Fungal and beetle diversity in deciduous fine woody debris in spruce-dominated forests in relation to substrate quantity and quality

Deciduous fine woody debris (DFWD) is a common deadwood substrate type in boreal conifer-dominated forests, but it is usually present in low volumes, and its importance for deadwood-dependent biodiversity is poorly understood. In this study, we investigated how DFWD-associated fungal and beetle diversity depends on local substrate availability and quality, and how species diversity differs between DFWD and coarse deciduous deadwood (birch logs) in boreal mixed spruce-dominated forests in southern Finland. We studied 25 forest plots (each 0.16 ha), measuring and sampling all pieces of DFWD with a diameter of 2–5 cm and a minimum length of 50 cm. Wood-inhabiting fungi were surveyed from wood samples by DNA metabarcoding and saproxylic beetles were surveyed by bark sieving. Our results showed a clear positive relationship between DFWD abundance and the diversity of fungi and beetles. Tree species and decay class diversity were not important in explaining fungal and beetle diversity or community composition, possibly due to the low degree of variation in DFWD quality among the study plots. DFWD hosted more diverse fungal assemblages than birch logs, including species of conservation concern, while no red-listed beetle species were observed on DFWD. Overall, species assemblages associated with fine and coarse deciduous deadwood were non-nested. Thus, DFWD represents a non-redundant complementary deadwood resource type alongside coarse deciduous deadwood in boreal forests.

Methods
Study sites (n=25) were located in Norway spruce dominated forests in the cities of Espoo, Helsinki, Vantaa, Lahti and Tampere in southern Finland. At each site, we collected and measured all pieces of deciduous fine woody debris with basal diameter between 2 and 5 cm and minimum length of 50 cm within a 0.16 ha sample plot (20 m × 80 m). For every piece, we recorded tree species, decay class, basal diameter, and length. Fragments originating from the same branch or stem were recorded as individual pieces if they had been fragmented before collection. We applied the following decay stage classification: 1 = hard, undecayed; 2 = knife penetrates through softened wood near the surface; 3 = piece holds its shape, but knife penetrates deeper into the wood; 4 = piece decayed and soft throughout, starting to lose shape. In addition to fine woody debris, we sampled 20 naturally downed birch logs (mean basal diameter 26.5 cm).

From fine woody debris, we extracted wood samples for fungal metabarcoding analysis by drilling at a distance of ca. 2-15 cm from the basal end of each piece. We removed bark and surface layer of the wood with a knife and drilled into the wood with a DNA-sterilized 6 mm drill head through the freshly exposed wood surface. We collected 7.5 ml portions of the resulting wood powder per piece and pooled them into composite plot-level samples. We then divided each pooled wood powder sample into two parallel subsamples. We extracted wood samples from birch logs (one per log) by drilling at five points with 1 m intervals starting at 50 cm from the basal end of each log. We drilled ca. 5 cm deep into the wood from one of the vertical sides of the log after removing bark. We ground the wood powder samples from DFWD and birch logs further into fine powder in a bead beater. Samples were beaten in 30 ml aliquots in 50 ml capsules with 8 mm steel bead for 2 min in 30 Hz. Tools were DNA sterilized with bleach solution between samples. We made two DNA extractions from each parallel subsample from fine woody debris, resulting in total of four replicate extractions per one plot-level sample of fine woody debris, and one DNA extraction per one birch log sample. DNA was extracted from ca. 90 mg of wood powder and eluted into 50 µl final elution volume with DNeasy Plant Pro extraction kit (Qiagen, Hilden, DE) according to the manufacturer’s instructions. For fungal metabarcoding, the internal transcribed spacer 2 of the nrRNA coding region was amplified with primers ITS3-2024F (GCATCGATGAAGAACGCAGC) and ITS4-2409R (TCCTCCGCTTATTGATATGC). Indexed amplicons were sequenced with Illumina NovaSeq 6000 (paired-end 250 bp) at Novogene Co (Cambridge, GB). Sequence reads were demultiplexed, and index and primer sequences were removed from paired-end reads. Further sequence processing was done with vsearch (v2.18.0). R1 and R2 reads were quality filtered, and then assembled. Assembled reads were chimera filtered de novo. Remaining reads were clustered into operational taxonomic units (OTUs) with 98% similarity threshold. OTUs were taxonomically assigned with 80% confidence cutoff using Naïve Bayesian Classifier trained with UNITE (v9, dynamic, all eucaryotes) database in mothur v1.36.1. For the analyses of fungal OTU community composition and richness, we converted read abundances into presence-absence by applying 1‰ relative abundance threshold. We checked for spurious sequencing results by comparing replicate samples of fine woody debris based on Jaccard distances. We identified three instances where one of the four replicates was notably discordant with the other three (Jaccard distance >0.7) and discarded those replicates from further analyses. For DFWD samples, OTU presence-absence data was then concatenated from individual replicates to sample level.

We collected saproxylic beetles by peeling and sifting bark. From fine woody debris, collected samples by peeling off loose bark from all pieces collected within the sample plot. From birch logs, we sampled 1 m² of bark per log. After sifting, the resulting wood litter was placed in plastic bags and studied later indoors, by spreading the litter in small lots on a white tray and collecting all beetle adults and larvae. We identified extracted adult beetles to species morphologically and larvae by sequencing the cytochrome c oxidase subunit I barcode gene region with the primers LCO1490 (GGTCAACAAATCATAAAGATATTGG) and HCO2198 (TAAACTTCAGGGTGACCAAAAAATCA). We used BOLD as the reference database for barcode sequence identification. In addition, a few species were recorded based on visual observations of characteristic exit holes and larval galleries in the wood.