The importance of within-log sampling replication in bark- and wood-inhabiting fungal metabarcoding studies

Despite the widespread use of metabarcoding approaches in wood-inhabiting fungal studies, there is currently no standardized procedure for sampling deadwood. How to sample and how much to replicate within logs has generally been decided arbitrarily. In this study, we provide quantitative results about how the bark- and wood-inhabiting fungi vary along the logs, compared to how they vary between logs. For this, we used two datasets, one representing variation in the sample's location along the logs (tree part; bottom, middle or top) and across tree species (pine or spruce), and another representing small-scale spatial variation (tens of centimeters) and in the sample type (wood or bark). Additionally, we conducted a visual survey of epiphytic lichens to assess the efficiency of bark metabarcoding to survey epiphytic lichens. Our results show that the diversity of fungi varies greatly between tree species and among the tree parts. Yet, within the tree parts, the fungal community composition is relatively homogeneous, although increasing the number of samples increases the number of species detected. Strikingly, bark was revealed to be highly species rich, holding threefold the diversity of the wood beneath it, which represents a subset of the diversity found in bark. Thus, our study shows that ignoring the bark-inhabiting fungal diversity in studies of saproxylic species overlooks a substantial part of the saproxylic diversity. Furthermore, we found a poor overlap between the lichen species identified morphologically and those detected via bark metabarcoding; however, both methods captured consistent patterns in how the lichen diversity varied across the logs. We conclude that to gain a representative view of the fungal community composition, bark should be included in fungal surveys and that replication within logs should focus on covering the different parts of the trees.