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Nanopore raw read accuracy has improved to over 99%, making it a potential tool for metabarcoding. For broad adoption, guidelines on quality filtering are needed to ensure reliable taxonomic unit recovery. This study aims to provide those guidelines for a fungal metabarcoding context and to apply them to a case study of ectomycorrhizae in the decaying bark of Fagus sylvatica. We introduce the eNano pipeline to test two standard metabarcoding approaches: (1) Reference-based mapping leveraging UNITE’s species hypothesis system (SH approach); (2) Constructing 98% OTUs (OTU approach). Our results demonstrate that both approaches are effective with Nanopore data. When using a reference database, we recommend strict mapping criteria rather than Phred-based filtering. Leveraging the SH-system further enhances reproducibility and facilitates cross-study communication. For the 98% OTUs, filtering reads at ≥Q25 is recommended. Our case study reveals that the decay gradient is a primary determinant of community composition and that specific mycorrhizal fungi colonize decaying bark. Complementing our metabarcoding results with root tip morphotypification, we identify Laccaria amethystina and Tomentella sublilacina as key ectomycorrhizae of saplings on decaying logs. These findings demonstrate that Nanopore sequencing can provide valuable ecological insights and support its broader use in fungal metabarcoding as read quality continues to improve.

IntroductionIn situ carbon sequestration in forests is important in the context of climate change mitigation, and setting aside managed forests has been proposed as an option for increased carbon sequestration. Comparing set-aside and managed forests may provide insights and rules of thumb on the potential for additional in situ carbon sequestration in set-aside forest.MethodsIn an observational study, we compared re-inventory data from the network of set-aside forest reserves in Flanders, which have been unmanaged for 17–66 years (2 surveys with a 10 years interval), with re-inventory data from the regional forest inventory, representing the overall forest area in Flanders (2 surveys with a 15 years interval).ResultsThe aboveground carbon pools and sequestration rates were higher in the set-aside forests compared to the average forest in Flanders. In the average Flemish forest, the aboveground carbon pool increased from 64.7 to 85.1 tC ha−1, over a period of 15 years. In the set-aside forests, the mean pool was higher at the first measurement and further increased from 84.8 to 102.4 tC ha−1, over a period of 10 years. The mean aboveground annual carbon sequestration rate was 1.3 tC ha−1 year−1 in the average forest in Flanders and 1.8 tC ha−1 year−1 in the set-aside forests. The stocks and fluxes depended on the soil conditions and were higher in set-aside forests on silt and sandy silt sites compared to wet and sand sites. The set-aside forests on dry sites showed additionality in in situ aboveground carbon sequestration. We saw no indication of approaching a culmination point in the first decades following set-aside: plots with high carbon pools did not show lower carbon sequestration. In conclusion, set-aside forests can combine high carbon pools with high sequestration rates on suitable sites. Under the current management policy, we expect Flemish forests—regular and set-aside—to further increase their carbon pools in the coming decades.

Forest biodiversity studies conducted across Europe use a multitude of forestry terms, often inconsistently. This hinders the comparability across studies and makes the assessment of the impacts of forest management on biodiversity highly context-dependent. Recent attempts to standardize forestry and stand description terminology mostly used a top-down approach that did not account for the perspectives and approaches of forest biodiversity experts. This work aims to establish common standards for silvicultural and vegetation definitions, creating a shared conceptual framework for a consistent study on the effects of forest management on biodiversity. We have identified both strengths and weaknesses of the silvicultural and vegetation information provided in forest biodiversity studies. While quantitative data on forest biomass and dominant tree species are frequently included, information on silvicultural activities and vegetation composition is often lacking, shallow, or based on broad and heterogeneous classifications. We discuss the existing classifications and their use in European forest biodiversity studies through a novel bottom-up and top-driven review process, and ultimately propose a common framework. This will enhance the comparability of forest biodiversity studies in Europe, and puts the basis for effective implementation and monitoring of sustainable forest management policies. The standards here proposed are potentially adaptable and applicable to other geographical areas and could be extended to other forest interventions.

Forests host most terrestrial biodiversity and their sustainable management is crucial to halt biodiversity loss. Although scientific evidence indicates that sustainable forest management (SFM) should be assessed by monitoring multi-taxon biodiversity, most current SFM criteria and indicators account only for trees or consider indirect biodiversity proxies. Several projects performed multi-taxon sampling to investigate the effects of forest management on biodiversity, but the large variability of their sampling approaches hampers the identification of general trends, and limits broad-scale inference for designing SFM. Here we address the need of common sampling protocols for forest structure and multi-taxon biodiversity to be used at broad spatial scales. We established a network of researchers involved in 41 projects on forest multi-taxon biodiversity across 13 European countries. The network data structure comprised the assessment of at least three taxa, and the measurement of forest stand structure in the same plots or stands. We mapped the sampling approaches to multi-taxon biodiversity, standing trees and deadwood, and used this overview to provide operational answers to two simple, yet crucial, questions: what to sample? How to sample? The most commonly sampled taxonomic groups are vascular plants (83% of datasets), beetles (80%), lichens (66%), birds (66%), fungi (61%), bryophytes (49%). They cover different forest structures and habitats, with a limited focus on soil, litter and forest canopy. Notwithstanding the common goal of assessing forest management effects on biodiversity, sampling approaches differed widely within and among taxonomic groups. Differences derive from sampling units (plots size, use of stand vs. plot scale), and from the focus on different substrates or functional groups of organisms. Sampling methods for standing trees and lying deadwood were relatively homogeneous and focused on volume calculations, but with a great variability in sampling units and diameter thresholds. We developed a handbook of sampling methods (SI 3) aimed at the greatest possible comparability across taxonomic groups and studies as a basis for European-wide biodiversity monitoring programs, robust understanding of biodiversity response to forest structure and management, and the identification of direct indicators of SFM.