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Article

Microbial Community Responses to Nitrogen Addition in Poplar Leaf and Branch Litter: Shifts in Taxonomic and Phylogeny

by
Yuan Gao
1,
Yiying Wang
1,
Haodong Zheng
1,
Rongkang Wang
1,
Zimei Miao
1,2,* and
Zhiwei Ge
1,2,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, College of Ecology and Environment, Nanjing Forestry University, Nanjing 210037, China
2
Academy of Chinese Ecological Progress and Forestry Development Studies, Nanjing Forestry University, Nanjing 210037, China
*
Authors to whom correspondence should be addressed.
Forests 2025, 16(9), 1446; https://doi.org/10.3390/f16091446
Submission received: 12 August 2025 / Revised: 31 August 2025 / Accepted: 10 September 2025 / Published: 11 September 2025
(This article belongs to the Section Forest Soil)
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Abstract

Poplar (Populus L. species), a fast-growing temperate species, forms plantations with high productivity and biomass, with its litter sustaining key functions in nutrient cycling, microbial diversity, and carbon storage. Litter microbial communities drive decomposition, particularly in early stages, this initial phase is characterized by the leaching of water-soluble carbon and nutrients from the litter, which creates a readily available resource pulse that facilitates rapid microbial colonization and activation. This process is followed by the activation of microbial enzymes and the immobilization of nutrients, collectively initiating the breakdown of more recalcitrant litter materials. Under rising global nitrogen deposition, we conducted a field randomized block experiment in 13-year-old pure poplar (Populus deltoides L. ‘35’) stands, with three nitrogen addition treatments: N0 (0 g N·m−2·yr−1), N2 (10 g N·m−2·yr−1), and N4 (30 g N·m−2·yr−1). In the initial phase of litter decomposition, we measured the soil properties and litter traits, the litter microbial community composition, and its taxonomic and phylogenetic diversity indices. The results indicate that nitrogen addition altered microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), soil NO3−-N, and accelerated litter decomposition rates. The microbial community in leaf litter responded to nitrogen addition with increased phylogenetic clustering (higher OTU richness and NRI), which suggests that environmental filtering exerted a homogenizing selective pressure linked to both soil and litter properties, whereas the microbial community in branch litter responded to nitrogen addition with increased taxonomic diversity (higher OTU richness, Shannon, ACE, and Chao1), a pattern associated with litter properties that likely alleviated nitrogen limitation and created opportunities for more taxa to coexist. The observed differences in response stem from distinct substrate properties of the litter. This study elucidates microbial taxonomic and phylogenetic diversity responses to nitrogen addition during litter decomposition, offering a scientific foundation for precise microbial community regulation and sustainable litter management.

1. Introduction

The genus Populus is a key species for ecological restoration and carbon sequestration in vulnerable coastal saline–alkali regions due to its remarkable tolerance to salt and alkaline stress [1]. Beyond providing substantial biomass, poplars significantly contribute to soil organic carbon (SOC) dynamics and sequestration through litter inputs from leaves and branches, forming a vital carbon pathway in these ecosystems [2]. The decomposition of this litter, driven by microbial communities, is the primary process transferring carbon and nutrients from vegetation to soil.
Litter decomposition is the primary process transferring carbon and nutrients from vegetation to soil, with microbial communities serving as the central drivers [3]. In forest ecosystems, poplar leaf and branch litter constitute major inputs. Studies specific to poplar litter decomposition highlight its relatively fast initial decay rate compared to many other tree species, influenced by its chemical composition [4]. Microbial communities colonizing this litter are fundamental agents, mediating decomposition through enzyme production, nutrient immobilization, and mineralization [5], thereby directly influencing the fate of carbon and nitrogen cycling within the ecosystem [6]. Since the early 20th century, global atmospheric nitrogen deposition has steadily increased, exerting complex effects on terrestrial ecosystems [7]. Research on nitrogen addition effects on terrestrial ecosystems has shown that elevated nitrogen availability can profoundly alter decomposition rates and pathways. Specifically for litter microbes, nitrogen addition has been documented to shift microbial community composition, particularly for high-lignin materials like woody litter. However, most studies on poplar litter–microbe–nitrogen interactions have concentrated on overall decomposition rates under nitrogen amendment, changes in broad functional groups or extracellular enzyme activities, and shifts in taxonomic composition within the microbial communities [8,9]. Some studies have also shown that nitrogen addition affects microbial communities either through environmental filtering [10] or by altering the severity of nitrogen limitation [11,12]. Moreover, the initial stage of decomposition is critical, beginning with the leaching of soluble compounds that provides a labile resource pulse to stimulate microbial colonization. Subsequent microbial processes, including enzyme activation and nutrient immobilization, are built upon this foundation, yet the role of leaching in shaping early microbial dynamics is often overlooked [13,14].
While nitrogen addition effects on broad microbial metrics during litter decomposition are increasingly recognized, critical gaps remain in understanding its impact on the phylogenetic dimension of these microbial communities. Most existing research has primarily focused on their functional diversity and relative abundance, with limited attention paid to phylogenetic diversity of populations or communities [15,16]. In particular, how nitrogen addition shapes the phylogenetic structure and diversity of microbial communities within specific litter types like poplar leaves and branches remains poorly understood. Analyzing phylogenetic patterns allows inference of the dominant ecological processes structuring communities [17]. Consequently, the lack of knowledge regarding how nitrogen addition shapes the phylogenetic structure and diversity of microbial communities within specific litter types like poplar leaves and branches represents a significant limitation. This knowledge gap impedes the anticipation of the stability and functional resilience of decomposer communities under nitrogen enrichment, limits our understanding of the evolutionary constraints that shape microbial responses to nitrogen-induced changes in litter chemistry and environmental conditions, and limits our assessment of the long-term implications for carbon and nutrient cycling in managed poplar ecosystems, especially in vulnerable saline–alkali areas where efficient decomposition is critical.
Therefore, this study aims to characterize how nitrogen addition affects microbial communities in poplar leaf and branch litter by examining both taxonomic shifts and, crucially, phylogenetic changes. By integrating analyses of community composition, phylogenetic diversity, and inferred assembly processes, we will clarify the mechanisms by which nitrogen deposition alters the microbes driving decomposition of this ecologically significant litter in coastal saline environments, notably in the initial phase of decomposition. Our research aims to explore the following: (i) As inferred from phylogenetic patterns, how nitrogen addition affects litter microbial taxonomic diversity and phylogenetic diversity. (ii) Whether leaf and branch litter, differing in substrate characteristics, exhibit distinct microbial taxonomic and phylogenetic diversity responses to nitrogen addition. We hypothesize that leaf and branch litter will differ in their taxonomic and phylogenetic diversity responses to nitrogen addition due to their distinct substrate characteristics. To explore these questions, a nitrogen addition trial was performed within a poplar plantation system. Specifically, we separately analyzed nitrogen addition effects on microbial taxonomic and phylogenetic diversity during initial decomposition phases in poplar leaf litter and branch litter fractions, rather than in mixed litter. Finally, we elucidated the potential drivers of the differing responses in microbial taxonomic and phylogenetic diversity between these two litter types. This study can help us further understand the mechanistic underpinnings of how nitrogen deposition reshapes microbial decomposer communities in distinct litter types within poplar plantations in coastal saline–alkali environments, specifically through the lens of taxonomic and phylogenetic diversity and assembly processes. It provides scientific support for the management of poplar plantations, emphasizing nitrogen deposition implications for key decomposing microorganisms to optimize management strategies, maintain soil health, and carbon sequestration potential, and offers a reference for formulating ecological monitoring standards based on microbial indicators.

2. Materials and Methods

2.1. Study Area

This study was undertaken in Huanghai Forest Park, located on the coast of the Yellow Sea in Dongtai City, Jiangsu Province, Eastern China (32°52′28.45″ N, 120°49′47.63″ E). Bordering the Yellow Sea to the east, it lies on a typical alluvial plain of the middle and lower Yangtze River. The soil is sandy-textured and slightly alkaline, with high porosity, an average soil bulk density of 1.26 g cm−3, and a moisture content of 28.93%. The pH was 8.03, TC was 15.8 g kg−1, TN was 1.17 g kg−1, and TP was 0.70 g kg−1 (Table 1). Northern subtropical monsoon climate prevails, marked by four-season cyclicity; rainfall and high temperatures coincide in summer, leading to concentrated precipitation during the growing season. The region experiences a mean annual temperature of 15.1 °C with approximately 1004 mm precipitation. Huanghai Forest Park covers approximately 2800 ha, of which 2186 ha are forested, yielding a forest coverage rate of about 80%.

2.2. Experimental Design

The study was conducted in a 13-year-old pure poplar (Populus deltoides L. ‘35’) plantation within the park. The stand was established at a density of 750 trees ha−1 with row and planting spacings of 5 × 6 m. At the time of the experiment, mean tree height was 25.6 m, mean DBH measured 27.5 cm with canopy density at approximately 0.6. Three experimental blocks (A, B, and C), each measuring 20 × 100 m, were selected based on uniform site conditions and management history. Within each block, three 20 × 20 m subplots were delineated, separated by buffer zones that were 20 m wide to minimize edge and nutrient transfer effects. A randomized block design assigned three nitrogen treatments: control (N0: 0 g N m−2 yr−1), low (N2: 10 g N m−2 yr−1), and high (N4: 30 g N m−2 yr−1) [18]. This simulated nitrogen deposition experiment began in May 2012, with nitrogen applications conducted annually during the growing season, from May to October. We dissolved ammonium nitrate (NH4NO3) at the prescribed concentrations in 20 L water and uniformly sprayed solutions onto assigned subplots, with control plots receiving 20 L of water only.
During the leaf abscission period, litter (branches and leaves) was collected from same-age poplar stands within the park that had not received nitrogen treatment. The collected litter was air-dried naturally for 15 days; afterward, branches were cut into 1–2 cm segments. Ten-gram subsamples of branch segments and leaves were placed separately into 15 × 15 cm, 200-mesh nylon litterbags. At each deployment point, surface debris was gently removed to expose the mineral soil layer. Litterbags were laid flat on the soil surface, debris was carefully replaced, and a PVC marker pipe was positioned adjacent to the bag. Each bag was secured with twine to prevent displacement. The experimental design resulted in a total of 18 litterbags: 3 nitrogen treatments × 2 litter types (leaf or branch) × 3 replicates per combination.
At the initial decomposition stage (62 days after deployment), litter branches and leaves were retrieved from the sampling points. Samples for microbial diversity analysis were promptly placed on dry ice and transferred to the laboratory. Litter leaves and branches were labeled “L” and “B”, respectively, and the remaining material was retained for subsequent analyses. Table 2 details initial chemistry of leaf and branch litter. Concurrently, prior to soil collection, the surface debris (including freshly fallen litter and fine root mats) was carefully removed to minimize the influence of un-decomposed organic matter. We then collected 0–10 cm soil cores from beneath each litterbag using a soil auger. This sampling depth (0–10 cm) was selected because it encompasses the primary zone of interactive biogeochemical activity between the decomposing litter and the mineral soil, where majority of the microbial processing and nutrient transformations occur. Soil samples from three replicate points within each plot were composited. The fresh composite sample was sieved (<2 mm) and subdivided: one portion underwent ambient air drying for subsequent soil pH measurement, and the second portion was preserved fresh for immediate soil water content analysis: NO3−-N, NH4+-N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN). The three composited samples from the three blocks were treated as independent replicates and replicates were retained for statistical analysis.

2.3. Soil Properties

Soil pH was quantified via potentiometric analysis of soil–water suspensions at a 1:2.5 (w/v) ratio, and soil water content (%) was determined gravimetrically [19]. Soil NO3−-N and NH4+-N concentrations were analyzed via UV spectrophotometry and are expressed in mg kg−1 [20]. The chloroform fumigation technique determined soil microbial biomass carbon and nitrogen, with results expressed in mg kg−1 [21].

2.4. Litter Properties

The retrieved litter samples were first freed of adhering soil particles and newly colonizing roots. Samples underwent oven drying at 65 °C until achieving constant mass, with resultant dry mass quantified. Finally, the litter decomposition rate was calculated and is expressed as the percentage of initial mass decomposed over the experimental period (%). Dried branch and leaf litter were subsequently processed through grinding followed by sequential fractionation using 0.48 mm and 0.149 mm sieves. Lignin concentration was determined by high-performance liquid chromatography (HPLC), and cellulose content by colorimetric assay; we have expressed the relative contents of litter components as g g−1 of dry mass [22,23].

2.5. Taxonomic and Phylogenetic Diversity Indices of Litter Microbes

The Illumina platform was used to perform high-throughput sequencing, enabling analysis of fungal and bacterial diversity in litter, with a sequencing depth of 50,000 Reads. Paired-end sequencing was employed to target the fungal ITS1 region and the bacterial 16S V4 region in the litter samples, the dominant fungal and bacterial taxa in the litter were identified separately; extraction of total DNA from the litter samples was performed using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). A NanoDrop2000 spectrophotometer was employed to determine DNA concentration, and purity was verified by electrophoresis on a 1% agarose gel. The purified DNA served as the template for PCR amplification of the fungal ITS1 and bacterial 16S V4 regions. Primers ITS5-1737F and ITS2-2043R were used to amplify the fungal ITS1 region, while bacterial 16S V4 was targeted using primers 515F and 806R. To purify the PCR products, the GeneJET™ Gel Extraction Kit (Thermo Scientific, Waltham, MA, USA) was employed. Sequencing libraries were then constructed with the Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) and quantified on a Qubit@ 2.0 Fluorometer. After sequencing, the raw data were subjected to data quality control. Reads were quality-filtered using Trimmomatic by scanning in a 50 bp window and truncating reads if the average quality score dropped below 20 and removing reads after trimming that were shorter than 50 bp. Barcodes were matched exactly, and primers were allowed up to 2 nucleotide mismatches. Sequences containing ambiguous bases were discarded. Paired-end reads were assembled using FLASH, requiring a minimum overlap of 10 bp, and unassembled reads were discarded. Then, OTUs (Operational Taxonomic Units) were clustered from quality-filtered reads at 97% sequence similarity. Taxonomic assignment was performed using the UNITE database for fungi and the SILVA database for bacteria, with a minimum confidence threshold of 70%. All samples were rarefied to an even depth based on the sample with the minimum number of high-quality sequences prior to calculating diversity indices.
The phylogenetic tree was constructed from OTU data using MEGA11 [24]. Phylogenetic trees were imported using the treeio [25] R packages. Microbial taxonomic and phylogenetic diversity indices were analyzed using the vegan [26] and picante [27] R packages.
The taxonomic diversity of litter-associated microbial communities was evaluated using several indices, including OTU richness, Shannon’s diversity index, Simpson’s diversity index, Chao1, and the Abundance-based Coverage Estimator (ACE) [28,29]. Phylogenetic diversity was assessed using Faith’s phylogenetic diversity index (PDI), net relatedness index (NRI), and nearest taxon index (NTI). The PDI quantifies the standardized relative magnitude of Faith’s phylogenetic diversity (PD) [30,31]. The NRI measures phylogenetic clustering or overdispersion across the entire phylogenetic tree by calculating the standardized effect size of the mean phylogenetic distance (MPD). In contrast, the NTI assesses clustering among closely related taxa near the tips of the phylogeny by calculating the standardized effect size of the mean nearest taxon distance (MNTD) [30,31]. For all three indices (PDI, NRI, and NTI), taxa labels were randomized 999 times to generate a null distribution. Positive NRI and NTI values indicate significant phylogenetic clustering compared to the null model, negative values indicate phylogenetic overdispersion (divergence), and values around zero indicate a random phylogenetic structure relative to the null model.

2.6. Data Analysis

The effects of nitrogen addition on soil properties (soil water content, pH, NO3−-N, NH4+-N, microbial biomass carbon, microbial biomass nitrogen), litter properties (lignin, cellulose, lignin/cellulose, decomposition rate), microbial litter taxonomic diversity (OTU richness, Shannon, Simpson, ACE, Chao1), and litter phylogenetic diversity (PD, MPD, MNTD, PDI, NRI, NTI) were assessed by one-way ANOVA conducted in SPSS Statistics 26.0 (SPSS Inc., Chicago, IL, USA). The associations between soil properties, litter properties, microbial litter taxonomic diversity, and litter phylogenetic diversity were determined by Pearson’s correlation coefficient analysis using the psych R package [32]. The influence of nitrogen addition on microbial taxonomic and phylogenetic diversity of litter were evaluated using structural equation modeling (SEM) with the plspm R package [33]. All statistical analyses were performed using the software R 4.4.1 [34].

3. Results

3.1. Soil and Litter Properties Responses to Nitrogen Addition

ANOVA analysis revealed that under nitrogen addition treatments (Table 3), soil water content (Figure 1a), pH (Figure 1b), and NH4+-N (Figure 1c) did not differ significantly among treatments; NO3−-N (Figure 1d) concentrations in N2 and N4 treatments demonstrated significant reductions relative to the N0 (p < 0.001). Microbial biomass carbon (MBC) (Figure 1e) under N2 was significantly higher than when under both N0 (p < 0.001) and N4 (p < 0.05), and MBC under N4 was significantly higher than when under N0 (p < 0.01). Microbial biomass nitrogen (MBN) (Figure 1f) under N4 was significantly higher than when under both N0 (p < 0.001) and N2 (p < 0.001), and MBN under N2 was significantly higher than when under N0 (p < 0.05).
Meanwhile, litter lignin, cellulose, and the ratio of lignin to cellulose remained unaffected by nitrogen addition (Table 4) (Figure 2a,b), nor did it alter the ratio of lignin to cellulose (Figure 2c). Furthermore, decomposition rates (Figure 2d) of both litter leaves and litter branches under N4 treatment were significantly higher than when under N0 (p < 0.05) and N2 (p < 0.05) treatments. Moreover, lignin content, cellulose content, and decomposition rates differed significantly between leaf litter and branch litter (Figure 2).

3.2. Nitrogen Addition Impacts on Abundance, Taxonomic and Phylogenetic Diversity of Litter Microbes

In both leaf and branch litter, bacterial communities (Figure 3a) were dominated by the phylum Proteobacteria (relative abundance 63.91%–77.40%) and Bacteroidetes (20.09%–33.62%). Subdominant phylum included Actinobacteria (0.99%–3.06%), Verrucomicrobia (0.08%–0.45%), and Firmicutes (0.06%–0.45%). In fungal communities (Figure 3b), Ascomycota (relative abundance 79.24%–99.64%) and Basidiomycota (0.35%–20.42%) comprised majority of the sequences. However, the dominant bacterial phyla differed in their relative abundances between leaf litter and branch litter.
In leaf litter (Figure 4a,b), N2 treatment significantly increased fungal OTU richness (p < 0.05) and significantly elevated the net relatedness index (NRI) of bacterial (p < 0.05) and fungal (p < 0.01) communities relative to N0 treatment. Moreover, the fungal NRI under N4 treatment was significantly lower than when under N2 treatment (p < 0.05). In branch litter (Figure 4c–f), N2 treatment significantly increased fungal Shannon (p < 0.05), OTU richness (p < 0.01), ACE (p < 0.05), Chao1 (p < 0.05), bacterial OTU richness (p < 0.05), and ACE (p < 0.05) indices relative to N0 treatment, and N4 treatment significantly increased fungal OTU richness (p < 0.05) and significantly reduced bacterial ACE indices (p < 0.01) relative to N0 treatment. Meanwhile, bacterial ACE indices demonstrated significant suppression under N4 treatment relative to N2 (p < 0.001); Table S1 shows microbial taxonomic and phylogenetic diversity metrics unaffected by nitrogen addition. The observed increase in multiple metrics of taxonomic diversity is consistent with a potential alleviation of resource limitation.

3.3. Environmental Influence on Taxonomic and Phylogenetic Diversity in Litter Microbial Communities

In leaf litter microbial communities (Figure 5), the net relatedness index (NRI) was more strongly correlated with microbial biomass carbon (MBC). In branch litter microbial communities (Figure 5), both bacterial and fungal OTU richness were significantly associated with microbial biomass carbon (MBC), NO3−-N, and lignin contents. The bacterial ACE richness was significantly correlated with microbial biomass nitrogen (MBN), NH4+-N, and decomposition rate. The fungal Shannon was significantly correlated with microbial biomass carbon (MBC) and NO3−-N, while ACE and Chao1 were significantly correlated with microbial biomass carbon (MBC).
We employed partial least squares structural equation modeling (PLS-SEM) to evaluate the pathways by which nitrogen addition affects microbial taxonomic and phylogenetic diversity during litter decomposition. We defined the latent variables “Soil Properties”, “Litter Properties”, “Taxonomic Diversity”, and “Phylogenetic Diversity”. These latent constructs were then integrated into the PLS-SEM framework to clarify both direct and indirect influence of nitrogen addition on the taxonomic and phylogenetic diversity of microbial communities within litter. After bootstrap validation (n = 500), all models demonstrated good fit values (GoF = 0.43, 0.46, 0.44, 0.47) (Figure 6).
Soil and litter properties were significantly associated with bacterial and fungal phylogenetic diversity in leaf litter, while litter properties were significantly associated with bacterial and fungal taxonomic diversity in branch litter. Specifically, in leaf litter (Figure 6a), nitrogen addition first alters soil properties (PC = 0.82, p < 0.01) and then influences the bacterial phylogenetic structure (PC = 0.57, p < 0.05). Meanwhile, those changes in soil properties further modify litter properties (PC = 1.16, p < 0.1), thereby affecting the bacterial phylogenetic structure (PC = 0.40, p < 0.05). For fungi (Figure 6c), nitrogen addition first alters soil properties (PC = −0.92, p < 0.001), then affects the fungal phylogenetic structure (PC = −0.67, p < 0.05). Concurrently, those changes in soil properties further modify litter properties (PC = −1.94, p < 0.05), thereby influencing fungal taxonomic diversity (PC = −0.80, p < 0.05) and phylogenetic structure (PC = 0.34, p < 0.05). In branch litter (Figure 6b), nitrogen addition alters soil properties (PC = −0.92, p < 0.001), which in turn modify litter properties (PC = −1.56, p < 0.05) and ultimately influence bacterial taxonomic diversity (PC = 0.47, p < 0.05). For fungi (Figure 6d), nitrogen addition first alters soil properties (PC = 0.92, p < 0.001), then modifies litter properties (PC = 1.60, p < 0.05), and ultimately affects fungal taxonomic diversity (PC = 0.26, p < 0.1). Nitrogen addition can also directly influence fungal taxonomic diversity (PC = 0.55, p < 0.05).

4. Discussion

4.1. Responses of Soil Physicochemical Properties and Litter Properties to Nitrogen Addition

Results reveal that soil water content remained unaffected by nitrogen addition (Figure 1a). Multiple field experiments have shown that soil’s moisture regime and physical architecture remain unaltered by direct nitrogen effects. Moreover, soils rich in organic matter and with high water-holding capacity can buffer the impacts of nitrogen addition [35]. In another temperate farmland study examining nitrogen addition and N2O emissions, nitrogen inputs failed to significantly affect soil pH [36]. This outcome corroborates our results (Figure 1b) and likely stems from the site’s alkaline soil properties. Carbonate compounds, such as calcium carbonate, can neutralize hydrogen ions. Additionally, alkaline soils typically exhibit relatively high cation exchange capacity, allowing acidic substances to be neutralized through the exchange of base cations like Ca2+ and Mg2+. This buffering capacity can mitigate the decrease in pH caused by nitrogen addition [37]. In this study, nitrogen addition did not significantly affect NH4+-N (Figure 1c). Following nitrogen addition, added N may rapidly converted to nitrate via nitrification, thereby maintaining stable NH4+-N levels [38]. Nitrogen addition may also increase microbial activity and promote the formation of anaerobic microsites, stimulating denitrification and nitrate reduction and thereby accelerating microbial consumption of NO3−-N (Figure 1d), which reduces its retention in the soil [39]. Furthermore, NO3−-N carries a negative charge and is less likely to be adsorbed by soil colloids, making it prone to leaching with water flow [40]. MBC and MBN can reflect microbial abundance and activity to some extent [41,42]. In this study, nitrogen addition promoted MBC at low N levels but inhibited it at high N levels (Figure 1e). A meta-analysis of 86 global studies found that low to moderate nitrogen addition significantly increased MBC, while high-level nitrogen fertilization resulted in significant MBC loss [43]. This response likely reflects that moderate nitrogen input alleviates microbial nitrogen limitation, enhances carbon use efficiency, and stimulates mineralization of labile organic carbon. However, as nitrogen concentrations continue to rise, soil organic matter may accumulate in less bioavailable forms, reducing its availability to microbes and thereby inhibiting microbial activity [44]. Meanwhile, high levels of nitrogen addition can deplete base cations and elevate aluminum ion concentrations, exerting toxic effects on microorganisms and restricting their nutrient uptake [45]. In this study, nitrogen addition affected MBN by causing it to continuously increase with nitrogen input (Figure 1f). This is because nitrogen input promotes microbial nitrogen immobilization, and the effect strengthens with increasing nitrogen application rates, thereby increasing MBN content [43]. Nitrogen addition directly increases the sources of available inorganic nitrogen, thereby promoting microbial nitrogen assimilation.
Our findings demonstrate that nitrogen addition markedly accelerates the decomposition rate (Figure 2). Litter decomposition proceeds in three stages: during the early stage, mass loss is driven primarily by the breakdown of water-soluble carbon and hemicellulose, while degradation of structural polymers such as lignin and cellulose is concentrated in the late decomposition stage. Additionally, the lignin and cellulose content in litter is mainly determined by plant synthesis and resorption during senescence. Short-term exogenous nitrogen addition does not alter pre-formed polymer structures. Thus, lignin and cellulose contents showed no significant changes in the early decomposition stage [46]; even if microorganisms are promoted by nitrogen addition, they can hardly alter their contents in the short term. Supplying abundant inorganic nitrogen promoted rapid bacterial proliferation and accelerated the decomposition of labile substrates, resulting in enhanced early-stage litter mass loss without substantially altering the overall content of structural polymers. Specifically, under the N4 treatment, the proportions of Proteobacteria and Actinomycetota, which are known from previous studies to harbor glycoside hydrolase genes [47], were markedly elevated (Figure 3), thereby improving the hydrolytic efficiency of substrates such as hemicellulose. Although the abundance of the lignin-degrading fungal phylum Basidiomycota remained unchanged (Figure 3), bacterial degradation of soluble compounds alone was sufficient to drive the observed reductions in litter mass.

4.2. Response Mechanisms of Leaf Litter Microbial Taxonomic and Phylogenetic Diversity to Nitrogen Addition

Our results reveal that nitrogen addition affects leaf litter microbial communities primarily by altering their phylogenetic structure. Specifically, bacterial and fungal NRI under the N2 treatment were markedly elevated compared with those in the other treatments (Figure 4b). Under conditions without nitrogen supplementation, microbial taxonomic and phylogenetic diversity are mainly regulated by resource competition and environmental heterogeneity [48]. However, nitrogen enrichment intensifies phosphorus limitation and alters the chemical environment [49], thereby selecting for microbial taxa capable of tolerating these altered chemical conditions. A limited number of nitrogen-tolerant lineages such as Proteobacteria can grow rapidly, reflecting strong environmental filtering. Meanwhile, microorganisms that share adaptive traits are often closely related. Consequently, the observed phylogenetic patterns are consistent with the action of environmental filtering, which is expected to favor the expansion of closely related taxa. Under consistent nitrogen conditions, these related lineages appear to expand collectively, resulting in the pronounced phylogenetic clustering observed within the community [50,51]. Simultaneously, nitrogen addition can alter resource availability and niche space, potentially shifting the relative ecological positions of species. These changes may correspondingly influence the community’s distribution within phylogenetic space [52].
Additionally, homogenizing selection describes a process in which a uniform environment exerts strong selective pressure on specific functional traits, which predominate under fertilization. This deterministic process promotes deep phylogenetic clustering of microbial lineages, as evidenced by studies in multiple rice paddies and mangroves which validated this assembly mechanism [53]. Our findings align with recent research demonstrating that nitrogen addition triggers environmental filtering through resource enrichment and intensified phosphorus limitation, confirming that nitrogen-mediated filtering is a universal driver of microbial community assembly [54,55,56]. As nitrogen levels continue to rise and stress becomes excessive, the tolerance limits of related species are reached and further environmental degradation no longer increases phylogenetic clustering; environmental stress has driven the local extinction of certain taxa. Instead, stochastic processes such as dispersal, drift, diversification, or speciation diminish phylogenetic clustering intensity [57]. Aligning with our findings, NRI peaks at the N2 treatment level before declining under N4 (Figure 4b). However, the process of phylogenetic clustering does not necessarily reduce species richness or substantially alter abundance distributions. Instead, the inherent functional redundancy within microbial communities allows for the replacement of taxa by closely related species occupying similar ecological niches. This mechanism maintains overall diversity while promoting phylogenetic clustering within the community [58].
Changes in soil and litter characteristics induced by nitrogen addition are inferred to be the driving force behind this process (Figure 6). Under experimental nitrogen enrichment, enhanced microbial nitrogen retention in soil and dynamic shifts in microbial biomass pools—specifically, elevated MBN (Figure 1f) alongside a unimodal response in MBC (Figure 1e and Figure 5)—intensify resource competition and thereby impose a strong environmental filter on colonizing taxa [59,60]. Simultaneously, leaf litter chemistry—characterized by low lignin and cellulose concentrations but a high lignin-to-cellulose ratio—defines a high-quality carbon substrate that functions as a biotic filter (Figure 2), selectively enriching microbial lineages endowed with efficient carbon–metabolic pathways [61]. Alterations in soil nutrient status amplify competitive intensity, while litter chemical traits delineate the selection criteria. Together, they are associated with the observed phylogenetic aggregation patterns in both bacterial and fungal communities. Thus, clarifying leaf litter responses to nitrogen deposition predicts microbial phylogenetic clustering and evidences environmental filtering in decomposer assembly, elucidates deterministic processes sustaining microbial functional stability in microhabitats, and provides a framework for assessing nutrient-cycling resilience under global nitrogen deposition.

4.3. Response Mechanisms of Branch Litter Microbial Taxonomic and Phylogenetic Diversity to Nitrogen Addition

Nitrogen addition altered microbial taxonomic diversity in branch litter. As nitrogen concentrations increased, OTU richness, Shannon, Chao1, and ACE index displayed a unimodal response—initially rising and subsequently declining (Figure 4c–f). Moderate nitrogen supply alleviates nitrogen limitation and is associated with increased taxonomic diversity. This pattern is consistent with the concept that enhanced nitrogen availability weakens the preeminence of highly competitive species, thereby fostering the establishment of a wider spectrum of taxa with diverse physiological adaptations and efficiencies of resource use to establish and persist within the community; it also promotes microbial taxonomic diversity by relaxing the stringent environmental filtering imposed under severe nitrogen limitation [62,63]. However, high nitrogen levels can cause metal toxicity and nutrient imbalances, ultimately suppressing microbial activity [64]. At the same time, excessive nitrogen input intensifies competition within the community, allowing dominant taxa to rapidly monopolize available resources [65]. These conditions further constrain the niche space of rare species, exacerbating competitive exclusion and contributes to accelerating species disappearance while contracting microbial taxonomic diversity [66]. Similarly, a six-year nitrogen addition experiment demonstrated that microbial taxonomic diversity was significantly higher under moderate nitrogen treatments compared to control conditions [67]. A recent study in a red pine plantation revealed that nitrogen addition enhanced fungal community richness, where the highest richness and Chao1 estimates occurred at low nitrogen doses (20 kg N ha−1 yr−1) [68]. Recent studies on the microbial response threshold in ecological environments constrained by nitrogen have shown that microbial diversity remains stable under nitrogen deposition before the nitrogen concentration exceeds the critical threshold but significantly decreases after exceeding the critical threshold [69]. These patterns validate both our experimental findings and the proposed explanatory framework. Despite nitrogen addition failing to significantly impact the phylogenetic structure of the microbial community, taxonomic diversity still changed through compensatory shifts among phylogenetic lineages [70]. Closely related clades tend to share conserved functional traits, leading to synchronous abundance shifts in response to nitrogen gradients. Simultaneously, functional redundancy across divergent lineages may allow declines in one clade to be offset by increases in another clade performing analogous ecological roles [71,72]. We propose that this dual mechanism, synchronized responses within clades and functional compensation between clades, could be responsible for maintaining phylogenetic structure stability despite fluctuations in taxonomic diversity [73].
Soil properties mediate this response to nitrogen addition, while litter traits primarily drive it. A high lignin concentration establishes a physical barrier that delays carbon release [74]. A high carbon-to-nitrogen ratio serves as a regulatory switch governing nitrogen limitation [75]. As exogenous nitrogen moves through the soil to the litter interface, the litter recalibrates resource allocation according to its intrinsic properties, triggering a unimodal trajectory in microbial diversity: an initial release of limitation reaching a peak, followed by competitive exclusion and metal toxicity driving a decline [76]. Under nitrogen addition, the independent regulation of both the intensity and timing of decomposer microbial community responses is driven by litter chemical properties, particularly the carbon-to-nitrogen ratio and lignin content, whereas soil primarily functions as a conduit for material transfer [77]. Critically, identifying the nitrogen level that maximizes branch litter microbial diversity (moderate nitrogen) enables the prediction of decomposition resilience under nitrogen deposition, a key parameter for modeling carbon turnover in anthropogenically fertilized ecosystems.

4.4. Differential Responses of Leaf and Branch Litter Microbial Taxonomic and Phylogenetic Diversity to Nitrogen Addition

Substrate differences between leaf and branch litter significantly regulate the microbial community’s response sensitivity by influencing substrate availability (Figure 2 and Figure 4). Lignin and cellulose possess complex structures composed of multiple reactive functional groups, and their degradation produces phenolic acid compounds [78]. These compounds may constrain certain microbial taxa but simultaneously select for specialized decomposers that are resistant to them [79]. Lignin and cellulose function as dual mediators in shaping microbial communities. Their recalcitrant structure imposes a potent environmental filter selecting for specialized decomposers, while their abundance as a carbon source simultaneously enriches these adapted taxa by providing sustained carbon substrates. Within bacterial communities, members of the phyla Firmicutes [80] and Proteobacteria [81] exhibit strong cellulolytic capabilities, while Bacteroidetes [82] predominantly metabolize aromatic compounds derived from lignin degradation. Together, these bacterial phyla contribute indirectly to promote the decomposition of lignin. Fungi are the most prominent microorganisms in degrading lignin and cellulose. Among them, brown-rot fungi are exclusively Basidiomycota, while white-rot fungi are primarily Basidiomycota [83,84], and soft-rot fungi are predominantly members of the Ascomycota [85]. Proteobacteria and Bacteroidetes dominate both leaf and branch litter, but branch litter harbors relatively higher abundances of Firmicutes (Figure 3). The relatively high abundance of Firmicutes in branch litter may reflect the proliferation of taxa within this phylum that possesses the enzymatic capabilities for cellulose degradation, suggesting that environments with high cellulose content may promote the enrichment of cellulolytic microorganisms [80]. Ascomycota dominate fungal communities in both leaf and branch litter (Figure 3), while Basidiomycota are significantly enriched in branch litter, indicating a stronger sensitivity to lignin and cellulose concentrations, as well as other key physicochemical properties [86].
Leaf litter generally contains abundant labile carbon compounds and lower lignin and cellulose concentrations, making it more susceptible to decomposition and facilitating rapid colonization by fungi and bacteria. This litter is rich in readily available carbon and relatively abundant in nitrogen. During the early stage of decomposition, leaf litter experiences less nitrogen limitation compared to branch litter [87,88]. In the beginning stages of decomposition, the release of large amounts of soluble carbon increases competition among microorganisms for resources. Nitrogen addition preferentially stimulates taxa capable of quickly using soluble carbon sources and through environmental filtering, favors closely related species adapted to more uniform conditions, thereby causing significant shifts in phylogenetic structure. Specifically, substrates low in lignin and cellulose are primarily influenced by carbon availability, with nitrogen addition mainly influencing carbon utilization rates, thereby reshaping the phylogenetic composition of dominant decomposer lineages through selective filtering and taxon replacement. Branch litter contains high concentrations of lignin and cellulose and has an initial C:N ratio nearly twice that of leaf litter (Table 2), making it especially prone to nitrogen limitation and more responsive to added inorganic nitrogen. Its decomposition relies on microbial taxa capable of degrading lignin and cellulose. Nitrogen addition directly alleviates nitrogen limitation by supplying inorganic nitrogen, thereby expanding resource availability and creating additional niche space. This promotes microbial proliferation and facilitates the recruitment of functionally complementary taxa as species supplements. The occupation of these newly available niches ultimately enhances taxonomic diversity. Studies have confirmed these differentiation mechanisms driven by the matrix, and owing to differences in the lignin-to-cellulose ratio and the carbon-to-nitrogen ratio, different litter types consistently show different microbial sensitivity thresholds to nitrogen addition [89,90]. These findings validate our proposed explanation for the distinct microbial responses to nitrogen addition observed between leaf and branch litter. Quantifying these differences in litter sensitivity enables refined predictions of decomposition trajectories under regional nitrogen deposition, providing a mechanistic basis for managing ecosystem nutrient cycling with heterogeneous litter inputs.
We recommend moderate nitrogen application in plantations within coastal saline–alkali environments, where recalcitrant branch litter dominates, to stimulate decomposition and nutrient cycling. Managers should avoid excessive doses to prevent suppressing microbial diversity. For nutrient-rich leaf litter, we advise against additional nitrogen input to preserve its natural rapid decomposition and prevent excessive phylogenetic clustering of microbial communities. Maintaining mixed litter types is advised to support diverse decomposer communities. This study focused solely on the initial stage of litter decomposition and did not analyze the entire process. Furthermore, it lacked functional gene analysis of litter microbes and did not incorporate environmental data such as daily mean air temperature, precipitation amount, and rainfall frequency during the experimental period. Future studies should therefore aim to monitor the entire decomposition continuum to fully understand temporal dynamics, employ metagenomic approaches to profile functional genes and clarify underlying mechanisms, and incorporate detailed monitoring of environmental variables such as temperature and precipitation to assess their interactions with nitrogen deposition.

5. Conclusions

Nitrogen addition significantly affected the soil NO3−-N concentrations, microbial biomass carbon (MBC) and nitrogen (MBN), and litter decomposition rates. In the early phase of litter decomposition, contrasting microbial taxonomic and phylogenetic diversity responses to nitrogen addition arise between leaf and branch litter due to their differing substrate properties. In leaf litter, nitrogen enrichment primarily reshapes the phylogenetic structure of microbial communities through environmental filtering, a process influenced by soil and litter properties. In contrast, in branch litter, nitrogen addition mainly affects taxonomic diversity by alleviating nitrogen limitation, this process is mediated by soil properties and linked to litter characteristics. Although their response patterns to nitrogen addition differ, neither treatment alters the dominant microbial taxa, and both facilitate litter decomposition. This study elucidates distinct patterns of microbial taxonomic and phylogenetic diversity responses to nitrogen addition in leaf and branch litter with differing substrate characteristics, providing a crucial microbial basis for accurately predicting forest litter decomposition dynamics and carbon cycle feedback under the scenario of increasing global nitrogen deposition.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f16091446/s1, Table S1: Effects of different nitrogen additions on the microbial taxonomic diversity and phylogenetic diversity index of litter bacteria and fungi.

Author Contributions

Conceptualization, formal analysis, methodology, writing—original draft, Y.G.; review and editing, Y.W.; data curation, H.Z. and R.W.; study conception and design, methodology development, and manuscript revision, Z.M.; project supervision, Z.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Jiangsu Provincial Innovation Research Program on Carbon Peaking and Carbon Neutrality (BT2024012, BE2022305), the Jiangsu Forestry Science and Technology Innovation and Extension Project (Project No: LYKJ [2022] 02), and the National Natural Science Foundation of China (31870506, 32171856).

Data Availability Statement

The datasets presented in this article are not publicly available, as they are part of ongoing studies.

Acknowledgments

We extend our gratitude to all members of the Biodiversity and Ecological Conservation Research Group at Nanjing Forestry University for their support in sample collection, data analysis, and insightful suggestions on experimental design and interpretation. We also wish to acknowledge the reviewers and editors for their insightful feedback, which substantially enhanced this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Responses of soil properties to nitrogen addition. (a) Soil water content (SWC); (b) pH; (c) ammonium nitrogen (NH4+-N); (d) nitrate nitrogen (NO3−-N); (e) microbial biomass carbon (MBC); (f) microbial biomass nitrogen (MBN). One-way analysis of variance (ANOVA) was conducted. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05.
Figure 1. Responses of soil properties to nitrogen addition. (a) Soil water content (SWC); (b) pH; (c) ammonium nitrogen (NH4+-N); (d) nitrate nitrogen (NO3−-N); (e) microbial biomass carbon (MBC); (f) microbial biomass nitrogen (MBN). One-way analysis of variance (ANOVA) was conducted. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05.
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Figure 2. Responses of litter properties to nitrogen addition. (a) Lignin content; (b) cellulose content; (c) lignin/cellulose; (d) decomposition rate; light blue represents leaf litter, and dark blue represents branch litter. One-way analysis of variance (ANOVA) was conducted. Different lowercase letters denote significant differences among treatments (p < 0.05), and different uppercase letters denote significant differences between leaf and branch litter (p < 0.05).
Figure 2. Responses of litter properties to nitrogen addition. (a) Lignin content; (b) cellulose content; (c) lignin/cellulose; (d) decomposition rate; light blue represents leaf litter, and dark blue represents branch litter. One-way analysis of variance (ANOVA) was conducted. Different lowercase letters denote significant differences among treatments (p < 0.05), and different uppercase letters denote significant differences between leaf and branch litter (p < 0.05).
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Figure 3. Horizontal distribution of microbial phylum in litter under different nitrogen treatments. (a) Bacterial; (b) fungal. LN0: the leaf litter under the N0 treatment, LN2: the leaf litter under the N2 treatment, LN4: the leaf litter under the N4 treatment, BN0: the branch litter under the N0 treatment, BN2: the branch litter under the N2 treatment, BN4: the branch litter under the N4 treatment.
Figure 3. Horizontal distribution of microbial phylum in litter under different nitrogen treatments. (a) Bacterial; (b) fungal. LN0: the leaf litter under the N0 treatment, LN2: the leaf litter under the N2 treatment, LN4: the leaf litter under the N4 treatment, BN0: the branch litter under the N0 treatment, BN2: the branch litter under the N2 treatment, BN4: the branch litter under the N4 treatment.
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Figure 4. Responses of litter microbial taxonomic and phylogenetic diversity indices to differential nitrogen addition. (a) Leaf litter OTU richness; (b) leaf litter net relatedness index (NRI); (c) branch litter Chao1; (d) branch litter OTU richness; (e) branch litter Shannon’s diversity index (Shannon); (f) branch litter Abundance-based Coverage Estimator (ACE). One-way analysis of variance (ANOVA) was conducted. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05. Table S1 shows microbial taxonomic and phylogenetic diversity metrics unaffected by nitrogen addition.
Figure 4. Responses of litter microbial taxonomic and phylogenetic diversity indices to differential nitrogen addition. (a) Leaf litter OTU richness; (b) leaf litter net relatedness index (NRI); (c) branch litter Chao1; (d) branch litter OTU richness; (e) branch litter Shannon’s diversity index (Shannon); (f) branch litter Abundance-based Coverage Estimator (ACE). One-way analysis of variance (ANOVA) was conducted. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05. Table S1 shows microbial taxonomic and phylogenetic diversity metrics unaffected by nitrogen addition.
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Figure 5. Pearson’s correlation analysis between environmental factors and taxonomic and phylogenetic diversity indices of litter microbial communities. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05.
Figure 5. Pearson’s correlation analysis between environmental factors and taxonomic and phylogenetic diversity indices of litter microbial communities. Asterisk notation designates significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05.
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Figure 6. Structural equation model of the relationships between soil properties, litter properties, and taxonomic and phylogenetic diversity of litter microbial communities. (a) Bacteria in leaf litter; (b) bacteria in branch litter; (c) fungi in leaf litter; (d) fungi in branch litter. Soil properties included soil water content (SWC), pH, NH4+-N, NO3−-N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN). Litter properties included lignin content, cellulose content, and lignin/cellulose. Taxonomic diversity included OTU richness, Shannon, Simpson, ACE, and Chao1 indices. Phylogenetic diversity included PD, MPD, MNTD, PDI, NRI, and NTI. Blue pathways: positive; red pathways: negative; solid lines: statistically significant paths; dashed lines: non-significant paths; the numbers above the lines: the path coefficients; GoF: goodness of fit; R2: the explained variance. Asterisks denote significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05; †: p < 0.1.
Figure 6. Structural equation model of the relationships between soil properties, litter properties, and taxonomic and phylogenetic diversity of litter microbial communities. (a) Bacteria in leaf litter; (b) bacteria in branch litter; (c) fungi in leaf litter; (d) fungi in branch litter. Soil properties included soil water content (SWC), pH, NH4+-N, NO3−-N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN). Litter properties included lignin content, cellulose content, and lignin/cellulose. Taxonomic diversity included OTU richness, Shannon, Simpson, ACE, and Chao1 indices. Phylogenetic diversity included PD, MPD, MNTD, PDI, NRI, and NTI. Blue pathways: positive; red pathways: negative; solid lines: statistically significant paths; dashed lines: non-significant paths; the numbers above the lines: the path coefficients; GoF: goodness of fit; R2: the explained variance. Asterisks denote significance levels: ***: p < 0.001; **: p < 0.01; *: p < 0.05; †: p < 0.1.
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Table 1. Baseline physicochemical properties of the soil at the study site.
Table 1. Baseline physicochemical properties of the soil at the study site.
Bulk DensitySWCpHTCTNTP
1.26 g cm−328.93%8.0315.8 g kg−11.17 g kg−10.70 g kg−1
Table 2. Initial content of chemical constituents in branch and leaf litter (Mean ± SD).
Table 2. Initial content of chemical constituents in branch and leaf litter (Mean ± SD).
The Composition of LitterBranch LitterLeaf Litter
Total carbon (mg g−1)487.27 ± 1.45429.87 ± 6.57
Total nitrigon (mg g−1)12.17 ± 2.0917.37 ± 2.29
Table 3. Soil properties responses to nitrogen addition (Mean ± SD).
Table 3. Soil properties responses to nitrogen addition (Mean ± SD).
Soil PropertiesN0N2N4
SWC (%)24.90 ± 0.77 a25.76 ± 0.70 a25.95 ± 1.31 a
pH7.70 ± 0.21 a7.66 ± 0.08 a7.65 ± 0.07 a
NH4+-N (mg kg−1)1.25 ± 0.10 a1.36 ± 0.09 a1.16 ± 0.20 a
NO3−-N (mg kg−1)7.78 ± 0.44 a4.58 ± 0.32 b4.61 ± 0.56 b
MBC (mg kg−1)310.04 ± 12.27 c610.87 ± 65.55 a469.36 ± 59.64 b
MBN (mg kg−1)3.22 ± 0.75 c13.85 ± 0.53 b43.34 ± 6.78 a
One-way analysis of variance (ANOVA) was conducted. Different lowercase letters represent significant differences (p < 0.05).
Table 4. Responses of litter properties to nitrogen addition (Mean ± SD).
Table 4. Responses of litter properties to nitrogen addition (Mean ± SD).
Litter PropertiesN0N2N4
Leaf Lignin (g g−1)0.33 ± 0.01 a0.33 ± 0.01 a0.33 ± 0.02 a
Leaf Cellulose (g g−1)0.24 ± 0.02 a0.22 ± 0.02 a0.23 ± 0.02 a
Leaf Lignin/Cellulose1.39 ± 0.13 a1.48 ± 0.05 a1.45 ± 0.18 a
Leaf Decomposition (%)8.87 ± 0.21 b8.98 ± 0.54 ab9.88 ± 0.60 a
Branch Lignin (g g−1)0.36 ± 0.00 a0.40 ± 0.04 a0.40 ± 0.03 a
Branch Cellulose (g g−1)0.32 ± 0.04 a0.30 ± 0.03 a0.31 ± 0.04 a
Branch Lignin/Cellulose1.15 ± 0.15 a1.34 ± 0.28 a1.30 ± 0.29 a
Branch Decomposition (%)6.39 ± 0.40 b4.77 ± 0.21 b11.97 ± 1.78 a
One-way analysis of variance (ANOVA) was conducted. Different lowercase letters represent significant differences (p < 0.05).
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Gao, Y.; Wang, Y.; Zheng, H.; Wang, R.; Miao, Z.; Ge, Z. Microbial Community Responses to Nitrogen Addition in Poplar Leaf and Branch Litter: Shifts in Taxonomic and Phylogeny. Forests 2025, 16, 1446. https://doi.org/10.3390/f16091446

AMA Style

Gao Y, Wang Y, Zheng H, Wang R, Miao Z, Ge Z. Microbial Community Responses to Nitrogen Addition in Poplar Leaf and Branch Litter: Shifts in Taxonomic and Phylogeny. Forests. 2025; 16(9):1446. https://doi.org/10.3390/f16091446

Chicago/Turabian Style

Gao, Yuan, Yiying Wang, Haodong Zheng, Rongkang Wang, Zimei Miao, and Zhiwei Ge. 2025. "Microbial Community Responses to Nitrogen Addition in Poplar Leaf and Branch Litter: Shifts in Taxonomic and Phylogeny" Forests 16, no. 9: 1446. https://doi.org/10.3390/f16091446

APA Style

Gao, Y., Wang, Y., Zheng, H., Wang, R., Miao, Z., & Ge, Z. (2025). Microbial Community Responses to Nitrogen Addition in Poplar Leaf and Branch Litter: Shifts in Taxonomic and Phylogeny. Forests, 16(9), 1446. https://doi.org/10.3390/f16091446

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