Mycobiomes of Young Beech Trees Are Distinguished by Organ Rather Than by Habitat, and Community Analyses Suggest Competitive Interactions Among Twig Fungi.

Beech trees (Fagus sylvatica) are prominent keystone species of great economic and environmental value for central Europe, hosting a diverse mycobiome. The composition of endophyte communities may depend on tree health, plant organ or tissue, and growth habitat. To evaluate mycobiome communalities at local scales, buds, and twigs were sampled from two young healthy mountain beech stands in Bavaria, Germany, four kilometers apart. With Illumina high-throughput sequencing, we found 113 fungal taxa from 0.7 million high-quality reads that mainly consisted of Ascomycota (52%) and Basidiomycota (26%) taxa. Significant correlations between richness and diversity indices were observed (p < 0.05), and mycobiomes did not differ between habitats in the current study. Species richness and diversity were higher in twigs compared to spring buds, and the assemblages in twigs shared most similarities. Interaction network analyses revealed that twig-bound fungi shared similar numbers of (interaction) links with others, dominated by negative co-occurrences, suggesting that competitive exclusion may be the predominant ecological interaction in the highly connected twig mycobiome. Combining community and network analyses strengthened the evidence that plant organs may filter endophytic communities directly through colonization access and indirectly by facilitating competitive interactions between the fungi.

Temporal dynamics in the taxonomic and functional profile of the Sphagnum-associated fungi (mycobiomes) in a Sphagnum farming field site in Northwestern Germany.

The drainage of peatlands for their agricultural use leads to huge emissions of greenhouse gases. One sustainable alternative is the cultivation of peat mosses after rewetting ('Sphagnum farming'). Environmental parameters of such artificial systems may differ from those of natural Sphagnum ecosystems which host a rich fungal community. We studied the fungal community at a 4 ha Sphagnum farming field site in Northwestern Germany and compared it with that of natural Sphagnum ecosystems. Additionally, we asked if any fungi occur with potentially negative consequences for the commercial production and/or use of Sphagnum biomass. Samples were collected every 3 months within 1 year. High-throughput sequencing of the fungal ITS2 barcode was used to obtain a comprehensive community profile of the fungi. The dominant taxa in the fungal community of the Sphagnum farming field site were all commonly reported from natural Sphagnum ecosystems. While the taxonomic composition showed clear differences between seasons, a stable functional community profile was identified across seasons. Additionally, nutrient supply seems to affect composition of fungal community. Despite a rather high abundance of bryophyte parasites, and the occurrence of both Sphagnum-species-specific and general plant pathogens, their impact on the productivity and usage of Sphagnum biomass as raw material for growing media was considered to be low.

Both plant genotype and herbivory shape aspen endophyte communities.

Salicinoid phenolic glycosides are common defence substances in salicaceous trees and specialist leaf beetles use these compounds for their own defence against predators. Salicinoids vary qualitatively and qualitatively in aspen (Populus tremula) and this variation has a genetic basis. The foliar endophyte mycobiome is plentiful and we hypothesised that it is related to plant genotype, potentially mediated by salicinoid composition, and that interactions with the leaf beetle Chrysomela tremula may alter this relationship. We studied these three-way interactions in controlled greenhouse experiments. Endophytic fungi were isolated from sterilised leaf tissues with and without beetle damage, and from beetles. We confirmed that endophyte composition was influenced by host genotype. Beetle activity added generalist morphs to the mycobiome that overrode the initial host association. Yeast-like genera (Cryptococcus and Rhodotorula) were isolated only from beetle-damaged tissues and from beetles, whereas fast-growing filamentous fungi dominated beetle-free control plants. Competition experiments between filamentous fungi of plant origin and beetle-related yeasts suggested interaction of both stimulating and inhibiting modes of action amongst the fungi. As a result, we detected examples of amensalism, commensalism, parasitism and competition between the morphs tested, but we found no evidence of mutualism, and consequently no co-evolutionary relationship could be demonstrated, between yeasts carried by beetles, host genotype and associated filamentous morphs. Endophyte studies are method-dependent and high-throughput sequencing technology best define the fungal mycobiome, culturing however continues to be a cheap way to provide fundamental ecological insights and it is also required for experimental studies.

Genetic barcoding of dark-spored myxomycetes (Amoebozoa)-Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies.

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.

Millions of reads, thousands of taxa: microbial community structure and associations analyzed via marker genes.

With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology.

Tuning the Voices of a Choir: Detecting Ecological Gradients in Time-Series Populations.

This paper introduces a new approach-the Principal Component Gradient Analysis (PCGA)-to detect ecological gradients in time-series populations, i.e. several time-series originating from different individuals of a population. Detection of ecological gradients is of particular importance when dealing with time-series from heterogeneous populations which express differing trends. PCGA makes use of polar coordinates of loadings from the first two axes obtained by principal component analysis (PCA) to define groups of similar trends. Based on the mean inter-series correlation (rbar) the gain of increasing a common underlying signal by PCGA groups is quantified using Monte Carlo Simulations. In terms of validation PCGA is compared to three other existing approaches. Focusing on dendrochronological examples, PCGA is shown to correctly determine population gradients and in particular cases to be advantageous over other considered methods. Furthermore, PCGA groups in each example allowed for enhancing the strength of a common underlying signal and comparably well as hierarchical cluster analysis. Our results indicate that PCGA potentially allows for a better understanding of mechanisms causing time-series population gradients as well as objectively enhancing the performance of climate transfer functions in dendroclimatology. While our examples highlight the relevance of PCGA to the field of dendrochronology, we believe that also other disciplines working with data of comparable structure may benefit from PCGA.

Habitat conditions and phenological tree traits overrule the influence of tree genotype in the needle mycobiome-Picea glauca system at an arctic treeline ecotone.

Plant-associated mycobiomes in extreme habitats are understudied and poorly understood. We analysed Illumina-generated ITS1 sequences from the needle mycobiome of white spruce (Picea glauca) at the northern treeline in Alaska (USA). Sequences were obtained from the same DNA that was used for tree genotyping. In the present study, fungal metabarcoding and tree microsatellite data were compared for the first time. In general, neighbouring trees shared more fungal taxa with each other than trees growing in further distance. Mycobiomes correlated strongly with phenological host traits and local habitat characteristics contrasting a dense forest stand with an open treeline site. Genetic similarity between trees did not influence fungal composition and no significant correlation existed between needle mycobiome and tree genotype. Our results suggest the pronounced influence of local habitat conditions and phenotypic tree traits on needle-inhabiting fungi. By contrast, the tree genetic identity cannot be benchmarked as a dominant driver for needle-inhabiting mycobiomes, at least not for white spruce in this extreme environment.

Diversity and Composition of the Leaf Mycobiome of Beech (Fagus sylvatica) Are Affected by Local Habitat Conditions and Leaf Biochemistry.

Comparative investigations of plant-associated fungal communities (mycobiomes) in distinct habitats and under distinct climate regimes have been rarely conducted in the past. Nowadays, high-throughput sequencing allows routine examination of mycobiome responses to environmental changes and results at an unprecedented level of detail. In the present study, we analysed Illumina-generated fungal ITS1 sequences from European beech (Fagus sylvatica) originating from natural habitats at two different altitudes in the German Alps and from a managed tree nursery in northern Germany. In general, leaf-inhabiting mycobiome diversity and composition correlated significantly with the origin of the trees. Under natural condition the mycobiome was more diverse at lower than at higher elevation, whereas fungal diversity was lowest in the artificial habitat of the tree nursery. We further identified significant correlation of leaf chlorophylls and flavonoids with both habitat parameters and mycobiome biodiversity. The present results clearly point towards a pronounced importance of local stand conditions for the structure of beech leaf mycobiomes and for a close interrelation of phyllosphere fungi and leaf physiology.

First insight into dead wood protistan diversity: a molecular sampling of bright-spored Myxomycetes (Amoebozoa, slime-moulds) in decaying beech logs.

Decaying wood hosts a large diversity of seldom investigated protists. Environmental sequencing offers novel insights into communities, but has rarely been applied to saproxylic protists. We investigated the diversity of bright-spored wood-inhabiting Myxomycetes by environmental sequencing. Myxomycetes have a complex life cycle culminating in the formation of mainly macroscopic fruiting bodies, highly variable in shape and colour that are often found on decaying logs. Our hypothesis was that diversity of bright-spored Myxomycetes would increase with decay. DNA was extracted from wood chips collected from 17 beech logs of varying decay stages from the Hainich-Dün region in Central Germany. We obtained 260 partial small subunit ribosomal RNA gene sequences of bright-spored Myxomycetes that were assembled into 29 OTUs, of which 65% were less than 98% similar to those in the existing database. The OTU richness revealed by molecular analysis surpassed that of a parallel inventory of fruiting bodies. We tested several environmental variables and identified pH, rather than decay stage, as the main structuring factor of myxomycete distribution.

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts.

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric-artificially joined-DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes of Diphasiastrum alpinum (Lycopodiaceae).

PREMISE OF THE STUDY: To understand the early evolution of mycorrhizal symbioses, it is important to know the fungal partners of gametophytes and sporophytes for basal lineages of vascular plants. Subterranean mycotrophic gametophytes of the clubmoss Diphasiastrum alpinum found at three localities gave an opportunity to study their morphology and anatomy and to identify and describe their hitherto unknown fungal endophytes. In addition, sporophytes were screened for fungal partners. METHODS: Gametophytes with attached young sporophytes were excavated, and their anatomy and their associated fungi were studied by light microscopy. DNA was isolated and amplified with both universal and group-specific fungal primers for the ITS region, the large subunit and small subunit of the nuclear rDNA, respectively, to identify the fungal partner. KEY RESULTS: Gametophytes were uniformly colonized by a fungus with septate hyphae forming coils and vesicles. Its morphology resembles that of the sebacinoid genus Piriformospora. Both ITS and LSU sequences were identified as Sebacinales group B, a basal clade of the Agaricomycetes (Basidiomycota). This fungus was detected in 11 gametophytes from two localities and in rootlets of adjacent Calluna vulgaris (Ericaceae) plants, but was absent in roots of sporophytes. In addition, several ascomycetes and glomeromycetes were found by DNA sequencing. CONCLUSIONS: Our study suggests a fungus belonging to Sebacinales group B as the main fungal host of the D. alpinum gametophytes. However, Sebacinales group B fungi occur as well in adjacent Ericaceae plants; therefore, we assume the mycoheterotrophic gametophyte to be epiparasitic on Ericaceae, which would explain the steady association of these plants.

Megraft: a software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes and similar environmental datasets.

Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of a community sequencing effort, rarefaction analysis of the ribosomal small subunit (SSU/16S/18S) gene in the metagenome is usually performed. The fragmentary, non-overlapping nature of SSU sequences in metagenomic libraries poses a problem for this analysis, however. We introduce a software package - Megraft - that grafts SSU fragments onto full-length SSU sequences, accounting for observed and unobserved variability, for accurate assessment of species richness and sequencing depth in metagenomics endeavors.

Species abundance distributions and richness estimations in fungal metagenomics--lessons learned from community ecology.

Results of diversity and community ecology studies strongly depend on sampling depth. Completely surveyed communities follow log-normal distribution, whereas power law functions best describe incompletely censused communities. It is arguable whether the statistics behind those theories can be applied to voluminous next generation sequencing data in microbiology by treating individual DNA sequences as counts of molecular taxonomic units (MOTUs). This study addresses the suitability of species abundance models in three groups of plant-associated fungal communities - phyllosphere, ectomycorrhizal and arbuscular mycorrhizal fungi. We tested the impact of differential treatment of molecular singletons on observed and estimated species richness and species abundance distribution models. The arbuscular mycorrhizal community of 48 MOTUs was exhaustively sampled and followed log-normal distribution. The ectomycorrhizal (153 MOTUs) and phyllosphere (327 MOTUs) communities significantly differed from log-normal distribution. The fungal phyllosphere community in particular was clearly undersampled. This undersampling bias resulted in strong sensitivity to the exclusion of molecular singletons and other rare MOTUs that may represent technical artefacts. The analysis of abundant (core) and rare (satellite) MOTUs clearly identified two species abundance distributions in the phyllosphere data - a log-normal model for the core group and a log-series model for the satellite group. The prominent log-series distribution of satellite phyllosphere fungi highlighted the ecological significance of an infrequent fungal component in the phyllosphere community.

Current state and perspectives of fungal DNA barcoding and rapid identification procedures.

Fungal research is experiencing a new wave of methodological improvements that most probably will boost mycology as profoundly as molecular phylogeny has done during the last 15 years. Especially the next generation sequencing technologies can be expected to have a tremendous effect on fungal biodiversity and ecology research. In order to realise the full potential of these exciting techniques by accelerating biodiversity assessments, identification procedures of fungi need to be adapted to the emerging demands of modern large-scale ecological studies. But how should fungal species be identified in the near future? While the answer might seem trivial to most microbiologists, taxonomists working with fungi may have other views. In the present review, we will analyse the state of the art of the so-called barcoding initiatives in the light of fungi, and we will seek to evaluate emerging trends in the field. We will furthermore demonstrate that the usability of DNA barcoding as a major tool for identification of fungi largely depends on the development of high-quality sequence databases that are thoroughly curated by taxonomists and systematists.

Dilution-to-extinction cultivation of leaf-inhabiting endophytic fungi in beech (Fagus sylvatica L.)--different cultivation techniques influence fungal biodiversity assessment.

Two cultivation-based isolation techniques - the incubation of leaf fragments (fragment plating) and dilution-to-extinction culturing on malt extract agar - were compared for recovery of foliar endophytic fungi from Fagus sylvatica near Greifswald, north-east Germany. Morphological-anatomical characters of vegetative and sporulating cultures and ITS sequences were used to assign morphotypes and taxonomic information to the isolates. Data analysis included species-accumulation curves, richness estimators, multivariate statistics and null model testing. Fragment plating and extinction culturing were significantly complementary with regard to species composition, because around two-thirds of the 35 fungal taxa were isolated with only one of the two cultivation techniques. The difference in outcomes highlights the need for caution in assessing fungal biodiversity based upon single isolation techniques. The efficiency of cultivation-based studies of fungal endophytes was significantly increased with the combination of the two isolation methods and estimations of species richness, when compared with a 20-years old reference study, which needed three times more isolates with fragment plating to attain the same species richness. Intensified testing and optimisation of extinction culturing in endophyte research is advocated.

Application of species richness estimators for the assessment of fungal diversity.

Species richness and distribution patterns of wood-inhabiting fungi and mycetozoans (slime moulds) were investigated in the canopy of a Central European temperate mixed deciduous forest. Species richness was described with diversity indices and species-accumulation curves. Nonmetrical multidimensional scaling was used to assess fungal species composition on different tree species. Different species richness estimators were used to extrapolate species richness beyond our own data. The reliability of the abundance-based coverage estimator, Chao, Jackknife and other estimators of species richness was evaluated for mycological surveys. While the species-accumulation curve of mycetozoans came close to saturation, that of wood-inhabiting fungi was continuously rising. The Chao 2 richness estimator was considered most appropriate to predict the number of species at the investigation site if sampling were continued. Gray's predictor of species richness should be used if statements of the number of species in larger areas are required. Multivariate analysis revealed the importance of different tree species for the conservation and maintenance of fungal diversity within forests, because each tree species possessed a characteristic fungal community. The described mathematical approaches of estimating species richness possess great potential to address fungal diversity on a regional, national, and global scale.

Species richness and ecological characterization of myxomycetes and myxomycete-like organisms in the canopy of a temperate deciduous forest.

The ecological community of myxomycetes and myxomycete-like organisms (MMLO) in the canopy of living deciduous trees was studied in a riparian deciduous forest at Leipzig, Germany. A systematic survey carried out with a total of 146 moist chamber cultures resulted in 386 records of 37 taxa, with 32 myxomycetes, two myxobacteria, two protostelids and the fruit body forming ciliate Sorogena stoianovitchae, the latter recorded for the first time for Europe. With 94% of all cultures positive for MMLO, these organisms are present consistently in the investigated sections of white-rotten twigs attached to living trees at 10-30 m above the ground. Our sampling recovered a majority of the likely species, with 37 out of the 42-45 predicted according to a species-accumulation curve and two other estimators of species richness. Nonmetric multidimensional scaling revealed pH, water-holding capacity and stage of decay to explain most of the variation in species distribution. Arcyria cinerea and Perichaena depressa as the most common species occurred in 32% and 29% of all samples, respectively. Viewing the sampled twigs as habitat islands and a single spore as sufficient to establish a population, a simulation program assuming a random spore rain estimated an average of 0.4 and 0.35 spore hits per twig as necessary to explain the observed frequencies. This is matched by the potential productivity of the substrate. All fruit bodies from the cultured twigs would be able to create a spore rain of 86 (A. cinerea) or 40 (P. depressa) spore hits per twig when dispersed evenly over the plot. The terminal fall velocity of spores was measured, revealing that it took about 5 h for a spore to land (30 m) in still air and indicating high dispersal ability for canopy-inhabiting MMLO.

Influence of small scale conditions on the diversity of wood decay fungi in a temperate, mixed deciduous forest canopy.

Studies on fungal richness and ecology have been largely disregarded since the first intensive efforts to investigate organismal diversity in forest canopies. We used the Leipzig Canopy Crane research facility to sample wood-decaying fungi in a mixed deciduous forest canopy 10-30 m in height. The structural complexity of the canopy was analysed using different methods, including meteorological measurements. With respect to temperature and relative humidity, marked differences existed between forest floor and upper canopy layers that persisted on smaller scales. Of the 118 taxa found in 128 sample units, pyrenomycetes and corticioid fungi outnumbered other macrofungal groups. Fungal communities showed distinct variations both in species richness and composition with respect to substrate (tree species), height in the canopy, stage of decay, and branch diameter. Pyrenomycetes and their anamorphs dominated the mycobiota on thin, exposed twigs at great heights, indicating their ability to overcome extended periods of drought and high levels of solar irradiance. Other taxa of Tremellales (Exidia spp.), Orbiliales (Hyalorbilia inflatula, Orbilia spp.) or Agaricales (Episphaeria fraxinicola, Cyphellopsis anomala, Lachnella spp.) also exhibited features that enabled them to develop in lesser protected habitats within tree crowns.