Morphological community analysis versus eDNA metabarcoding: diversity and ecological functions of fungal communities in differently managed stages along a forest conversion of Norway spruce towards old-growth European beech : distinct differences in fungal species richness and community composition in differently managed forest conversion stages of the Eifel National Park in Germany

Heine, Peggy; Schäffer, Andreas (Thesis advisor); van Dongen, Joost Thomas (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


In forest ecosystems, fungi are among the most important organismgroups due to their specialized functional roles in nutrient recycling, symbiotic associations and plant community dynamics. Fungi drive fundamental ecological functions in various ecosystem processes, indicating their highly relevant contribution to energy flows and nutrient cycles in forest ecosystems. Fungi are endangered and require attention in terms of protection and conservation. In addition, fungi are very sensitive to natural or anthropogenic disturbances and thus are a promising bioindicator in environmental studies of forest ecosystems. This study investigated response patterns of macrofungal communities to differently managed habitat stages representing a forest conversion from Norway spruce (Picea abies) to European beech (Fagus sylvatica) in the Eifel National Park in Germany. Thereby, a space-for-time substitution approach with three replicated study sites of each a priori classified forest stage was used for the study design: (I) even-aged single species spruce, (II) unmanaged spruce windthrow, (III) salvage-logged spruce windthrow, (IV) single spruce tree selection cutting with beech underplanting (close-to-nature management), and (V) old-growth, uneven-aged beech. The present study combined a traditional morphological fungal assessment over a 3-year sporocarp survey period with environmental DNA (eDNA) metabarcoding and high-throughput sequencing by a HiSeq Illumina platform of topsoil samples at the same study sites in the Eifel National Park. Above the soil ground, all visible sporocarps of Basidiomycetes and Ascomycetes were identified to species level. Below the soil ground through topsoil sampling, all obtained fungal operational taxonomic units (OTUs) of the fungal ITS1 (internal transcribed spacer 1) region were aligned through the UNITE reference database to reveal a molecular dataset at OTU level and species level. For a better understanding of the effects of biotic and abiotic impacts during this forest conversion process, various environmental variables (e.g. pH-value, Carbon:Nitrogen ratio, microclimate) were measured to find correlations with the soil fungal community. Further, all fungal species were categorized into functional groups to link taxonomic information to potential ecosystem functions. Comparing the results of the fungal eDNA metabarcoding with the traditional morphological assessment method revealed 406 unique fungi including 27 shared fungi. In detail, 235 fungi were observed through morphological identification, while eDNA metabarcoding revealed 869 fungal OTUs which led to 198 fungal species after ITS alignment provided by the UNITE reference database. For a better interpretation and comparison of both datasets, presence-absence data was used for generalized linear models (GLMs), as well as for classification and the ordination analyses, to predict how the soil fungal community will respond to the spruce forest conversion process and what consequences for forest ecosystem functioning are measurable. Thereby, individual fungal species were discussed and dominant functional groups were highlighted. The most dominant functional group contains the wood-decaying fungi. The morphological dataset revealed distinct fungal community compositions among the forest conversion stages, each with a different species richness, while similar values were found within the same a priori classified forest conversion stage. Thus, the species composition of the European beech forests is clearly distinct from all other investigated habitats, mirroring a clear shift due to the tree species composition change. During the forest conversion, spruce-associated and more fungal generalists were replaced by beech-associated fungal specialists, and leading to a higher fungal diversity. Further, the spore-based dataset revealed that different functional groups of fungi responded differently to the forest management, tree hosts, and environmental variables. Although the molecular dataset shared only 27 species with the morphological dataset and showed an opposite response pattern in its species richness within each forest conversion stage, the community composition indicated similar results. Both datasets revealed that the beech reference forest showed totally distinct community compositions compared to the other habitats, but the communities below the ground were less distinct among the forest conversion stages. All forest conversion stages, also the spruce-inhabiting stages, shared beech-associated fungi identified through eDNA metabarcoding, whereas the morphological dataset showed only unique, beech-associated fungal species within the beech-inhabiting stages. These results indicated that the spruce forest conversion towards beech is suitable and may support the dominant tree species change by establishing active beech-associated fungal communities. Overall, both datasets of this study showed that the host trees, European beech and Norway spruce, significantly shape the soil fungal community. Beside the tree species composition change and the forests management, the nutrient availability was a driver for the fungal species composition change. The differently managed windthrow stages did not differ from each other but were distinct to the forested stages (either spruce- or beech-inhabiting areas) probably due to the natural and anthropogenic disturbance as well as due to related environmental changes such as higher microclimate fluctuations through open canopy. The results of the morphological community analysis indicated that single tree species cutting of Norway spruce with further introduction of European beech trees (close-to-nature management) can be an adequate forest management strategy (in this study area) to allow a spruce forest conversion process without necessarily reducing the fungal species richness and its functional structure and redundancy of the fungal community. Further, the results of the molecular analysis demonstrated the importance of spruce forest conversion towards beech-inhabiting forests to increase and support landscape level habitats and fungal red-listed species. Thereby, the close-to-nature spruce-beech mixed forest could constitute a key role for maintaining high fungal biodiversity and a continuous forest structure in temperate European beech forest ecosystem conversion projects. Fungal eDNA metabarcoding generated results in a shorter timeframe and thus brought light in the fungal "black box" below the ground. Nevertheless, taxonomic expert knowledge together with habitat information through sporocarp surveys are highly needed to interpret sequencing data for planning and evaluating of forest management strategies in regard to fungal conservation. Both approaches cannot be used interchangeably while fungal eDNA metabarcoding with high-throughput sequencing should be integrated in traditional morphological community analyses.