Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

Tidying up international nucleotide sequence databases: ecological, geographical and sequence quality annotation of its sequences of mycorrhizal fungi

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi.     

Fungal‐host diversity among mycoheterotrophic plants increases proportionally to their fungal‐host overlap

The vast majority of plants obtain an important proportion of vital resources from soil through mycorrhizal fungi. Generally, this happens in exchange of photosynthetically fixed carbon, but occasionally the interaction is mycoheterotrophic, and plants obtain carbon from mycorrhizal fungi. This process results in an antagonistic interaction between mycoheterotrophic plants and their fungal hosts. Importantly, the fungal‐host diversity available for plants is restricted as mycoheterotrophic interactions often involve narrow lineages of fungal hosts. Unfortunately, little is known whether fungal‐host diversity may be additionally modulated by plant–plant interactions through shared hosts. Yet, this may have important implications for plant competition and coexistence. Here, we use DNA sequencing data to investigate the interaction patterns between mycoheterotrophic plants and arbuscular mycorrhizal fungi. We find no phylogenetic signal on the number of fungal hosts nor on the fungal hosts shared among mycoheterotrophic plants. However, we observe a potential trend toward increased phylogenetic diversity of fungal hosts among mycoheterotrophic plants with increasing overlap in their fungal hosts. While these patterns remain for groups of plants regardless of location, we do find higher levels of overlap and diversity among plants from the same location. These findings suggest that species coexistence cannot be fully understood without attention to the two sides of ecological interactions.     

Consideration of the taxonomy and biodiversity of Australian ectomycorrhizal fungi

Mycorrhiza management in forestry must be predicated on an understanding of fungal biology and ecology. A fundamental building block of the biology and ecology of any organism is accurate identification and an understanding of its relationship to other organisms. The taxonomy of the larger fungi has been largely based on morphological classification of sexual structures but now Taxonomy routinely incorporates mating studies, and biochemical and molecular data. Taxonomy may not revolutionize theories on mycorrhiza but can clarify some of the inconsistencies due to misrepresentation or over-generalizations and inappropriate conclusions drawn from studies with inaccurately identified fungi. To illustrate this, we discuss and example where incorrect fungal names were repeatedly erroneously applied in morphological and physiological research reports on this fungus, e.g. Laccaria laccata. In this case subsequent taxonomic study revealed the reason for the conflicting research results reported for this fungus. We discuss the status of identifying the ectomycorrhizal fungi in various forest communities in Australia and the relationships of this process to assessing their use in forestry. Recent intensive efforts to collect, isolate and identify Australian ectomycorrhizal fungi have revealed an enormous and unique species diversity, e.g., for truffle-like fungi, over 2000 collections from the last five years alone have yielded 2 new families, 24 new genera, and about 184 new species. Nearly 95% of the described and undescribed fungi from Australia are novel, with some 22 genera and 3 families endemic. In most cases the current systematic knowledge of mycorrhizal fungi is inadequate to support clear framework for Australian taxa. This reflects the traditional Northern Hemisphere view of the world, the uniqueness of the Australian fungal flora, and how poorly it is known. For example, the genus Hymenogaster had been widely acknowledged as the most reduced member of the Cortinariaceae. However recent work on Hymenogaster species from the Southern Hemisphere has offered a number of alternative affinities to various species. We also discuss the role proper identification of the organisms involved plays in understanding the ecosystem. Emphasis should be placed on how species diversity equates with physiological and genetic diversity and how a sound taxonomic understanding of species and their systematic position is essential to properly manage them. Accurate taxonomic information will continue to be required as the basis for assessing the role of ectomycorrhizal fungi in sustained ecological development. Of particular significance is the role of ectomycorrhizal fungi in maintenance of plant diversity in natural ecosystems and those disturbed by management. In conclusion, we present some key research areas involving the use of taxonomy that need priority attention.     

Effects of an introduced mustard,Thlaspi arvense,on soil fungal communities in subalpine meadows

The subalpine meadows of the Rocky Mountains, USA, are at the advancing front of global change; however, little is known about the sensitivities of high-elevation soil fungal communities to ongoing ecological changes. Soil fungi are sensitive to abiotic and biotic environmental stressors, including climate change, soil disturbance, and the presence of introduced, non-native plants. Invasive plants in the Brassicaceae (mustard family) are known to alter fungal community structure, suppress arbuscular mycorrhizal fungi, and change their relationship with native plant hosts in forest ecosystems, but these phenomena have not been studied in the subalpine zone where non-native mustard plants are becoming established. Here, we investigated whether the presence of the introduced mustard plant, Thlaspi arvense, is associated with distinct properties of the whole fungal and arbuscular mycorrhizal fungal communities in subalpine meadow ecosystems. We observed clear differences in the composition, relative abundance of core taxa, and mean taxon relatedness of soil fungal communities in plots with T. arvense relative to those with only native vegetation. A suite of novel fungi were associated with T. arvense, and overall patterns of AMF phylogenetic diversity were drastically reduced in association with its presence. Our results suggest that T. arvense introduction impacts the soil fungal community, with potential implications for native plant communities and soil nutrient cycling in high elevation meadows of the Rocky Mountains.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

Phylogeny, phylogeography, phylobetadiversity and the molecular analysis of biological communities

There has been much recent interest and progress in the characterization of community structure and community assembly processes through the application of phylogenetic methods. To date most focus has been on groups of taxa for which some relevant detail of their ecology is known, for which community composition is reasonably easily quantified and where the temporal scale is such that speciation is not likely to feature. Here, we explore how we might apply a molecular genetic approach to investigate community structure and assembly at broad taxonomic and geographical scales, where we have little knowledge of species ecology, where community composition is not easily quantified, and where speciation is likely to be of some importance. We explore these ideas using the class Collembola as a focal group. Gathering molecular evidence for cryptic diversity suggests that the ubiquity of many species of Collembola across the landscape may belie greater community complexity than would otherwise be assumed. However, this morphologically cryptic species-level diversity poses a challenge for attempts to characterize diversity both within and among local species assemblages. Recent developments in high throughput parallel sequencing technology, combined with mtDNA barcoding, provide an advance that can bring together the fields of phylogenetic and phylogeographic analysis to bear on this problem. Such an approach could be standardized for analyses at any geographical scale for a range of taxonomic groups to quantify the formation and composition of species assemblages.     

Plant traits determine the phylogenetic structure of arbuscular mycorrhizal fungal communities

Functional diversity in ecosystems has traditionally been studied using aboveground plant traits. Despite the known effect of plant traits on the microbial community composition, their effects on the microbial functional diversity are only starting to be assessed. In this study, the phylogenetic structure of arbuscular mycorrhizal (AM) fungal communities associated with plant species differing in life cycle and growth form, that is, plant life forms, was determined to unravel the effect of plant traits on the functional diversity of this fungal group. The results of the 454 pyrosequencing showed that the AM fungal community composition differed across plant life forms and this effect was dependent on the soil collection date. Plants with ruderal characteristics tended to associate with phylogenetically clustered AM fungal communities. By contrast, plants with resource‐conservative traits associated with phylogenetically overdispersed AM fungal communities. Additionally, the soil collected in different seasons yielded AM fungal communities with different phylogenetic dispersion. In summary, we found that the phylogenetic structure, and hence the functional diversity, of AM fungal communities is dependent on plant traits. This finding adds value to the use of plant traits for the evaluation of belowground ecosystem diversity, functions and processes.     

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.     

Biological invasions increase the richness of arbuscular mycorrhizal fungi from a Hawaiian subtropical ecosystem

Biological invasions can have various impacts on the diversity of important microbial mutualists such as mycorrhizal fungi, but few studies have tested whether the effects of invasions on mycorrhizal diversity are consistent across spatial gradients. Furthermore, few of these studies have taken place in tropical ecosystems that experience an inordinate rate of invasions into native habitats. Here, we examined the effects of plant invasions dominated by non-native tree species on the diversity of arbuscular mycorrhizal (AM) fungi in Hawaii. To test the hypothesis that invasions result in consistent changes in AM fungal diversity across spatial gradients relative to native forest habitats, we sampled soil in paired native and invaded sites from three watersheds and used amplicon sequencing to characterize AM fungal communities. Whether our analyses considered phylogenetic relatedness or not, we found that invasions consistently increased the richness of AM fungi. However, AM fungal species composition was not related to invasion status of the vegetation nor local environment, but stratified by watershed. Our results suggest that while invasions can lead to an overall increase in the diversity of microbial mutualists, the effects of plant host identity or geographic structuring potentially outweigh those of invasive species in determining the community membership of AM fungi. Thus, host specificity and spatial factors such as dispersal need to be taken into consideration when examining the effects of biological invasions on symbiotic microbes.     

Fungal associates of <Emphasis Type="Italic">Pyrola rotundifolia</Emphasis>, a mixotrophic Ericaceae,from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.     

Against the naming of fungi

The use of molecular bar-coding and consensus on nomenclatural practices has encouraged optimism about the future of fungal taxonomy and systematics. There are, however, profound deficiencies in our understanding of fungal diversity and broader problems with the taxonomic enterprise that deserve greater attention. For 250 years mycologists have tried to reconcile fungal diversity with the Linnean fantasy of a divine order throughout nature that included unambiguous species. This effort has failed and today's taxonomy rests on an unstable philosophical foundation. Rather than persisting with the present endeavour, it may be more fruitful to abandon the notion of fungal species pending further basic research. In the meantime, mycologists should consider tagging collections with digital codes and assigning these operational taxonomic units to higher taxonomic ranks whose objective reality is corroborated by strong phylogenetic evidence.     

Molecular phylogeny,taxonomy, and evolution of Geosiphon pyriformis and arbuscular mycorrhizal fungi

Geosiphon pyriformis(Kütz.) v. Wettstein is the only known example of a fungus living in endocytobiotic association with a cyanobacterium. The close phylogenetic relationship of Geosiphonwith some arbuscular mycorrhizal fungi (AMF) and the phylogenetic position of Geosiphonare shown in detail. Comprehensive small subunit (SSU) rRNA sequence analyses allow the erection of a new, molecular phylogeny-based taxonomic system for the AMF, including Geosiphon (Geosiphonaceae). Within the recently described phylum Glomeromycota (with one class, Glomeromycetes), a system including four orders was proposed. The erection of several new families will also be necessary. Evolutionary implications are discussed, referring to different possibilities of the influence of AMF on the colonization of the terrestrial habitat by plants.     

Climate and geographic distance are more influential than rivers on the beta diversity of passerine birds in Amazonia

Variation in the spatial structure of communities in terms of species composition (beta diversity) is affected by different ecological processes, such as environmental filtering and dispersal limitation. Large rivers are known as barriers for species dispersal (riverine hypothesis) in tropical regions. However, when organisms are not dispersal limited by geographic barriers, other factors, such as climatic conditions and geographic distance per se, may affect species distribution. In order to investigate the relative contribution of major rivers, climate and geographic distance on Passeriformes beta diversity, we divided Amazonia into 549 grid cells (1° of latitude and longitude) and obtained data of species occurrence, climate and geographic position for each cell. Beta diversity was measured using taxonomic, phylogenetic and functional metrics of composition. The influence of climatic variables, geographic distance and rivers on these metrics was tested using regression analyses. Passerine beta diversity is characterized mainly by the change in species taxonomic identity and in phylogenetic lineages across climatic gradients and over geographic distance. However, species with similar traits are found throughout the entire Amazonia. The size of rivers was proportional to their effect on species composition. However, climate and geographic distance are relatively more important than rivers for Amazonian taxonomic and phylogenetic species composition.     

Fungal diversity living in the root and sporophore of the endemic Korean fern Mankyua chejuense

Ferns represent the basal group of vascular plants and are known to have fungal interactions with arbuscular mycorrhizal fungi, but diversity of endophytic fungi from ferns is rarely studied. Moreover, fungal diversity associated with ferns is likely underestimated as most studies have been performed based on a microscopic or culture-dependent approach. In this study, we investigated the endophytic fungal diversity within roots and sporophore of an endangered Korean fern (Mankyua chejuense), and compared it to fungi in surrounding soil using a metabarcoding approach. A high diversity of endophytic fungi (236 OTUs), mostly belonging to Ascomycota, was detected and fungal richness and composition were significantly different between habitats. Indicator species analysis showed that endophytic fungi have similar ecological characteristics to fungal species found from other land plants. Our results suggest that various fungal species are associated with ferns, thus understanding fern-associated fungal diversity can have a great implication for fern biology and conservation.