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 varies among studies, making comparisons difficult. Here, we provide quantitative results about how bark- and wood-inhabiting fungi vary along logs in early decay stages, 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 variation (tens of centimeters) in sampling location and variation in sample type (wood or bark). Additionally, we conducted a visual survey of epiphytic lichens to assess the efficiency of bark metabarcoding for surveying 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, fungal community composition is relatively homogeneous, with an increasing number of samples only moderately increasing the number of species detected. Strikingly, our results reveal bark samples to be especially species-rich, holding threefold the diversity of the wood beneath it, which represents a subset of the diversity found in bark. This finding suggests that the common practice of excluding bark-inhabiting fungal diversity in studies of saproxylic species in early decay-stage logs overlooks a substantial part of the saproxylic diversity. We found a poor overlap between lichen species identified morphologically and those detected via bark metabarcoding; however, both methods captured consistent patterns in how lichen diversity varied across the logs. We conclude that to gain a representative view of the fungal community composition in early decay-stage deadwood, bark should be included in fungal surveys and that replication within logs should focus on covering the different tree parts.