Sampling fungal communities

Sampling fungal communities by environmental DNA

High throughput sequencing of eDNA (DNA metabarcoding) has become a standard technique for characterising fungal communities. The technique holds great potential to resolve fungal conservation challenges, as it neither requires detectable fruit bodies nor taxonomic field expertise. Yet, in order to fully exploit its potential, the method must be optimised specifically for questions relevant for fungal conservation, which so far has mainly been based on sporocarp data. In WP4, we will establish ~100 study plots in forests belonging to the EU Natura 2000 habitat network (or similar protected areas in non-EU countries). We will collect eDNA samples from soil, litter, deadwood and air, and in parallel conduct traditional fruit body surveys, and analyse strengths and drawbacks of the two approaches. The unique dataset will further yield insights into diversity, uniqueness and conservation value of the different forest types and help practitioners to guide fungal conservation.


How can we best capture fungal diversity? We will explore how much of the fungal diversity can be detected by eDNA barcoding versus sporocarp/thallus sampling at plot level, and the overlap among the two approaches, with special reference to conservation relevant species. Further, we will include citizen science data from WP3 to explore the added value of exploring larger areas within comparable habitat types using voluntary approaches.

Which substrate types should we sample by eDNA to best capture fungal diversity? We will compare the ability of eDNA sampling of various substrate types as tools to capture fungal biodiversity in forest habitats, with a focus on conservation relevant species. We hypothesise that each substrate will give unique insights to fungal diversity, but with considerable variation in the relative contribution to total revealed diversity, depending on forest type.

Which metabarcoding method should we use to best capture fungal diversity? We will combine short- and long-read sequencing pipelines to compare their cost-effectiveness and ability to reliably capture fungal diversity. We hypothesise that overall fungal diversity is best captured with short-read, whereas taxonomic assignments and phylogenetic placements are more precise using long read methods .

What are the patterns of fungal alpha diversity, uniquity, and phylogenetic diversity across European forests? Based on the full dataset we will explore fungal biodiversity patterns across the forest types sampled. We will focus especially on threatened fungi and uniquity patterns to identify plots and forest types with unique value for fungal conservation in Europe. This work will be tightly coordinated with WP5.


Establish study plots. We will establish around 100 study plots (40 x 40 m2) in European Natura 2000 types / protected forests, covering an extensive environmental gradient ranging from subarctic pine and birch forests in Northern Norway, over temperate, boreal and alpine forest in Western and Central Europe to Mediterranean forests spanning from Portugal to Greece.

Obtain environmental samples. We will in each plot collect 5-6 different substrates for metabarcoding analyses, including: (i) aerial spore samples, (ii) litter, (iii) humus, (iv) dead wood, (v) fruit bodies, and (iv) lichens. We will use a passive spore sampler to collect spores over a two-week period, twice per plot. The other sample types (ii-iv) will represent pools of numerous sub-samples systematically obtained per plots, in order to cover the microscale fungal diversity.

Collect sporocarps and lichen thalli. Fruit bodies within the 40 x 40 m2 plot will be recorded three times during the sampling season and determined based on morphology. In addition, small pieces of each sporocarp will be pooled into a fruit body sample (v) for DNA sequencing, enabling direct comparison of sporocarp and eDNA diversity. In a subset of the forest types, lichens will be registered, and a corresponding pooled lichen sample will be obtained. Hence, for each plot, 8-9 samples will be obtained, providing a total of 800-900 samples to be sequenced. Suitable fungal collections will be documented individually and included, linking to WP2 (model training) and WP3 (discovery mission).

DNA library preparation and sequencing. All samples will be frozen upon sampling and later sent to one lab (WSL), where DNA extraction and library preparation of the fungal ITS2 metabarcoding marker will be conducted. The ITS2 libraries will be analysed using short-read Illumina sequencing, and state-of-the art bioinformatics approaches will be used to produce fungal OTU-matrices that will be taxonomically annotated. DNA extracts from all samples will also be sent to another lab (UiO), where long-read sequencing of the entire rDNA operon will be conducted using circular PacBio-sequencing. Corresponding OTU-matrices will be produced from the long-read sequences, as well as comprehensive sequence alignments for phylogenetic analyses.

Analyse substrate differences. Assess which substrate types best capture fungal diversity / threatened species in different European forests, and what are the overlap in fungal diversity among substrate types.

Compare short and long-read sequences. Assess to what extent long-read sequencing can reveal the same fungal diversity as short read sequencing, and to what degree long-read sequencing provides improved taxonomic annotation.

Analyse eDNA vs sporocarp diversity. We will compare how much of the fungal diversity can be detected through sporocarp/thallus surveys versus eDNA, and evaluate to what degree threatened fungi can be targeted with the two sequencing approaches, versus sporocarps/thalli.

Compare fungal diversity patterns across European forests. In order to better understand the distribution of European threatened fungi.


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