Morphological-anatomical characterization and molecular identification of Tomentella stuposa ectomycorrhizae and related anatomotypes

Species in the genus Tomentella (Thelephoraceae) belong to the most frequent and widespread ectomycorrhizal (EM) fungi found in temperate and boreal forests. Although several unidentified tomentelloid morphotypes have been presented as common members of EM communities in coniferous and broad-leaved forests, few tomentelloid EM have been identified and described in detail. In this study, ten tomentelloid EM isolates collected from Populus alba, Quercus cerris and Picea abies stands in Hungary and Germany are characterized and documented by morphological-anatomical methods using light microscopy. The investigated ectomycorrhizae belong to the same brown-black tomentelloid morphotype but form two different anatomotype groups (At I and At II). Molecular taxonomical identification was accomplished using phylogenetic analysis (neighbor joining method) of 49 Tomentella nrDNA-ITS nucleotide sequences including the 10 new and 39 GenBank sequences. The EM isolates clustered into two adjoining clades identical with the two anatomotypes. At II clustered with Tomentella stuposa while At I could not be identified to species. Based on the morphological similarity and the low genetic difference it must be a closely related taxon. A comparison of the recently known tomentelloid EM to T. stuposa is presented. Ecological questions involving abundance and host relationships are discussed.     

Three new <Emphasis Type="Italic">Tomentella</Emphasis> species from West Africa identified by anatomical and molecular data

We used a combination of molecular-phylogenetic inference of 82 ITS rDNA sequences and anatomical approach to describe three new west African thelephoroid species, namely Tomentella afrostuposa, T. guineensis and T. guinkoi. Anatomically, T. afrostuposa is reminiscent of T. stuposa with globose to broadly ellipsoid large basidiospores of 8–14 μm, long aculei of up to 3 μm and prominent apiculi of 2 μm width. Molecular-phylogenetically, it falls within the T. stuposa complex. However, T. afrostuposa deviates by at least 7.80–10.74% from T. stuposa in regard with the ITS rDNA sequences. Tomentella guineensis is characterised by long (up to 85 μm) utriform basidia, the presence of reniform basidiospores in lateral view (up to 9 μm) with aculei not exceeding 1 μm and a strong cyanescent reaction of the subhymenial hyphae and basidia in 2.5% KOH. It forms a sister species of the newly described species Tomentella maroana; however, deviating from the last species by at least 9.75–10.04%. The very short, inflated (up to 14 μm) and thick-walled septate (septa up to 1.5 μm) subhymenial hyphae combined with ellipsoid basidiospores (up to 8 μm) and short aculei not exceeding 0.5 μm characterise Tomentella guinkoi. Anatomically, T. guinkoi recalls T. ellisii. Genetic distance between both species ranges from 12.67 to 13.73% according to ITS rDNA sequences analyses. Tomentella guinkoi forms a sister species of the group composed of T. ellisii, T. hjortstamiana and T. pisoniae. Detailed anatomical comparisons between the newly described species and their close relatives are given.     

Studies in African thelephoroid fungi: 1. <Emphasis Type="Italic">Tomentella capitata</Emphasis> and <Emphasis Type="Italic">Tomentella brunneocystidia</Emphasis>, two new species from Benin (West Africa) with capitate cystidia

This paper presents Tomentella capitata and Tomentella brunneocystidia as new species based on molecular data and anatomical features. Both T. capitata and T. brunneocystidia form sister species with Tomentella pilosa. All three taxa are well supported by bootstrap values. Anatomically, T. capitata and T. brunneocystidia are very close and are similar in shape, size, ornamentation of basidiospores, and size and colour of subicular hyphae. Monomitic rhizomorphs sometimes covered by irregularly shaped thin hyphae are present in both species. Shape and pigmentation of the cystidia are the most discriminating features between T. capitata and T. brunneocystidia. The cystidia of T. capitata are maximum 35 μm long, show a distinctive globose apex and are sometimes covered with dark brown pigmentation and/or encrustation, whereas cystidia of T. brunneocystidia are bigger, up to 55 μm long, with a sub-capitate shape and dark blue to dark green contents all over their length. The differences to species, already described as having capitate and clavate cystidia, are discussed. A key for the identification of cystidioid Tomentella species is given. Taxonomical novelties: Tomentella capitata Yorou & Agerer, Tomentella brunneocystidia Yorou & Agerer.     

Ectomycorrhizae of Tomentella albomarginata (Thelephoraceae) on Scots pine

 The ectomycorrhizae of Tomentella albomarginata are comprehensively described and compared to ectomycorrhizae of other Tomentella species and to ectomycorrhizae of some members of Thelephoraceae ss. Stalpers and Bankeraceae ss. Stalpers. The ectomycorrhizae of T. albomarginata are characterized by a hyphal net lying on a pseudoparenchymatous mantle surface, by tubular outgrowths of irregularly angular mantle cells, and by clamp-bearing emanating hyphae. Accepted: 15 July 1995     

Resistance to photoinhibition of photosystem II and catalase and antioxidative protection in high mountain plants

In leaves of three alpine high mountain plants, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, both photosystem II (PSII) and the enzyme catalase appeared to he highly resistant to photoinactivation under natural field conditions. While the Dl protein of PSII and catalase have a rapid turnover in light and require continuous new protein synthesis in non-adapted plants, little apparent photoinactivation of PSII or catalase was induced in the alpine plants by translation inhibitors or at low temperature, suggesting that turnover of the Dl protein and catalase was slow in these leaves. In vitro PSII was rapidly inactivated in light in isolated thylakoids from H. alpina and R. glacialis. In isolated intact chloroplasts from R. glacialis, photoinactivation of PSII was slower than in thylakoids. Partially purified catalase from R. glacialis and S. alpina was as sensitive to photoinactivation in vitro as catalases from other sources. Catalase from H. alpina had, however, a 10-fold higher stability in light. The levels of xanthophyll cycle carotenoids, of the antioxidants ascorbate and glulathione, and of the activities of catalase, superoxide dismutase and glutathione reductase were very high in S. alpina, intermediate in H. alpina, but very low in R. glacialis. However, isolated chloroplasts from all three alpine species contained much higher concentrations of ascorbate and glutathione than chloroplasts from lowland plants.     

<Emphasis Type="Italic">Tomentella furcata</Emphasis>, a new species from Benin (West Africa) with basidia forming internal hyphae

In this study, a new tropical African resupinate thelephoroid species, namely Tomentella furcata, is described. Microscopically, it is characterised by mostly simple septate and thick-walled (1–2 μm) subicular and subhymenial hyphae, suburniform basidia with transverse septa and/or infrequently with an internal hypha, large sterigmata of 2–4 μm at base, and distinctly yellow, subglobose to globose basidiospores (both in frontal and lateral view) with long (1–2 μm) and dense, simple or forked spines. Phylogenetically, T. furcata falls within terminal clades of investigated Tomentella species. We discuss ornamentation types of basidiospores for discrimination of resupinate thelepohoroid genera and confirm the limitation of ornament bifurcation as a diagnostic feature of Pseudotomentella. Anatomical dissimilarities between T. furcata and close species are given. Taxonomical novelty: Tomentella furcata Yorou & Agerer.     

Phylogenetic placement and anatomical characterisation of two new West African Tomentella (Basidiomycota, Fungi) species

Tomentella amyloapiculata and T. agbassaensis are described as new species within the genus Tomentella based on materials we collected in the West African, northern Guinean seasonal forests. We used a combination of anatomical characters, sequence analyses and phylogenetic inference of 71 ITS rDNA sequences to characterise the two new species. Anatomically, T. amyloapiculata is characterised by simple septate brown to dark brown, thick-walled subicular and subhymenial hyphae and triangular to slightly lobed brown basidiospores (in frontal view), with isolate aculei of 1–2 μm. Phylogenetically, T. amyloapiculata forms a sister species of T. fuscocinerea with a moderate bootstrap support of 70%. T. amyloapiculata deviates from T. fuscocinerea by 10.07–11.73% in their sequence similarities. As far as T. agbassaensis is concerned, it clusters phylogenetically together with T. bryophila with a strong bootstrap support of 99%. The species is characterised by slightly differentiated rhizomorphs with yellowish hyphae, clamped, thick-walled and yellow to dark yellow subicular hyphae and pale yellow, small basidiospores of 6–8(8.5) μm with aculei of up to 0.5 μm. Both new species deviated from each other by 11.0–11.60% with regard to the ITS rDNA nucleotides.     

Ectomycorrhizal fungi associated with two species of Kobresia in an alpine meadow in the eastern Himalaya

The diversity of ectomycorrhizal fungi (EMF) on Kobresia filicina and Kobresia capillifolia in an alpine meadow in China’s southwestern mountains, one of the word’s hotspots of biodiversity, was estimated based on internal transcribed spacer rDNA sequence analysis of root tips. Seventy EMF operational taxonomical units (OTUs) were found in the two plant species. Dauciform roots with EMF were detected in species of Kobresia for the first time. OTU richness of EMF was high in Tomentella/Thelophora and Inocybe, followed by Cortinarius, Sebacina, the Cenococcum geophilum complex, and Russula. Tomentella/Thelophora and Inocybe were general and dominant mycobiont genera of the two sedges. Besides the C. geophilum complex, the ascomycete components Hymenoscyphus and Lachnum were also detected on the two plants. Alpine plants in different geographical regions share similar main genera and/or families of EMF while harboring predominantly different mycobiont species; most of the members detected by us have not been found elsewhere. Significant differences in the profile of EMF occurrences were not found between the two plant species and among the three sampling seasons in our sample size.     

Growth and reproduction of the alpine grasshopper <Emphasis Type="Italic">Miramella alpina</Emphasis> feeding on CO<Subscript>2</Subscript>-enriched dwarf shrubs at treeline

The consequences for plant-insect interactions of atmospheric changes in alpine ecosystems are not well understood. Here, we tested the effects of elevated CO₂ on leaf quality in two dwarf shrub species (Vaccinium myrtillus and V. uliginosum) and the response of the alpine grasshopper (Miramella alpina) feeding on these plants in a field experiment at the alpine treeline (2,180 m a.s.l.) in Davos, Switzerland. Relative growth rates (RGR) of M. alpina nymphs were lower when they were feeding on V. myrtillus compared to V. uliginosum, and were affected by elevated CO₂ depending on plant species and nymph developmental stage. Changes in RGR correlated with CO₂-induced changes in leaf water, nitrogen, and starch concentrations. Elevated CO₂ resulted in reduced female adult weight irrespective of plant species, and prolonged development time on V. uliginosum only, but there were no significant differences in nymphal mortality. Newly molted adults of M. alpina produced lighter eggs and less secretion (serving as egg protection) under elevated CO₂. When grasshoppers had a choice among four different plant species grown either under ambient or elevated CO₂, V. myrtillus and V. uliginosum consumption increased under elevated CO₂ in females while it decreased in males compared to ambient CO₂-grown leaves. Our findings suggest that rising atmospheric CO₂ distinctly affects leaf chemistry in two important dwarf shrub species at the alpine treeline, leading to changes in feeding behavior, growth, and reproduction of the most important insect herbivore in this system. Changes in plant-grasshopper interactions might have significant long-term impacts on herbivore pressure, community dynamics and ecosystem stability in the alpine treeline ecotone.     

Contrasting phylogeographies inferred for the two alpine sister species Cardamine resedifolia and C. alpina (Brassicaceae)

Aim We use Cardamine alpina and C. resedifolia as models to address the detailed history of disjunctions in the European alpine system. These species grow on siliceous bedrock: C. alpina in the Alps and Pyrenees, and C. resedifolia in several mountain ranges from the Sierra Nevada to the Balkans. We explore differentiation among their disjunct populations as well as within the contiguous Alpine and Pyrenean ranges, and compare the phylogeographical histories of these diploid sister species. We also include samples of the closely related, arctic diploid C. bellidifolia in order to explore its origin and post‐glacial establishment. Location European alpine system, Norway and Iceland. Methods We employed amplified fragment length polymorphisms (AFLPs). AFLP data were analysed using principal coordinates analysis, neighbour joining and Bayesian clustering, and measures of diversity and differentiation were computed. Results For the snow‐bed species C. alpina (27 populations, 203 plants) we resolved two strongly divergent lineages, corresponding to the Alps and the Pyrenees. Although multiple glacial refugia were invoked in the Pyrenees, we inferred only a single one in the Maritime Alps – from which rapid post‐glacial colonization of the entire Alps occurred, accompanied by a strong founder effect. For C. resedifolia (33 populations, 247 plants), which has a broader ecological amplitude and a wider distribution, the genetic structuring was rather weak and did not correspond to the main geographical disjunctions. This species consists of two widespread and largely sympatric main genetic groups (one of them subdivided into four geographically more restricted groups), and frequent secondary contacts exist between them. Main conclusions The conspicuously different histories of these two sister species are likely to be associated with their different ecologies. The more abundant habitats available for C. resedifolia may have increased the probability of its gradual migration during colder periods and also of successful establishment after long‐distance dispersal, whereas C. alpina has been restricted by its dependence on snow‐beds. Surprisingly, the arctic C. bellidifolia formed a very divergent lineage with little variation, contradicting a scenario of recent, post‐glacial migration from the Alps or Pyrenees.     

Morphological and molecular diversity and abundance of tomentelloid ectomycorrhizae in broad-leaved forests of the Hungarian Plain

The most common representatives of tomentelloid ectomycorrhizae (EM) collected in broad-leaved forests (Populus and Quercus) of the Hungarian Plain during a 4-year project are demonstrated. Eighteen specimens of nine tomentelloid EM morphotypes were investigated. Five of these, introduced here for the first time, were characterized by microscopical–morphological and anatomical methods. Molecular identification was carried out using sequence analysis of the nrDNA ITS region. Altogether, 54 ITS sequences (6 previously published and 12 new sequences from our mycorrhizae and 36 from GenBank derived from fruitbodies) were compared by phylogenetic analyses using neighbor-joining, maximum parsimony and maximum likelihood methods. Identification to species level was successful in the case of six EM morphotypes (Tomentella galzinii, T. subtestacea, T. sublilacina, T. pilosa, T. ferruginea and T. stuposa), and the possible taxonomic position of the remaining three was approached. These results are supported by morphology, as compared with literature data. Relative abundance of the EM morphotypes within the soil samples was estimated. Our results confirm that tomentelloid EM are constant, diverse and abundant members of the EM communities in temperate-continental broad-leaved forests.     

Life-form composition of alpine plant communities at the Eastern Qinghai-Tibetan plateau

Alpine plants of the eastern Qinghai-Tibetan plateau (Sichuan, China) are developed under long-lasting grazing by wild and domestic yaks. Among morphological features of plants, life forms may reflect their adaptation to grazing. We studied life-form composition of four typical communities within the alpine belt (3930–3960 m a.s.l.) subjected to grazing of various intensity: alpine fen (heavily grazed), alpine shrub meadow (heavily grazed), Spiraea alpina thicket (grazed), and Rhododendron thicket (practically not grazed). The following morphological traits were studied: (1) life form according to Raunkiaer, (2) life form according to Serebryakov, (3) canopy structure, and (4) rate of lateral spreading. We derived life-form spectra based on (1) the number of species per life form and (2) the cumulative abundance of species which have the same life form. One-way ANOVA and nonparametric ANOVA were run to test for significance of differences between spectra. The studied communities differed significantly by the proportion of different life forms. The main life forms are caudex and short rhizome hemicryptophytes, nonclonal species, or species with a low rate of lateral spreading. Therophytes made up 10–11% of the communities except in Rhododendron thickets, where such were absent. These life forms can indicate grazing in the study area.     

Weak habitat specificity in ectomycorrhizal communities associated with Salix herbacea and Salix polaris in alpine tundra

This study explores mid-alpine ectomycorrhizal communities on Salix herbacea and Salix polaris in plant communities differing in nutrient status and snow conditions. Plant species were identified by tracking roots back to above ground structures while fungal species were identified using molecular methods. The fungi were identified to 34 molecular operational taxonomic units (MOTUs)/species but species accumulation curves indicated that the communities were only partially sampled. The estimated total species richness was 49 (±9 SD) MOTUs/species. No significant ectomycorrhizal community specificity was found between the two plant species and only weak specificity between different plant communities. Furthermore, no difference in proportion of colonized root tips could be demonstrated between plant communities. However, some fungal taxa showed tendencies to associate with specific environmental conditions. Sebacinaceae, Inocybe egenula, Russula cf. emetica, and a Tomentella sp. were found in meadow communities but not in the heath communities. Sistotrema cf. alboluteum and Tomentella cf. terrestris were only found in the dry and mesic heath communities. Classifications into exploration types showed that the contact type is more abundant in the dry heath community than the other communities. Cenococcum geophilum was the most common species but Cortinarius spp., Russula spp., Tomentella spp., and Lactarius spp. were also common. This study confirms that alpine communities are rich in ectomycorrhizal fungi including species from a wide variety of fungal lineages and also show that many dominant species have wide ecological amplitude.     

Stable isotope analysis,field observations and synthesis experiments suggest that Odontia is a non-mycorrhizal sister genus of Tomentella and Thelephora

Ectomycorrhizal symbiosis has evolved multiple times in plants and fungi, but the trophic status of certain fungal groups remains poorly understood due to their unculturability or ambiguous interpretation of biotrophic associations. Combining field observations, molecular identification of root tips, synthesis experiments and analysis of stable isotopes, we address the lifestyle of Tomentella crinalis and another species closely related to T. fibrosa that represents a sister group to the ectomycorrhizal genera Tomentella and Thelephora. Based on molecular analyses these two and other related species are moved to the genus Odontia. In Odontia species, ectomycorrhizal associations were not observed in nature or in various synthesis experiments. Although Odontia species normally fruit in old forests, Odontia ferruginea has also been identified from a deep belowground mine. Unlike saprotrophs, Odontia spp. and ectomycorrhizal fungi were not enriched in ¹³C compared with their woody fruiting substratum, suggesting that wood is not their major energy source. In contrast to ectomycorrhizal fungi, Odontia species and saprotrophs were not enriched in ¹⁵N relative to their substratum. Taken together, we suggest that Odontia spp. are non-mycorrhizal, but their nutrition differs from typical wood-rotting Basidiomycota.     

Contribution of nitrogen fixation to nitrogen nutrition in an alpine sedge community (Caricetum curvulae)

Summary In situ acetylene reduction assays (ARA) were carried out over two growing seasons at 2550 m in the upper alpine zone of the Tyrolean Central Alps of Austria. For comparative purposes, some Fabaceae species introduced into the upper alpine zone from lower elevation (2000 m) were subjected to ARA. At the end of the growing season the potted plants were transferred to the laboratory where their acetylene reducing activities were measured again. In situ nitrogenase activity is very low. The highest values were found in association with Leucanthemopsis alpina and Veronica bellidioides (150 and 217 nmol ethylene 24 h^-1 per pot respectively). Higher levels of activity were detected in pots transferred to the laboratory (maximum value 750 nmol ethylene 24 h^-1 per pot; assay temperature about 12°C higher than in the field) and in the Fabaceae transferred to the upper alpine zone (14×10³ nmol ethylene 24 h^-1 per pot of Trifolium badium and T. pallescens). Maximum nitrogen input in the field is in the range of 8 mg m^-2 a^-1. Therefore, under natural circumstances biological nitrogen fixation contributes only very small amounts of nitrogen to this alpine vegetation system, the process being inhibited by low soil temperatures. Possible alternative sources and patterns of N acquisition are discussed in relation to the overall nitrogen economics of plants of the upper alpine zone.     

Parallel evolution of flower reduction in two alpine Soldanella species (Primulaceae)

The European endemic Soldanella has traditionally been divided into two morphologically well‐defined sections. Section Tubiflores contains two species growing in high‐elevation habitats, whereas most of the 14 species of section Soldanella inhabit montane forests. Section Tubiflores has a reduced floral morphology compared with section Soldanella. A previous phylogenetic study based on internal transcribed spacer (ITS) and AFLP data has revealed that, although the genus Soldanella itself is monophyletic, both sections are paraphyletic. Soldanella alpina (section Soldanella) forms a clade with S. minima and S. pusilla (section Tubiflores), and the grouping of S. alpina with S. pusilla has been hypothesized to be the result of hybridization between S. pusilla and an unidentified species of section Soldanella. We re‐examined the phylogenetic relationships among the above species using additional sequence data (plastid DNA) and increasing the sample for ITS and AFLP data. Our new data confirmed that S. alpina is most closely related to S. pusilla. However, our data do not provide any evidence in support of the hypothesis that S. alpina is a hybrid species. In consequence, we consider it likely that the two species of section Tubiflores originated independently. The reduced floral morphology of these two alpine species is probably evidence for increased levels of self‐pollination, ensuring reproductive success in a high‐altitude habitat. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 409–422.     

Divergent strategies of photoprotection in high-mountain plants

Leaves of high-mountain plants were highly resistant to photoinhibitory damage at low temperature. The roles of different photoprotective mechanisms were compared. Mainly, the alpine species Ranunculus glacialis (L.) and Soldanella alpina were investigated because they appeared to apply greatly divergent strategies of adaptation. The ratio of electron transport rates of photosystem II/photosystem I measured in thylakoids from R. glacialis did not indicate a specific acclimation to high irradiance. Low rates of a chloroplast-mediated inactivation of catalase (EC 1.11.1.6) in red light indicated, however, that less reactive oxygen was released by isolated chloroplasts from R. glacialis than by chloroplasts from lowland plants. Leaves of S. alpina and of Homogyne alpina (L.) Cass, but not those of R. glacialis, had a very high capacity for antioxidative protection, relative to lowland plants, as indicated by a much higher tolerance against paraquat-mediated photooxidative damage and a higher -tocopherol content. Accordingly, ascorbate and glutathione were strongly oxidized and already largely destroyed at low paraquat concentrations in leaves of R. glacialis, but were much less affected in leaves of  S. alpina. Non-radiative dissipation of excitation energy was essential for photoprotection of leaves of  S. alpina and depended on the operation of the xanthophyll cycle. Strong non-photochemical quenching of chlorophyll fluorescence occurred also in R. glacialis leaves at high irradiance, but was largely independent of the presence of zeaxanthin or antheraxanthin. For R. glacialis, photorespiration appeared to provide a strong electron sink and a most essential means of photoprotection, even at low temperature. Application of phosphinothricin, which interferes with photorespiration by inhibition of glutamine synthetase, caused a striking reduction of electron transport through photosystem II and induced marked photoinhibition at both ambient and low temperature in leaves of R. glacialis, while  S. alpina was less affected. Received: 18 March 1998 / Accepted: 7 August 1998     

Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate?

Summary. The cytology of leaf cells from five different high-alpine plants was studied and compared with structures in chloroplasts from the typical high-alpine plant Ranunculus glacialis previously described as having frequent envelope plus stroma protrusions. The plants under investigation ranged from subalpine/alpine Geum montanum through alpine Geum reptans, Poa alpina var. vivipara, and Oxyria digyna to nival Cerastium uniflorum and R. glacialis. The general leaf structure (by light microscopy) and leaf mesophyll cell ultrastructure (by transmission electron microscopy [TEM]) did not show any specialized structures unique to these mountain species. However, chloroplast protrusion formation could be found in G. reptans and, to a greater extent, in O. digyna. The other species exhibited only a low percentage of such chloroplast structural changes. Occurrence of protrusions in samples of G. montanum and O. digyna growing in a mild climate at about 50 m above sea level was drastically reduced. Serial TEM sections of O. digyna cells showed that the protrusions can appear as rather broad and long appendices of plastids, often forming pocketlike structures where mitochondria and microbodies are in close vicinity to the plastid and to each other. It is suggested that some high-alpine plants may form such protrusions to facilitate fast exchange of molecules between cytoplasm and plastid as an adaptation to the short, often unfavorable vegetation period in the Alps, while other species may have developed different types of adaptation that are not expressed in ultrastructural changes of the plastids. Correspondence and reprints: Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.