Soil faunal diversity as an indicator of Ginseng cultivation duration in Northeast China

Soil faunal diversity as an indicator of Ginseng cultivation duration in Northeast China | 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 Soil faunal diversity as an indicator of Ginseng cultivation duration in Northeast China Cheng Wang, Mengqi Jiang, Zhiwei Gu, Luxin Li, Tianyue Yang, Hongyu Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8213596/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and aims Continuous cultivation in temperate "ginseng-forest" systems leads to progressive soil degradation, yet the response of soil fauna - a key bioindicator - to cultivation chronosequences remains poorly understood. This study investigates the response patterns of soil faunal communities to ginseng cultivation duration and identifies the key environmental drivers at different stages. Methods Selected forest-cultivated ginseng sites with cultivation durations of 5, 10, and 15 years in a temperate broad-leaved forest of Northeast China. Within plots of each duration, we systematically collected soil fauna and soil samples to analyze community composition, diversity, and physicochemical properties. Results Soil fauna diversity and richness declined significantly with prolonged cultivation, accompanied by structural simplification and functional homogenization. Dominant taxa were mainly small-to-medium sized (300–400 µm in length, 100–200 µm in width). Redundancy analysis revealed a temporal shift in key environmental drivers: from soil moisture in 5-year habitats, to ammonium nitrogen and pH in 10-year habitats, and finally to nitrate nitrogen limitation in long-term (15-year) habitats. Prolonged cultivation also induced progressive soil acidification and nutrient depletion. Conclusions Our findings underscore that soil faunal diversity serves as a sensitive indicator of cultivation-induced soil change and highlight the stage-specific environmental constraints in "ginseng-forest" ecosystems. This study provides a scientific basis for developing temporally tailored soil management strategies to enhance the sustainability of ginseng cultivation. Soil fauna Forest-cultivated ginseng Years of planting Soil acidification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Forest ecosystems provide critical habitats for a vast array of biodiversity and offer a range of ecosystem services. In recent decades, the cultivation of high-value medicinal plants under forest canopies, such as ginseng ( Panax ginseng C.A. Mey.), has emerged as a widespread agroforestry practice, particularly in Northeast Asia (Dai et al. 2018). Ginseng, a perennial herbaceous plant belonging to the Araliaceae family, thrives in cool, shaded environments. Ginseng cultivated in Jilin Province, China, is recognized for its superior quality (Shin et al. 2021). However, persistent ecological destruction and intense anthropogenic harvesting have led to the rapid depletion of wild ginseng resources, hindering the sustainable development of forest ecosystems. Consequently, the forest-cultivated ginseng industry, characterized by artificial seeding under forest canopies, has flourished in response to national policies promoting understory economies (Li et al. 2019). Forest-cultivated ginseng, involves sowing ginseng seeds within mixed coniferous-broadleaf forests composed of trees, shrubs, and weeds, without subsequent human intervention, relying solely on natural growth. Harvesting occurs after 10–15 years of growth, with studies confirming that its characteristics and nutritional composition rival those of wild mountain ginseng (Wang et al. 2021). This cultivation mode effectively alleviates land-use conflicts between ginseng farming and forestry, significantly contributing to controlling and reducing deforestation for ginseng plantations. Thus, it plays a vital role in promoting the sustainable utilization of forest resources. Continuous cultivation of ginseng at the same site leads to selective uptake of soil nutrients, thereby progressively altering soil physicochemical properties. This disruption of intrinsic nutrient equilibrium becomes a primary contributing factor to disease and pest infestation as well as quality decline in ginseng (Liu et al. 2022). Long-term ginseng cultivation results in a progressive decline in soil environmental quality, with soil properties becoming increasingly susceptible to environmental stresses (Matsumoto et al. 2022). Among these, soil acidification has emerged as the most prominent issue. As an acidophilic crop, ginseng prefers a rhizosphere soil pH range of 5.5–6.5 (Kim et al. 2023). Optimal soil pH facilitates the absorption of both cations and anions by various crops, including ginseng, promotes nutrient cycling and transfer, and enhances the utilization efficiency of inherent soil nutrients. (Marschner et al. 2004). Existing research demonstrates that soil pH progressively decreases with increasing cultivation duration, leading to heightened soil acidity and increasingly severe soil acidification in ginseng-growing areas (Dong et al. 2018; Fang et al. 2024). Soil acidification induces deficiencies in essential nutrients for ginseng growth and development, thereby impeding normal physiological processes and reducing both ginseng quality and yield (Lv et al. 2024). This poses a significant challenge not only to crop yield but also to the long-term health of the forest soil ecosystem. Soil fauna are highly sensitive to environmental shifts and serve as reliable bioindicators for monitoring forest management and ecosystem changes (Elie et al. 2018). Studies have shown that its community structure and diversity are governed by the collective influence of multiple soil physicochemical properties, including soil acidity, moisture regime, organic matter content, and the availability of nutrients such as nitrogen (Fang et al. 2024; Santorufo et al, 2024). This highlights the significant response of soil fauna to soil acidification and the consequently altered availability of nitrogen (Hu et al. 2024; Ma et al. 2023). Existing studies have shown that continuous ginseng monoculture will lead to an increase in pathogens in the later stages, while other microbial communities will decrease (Cho et al. 2024). Simultaneously, soil acidification negatively impacts soil fauna by inhibiting microbial activity and releasing toxic elements (Hu et al. 2024; Wang and Kuzyakov 2024). Hence, to address the current challenge of soil acidification in ginseng fields, utilizing the indicative function of soil fauna is essential for indirectly assessing the degree of soil acidification and overall health. This approach can inform strategies to mitigate adverse effects on soil fauna and the broader soil ecosystem. However, how these key environmental factors evolve with the duration of cultivation, and which factor acts as the primary constraint at each specific stage in acidic soils under continuous ginseng monoculture, remains unclear. This study investigates soil faunal communities across a ginseng cultivation chronosequence (5-, 10-, and 15-year stands) in a temperate forest of Northeast China., we propose the following hypotheses: (1) Soil fauna diversity will progressively decline with increasing ginseng cultivation duration, and (2) The composition of soil faunal communities under different cultivation durations is primarily governed by soil pH. Materials and methods General description of the study area The study area is located within a disturbed natural forest used for cultivating forest-grown ginseng in Ji'an City, Jilin Province, China (41°14'29"N, 126°10'36"E). Situated in the mountainous southeastern region of Jilin Province, it lies within the Changbai-Laoeling mountain range, with a mean elevation of 519 m a.s.l. Ji'an City experiences a distinct temperate continental climate with four clearly defined seasons. The mean annual temperature is approximately 5.3℃, and the average annual precipitation reaches 982 mm. The accumulated temperature (≥ 10℃) peaks at 3,650℃ annually, while the frost-free period lasts about 150 days. The forest vegetation represents a transitional zone between the Northeast China floristic regions. It is dominated by temperate broad-leaved forest, with key species including Larix olgensis , Quercus mongolica , Fraxinus mandshurica , and Juglans mandshurica . The predominant soil type is Haplic Luvisols (FAO classification), corresponding to dark brown forest soils in the Chinese soil taxonomy. Experimental design and sampling This study examined ginseng cultivation chronosequences of 5, 10, and 15 years using a randomized block design. For each cultivation duration, two independent 10 m×10 m plots were established, with all management practices uniformly applied by local farmers to ensure consistency in all agricultural practices. Within each plot, three 2 m×2 m subplots spaced approximately 2 m apart were positioned in areas of uniform forest composition and ginseng density, the micro-topography and altitude remain consistent. Sampling was conducted in both May and June 2023, employing a three-point sampling method with 1 m spacing within each subplot. Each cultivation duration was sampled monthly at 18 locations, yielding 54 samples per month across all durations and a grand total of 108 samples over the two-month study period. At each sampling point, soil cores were collected from the 0–20 cm depth. The samples were subsequently partitioned for soil fauna extraction and physicochemical analysis, with the latter obtained from locations immediately adjacent to the fauna sampling points. Samples for soil fauna extraction were placed in perforated plastic containers for transport to the laboratory. To account for peak biological activity during the sampling period (coinciding with optimal growth phases for both ginseng and soil fauna), data from both sampling campaigns were combined for analysis. Taxonomic identification of soil fauna Macrofauna (> 2 mm) were collected through manual sorting, mesofauna (0.1-2 mm) were collected through Tullgren dry-funnel extraction respectively. The Tullgren extraction was conducted at 40 ± 1°C for 24 hours (Wallwork at al. 1976). All specimens were preserved in 75% ethanol immediately after collection. Morphological identification to family level, abundance counting, and morphometric measurements (body length/width) were performed using an stereomicroscope (SZX16 Research Stereo Microscope, Shinjuku, Tokyo, Japan). The methodology primarily referenced Pictorial Keys to Soil Animals of China (Yin et al. 1998) for taxonomic identification. Adults and larvae were enumerated separately during identification due to their distinct ecological roles. Body size (length and width), recognized as pivotal functional traits mediating resource utilization, energy transfer efficiency, predation, and dispersal capacity in soil ecosystems (Ferrín et al. 2023; Moretti et al. 2017), was quantified following standardized protocols: (1) Body length: Linear distance in fully extended specimens, excluding chelicera; (2) Body width: In dorsal view, the maximum width at the widest point of the fully extended body (Douce 1976). Statistical analyses focused on dominant taxa (Isotomidae, Onychiuridae, Lohmanniidae) shared across all three habitats (5, 10, and 15-year sites), with particular emphasis on size-dependent functional responses. Soil sample treatment and analysis Soil bulk density was determined using the cutting ring method. At each sampling point, a flat area was selected adjacent to the core location. The cutting ring was vertically inserted into the soil until fully packed, carefully extracted, and excess soil was trimmed from both ends. The ring's exterior was wiped clean, sealed with lids to minimize moisture evaporation, and transported to the laboratory. Samples were oven-dried at 105°C until constant weight was achieved. After cooling in a desiccator, the weight was recorded to the nearest 0.01g. Soil moisture content was calculated from the mass difference before and after drying. After transport to the laboratory, soil samples were air-dried naturally for 5–7 days, manually cleared of visible stones and plant debris, then divided into two subsamples and sieved through 18-mesh (1 mm) and 100-mesh (0.15 mm) screens for physicochemical analyses. For soil organic carbon (SOC) determination, 1.0 g of 100-mesh soil was decarbonated with 3 mol·L⁻¹ HCl in a 100-mL beaker, dried at 80°C to constant weight, and precisely weighed subsamples (0.025 ± 0.0005 g) were analyzed using an elemental TOC analyzer (vario TOC cube, Elementar, Germany). Nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N) was measured by KCl extraction (2 mol·L⁻¹), filtration, and filtrate analysis via continuous flow analyzer (AutoAnalyzer 3 HR, SEAL Analytical, Norderstedt, Germany) (Kou et al., 2020). Total carbon (TC) and total nitrogen (TN) were determined on unacidified 100-mesh samples using an elemental analyzer (2400 Series II, PerkinElmer, USA), while soil pH, electrical conductivity (EC), and soluble salts were measured in 1:5 soil-water suspensions with a pH meter (PHS-3E, INESA, Shanghai, China) and conductivity meter (CON200, conductivity meter, Suzhou, China). Table 1 Soil physico-chemical properties (0–20 cm) under different ginseng cultivation durations. Years of planting 5-year 10-year 15-year Soil pH 5.83 ± 0.05a 5.76 ± 0.05a 5.52 ± 0.03b EC (µS/m) 230.42 ± 15.49a 165.61 ± 12.21b 146.91 ± 19.35b Soil soluble salt (mg/L) 138.09 ± 9.42a 100.03 ± 7.86b 88.29 ± 11.82b SOC (g/kg) 1.95 ± 0.02a 1.60 ± 0.03c 1.79 ± 0.13b TN (g/kg) 15.29 ± 0.26a 11.77 ± 0.31b 13.26 ± 0.47c TC (g/kg) 106.56 ± 3.85a 57.23 ± 4.26b 81.81 ± 6.78c NO₃⁻-N (mg/kg) 8.66 ± 0.63a 4.21 ± 2.81b 3.92 ± 0.49b NH₄⁺-N (mg/kg) 4.12 ± 0.97ab 3.78 ± 0.99a 4.69 ± 0.89b Soil bulk density (g/m 3 ) 0.84 ± 0.03b 1.05 ± 0.01a 0.91 ± 0.02c Soil moisture content % 23.99 ± 1.09b 32.31 ± 0.60a 31.31 ± 1.14a Different lowercase letters within a row indicate statistically significant differences ( p < 0.05) in soil environmental factors across ginseng cultivation durations. EC Electric conductivity; SOC Soil organic carbon; TN Total nitrogen; TC Total carbon; NO₃⁻-N Nitrate nitrogen; NH₄⁺-N Ammonium nitrogen. Data processing and statistical analysis Community characteristics were quantified using four biodiversity indices: Shannon index (H`), Pielou's index (E), Margalef index(D), Simpson index(C). The respective formulas are: (Steinwandter and Seeber 2023): $$\:\begin{array}{c}H`=\:-{\sum\:}_{i=1}^{S}{P}_{i}ln{P}_{i}\#\left(1\right)\end{array}$$ $$\:\begin{array}{c}E=\:{H}^{\prime }/ln\left(S\right)\#（2）\end{array}$$ $$\:\begin{array}{c}D=(S-1)/ln\left(N\right)\#（3）\end{array}$$ $$\:\begin{array}{c}C=\:{\sum\:}_{i=1}^{S}{\left({n}_{i}/N\right)}^{2}\#（4）\end{array}$$ Data organization was performed using Microsoft Excel 2021. Statistical analyses were conducted in IBM SPSS Statistics 13: One-way ANOVA tested differences in soil fauna abundance (individual counts), taxon richness, diversity indices, body length/width of dominant taxa (Isotomidae, Onychiuridae, Lohmanniidae) and soil environmental factors across ginseng cultivation durations. Differences were tested using Fisher's Least Significant Difference (LSD) method (α = 0.05). Data violating homogeneity of variance underwent log₁₀ or natural logarithmic transformation prior to analysis. Venn diagrams illustrating shared and unique taxa across ginseng cultivation durations were generated using the Venn Diagram plugin in Origin 2021. Redundancy analysis (RDA) implemented in Canoco 5.0 for Windows (Microcomputer Power, Ithaca, NY, USA) quantified relationships between soil faunal communities and environmental factors under different cultivation periods. The results are presented as conditional effects derived from forward selection. To examine the effects of different habitats (5-year, 10-year and 15-year) on soil fauna community structure, we performed permutational multivariate analysis of variance (PERMANOVA) analysis based on Bray-Curtis distance matrices using the adonis2 function in the R vegan package (999 permutations) to assess significant differences in community composition attributable to habitat type. We further calculated the average distances within and between groups to analyse community similarity and heterogeneity across habitats. Non-metric multidimensional scaling (NMDS) was used to analyze the similarity of soil fauna communities across different habitats. Statistical analysis of correlations between environmental factors and soil faunal functional groups using Pearson’s method, implemented in R (version 4.4.3) with Hmisc, pheatmap, and RColorBrewer packages for correlation computation and heatmap visualization. Bar and chord diagrams were generated using Origin 2021 (OriginLab Corporation, Northampton, MA, USA). Statistical analysis and visualization of box plots were performed in R 4.4.3 (R Foundation for Statistical Computing, Vienna, Austria) with the "ggplot2", "dplyr", and "tidyr" packages. Result Compositional dynamics of soil faunal communities across ginseng cultivation durations Significant compositional differences were observed in soil fauna communities across ginseng cultivation durations. A total of 4,069 soil fauna individuals were collected from the 5, 10, and 15-year sites, representing 81 families within 18 taxonomic orders (Fig. 1 ). Distinct shifts in soil faunal composition were recorded along the cultivation chronosequence. In the 5-year habitat, a total of 1,348 soil fauna individuals were captured, representing 59 taxa. Dominant taxa comprised two families: Isotomidae and Lohmanniidae, accounting for 29.67% of total captures. Common taxa included Neanuridae, Entomobryidae, Oppiidae, and 12 additional families (15 taxa total), representing 58.53% of captures. Rare taxa consisted of 43 families (Fig. 1 ). In the 10-year habitat, 1,203 soil fauna individuals were captured, representing 55 taxa. Dominant taxa comprised Isotomidae, Onychiuridae and Lohmanniidae, accounting for 46.38% of total captures. Common taxa included Cyphoderidae, Parasitidae, Hypoaspididae, and 12 additional families (15 taxa total), representing 45.61% of captures. Rare taxa consisted of 37 families (Fig. 1 ). In the 15-year habitat, 1,557 individuals were captured across 49 taxa. Dominant taxa were Isotomidae and Lohmanniidae (37.51% of captures). Common taxa included Poduridae, Sminthuridae, Oribatulidae, and 12 additional families (15 taxa total), constituting 54.78% of captures. Rare taxa comprised 32 families (Fig. 1 ). Unique and shared soil faunal taxa across ginseng cultivation durations, including species composition similarity and overlap metrics (Fig. 2 ). In the 5-year habitat, unique taxa comprised Tomoceridae, Dolichocybidae, Porcellionidae, and 8 additional families (11 taxa total) (Fig. 2 ). In the 10-year habitat, unique taxa included Phlaeothripidae, Carabidae larvae, Lucanidae, and 11 additional families (14 taxa total) (Fig. 2 ). In the 15-year habitat, unique taxa consisted of Nanhermanniidae, Galumnidae, Pselaphinae larvae, and 2 additional families (5 taxa total) (Fig. 2 ). Shared taxa across all three habitats totaled 32 taxa (e.g., Isotomidae, Carabodidae, Hypochthoniidae); between 5 and 10-year habitats, 7 taxa (e.g., Ameroseiidae, Geophilidae, Elateridae); between 5 and 15-year habitats, 10 taxa (e.g., Mycobatidae, Ptychacaridae, Trachytoidae); and between 10 and 15-year habitats, 2 taxa (Pachylaelapidae, Curculionidae) (Fig. 2 ). Among the 32 shared taxa, individual densities of Ceratozetidae and Suctobelbidae were significantly higher in the 5-year habitat than in 10- and 15-year habitats ( p < 0.05); Rhodacaridae density was significantly higher in 5- and 15-year habitats than in the 10-year habitat ( p < 0.05); Macrochelidae density was significantly higher in the 5-year than 10-year habitat ( p 0.05); no significant differences ( p > 0.05) occurred in the remaining 28 shared taxa across habitats (Fig. 3 ). Shared rare taxa Carabodidae and Phthiracaridae exhibited declining density trends without statistical significance ( p > 0.05). Statistically significant differences were obtained by LSD analysis (Fig. 3 ). For smaller Isotomidae ( 0.05). Significant variations ( p 0.05) in other size ranges. The predominant body length (300–400µm) comprised > 50% of individuals in all habitats, exceeding 50% in 5-year sites. Similarly, the dominant body width (100–200µm) consistently exceeded 50% across habitats. Structural dynamics of soil fauna communities across ginseng cultivation durations Community structure of identical species may vary across cultivation durations. Non-metric multidimensional scaling (NMDS) analysis of soil faunal communities in the three habitats revealed high similarity (Fig. 4 ; Stress = 0.0849 < 0.2, indicating excellent model fit with strong representativeness and ecological interpretability). PERMANOVA results revealed no significant effect of cultivation duration on soil fauna community composition (R² = 0.070, F = 0.93, p = 0.681). Substantial overlap among 5, 10, and 15-year habitats demonstrates non-significant structural divergence, while complete clustering of communities in the ordination space reflects high similarity driven by shared taxa. Non-overlapping sections denote compositional differences, with notably greater dispersion in the 15-year habitat suggesting heightened compositional distinctiveness, though structural convergence trends were observed (Fig. 4 ). Soil faunal community evenness, Margalef index, and diversity exhibited declining trends with increasing cultivation duration, while dominance initially decreased then increased (Fig. 5 a, 5 c, 5 d). No significant differences ( p > 0.05) were detected across durations for any diversity indices, indicating progressive structural homogenization and simplification that disrupted initial equilibrium (Fig. 5 ). This shift manifested through: (1) Reduced microarthropod diversity due to declines in Collembola (Isotomidae, Onychiuridae, Entomobryidae), Oribatida (Ceratozetidae, Suctobelbidae) andMesostigmata (Parasitidae, Rhodacaridae, Ameroseiidae, Macrochelidae); (2) Elevated dominance driven by increased Oribatida (Lohmanniidae, Oribatulidae), where proliferation of functionally dominant oribatid mites counterbalanced collembolan losses, buffering significant evenness and dominance declines despite progressive reductions (Fig. 1 , 3 ). Coupling between soil faunal communities and environmental determinants across ginseng cultivation durations Soil environmental factors exhibited significant differences ( p 15-year > 10-year habitats; pH, EC, water-soluble salts, and NO₃⁻-N progressively decreased with cultivation duration; NH₄⁺-N ranked 15-year > 5-year > 10-year; while bulk density and moisture content peaked in 10-year habitats and minimized in 5-year habitats (Table 1 ).Soil fauna individual density showed significant positive correlations with EC, soluble salts, and NO₃⁻-N ( p < 0.05) (Fig. 6 ). Notably, however, extremely significant positive correlations were detected between EC and soluble salts, as well as between each of these two parameters and NO₃⁻-N ( p < 0.001), indicating collinearity among these three variables, NO₃⁻-N and NH₄⁺-N showed a very significant negative correlation( p < 0.01) (Fig. 6 ). RDA ordination effectively visualized and interpreted bidirectional linkages between soil faunal communities and environmental determinants (Fig. 7 ). In 5 and 10-year habitats, soil moisture content emerged as the primary environmental driver, with RDA1 explaining 36.37% and 44.74% of fauna-environment covariation respectively (Fig. 7 a,b). Specifically: (1) 5-year habitat: dominant/common taxa showed negative correlations with moisture but strong positive correlations with pH, TC, TN, and SOC; (Fig. 7 a) (2) 10-year habitat: Ceratozetidae, Lumbricidae, and Formicidae exhibited positive moisture correlations while other taxa correlated negatively, with NH₄⁺-N and TN being key determinants (Fig. 7 b). EC and water-soluble salts superseded moisture as primary determinants of soil faunal communities in 15-year habitats, with RDA1 explaining 39.04% of fauna-environment covariation (Fig. 7 c). Crucially, only Lumbricidae exhibited positive correlations with EC, salts, NO 3 - -N, while all other dominant/common taxa showed negative responses; communities maintained strong correlations with pH, TC, and TN (Fig. 7 c). Analysis of conditional effects following forward selection in the 15-year cultivation habitat revealed that (NO₃⁻-N contributed 25.4% of the explained variance, substantially exceeding the influence of other environmental factors, and demonstrated a significant independent effect ( p < 0.05). Discussion Implications of ginseng cultivation chronosequence for soil faunal structural configuration Our results confirm our first hypothesis, demonstrating a progressive decline in both the diversity and richness of soil fauna with continuous ginseng cultivation. Soil fauna dominant species play a significant regulatory role in shaping community structure and the adjacent habitat, constituting a fundamental component of community characteristics (Praeg et al. 2025). Different taxa of soil fauna exhibit certain selectivity towards distinct soil environments (Soininen et al. 2024). Collembola and Acari were the predominant soil fauna groups in the study area, this finding aligns with most previous studies conducted in temperate forest ecosystems. (Chi et al. 2025; Dumas et al. 2025; Junggebauer et al. 2025). In the early cultivation stage (5-year), the soil fauna community was dominated by Isotomidae and Lohmanniidae, The composition of the soil springtail community was largely consistent with findings from investigations in mixed coniferous-broadleaf forests dominated by Quercus mongolica and Fraxinus mandshurica in the Changbai Mountains (Chi et al. 2025; Xie et al. 2022). As a saprophagy, the high abundance of Lohmanniidae correlates with an active phase of litter decomposition and an abundant organic resource base (Liu et al. 2025a). In the 10-year habitat, Isotomidae and Lohmanniidae maintained their dominance. Concurrently, Onychiuridae proliferated and emerged as a new dominant taxon. The two dominant collembolan species are both primarily consumers of saprotrophic microorganisms, forming a key trophic link by channeling microbial energy through the soil food web (Gao et al. 2018; Li et al. 2022), that saprotrophic microorganisms are the primary agents of litter decomposition (Chen et al. 2025). Under the high C:N ratio conditions in this habitat, the accelerated litter decomposition pathway supported a flourishing community of saprotrophic microorganisms (Dang et al. 2025; Xiao et al. 2025). Concurrently, abundances of several predatory families, including Oppiidae, Ceratozetidae, and Suctobelbidae, decreased substantially (Lu et al. 2022). The observed changes were closely linked to deteriorating physical soil conditions, specifically increased soil moisture and bulk density (Lami et al. 2020). The dominant taxa in the 15-year habitat were identical to those in the 5-year habitat: Isotomidae and Lohmanniidae, two dominant taxa showed divergent trajectories: while Isotomidae numbers decreased, Lohmanniidae proliferated markedly. Isotomidae maintained its dominant status across all three cultivation durations, a resilience potentially owing to its ancient Mesozoic origin and considerable adaptive capacity (Saltzwedel et al. 2016). Then, soil organic matter (SOM), TC and TN all showed a significant increase, led to a substantial reduction in saprophagous oribatid mites, including families such as Lohmanniidae, Oribatulidae and Nothridae (Lu et al. 2022; Kamczyc et al. 2019; Potapov at al. 2022). Simultaneously, there was a substantial decline in the number of meso- and macro-soil animal taxa, excluding mites and collembola, aligns with the results reported by Wen et al (Wen et al. 2025). Body size, a key functional trait in soil fauna, significantly influences critical ecological processes such as foraging efficiency, predatory success, energy transfer, and dispersal capacity (Sun et al. 2024). Previous studies have demonstrated that collembolans with smaller body sizes exhibit enhanced tolerance to environmental stress and resource limitation (Santorufo et al. 2014), thereby enhancing their adaptation to the soil degradation caused by continuous ginseng cropping. Furthermore, in the 15-year habitat, the proportion of soil fauna with a body width > 900 µm decreased significantly. Diminution of soil macrofauna, it shows a severely diminished flux of mineral nitrogen into the soil food web, thereby constraining the base of the trophic system (Zhong et al. 2025). Our results demonstrated a progressive reduction in both the diversity and population size of rare soil animal taxa across the ginseng cultivation chronosequence. Rare taxonomic groups are known to be more responsive to soil microenvironment shifts than their abundant counterparts (Coudrain et al. 2016; Liu et al. 2025b). In summary, the soil faunal community in our ginseng chronosequence underwent a systematic transition from a taxonomically and functionally richer state to a simplified and homogenized one. These findings provide strong support for our first hypothesis, soil faunal diversity declined progressively with continuous ginseng cropping. Key drivers of soil faunal communities in soils under continuous ginseng cropping Our study reveals a critical shift in the primary environmental drivers governing soil faunal communities along the ginseng cultivation chronosequence. These results partially support our second hypothesis, linking the decline in soil faunal diversity to soil pH, though driven more directly by pH-regulated labile nitrogen availability. The majority of soil fauna inhabit th soil, exhibiting strong sensitivity to and dependence on the soil environment. Significant associations exist between soil fauna and the soil environment (Wu et al. 2024). The life activities of soil fauna facilitate the cycling of nutrient elements within the litter and soil ecosystem, thereby influencing soil physicochemical properties (Dou et al. 2025). Simultaneously, the distribution of soil fauna is constrained by soil physicochemical properties and nutrient conditions (Gu et al. 2025). Soil moisture content was a primary environmental factor influencing soil fauna community composition in both the 5-year and 10-year habitats. This finding aligns with the results reported by Martin et al (2024). Studies have indicated that while moist soil conditions are favorable for the habitat of some moisture-tolerant taxa, such as certain mites and collembolans (Yang et al. 2021). In contrast, elevated soil moisture can suppress the overall soil faunal community through the reduction of soil pore space and the establishment of saturated conditions (Castillo-Figueroa and Castillo-Avila 2025). Significantly higher soil moisture in the 10-year habitat was accompanied by marked declines in salinity and TN, indicative of intensified leaching (Zhu et al. 2024). In the 10-year habitat, NH₄⁺-N, TN and pH emerged as key factors correlated with soil fauna community structure. The lowest concentrations of TN and NH₄⁺-N coincided in the 10-year habitat, this "dual nitrogen stress" epitomizes the extensive nutrient depletion caused by continuous ginseng cropping (Geem et al. 2023; Liu et al. 2021). The depletion of TN adversely affects the physiological processes of soil fauna, consequently impairing the long-term sustainability of their populations (Li et al. 2021), as NH₄⁺-N serves as a key nitrogen source for ginseng, soil fauna, and microorganisms (Geisseler et al. 2010; Liu et al. 2021). Consequently, this nitrogen limitation exerted a bottom-up control (Bodur et al. 2024), this limitation cascaded through the soil food web, consequently diminishing the overall diversity of the soil fauna. (Zhang et al. 2025a). However, existing studies have found that in moderately acidified soils (pH = 5.5-6.0), soil acidification inhibits nitrification and ammonia oxidation processes, leading to a decrease in available nitrogen content (Qiu et al. 2024). Thereby suppressing key functional microbial groups such as Ammonium-Oxidizing Archaea (AOA), Ammonium-Oxidizing Bacteria (AOB), and Arbuscular Mycorrhizal Fungi (AMF) (Balume et al. 2022; Zhai et al. 2023), and indirectly altering the structure of the mesofaunal community (Creamer et al. 2016). The heatmap indicates an overall positive correlation between soil fauna abundance, nitrate nitrogen, and soil salinity. An increase in soil available nitrogen promotes a bacterial-dominated energy channel and triggers a bottom-up trophic cascade that influences the mesofauna community (Peguero et al. 2021). However, the long-term (15-year) habitat was primarily governed by the extreme depletion of NO₃⁻-N, soluble salts, and EC, whose collective minima signify a state of nutrient exhaustion and salinity stress. The decline in soil health observed here aligns with the broad phenomenon of soil degradation under continuous monoculture (Tan et al. 2021; Wang et al. 2025). Simultaneously, the significant rise in NH₄⁺-N indicates a fundamental shift in the composition of soil available nitrogen, moving from a nitrate-dominated to an ammonium-dominated regime. This alteration in the primary nitrogen form has profound implications for the structure of both soil fauna and microbial communities (Chertov et al. 2022; Francesca et al. 2021). Concurrently, Wakelin et al demonstrated that low nitrate-N environments select for a distinct microbial community, characterized by an increased abundance of specific taxa such as Firmicutes, Actinobacteria, certain Proteobacteria, and fungal groups (Wakelin et al. 2019). Additionally, an increase in the ammonium-to-nitrate ratio exacerbates soil acidification and suppresses microbial abundance and diversity (Bosman et al. 2024; Wang et al. 2023). Collectively, the sequential shift in primary environmental drivers along the cultivation chronosequence underscores a reorganization of the soil ecosystem. In moderately acidified soils, the inhibition of nitrification redirects nitrogen cycling, leading to a regime dominated by ammonium. However, under the prolonged and intensified acidification observed in the 15-year habitat, the system transitions into a state of extreme nitrate-N depletion. This critical shift in the dominant available nitrogen form, driven by pH, serves as the proximate mechanism structuring the soil faunal community. The successional pattern of driving factors shifting from soil acidification to nitrogen stoichiometric stress, as revealed in this study, is consistent with observations in other forest-based continuous cropping, such as Panax notoginseng , mushroom and cacao (Hei at al. 2024; Lou et al. 2017; Morales-Belpaire et al. 2024). However, unlike monoculture plantation systems, the natural mixed-forest environment examined here is endowed with higher ecological stability and species diversity (Qian et al. 2025), 15 years of continuous ginseng cultivation was enough to push the system past its ecological tipping point. Consequently, the management of land cultivated for over 10 years must focus on countering acidification with amendments like lime and biochar, and correcting nitrogen imbalance via nitrate-based fertilizer application (Hao et al. 2022; Zhang et al. 2025b). Conclusions Our study demonstrates that continuous ginseng cultivation under a natural forest canopy leads to a progressive decline in soil faunal diversity and a structural simplification of the community. Crucially, we identified a dynamic succession in the primary environmental drivers governing these communities: early-stage assemblages were primarily limited by soil moisture, which transitioned to control by NH₄⁺-N and pH at the mid-stage, and finally to severe NO₃⁻-N limitation under long-term cultivation. This successional pattern, driven by progressive soil acidification, reveals that pH governs the community structure indirectly by regulating the availability of different nitrogen forms. However, this study primarily focused on the overall community response. Future research should delve into the specific responses of key functional groups (e.g., predators, decomposers) and their cascading effects on ecosystem processes. Abbreviations EC Electric conductivity SOC Soil organic carbon TN Total nitrogen TC Total carbon NO₃⁻-N Nitrate nitrogen NH₄⁺-N Ammonium nitrogen SOM Soil organic matter AOA Ammonium-Oxidizing Archaea AOB Ammonium-Oxidizing Bacteria AMF Arbuscular Mycorrhizal Fungi Declarations The authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare that are relevant to the content of this article. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. Acknowledgments The authors would like to thank all those who assisted during the field work. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Cheng Wang, Mengqi Jiang, Zhiwei Gu, Luxin Li, Tianyue Yang, Hongyu Zhao, Xiaoqiang Li and Weihua Dong. The first draft of the manuscript was written by Cheng Wang and Mengqi Jiang. The academic review and writing supervision were conducted by Xiaoqiang Li and Weihua Dong. All authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (41601263), the Science and Technology Development Program of Jilin Province (20220101188JC), Natural Science Foundation Projects of CCNU (CSJJ2023003GZR), Institute of Innovation Science and Technology, Changchun Normal University (YJYKF-001). Data availability The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no relevant financial or non-financial interests to disclose. 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1","display":"","copyAsset":false,"role":"figure","size":1222903,"visible":true,"origin":"","legend":"\u003cp\u003eSoil faunal community composition under different planting years. \u003cstrong\u003eDominant taxa\u003c/strong\u003e10% or more of the individuals; \u003cstrong\u003eCommon taxa\u003c/strong\u003e 1% to 10% of the individuals; \u003cstrong\u003eRare taxa\u003c/strong\u003e Less than 1% of the individuals.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/89b958ad01c4a824879a5d0e.png"},{"id":98528214,"identity":"5b2f14a0-ecef-41c0-9871-e50cef5f63fd","added_by":"auto","created_at":"2025-12-18 14:57:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149041,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of common and unique taxa of soil fauna under different planting years of ginseng\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/f57ee96bceff2aae1ca84afe.png"},{"id":98625360,"identity":"0b517625-3923-4d6e-9c88-a71cb6a2a3b6","added_by":"auto","created_at":"2025-12-19 17:09:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":194476,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual density difference of common taxa of soil fauna in different planting years of ginseng.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/d70b4a12d8d729b303a1aaeb.png"},{"id":98624742,"identity":"33866dd4-02df-44d1-ae6b-5dc3b2df67f3","added_by":"auto","created_at":"2025-12-19 17:08:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88398,"visible":true,"origin":"","legend":"\u003cp\u003eNMDS analysis of soil fauna communities under different planting years.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/99ef398c6a249ce6c3f720af.png"},{"id":98528218,"identity":"ad2c2a69-3837-4b5a-adc6-8e1eaf3e988b","added_by":"auto","created_at":"2025-12-18 14:57:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":948479,"visible":true,"origin":"","legend":"\u003cp\u003ePielou index (a), Simpson index(b), Margalef index(c), Shannon index(d) of soil faunal community in different planting years. (mean ± SD)\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/bb228c60a69f358cd4582c39.png"},{"id":98625207,"identity":"daf8b424-330f-4bbc-bdd1-07a15efaaeba","added_by":"auto","created_at":"2025-12-19 17:08:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":276032,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions among soil environmental factors and soil fauna individual density. (*)\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.05, (**) \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and (***) \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eEC\u003c/strong\u003e Electric conductivity; \u003cstrong\u003eSS\u003c/strong\u003e Soluble salt; \u003cstrong\u003eSOC\u003c/strong\u003eSoil organic carbon; \u003cstrong\u003eTN\u003c/strong\u003e Total nitrogen; \u003cstrong\u003eTC\u003c/strong\u003e Total carbon; \u003cstrong\u003eBD\u003c/strong\u003eBulk density; \u003cstrong\u003eMC\u003c/strong\u003e Moisture content.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/db69f34d27ef450bdb6ff3e5.png"},{"id":98528231,"identity":"86eb3370-7d90-419e-823b-cda5f4f9e0d5","added_by":"auto","created_at":"2025-12-18 14:57:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1049852,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis (RDA) of soil faunal communities and soil environmental factors across (a) 5-year, (b) 10-year, and (c) 15-year habitats. \u003cstrong\u003eEC\u003c/strong\u003eElectric conductivity; \u003cstrong\u003eSS\u003c/strong\u003e Soluble salt; \u003cstrong\u003eSOC\u003c/strong\u003e Soil organic carbon; \u003cstrong\u003eTN\u003c/strong\u003eTotal nitrogen; \u003cstrong\u003eTC\u003c/strong\u003e Total carbon; \u003cstrong\u003eAN\u003c/strong\u003e Ammonium nitrogen; \u003cstrong\u003eNN\u003c/strong\u003e Nitrate nitrogen; \u003cstrong\u003eBD\u003c/strong\u003e Bulk density; \u003cstrong\u003eMC\u003c/strong\u003e Moisture content.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/bda8500d0df4fde7d971d7ed.png"},{"id":104398865,"identity":"2fa0769c-c7cd-4079-b2cd-440be0ae8656","added_by":"auto","created_at":"2026-03-11 12:04:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3815590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8213596/v1/ed6cbb8e-459c-4a61-8e24-8f76de3a843a.pdf"}],"financialInterests":"","formattedTitle":"Soil faunal diversity as an indicator of Ginseng cultivation duration in Northeast China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eForest ecosystems provide critical habitats for a vast array of biodiversity and offer a range of ecosystem services. In recent decades, the cultivation of high-value medicinal plants under forest canopies, such as ginseng (\u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Mey.), has emerged as a widespread agroforestry practice, particularly in Northeast Asia (Dai et al. 2018). Ginseng, a perennial herbaceous plant belonging to the Araliaceae family, thrives in cool, shaded environments. Ginseng cultivated in Jilin Province, China, is recognized for its superior quality (Shin et al. 2021). However, persistent ecological destruction and intense anthropogenic harvesting have led to the rapid depletion of wild ginseng resources, hindering the sustainable development of forest ecosystems. Consequently, the forest-cultivated ginseng industry, characterized by artificial seeding under forest canopies, has flourished in response to national policies promoting understory economies (Li et al. 2019). Forest-cultivated ginseng, involves sowing ginseng seeds within mixed coniferous-broadleaf forests composed of trees, shrubs, and weeds, without subsequent human intervention, relying solely on natural growth. Harvesting occurs after 10\u0026ndash;15 years of growth, with studies confirming that its characteristics and nutritional composition rival those of wild mountain ginseng (Wang et al. 2021). This cultivation mode effectively alleviates land-use conflicts between ginseng farming and forestry, significantly contributing to controlling and reducing deforestation for ginseng plantations. Thus, it plays a vital role in promoting the sustainable utilization of forest resources.\u003c/p\u003e \u003cp\u003eContinuous cultivation of ginseng at the same site leads to selective uptake of soil nutrients, thereby progressively altering soil physicochemical properties. This disruption of intrinsic nutrient equilibrium becomes a primary contributing factor to disease and pest infestation as well as quality decline in ginseng (Liu et al. 2022). Long-term ginseng cultivation results in a progressive decline in soil environmental quality, with soil properties becoming increasingly susceptible to environmental stresses (Matsumoto et al. 2022). Among these, soil acidification has emerged as the most prominent issue. As an acidophilic crop, ginseng prefers a rhizosphere soil pH range of 5.5\u0026ndash;6.5 (Kim et al. 2023). Optimal soil pH facilitates the absorption of both cations and anions by various crops, including ginseng, promotes nutrient cycling and transfer, and enhances the utilization efficiency of inherent soil nutrients. (Marschner et al. 2004). Existing research demonstrates that soil pH progressively decreases with increasing cultivation duration, leading to heightened soil acidity and increasingly severe soil acidification in ginseng-growing areas (Dong et al. 2018; Fang et al. 2024). Soil acidification induces deficiencies in essential nutrients for ginseng growth and development, thereby impeding normal physiological processes and reducing both ginseng quality and yield (Lv et al. 2024). This poses a significant challenge not only to crop yield but also to the long-term health of the forest soil ecosystem.\u003c/p\u003e \u003cp\u003eSoil fauna are highly sensitive to environmental shifts and serve as reliable bioindicators for monitoring forest management and ecosystem changes (Elie et al. 2018). Studies have shown that its community structure and diversity are governed by the collective influence of multiple soil physicochemical properties, including soil acidity, moisture regime, organic matter content, and the availability of nutrients such as nitrogen (Fang et al. 2024; Santorufo et al, 2024). This highlights the significant response of soil fauna to soil acidification and the consequently altered availability of nitrogen (Hu et al. 2024; Ma et al. 2023). Existing studies have shown that continuous ginseng monoculture will lead to an increase in pathogens in the later stages, while other microbial communities will decrease (Cho et al. 2024). Simultaneously, soil acidification negatively impacts soil fauna by inhibiting microbial activity and releasing toxic elements (Hu et al. 2024; Wang and Kuzyakov 2024). Hence, to address the current challenge of soil acidification in ginseng fields, utilizing the indicative function of soil fauna is essential for indirectly assessing the degree of soil acidification and overall health. This approach can inform strategies to mitigate adverse effects on soil fauna and the broader soil ecosystem. However, how these key environmental factors evolve with the duration of cultivation, and which factor acts as the primary constraint at each specific stage in acidic soils under continuous ginseng monoculture, remains unclear.\u003c/p\u003e \u003cp\u003eThis study investigates soil faunal communities across a ginseng cultivation chronosequence (5-, 10-, and 15-year stands) in a temperate forest of Northeast China., we propose the following hypotheses: (1) Soil fauna diversity will progressively decline with increasing ginseng cultivation duration, and (2) The composition of soil faunal communities under different cultivation durations is primarily governed by soil pH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eGeneral description of the study area\u003c/p\u003e \u003cp\u003eThe study area is located within a disturbed natural forest used for cultivating forest-grown ginseng in Ji'an City, Jilin Province, China (41°14'29\"N, 126°10'36\"E). Situated in the mountainous southeastern region of Jilin Province, it lies within the Changbai-Laoeling mountain range, with a mean elevation of 519 m a.s.l. Ji'an City experiences a distinct temperate continental climate with four clearly defined seasons. The mean annual temperature is approximately 5.3℃, and the average annual precipitation reaches 982 mm. The accumulated temperature (≥ 10℃) peaks at 3,650℃ annually, while the frost-free period lasts about 150 days.\u003c/p\u003e \u003cp\u003eThe forest vegetation represents a transitional zone between the Northeast China floristic regions. It is dominated by temperate broad-leaved forest, with key species including \u003cem\u003eLarix olgensis\u003c/em\u003e, \u003cem\u003eQuercus mongolica\u003c/em\u003e, \u003cem\u003eFraxinus mandshurica\u003c/em\u003e, and \u003cem\u003eJuglans mandshurica\u003c/em\u003e. The predominant soil type is Haplic Luvisols (FAO classification), corresponding to dark brown forest soils in the Chinese soil taxonomy.\u003c/p\u003e \u003cp\u003eExperimental design and sampling\u003c/p\u003e \u003cp\u003eThis study examined ginseng cultivation chronosequences of 5, 10, and 15 years using a randomized block design. For each cultivation duration, two independent 10 m×10 m plots were established, with all management practices uniformly applied by local farmers to ensure consistency in all agricultural practices. Within each plot, three 2 m×2 m subplots spaced approximately 2 m apart were positioned in areas of uniform forest composition and ginseng density, the micro-topography and altitude remain consistent. Sampling was conducted in both May and June 2023, employing a three-point sampling method with 1 m spacing within each subplot. Each cultivation duration was sampled monthly at 18 locations, yielding 54 samples per month across all durations and a grand total of 108 samples over the two-month study period.\u003c/p\u003e \u003cp\u003eAt each sampling point, soil cores were collected from the 0–20 cm depth. The samples were subsequently partitioned for soil fauna extraction and physicochemical analysis, with the latter obtained from locations immediately adjacent to the fauna sampling points. Samples for soil fauna extraction were placed in perforated plastic containers for transport to the laboratory. To account for peak biological activity during the sampling period (coinciding with optimal growth phases for both ginseng and soil fauna), data from both sampling campaigns were combined for analysis.\u003c/p\u003e \u003cp\u003eTaxonomic identification of soil fauna\u003c/p\u003e \u003cp\u003eMacrofauna (\u0026gt; 2 mm) were collected through manual sorting, mesofauna (0.1-2 mm) were collected through Tullgren dry-funnel extraction respectively. The Tullgren extraction was conducted at 40 ± 1°C for 24 hours (Wallwork at al. 1976). All specimens were preserved in 75% ethanol immediately after collection. Morphological identification to family level, abundance counting, and morphometric measurements (body length/width) were performed using an stereomicroscope (SZX16 Research Stereo Microscope, Shinjuku, Tokyo, Japan). The methodology primarily referenced Pictorial Keys to Soil Animals of China (Yin et al. 1998) for taxonomic identification. Adults and larvae were enumerated separately during identification due to their distinct ecological roles.\u003c/p\u003e \u003cp\u003eBody size (length and width), recognized as pivotal functional traits mediating resource utilization, energy transfer efficiency, predation, and dispersal capacity in soil ecosystems (Ferrín et al. 2023; Moretti et al. 2017), was quantified following standardized protocols: (1) Body length: Linear distance in fully extended specimens, excluding chelicera; (2) Body width: In dorsal view, the maximum width at the widest point of the fully extended body (Douce 1976). Statistical analyses focused on dominant taxa (Isotomidae, Onychiuridae, Lohmanniidae) shared across all three habitats (5, 10, and 15-year sites), with particular emphasis on size-dependent functional responses.\u003c/p\u003e \u003cp\u003eSoil sample treatment and analysis\u003c/p\u003e \u003cp\u003eSoil bulk density was determined using the cutting ring method. At each sampling point, a flat area was selected adjacent to the core location. The cutting ring was vertically inserted into the soil until fully packed, carefully extracted, and excess soil was trimmed from both ends. The ring's exterior was wiped clean, sealed with lids to minimize moisture evaporation, and transported to the laboratory. Samples were oven-dried at 105°C until constant weight was achieved. After cooling in a desiccator, the weight was recorded to the nearest 0.01g. Soil moisture content was calculated from the mass difference before and after drying.\u003c/p\u003e \u003cp\u003eAfter transport to the laboratory, soil samples were air-dried naturally for 5–7 days, manually cleared of visible stones and plant debris, then divided into two subsamples and sieved through 18-mesh (1 mm) and 100-mesh (0.15 mm) screens for physicochemical analyses. For soil organic carbon (SOC) determination, 1.0 g of 100-mesh soil was decarbonated with 3 mol·L⁻¹ HCl in a 100-mL beaker, dried at 80°C to constant weight, and precisely weighed subsamples (0.025 ± 0.0005 g) were analyzed using an elemental TOC analyzer (vario TOC cube, Elementar, Germany). Nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N) was measured by KCl extraction (2 mol·L⁻¹), filtration, and filtrate analysis via continuous flow analyzer (AutoAnalyzer 3 HR, SEAL Analytical, Norderstedt, Germany) (Kou et al., 2020). Total carbon (TC) and total nitrogen (TN) were determined on unacidified 100-mesh samples using an elemental analyzer (2400 Series II, PerkinElmer, USA), while soil pH, electrical conductivity (EC), and soluble salts were measured in 1:5 soil-water suspensions with a pH meter (PHS-3E, INESA, Shanghai, China) and conductivity meter (CON200, conductivity meter, Suzhou, China).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSoil physico-chemical properties (0–20 cm) under different ginseng cultivation durations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYears of planting\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5-year\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10-year\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15-year\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil pH\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.83 ± 0.05a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.76 ± 0.05a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.52 ± 0.03b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC (µS/m)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e230.42 ± 15.49a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e165.61 ± 12.21b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e146.91 ± 19.35b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil soluble salt (mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138.09 ± 9.42a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.03 ± 7.86b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.29 ± 11.82b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOC (g/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.95 ± 0.02a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.60 ± 0.03c\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.79 ± 0.13b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTN (g/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.29 ± 0.26a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.77 ± 0.31b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.26 ± 0.47c\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC (g/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106.56 ± 3.85a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.23 ± 4.26b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.81 ± 6.78c\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO₃⁻-N (mg/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.66 ± 0.63a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.21 ± 2.81b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.92 ± 0.49b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH₄⁺-N (mg/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.12 ± 0.97ab\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.78 ± 0.99a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.69 ± 0.89b\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil bulk density (g/m\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.84 ± 0.03b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05 ± 0.01a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.91 ± 0.02c\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil moisture content %\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.99 ± 1.09b\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.31 ± 0.60a\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.31 ± 1.14a\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eDifferent lowercase letters within a row indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) in soil environmental factors across ginseng cultivation durations. \u003cb\u003eEC\u003c/b\u003e Electric conductivity; \u003cb\u003eSOC\u003c/b\u003e Soil organic carbon; \u003cb\u003eTN\u003c/b\u003e Total nitrogen; \u003cb\u003eTC\u003c/b\u003e Total carbon; \u003cb\u003eNO₃⁻-N\u003c/b\u003e Nitrate nitrogen; \u003cb\u003eNH₄⁺-N\u003c/b\u003e Ammonium nitrogen.\u003c/p\u003e \u003cp\u003eData processing and statistical analysis\u003c/p\u003e \u003cp\u003eCommunity characteristics were quantified using four biodiversity indices: Shannon index (H`), Pielou's index (E), Margalef index(D), Simpson index(C). The respective formulas are: (Steinwandter and Seeber 2023):\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}H`=\\:-{\\sum\\:}_{i=1}^{S}{P}_{i}ln{P}_{i}\\#\\left(1\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}E=\\:{H}^{\\prime }/ln\\left(S\\right)\\#（2）\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}D=(S-1)/ln\\left(N\\right)\\#（3）\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}C=\\:{\\sum\\:}_{i=1}^{S}{\\left({n}_{i}/N\\right)}^{2}\\#（4）\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003eData organization was performed using Microsoft Excel 2021. Statistical analyses were conducted in IBM SPSS Statistics 13: One-way ANOVA tested differences in soil fauna abundance (individual counts), taxon richness, diversity indices, body length/width of dominant taxa (Isotomidae, Onychiuridae, Lohmanniidae) and soil environmental factors across ginseng cultivation durations. Differences were tested using Fisher's Least Significant Difference (LSD) method (α = 0.05). Data violating homogeneity of variance underwent log₁₀ or natural logarithmic transformation prior to analysis. Venn diagrams illustrating shared and unique taxa across ginseng cultivation durations were generated using the Venn Diagram plugin in Origin 2021. Redundancy analysis (RDA) implemented in Canoco 5.0 for Windows (Microcomputer Power, Ithaca, NY, USA) quantified relationships between soil faunal communities and environmental factors under different cultivation periods. The results are presented as conditional effects derived from forward selection. To examine the effects of different habitats (5-year, 10-year and 15-year) on soil fauna community structure, we performed permutational multivariate analysis of variance (PERMANOVA) analysis based on Bray-Curtis distance matrices using the adonis2 function in the R vegan package (999 permutations) to assess significant differences in community composition attributable to habitat type. We further calculated the average distances within and between groups to analyse community similarity and heterogeneity across habitats. Non-metric multidimensional scaling (NMDS) was used to analyze the similarity of soil fauna communities across different habitats. Statistical analysis of correlations between environmental factors and soil faunal functional groups using Pearson’s method, implemented in R (version 4.4.3) with Hmisc, pheatmap, and RColorBrewer packages for correlation computation and heatmap visualization. Bar and chord diagrams were generated using Origin 2021 (OriginLab Corporation, Northampton, MA, USA). Statistical analysis and visualization of box plots were performed in R 4.4.3 (R Foundation for Statistical Computing, Vienna, Austria) with the \"ggplot2\", \"dplyr\", and \"tidyr\" packages.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003eCompositional dynamics of soil faunal communities across ginseng cultivation durations\u003c/p\u003e\u003cp\u003eSignificant compositional differences were observed in soil fauna communities across ginseng cultivation durations. A total of 4,069 soil fauna individuals were collected from the 5, 10, and 15-year sites, representing 81 families within 18 taxonomic orders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Distinct shifts in soil faunal composition were recorded along the cultivation chronosequence. In the 5-year habitat, a total of 1,348 soil fauna individuals were captured, representing 59 taxa. Dominant taxa comprised two families: Isotomidae and Lohmanniidae, accounting for 29.67% of total captures. Common taxa included Neanuridae, Entomobryidae, Oppiidae, and 12 additional families (15 taxa total), representing 58.53% of captures. Rare taxa consisted of 43 families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the 10-year habitat, 1,203 soil fauna individuals were captured, representing 55 taxa. Dominant taxa comprised Isotomidae, Onychiuridae and Lohmanniidae, accounting for 46.38% of total captures. Common taxa included Cyphoderidae, Parasitidae, Hypoaspididae, and 12 additional families (15 taxa total), representing 45.61% of captures. Rare taxa consisted of 37 families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the 15-year habitat, 1,557 individuals were captured across 49 taxa. Dominant taxa were Isotomidae and Lohmanniidae (37.51% of captures). Common taxa included Poduridae, Sminthuridae, Oribatulidae, and 12 additional families (15 taxa total), constituting 54.78% of captures. Rare taxa comprised 32 families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eUnique and shared soil faunal taxa across ginseng cultivation durations, including species composition similarity and overlap metrics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the 5-year habitat, unique taxa comprised Tomoceridae, Dolichocybidae, Porcellionidae, and 8 additional families (11 taxa total) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the 10-year habitat, unique taxa included Phlaeothripidae, Carabidae larvae, Lucanidae, and 11 additional families (14 taxa total) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the 15-year habitat, unique taxa consisted of Nanhermanniidae, Galumnidae, Pselaphinae larvae, and 2 additional families (5 taxa total) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Shared taxa across all three habitats totaled 32 taxa (e.g., Isotomidae, Carabodidae, Hypochthoniidae); between 5 and 10-year habitats, 7 taxa (e.g., Ameroseiidae, Geophilidae, Elateridae); between 5 and 15-year habitats, 10 taxa (e.g., Mycobatidae, Ptychacaridae, Trachytoidae); and between 10 and 15-year habitats, 2 taxa (Pachylaelapidae, Curculionidae) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eAmong the 32 shared taxa, individual densities of Ceratozetidae and Suctobelbidae were significantly higher in the 5-year habitat than in 10- and 15-year habitats (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); Rhodacaridae density was significantly higher in 5- and 15-year habitats than in the 10-year habitat (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); Macrochelidae density was significantly higher in the 5-year than 10-year habitat (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) but showed no significant differences between 15-year and other habitats (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05); no significant differences (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) occurred in the remaining 28 shared taxa across habitats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Shared rare taxa Carabodidae and Phthiracaridae exhibited declining density trends without statistical significance (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Statistically significant differences were obtained by LSD analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eFor smaller Isotomidae (\u0026lt; 200µm) were more abundant in 15-year than 5 and 10-year habitats; Onychiuridae and Lohmanniidae exhibited no significant differences (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Significant variations (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) occurred only in 700–800 µm body length and the class of body width exceeding 900 µm body width size classes, with no differences (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) in other size ranges. The predominant body length (300–400µm) comprised \u0026gt; 50% of individuals in all habitats, exceeding 50% in 5-year sites. Similarly, the dominant body width (100–200µm) consistently exceeded 50% across habitats.\u003c/p\u003e\u003cp\u003eStructural dynamics of soil fauna communities across ginseng cultivation durations\u003c/p\u003e\u003cp\u003eCommunity structure of identical species may vary across cultivation durations. Non-metric multidimensional scaling (NMDS) analysis of soil faunal communities in the three habitats revealed high similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Stress = 0.0849 \u0026lt; 0.2, indicating excellent model fit with strong representativeness and ecological interpretability). PERMANOVA results revealed no significant effect of cultivation duration on soil fauna community composition (R² = 0.070, F = 0.93, \u003cem\u003ep\u003c/em\u003e = 0.681). Substantial overlap among 5, 10, and 15-year habitats demonstrates non-significant structural divergence, while complete clustering of communities in the ordination space reflects high similarity driven by shared taxa. Non-overlapping sections denote compositional differences, with notably greater dispersion in the 15-year habitat suggesting heightened compositional distinctiveness, though structural convergence trends were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eSoil faunal community evenness, Margalef index, and diversity exhibited declining trends with increasing cultivation duration, while dominance initially decreased then increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). No significant differences (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) were detected across durations for any diversity indices, indicating progressive structural homogenization and simplification that disrupted initial equilibrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This shift manifested through: (1) Reduced microarthropod diversity due to declines in Collembola (Isotomidae, Onychiuridae, Entomobryidae), Oribatida (Ceratozetidae, Suctobelbidae) andMesostigmata (Parasitidae, Rhodacaridae, Ameroseiidae, Macrochelidae); (2) Elevated dominance driven by increased Oribatida (Lohmanniidae, Oribatulidae), where proliferation of functionally dominant oribatid mites counterbalanced collembolan losses, buffering significant evenness and dominance declines despite progressive reductions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eCoupling between soil faunal communities and environmental determinants across ginseng cultivation durations\u003c/p\u003e\u003cp\u003eSoil environmental factors exhibited significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) across the three ginseng cultivation durations, with these variations substantially modulating soil faunal community structure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). SOC, TN, and TC followed the order 5-year \u0026gt; 15-year \u0026gt; 10-year habitats; pH, EC, water-soluble salts, and NO₃⁻-N progressively decreased with cultivation duration; NH₄⁺-N ranked 15-year \u0026gt; 5-year \u0026gt; 10-year; while bulk density and moisture content peaked in 10-year habitats and minimized in 5-year habitats (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).Soil fauna individual density showed significant positive correlations with EC, soluble salts, and NO₃⁻-N (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, however, extremely significant positive correlations were detected between EC and soluble salts, as well as between each of these two parameters and NO₃⁻-N (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), indicating collinearity among these three variables, NO₃⁻-N and NH₄⁺-N showed a very significant negative correlation(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eRDA ordination effectively visualized and interpreted bidirectional linkages between soil faunal communities and environmental determinants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In 5 and 10-year habitats, soil moisture content emerged as the primary environmental driver, with RDA1 explaining 36.37% and 44.74% of fauna-environment covariation respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). Specifically: (1) 5-year habitat: dominant/common taxa showed negative correlations with moisture but strong positive correlations with pH, TC, TN, and SOC; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) (2) 10-year habitat: Ceratozetidae, Lumbricidae, and Formicidae exhibited positive moisture correlations while other taxa correlated negatively, with NH₄⁺-N and TN being key determinants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eEC and water-soluble salts superseded moisture as primary determinants of soil faunal communities in 15-year habitats, with RDA1 explaining 39.04% of fauna-environment covariation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Crucially, only Lumbricidae exhibited positive correlations with EC, salts, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, while all other dominant/common taxa showed negative responses; communities maintained strong correlations with pH, TC, and TN (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Analysis of conditional effects following forward selection in the 15-year cultivation habitat revealed that (NO₃⁻-N contributed 25.4% of the explained variance, substantially exceeding the influence of other environmental factors, and demonstrated a significant independent effect (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImplications of ginseng cultivation chronosequence for soil faunal structural configuration\u003c/p\u003e \u003cp\u003eOur results confirm our first hypothesis, demonstrating a progressive decline in both the diversity and richness of soil fauna with continuous ginseng cultivation. Soil fauna dominant species play a significant regulatory role in shaping community structure and the adjacent habitat, constituting a fundamental component of community characteristics (Praeg et al. 2025). Different taxa of soil fauna exhibit certain selectivity towards distinct soil environments (Soininen et al. 2024). Collembola and Acari were the predominant soil fauna groups in the study area, this finding aligns with most previous studies conducted in temperate forest ecosystems. (Chi et al. 2025; Dumas et al. 2025; Junggebauer et al. 2025).\u003c/p\u003e \u003cp\u003eIn the early cultivation stage (5-year), the soil fauna community was dominated by Isotomidae and Lohmanniidae, The composition of the soil springtail community was largely consistent with findings from investigations in mixed coniferous-broadleaf forests dominated by \u003cem\u003eQuercus mongolica\u003c/em\u003e and \u003cem\u003eFraxinus mandshurica\u003c/em\u003e in the Changbai Mountains (Chi et al. 2025; Xie et al. 2022). As a saprophagy, the high abundance of Lohmanniidae correlates with an active phase of litter decomposition and an abundant organic resource base (Liu et al. 2025a).\u003c/p\u003e \u003cp\u003eIn the 10-year habitat, Isotomidae and Lohmanniidae maintained their dominance. Concurrently, Onychiuridae proliferated and emerged as a new dominant taxon. The two dominant collembolan species are both primarily consumers of saprotrophic microorganisms, forming a key trophic link by channeling microbial energy through the soil food web (Gao et al. 2018; Li et al. 2022), that saprotrophic microorganisms are the primary agents of litter decomposition (Chen et al. 2025). Under the high C:N ratio conditions in this habitat, the accelerated litter decomposition pathway supported a flourishing community of saprotrophic microorganisms (Dang et al. 2025; Xiao et al. 2025). Concurrently, abundances of several predatory families, including Oppiidae, Ceratozetidae, and Suctobelbidae, decreased substantially (Lu et al. 2022). The observed changes were closely linked to deteriorating physical soil conditions, specifically increased soil moisture and bulk density (Lami et al. 2020).\u003c/p\u003e \u003cp\u003eThe dominant taxa in the 15-year habitat were identical to those in the 5-year habitat: Isotomidae and Lohmanniidae, two dominant taxa showed divergent trajectories: while Isotomidae numbers decreased, Lohmanniidae proliferated markedly. Isotomidae maintained its dominant status across all three cultivation durations, a resilience potentially owing to its ancient Mesozoic origin and considerable adaptive capacity (Saltzwedel et al. 2016). Then, soil organic matter (SOM), TC and TN all showed a significant increase, led to a substantial reduction in saprophagous oribatid mites, including families such as Lohmanniidae, Oribatulidae and Nothridae (Lu et al. 2022; Kamczyc et al. 2019; Potapov at al. 2022). Simultaneously, there was a substantial decline in the number of meso- and macro-soil animal taxa, excluding mites and collembola, aligns with the results reported by Wen et al (Wen et al. 2025).\u003c/p\u003e \u003cp\u003eBody size, a key functional trait in soil fauna, significantly influences critical ecological processes such as foraging efficiency, predatory success, energy transfer, and dispersal capacity (Sun et al. 2024). Previous studies have demonstrated that collembolans with smaller body sizes exhibit enhanced tolerance to environmental stress and resource limitation (Santorufo et al. 2014), thereby enhancing their adaptation to the soil degradation caused by continuous ginseng cropping. Furthermore, in the 15-year habitat, the proportion of soil fauna with a body width\u0026thinsp;\u0026gt;\u0026thinsp;900 \u0026micro;m decreased significantly. Diminution of soil macrofauna, it shows a severely diminished flux of mineral nitrogen into the soil food web, thereby constraining the base of the trophic system (Zhong et al. 2025). Our results demonstrated a progressive reduction in both the diversity and population size of rare soil animal taxa across the ginseng cultivation chronosequence. Rare taxonomic groups are known to be more responsive to soil microenvironment shifts than their abundant counterparts (Coudrain et al. 2016; Liu et al. 2025b).\u003c/p\u003e \u003cp\u003eIn summary, the soil faunal community in our ginseng chronosequence underwent a systematic transition from a taxonomically and functionally richer state to a simplified and homogenized one. These findings provide strong support for our first hypothesis, soil faunal diversity declined progressively with continuous ginseng cropping.\u003c/p\u003e \u003cp\u003eKey drivers of soil faunal communities in soils under continuous ginseng cropping\u003c/p\u003e \u003cp\u003eOur study reveals a critical shift in the primary environmental drivers governing soil faunal communities along the ginseng cultivation chronosequence. These results partially support our second hypothesis, linking the decline in soil faunal diversity to soil pH, though driven more directly by pH-regulated labile nitrogen availability. The majority of soil fauna inhabit th soil, exhibiting strong sensitivity to and dependence on the soil environment. Significant associations exist between soil fauna and the soil environment (Wu et al. 2024). The life activities of soil fauna facilitate the cycling of nutrient elements within the litter and soil ecosystem, thereby influencing soil physicochemical properties (Dou et al. 2025). Simultaneously, the distribution of soil fauna is constrained by soil physicochemical properties and nutrient conditions (Gu et al. 2025).\u003c/p\u003e \u003cp\u003eSoil moisture content was a primary environmental factor influencing soil fauna community composition in both the 5-year and 10-year habitats. This finding aligns with the results reported by Martin et al (2024). Studies have indicated that while moist soil conditions are favorable for the habitat of some moisture-tolerant taxa, such as certain mites and collembolans (Yang et al. 2021). In contrast, elevated soil moisture can suppress the overall soil faunal community through the reduction of soil pore space and the establishment of saturated conditions (Castillo-Figueroa and Castillo-Avila 2025). Significantly higher soil moisture in the 10-year habitat was accompanied by marked declines in salinity and TN, indicative of intensified leaching (Zhu et al. 2024).\u003c/p\u003e \u003cp\u003eIn the 10-year habitat, NH₄⁺-N, TN and pH emerged as key factors correlated with soil fauna community structure. The lowest concentrations of TN and NH₄⁺-N coincided in the 10-year habitat, this \"dual nitrogen stress\" epitomizes the extensive nutrient depletion caused by continuous ginseng cropping (Geem et al. 2023; Liu et al. 2021). The depletion of TN adversely affects the physiological processes of soil fauna, consequently impairing the long-term sustainability of their populations (Li et al. 2021), as NH₄⁺-N serves as a key nitrogen source for ginseng, soil fauna, and microorganisms (Geisseler et al. 2010; Liu et al. 2021). Consequently, this nitrogen limitation exerted a bottom-up control (Bodur et al. 2024), this limitation cascaded through the soil food web, consequently diminishing the overall diversity of the soil fauna. (Zhang et al. 2025a). However, existing studies have found that in moderately acidified soils (pH\u0026thinsp;=\u0026thinsp;5.5-6.0), soil acidification inhibits nitrification and ammonia oxidation processes, leading to a decrease in available nitrogen content (Qiu et al. 2024). Thereby suppressing key functional microbial groups such as Ammonium-Oxidizing Archaea (AOA), Ammonium-Oxidizing Bacteria (AOB), and Arbuscular Mycorrhizal Fungi (AMF) (Balume et al. 2022; Zhai et al. 2023), and indirectly altering the structure of the mesofaunal community (Creamer et al. 2016).\u003c/p\u003e \u003cp\u003eThe heatmap indicates an overall positive correlation between soil fauna abundance, nitrate nitrogen, and soil salinity. An increase in soil available nitrogen promotes a bacterial-dominated energy channel and triggers a bottom-up trophic cascade that influences the mesofauna community (Peguero et al. 2021). However, the long-term (15-year) habitat was primarily governed by the extreme depletion of NO₃⁻-N, soluble salts, and EC, whose collective minima signify a state of nutrient exhaustion and salinity stress. The decline in soil health observed here aligns with the broad phenomenon of soil degradation under continuous monoculture (Tan et al. 2021; Wang et al. 2025). Simultaneously, the significant rise in NH₄⁺-N indicates a fundamental shift in the composition of soil available nitrogen, moving from a nitrate-dominated to an ammonium-dominated regime. This alteration in the primary nitrogen form has profound implications for the structure of both soil fauna and microbial communities (Chertov et al. 2022; Francesca et al. 2021). Concurrently, Wakelin et al demonstrated that low nitrate-N environments select for a distinct microbial community, characterized by an increased abundance of specific taxa such as Firmicutes, Actinobacteria, certain Proteobacteria, and fungal groups (Wakelin et al. 2019). Additionally, an increase in the ammonium-to-nitrate ratio exacerbates soil acidification and suppresses microbial abundance and diversity (Bosman et al. 2024; Wang et al. 2023).\u003c/p\u003e \u003cp\u003eCollectively, the sequential shift in primary environmental drivers along the cultivation chronosequence underscores a reorganization of the soil ecosystem. In moderately acidified soils, the inhibition of nitrification redirects nitrogen cycling, leading to a regime dominated by ammonium. However, under the prolonged and intensified acidification observed in the 15-year habitat, the system transitions into a state of extreme nitrate-N depletion. This critical shift in the dominant available nitrogen form, driven by pH, serves as the proximate mechanism structuring the soil faunal community.\u003c/p\u003e \u003cp\u003eThe successional pattern of driving factors shifting from soil acidification to nitrogen stoichiometric stress, as revealed in this study, is consistent with observations in other forest-based continuous cropping, such as \u003cem\u003ePanax notoginseng\u003c/em\u003e, mushroom and cacao (Hei at al. 2024; Lou et al. 2017; Morales-Belpaire et al. 2024). However, unlike monoculture plantation systems, the natural mixed-forest environment examined here is endowed with higher ecological stability and species diversity (Qian et al. 2025), 15 years of continuous ginseng cultivation was enough to push the system past its ecological tipping point. Consequently, the management of land cultivated for over 10 years must focus on countering acidification with amendments like lime and biochar, and correcting nitrogen imbalance via nitrate-based fertilizer application (Hao et al. 2022; Zhang et al. 2025b).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study demonstrates that continuous ginseng cultivation under a natural forest canopy leads to a progressive decline in soil faunal diversity and a structural simplification of the community. Crucially, we identified a dynamic succession in the primary environmental drivers governing these communities: early-stage assemblages were primarily limited by soil moisture, which transitioned to control by NH₄⁺-N and pH at the mid-stage, and finally to severe NO₃⁻-N limitation under long-term cultivation. This successional pattern, driven by progressive soil acidification, reveals that pH governs the community structure indirectly by regulating the availability of different nitrogen forms. However, this study primarily focused on the overall community response. Future research should delve into the specific responses of key functional groups (e.g., predators, decomposers) and their cascading effects on ecosystem processes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEC \u0026nbsp; \u0026nbsp;Electric conductivity\u003c/p\u003e\n\u003cp\u003eSOC \u0026nbsp; \u0026nbsp;Soil organic carbon\u003c/p\u003e\n\u003cp\u003eTN \u0026nbsp; \u0026nbsp;Total nitrogen\u003c/p\u003e\n\u003cp\u003eTC \u0026nbsp; \u0026nbsp;Total carbon\u003c/p\u003e\n\u003cp\u003eNO₃⁻-N \u0026nbsp; \u0026nbsp;Nitrate nitrogen\u003c/p\u003e\n\u003cp\u003eNH₄⁺-N \u0026nbsp; \u0026nbsp;Ammonium nitrogen\u003c/p\u003e\n\u003cp\u003eSOM \u0026nbsp; \u0026nbsp;Soil organic matter\u003c/p\u003e\n\u003cp\u003eAOA \u0026nbsp; \u0026nbsp;Ammonium-Oxidizing Archaea\u003c/p\u003e\n\u003cp\u003eAOB \u0026nbsp; \u0026nbsp;Ammonium-Oxidizing Bacteria\u003c/p\u003e\n\u003cp\u003eAMF \u0026nbsp; \u0026nbsp;Arbuscular Mycorrhizal Fungi\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare that are relevant to the content of this article. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors would like to thank all those who assisted during the field work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Cheng Wang, Mengqi Jiang, Zhiwei Gu, Luxin Li, Tianyue Yang, Hongyu Zhao, Xiaoqiang Li and Weihua Dong. The first draft of the manuscript was written by Cheng Wang and Mengqi Jiang. The academic review and writing supervision were conducted by Xiaoqiang Li and Weihua Dong. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by the National Natural Science Foundation of China (41601263), the Science and Technology Development Program of Jilin Province (20220101188JC), Natural Science Foundation Projects of CCNU (CSJJ2023003GZR), Institute of Innovation Science and Technology, Changchun Normal University (YJYKF-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBalume I, Agumas B, Musyoki M, Marhan S, Cadisch G, Rasche F (2022) Potential proteolytic enzyme activities modulate archaeal and bacterial nitrifier abundance in soils differing in acidity and organic residue treatment. 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Sci Total Environ 918:170789. https://doi.org/10.1016/j.scitotenv.2024.170789\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Soil fauna, Forest-cultivated ginseng, Years of planting, Soil acidification","lastPublishedDoi":"10.21203/rs.3.rs-8213596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8213596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and aims\u003c/h2\u003e \u003cp\u003eContinuous cultivation in temperate \"ginseng-forest\" systems leads to progressive soil degradation, yet the response of soil fauna - a key bioindicator - to cultivation chronosequences remains poorly understood. This study investigates the response patterns of soil faunal communities to ginseng cultivation duration and identifies the key environmental drivers at different stages.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSelected forest-cultivated ginseng sites with cultivation durations of 5, 10, and 15 years in a temperate broad-leaved forest of Northeast China. Within plots of each duration, we systematically collected soil fauna and soil samples to analyze community composition, diversity, and physicochemical properties.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSoil fauna diversity and richness declined significantly with prolonged cultivation, accompanied by structural simplification and functional homogenization. Dominant taxa were mainly small-to-medium sized (300\u0026ndash;400 \u0026micro;m in length, 100\u0026ndash;200 \u0026micro;m in width). Redundancy analysis revealed a temporal shift in key environmental drivers: from soil moisture in 5-year habitats, to ammonium nitrogen and pH in 10-year habitats, and finally to nitrate nitrogen limitation in long-term (15-year) habitats. Prolonged cultivation also induced progressive soil acidification and nutrient depletion.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings underscore that soil faunal diversity serves as a sensitive indicator of cultivation-induced soil change and highlight the stage-specific environmental constraints in \"ginseng-forest\" ecosystems. This study provides a scientific basis for developing temporally tailored soil management strategies to enhance the sustainability of ginseng cultivation.\u003c/p\u003e","manuscriptTitle":"Soil faunal diversity as an indicator of Ginseng cultivation duration in Northeast China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 14:57:33","doi":"10.21203/rs.3.rs-8213596/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a952f2aa-1270-4311-83d8-39cd9c5bb29c","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T07:27:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 14:57:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8213596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8213596","identity":"rs-8213596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}