Effects of oxygen availability on mycobenthic communities of marine coastal sediments.

In coastal marine sediments, oxygen availability varies greatly, and anoxic conditions can develop quickly over low spatial resolution. Although benthic fungi are important players in the marine carbon cycle, little is known about their adaptation to fluctuating availability of oxygen as terminal electron acceptor. Here, we study which part of a mycobenthic community from oxic coastal sediments can thrive under temporarily anoxic conditions. We test whether phylogeny or certain fungal traits promote plasticity in respect to changes in oxygen availability. Therefore, we incubated mycobenthos under oxic and anoxic conditions, performed ITS2 Illumina tag-sequencing and an additional meta-analysis on a literature survey. Half of all OTUs showed a plasticity towards changing oxygen availability and exhibited different strategies towards anoxic conditions, with rapid response within hours or a delayed one after several days. The strategy of dimorphism and facultative yeasts were significantly linked to OTU occurrence in anoxic conditions, while phylogeny and other traits had less effect. Our results suggest that different fungal niches are formed over the duration of prolonged anoxic conditions. The taxon-specific proliferation seems to be regulated by the fine-tuning of various traits and factors. It is essential to take these results into account when conducting conceptual work on the functionality of the marine benthos.

Mycoplankton Biome Structure and Assemblage Processes Differ Along a Transect From the Elbe River Down to the River Plume and the Adjacent Marine Waters.

Rivers are transport systems and supply adjacent ecosystems with nutrients. They also serve human well-being, for example as a source of food. Microorganism biodiversity is an important parameter for the ecological balance of river ecosystems. Despite the knowledge that fungi are key players in freshwater nutrient cycling and food webs, data on planktonic fungi of streams with higher stream order are scarce. This study aims to fill this knowledge gap by a fungi-specific 18S ribosomal RNA (rRNA) gene tag sequencing approach, investigating mycoplankton diversity in the Elbe River along a transect from shallow freshwater, to the estuary and river plume down to the adjacent marine waters (sections of seventh stream order number). Using multivariate analyses and the quantitative process estimates (QPEs) method, questions (i) of how mycoplankton communities as part of the river continuum change along the transect, (ii) what factors, spatial and environmental, play a role, and (iii) what assembly processes, such as selection or dispersion, operate along the transect, were addressed. The partitioning of mycoplankton communities into three significant distant biomes was mainly driven by local environmental conditions that were partly under spatial control. The assembly processes underlying the biomes also differed significantly. Thus, variable selection dominated the upstream sections, while undominated processes like ecological drift dominated the sections close to the river mouth and beyond. Dispersal played a minor role. The results suggest that the ecological versatility of the mycoplankton communities changes along the transect as response, for example, to a drastic change from an autotrophic to a heterotrophic system caused by an abrupt increase in the river depth. Furthermore, a significant salinity-dependent occurrence of diverse basal fungal groups was observed, with no clade found exclusively in marine waters. These results provide an important framework to help understand patterns of riverine mycoplankton communities and serve as basis for a further in-depth work so that fungi, as an important ecological organism group, can be integrated into models of, e.g., usage-balance considerations of rivers.

Diversity and biomass dynamics of unicellular marine fungi during a spring phytoplankton bloom.

Microbial communities have important functions during spring phytoplankton blooms, regulating bloom dynamics and processing organic matter. Despite extensive research into such processes, an in-depth assessment of the fungal component is missing, especially for the smaller size fractions. We investigated the dynamics of unicellular mycoplankton during a spring phytoplankton bloom in the North Sea by 18S rRNA gene tag sequencing and a modified CARD-FISH protocol. Visualization and enumeration of dominant taxa revealed unique cell count patterns that varied considerably over short time scales. The Rozellomycota sensu lato (s.l.) reached a maximum of 105 cells L-1 , being comparable to freshwater counts. The abundance of Dikarya surpassed previous values by two orders of magnitude (105 cells L-1 ) and the corresponding biomass (maximum of 8.9 mg C m-3 ) was comparable to one reported for filamentous fungi with assigned ecological importance. Our results show that unicellular fungi are an abundant and, based on high cellular ribosome content and fast dynamics, active part of coastal microbial communities. The known ecology of the visualized taxa and the observed dynamics suggest the existence of different ecological niches that link primary and secondary food chains, highlighting the importance of unicellular fungi in food web structures and carbon transfer.

Seasonal Dynamics of Pelagic Mycoplanktonic Communities: Interplay of Taxon Abundance, Temporal Occurrence, and Biotic Interactions.

Marine fungi are an important component of pelagic planktonic communities. However, it is not yet clear how individual fungal taxa are integrated in marine processes of the microbial loop and food webs. Most likely, biotic interactions play a major role in shaping the fungal community structure. Thus, the aim of our work was to identify possible biotic interactions of mycoplankton with phytoplankton and zooplankton groups and among fungi, and to investigate whether there is coherence between interactions and the dynamics, abundance and temporal occurrence of individual fungal OTUs. Marine surface water was sampled weekly over the course of 1 year, in the vicinity of the island of Helgoland in the German Bight (North Sea). The mycoplankton community was analyzed using 18S rRNA gene tag-sequencing and the identified dynamics were correlated to environmental data including phytoplankton, zooplankton, and abiotic factors. Finally, co-occurrence patterns of fungal taxa were detected with network analyses based on weighted topological overlaps (wTO). Of all abundant and persistent OTUs, 77% showed no biotic relations suggesting a saprotrophic lifestyle. Of all other fungal OTUs, nearly the half (44%) had at least one significant negative relationship, especially with zooplankton and other fungi, or to a lesser extent with phytoplankton. These findings suggest that mycoplankton OTUs are embedded into marine food web chains via highly complex and manifold relationships such as parasitism, predation, grazing, or allelopathy. Furthermore, about one third of all rare OTUs were part of a dense fungal co-occurrence network probably stabilizing the fungal community against environmental changes and acting as functional guilds or being involved in fungal cross-feeding. Placed in an ecological context, strong antagonistic relationships of the mycoplankton community with other components of the plankton suggest that: (i) there is a top-down control by fungi on zooplankton and phytoplankton; (ii) fungi serve as a food source for zooplankton and thereby transfer nutrients and organic material; (iii) the dynamics of fungi harmful to other plankton groups are controlled by antagonistic fungal taxa.

Correction to: A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

Following publication of the original article [1], we have been notified that three of the primer names identified as most promising candidates for fungal community surveys were incorrectly renamed following the primer nomenclature system proposed by Gargas & DePriest [2].

A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

BACKGROUND: Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. RESULTS: We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5'/nu-SSU-1647-3' (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. CONCLUSIONS: The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer's characteristics and performance to facilitate primer pair selection.

How to boost marine fungal research: A first step towards a multidisciplinary approach by combining molecular fungal ecology and natural products chemistry.

Marine fungi have attracted attention in recent years due to increased appreciation of their functional role in ecosystems and as important sources of new natural products. The concomitant development of various "omic" technologies has boosted fungal research in the fields of biodiversity, physiological ecology and natural product biosynthesis. Each of these research areas has its own research agenda, scientific language and quality standards, which have so far hindered an interdisciplinary exchange. Inter- and transdisciplinary interactions are, however, vital for: (i) a detailed understanding of the ecological role of marine fungi, (ii) unlocking their hidden potential for natural product discovery, and (iii) designing access routes for biotechnological production. In this review and opinion paper, we describe the two different "worlds" of marine fungal natural product chemists and marine fungal molecular ecologists. The individual scientific approaches and tools employed are summarised and explained, and enriched with a first common glossary. We propose a strategy to find a multidisciplinary approach towards a comprehensive view on marine fungi and their chemical potential.

A phylogenetic framework for the kingdom Fungi based on 18S rRNA gene sequences.

The usage of molecular phylogenetic approaches is critical to advance the understanding of systematics and community processes in the kingdom Fungi. Among the possible phylogenetic markers (or combinations of them), the 18S rRNA gene appears currently as the most prominent candidate due to its large availability in public databases and informative content. The purpose of this work was the creation of a reference phylogenetic framework that can serve as ready-to-use package for its application on fungal classification and community analysis. The current database contains 9329 representative 18S rRNA gene sequences covering the whole fungal kingdom, a manually curated alignment, an annotated and revised phylogenetic tree with all the sequence entries, updated information on current taxonomy, and recommendations of use. Out of 201 total fungal taxa with more than two sequences in the dataset, 179 were monophyletic. From another perspective, 66% of the entries had a tree-derived classification identical to that obtained from the NCBI taxonomy, whereas 34% differed in one or the other rank. Most of the differences were associated to missing taxonomic assignments in NCBI taxonomy, or the unexpected position of sequences that positioned out of their theoretically corresponding clades. The strong correlation observed with current fungal taxonomy evidences that 18S rRNA gene sequence-based phylogenies are adequate to reflect genealogy of Fungi at the levels of order and above, and justify their further usage and exploration.

Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data.

Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.

Analysis of the floral transcriptome of Tarenaya hassleriana (Cleomaceae), a member of the sister group to the Brassicaceae: towards understanding the base of morphological diversity in Brassicales.

BACKGROUND: Arabidopsis thaliana, a member of the Brassicaceae family is the dominant genetic model plant. However, while the flowers within the Brassicaceae members are rather uniform, mainly radially symmetrical, mostly white with fixed organ numbers, species within the Cleomaceae, the sister family to the Brassicaceae show a more variable floral morphology. We were interested in understanding the molecular basis for these morphological differences. To this end, the floral transcriptome of a hybrid Tarenaya hassleriana, a Cleomaceae with monosymmetric, bright purple flowers was sequenced, annotated and analyzed in respect to floral regulators. RESULTS: We obtained a comprehensive floral transcriptome with high depth and coverage close to saturation analyzed using rarefaction analysis a method well known in biodiversity studies. Gene expression was analyzed by calculating reads per kilobase gene model per million reads (RPKM) and for selected genes in silico expression data was corroborated by qRT-PCR analysis. Candidate transcription factors were identified based on differences in expression pattern between A. thaliana and T. hassleriana, which are likely key regulators of the T. hassleriana specific floral characters such as coloration and male sterility in the hybrid plant used. Analysis of lineage specific genes was carried out with members of the fabids and malvids. CONCLUSIONS: The floral transcriptome of T. hassleriana provides insights into key pathways involved in the regulation of late anthocyanin biosynthesis, male fertility, flowering time and organ growth regulation which are unique traits compared the model organism A. thaliana. Analysis of lineage specific genes carried out with members of the fabids and malvids suggests an extensive gene birth rate in the lineage leading to core Brassicales while only few genes were potentially lost during core Brassicales evolution, which possibly reflects the result of the At-ß whole genome duplication. Our analysis should facilitate further analyses into the molecular mechanisms of floral morphogenesis and pigmentation and the mechanisms underlying the rather diverse floral morphologies in the Cleomaceae.

Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities.

BACKGROUND: In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. RESULTS: We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. CONCLUSION: For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.

Fatty acid metabolism in the ectomycorrhizal fungus Laccaria bicolor.

Here, the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolorwas explored with the aim of constructing a genome-wide inventory of genes involved in fatty acid metabolism. Sixty-three genes of the major pathways were annotated and validated by the detection of the corresponding transcripts. Seventy-one per cent belonged to multigene families of up to five members. In the mycelium of L. bicolor, 19 different fatty acids were detected, including at low concentrations palmitvaccenic acid (16:1(11Z)), which is known to be a marker for arbuscular mycorrhizal fungi. The pathways of fatty acid biosynthesis and degradation in L. bicolor were reconstructed using lipid composition, gene annotation and transcriptional analysis. Annotation results indicated that saturated fatty acids were degraded in mitochondria, whereas degradation of modified fatty acids was confined to peroxisomes. Fatty acid synthase (FAS) was the second largest protein annotated in L. bicolor. Phylogenetic analysis indicated that L. bicolor, Ustilago maydis and Coprinopsis cinerea have a vertebrate-like type I FAS encoded as a single protein, whereas in other basidiomycetes, including the human pathogenic basidiomycete Cryptococcus neoformans, and in most ascomycetes FAS is composed of the two structurally distinct subunits α and ß.

Sugar for my honey: carbohydrate partitioning in ectomycorrhizal symbiosis.

Simple, readily utilizable carbohydrates, necessary for growth and maintenance of large numbers of microbes are rare in forest soils. Among other types of mutualistic interactions, the formation of ectomycorrhizas, a symbiosis between tree roots and certain soil fungi, is a way to overcome nutrient and carbohydrate limitations typical for many forest ecosystems. Ectomycorrhiza formation is typical for trees in boreal and temperate forests of the northern hemisphere and alpine regions world-wide. The main function of this symbiosis is the exchange of fungus-derived nutrients for plant-derived carbohydrates, enabling the colonization of mineral nutrient-poor environments. In ectomycorrhizal symbiosis up to 1/3 of plant photoassimilates could be transferred toward the fungal partner. The creation of such a strong sink is directly related to the efficiency of fungal hexose uptake at the plant/fungus interface, a modulated fungal carbohydrate metabolism in the ectomycorrhiza, and the export of carbohydrates towards soil growing hyphae. However, not only the fungus but also the plant partner increase its expression of hexose importer genes at the plant/fungus interface. This increase in hexose uptake capacity of plant roots in combination with an increase in photosynthesis may explain how the plant deals with the growing fungal carbohydrate demand in symbiosis and how it can restrict this loss of carbohydrates under certain conditions to avoid fungal parasitism.