Fragile foundations: Succession patterns of bacterial communities in fine woody debris and soil under long-term microclimate influence

Fragile foundations: Succession patterns of bacterial communities in fine woody debris and soil under long-term microclimate influence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fragile foundations: Succession patterns of bacterial communities in fine woody debris and soil under long-term microclimate influence Vojtěch Tláskal, Jason Bosch, Priscila Thiago Dobbler, Jörg Müller, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6118769/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Environmental Microbiome → Version 1 posted 11 You are reading this latest preprint version Abstract Background Fine woody debris (FWD) is a crucial yet often overlooked component of forest ecosystems, providing a dynamic habitat for microbial communities and playing a key role in carbon and nutrient cycling. In managed forests with low deadwood stocks, FWD decomposition enhances soil fertility by facilitating microbial nutrient cycling. Climate change increases the prevalence of forest disturbances enhancing the area of early succession forests with low canopy cover, but the consequences on the microbial communities and related processes is insufficiently understood. Results Here we conducted a ten-year experiment manipulating canopy cover to examine the decomposition of FWD of Fagus sylvatica and Abies alba . Our study revealed that canopy openness significantly affected bacterial diversity in the decomposing wood as well as in the surrounding soil. While community structure in FWD was primarily influenced by decomposition time, tree species and canopy density also played a role. We identified bacterial taxa associated with carbohydrate utilization, fungal biomass degradation, and nitrogen fixation, highlighting the diverse functional roles of FWD bacteria in nutrient cycling. Bacterial community in almost completely decomposed FWD remains clearly distinct from soil bacterial communities. Conclusions Complex ecological interactions shape deadwood decomposition and nutrient cycling. The interplay between FWD decomposition time, tree species, and microclimatic variability influences microbial community dynamics, with bacteria acting as a more stable component of the decomposer community compared previously studied fungi. This stability may be critical for sustaining decomposition and nutrient turnover despite environmental fluctuations associated with global change. decomposition deadwood bacterial community succession canopy cover microclimate temperate forest ecology fine woody debris Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Forests are essential for nutrient cycling and serve as a major global carbon sink, storing approximately 45% of the carbon (C) present in terrestrial ecosystems [ 1 , 2 ]. Soil is the largest terrestrial carbon pool storing roughly 44% of the forest C, while deadwood accounts for another 8%. In natural forests with numerous snags and coarse wood from fallen trees, fine woody debris (FWD, deadwood with a diameter of less than 10 cm) constitutes only a small fraction of the total deadwood stock [ 3 , 4 ]. In contrast, in managed forests where the major part of trees is typically extracted, FWD represent an important pool of considerable amount. For example, within the management zone of the Bavarian Forest National Park, the amount of FWD was estimated at ∼17.5 m 3 ha − 1 [ 5 ], roughly corresponding to the recently reported quantity of coarse wood in European forests across all management types [ 6 ] affected by strong deadwood extraction [ 7 ]. FWD shows rapid turnover due to fast decomposition [ 5 , 7 ]. The carbon flux through FWD is approximately five times faster than that through large deadwood (coarse woody debris, CWD) and in total it corresponds to the flow of a stock of approximately 90 m 3 ha − 1 of CWD [ 5 ]. FWD thus represent a key part of the carbon and nitrogen flow in managed forests. Deadwood serves a key source of forest soil nutrient pool [ 8 – 11 ] and plays a crucial role in maintaining forest biodiversity [ 12 , 13 ], providing both habitat and nutrients for a diverse array of organisms, including microorganisms, such as fungi and bacteria [ 14 – 17 ]. Different deadwood size, turnover dynamic and frequent fragmentation of the FWD creates a high variety of habitats and a high number of habitat patches, leading to a high diversity in detected fungal communities [ 18 ] and increase in fungal species richness as a consequence of their competitive life style [ 19 ]. Deadwood decomposition is steered by fungi, thanks to broad enzymatic portfolio enabling them to efficiently colonize large sections of wood and decompose it as nutrient source, despite the recalcitrance, fluctuating moisture content and low nitrogen content of this substrate. Studies from forest topsoil and deadwood, however, pointed to the prevalence of bacterial taxa with potential to contribute to decomposition of wood structural compounds like cellulose and hemicellulose [ 20 – 22 ]. These include Bacteroidota (e.g. Mucilaginibacter ) and Acidobacteriota that incorporate C from 13 C-labelled cellulose [ 23 , 24 ]. Similar taxa were characterised as versatile decomposers based on their rich gene toolkit for carbohydrate decomposition [ 21 , 25 ]. Gammaproteobacteria (e.g. Hermiinimonas , Variovorax ) were also associated with degradation of complex polymers [ 24 , 26 , 27 ]. Contrary to the typically high abundance of Alphaproteobacteria, these decomposers might be actively growing while utilizing labile carbon sources, as they exhibit limited potential for complex carbohydrate decomposition [ 25 ]. Acidobacteriota are furthermore reported to be associated with fungal biomass [ 28 ], which represents a more accessible substrate from a decomposer's perspective owing to its lower C:N ratio. Indeed, several acidobacterial strains were shown to be able to perform chitin degradation to cover nitrogen and carbon demand [ 29 , 30 ]. While mycophagy represents one approach how obtain nitrogen at a lower resource cost for microbes, there are other approaches to overcome the recalcitrance of deadwood. Nitrogen might be translocated from surrounding soil [ 31 ] or its pool might be enriched in situ by specialised diazotrophic bacteria dwelling in deadwood and performing nitrogen fixation [ 22 , 29 ]. Increased nitrogen level enables further microbial colonization while it also enables nitrogen immobilization in microbial biomass with potential importance for subsequent soil formation. Although the majority of studies have targeted the decomposition of coarse wood [ 17 , 32 – 34 ] and its influence on surrounding soil, FWD has been shown to be reservoir of fungal forest diversity [ 5 , 18 , 19 , 35 , 36 ]. The high surface-to-volume ratio of FWD means higher a sensitivity of FWD to changes in temperature and moisture [ 35 ]. These microclimatic fluctuations are strongly related to both forest management practice and forest stand damage. Importantly, disturbance rates increase with the ongoing global change and damage to canopy cover is expected to be an important factor affecting ecosystem processes [ 2 ]. The canopy openness of forests is strongly linked to their temperature buffering capacity [ 37 , 38 ]. Increased respiration rate [ 39 ], elevated summer temperatures, intensive solar radiation, and fluctuating water content are characteristic for forest gaps [ 40 ]. The microbial community in FWD is thus subjected to greater fluctuations of these environmental variables. In previous research, focused on characterisation of microbial community throughout the lifetime of FWD, the fungal community was showed to be influenced by both tree species of FWD and microclimatic conditions (canopy openness) [ 5 ]. In contrast, the bacterial community is typically considered less tightly bound to the deadwood of the preferential tree species [ 17 ], although other studies indicate the close relationship [ 41 , 42 ]. Moreover, bacteria are very sensitive to pH and moisture fluctuations [ 11 , 17 , 43 ]. Thus, bacterial community assembly in deadwood is likely affected by multiple driving factors of varying intensity and its development during FWD decomposition remains elusive. In this study, we follow up on an experiment where the decomposition successional patterns of the FWD of beech ( Fagus sylvatica ) and fir ( Abies alba ), the two main tree species of the temperate forests of central Europe [ 44 ], were followed throughout its decomposition lifetime under open and closed canopies. The aim was to determine the factors affecting bacterial succession during decomposition with the open canopy treatment serving as a proxy of a disturbed ecosystem. Moreover, canopy openness and deadwood source selection allowed us to assess the size and relative importance of the effects of microclimate and the host tree. The frequency of canopy disturbance events has increased in the temperate zone, and they have more pronounced effects in managed forests than in unmanaged forests [ 45 ]. Climatic factors are important in influencing fungal community composition [ 46 ], sun exposure and canopy openness influence inner temperature of larger deadwood objects [ 36 ], composition of saproxylic beetles [ 47 ] and assembly patterns of fungi in FWD [ 5 ]. As the wood decomposer strategies may exert significant selection effects [ 48 ] determining the bacterial community structure [ 49 ], all these environmental factors likely contribute to shape bacterial community development. We hypothesize that bacterial community will undergo directional succession. During complete decomposition of FWD, it should be possible to track the decrease of endophytic taxa, steady prevalence of bacterial generalists, and increase in specialists with the ability to degrade recalcitrant substrates. Based on previous research on CWD [ 17 ], we hypothesize that the bacterial community at the late stage of decomposition with almost decomposed FWD, resembles the soil community. To enable such comparison, we performed characterization of the soil bacterial community at the end of experiment to be able to characterise the overlap of the FWD and soil bacterial communities. In addition to carbon utilisation, we expect to record bacterial taxa involved in nitrogen cycling including taxa known for nitrogen fixation and mycophagous bacteria which target fungal biomass for its less restrictive C:N ratio. Previous studies indicate that forest management does not significantly affect soil and rhizosphere bacterial communities in short term scheme [ 50 ]. Here, we specifically focused on long-term changes in soil properties and soil bacterial community composition in Bavarian Forest National park, where natural vegetation recovery rate is very slow and we expect detectable changes in soil properties due to the limited primary productivity of open canopy sites. Materials and Methods Study area and experimental design The experimental sites were located in the management zone of the Bavarian Forest National Park in Germany (48.9° N, 13.3°E). The management zone covers an area of 6,000 ha that surrounds the 18,000 ha core zone of the national park. The area is characterized by montane mixed forest consisting of European beech ( Fagus sylvatica L.), Silver fir ( Abies alba Mill.) and Norway spruce ( Picea abies (L.) H. Karst) [ 35 ]. The sampling design was a part of the broader experimental design described in detail in previous publications [ 5 , 51 ]. In autumn 2011, freshly cut branches of fir and beech were deposited on 64 plots and arranged in a random block design with four spatially independent blocks. The branches (fine woody debris, FWD) had diameters of 3.2 ± 1.3 cm and lengths of 2.7 ± 0.9 m. These branches were taken from trees of the same age that were harvested from the same forest stand. The origin of the branches was identical and branches were randomly distributed across study sites to mitigate the potential effect of communities of fungal endophytes inhabiting individual branches on microbial community development. Each block contained randomly located sets of plots with either fir or beech branches or both. The mixture of fir and beech deadwood represented the factor of forest stand tree diversity. Within each block, two plots per treatment (fir, beech, or mixed) were set under open or closed canopies (Supplementary Fig. 1). Canopy openness was used as a surrogate for stand microclimates [ 13 , 38 , 51 , 52 ]. The open canopy plots were the result of clearings where an area of 0.1 ha was freed from living and dead trees. To avoid shading by a dense grass layer surrounding the deadwood on open canopy plots, each plot was mowed once a year during the growing season as described previously [ 53 , 54 ]. The daily peak temperatures of the deadwood surfaces in summer were measured in the open and closed plots. The mean values were much higher in open canopy plots (~ 30°C) than in the closed canopy plots (~ 15°C) [ 55 ]. All experimental plots were sampled annually in September / October from 2012–2018 and in 2021 and 2022. Sampling, sample processing and analysis One composite FWD sample was obtained from each selected branch. It was obtained from two vertical drillings of the branch in its centre using an electric drill equipped with a 10 mm diameter auger across the entire diameter of the branch. The drilling points were placed evenly along the branch, avoiding the close proximity of the ends of the branch. The auger was sterilized between drillings, and the dust from all drilling points was collected in sterile plastic bags and frozen within a few hours after drilling. In total, 2 beech or 2 fir samples were taken from each block from plots containing only beech or fir, and 2 beech and 2 fir samples were taken from plots containing mixed deadwood, resulting in 4 beech and 4 fir composite samples per canopy type. Thus 64 samples in total were taken annually, (Supplementary Fig. 1) resulting in a total of 512 samples available for this study. The drilled materials were weighed in the laboratory and freeze-dried to estimate the deadwood dry mass. Next, it was milled using an Ultra Centrifugal Mill ZM 200 (Retsch, Germany), and the resulting fine powder was used for the subsequent analyses. Dry mass content was based on mass loss during freeze-drying, and pH was measured after mixing with distilled water (1:10 w: vol). The wood carbon and nitrogen contents were measured using an elemental analyser in an external laboratory of the Institute of Botany of the Czech Academy of Sciences, Průhonice, Czech Republic, as described previously [ 56 ]. Carbon was measured using sulfochromic oxidation, and the nitrogen content was estimated by sulfuric acid mineralization with the addition of selenium and sodium sulfate and conversion to ammonium ions, which were measured by a segmented flow analyser (SFA), Skalar. To quantify fungal biomass, total ergosterol was extracted using 10% KOH in methanol and analysed by HPLC [ 57 ]. Soil from all sampling sites was collected in September 2022 in one sampling campaign. The sampling point was located in the close proximity of tested FWD object (proximately 1-1.5 m). Plastic soil cores (4.5 cm in diameter) were hammered in central point and 0.5 m from central a point to the North, South, West and East and the full core were placed in plastic bag. Soil was immediately transferred into laboratory and stored prior to processing for maximum of 24 hours at 4°C. When processed, litter layer was removed and discarded. Only the top 10 cm of soil was used as a composite sample after homogenization and sieving through a 5 mm sieve. The homogenized soil sample was immediately frozen, freeze-dried and stored at -20°C prior to subsequent analyses. All further analyses were performed the same way as for deadwood samples. Extraction and analysis of environmental DNA Total genomic DNA was extracted from 200 mg of freeze-dried material using the NucleoSpin Soil Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. Briefly, cells were lysed using SL1 lysis buffer. Enhancer SX was added prior to lysis. The samples were homogenized using FastPrep-24 (MP Biomedicals, Santa Anna, USA) at 5 m s − 1 for 2 × 30 s. In the last step, DNA was eluted from the columns using 50 µl of deionized water. One extraction per sample was performed [ 58 ]. For the bacterial community analysis, PCR amplification of the prokaryotic hypervariable V4 region of the 16S rRNA gene was performed using barcoded 515F and 806R primers [ 59 ] in triplicate PCRs per sample as described previously [ 17 ]. PCRs contained 5 µl of 5× Q5 reaction buffer, 1.5 µl of BSA (10 mg ml − 1 ), 1 µl of each primer (0.01 mM), 0.5 µl of PCR Nucleotide Mix (10 mM each), 0.25 µl of Q5 High Fidelity DNA polymerase (2 U µl − 1 , New England Biolabs, Inc.), 5 µl of 5× Q5 HighGC Enhancer and 1 µl of template DNA (approx. 50 ng µl − 1 ). Cycling conditions were 98°C for 30 sec, 25 cycles of 94°C for 10 sec, 56°C for 30 sec, and 72°C for 20 sec, and a final extension at 72°C for 2 min. PCR triplicate reaction products were pooled and purified (MinElute PCR Purification Kit, Quiagen), and amplicon libraries prepared with the TruSeq DNA PCR-Free Kit LP (Illumina) were sequenced in house on the Illumina MiSeq (2 × 250-base reads). The amplicon sequencing data were processed using the pipeline SEED 2.1.3 [ 60 ]. Briefly, paired-end reads were merged using fastq-join [ 61 ]. Chimeric sequences were detected using Usearch 11.0.667 [ 62 ] and deleted, and sequences were clustered using UPARSE implemented within Usearch 8.1.1861 [ 63 ] at a 97% similarity level. The most abundant sequence was selected from each cluster, and the closest hits at the species level were identified using BLASTn against an edited version of the SILVA 138.1 database including mitochondria and chloroplasts. Where the best hit showed lower similarity than 97% with 95% coverage, the best genus-level hit was identified. The sequences used in all community structure analyses were rarefied at 11400 sequence (median of sequence count for all samples). Sequences from FWD and soil were processed together and the dataset was split as needed. The species-level analyses were performed on a dataset where OTUs belonging to the same species were combined and all other OTUs were combined into the genus of the best hit and designated “sp.” Only sequences identified as bacterial were used in the analysis. Sequencing data have been deposited in the SRA database under BioProject accession number PRJNA1228314 and in Zenodo repository ( https://doi.org/10.5281/zenodo.14900894 ). Data processing and statistics Succession time or so called temporal niche position was defined as the average position of a taxon in succession considering its relative abundance over time, and the duration of occurrence was defined as the time span covering 90% of the taxon relative abundance as defined and calculated previously [ 64 ]. Tree or canopy specificity was defined as the strength of association of the taxon with one particular tree or canopy type and calculated as the sum of abundances in beech deadwood (closed canopy deadwood) divided by the sum of abundances in all samples. A value of 1 corresponds to a taxon exclusively found on beech deadwood. A value of 0.5 assigned to a taxon indicates that it is equally abundant on beech and fir deadwood. A value of 0 assigned to a taxon indicates that it is exclusively found on fir deadwood. For canopy openness, a value of 1 corresponds to a taxon exclusively found under the closed canopy and 0 corresponds to a taxon exclusively found under the open canopy. Taxa with tree specificities between 1.0 and 0.95 were considered beech-specific (or closed canopy-specific, respectively), and those with specificities between 0.05 and 0.00 were considered fir-specific (or open canopy-specific, respectively). Statistical analyses were performed in R [ 65 ]. Two-dimensional nonmetric multidimensional scaling (NMDS) ordination analysis on Bray-Curtis distances was used to address the dissimilarity of the fungal community compositions based on Hellinger-transformed relative abundances (package vegan , function metaMDS [ 65 , 66 ]. Variables were fitted to the ordination diagram as vectors with 999 permutations and included pH, as well as the carbon and nitrogen and ergosterol contents. Diversity estimates (Shannon–Wiener index, OTU richness, Chao 1 and evenness) were calculated for a dataset containing the relative abundance of 2,100 randomly selected sequences from each sample in SEED 2.1.3. [ 60 ]. Differences in the environmental variables (pH, carbon, nitrogen, C:N, ergosterol and water contents) were tested using a linear mixed model (LMM, function lmer) with log-transformed data. For the LMM, the effect of explanatory variables, i.e., tree species, canopy openness and their interaction, over decomposition time were tested by considering time and plot identifiers (the same plots were repeatedly measured over time, the objects are nested in block/plot) as random effects. One-way or two-way PERMANOVA tests with 9,999 permutations were used to examine the effects of treatments on bacterial communities. Spearman rank correlations were used as a measure of the relationships between variables. Variation partitioning analyses on Hellinger-transformed OTU abundances were performed to identify the parts of the variance explained by tree species and canopy openness for the whole dataset and with length of decomposition, canopy openness and wood chemistry (i.e., nitrogen, carbon, C:N and pH) for each tree species independently (package vegan , function varpart [ 66 ]. The importance values of the obtained variances were determined with Monte Carlo permutation tests. In all cases, differences at P < 0.05 were considered statistically significant. Results Properties of fine woody debris during ten years of decomposition The physicochemical properties of decomposing fine woody debris (FWD) over a decade revealed dynamic development similar to the early decomposition phases [ 5 ]. Carbon content increased during the initial stages of decomposition but seems to stabilize thereafter (LMM: χ² = 2.37, P = 0.12). Fine woody debris of fir ( Abies alba ) exhibited significantly higher carbon content than beech ( Fagus sylvaticus , LMM: χ² = 49.85, P < 0.001), while canopy cover had no significant effect (Fig. 1 ). Nitrogen content showed a gradual increase over time (LMM: χ² = 5.26, P = 0.02). Beech FWD had higher nitrogen content than fir (LMM: χ² = 52.39, P < 0.001), and nitrogen levels were generally lower under canopy gaps, although the effect of canopy was significant only for fir deadwood (LMM: χ² = 4.35, P = 0.00). As nitrogen content increased, particularly since year 7, it approached levels observed in soil. Consequently, the C:N ratio of decomposing FWD declined, though this trend was not statistically significant due to high variability (Fig. 1 ). The pH of FWD decreased over time, reaching its minimum in 2018, followed by a slight increase in 2021 (LMM: χ² = 8.49, P = 0.00). pH values were higher under open canopy conditions (LMM: χ² = 6.68, P = 0.01) but did not differ significantly between tree species (LMM: χ² = 0.23, P = 0.63). Water content increased during the later stages of decomposition (LMM: χ² = 11.88, P < 0.001), although variability was high. Beech FWD consistently exhibited higher moisture levels compared to fir (LMM: χ² = 47.32, P < 0.001) (Supplementary Fig. 2). Fungal biomass, measured via ergosterol content, increased during the first 6–7 years of decomposition and subsequently declined (LMM: χ² = 7.50, P = 0.00). Beech FWD contained approximately twice as much ergosterol as fir (LMM: χ² = 76.78, P < 0.001), and ergosterol levels were higher under closed canopy conditions (LMM: χ² = 94.59, P < 0.001). These trends suggest that the depletion of accessible nutrients characterizes advanced decomposition stages. Longtime changes in soil physicochemical properties caused by canopy openness The analysis of soil physicochemical properties revealed significant long-term changes resulting associated with the differences in canopy cover (Fig. 2 ). The overall carbon-to-nitrogen (C:N) ratio increased markedly in response to canopy opening, rising from 18.7 under closed canopy conditions to 21.2 under open canopy conditions (ANOVA: F = 35.36, df = 1, P < 0.001). This shift was not hidden behind notable spatial variability in the C:N ratio (ANOVA: F = 1.48, df = 3, P = 0.24). Soil carbon content increased substantially following canopy opening (ANOVA: F = 9.05, df = 1, P < 0.001), but was only marginally affected by site-specific factors. In contrast, detected variations in soil nitrogen content and fungal biomass content were predominantly influenced by site-specific factors rather than the effects of canopy openness. Soil moisture levels, which also increased significantly after canopy opening (ANOVA: F = 3.03, df = 1, P = 0.04), showed a strong dependence on both canopy openness and site-specific conditions (Supplementary table 1 ). Soil pH parameters were identical nonetheless the canopy stage or site specificity. All measured soil parameters differed significantly from the values observed in decomposing fine woody debris (ANOVA: P < 0.002, see Fig. 2 and Supplementary table 1 ) from these sites. Impact of canopy openness on bacterial community composition and diversity in soil Bacterial diversity in soil and fine woody debris (FWD, described on OTU level), assessed through species richness and the Chao-1 index, differed significantly (Kruskal-Wallis test, P = 0.000). On average, species richness in soil was higher, with 500 ± 11 species compared to 405 ± 29 species in FWD. Similarly, the Chao-1 index was higher in soil (1,043 ± 34) than in FWD (776 ± 76). While the overall bacterial diversity in soil remained unaffected by canopy openness (ANOVA, species richness F = 1.53, df = 1, P = 0.22 and Chao-1 index F = 1.06, df = 1, P = 0.319), the composition of the soil bacterial community was significantly influenced by canopy cover and environmental properties at the sampling locations (PERMANOVA: F = 6.97 and F = 2.22, P = 0.00 for both). These factors operated independently, with canopy cover accounting for 1.7% of community variability, and environmental parameters explaining 24.8% (variation partitioning: P = 0.002 and P = 0.00). Among the tested environmental factors, soil moisture had the largest impact, explaining 14.8% of the variability (P = 0.001), followed by the C:N ratio (4.2%, P = 0.00) and soil pH (8.3%, P = 0.00, see Fig. 3 ). Bacterial communities in soil were distinct from those in FWD (PERMANOVA: F = 151.3, df = 1, P = 0.001). Although soil bacterial communities were canopy-specific, the differences were less pronounced in the NMDS plot due to strong successional turnover (Fig. 4 ). The bacterial composition in soil was characterized by a high abundance of Pseudomonadota (28.5%, mainly Alphaproteobacteria, 22.7%), Acidobacteriota (22.2%), and Planctomycetota (10.1%) (Fig. 5 ). The most abundant soil genera included undefined_Subgroup_2 Acidobacterium , Acidothermus , undefined Xanthobacteraceae bacterium , undefined Elsterales bacterium , and undefined Acidobacteriales bacterium , each with an average relative abundance exceeding 5%. Together, dominant bacterial taxa accounted for as much as 85% of soil bacterial abundance, but these same taxa represented only 30% of bacterial abundance in FWD. The observed differences in bacterial community structure between soil and FWD were largely driven by variations in bacterial abundance (Supplementary Fig. 3). Highly abundant soil bacteria (those with > 0.5% relative abundance) were generally not canopy-specific. However, the Burkholderia-Caballeronia-Paraburkholderia group was significantly more prevalent under closed canopies, while Occallatibacter and Bacillus sequences were most frequently detected under open canopies, with more than 75% of their sequences occurring in these conditions (Stable2:Soil_bacteria_abundance). Bacterial community compositions in the different types of fine woody debris The diversity of bacterial communities in decomposing fine woody debris (FWD) was markedly lower than in soil, as noted earlier. However, the impact of canopy cover on bacterial communities in FWD was more pronounced. Bacterial diversity, estimated using the Chao-1 index and species richness, was significantly higher under closed canopy conditions and in fir FWD compared to beech (Kruskal-Wallis test, P = 0.00). Species richness in fir FWD reached its highest levels under open canopy conditions during the third and fifth years of decomposition and declined in later stages. Despite this decline, richness levels remained above those observed during the first year of decomposition (Supplementary Fig. 4). Throughout the decomposition process, bacterial diversity exhibited substantial variability across FWD samples. Bacterial communities in both beech and fir FWD underwent clear, continuous changes over time (PERMANOVA: F = 166.5, P = 0.00,) throughout the whole decomposition period (Fig. 4 , analysis including only deadwood samples). While these temporal changes were less pronounced than those observed in fungal communities [ 5 ], canopy cover and the origin of deadwood both significantly influenced bacterial community structure (PERMANOVA: F = 34.43 and F = 22.93, respectively, P = 0.00,). However, these effects were not clearly distinguishable in the NMDS plot (Fig. 4 ). Increased heterogeneity of deadwood origin had no significant impact on bacterial community composition (PERMANOVA: F = 0.87, P = 0.84). Variation partitioning analysis identified temporal development as the primary driver of bacterial community composition, uniquely explaining 29.3% of the observed variability (PERMANOVA: F = 32.63, P = 0.00, Fig. 3 ). Tree species and canopy cover explained much smaller proportions, accounting for 3.1% (F = 24.44, P = 0.00) and 4.7% (F = 36.22, P = 0.00) of the variability, respectively. Bacterial communities in soil generally show strong responses to the physicochemical conditions of the substrate, prompting additional analysis of environmental parameters in the context of forest management treatments. However, environmental factors alone accounted for only 1.3% of the total variability, and their impact on bacterial community structure was not statistically significant (PERMANOVA: F = 1.28, P = 0.126). When combined with temporal development, environmental factors increased the share of explained variability to 17.4%, while temporal development as a standalone factor explained 11.7%. Despite all these findings, a significant portion of the variability in bacterial community composition (62%) remained unexplained, highlighting the complexity of the ecological processes influencing bacterial communities in decomposing FWD. Overall, FWD bacterial communities were dominated by Pseudomonadota (46.7%), particularly Alphaproteobacteria (27.5%) and Gammaproteobacteria (19.2%), with high share of Actinomycetota (13.6%) and Acidobacteriota (11.7%). Most abundant bacterial phyla underwent continuous successional changes, trending towards soil-like community composition. Specifically, Pseudomonadota and Bacteroidota decreased over time, Acidobacteriota increased, and Actinomycetota exhibited fluctuations. Verrucomicrobiota and Planctomycetota became more prominent as decomposition advanced. Despite these shifts, the bacterial community composition in FWD remained distinct from that in soil even after 10 years of decomposition. Differences in bacterial communities under closed canopies and in canopy gaps were evident only at lower taxonomic levels (Fig. 6 , Supplementary Fig. 5). Highly abundant bacterial genera clustered into early (2012–2014) and late (2015–2021) phases of decomposition, with the final years clustering more closely. During the early phase, dominant genera included Sphingomonas (6.4% average abundance), Galbitalea (5.6%), Granulicella (4.4%), Mucilaginibacter (4.3%), Burkholderia - Caballeronia - Paraburkholderia group (4.0%), Dyella (3.4%), Pseudomonas (2.8%), and Pedobacter (2.7%). In the later phase, the community shifted towards dominance of Burkholderia - Caballeronia - Paraburkholderia group (8.8%) alongside Granulicella (6.1%). Other taxa with higher contributions included undefined Methylacidiphilaceae (3.4%), Mucilaginibacter (3.1%), Conexibacter (2.9%), and Jatrophihabitans (2.9%). Bacterial communities in FWD exhibited clear successional dynamics over the course of decomposition. The majority of bacterial taxa persisted for extended periods, reflecting a slow turnover rate, with the exception of certain taxa present during the initial years of decomposition, which demonstrated faster turnover rates (Fig. 6 ). Most bacterial taxa were detected in both beech and fir FWD and were not specific to canopy cover, based on the defined criteria (95% of total abundance restricted to a selected group). Among the highly abundant bacteria, only Dyella (Gammaproteobacteria), Gryllotalpicola (Actinobacteriota ), Endobacter (Alphaproteobacteria) and Pleomorphomonas (Alphaproteobacteria) were open canopy specific bacterial genera. Moreover, Luteibacter and Comamonas (Gammaproteobacteria) were in roughly 74% recovered from sites under closed canopy and can be described as “preferring close canopy”. Similarly, Pseudomonas (75%, Gammaproteobacteria) and Methylovirgula (81%, Alphaproteobacteria) preferred beech FWD and Nocardioides (75%, Actinobacteriota), undefined Chitinophagaceae bacteria (78%, Bacteroidota) and Kineosporia (87%, Actinobacteriota) belonged to fir FWD preferring bacterial genera. Despite these variations, the calculated succession times of commonly present bacterial taxa did not differ significantly between beech and fir FWD or between open and closed canopies (Fig. 6 ). Discussion Our study highlights key factors influencing fine woody debris (FWD) decomposition and associated bacterial communities, including the effects of microclimatic conditions caused by canopy openness on surrounding soil bacterial communities. We observed a rapid initial decomposition phase followed by a slowdown, linked to fungal activity decline and pH stabilization. Canopy openness affected FWD and soil properties but had limited long-term effects on fungal biomass, while bacterial diversity in FWD and soil responded primarily to substrate characteristics rather than canopy conditions. Bacterial community succession in decomposing FWD revealed a continuous shift, with a few canopy- and tree-specific taxa. Nonetheless, even in the highly advanced decomposition stage, bacteria inhabiting decomposing FWD—spanning a broad range of functional roles—formed a distinct community compared to the surrounding soil. These findings underscore the complex interplay between canopy structure, soil properties, and microbial community dynamics in forest ecosystems. Fine woody debris decomposition The extension of the FWD decomposing experiment [ 5 ] allowed for the observation of the complete lifespan of FWD. While FWD decomposition progressed rapidly in the early years, the rate slowed after seven years, as indicated by a gradual decrease in ergosterol content and an increase in pH values. This pattern may reflect a decline in fungal wood decomposer activity typical for coarse deadwood [ 58 , 67 ]. Fungi actively lower pH through the production of organic acids, so their decline in later decomposition stages leads to pH stabilization or even an increase. This shift suggests weakening of fungal control over the decomposition process. The higher ergosterol content and moisture levels in beech FWD are consistent with the faster decomposition of beech deadwood, as previously reported [ 67 – 69 ]. This trend further supports the presence of ectomycorrhizal species [ 5 ], which typically appear in the final stages of succession [ 58 , 70 ]. The increase in nitrogen content in the late decomposition phase, partially due to the accumulation of nitrogen-rich microbial biomass and biological nitrogen fixation, suggests a gradual convergence with soil nutrient profiles. Despite variations in decomposition time, changes in deadwood chemistry were largely consistent with initial wood composition, leading to a relatively uniform decomposition process across different deadwood origins and microclimatic conditions. Fluctuations in FWD pH and moisture content [ 71 ], along with shifts in fungal community composition and development [ 48 , 49 ], were likely key factors influencing bacterial community assembly, as these parameters strongly predict bacterial composition on decomposing deadwood. Longtime changes in soil physicochemical properties caused by canopy openness Canopy openings influence forest microclimates [ 37 , 38 ] and affect soil properties, especially carbon content and nutrient availability [ 72 ]. These effects vary with gap size, age, and forest stand characteristics [ 73 ]. Contrary to expectations, we observed an increase in total soil carbon, C:N ratio, likely due to altered organic matter inputs and the slow recovery of forest floor vegetation. Dense and intense grass layer and no clear structured organic layer was frequent under open canopy in contrast to the closed forest. Increase in soil moisture was observed also in other forest clearcutting experiments (data not published). Despite changes in soil conditions, fungal biomass remained unaffected by canopy opening in the long term. This aligns with previous findings showing that fungal biomass declines immediately after clear-cutting but recovers rapidly in regenerating forests within 2 years, mainly due to an increase in nonmycorrhizal fungi [ 74 ], Awokunle-Holla, in preparation). Further support comes from the lack of a relationship between soil CO₂ efflux and canopy openness. Instead, microbial activity, as indicated by CO₂ efflux, was more influenced by soil properties than by microclimatic factors [ 75 ], though increased respiration has been reported in some gap studies [ 39 ]. Higher soil moisture in gaps, detected in our study and repeatedly previously reported [ 40 ], likely results from reduced transpiration-driven water loss. These observations have important ecological implications, as prolonged droughts due to climate change can severely impact forests, which are less drought-resistant than agricultural and grassland systems [ 76 ]. However, small canopy gaps, with reduced tree water uptake, may serve as refugee for moisture-sensitive soil organisms. While fungi are generally more drought-resistant, bacterial communities are more vulnerable to prolonged moisture deficits [ 77 ]. Soil pH plays a pivotal role in shaping microbial communities, influencing key processes such as nutrient cycling and organic matter decomposition. However, no significant changes in soil pH were detected in our study or a recent meta-analysis on canopy gaps [ 72 ]. The observed changes in soil properties and microbial community composition thus underscore the complex interactions between canopy structure, soil conditions, and microbial communities in forest ecosystems. Drivers of bacterial community composition in fine deadwood and soil, effect of canopy openness Bacterial diversity reached its peak between the third and the fifth year of decomposition, showing the steep increase after the initial two years before declining towards the end of the decomposition process. The low diversity values during initial decomposition can be attributed to a less developed microbial community that utilizes labile carbon sources and includes potential plant pathogens known from living trees (genus Erwinia , [ 78 ] and taxa typically detected in early decomposition phase of CWD (genera Sphingomonas , Sodalis and Pseudomonas, [ 25 ]. The priority effect, which favours already present taxa, likely shapes the initial decomposition phase [ 79 , 80 ]. The middle stage of decomposition is characterized by increasing bacterial diversity [ 81 ], suggesting that the complex community might be reaching the maximum carrying capacity of FWD as a growth substrate. During the final decomposition stage, the bacterial community shows decreasing diversity and structurally approaches the composition of soil communities. This convergence occurs as FWD disintegrates and mixes with the surrounding soil, leading to increasingly similar habitat variables and, consequently, similar communities. Despite the convergence trend, structural differences between FWD and soil bacterial communities remained significant throughout the decomposition process in our experiment, with FWD communities consistently showing relatively high diversity, yet significantly lower than soil bacterial communities. This lower diversity can be attributed to less favourable conditions for bacterial growth in deadwood, including high C:N ratios, lower pH and likely increased competition for nutrients with fungi in a habitat lacking carbon input through rhizodeposition [ 70 , 82 ]. While soil serves as a reservoir of bacterial diversity for deadwood colonization [ 83 ], the selective pressures within deadwood drive the community through specific developmental stages. The decomposition process itself continuously influences pedological properties and microbial community in the upper horizons of underlying soil, creating an interplay between these two physically connected habitats [ 68 ]. Similarly to fungal diversity [ 5 ], fir deadwood supported higher bacterial diversity than beech, extending our understanding of substrate-specific community development during FWD decomposition. While previous studies have documented comparable or lower bacterial richness in beech compared to other tree species [ 17 , 71 ], the diversity patterns in fir remained unexplored. The contrasting responses of bacterial and fungal communities to canopy conditions - with bacterial diversity flourishing under closed canopy while fungal diversity in fir FWD increased with canopy opening [ 5 ] - suggest distinct niche differentiation mechanisms. Despite the observed influence of tree species and canopy openness on community structure, the major bacterial taxa in FWD displayed remarkable habitat generalism, showing no strong preferences for either tree species or canopy conditions. This pattern of environmental response extended to the soil bacterial communities, where canopy openness induced subtle yet significant structural changes likely through altered forest floor dynamics, including shifts in vegetation, litter input, and root exudate profiles [ 84 ]. The absence of canopy-specific associations among dominant soil bacterial taxa, combined with their stronger response to abiotic factors such as soil moisture, C:N ratio, and pH [ 85 ], indicates that bacterial community assembly in both FWD and soil is primarily driven by fundamental habitat conditions rather than canopy-related variables, confirming the previous results from forest clearcuts [ 50 ]. Generalists versus Specialists The genus Granulicella (Acidobacteriota) and Mucilaginibacter (Bacteroidota) showed genomes containing rich toolkit of CAZymes which suggests their broad substrate specificity [ 25 , 86 ]. Indeed, these taxa were identified at high relative abundances throughout the whole FWD decomposition which might indicate that they may modulate their metabolism to maintain high population density even during the changes in substrate availability. The role of Acidobacteriota in the decomposition was confirmed by the fact that their carbohydrate-active enzymes were widely expressed on coarse deadwood [ 20 ]. In addition to Acidobacteriota, potential for versatility in carbohydrate utilization was shown in a global genome comparison also for Planctomycetota and Verrucomicrobiota [ 21 ] which appeared as Tundrisphaera (Planctomycetota), Chthoniobacter and Methylacidiphilaceae bacterium (Verrucomicrobiota) in the advanced FWD decomposition phase. In contrast to generalists, several taxa showed preference for a specific phase of FWD decomposition. This includes Erwinia (Gammaproteobacteria) known as a potential pathogen from live plant tissues [ 78 ] and from association with insect pests [ 87 ]. Their high abundances at the beginning of decomposition might represent a diminishing community which is not adapted for long-term survival in decomposing wood. Luteibacter displayed the preference for the early FWD decomposition and was shown to degrade cellulose in soil [ 88 ]. In a pure culture, Luteibacter carbohydrate utilization was performed mainly through the cell-associated enzymes [ 89 ] which might prevent resource loss through diffusion of decomposition products but also represents disadvantage in the advanced decomposition when proximal resources might be exhausted. Pedobacter abundant at the beginning of FWD decomposition was shown to degrade simple compounds [ 26 ] and thus this taxon might be outcompeted in later stages of FWD decomposition. Further possible bacterial functions Despite less pronounced bacterial potential to degrade complex compounds in comparison with fungal activity owing to a limited abilities to colonize and change large patches of the substrate, bacteria still hold potential to contribute to the process of decomposition with decisive roles. Their roles are represented by functional traits described in the following paragraphs. While 16S rRNA sequencing has a limited ability to identify specific bacterial functions, bacterial taxa identified here have repeatedly been observed in association with decomposing wood; Acidobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes were previously identified in deadwood by 16S rRNA sequencing and cultivation [ 16 , 25 ]. Members of the Alphaproteobacteria cultivated from deadwood showed lower potential for carbohydrate utilization as seen in their limited CAZyme gene content and thus narrow spectrum of carbohydrates which they can utilize [ 25 ]. On the other hand, Alphaproteobacteria together with Gammaproteobacteria are able to utilize C1 compounds. Most importantly, they can oxidize methane and subsequently methanol, both of which are produced during deadwood decomposition (Lennart et al., 2012). Part of the carbon from these compounds is also assimilated and thus stabilized which prevents its loss from the system. The potential taxa feeding on C1 compounds in decomposing FWD are represented by Methylorosula (Alphaproteobacteria) [ 91 ], Methylovirgula (Alphaproteobacteria) [ 92 ] and Methylacidiphilales (Verrucomicrobiota), although the methanotrophic abilities of the latter probably depend on the habitat [ 93 , 94 ]. While deadwood is carbon-rich substrate, its nitrogen limitation makes its colonization and decomposition difficult. Community of diazotrophs alleviate nitrogen limitation in deadwood [ 32 ] which has the strongest effect at the beginning of decomposition. In situ expression of nitrogen-fixation genes was shown to be strong on coarse deadwood [ 29 ]. Transcripts of nitrogen fixation genes mapped to the genomes/metagenome-assembled genomes have unveiled potential diazotrophs which include also genomes of cultivated enterobacteria from the genus Sodalis (identified as Pectobacteriaceae bacterium in decomposing FWD Here Sodalis appears throughout the decomposition of FWD with the highest values at the beginning of FWD decomposition which is in line with its putative role in nitrogen fixation. In terms of FWD decomposition the initial years were characterized by low nitrogen content which increased only after four years as nitrogen enrichment by diazotrophs and nitrogen retention in microbial, mainly fungal, biomass took place. Recycling of microbial biomass is among the major factors sustaining bacterial growth in forest soil [ 26 , 27 ]. Given the high amount of fungal biomass in deadwood, mycophagous bacterial lifestyle can represent a successful strategy in obtaining the energy and covering carbon and nitrogen demand. Several chitinolytic bacteria were identified in the phylum Acidobacteriota [ 24 , 95 ] and members of this group were identified as either being present throughout the FWD decomposition (genus Granulicella , Terriglobus ) or at the advanced phase of decomposition ( Bryocella , Silvibacterium , Occallatibacter ). Conclusions This study demonstrates that microclimatic changes induced by forest canopy gaps significantly influence soil bacterial communities and those involved in the decomposition of fine woody debris (FWD). Although FWD bacterial communities originate from the soil, they undergo rapid and dynamic development, shaped in part by microclimatic factors. As FWD plays a crucial role in nutrient flow, particularly in managed forests, disturbances to the canopy could disrupt nutrient dynamics and ecosystem functions. While fungi are the primary decomposers of dead plant biomass, their function is heavily influenced by substrate quality and microclimatic conditions. In contrast, bacteria appear to represent a more stable component of the microbial community, even in such fluctuating environments. Environmental factors not only impact the structure of microbial communities but can also modulate the metabolic activities of their members [ 96 ]. Future research should explore functional gene activity related to nutrient cycling, microbial resilience, and bacterial-fungal interactions across different canopy conditions. Additionally, examining disturbance-specific bacterial functions and their impact on carbon dynamics could provide valuable insights for sustainable forest management. Declarations Authors contributions: Conceptualization C. B., P. B., J. M, R. B.; experimental design and methodology C. B., V. T., J. B., T. D. P., P. B. and V. B.; performance of experimental work, data evaluation and statistical analyses, V. T., T.D. P. and V. B.; validation V. T. , C. B., P. B. and V. B.; writing-original draft preparation V. T. and V. B.; writing – review and editing, V. T., J. B. , C. B., J. M., P. B. and V. B., supervision, project administration and funding acquisition, P. B. and V. B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Czech Science Foundation (20-14961S). PB and PTD were supported by the Ministry of Education, Youth and Sports of the Czech Republic (MŠMT CZ.02.01.01/00/22_008/0004635). Data availability statement: Sequencing data presented in this study have been deposited in the SRA database under BioProject accession number PRJNA1228314 and in Zenodo repository (https://doi.org/10.5281/zenodo.14900894). Acknowledgements: We thank Clementine Lepinay for sharing R scripts used for LMM models created in former paper of the authors. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Front Glob Chang. 2021;4. Lladó S, Větrovský T, Baldrian P. Tracking of the activity of individual bacteria in temperate forest soils shows guild-specific responses to seasonality. Soil Biol Biochem. 2019;135:275–82. Fabryová A, Kostovčík M, Díez-Méndez A, Jiménez-Gómez A, Celador-Lera L, Saati-Santamaría Z, et al. On the bright side of a forest pest-the metabolic potential of bark beetles’ bacterial associates. Sci Total Environ. 2018;619–620:9–17. López-Mondéjar R, Zühlke D, Becher D, Riedel K, Baldrian P. Cellulose and hemicellulose decomposition by forest soil bacteria proceeds by the action of structurally variable enzymatic systems. Sci Rep. 2016;6:25279. Lasa AV, Mašínová T, Baldrian P, Fernández-López M. Bacteria from the endosphere and rhizosphere of Quercus spp. Use mainly cell wall-associated enzymes to decompose organic matter. PLoS ONE. 2019;14. Lenhart K, Bunge M, Ratering S, Neu TR, Schüttmann I, Greule M et al. Evidence for methane production by saprotrophic fungi. Nat Commun. 2012;3. Berestovskaya JJ, Kotsyurbenko OR, Tourova TP, Kolganova TV, Doronina NV, Golyshin PN, et al. Methylorosula polaris gen. nov., sp. nov., an aerobic, facultatively methylotrophic psychrotolerant bacterium from tundra wetland soil. Int J Syst Evol Microbiol. 2012;62:638–46. Vorob’ev AV, de Boer W, Folman LB, Bodelier PLE, Doronina NV, Suzina NE, et al. Methylovirgula ligni gen. nov., sp. nov., a nobligately acidophilic, facultatively methylotrophic bacterium with a highly divergent mxaF gene. Int J Syst Evol Microbiol. 2009;59:2538–45. Op den Camp HJM, Islam T, Stott MB, Harhangi HR, Hynes A, Schouten S et al. Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia. Environ Microbiol Rep. 2009. pp. 293–306. Dedysh SN, Beletsky AV, Ivanova AA, Danilova OV, Begmatov S, Kulichevskaya IS et al. Peat-inhabiting Verrucomicrobia of the order Methylacidiphilales do not possess methanotrophic capabilities. Microorganisms. 2021;9. Lladó S, Žifčáková L, Větrovský T, Eichlerová I, Baldrian P. Functional screening of abundant bacteria from acidic forest soil indicates the metabolic potential of Acidobacteria subdivision 1 for polysaccharide decomposition. Biol Fertil Soils. 2016;52:251–60. Kopecky J, Kamenik Z, Omelka M, Novotna Jitkaand Stefani T, Sagova-Mareckova M. Phylogenetically related soil actinomycetes distinguish isolation sitesby their metabolic activities. FEMS Microbiol Ecol. 2023;99. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1sampledesign.pdf Supplementary Figure 1:Exp_design: Experimental design, based on a figure from (Krah et al., 2018) SupplementaryFigure2chemdetail.pdf Supplementary Figure 2: Changes in chemical parameters in fine woody debris and surrounding soil under different canopy management during the decomposition in temperate forest. Data represent the mean ± SE of 8 samples per treatment and timepoint (years) for FWD in red (beech) and blue (fir) and 23 samples per treatment for soil (grey). SupplementaryFigure3soilbacgenera.pdf Supplementary Figure 3 Bacterial genera in soil with average relative abundance over 0.5% under open or closed canopy (n = 23 in each group). Average relative abundance of these bacterial genera in fine deadwood in all samples across whole decomposition (n = 486). SupplementaryFigure4diversitynew.pdf Supplementary Figure 4 Diversity of bacteria during the decomposition of the FWD of beech and fir and in soil under different forest management in a temperate natural forest expressed as Shannon Wiener diversity index (A), evenness (B), species richness (C) and Chao-1 estimates (D). Development of Chao-1 estimate (E) and bacterial species richness (F) in beech and fir fine deadwood estimated over time under open and closed canopy (n = 16 for each time point – canopy and tree species). SupplementaryFigure5bacterialgenera.pdf Supplementary Figure 5 Abundant bacterial genera in decomposing fine deadwood of beech ( Fagus sylvatica ) and fir ( Abies alba ) with average relative abundance over 1 % under open or closed canopy (n = 8 in each group and time point ). Average relative abundance of abundant soil taxa is presented to highlight the differences in the communities (n = 486). SupplementaryTable1chemism.pdf Supplementary table 1: Effect of canopy openness and area specificity on soil physico-chemical properties. Tested by ANOVA after log10 transformation of data to reach the assumptions of the normal distribution. SupplementaryTable2soilbacteriaabundancenew.pdf Supplementary table 2: Relative average abundance of soil bacterial taxa and their specificity in habitat and canopy. Specificity cut off: 95% of total number of sequences present in one of the groups, value reflect the ratio of sequence abundance sum under closed canopy. If over 75% of sequences were detected in any of groups, signed as "preferred". Cite Share Download PDF Status: Published Journal Publication published 06 Aug, 2025 Read the published version in Environmental Microbiome → Version 1 posted Editorial decision: Revision requested 24 May, 2025 Reviews received at journal 23 May, 2025 Reviewers agreed at journal 07 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviews received at journal 01 Apr, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers invited by journal 26 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 22 Mar, 2025 First submitted to journal 20 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6118769","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434299207,"identity":"47d2c640-b8d8-4dfe-b15c-3713d2ca4365","order_by":0,"name":"Vojtěch Tláskal","email":"","orcid":"","institution":"Institute of Microbiology of the Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Vojtěch","middleName":"","lastName":"Tláskal","suffix":""},{"id":434299209,"identity":"fb2df516-063e-4a38-8a8f-af90c91999f1","order_by":1,"name":"Jason Bosch","email":"","orcid":"","institution":"Institute of Microbiology of the Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Bosch","suffix":""},{"id":434299212,"identity":"ca92565d-9860-448f-98f3-41a211db2032","order_by":2,"name":"Priscila Thiago Dobbler","email":"","orcid":"","institution":"Institute of Microbiology of the Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Priscila","middleName":"Thiago","lastName":"Dobbler","suffix":""},{"id":434299213,"identity":"2261d06d-8aa5-4418-9c53-52fb033fd5bf","order_by":3,"name":"Jörg Müller","email":"","orcid":"","institution":"University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Müller","suffix":""},{"id":434299214,"identity":"6d412e88-0807-415d-b0ba-cc22d42177aa","order_by":4,"name":"Roland Brandl","email":"","orcid":"","institution":"Philipps University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Roland","middleName":"","lastName":"Brandl","suffix":""},{"id":434299215,"identity":"11f2c403-20a0-4eb5-a235-65c8b109db2a","order_by":5,"name":"Claus Bässler","email":"","orcid":"","institution":"Bavarian Forest National Park","correspondingAuthor":false,"prefix":"","firstName":"Claus","middleName":"","lastName":"Bässler","suffix":""},{"id":434299216,"identity":"e906ccd9-40ae-4430-a95c-3e1e2e34ea07","order_by":6,"name":"Petr Baldrian","email":"","orcid":"","institution":"Institute of Microbiology of the Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Petr","middleName":"","lastName":"Baldrian","suffix":""},{"id":434299217,"identity":"cdf8336e-6f9e-48fd-bbec-322e40a4441b","order_by":7,"name":"Vendula Brabcová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBACAwYGNjCDX4KxAUgxk6BFcgbJWgxugCkitJhLH3724EfFYXnj282Nj25UWDPwtx9/wPDjD24tln1p5oY9Zw4bbrtzsNk450w6g8SZHAPGHh48DjvDYCbN2JbGuO1GYpt0btthoFNzgM6TwKeF/RtIi/3mGSAt/4Ba+J8/YAb5EbcWHpAtNokbJEBaGoBaJBIMmBkS8Pilh6cc6Beb5BlgvxxL55G48cbgYM8B3FrMedi3AUNMwrZ/dvvDxzk11nL8/ekPH+ALMQwADio8doyCUTAKRsEoIAYAALVlTmVzZqVjAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Microbiology of the Czech Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Vendula","middleName":"","lastName":"Brabcová","suffix":""}],"badges":[],"createdAt":"2025-02-27 08:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6118769/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6118769/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40793-025-00756-9","type":"published","date":"2025-08-06T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79631255,"identity":"b0fa6e8c-7363-4c06-83e2-23a7727b59c2","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43037,"visible":true,"origin":"","legend":"\u003cp\u003eFungal biomass and changes in physicochemical compositions of fine woody debris during decomposition in a natural temperate forest. Boxplots show combined data of fir and beech FWD and are based on 16 samples per treatment and timepoint (years) for FWD. Red line represents the values in surrounding soil (average of 46 samples).\u003c/p\u003e","description":"","filename":"Figure161.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/98903e2dc0dd10e19474a6bc.png"},{"id":79631333,"identity":"ba6aeda6-a5b8-4fca-a7d5-fd2f6445159d","added_by":"auto","created_at":"2025-04-01 03:21:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5692,"visible":true,"origin":"","legend":"\u003cp\u003eFungal biomass and changes in physicochemical compositions in soil caused by 10 years of development under different microclimatic condition caused by forest stand canopy openness. Boxplots show data based on 23 soil samples per treatment.\u003c/p\u003e","description":"","filename":"Figure163.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/77ce3735153ed618a77e7605.png"},{"id":79631334,"identity":"dc9d72fa-dc87-422d-ab27-55aabcc0166e","added_by":"auto","created_at":"2025-04-01 03:21:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59575,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram representing the results of variation partitioning analyses on Hellinger-transformed OTU abundances. (A) Treatment specific effect: length of decomposition, tree species, canopy cover and deadwood heterogeneity of origin on site calculated using all FWD samples, (B) Effect of environmental factors on FWD bacterial community composition calculated using all FWD samples, (C) Effect of canopy openness and environmental factors on soil bacteria calculated using all soil samples, (D) Effect of selected environmental factors on soil bacterial community composition calculated using all soil samples.\u003c/p\u003e","description":"","filename":"Figure162.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/c6c2610772d34f3ace11e4a0.png"},{"id":79631906,"identity":"960b3704-c379-4f99-863d-924f94b38c50","added_by":"auto","created_at":"2025-04-01 03:29:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49945,"visible":true,"origin":"","legend":"\u003cp\u003eNon-metric multidimensional scaling of bacterial community in decomposing fine deadwood of beech and fir and surrounding soil under different microclimatic conditions in a natural temperate forest based on dissimilarities among samples. Analysis was based on Euclidean distances of Hellinger-transformed relative abundances. Vectors indicate the potential effect of environmental variables. OTUs with relative abundances over 0.5% in at least three samples were included.\u003c/p\u003e","description":"","filename":"Figure164.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/b66b8443f1c6e008ed5b00e3.png"},{"id":79631257,"identity":"7191578d-83f2-4f44-a653-9c21eb638743","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41204,"visible":true,"origin":"","legend":"\u003cp\u003eAbundant bacterial genera (A) and phyla (B) in decomposing fine beech and fir deadwoods and soil in a natural temperate forest. The data represent the means of 32 samples of FWD or 46 samples of soil. Bacterial genera and phyla with average abundances over 1 % are included.\u003c/p\u003e","description":"","filename":"Figure165.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/5d65bedff4b93d6feb1c0a7b.png"},{"id":79631260,"identity":"8a915e24-60f8-4b2d-bfd0-d2e08d8b7db4","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":196656,"visible":true,"origin":"","legend":"\u003cp\u003eSuccessional development of the bacterial communities in beech \u003cem\u003e(Fagus sylvatica)\u003c/em\u003e and fir \u003cem\u003e(Abies alba)\u003c/em\u003e fine deadwoods decomposing under closed and open canopies in a natural temperate forest. All taxa with abundances above 1% in 3 yearly observations were listed.\u003c/p\u003e","description":"","filename":"Figure166.png","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/b1d2e4a75b1f13b71b87aba8.png"},{"id":88814931,"identity":"af9a868f-52d8-4a28-b677-bfb7781441ed","added_by":"auto","created_at":"2025-08-11 16:10:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1393562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/edfa9997-8329-4039-a255-b59fb8ccff30.pdf"},{"id":79631264,"identity":"a4d220d9-8b94-4610-a774-64eb866f09f7","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1708697,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 1:Exp_design: Experimental design, based on a figure from (Krah et al., 2018)\u003c/p\u003e","description":"","filename":"SupplementaryFigure1sampledesign.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/e0ca86c317d7c6ab83afe960.pdf"},{"id":79631337,"identity":"5548a737-4fd3-4f8b-89ad-e5ef5ec021a4","added_by":"auto","created_at":"2025-04-01 03:21:04","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1412025,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 2: Changes in chemical parameters in fine woody debris and surrounding soil under different canopy management during the decomposition in temperate forest. Data represent the mean ± SE of 8 samples per treatment and timepoint (years) for FWD in red (beech) and blue (fir) and 23 samples per treatment for soil (grey).\u003c/p\u003e","description":"","filename":"SupplementaryFigure2chemdetail.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/ade961d5ffebcb6adb8523df.pdf"},{"id":79632549,"identity":"264f5f22-d6ff-42c8-8226-b7e4317d0900","added_by":"auto","created_at":"2025-04-01 03:37:04","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":28325,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 3 Bacterial genera in soil with average relative abundance over 0.5% under open or closed canopy (n = 23 in each group). Average relative abundance of these bacterial genera in fine deadwood in all samples across whole decomposition (n = 486).\u003c/p\u003e","description":"","filename":"SupplementaryFigure3soilbacgenera.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/a83eda243e76b77198245048.pdf"},{"id":79631267,"identity":"0fed3139-d2b2-435d-bc21-7fea0e74fd52","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1558665,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 4 Diversity of bacteria during the decomposition of the FWD of beech and fir and in soil under different forest management in a temperate natural forest expressed as Shannon Wiener diversity index (A), evenness (B), species richness (C) and Chao-1 estimates (D). Development of \u0026nbsp;Chao-1 estimate (E) and bacterial species richness (F) in beech and fir fine deadwood estimated over time under open and closed canopy (n = 16 for each time point – canopy and tree species).\u003c/p\u003e","description":"","filename":"SupplementaryFigure4diversitynew.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/189c91d202f93914e0a2c7a6.pdf"},{"id":79631265,"identity":"f80426f8-57f8-4ea4-82f2-faf9a0462dc3","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1417967,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure 5 Abundant bacterial genera in decomposing fine deadwood of beech (\u003cem\u003eFagus\u003c/em\u003e \u003cem\u003esylvatica\u003c/em\u003e) and fir (\u003cem\u003eAbies\u003c/em\u003e \u003cem\u003ealba\u003c/em\u003e) with average relative abundance over 1 % under open or closed canopy (n = 8 in each group and time point ). Average relative abundance of abundant soil taxa is presented to highlight the differences in the communities (n = 486).\u003c/p\u003e","description":"","filename":"SupplementaryFigure5bacterialgenera.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/c5224e7ab7c1f38ad4d11ce5.pdf"},{"id":79631271,"identity":"6babc410-8dfc-4390-9ba9-295242e88655","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":411550,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 1: Effect of canopy openness and area specificity on soil physico-chemical properties. Tested by ANOVA after log10 transformation of data to reach the assumptions of the normal distribution.\u003c/p\u003e","description":"","filename":"SupplementaryTable1chemism.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/bf307833442dfcbeaf5f0319.pdf"},{"id":79631270,"identity":"971ea1df-d48d-45cb-afdc-a473936dd8e2","added_by":"auto","created_at":"2025-04-01 03:13:04","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":198592,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table 2: Relative average abundance of soil bacterial taxa and their specificity in habitat and canopy. Specificity cut off: 95% of total number of sequences present in one of the groups, value reflect the ratio of sequence abundance sum under closed canopy. If over 75% of sequences were detected in any of groups, signed as \"preferred\".\u003c/p\u003e","description":"","filename":"SupplementaryTable2soilbacteriaabundancenew.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118769/v1/5235a1a3159f1bd7b8de0bf5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fragile foundations: Succession patterns of bacterial communities in fine woody debris and soil under long-term microclimate influence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eForests are essential for nutrient cycling and serve as a major global carbon sink, storing approximately 45% of the carbon (C) present in terrestrial ecosystems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Soil is the largest terrestrial carbon pool storing roughly 44% of the forest C, while deadwood accounts for another 8%. In natural forests with numerous snags and coarse wood from fallen trees, fine woody debris (FWD, deadwood with a diameter of less than 10 cm) constitutes only a small fraction of the total deadwood stock [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, in managed forests where the major part of trees is typically extracted, FWD represent an important pool of considerable amount. For example, within the management zone of the Bavarian Forest National Park, the amount of FWD was estimated at \u0026sim;17.5 m\u003csup\u003e3\u003c/sup\u003e ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], roughly corresponding to the recently reported quantity of coarse wood in European forests across all management types [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] affected by strong deadwood extraction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. FWD shows rapid turnover due to fast decomposition [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The carbon flux through FWD is approximately five times faster than that through large deadwood (coarse woody debris, CWD) and in total it corresponds to the flow of a stock of approximately 90 m\u003csup\u003e3\u003c/sup\u003e ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of CWD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. FWD thus represent a key part of the carbon and nitrogen flow in managed forests.\u003c/p\u003e \u003cp\u003eDeadwood serves a key source of forest soil nutrient pool [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and plays a crucial role in maintaining forest biodiversity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], providing both habitat and nutrients for a diverse array of organisms, including microorganisms, such as fungi and bacteria [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Different deadwood size, turnover dynamic and frequent fragmentation of the FWD creates a high variety of habitats and a high number of habitat patches, leading to a high diversity in detected fungal communities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and increase in fungal species richness as a consequence of their competitive life style [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Deadwood decomposition is steered by fungi, thanks to broad enzymatic portfolio enabling them to efficiently colonize large sections of wood and decompose it as nutrient source, despite the recalcitrance, fluctuating moisture content and low nitrogen content of this substrate. Studies from forest topsoil and deadwood, however, pointed to the prevalence of bacterial taxa with potential to contribute to decomposition of wood structural compounds like cellulose and hemicellulose [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These include Bacteroidota (e.g. \u003cem\u003eMucilaginibacter\u003c/em\u003e) and Acidobacteriota that incorporate C from \u003csup\u003e13\u003c/sup\u003eC-labelled cellulose [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similar taxa were characterised as versatile decomposers based on their rich gene toolkit for carbohydrate decomposition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Gammaproteobacteria (e.g. \u003cem\u003eHermiinimonas\u003c/em\u003e, \u003cem\u003eVariovorax\u003c/em\u003e) were also associated with degradation of complex polymers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Contrary to the typically high abundance of Alphaproteobacteria, these decomposers might be actively growing while utilizing labile carbon sources, as they exhibit limited potential for complex carbohydrate decomposition [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Acidobacteriota are furthermore reported to be associated with fungal biomass [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which represents a more accessible substrate from a decomposer's perspective owing to its lower C:N ratio. Indeed, several acidobacterial strains were shown to be able to perform chitin degradation to cover nitrogen and carbon demand [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While mycophagy represents one approach how obtain nitrogen at a lower resource cost for microbes, there are other approaches to overcome the recalcitrance of deadwood. Nitrogen might be translocated from surrounding soil [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or its pool might be enriched in situ by specialised diazotrophic bacteria dwelling in deadwood and performing nitrogen fixation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Increased nitrogen level enables further microbial colonization while it also enables nitrogen immobilization in microbial biomass with potential importance for subsequent soil formation.\u003c/p\u003e \u003cp\u003eAlthough the majority of studies have targeted the decomposition of coarse wood [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and its influence on surrounding soil, FWD has been shown to be reservoir of fungal forest diversity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The high surface-to-volume ratio of FWD means higher a sensitivity of FWD to changes in temperature and moisture [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These microclimatic fluctuations are strongly related to both forest management practice and forest stand damage. Importantly, disturbance rates increase with the ongoing global change and damage to canopy cover is expected to be an important factor affecting ecosystem processes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The canopy openness of forests is strongly linked to their temperature buffering capacity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Increased respiration rate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], elevated summer temperatures, intensive solar radiation, and fluctuating water content are characteristic for forest gaps [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The microbial community in FWD is thus subjected to greater fluctuations of these environmental variables. In previous research, focused on characterisation of microbial community throughout the lifetime of FWD, the fungal community was showed to be influenced by both tree species of FWD and microclimatic conditions (canopy openness) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, the bacterial community is typically considered less tightly bound to the deadwood of the preferential tree species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], although other studies indicate the close relationship [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, bacteria are very sensitive to pH and moisture fluctuations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Thus, bacterial community assembly in deadwood is likely affected by multiple driving factors of varying intensity and its development during FWD decomposition remains elusive.\u003c/p\u003e \u003cp\u003eIn this study, we follow up on an experiment where the decomposition successional patterns of the FWD of beech (\u003cem\u003eFagus sylvatica\u003c/em\u003e) and fir (\u003cem\u003eAbies alba\u003c/em\u003e), the two main tree species of the temperate forests of central Europe [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], were followed throughout its decomposition lifetime under open and closed canopies. The aim was to determine the factors affecting bacterial succession during decomposition with the open canopy treatment serving as a proxy of a disturbed ecosystem. Moreover, canopy openness and deadwood source selection allowed us to assess the size and relative importance of the effects of microclimate and the host tree. The frequency of canopy disturbance events has increased in the temperate zone, and they have more pronounced effects in managed forests than in unmanaged forests [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Climatic factors are important in influencing fungal community composition [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], sun exposure and canopy openness influence inner temperature of larger deadwood objects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], composition of saproxylic beetles [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and assembly patterns of fungi in FWD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As the wood decomposer strategies may exert significant selection effects [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] determining the bacterial community structure [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], all these environmental factors likely contribute to shape bacterial community development.\u003c/p\u003e \u003cp\u003eWe hypothesize that bacterial community will undergo directional succession. During complete decomposition of FWD, it should be possible to track the decrease of endophytic taxa, steady prevalence of bacterial generalists, and increase in specialists with the ability to degrade recalcitrant substrates. Based on previous research on CWD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we hypothesize that the bacterial community at the late stage of decomposition with almost decomposed FWD, resembles the soil community. To enable such comparison, we performed characterization of the soil bacterial community at the end of experiment to be able to characterise the overlap of the FWD and soil bacterial communities. In addition to carbon utilisation, we expect to record bacterial taxa involved in nitrogen cycling including taxa known for nitrogen fixation and mycophagous bacteria which target fungal biomass for its less restrictive C:N ratio. Previous studies indicate that forest management does not significantly affect soil and rhizosphere bacterial communities in short term scheme [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Here, we specifically focused on long-term changes in soil properties and soil bacterial community composition in Bavarian Forest National park, where natural vegetation recovery rate is very slow and we expect detectable changes in soil properties due to the limited primary productivity of open canopy sites.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and experimental design\u003c/h2\u003e \u003cp\u003eThe experimental sites were located in the management zone of the Bavarian Forest National Park in Germany (48.9\u0026deg; N, 13.3\u0026deg;E). The management zone covers an area of 6,000 ha that surrounds the 18,000 ha core zone of the national park. The area is characterized by montane mixed forest consisting of European beech (\u003cem\u003eFagus sylvatica\u003c/em\u003e L.), Silver fir (\u003cem\u003eAbies alba\u003c/em\u003e Mill.) and Norway spruce (\u003cem\u003ePicea abies\u003c/em\u003e (L.) H. Karst) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The sampling design was a part of the broader experimental design described in detail in previous publications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In autumn 2011, freshly cut branches of fir and beech were deposited on 64 plots and arranged in a random block design with four spatially independent blocks. The branches (fine woody debris, FWD) had diameters of 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 cm and lengths of 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 m. These branches were taken from trees of the same age that were harvested from the same forest stand. The origin of the branches was identical and branches were randomly distributed across study sites to mitigate the potential effect of communities of fungal endophytes inhabiting individual branches on microbial community development. Each block contained randomly located sets of plots with either fir or beech branches or both. The mixture of fir and beech deadwood represented the factor of forest stand tree diversity. Within each block, two plots per treatment (fir, beech, or mixed) were set under open or closed canopies (Supplementary Fig.\u0026nbsp;1). Canopy openness was used as a surrogate for stand microclimates [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The open canopy plots were the result of clearings where an area of 0.1 ha was freed from living and dead trees. To avoid shading by a dense grass layer surrounding the deadwood on open canopy plots, each plot was mowed once a year during the growing season as described previously [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The daily peak temperatures of the deadwood surfaces in summer were measured in the open and closed plots. The mean values were much higher in open canopy plots (~\u0026thinsp;30\u0026deg;C) than in the closed canopy plots (~\u0026thinsp;15\u0026deg;C) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. All experimental plots were sampled annually in September / October from 2012\u0026ndash;2018 and in 2021 and 2022.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSampling, sample processing and analysis\u003c/h3\u003e\n\u003cp\u003eOne composite FWD sample was obtained from each selected branch. It was obtained from two vertical drillings of the branch in its centre using an electric drill equipped with a 10 mm diameter auger across the entire diameter of the branch. The drilling points were placed evenly along the branch, avoiding the close proximity of the ends of the branch. The auger was sterilized between drillings, and the dust from all drilling points was collected in sterile plastic bags and frozen within a few hours after drilling. In total, 2 beech or 2 fir samples were taken from each block from plots containing only beech or fir, and 2 beech and 2 fir samples were taken from plots containing mixed deadwood, resulting in 4 beech and 4 fir composite samples per canopy type. Thus 64 samples in total were taken annually, (Supplementary Fig.\u0026nbsp;1) resulting in a total of 512 samples available for this study.\u003c/p\u003e \u003cp\u003eThe drilled materials were weighed in the laboratory and freeze-dried to estimate the deadwood dry mass. Next, it was milled using an Ultra Centrifugal Mill ZM 200 (Retsch, Germany), and the resulting fine powder was used for the subsequent analyses. Dry mass content was based on mass loss during freeze-drying, and pH was measured after mixing with distilled water (1:10 w: vol). The wood carbon and nitrogen contents were measured using an elemental analyser in an external laboratory of the Institute of Botany of the Czech Academy of Sciences, Průhonice, Czech Republic, as described previously [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Carbon was measured using sulfochromic oxidation, and the nitrogen content was estimated by sulfuric acid mineralization with the addition of selenium and sodium sulfate and conversion to ammonium ions, which were measured by a segmented flow analyser (SFA), Skalar. To quantify fungal biomass, total ergosterol was extracted using 10% KOH in methanol and analysed by HPLC [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoil from all sampling sites was collected in September 2022 in one sampling campaign. The sampling point was located in the close proximity of tested FWD object (proximately 1-1.5 m). Plastic soil cores (4.5 cm in diameter) were hammered in central point and 0.5 m from central a point to the North, South, West and East and the full core were placed in plastic bag. Soil was immediately transferred into laboratory and stored prior to processing for maximum of 24 hours at 4\u0026deg;C. When processed, litter layer was removed and discarded. Only the top 10 cm of soil was used as a composite sample after homogenization and sieving through a 5 mm sieve. The homogenized soil sample was immediately frozen, freeze-dried and stored at -20\u0026deg;C prior to subsequent analyses. All further analyses were performed the same way as for deadwood samples.\u003c/p\u003e\n\u003ch3\u003eExtraction and analysis of environmental DNA\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA was extracted from 200 mg of freeze-dried material using the NucleoSpin Soil Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. Briefly, cells were lysed using SL1 lysis buffer. Enhancer SX was added prior to lysis. The samples were homogenized using FastPrep-24 (MP Biomedicals, Santa Anna, USA) at 5 m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 2 \u0026times; 30 s. In the last step, DNA was eluted from the columns using 50 \u0026micro;l of deionized water. One extraction per sample was performed [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the bacterial community analysis, PCR amplification of the prokaryotic hypervariable V4 region of the 16S rRNA gene was performed using barcoded 515F and 806R primers [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] in triplicate PCRs per sample as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. PCRs contained 5 \u0026micro;l of 5\u0026times; Q5 reaction buffer, 1.5 \u0026micro;l of BSA (10 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 1 \u0026micro;l of each primer (0.01 mM), 0.5 \u0026micro;l of PCR Nucleotide Mix (10 mM each), 0.25 \u0026micro;l of Q5 High Fidelity DNA polymerase (2 U \u0026micro;l \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, New England Biolabs, Inc.), 5 \u0026micro;l of 5\u0026times; Q5 HighGC Enhancer and 1 \u0026micro;l of template DNA (approx. 50 ng \u0026micro;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Cycling conditions were 98\u0026deg;C for 30 sec, 25 cycles of 94\u0026deg;C for 10 sec, 56\u0026deg;C for 30 sec, and 72\u0026deg;C for 20 sec, and a final extension at 72\u0026deg;C for 2 min. PCR triplicate reaction products were pooled and purified (MinElute PCR Purification Kit, Quiagen), and amplicon libraries prepared with the TruSeq DNA PCR-Free Kit LP (Illumina) were sequenced in house on the Illumina MiSeq (2 \u0026times; 250-base reads).\u003c/p\u003e \u003cp\u003eThe amplicon sequencing data were processed using the pipeline SEED 2.1.3 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Briefly, paired-end reads were merged using fastq-join [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Chimeric sequences were detected using Usearch 11.0.667 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and deleted, and sequences were clustered using UPARSE implemented within Usearch 8.1.1861 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] at a 97% similarity level. The most abundant sequence was selected from each cluster, and the closest hits at the species level were identified using BLASTn against an edited version of the SILVA 138.1 database including mitochondria and chloroplasts. Where the best hit showed lower similarity than 97% with 95% coverage, the best genus-level hit was identified. The sequences used in all community structure analyses were rarefied at 11400 sequence (median of sequence count for all samples). Sequences from FWD and soil were processed together and the dataset was split as needed. The species-level analyses were performed on a dataset where OTUs belonging to the same species were combined and all other OTUs were combined into the genus of the best hit and designated \u0026ldquo;sp.\u0026rdquo; Only sequences identified as bacterial were used in the analysis. Sequencing data have been deposited in the SRA database under BioProject accession number PRJNA1228314 and in Zenodo repository (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.14900894\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.14900894\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eData processing and statistics\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eSuccession time\u003c/em\u003e or so called \u003cem\u003etemporal niche position\u003c/em\u003e was defined as the average position of a taxon in succession considering its relative abundance over time, and \u003cem\u003ethe duration of occurrence\u003c/em\u003e was defined as the time span covering 90% of the taxon relative abundance as defined and calculated previously [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Tree or canopy specificity was defined as the strength of association of the taxon with one particular tree or canopy type and calculated as the sum of abundances in beech deadwood (closed canopy deadwood) divided by the sum of abundances in all samples. A value of 1 corresponds to a taxon exclusively found on beech deadwood. A value of 0.5 assigned to a taxon indicates that it is equally abundant on beech and fir deadwood. A value of 0 assigned to a taxon indicates that it is exclusively found on fir deadwood. For canopy openness, a value of 1 corresponds to a taxon exclusively found under the closed canopy and 0 corresponds to a taxon exclusively found under the open canopy. Taxa with tree specificities between 1.0 and 0.95 were considered beech-specific (or closed canopy-specific, respectively), and those with specificities between 0.05 and 0.00 were considered fir-specific (or open canopy-specific, respectively).\u003c/p\u003e \u003cp\u003eStatistical analyses were performed in R [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Two-dimensional nonmetric multidimensional scaling (NMDS) ordination analysis on Bray-Curtis distances was used to address the dissimilarity of the fungal community compositions based on Hellinger-transformed relative abundances (package \u003cem\u003evegan\u003c/em\u003e, function metaMDS [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Variables were fitted to the ordination diagram as vectors with 999 permutations and included pH, as well as the carbon and nitrogen and ergosterol contents. Diversity estimates (Shannon\u0026ndash;Wiener index, OTU richness, Chao 1 and evenness) were calculated for a dataset containing the relative abundance of 2,100 randomly selected sequences from each sample in SEED 2.1.3. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Differences in the environmental variables (pH, carbon, nitrogen, C:N, ergosterol and water contents) were tested using a linear mixed model (LMM, function lmer) with log-transformed data. For the LMM, the effect of explanatory variables, i.e., tree species, canopy openness and their interaction, over decomposition time were tested by considering time and plot identifiers (the same plots were repeatedly measured over time, the objects are nested in block/plot) as random effects. One-way or two-way PERMANOVA tests with 9,999 permutations were used to examine the effects of treatments on bacterial communities. Spearman rank correlations were used as a measure of the relationships between variables. Variation partitioning analyses on Hellinger-transformed OTU abundances were performed to identify the parts of the variance explained by tree species and canopy openness for the whole dataset and with length of decomposition, canopy openness and wood chemistry (i.e., nitrogen, carbon, C:N and pH) for each tree species independently (package \u003cem\u003evegan\u003c/em\u003e, function varpart [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The importance values of the obtained variances were determined with Monte Carlo permutation tests. In all cases, differences at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProperties of fine woody debris during ten years of decomposition\u003c/h2\u003e \u003cp\u003eThe physicochemical properties of decomposing fine woody debris (FWD) over a decade revealed dynamic development similar to the early decomposition phases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Carbon content increased during the initial stages of decomposition but seems to stabilize thereafter (LMM: χ\u0026sup2; = 2.37, P\u0026thinsp;=\u0026thinsp;0.12). Fine woody debris of fir (\u003cem\u003eAbies alba\u003c/em\u003e) exhibited significantly higher carbon content than beech (\u003cem\u003eFagus sylvaticus\u003c/em\u003e, LMM: χ\u0026sup2; = 49.85, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while canopy cover had no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nitrogen content showed a gradual increase over time (LMM: χ\u0026sup2; = 5.26, P\u0026thinsp;=\u0026thinsp;0.02). Beech FWD had higher nitrogen content than fir (LMM: χ\u0026sup2; = 52.39, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and nitrogen levels were generally lower under canopy gaps, although the effect of canopy was significant only for fir deadwood (LMM: χ\u0026sup2; = 4.35, P\u0026thinsp;=\u0026thinsp;0.00). As nitrogen content increased, particularly since year 7, it approached levels observed in soil. Consequently, the C:N ratio of decomposing FWD declined, though this trend was not statistically significant due to high variability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The pH of FWD decreased over time, reaching its minimum in 2018, followed by a slight increase in 2021 (LMM: χ\u0026sup2; = 8.49, P\u0026thinsp;=\u0026thinsp;0.00). pH values were higher under open canopy conditions (LMM: χ\u0026sup2; = 6.68, P\u0026thinsp;=\u0026thinsp;0.01) but did not differ significantly between tree species (LMM: χ\u0026sup2; = 0.23, P\u0026thinsp;=\u0026thinsp;0.63). Water content increased during the later stages of decomposition (LMM: χ\u0026sup2; = 11.88, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), although variability was high. Beech FWD consistently exhibited higher moisture levels compared to fir (LMM: χ\u0026sup2; = 47.32, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Supplementary Fig.\u0026nbsp;2). Fungal biomass, measured via ergosterol content, increased during the first 6\u0026ndash;7 years of decomposition and subsequently declined (LMM: χ\u0026sup2; = 7.50, P\u0026thinsp;=\u0026thinsp;0.00). Beech FWD contained approximately twice as much ergosterol as fir (LMM: χ\u0026sup2; = 76.78, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and ergosterol levels were higher under closed canopy conditions (LMM: χ\u0026sup2; = 94.59, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These trends suggest that the depletion of accessible nutrients characterizes advanced decomposition stages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLongtime changes in soil physicochemical properties caused by canopy openness\u003c/h3\u003e\n\u003cp\u003eThe analysis of soil physicochemical properties revealed significant long-term changes resulting associated with the differences in canopy cover (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall carbon-to-nitrogen (C:N) ratio increased markedly in response to canopy opening, rising from 18.7 under closed canopy conditions to 21.2 under open canopy conditions (ANOVA: F\u0026thinsp;=\u0026thinsp;35.36, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This shift was not hidden behind notable spatial variability in the C:N ratio (ANOVA: F\u0026thinsp;=\u0026thinsp;1.48, df\u0026thinsp;=\u0026thinsp;3, P\u0026thinsp;=\u0026thinsp;0.24). Soil carbon content increased substantially following canopy opening (ANOVA: F\u0026thinsp;=\u0026thinsp;9.05, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but was only marginally affected by site-specific factors. In contrast, detected variations in soil nitrogen content and fungal biomass content were predominantly influenced by site-specific factors rather than the effects of canopy openness. Soil moisture levels, which also increased significantly after canopy opening (ANOVA: F\u0026thinsp;=\u0026thinsp;3.03, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.04), showed a strong dependence on both canopy openness and site-specific conditions (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Soil pH parameters were identical nonetheless the canopy stage or site specificity. All measured soil parameters differed significantly from the values observed in decomposing fine woody debris (ANOVA: P\u0026thinsp;\u0026lt;\u0026thinsp;0.002, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) from these sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpact of canopy openness on bacterial community composition and diversity in soil\u003c/h3\u003e\n\u003cp\u003eBacterial diversity in soil and fine woody debris (FWD, described on OTU level), assessed through species richness and the Chao-1 index, differed significantly (Kruskal-Wallis test, P\u0026thinsp;=\u0026thinsp;0.000). On average, species richness in soil was higher, with 500\u0026thinsp;\u0026plusmn;\u0026thinsp;11 species compared to 405\u0026thinsp;\u0026plusmn;\u0026thinsp;29 species in FWD. Similarly, the Chao-1 index was higher in soil (1,043\u0026thinsp;\u0026plusmn;\u0026thinsp;34) than in FWD (776\u0026thinsp;\u0026plusmn;\u0026thinsp;76).\u003c/p\u003e \u003cp\u003eWhile the overall bacterial diversity in soil remained unaffected by canopy openness (ANOVA, species richness F\u0026thinsp;=\u0026thinsp;1.53, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.22 and Chao-1 index F\u0026thinsp;=\u0026thinsp;1.06, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.319), the composition of the soil bacterial community was significantly influenced by canopy cover and environmental properties at the sampling locations (PERMANOVA: F\u0026thinsp;=\u0026thinsp;6.97 and F\u0026thinsp;=\u0026thinsp;2.22, P\u0026thinsp;=\u0026thinsp;0.00 for both). These factors operated independently, with canopy cover accounting for 1.7% of community variability, and environmental parameters explaining 24.8% (variation partitioning: P\u0026thinsp;=\u0026thinsp;0.002 and P\u0026thinsp;=\u0026thinsp;0.00). Among the tested environmental factors, soil moisture had the largest impact, explaining 14.8% of the variability (P\u0026thinsp;=\u0026thinsp;0.001), followed by the C:N ratio (4.2%, P\u0026thinsp;=\u0026thinsp;0.00) and soil pH (8.3%, P\u0026thinsp;=\u0026thinsp;0.00, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBacterial communities in soil were distinct from those in FWD (PERMANOVA: F\u0026thinsp;=\u0026thinsp;151.3, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.001). Although soil bacterial communities were canopy-specific, the differences were less pronounced in the NMDS plot due to strong successional turnover (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The bacterial composition in soil was characterized by a high abundance of Pseudomonadota (28.5%, mainly Alphaproteobacteria, 22.7%), Acidobacteriota (22.2%), and Planctomycetota (10.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The most abundant soil genera included \u003cem\u003eundefined_Subgroup_2 Acidobacterium\u003c/em\u003e, \u003cem\u003eAcidothermus\u003c/em\u003e, \u003cem\u003eundefined Xanthobacteraceae bacterium\u003c/em\u003e, \u003cem\u003eundefined Elsterales bacterium\u003c/em\u003e, and \u003cem\u003eundefined Acidobacteriales bacterium\u003c/em\u003e, each with an average relative abundance exceeding 5%. Together, dominant bacterial taxa accounted for as much as 85% of soil bacterial abundance, but these same taxa represented only 30% of bacterial abundance in FWD. The observed differences in bacterial community structure between soil and FWD were largely driven by variations in bacterial abundance (Supplementary Fig.\u0026nbsp;3). Highly abundant soil bacteria (those with \u0026gt;\u0026thinsp;0.5% relative abundance) were generally not canopy-specific. However, the \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e group was significantly more prevalent under closed canopies, while \u003cem\u003eOccallatibacter\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e sequences were most frequently detected under open canopies, with more than 75% of their sequences occurring in these conditions (Stable2:Soil_bacteria_abundance).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBacterial community compositions in the different types of fine woody debris\u003c/h2\u003e \u003cp\u003eThe diversity of bacterial communities in decomposing fine woody debris (FWD) was markedly lower than in soil, as noted earlier. However, the impact of canopy cover on bacterial communities in FWD was more pronounced. Bacterial diversity, estimated using the Chao-1 index and species richness, was significantly higher under closed canopy conditions and in fir FWD compared to beech (Kruskal-Wallis test, P\u0026thinsp;=\u0026thinsp;0.00). Species richness in fir FWD reached its highest levels under open canopy conditions during the third and fifth years of decomposition and declined in later stages. Despite this decline, richness levels remained above those observed during the first year of decomposition (Supplementary Fig.\u0026nbsp;4). Throughout the decomposition process, bacterial diversity exhibited substantial variability across FWD samples.\u003c/p\u003e \u003cp\u003eBacterial communities in both beech and fir FWD underwent clear, continuous changes over time (PERMANOVA: F\u0026thinsp;=\u0026thinsp;166.5, P\u0026thinsp;=\u0026thinsp;0.00,) throughout the whole decomposition period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, analysis including only deadwood samples). While these temporal changes were less pronounced than those observed in fungal communities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], canopy cover and the origin of deadwood both significantly influenced bacterial community structure (PERMANOVA: F\u0026thinsp;=\u0026thinsp;34.43 and F\u0026thinsp;=\u0026thinsp;22.93, respectively, P\u0026thinsp;=\u0026thinsp;0.00,). However, these effects were not clearly distinguishable in the NMDS plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Increased heterogeneity of deadwood origin had no significant impact on bacterial community composition (PERMANOVA: F\u0026thinsp;=\u0026thinsp;0.87, P\u0026thinsp;=\u0026thinsp;0.84). Variation partitioning analysis identified temporal development as the primary driver of bacterial community composition, uniquely explaining 29.3% of the observed variability (PERMANOVA: F\u0026thinsp;=\u0026thinsp;32.63, P\u0026thinsp;=\u0026thinsp;0.00, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Tree species and canopy cover explained much smaller proportions, accounting for 3.1% (F\u0026thinsp;=\u0026thinsp;24.44, P\u0026thinsp;=\u0026thinsp;0.00) and 4.7% (F\u0026thinsp;=\u0026thinsp;36.22, P\u0026thinsp;=\u0026thinsp;0.00) of the variability, respectively.\u003c/p\u003e \u003cp\u003eBacterial communities in soil generally show strong responses to the physicochemical conditions of the substrate, prompting additional analysis of environmental parameters in the context of forest management treatments. However, environmental factors alone accounted for only 1.3% of the total variability, and their impact on bacterial community structure was not statistically significant (PERMANOVA: F\u0026thinsp;=\u0026thinsp;1.28, P\u0026thinsp;=\u0026thinsp;0.126). When combined with temporal development, environmental factors increased the share of explained variability to 17.4%, while temporal development as a standalone factor explained 11.7%. Despite all these findings, a significant portion of the variability in bacterial community composition (62%) remained unexplained, highlighting the complexity of the ecological processes influencing bacterial communities in decomposing FWD.\u003c/p\u003e \u003cp\u003eOverall, FWD bacterial communities were dominated by Pseudomonadota (46.7%), particularly Alphaproteobacteria (27.5%) and Gammaproteobacteria (19.2%), with high share of Actinomycetota (13.6%) and Acidobacteriota (11.7%). Most abundant bacterial phyla underwent continuous successional changes, trending towards soil-like community composition. Specifically, Pseudomonadota and Bacteroidota decreased over time, Acidobacteriota increased, and Actinomycetota exhibited fluctuations. Verrucomicrobiota and Planctomycetota became more prominent as decomposition advanced. Despite these shifts, the bacterial community composition in FWD remained distinct from that in soil even after 10 years of decomposition. Differences in bacterial communities under closed canopies and in canopy gaps were evident only at lower taxonomic levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary Fig.\u0026nbsp;5). Highly abundant bacterial genera clustered into early (2012\u0026ndash;2014) and late (2015\u0026ndash;2021) phases of decomposition, with the final years clustering more closely. During the early phase, dominant genera included \u003cem\u003eSphingomonas\u003c/em\u003e (6.4% average abundance), \u003cem\u003eGalbitalea\u003c/em\u003e (5.6%), \u003cem\u003eGranulicella\u003c/em\u003e (4.4%), \u003cem\u003eMucilaginibacter\u003c/em\u003e (4.3%), \u003cem\u003eBurkholderia\u003c/em\u003e-\u003cem\u003eCaballeronia\u003c/em\u003e-\u003cem\u003eParaburkholderia\u003c/em\u003e group (4.0%), \u003cem\u003eDyella\u003c/em\u003e (3.4%), \u003cem\u003ePseudomonas\u003c/em\u003e (2.8%), and \u003cem\u003ePedobacter\u003c/em\u003e (2.7%). In the later phase, the community shifted towards dominance of \u003cem\u003eBurkholderia\u003c/em\u003e-\u003cem\u003eCaballeronia\u003c/em\u003e-\u003cem\u003eParaburkholderia\u003c/em\u003e group (8.8%) alongside \u003cem\u003eGranulicella\u003c/em\u003e (6.1%). Other taxa with higher contributions included undefined \u003cem\u003eMethylacidiphilaceae\u003c/em\u003e (3.4%), \u003cem\u003eMucilaginibacter\u003c/em\u003e (3.1%), \u003cem\u003eConexibacter\u003c/em\u003e (2.9%), and \u003cem\u003eJatrophihabitans\u003c/em\u003e (2.9%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBacterial communities in FWD exhibited clear successional dynamics over the course of decomposition. The majority of bacterial taxa persisted for extended periods, reflecting a slow turnover rate, with the exception of certain taxa present during the initial years of decomposition, which demonstrated faster turnover rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Most bacterial taxa were detected in both beech and fir FWD and were not specific to canopy cover, based on the defined criteria (95% of total abundance restricted to a selected group). Among the highly abundant bacteria, only \u003cem\u003eDyella\u003c/em\u003e (Gammaproteobacteria), \u003cem\u003eGryllotalpicola\u003c/em\u003e (Actinobacteriota\u003cem\u003e), Endobacter\u003c/em\u003e (Alphaproteobacteria) and \u003cem\u003ePleomorphomonas\u003c/em\u003e (Alphaproteobacteria) were open canopy specific bacterial genera. Moreover, \u003cem\u003eLuteibacter\u003c/em\u003e and \u003cem\u003eComamonas\u003c/em\u003e (Gammaproteobacteria) were in roughly 74% recovered from sites under closed canopy and can be described as \u0026ldquo;preferring close canopy\u0026rdquo;. Similarly, \u003cem\u003ePseudomonas\u003c/em\u003e (75%, Gammaproteobacteria) and \u003cem\u003eMethylovirgula\u003c/em\u003e (81%, Alphaproteobacteria) preferred beech FWD and \u003cem\u003eNocardioides\u003c/em\u003e (75%, Actinobacteriota), undefined \u003cem\u003eChitinophagaceae\u003c/em\u003e bacteria (78%, Bacteroidota) and \u003cem\u003eKineosporia\u003c/em\u003e (87%, Actinobacteriota) belonged to fir FWD preferring bacterial genera. Despite these variations, the calculated succession times of commonly present bacterial taxa did not differ significantly between beech and fir FWD or between open and closed canopies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study highlights key factors influencing fine woody debris (FWD) decomposition and associated bacterial communities, including the effects of microclimatic conditions caused by canopy openness on surrounding soil bacterial communities. We observed a rapid initial decomposition phase followed by a slowdown, linked to fungal activity decline and pH stabilization. Canopy openness affected FWD and soil properties but had limited long-term effects on fungal biomass, while bacterial diversity in FWD and soil responded primarily to substrate characteristics rather than canopy conditions. Bacterial community succession in decomposing FWD revealed a continuous shift, with a few canopy- and tree-specific taxa. Nonetheless, even in the highly advanced decomposition stage, bacteria inhabiting decomposing FWD\u0026mdash;spanning a broad range of functional roles\u0026mdash;formed a distinct community compared to the surrounding soil. These findings underscore the complex interplay between canopy structure, soil properties, and microbial community dynamics in forest ecosystems.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFine woody debris decomposition\u003c/h2\u003e \u003cp\u003eThe extension of the FWD decomposing experiment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] allowed for the observation of the complete lifespan of FWD. While FWD decomposition progressed rapidly in the early years, the rate slowed after seven years, as indicated by a gradual decrease in ergosterol content and an increase in pH values. This pattern may reflect a decline in fungal wood decomposer activity typical for coarse deadwood [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Fungi actively lower pH through the production of organic acids, so their decline in later decomposition stages leads to pH stabilization or even an increase. This shift suggests weakening of fungal control over the decomposition process. The higher ergosterol content and moisture levels in beech FWD are consistent with the faster decomposition of beech deadwood, as previously reported [\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. This trend further supports the presence of ectomycorrhizal species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which typically appear in the final stages of succession [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The increase in nitrogen content in the late decomposition phase, partially due to the accumulation of nitrogen-rich microbial biomass and biological nitrogen fixation, suggests a gradual convergence with soil nutrient profiles.\u003c/p\u003e \u003cp\u003eDespite variations in decomposition time, changes in deadwood chemistry were largely consistent with initial wood composition, leading to a relatively uniform decomposition process across different deadwood origins and microclimatic conditions. Fluctuations in FWD pH and moisture content [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], along with shifts in fungal community composition and development [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], were likely key factors influencing bacterial community assembly, as these parameters strongly predict bacterial composition on decomposing deadwood.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLongtime changes in soil physicochemical properties caused by canopy openness\u003c/h2\u003e \u003cp\u003eCanopy openings influence forest microclimates [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and affect soil properties, especially carbon content and nutrient availability [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. These effects vary with gap size, age, and forest stand characteristics [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Contrary to expectations, we observed an increase in total soil carbon, C:N ratio, likely due to altered organic matter inputs and the slow recovery of forest floor vegetation. Dense and intense grass layer and no clear structured organic layer was frequent under open canopy in contrast to the closed forest. Increase in soil moisture was observed also in other forest clearcutting experiments (data not published). Despite changes in soil conditions, fungal biomass remained unaffected by canopy opening in the long term. This aligns with previous findings showing that fungal biomass declines immediately after clear-cutting but recovers rapidly in regenerating forests within 2 years, mainly due to an increase in nonmycorrhizal fungi [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], Awokunle-Holla, in preparation). Further support comes from the lack of a relationship between soil CO₂ efflux and canopy openness. Instead, microbial activity, as indicated by CO₂ efflux, was more influenced by soil properties than by microclimatic factors [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], though increased respiration has been reported in some gap studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigher soil moisture in gaps, detected in our study and repeatedly previously reported [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], likely results from reduced transpiration-driven water loss. These observations have important ecological implications, as prolonged droughts due to climate change can severely impact forests, which are less drought-resistant than agricultural and grassland systems [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. However, small canopy gaps, with reduced tree water uptake, may serve as refugee for moisture-sensitive soil organisms. While fungi are generally more drought-resistant, bacterial communities are more vulnerable to prolonged moisture deficits [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Soil pH plays a pivotal role in shaping microbial communities, influencing key processes such as nutrient cycling and organic matter decomposition. However, no significant changes in soil pH were detected in our study or a recent meta-analysis on canopy gaps [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The observed changes in soil properties and microbial community composition thus underscore the complex interactions between canopy structure, soil conditions, and microbial communities in forest ecosystems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDrivers of bacterial community composition in fine deadwood and soil, effect of canopy openness\u003c/h2\u003e \u003cp\u003eBacterial diversity reached its peak between the third and the fifth year of decomposition, showing the steep increase after the initial two years before declining towards the end of the decomposition process. The low diversity values during initial decomposition can be attributed to a less developed microbial community that utilizes labile carbon sources and includes potential plant pathogens known from living trees (genus \u003cem\u003eErwinia\u003c/em\u003e, [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and taxa typically detected in early decomposition phase of CWD (genera \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eSodalis\u003c/em\u003e and Pseudomonas, [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The priority effect, which favours already present taxa, likely shapes the initial decomposition phase [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The middle stage of decomposition is characterized by increasing bacterial diversity [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e], suggesting that the complex community might be reaching the maximum carrying capacity of FWD as a growth substrate. During the final decomposition stage, the bacterial community shows decreasing diversity and structurally approaches the composition of soil communities. This convergence occurs as FWD disintegrates and mixes with the surrounding soil, leading to increasingly similar habitat variables and, consequently, similar communities.\u003c/p\u003e \u003cp\u003eDespite the convergence trend, structural differences between FWD and soil bacterial communities remained significant throughout the decomposition process in our experiment, with FWD communities consistently showing relatively high diversity, yet significantly lower than soil bacterial communities. This lower diversity can be attributed to less favourable conditions for bacterial growth in deadwood, including high C:N ratios, lower pH and likely increased competition for nutrients with fungi in a habitat lacking carbon input through rhizodeposition [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. While soil serves as a reservoir of bacterial diversity for deadwood colonization [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], the selective pressures within deadwood drive the community through specific developmental stages. The decomposition process itself continuously influences pedological properties and microbial community in the upper horizons of underlying soil, creating an interplay between these two physically connected habitats [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilarly to fungal diversity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], fir deadwood supported higher bacterial diversity than beech, extending our understanding of substrate-specific community development during FWD decomposition. While previous studies have documented comparable or lower bacterial richness in beech compared to other tree species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], the diversity patterns in fir remained unexplored. The contrasting responses of bacterial and fungal communities to canopy conditions - with bacterial diversity flourishing under closed canopy while fungal diversity in fir FWD increased with canopy opening [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] - suggest distinct niche differentiation mechanisms. Despite the observed influence of tree species and canopy openness on community structure, the major bacterial taxa in FWD displayed remarkable habitat generalism, showing no strong preferences for either tree species or canopy conditions.\u003c/p\u003e \u003cp\u003eThis pattern of environmental response extended to the soil bacterial communities, where canopy openness induced subtle yet significant structural changes likely through altered forest floor dynamics, including shifts in vegetation, litter input, and root exudate profiles [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The absence of canopy-specific associations among dominant soil bacterial taxa, combined with their stronger response to abiotic factors such as soil moisture, C:N ratio, and pH [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e], indicates that bacterial community assembly in both FWD and soil is primarily driven by fundamental habitat conditions rather than canopy-related variables, confirming the previous results from forest clearcuts [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGeneralists versus Specialists\u003c/h2\u003e \u003cp\u003eThe genus \u003cem\u003eGranulicella\u003c/em\u003e (Acidobacteriota) and \u003cem\u003eMucilaginibacter\u003c/em\u003e (Bacteroidota) showed genomes containing rich toolkit of CAZymes which suggests their broad substrate specificity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Indeed, these taxa were identified at high relative abundances throughout the whole FWD decomposition which might indicate that they may modulate their metabolism to maintain high population density even during the changes in substrate availability. The role of Acidobacteriota in the decomposition was confirmed by the fact that their carbohydrate-active enzymes were widely expressed on coarse deadwood [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition to Acidobacteriota, potential for versatility in carbohydrate utilization was shown in a global genome comparison also for Planctomycetota and Verrucomicrobiota [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] which appeared as \u003cem\u003eTundrisphaera\u003c/em\u003e (Planctomycetota), \u003cem\u003eChthoniobacter\u003c/em\u003e and \u003cem\u003eMethylacidiphilaceae\u003c/em\u003e bacterium (Verrucomicrobiota) in the advanced FWD decomposition phase.\u003c/p\u003e \u003cp\u003eIn contrast to generalists, several taxa showed preference for a specific phase of FWD decomposition. This includes \u003cem\u003eErwinia\u003c/em\u003e (Gammaproteobacteria) known as a potential pathogen from live plant tissues [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and from association with insect pests [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Their high abundances at the beginning of decomposition might represent a diminishing community which is not adapted for long-term survival in decomposing wood.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLuteibacter\u003c/em\u003e displayed the preference for the early FWD decomposition and was shown to degrade cellulose in soil [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. In a pure culture, \u003cem\u003eLuteibacter\u003c/em\u003e carbohydrate utilization was performed mainly through the cell-associated enzymes [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] which might prevent resource loss through diffusion of decomposition products but also represents disadvantage in the advanced decomposition when proximal resources might be exhausted. \u003cem\u003ePedobacter\u003c/em\u003e abundant at the beginning of FWD decomposition was shown to degrade simple compounds [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and thus this taxon might be outcompeted in later stages of FWD decomposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFurther possible bacterial functions\u003c/h2\u003e \u003cp\u003eDespite less pronounced bacterial potential to degrade complex compounds in comparison with fungal activity owing to a limited abilities to colonize and change large patches of the substrate, bacteria still hold potential to contribute to the process of decomposition with decisive roles. Their roles are represented by functional traits described in the following paragraphs. While 16S rRNA sequencing has a limited ability to identify specific bacterial functions, bacterial taxa identified here have repeatedly been observed in association with decomposing wood; Acidobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes were previously identified in deadwood by 16S rRNA sequencing and cultivation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Members of the Alphaproteobacteria cultivated from deadwood showed lower potential for carbohydrate utilization as seen in their limited CAZyme gene content and thus narrow spectrum of carbohydrates which they can utilize [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, Alphaproteobacteria together with Gammaproteobacteria are able to utilize C1 compounds. Most importantly, they can oxidize methane and subsequently methanol, both of which are produced during deadwood decomposition (Lennart et al., 2012). Part of the carbon from these compounds is also assimilated and thus stabilized which prevents its loss from the system. The potential taxa feeding on C1 compounds in decomposing FWD are represented by \u003cem\u003eMethylorosula\u003c/em\u003e (Alphaproteobacteria) [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e], \u003cem\u003eMethylovirgula\u003c/em\u003e (Alphaproteobacteria) [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e] and \u003cem\u003eMethylacidiphilales\u003c/em\u003e (Verrucomicrobiota), although the methanotrophic abilities of the latter probably depend on the habitat [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile deadwood is carbon-rich substrate, its nitrogen limitation makes its colonization and decomposition difficult. Community of diazotrophs alleviate nitrogen limitation in deadwood [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] which has the strongest effect at the beginning of decomposition. \u003cem\u003eIn situ\u003c/em\u003e expression of nitrogen-fixation genes was shown to be strong on coarse deadwood [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Transcripts of nitrogen fixation genes mapped to the genomes/metagenome-assembled genomes have unveiled potential diazotrophs which include also genomes of cultivated enterobacteria from the genus \u003cem\u003eSodalis\u003c/em\u003e (identified as Pectobacteriaceae bacterium in decomposing FWD Here \u003cem\u003eSodalis\u003c/em\u003e appears throughout the decomposition of FWD with the highest values at the beginning of FWD decomposition which is in line with its putative role in nitrogen fixation. In terms of FWD decomposition the initial years were characterized by low nitrogen content which increased only after four years as nitrogen enrichment by diazotrophs and nitrogen retention in microbial, mainly fungal, biomass took place.\u003c/p\u003e \u003cp\u003eRecycling of microbial biomass is among the major factors sustaining bacterial growth in forest soil [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Given the high amount of fungal biomass in deadwood, mycophagous bacterial lifestyle can represent a successful strategy in obtaining the energy and covering carbon and nitrogen demand. Several chitinolytic bacteria were identified in the phylum Acidobacteriota [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e] and members of this group were identified as either being present throughout the FWD decomposition (genus \u003cem\u003eGranulicella\u003c/em\u003e, \u003cem\u003eTerriglobus\u003c/em\u003e) or at the advanced phase of decomposition (\u003cem\u003eBryocella\u003c/em\u003e, \u003cem\u003eSilvibacterium\u003c/em\u003e, \u003cem\u003eOccallatibacter\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that microclimatic changes induced by forest canopy gaps significantly influence soil bacterial communities and those involved in the decomposition of fine woody debris (FWD). Although FWD bacterial communities originate from the soil, they undergo rapid and dynamic development, shaped in part by microclimatic factors. As FWD plays a crucial role in nutrient flow, particularly in managed forests, disturbances to the canopy could disrupt nutrient dynamics and ecosystem functions. While fungi are the primary decomposers of dead plant biomass, their function is heavily influenced by substrate quality and microclimatic conditions. In contrast, bacteria appear to represent a more stable component of the microbial community, even in such fluctuating environments. Environmental factors not only impact the structure of microbial communities but can also modulate the metabolic activities of their members [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Future research should explore functional gene activity related to nutrient cycling, microbial resilience, and bacterial-fungal interactions across different canopy conditions. Additionally, examining disturbance-specific bacterial functions and their impact on carbon dynamics could provide valuable insights for sustainable forest management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contributions:\u003c/strong\u003e Conceptualization C. B., P. B., J. M, R. B.; experimental design and methodology C. B., V. T., J. B., T. D. P., P. B. and V. B.; performance of experimental work, data evaluation and statistical analyses, V. T., T.D. P. and V. B.; validation V. T. , C. B., P. B. and V. B.; writing-original draft preparation V. T. and V. B.; writing \u0026ndash; review and editing, V. T., J. B. , C. B., J. M., P. B. and V. B., supervision, project administration and funding acquisition, P. B. and V. B. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the Czech Science Foundation (20-14961S). PB and PTD were supported by the Ministry of Education, Youth and Sports of the Czech Republic (M\u0026Scaron;MT CZ.02.01.01/00/22_008/0004635).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e Sequencing data presented in this study have been deposited in the SRA database under BioProject accession number PRJNA1228314 and in Zenodo repository (https://doi.org/10.5281/zenodo.14900894).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We thank Clementine Lepinay for sharing R scripts used for LMM models created in former paper of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarris NL, Gibbs DA, Baccini A, Birdsey RA, de Bruin S, Farina M, et al. 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FEMS Microbiol Ecol. 2023;99.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"decomposition, deadwood, bacterial community, succession, canopy cover, microclimate, temperate forest, ecology, fine woody debris","lastPublishedDoi":"10.21203/rs.3.rs-6118769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6118769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFine woody debris (FWD) is a crucial yet often overlooked component of forest ecosystems, providing a dynamic habitat for microbial communities and playing a key role in carbon and nutrient cycling. In managed forests with low deadwood stocks, FWD decomposition enhances soil fertility by facilitating microbial nutrient cycling. Climate change increases the prevalence of forest disturbances enhancing the area of early succession forests with low canopy cover, but the consequences on the microbial communities and related processes is insufficiently understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere we conducted a ten-year experiment manipulating canopy cover to examine the decomposition of FWD of \u003cem\u003eFagus sylvatica\u003c/em\u003e and \u003cem\u003eAbies alba\u003c/em\u003e. Our study revealed that canopy openness significantly affected bacterial diversity in the decomposing wood as well as in the surrounding soil. While community structure in FWD was primarily influenced by decomposition time, tree species and canopy density also played a role. We identified bacterial taxa associated with carbohydrate utilization, fungal biomass degradation, and nitrogen fixation, highlighting the diverse functional roles of FWD bacteria in nutrient cycling. Bacterial community in almost completely decomposed FWD remains clearly distinct from soil bacterial communities.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eComplex ecological interactions shape deadwood decomposition and nutrient cycling. The interplay between FWD decomposition time, tree species, and microclimatic variability influences microbial community dynamics, with bacteria acting as a more stable component of the decomposer community compared previously studied fungi. This stability may be critical for sustaining decomposition and nutrient turnover despite environmental fluctuations associated with global change.\u003c/p\u003e","manuscriptTitle":"Fragile foundations: Succession patterns of bacterial communities in fine woody debris and soil under long-term microclimate influence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 03:12:59","doi":"10.21203/rs.3.rs-6118769/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-24T05:12:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T12:20:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43203069181958333536879976774627659268","date":"2025-05-07T13:20:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168583182825323702623250434995423470678","date":"2025-05-06T20:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T08:46:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75469162402531507049440495188296442385","date":"2025-03-28T07:52:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281694844869521255291938548253295567512","date":"2025-03-26T05:19:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-26T04:51:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T12:34:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-22T05:41:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2025-03-20T14:06:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"15a268f2-4b69-4059-b325-184e01ad6b07","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T16:09:13+00:00","versionOfRecord":{"articleIdentity":"rs-6118769","link":"https://doi.org/10.1186/s40793-025-00756-9","journal":{"identity":"environmental-microbiome","isVorOnly":false,"title":"Environmental Microbiome"},"publishedOn":"2025-08-06 15:57:59","publishedOnDateReadable":"August 6th, 2025"},"versionCreatedAt":"2025-04-01 03:12:59","video":"","vorDoi":"10.1186/s40793-025-00756-9","vorDoiUrl":"https://doi.org/10.1186/s40793-025-00756-9","workflowStages":[]},"version":"v1","identity":"rs-6118769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6118769","identity":"rs-6118769","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}