be considered unambiguously as mycorrhizae. There was no difference between the RFLP profiles of the nr DNA ITS of nineteen fruit bodies collected at the same time from the habitat and the ITS of three randomly chosen specimens were identical on sequence level, too. These invariability makes to design species specific PCR primers possible to check unambiguously the host plants.

Ultrastructural element localization by EDXS in Empetrum nigrum.

Empetrum nigrum L. is one of the few species growing on highly polluted areas in the northern boreal forests and it accumulates considerable amounts of heavy metals especially in its older stems. Previous-year stems of Empetrum nigrum were collected from two different sites located at distances of 0.5km (highly contaminated) and 8km (low contaminated) from a Cu--Ni smelter at Harjavalta, SW Finland. The element (Al, As, Cu, Fe, Mn, Zn, Ca, K, P, S, Mg, Na) localization was performed by energy-dispersive X-ray spectroscopy (EDXS) after cryofixation, freeze-drying and pressure infiltration of the material. The results showed higher levels of Cu, As and Fe in cell compartments of E. nigrum close to the smelter than at further distance. The Al and Zn levels, in contrast, showed no clear differences between the sites. Cu was distributed homogeneously in the tissue and occurred in vacuoles, cytoplasm, cell walls as well as in lumens of the vascular tissue. The higher amounts of As were localized in the outer regions of the stem cross-section and the amounts were higher in the primary cell walls of living (ray cells, phloem) than dead cells (xylem, sclereids). Ray cells, phloem and sclereids had elevated Fe amounts compared to the other tissues in the contaminated stem samples but owing to the high variation between the replicates, no significant differences were found. Based on the rather homogeneous localization of Cu, As and Fe in the living tissue and increased levels of Cu, As and Fe in vacuoles, cell walls and cytoplasm near the smelter, it seems that more than one specific mechanism contribute to the heavy metal tolerance of E. nigrum. Macronutrients did not show clear differences between the two distances or connection to heavy metal localization. Neither the role of complexing agents in heavy metal tolerance in the cytoplasm or vacuoles could be shown by this study. Because of the more frequent localization of electron dense phenolic material in the polluted samples, it might also have a function in the heavy metal tolerance of E. nigrum.