Higher soil organic carbon in diverse plant communities linked to sustained biomass production during short-term drought

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Verschoor, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9141925/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background and Aims Soil organic carbon (SOC) plays a central role in global carbon cycling and is a key component of nature-based climate solutions. However, climate change and biodiversity loss are interrelated threats that may jointly undermine SOC. While plant diversity is known to enhance ecosystem functioning, its potential to buffer SOC under climate extremes such as drought, remains poorly understood. This study investigates whether plants growing in mixtures can mitigate SOC losses under drought by maintaining biomass production and moderating soil microclimate conditions. Methods This study was conducted within a large-scale factorial grassland biodiversity and climate variability experiment (UU-BioCliVE) at Utrecht University, manipulating planted species richness (1, 4, 8, 12 species) and precipitation to impose drought. We used structural equation modeling (SEM) to understand how plant diversity alters and/or maintains SOC via aboveground biomass, soil temperature, and soil moisture under drought conditions. Results SOC was higher in soils under plant mixtures than under monocultures across both control and drought treatments. Under drought, SEM showed a positive association between aboveground biomass and SOC in mixtures, while soil microclimate variables were not related to SOC. Under control conditions, mixtures increased biomass and reduced soil temperature, but neither factor explained SOC. Conclusion Our findings show that plant diversity supports SOC retention under short-term drought by sustaining biomass production. This underscores the importance of conserving plant diversity to sustain carbon storage under future drought stress. Plant diversity Soil organic carbon Soil microclimate Aboveground biomass Drought stress Figures Figure 1 Figure 2 Figure 3 Introduction Soil organic carbon (SOC) is fundamental to ecosystem functioning and has become a key focus of climate mitigation efforts (Minasny et al. 2023; Amelung et al. 2020; Rumpel and Chabbi 2021). Globally, soils store more carbon (C) than the atmosphere and terrestrial vegetation combined, making even small changes in SOC highly consequential for the global C cycle (Lal 2004). However, SOC stocks are increasingly threatened by drought (Zhou et al. 2026), which reduces plant-derived C inputs, accelerates microbial decomposition, and exacerbates soil degradation (Schimel et al. 2007; Díaz-Martínez et al. 2024). Recent declines in biodiversity (IPBES 2019; Eichenberg et al. 2021) may even further compromise ecosystem C storage capacity (Weiskopf et al. 2024), as climate change and biodiversity loss are interconnected global challenges. Soil C content is determined by multiple interacting factors, including climate, soil properties, and vegetation characteristics (Jobbágy and Jackson 2000). Among these factors, plant diversity has been recognized as an important driver of SOC accumulation (Xu et al. 2020;Winterfeldt et al. 2026). Evidence from grassland biodiversity experiments suggests that more diverse communities store more C than monocultures by increasing organic matter inputs and reducing C losses (Fornara and Tilman 2008; De Deyn et al. 2011; Conti and Díaz 2013; Lange et al. 2015; Chen et al. 2018). These diversity effects on SOC often strengthen over time as greater and more continuous organic inputs accumulate and interact with microbial and mineral stabilization processes (Lange et al. 2023). Drought can modify these diversity effects on SOC by altering both plant productivity (Smith et al. 2024) and soil microclimatic conditions, ultimately influencing C inputs and decomposition (Isbell et al., 2015; Craven et al., 2016). Whether and how plant diversity regulates SOC under drought remains unclear, constraining our understanding of soil C dynamics under climate change. Plant diversity can influence SOC through two potential pathways: biomass production and regulation of the soil microclimate. First, diverse plant communities often produce more aboveground biomass and have higher standing root biomass than monocultures (Hooper et al. 2005; Tilman et al. 2014; Ravenek et al., 2014; Barry et al. 2019). Higher plant productivity increases both the quantity and continuity of C inputs to soil through leaf litter and root turnover, which are key determinants of SOC formation and storage in grassland systems (Fornara and Tilman 2008; Cong et al. 2014). The quality and quantity of these inputs influence both the rate of C incorporation into soil and its subsequent stabilization through microbial processing and mineral association (Cotrufo et al. 2013). Second, plant diversity can regulate soil microclimate through facilitative interactions among species (Wright et al. 2017). Higher diversity increases structural complexity, creating more dense and heterogeneous canopies that reduce soil temperature extremes and buffer moisture conditions (Beugnon et al. 2024; Schnabel et al., 2025). This happens via multiple mechanisms: greater canopy cover reduces solar radiation reaching the soil's surface, and litter accumulation insulates the soil and maintains soil moisture (Zavaleta et al. 2003; De Boeck et al. 2008). These microclimatic effects reduce evaporation rates and dampen both temperature and moisture fluctuations, creating more favorable conditions for SOC storage (Huang et al. 2024). These pathways, however, are not independent; biomass and microclimate can have synergistic effects on SOC. Greater biomass creates canopy cover and litter inputs, which cool and shade the soil surface, reduce evaporation, and maintain soil moisture (Guimarães-Steinicke et al. 2021; Huang et al. 2024). In turn, favorable microclimate conditions can further promote biomass production (De Boeck et al. 2008), creating a positive feedback loop that supports SOC storage. Understanding these interconnected pathways is essential for predicting how SOC will respond to both diversity loss and climate change effects such as drought. Drought typically suppresses plant productivity, reduces organic matter inputs through decreased litter production and root turnover, and alters microbial activity, ultimately leading to SOC losses (Schimel et al. 2007; Deng et al. 2021). The magnitude of these losses, however, can depend on plant community diversity. Grassland communities with higher species richness lose less biomass during drought and recover more rapidly following stress (Van Ruijven and Berendse 2010; Wagg et al. 2017; Kreyling et al. 2017). High-diversity communities are more likely to contain species that facilitate microclimate amelioration under stressful conditions by maintaining canopy cover and soil moisture (Wright et al. 2017). While plant diversity enhances SOC under ambient conditions, whether these diversity-driven gains persist under drought remains unclear. The relative importance of biomass production and soil microclimate regulation in governing SOC dynamics under water limitation has not been explicitly tested. Understanding these interactions is essential for assessing soil C vulnerability to climate extremes and the efficacy of diversity-based mitigation strategies (Bradford et al. 2016). Here, we study the relationship between plant diversity and SOC under drought by examining the dual roles of aboveground biomass production and microclimate regulation. We hypothesize that (H1) plant diversity increases SOC by (H2) biomass production and microclimate amelioration, and that (H3) plant diversity mitigates the adverse effects of drought on SOC. We test these hypotheses in a long-term grassland biodiversity and climate variability experiment using structural equation modeling (SEM). Materials and Methods Experimental design This study was conducted in the Utrecht University Biodiversity Climate Variability Experiment (UU BioCliVE) in the Netherlands (52 °13’ N, 5°29’ E), a large-scale common garden experiment designed to test the responses of grassland communities to future climate scenarios (see Hautier et al. 2024 for full experimental details). In short, the biodiversity experiment was established in 2017 and consists of a randomized block design with two blocks comprising 88 plots. Each plot is composed of four subplots for a total of 352 subplots. Subplots are containers measuring 1.2 m × 1 m × 1 m (l × w × h) and are filled with river sand and topsoil excavated from a nearby nature reserve in the Rammelwaard (52°10’ N, 6°11’ E). The soils used in this experiment are Sandy (90.98% Sand, 8.17%, Silt, 0.85% Clay). The site is characterized by a typical temperate grassland vegetation, namely Arrhenatheretum elatioris association. Six grass species ( Arrhenaterum elatius, Festuca rubra, Holcus lanatus, Poa trivialis, Anthoxanthum odoratum, Luzula campestris ) and six forb species ( Rumex acetosa, Ranunculus repens, Tragopogon pratensis, Veronica chamaedrys, Knautia arvensis, Origanum vulgare ) were sown in monocultures and mixtures of 4, 8, and 12 species. This includes monocultures of each of the 12 selected species, as well as 12 combinations of 4 species, 12 combinations of 8 species, and 8 plots with all 12 species per block. To maintain the intended diversity gradient, all plots were weeded manually three times a year (in March, June, and September). Data for the current study were collected in two of the precipitation treatments: historical control (control) and extreme treatment (drought). In the control treatment, precipitation is provided based on climatic records spanning 50 years (1920 to 1971) from the KNMI (Attema et al. 2014). In May and June 2023, we decreased rainfall events by 75% compared to the control in the extreme treatment, creating conditions representative of an extreme drought. During this time, the retractable roof automatically closes to prevent plots from unintentional watering. The roof remains otherwise open to allow for nearly natural conditions. To test the effect of plant diversity and the drought treatment on our hypothesized relationships, we here used data collected during spring 2023. Aboveground biomass We collected the aboveground plant biomass in July 2023 by harvesting two 12.5 cm by 50 cm clip strips in the inner 50 cm by 50 cm area of the plot at a height of 8 cm. The harvested biomass was then oven-dried at 70°C for 48 hours and weighed. Microclimate measurements Microclimate conditions were recorded using TOMST TMS-4 probes: soil temperature (°C) at -6 cm depth and soil water content (m³/m³) measured across the upper 10 cm of soil (Wild et al. 2019). The probes were positioned with the surface temperature sensor approximately 1 cm above the soil and placed at a consistent distance from both the watering system and plot edges. Sensors were placed facing north to prevent direct sunlight exposure. Data collection was automated using Lolly software (version 1.52) during the initial drought period from May 13 to June 30, 2023. In data cleaning, we removed extreme values likely to represent measurement errors, excluding temperature readings below -20°C or above 45°C and soil water content readings below 0.01 m³/m³. These values were validated with data from the nearby weather station (KNMI, De Bilt). Nighttime data (21:00 – 06:00) were also excluded, as the study focused on microclimate amelioration during warm periods. Soil water content was calculated from the raw moisture count using the TMS4 universal calibration. After data cleaning, we computed the mean per sampled subplot over the drought period. We did this for both soil temperature and soil water content, and used these mean values in all subsequent analyses. SOC measurements Soil samples were collected by taking five subsamples from the central 25 by 50 cm sampling area of each subplot with a soil auger (2.5 cm diameter) (Fig. S1). Samples were sieved (2mm) and stored at -16 °C until further analysis. The SOC for each soil sample was calculated by measuring total C content and correcting for inorganic C as follows. First, soil samples were oven-dried at 40°C for a minimum of 24 hours. After drying, ~1.5 grams of each sample was milled at 25 Hz for 30 seconds. Next, the total C content of the ground samples was determined using an elemental analyzer after combustion at 1,150 °C with a CHN Elemental Analyzer (FlashSmart, Interscience Instruments). For the CHN analysis, samples were weighed to approximately 40mg in 8 × 5 mm tin foil containers. The analysis employed both solid and liquid standards, including Atropine (4.84% N, 70.56% C), BBOT (6.51% N, 72.53% C), Acetanilide (10.36% N, 71.09% C), and Imidazole (41.15% N, 52.93% C). Solid standards were weighed to approximately 3 mg in 5 × 3.5 mm tin foil containers. For liquid standards, an Imidazole solution (1.215 g/100 ml) was prepared, and specific quantities (8, 12, 16, 20, or 25 µl) were added to 5 mg of Chromosorb W. Then, inorganic Concentration was measured by elemental analysis as described above, after removing organic C for 2.5 h at 550 °C in a furnace (SNOL 30/1100 LSF01). Finally, the organic C concentration was calculated by subtracting the inorganic C concentration from the total C concentration and expressed as (g kg -1 ). Data Analysis All statistical analyses were conducted in R version 4.4.0 (R Core Team 2024). For our analysis, we examined the effects of plant diversity by comparing all mixtures (4, 8, and 12 species) versus monocultures based on results from a first data exploration (Supplementary Information). First, we tested how SOC differed between the four diversity levels (1, 4, 8, and 12 species) and precipitation treatments (control vs drought) using a two-way ANOVA from the ‘car’ package (Fox and Weisberg 2019). Although a two-way ANOVA revealed a significant overall effect of diversity ( F 1,167 = 4.24, p = 0.007), no main drought effect ( F 1,163 = 0.61, p = 0.43) and no interaction between diversity × drought ( F 3,163 = 1.30, p = 0.28) were found (Table S1). Post-hoc comparisons using Tukey's HSD revealed that SOC was lower in monocultures, with a significant diversity effect observed between 1- and 4-species communities ( p = 0.006). In contrast, SOC did not differ among 4, 8, and 12 species mixtures (all p > 0.11) (Fig. S2, Table S2). Because the 4, 8, and 12 species mixtures did not differ from each other, we grouped them into a single “mixture” category for subsequent analyses. The experimental factor 'plant diversity' thus contained two levels: monoculture ( n = 24 per treatment; 48 total) and mixture ( n = 64 per treatment; 128 total). We then tested the effect of plant diversity (monoculture vs mixture) and precipitation treatment (control vs drought) on SOC using a two-way ANOVA including the diversity × drought interaction term (Fig. 2). We tested the hypothesized pathways by which plant diversity could drive SOC (Fig. 1C) with structural equation modelling (SEM). We first conducted individual bivariate analyses (Fig. S3-S4) to explore the relationships among plant diversity and drought to microclimate (soil moisture, soil temperature) and aboveground biomass, as well as microclimate and biomass to SOC, with Generalized Least Squares (GLS) models using the 'nlme' package (v3.1-168; (Pinheiro et al. 2025). The GLS approach allowed us to account for heteroskedasticity. Next, we conducted multigroup SEM using the ‘lavaan’ package (v0.6-7; Rosseel 2012) to compare pathways between drought and control conditions. These bivariate GLS models informed the SEM structure but are presented only in Fig. S3-S4; all path coefficients and significance levels reported in the Results are derived from the final multigroup SEM. Multigroup SEM analyses were conducted separately for control and drought treatments, with approximately 80-82 plots per treatment group after outlier removal. Outliers were identified as data points exceeding two standard deviations from the mean for each response variable (within the SEM) and removed during diagnostic assessment. The SEM structure was based on the hypothesized relationships outlined in the introduction (Fig. 1C). The output summarized the modeled pathways, identifying both predefined relationships and significant pathways that influenced SOC under control and drought conditions. Model fit was evaluated based on χ² ( p > 0.05), CFI > 0.90, RMSEA < 0.08, and SRMR < 0.08, all of which indicated acceptable model fit. Standardized path coefficients (Std.all) and associated p -values were reported for each treatment group to compare the strength and significance of pathways between control and drought conditions. Results Mixtures have higher SOC regardless of drought treatment SOC was consistently higher in mixtures, regardless of the drought treatment ( F 1,167 = 0.48, p = 0.49, Table S3). Compared to monocultures, SOC in mixtures was ~10% higher overall ( F 1,167 = 8.90, p = 0.003; Fig. 2). In contrast, SOC did not differ between control and drought treatments ( F 1,167 = 0.13, p = 0.72). Overall, the model explained ~4% of the variation in SOC (adjusted R² = 0.04). Mixtures boost biomass and cool soils both directly and via biomass. These effects do not translate into SOC gains under control conditions. Under control conditions, increasing planted species richness significantly increased aboveground biomass ( p < 0.001, df = 80, std. est. = 0.57) and reduced soil temperature ( p = 0.006, df = 80, std. est. = −0.33). Biomass also contributed to cooler soils, although this effect was weaker ( p = 0.024, df = 80, std. est. = −0.27). In contrast, neither richness nor biomass influenced soil moisture ( p = 0.23–0.37). Despite these strong effects on biomass and microclimate, we found no evidence that richness, biomass, or microclimate variables were associated with SOC under control conditions (all p > 0.10). Overall, the model explained 32% of the variance in biomass and 28% of the variance in soil temperature, but only 8% of the variance in SOC (Fig. 3a). Mixtures boost biomass and cool soils while also slightly drying soils. Biomass increases SOC under drought conditions. Our SEM analysis showed that in drought conditions, mixtures had higher aboveground biomass ( p < 0.001, df = 82, std. est = 0.461). Mixtures had cooler and drier soils ( p = 0.002, df = 82, std. est. = -0.373) ( p = 0.036, df = 82, std. est. = -0.256). These microclimate effects, however, did not appear to be driven by the accumulation of aboveground biomass in the drought treatment ( p = 0.780, p = 0.295). We did not find a direct relationship between planted species richness and SOC ( p = 0.340). Similarly, SOC did not appear to be linked to changes in microclimate ( p = 0.463, p = 0.915). However, increased plant diversity increased aboveground biomass, which in turn increased SOC under drought ( p = 0.006, df = 82, std. est = 0.319). The model explained 21% of the variance in biomass, 15% of soil temperature, 11% of soil moisture, and 14% of SOC (Fig. 3b). Discussion Understanding whether plant diversity can sustain SOC under increasing drought stress represents a critical next step in biodiversity–ecosystem functioning research, particularly given the central role of SOC in climate regulation and soil functioning. Here, we examined whether plant diversity promotes SOC and whether this relationship is maintained under short-term drought through effects on aboveground biomass production and soil microclimate regulation. We found that mixtures stored on average 10% more SOC than monocultures, regardless of drought conditions. This is consistent with our first hypothesis and aligns with the results of previous biodiversity ecosystem functioning studies (Lange et al. 2015; Chen et al. 2018; Spohn et al. 2023). However, contrary to our expectation that drought would strengthen diversity effects, we found no diversity × drought interaction; mixtures maintained higher SOC regardless of treatment. Our SEM revealed that under control conditions, species richness increased aboveground biomass and decreased soil temperature both directly and indirectly through biomass. However, these effects did not translate into higher SOC. In contrast, under drought, plant diversity was linked to decreased soil temperature and moisture, as well as increased aboveground biomass. Aboveground biomass, in turn, was positively related to SOC in the drought treatment. Our findings suggest that the proposed role of aboveground biomass in supporting soil C is particularly important under environmental stress. Soil C levels are higher in mixtures Plant diversity has been shown to increase SOC across a wide range of experiments from temperate grasslands such as ours, to tropical forests (Schnabel et al. 2025). This biodiversity effect has largely been attributed to increased organic matter inputs driven by higher biomass production and, in other studies, root turnover in more diverse plant communities (Fornara and Tilman 2008; Mueller et al. 2013). Previous studies documenting this biodiversity-SOC relationship measured this effect in older experiments. For example, Lange et al. (2015) observed these patterns in the Jena Experiment after approximately 10–11 years. In our experiment, SOC levels were ~10% higher in diverse mixtures than in monocultures after only 7 years. Mixtures in BioCliVE produced substantially more aboveground biomass than monocultures under both control and drought conditions, indicating greater potential C inputs to soil through litter production. Moreover, under drought, aboveground biomass was positively associated with SOC, providing evidence that biomass inputs contributed to SOC maintenance under stress. These results reinforce the view that species richness is a key driver of C storage in grassland systems and that these benefits start accumulating early. Plant diversity-driven SOC gains remain intact under drought Former studies have shown that drought can have differing effects on SOC depending on ecosystem type and drought intensity (Deng et al. 2021; Shi et al. 2024). Drought can suppress microbial activity, reduce productivity, and decrease organic matter inputs to soil, processes that may lead to decreased SOC (Schimel et al. 2007; Oram et al. 2023). Contrary to this expectation, we did not observe SOC declines under drought, and mixtures consistently maintained higher SOC than monocultures across treatments. This pattern suggests that plant diversity may act as a buffer against short-term stress, maintaining soil C even when water availability is reduced. The relatively short duration of our drought treatment (< 2 months) likely limited the potential for detectable SOC losses. Since this study focused on a single drought event, reoccurring drought events of varying intensities could produce more compounded effects not captured in our experiment. Established soil C pools are often resistant to brief disturbances, particularly in sandy soils with inherently low SOC concentrations (Yost and Hartemink 2019). Nevertheless, this temporal scale provides insight into the early-stage responses of soil C dynamics to climatic stress. The persistence of higher SOC in mixtures during this initial drought period suggests that diversity-driven C gains are not immediately reversed under short-term stress. Other studies have similarly shown that ecosystem responses to drought can lag or require cumulative stress across years. For instance, Frank et al. (2015) showed that C cycle responses to climate extremes may manifest only after repeated events, and Hoover and Rogers (2016) reported progressive declines in C inputs following recurrent drought episodes. While our short-term drought provided insights into initial ecosystem response, further research over extended temporal scales is crucial for capturing the cumulative effects of repeated or prolonged drought events on SOC dynamics. Our SEM, however, revealed a significant positive pathway from aboveground biomass to SOC under drought conditions. By maintaining higher biomass than monocultures under drought, plant diversity likely contributed to sustaining SOC and preventing potential C losses. Similar mechanisms have been reported in other studies, where diverse communities maintained ecosystem functioning and buffered the effects of environmental stress (Isbell et al. 2015; Craven et al. 2016; Wagg et al. 2017). Plant diversity increases biomass and buffers temperature but reduces soil moisture under drought Consistent with previous BEF research (Van Ruijven and Berendse 2005; Isbell et al. 2011; Cardinale et al. 2012; Tilman et al. 2014; Wright et al. 2015; Wright et al. 2021), our findings confirm that plant diversity significantly increases aboveground biomass. Under both control and drought conditions, plant mixtures produced substantially more biomass compared to monocultures. Under control conditions, plant diversity reduced soil temperature both directly and indirectly through aboveground biomass, although the biomass effect was weaker. Under drought, plant diversity directly reduced soil temperature, but the indirect pathway via biomass was no longer significant. This aligns with reported observations that apart from the increase in biomass, the greater trait diversity and structural complexity associated with diverse plant communities can buffer microclimates by shading the soil surface and trapping humidity (Wright and Francia 2024; Schnabel et al. 2025). Based on observed soil cooling effects, one would expect plant diversity to increase soil moisture by reducing evaporation. Instead, we observed the opposite pattern: plant mixtures had lower soil moisture than monocultures under drought. This result suggests that diverse communities utilize available soil water more completely, as greater total plant biomass and complementary rooting strategies enable more thorough water extraction from the soil profile (Maestre et al. 2009). Aboveground mechanisms become significant under drought While mixtures had higher SOC than monocultures overall (Fig. 2), the SEM revealed that the mechanisms underlying this effect differed between control and drought conditions. Under control conditions, plant diversity increased biomass and reduced soil temperature (both directly and indirectly via biomass), but neither pathway translated into higher SOC. Under drought, plant diversity increased biomass, which in turn positively influenced SOC, a pathway absent under control conditions. Aboveground processes alone were insufficient to explain SOC variation under control conditions but became important during drought. This indicates that under environmental stress, aboveground productivity becomes a key short-term determinant of soil C dynamics. Over longer time scales, consistent aboveground inputs should accumulate, potentially leading to more pronounced increases in SOC (Bardgett and van der Putten 2014). Belowground processes are also fundamental to how plant diversity influences SOC. Root-derived C contributes disproportionately to SOC formation through direct soil entry and coupling with microbial and mineral interactions (De Deyn et al. 2008; Lange et al. 2015; Kravchenko et al. 2021), with plant diversity influencing these rhizosphere processes (Bardgett et al. 2014). Future studies should therefore integrate belowground processes and repeated drought events to determine whether plant diversity can sustain soil C storage under increasingly variable climatic conditions. Conclusion Our study shows that diversity-driven SOC gains are maintained under short-term drought. After seven years, mixtures maintained approximately 10% higher SOC than monocultures regardless of water availability, and, under drought, this was driven indirectly by sustained aboveground biomass production. This suggests that the role of productivity in maintaining soil C becomes particularly important when ecosystems face stress. As drought events intensify and become more frequent with climate change, maintaining diverse plant communities may be critical for preserving soil C stocks and the ecosystem services they support. Our findings highlight the importance of long-term experiments that integrate multiple stressors and examine both above- and belowground C pathways, particularly as the mechanisms driving SOC accumulation under ambient conditions remain unclear. Abbreviations SOC soil organic carbon SEM structural equation model C carbon Declarations Author contributions: Alya Kingsland-Mengi : conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing – original draft, writing – review & editing. Julia Mayr: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing – original draft, writing – review & editing. Yuheng Chen: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing – original draft, writing – review & editing. Mink R. Verschoor: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing – original draft, writing – review & editing. Diana Hoekstra: data curation, methodology. Nehizena Osagie: data curation, methodology. Rola E. Johannes: methodology, project administration, resources. Betty P. Verduyn: methodology, project administration, resources. Peter Veenhuizen: methodology, project administration, resources. Janna M. Barel: methodology, writing – review & editing. Yann Hautier: conceptualization, funding acquisition, investigation, methodology, writing – review & editing. George A. Kowalchuk: conceptualization, funding acquisition, investigation, methodology, writing – review & editing. Kathryn E. Barry: conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, resources, writing – original draft, writing – review & editing. All authors read and approved the final version of the manuscript. Acknowledgements : We are deeply grateful to all the field workers of the BIOCLIVE whose efforts made this experiment and its extensive measurements possible. The BIOCLIVE experiment is supported by the Ecology & Biodiversity Group, the Department of Biology of Utrecht University, and the Utrecht University Fund. We extend our sincere thanks to Ali Miedema and Laurens Gaarenstroom for their invaluable contributions. We are also grateful for the financial support from the trusts and foundations associated with the Utrecht University Fund: K.F. Hein Fonds, WNF INNO-Fonds, Ars Donandi, Stichting Thurkowfonds, and the M.A.O.C. Gravin van Bylandt Stichting. Alya Kingsland-Mengi was funded by the Startersbeurs Barry 2022 Grant (BB.000725.1). Conflicts of Interest : The authors declare no conflicts of interest. Data Availability Statement : The data and code that support the findings of this study are openly available in Zenodo at https://zenodo.org/records/19056618. References Amelung, W., Bossio, D., de Vries, W., Kögel-Knabner, I., Lehmann, J., Amundson, R., Bol, R., Collins, C., Lal, R., Leifeld, J., Minasny, B., Pan, G., Paustian, K., Rumpel, C., Sanderman, J., van Groenigen, J. W., Mooney, S., van Wesemael, B., Wander, M., & Chabbi, A. (2020). Towards a global-scale soil climate mitigation strategy. Nature Communications , 11 (1), 5427. https://doi.org/10.1038/s41467-020-18887-7 Attema, J. 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Barry","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kathryn","middleName":"E.","lastName":"Barry","suffix":""}],"badges":[],"createdAt":"2026-03-16 21:40:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9141925/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9141925/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106960551,"identity":"23d84e2b-dd44-4ea2-bd03-0d0af4d2f0b5","added_by":"auto","created_at":"2026-04-15 09:21:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222365,"visible":true,"origin":"","legend":"\u003cp\u003eConceptual framework for plant diversity effects on SOC under ambient and drought conditions.\u003cstrong\u003e \u003c/strong\u003eHigher plant diversity is expected to increase aboveground biomass production and improve the microclimate by buffering soil temperature and conserving soil moisture (a). These two mechanisms, biomass inputs and microclimatic stabilization, jointly contribute to SOC accumulation by increasing organic matter inputs and supporting processes that stabilize C in the soil (c). Under drought conditions, plant diversity ameliorates SOC reductions (b). In (c), arrows represent hypothesized relationships (solid = positive; dashed = negative) between plant diversity, the mediating factors, and SOC\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9141925/v1/5319429a2b9c6adbbcce56d2.jpg"},{"id":106772198,"identity":"195dfb40-4db1-42b1-97a2-e46ffa309541","added_by":"auto","created_at":"2026-04-13 10:21:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":196783,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of mixture and monoculture on SOC under drought and control conditions. Boxplots represent the distribution of SOC values, with boxes showing the interquartile range, horizontal lines indicating median values, and whiskers extending to 1.5× the interquartile range. Open circles represent individual plot measurements. Different letters indicate significant differences between treatments based on Tukey’s HSD post-hoc test (α = 0.05)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9141925/v1/54b6958cf35cd90857c71a47.jpg"},{"id":106772199,"identity":"d6a43c3d-2004-4455-b969-87507eeae2e1","added_by":"auto","created_at":"2026-04-13 10:21:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66233,"visible":true,"origin":"","legend":"\u003cp\u003eStructural equation model (SEM) of plant diversity, aboveground biomass, microclimate (soil temperature and soil moisture), and soil organic carbon (SOC) under control (a) and drought (b) conditions. Solid arrows indicate positive paths, and dashed arrows indicate negative paths. Only paths with \u003cem\u003ep\u003c/em\u003e \u0026lt;0.1 are shown with asterisks denoting significance levels (* = \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** = \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, *** = \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001). Global model fit indices: χ² = 3.79, \u003cem\u003ep\u003c/em\u003e = 0.15; CFI = 0.95; RMSEA = 0.11; SRMR = 0.03\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9141925/v1/f51aaeb4b6cc6ebe93634b76.jpg"},{"id":106963143,"identity":"62bcaa19-d9ba-44ab-93ae-fe0ee558825a","added_by":"auto","created_at":"2026-04-15 09:42:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1433211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9141925/v1/99fad9dc-f01a-4793-9548-525e3f902309.pdf"},{"id":106772196,"identity":"a06feb1b-e0a5-4502-87e6-e02c1c9f6fa5","added_by":"auto","created_at":"2026-04-13 10:21:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1318149,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-9141925/v1/7bc8ed1e6143ebb5c8a23eaf.docx"}],"financialInterests":"","formattedTitle":"Higher soil organic carbon in diverse plant communities linked to sustained biomass production during short-term drought","fulltext":[{"header":"Introduction ","content":"\u003cp\u003eSoil organic carbon (SOC) is fundamental to ecosystem functioning and has become a key focus of climate mitigation efforts (Minasny et al. 2023; Amelung et al. 2020; Rumpel and Chabbi 2021). Globally, soils store more carbon (C) than the atmosphere and terrestrial vegetation combined, making even small changes in SOC highly consequential for the global C cycle (Lal 2004). However, SOC stocks are increasingly threatened by drought (Zhou et al. 2026), which reduces plant-derived C inputs, accelerates microbial decomposition, and exacerbates soil degradation (Schimel et al. 2007; D\u0026iacute;az-Mart\u0026iacute;nez et al. 2024). Recent declines in biodiversity (IPBES 2019; Eichenberg et al. 2021) may even further compromise ecosystem C storage capacity (Weiskopf et al. 2024), as climate change and biodiversity loss are interconnected global challenges.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSoil C content is determined by multiple interacting factors, including climate, soil properties, and vegetation characteristics (Jobb\u0026aacute;gy and Jackson 2000). Among these factors, plant diversity has been recognized as an important driver of SOC accumulation (Xu et al. 2020;Winterfeldt et al. 2026). Evidence from grassland biodiversity experiments suggests that more diverse communities store more C than monocultures by increasing organic matter inputs and reducing C losses (Fornara and Tilman 2008; De Deyn et al. 2011; Conti and D\u0026iacute;az 2013; Lange et al. 2015; Chen et al. 2018). These diversity effects on SOC often strengthen over time as greater and more continuous organic inputs accumulate and interact with microbial and mineral stabilization processes (Lange et al. 2023). Drought can modify these diversity effects on SOC by altering both plant productivity (Smith et al. 2024) and soil microclimatic conditions, ultimately influencing C inputs and decomposition (Isbell et al., 2015; Craven et al., 2016). Whether and how plant diversity regulates SOC under drought remains unclear, constraining our understanding of soil C dynamics under climate change.\u003c/p\u003e\n\u003cp\u003ePlant diversity can influence SOC through two potential pathways: biomass production and regulation of the soil microclimate. First, diverse plant communities often produce more aboveground biomass and have higher standing root biomass than monocultures (Hooper et al. 2005; Tilman et al. 2014; Ravenek et al., 2014; Barry et al. 2019). Higher plant productivity increases both the quantity and continuity of C inputs to soil through leaf litter and root turnover, which are key determinants of SOC formation and storage in grassland systems (Fornara and Tilman 2008; Cong et al. 2014). The quality and quantity of these inputs influence both the rate of C incorporation into soil and its subsequent stabilization through microbial processing and mineral association (Cotrufo et al. 2013). Second, plant diversity can regulate soil microclimate through facilitative interactions among species (Wright et al. 2017). Higher diversity increases structural complexity, creating more dense and heterogeneous canopies that reduce soil temperature extremes and buffer moisture conditions (Beugnon et al. 2024; Schnabel et al., 2025). This happens via multiple mechanisms: greater canopy cover reduces solar radiation reaching the soil\u0026apos;s surface, and litter accumulation insulates the soil and maintains soil moisture (Zavaleta et al. 2003; De Boeck et al. 2008). These microclimatic effects reduce evaporation rates and dampen both temperature and moisture fluctuations, creating more favorable conditions for SOC storage (Huang et al. 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese pathways, however, are not independent; biomass and microclimate can have synergistic effects on SOC. Greater biomass creates canopy cover and litter inputs, which cool and shade the soil surface, reduce evaporation, and maintain soil moisture (Guimar\u0026atilde;es-Steinicke et al. 2021; Huang et al. 2024). In turn, favorable microclimate conditions can further promote biomass production (De Boeck et al. 2008), creating a positive feedback loop that supports SOC storage. Understanding these interconnected pathways is essential for predicting how SOC will respond to both diversity loss and climate change effects such as drought.\u003c/p\u003e\n\u003cp\u003eDrought typically suppresses plant productivity, reduces organic matter inputs through decreased litter production and root turnover, and alters microbial activity, ultimately leading to SOC losses (Schimel et al. 2007; Deng et al. 2021). The magnitude of these losses, however, can depend on plant community diversity. Grassland communities with higher species richness lose less biomass during drought and recover more rapidly following stress (Van Ruijven and Berendse 2010; Wagg et al. 2017; Kreyling et al. 2017). High-diversity communities are more likely to contain species that facilitate microclimate amelioration under stressful conditions by maintaining canopy cover and soil moisture (Wright et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile plant diversity enhances SOC under ambient conditions, whether these diversity-driven gains persist under drought remains unclear. The relative importance of biomass production and soil microclimate regulation in governing SOC dynamics under water limitation has not been explicitly tested. Understanding these interactions is essential for assessing soil C vulnerability to climate extremes and the efficacy of diversity-based mitigation strategies (Bradford et al. 2016). Here, we study the relationship between plant diversity and SOC under drought by examining the dual roles of aboveground biomass production and microclimate regulation. We hypothesize that (H1) plant diversity increases SOC by (H2) biomass production and microclimate amelioration, and that (H3) plant diversity mitigates the adverse effects of drought on SOC. We test these hypotheses in a long-term grassland biodiversity and climate variability experiment using structural equation modeling (SEM).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in the Utrecht University Biodiversity Climate Variability Experiment (UU BioCliVE) in the Netherlands (52 \u0026deg;13\u0026rsquo; N, 5\u0026deg;29\u0026rsquo; E), a large-scale common garden experiment designed to test the responses of grassland communities to future climate scenarios (see Hautier et al. 2024 for full experimental details). In short, the biodiversity experiment was established in 2017 and consists of a randomized block design with two blocks comprising 88 plots. Each plot is composed of four subplots for a total of 352 subplots. Subplots are containers measuring 1.2 m \u0026times; 1 m \u0026times; 1 m (l \u0026times; w \u0026times; h) and are filled with river sand and topsoil excavated from a nearby nature reserve in the Rammelwaard (52\u0026deg;10\u0026rsquo; N, 6\u0026deg;11\u0026rsquo; E). The soils used in this experiment are Sandy (90.98% Sand, 8.17%, Silt, 0.85% Clay). The site is characterized by a typical temperate grassland vegetation, namely Arrhenatheretum elatioris association. Six grass species (\u003cem\u003eArrhenaterum\u0026nbsp;elatius, Festuca\u0026nbsp;rubra, Holcus\u0026nbsp;lanatus, Poa\u0026nbsp;trivialis, Anthoxanthum\u0026nbsp;odoratum, Luzula\u0026nbsp;campestris\u003c/em\u003e) and six forb species (\u003cem\u003eRumex acetosa, Ranunculus repens, Tragopogon pratensis, Veronica chamaedrys, Knautia arvensis, Origanum vulgare\u003c/em\u003e) were sown in monocultures and mixtures of 4, 8, and 12 species. This includes monocultures of each of the 12 selected species, as well as 12 combinations of 4 species, 12 combinations of 8 species, and 8 plots with all 12 species per block. To maintain the intended diversity gradient, all plots were weeded manually three times a year (in March, June, and September).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData for the current study were collected in two of the precipitation treatments: historical control (control) and extreme treatment (drought). In the control treatment, precipitation is provided based on climatic records spanning 50 years (1920 to 1971) from the KNMI (Attema et al. 2014). In May and June 2023, we decreased rainfall events by 75% compared to the control in the extreme treatment, creating conditions representative of an extreme drought. During this time,\u0026nbsp;the retractable roof automatically closes to prevent plots from unintentional watering. The roof remains otherwise open to allow for nearly natural conditions.\u0026nbsp;To test the effect of plant diversity and the drought treatment on our hypothesized relationships, we here used data collected during spring 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAboveground biomass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe collected the aboveground plant biomass in July 2023 by harvesting two 12.5 cm by 50 cm clip strips in the inner 50 cm by 50 cm area of the plot at a height of 8 cm. The harvested biomass was then oven-dried at 70\u0026deg;C for 48 hours and weighed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroclimate measurements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroclimate conditions were recorded using TOMST TMS-4 probes: soil temperature (\u0026deg;C) at -6 cm depth and soil water content (m\u0026sup3;/m\u0026sup3;) measured across the upper 10 cm of soil (Wild et al. 2019). The probes were positioned with the surface temperature sensor approximately 1 cm above the soil and placed at a consistent distance from both the watering system and plot edges. Sensors were placed facing north to prevent direct sunlight exposure. Data collection was automated using Lolly software (version 1.52) during the initial drought period from May 13 to June 30, 2023.\u003c/p\u003e\n\u003cp\u003eIn data cleaning, we removed extreme values likely to represent measurement errors, excluding temperature readings below -20\u0026deg;C or above 45\u0026deg;C and soil water content readings below 0.01 m\u0026sup3;/m\u0026sup3;. These values were validated with data from the nearby weather station (KNMI, De Bilt). Nighttime data (21:00 \u0026ndash; 06:00) were also excluded, as the study focused on microclimate amelioration during warm periods. Soil water content was calculated from the raw moisture count using the TMS4 universal calibration. After data cleaning, we computed the mean per sampled subplot over the drought period. We did this for both soil temperature and soil water content, and used these mean values in all subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOC measurements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil samples were collected by taking five subsamples from the central 25 by 50 cm sampling area of each subplot with a soil auger (2.5 cm diameter) (Fig. S1). Samples were sieved (2mm) and stored at -16 \u0026deg;C until further analysis.\u003c/p\u003e\n\u003cp\u003eThe SOC for each soil sample was calculated by measuring total C content and correcting for inorganic C as follows. First, soil samples were oven-dried at 40\u0026deg;C for a minimum of 24 hours. After drying, ~1.5 grams of each sample was milled at 25 Hz for 30 seconds. Next, the total C content of the ground samples was determined using an elemental analyzer after combustion at 1,150\u0026thinsp;\u0026deg;C with a CHN Elemental Analyzer (FlashSmart, Interscience Instruments). For the CHN analysis, samples were weighed to approximately 40mg in 8 \u0026times; 5 mm tin foil containers. The analysis employed both solid and liquid standards, including Atropine (4.84% N, 70.56% C), BBOT (6.51% N, 72.53% C), Acetanilide (10.36% N, 71.09% C), and Imidazole (41.15% N, 52.93% C). Solid standards were weighed to approximately 3 mg in 5 \u0026times; 3.5 mm tin foil containers. For liquid standards, an Imidazole solution (1.215 g/100 ml) was prepared, and specific quantities (8, 12, 16, 20, or 25 \u0026micro;l) were added to 5 mg of Chromosorb W. Then, inorganic Concentration was measured by elemental analysis as described above, after removing organic C for 2.5\u0026thinsp;h at 550\u0026thinsp;\u0026deg;C in a furnace (SNOL 30/1100 LSF01). Finally, the organic C concentration was calculated by subtracting the inorganic C concentration from the total C concentration and expressed as (g kg\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted in R version 4.4.0 (R Core Team 2024). For our analysis, we examined the effects of plant diversity by comparing all mixtures (4, 8, and 12 species) versus monocultures based on results from a first data exploration (Supplementary Information). First, we tested how SOC differed between the four diversity levels (1, 4, 8, and 12 species) and precipitation treatments (control vs drought) using a two-way ANOVA from the \u0026lsquo;car\u0026rsquo; package (Fox and Weisberg 2019). Although a two-way ANOVA revealed a significant overall effect of diversity (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,167\u0026nbsp;\u003c/sub\u003e= 4.24, \u003cem\u003ep\u003c/em\u003e = 0.007), no main drought effect (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,163\u003c/sub\u003e = 0.61, \u003cem\u003ep\u003c/em\u003e = 0.43) and no interaction between diversity \u0026times; drought (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e3,163\u003c/sub\u003e = 1.30, \u003cem\u003ep\u003c/em\u003e = 0.28) were found (Table S1). Post-hoc comparisons using Tukey\u0026apos;s HSD revealed that SOC was lower in monocultures, with a significant diversity effect observed between 1- and 4-species communities (\u003cem\u003ep\u003c/em\u003e = 0.006). In contrast, SOC did not differ among 4, 8, and 12 species mixtures (all \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.11) (Fig. S2, Table S2). Because the 4, 8, and 12 species mixtures did not differ from each other, we grouped them into a single \u0026ldquo;mixture\u0026rdquo; category for subsequent analyses. The experimental factor \u0026apos;plant diversity\u0026apos; thus contained two levels: monoculture (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 24 per treatment; 48 total) and mixture (\u003cem\u003en\u003c/em\u003e = 64 per treatment; 128 total). We then tested the effect of plant diversity (monoculture vs mixture) and precipitation treatment (control vs drought) on SOC using a two-way ANOVA including the diversity \u0026times; drought interaction term (Fig. 2).\u003c/p\u003e\n\u003cp\u003eWe tested the hypothesized pathways by which plant diversity could drive SOC (Fig. 1C) with structural equation modelling (SEM). We first conducted individual bivariate analyses (Fig. S3-S4) to explore the relationships among plant diversity and drought to microclimate (soil moisture, soil temperature) and aboveground biomass, as well as microclimate and biomass to SOC, with Generalized Least Squares (GLS) models using the \u0026apos;nlme\u0026apos; package (v3.1-168; (Pinheiro et al. 2025). The GLS approach allowed us to account for heteroskedasticity. Next, we conducted multigroup SEM using the \u0026lsquo;lavaan\u0026rsquo; package (v0.6-7; Rosseel 2012) to compare pathways between drought and control conditions. These bivariate GLS models informed the SEM structure but are presented only in Fig. S3-S4; all path coefficients and significance levels reported in the Results are derived from the final multigroup SEM. Multigroup SEM analyses were conducted separately for control and drought treatments, with approximately 80-82 plots per treatment group after outlier removal. Outliers were identified as data points exceeding two standard deviations from the mean for each response variable (within the SEM) and removed during diagnostic assessment. The SEM structure was based on the hypothesized relationships outlined in the introduction (Fig. 1C). The output summarized the modeled pathways, identifying both predefined relationships and significant pathways that influenced SOC under control and drought conditions. Model fit was evaluated based on \u0026chi;\u0026sup2; (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), CFI \u0026gt; 0.90, RMSEA \u0026lt; 0.08, and SRMR \u0026lt; 0.08, all of which indicated acceptable model fit. Standardized path coefficients (Std.all) and associated \u003cem\u003ep\u003c/em\u003e-values were reported for each treatment group to compare the strength and significance of pathways between control and drought conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMixtures have higher SOC regardless of drought treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOC was consistently higher in mixtures, regardless of the drought treatment (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,167\u003c/sub\u003e = 0.48, \u003cem\u003ep\u003c/em\u003e = 0.49, Table S3). Compared to monocultures, SOC in mixtures was ~10% higher overall (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,167\u003c/sub\u003e = 8.90, \u003cem\u003ep\u003c/em\u003e = 0.003; Fig. 2). In contrast, SOC did not differ between control and drought treatments (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e1,167\u0026nbsp;\u003c/sub\u003e= 0.13, \u003cem\u003ep\u003c/em\u003e = 0.72). Overall, the model explained ~4% of the variation in SOC (adjusted R\u0026sup2; = 0.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMixtures boost biomass and cool soils both directly and via biomass. These effects do not translate into SOC gains under control conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder control conditions, increasing planted species richness significantly increased aboveground biomass (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, df = 80, std. est. = 0.57) and reduced soil temperature (\u003cem\u003ep\u003c/em\u003e = 0.006, df = 80, std. est. = \u0026minus;0.33). Biomass also contributed to cooler soils, although this effect was weaker (\u003cem\u003ep\u003c/em\u003e = 0.024, df = 80, std. est. = \u0026minus;0.27). In contrast, neither richness nor biomass influenced soil moisture (\u003cem\u003ep\u003c/em\u003e = 0.23\u0026ndash;0.37). Despite these strong effects on biomass and microclimate, we found no evidence that richness, biomass, or microclimate variables were associated with SOC under control conditions (all \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.10). Overall, the model explained 32% of the variance in biomass and 28% of the variance in soil temperature, but only 8% of the variance in SOC (Fig. 3a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMixtures boost biomass and cool soils while also slightly drying soils. Biomass increases SOC under drought conditions.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur SEM analysis showed that in drought conditions, mixtures had higher aboveground biomass (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, df = 82, std. est = 0.461). Mixtures had cooler and drier soils (\u003cem\u003ep\u003c/em\u003e = 0.002, df = 82, std. est. = -0.373) (\u003cem\u003ep\u003c/em\u003e = 0.036, df = 82, std. est. = -0.256). These microclimate effects, however, did not appear to be driven by the accumulation of aboveground biomass in the drought treatment (\u003cem\u003ep\u003c/em\u003e = 0.780, \u003cem\u003ep\u003c/em\u003e = 0.295). We did not find a direct relationship between planted species richness and SOC (\u003cem\u003ep\u003c/em\u003e = 0.340). Similarly, SOC did not appear to be linked to changes in microclimate (\u003cem\u003ep\u003c/em\u003e = 0.463, \u003cem\u003ep\u003c/em\u003e = 0.915). However, increased plant diversity increased aboveground biomass, which in turn increased SOC under drought (\u003cem\u003ep\u003c/em\u003e = 0.006, df = 82, std. est = 0.319). The model explained 21% of the variance in biomass, 15% of soil temperature, 11% of soil moisture, and 14% of SOC (Fig. 3b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding whether plant diversity can sustain SOC under increasing drought stress represents a critical next step in biodiversity\u0026ndash;ecosystem functioning research, particularly given the central role of SOC in climate regulation and soil functioning. Here, we examined whether plant diversity promotes SOC and whether this relationship is maintained under short-term drought through effects on aboveground biomass production and soil microclimate regulation. We found that mixtures stored on average 10% more SOC than monocultures, regardless of drought conditions. This is consistent with our first hypothesis and aligns with the results of previous biodiversity ecosystem functioning studies (Lange et al. 2015; Chen et al. 2018; Spohn et al. 2023). However, contrary to our expectation that drought would strengthen diversity effects, we found no diversity \u0026times; drought interaction; mixtures maintained higher SOC regardless of treatment. Our SEM revealed that under control conditions, species richness increased aboveground biomass and decreased soil temperature both directly and indirectly through biomass. However, these effects did not translate into higher SOC. In contrast, under drought, plant diversity was linked to decreased soil temperature and moisture, as well as increased aboveground biomass. Aboveground biomass, in turn, was positively related to SOC in the drought treatment. Our findings suggest that the proposed role of aboveground biomass in supporting soil C is particularly important under environmental stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil C levels are higher in mixtures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant diversity has been shown to increase SOC across a wide range of experiments from temperate grasslands such as ours, to tropical forests (Schnabel et al. 2025). This biodiversity effect has largely been attributed to increased organic matter inputs driven by higher biomass production and, in other studies, root turnover in more diverse plant communities (Fornara and Tilman 2008; Mueller et al. 2013). Previous studies documenting this biodiversity-SOC relationship measured this effect in older experiments. For example, Lange et al. (2015) observed these patterns in the Jena Experiment after approximately 10\u0026ndash;11 years. In our experiment, SOC levels were ~10% higher in diverse mixtures than in monocultures after only 7 years. Mixtures in BioCliVE produced substantially more aboveground biomass than monocultures under both control and drought conditions, indicating greater potential C inputs to soil through litter production. Moreover, under drought, aboveground biomass was positively associated with SOC, providing evidence that biomass inputs contributed to SOC maintenance under stress. These results reinforce the view that species richness is a key driver of C storage in grassland systems and that these benefits start accumulating early.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant diversity-driven SOC gains remain intact under drought\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormer studies have shown that drought can have differing effects on SOC depending on ecosystem type and drought intensity (Deng et al. 2021; Shi et al. 2024). Drought can suppress microbial activity, reduce productivity, and decrease organic matter inputs to soil, processes that may lead to decreased SOC (Schimel et al. 2007; Oram et al. 2023). Contrary to this expectation, we did not observe SOC declines under drought, and mixtures consistently maintained higher SOC than monocultures across treatments. This pattern suggests that plant diversity may act as a buffer against short-term stress, maintaining soil C even when water availability is reduced.\u003c/p\u003e\n\u003cp\u003eThe relatively short duration of our drought treatment (\u0026lt; 2 months) likely limited the potential for detectable SOC losses. Since this study focused on a single drought event, reoccurring drought events of varying intensities could produce more compounded effects not captured in our experiment. Established soil C pools are often resistant to brief disturbances, particularly in sandy soils with inherently low SOC concentrations (Yost and Hartemink 2019). Nevertheless, this temporal scale provides insight into the early-stage responses of soil C dynamics to climatic stress. The persistence of higher SOC in mixtures during this initial drought period suggests that diversity-driven C gains are not immediately reversed under short-term stress. Other studies have similarly shown that ecosystem responses to drought can lag or require cumulative stress across years. For instance, Frank et al. (2015) showed that C cycle responses to climate extremes may manifest only after repeated events, and Hoover and Rogers (2016) reported progressive declines in C inputs following recurrent drought episodes. While our short-term drought provided insights into initial ecosystem response, further research over extended temporal scales is crucial for capturing the cumulative effects of repeated or prolonged drought events on SOC dynamics.\u003c/p\u003e\n\u003cp\u003eOur SEM, however, revealed a significant positive pathway from aboveground biomass to SOC under drought conditions. By maintaining higher biomass than monocultures under drought, plant diversity likely contributed to sustaining SOC and preventing potential C losses. Similar mechanisms have been reported in other studies, where diverse communities maintained ecosystem functioning and buffered the effects of environmental stress (Isbell et al. 2015; Craven et al. 2016; Wagg et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant diversity increases biomass and buffers temperature but reduces soil moisture under drought\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with previous BEF research (Van Ruijven and Berendse 2005; Isbell et al. 2011; Cardinale et al. 2012; Tilman et al. 2014; Wright et al. 2015; Wright et al. 2021), our findings confirm that plant diversity significantly increases aboveground biomass. Under both control and drought conditions, plant mixtures produced substantially more biomass compared to monocultures. Under control conditions, plant diversity reduced soil temperature both directly and indirectly through aboveground biomass, although the biomass effect was weaker. Under drought, plant diversity directly reduced soil temperature, but the indirect pathway via biomass was no longer significant. This aligns with reported observations that apart from the increase in biomass, the greater trait diversity and structural complexity associated with diverse plant communities can buffer microclimates by shading the soil surface and trapping humidity (Wright and Francia 2024; Schnabel et al. 2025).\u003c/p\u003e\n\u003cp\u003eBased on observed soil cooling effects, one would expect plant diversity to increase soil moisture by reducing evaporation. Instead, we observed the opposite pattern: plant mixtures had lower soil moisture than monocultures under drought. This result suggests that diverse communities utilize available soil water more completely, as greater total plant biomass and complementary rooting strategies enable more thorough water extraction from the soil profile (Maestre et al. 2009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAboveground mechanisms become significant under drought\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile mixtures had higher SOC than monocultures overall (Fig. 2), the SEM revealed that the mechanisms underlying this effect differed between control and drought conditions. Under control conditions, plant diversity increased biomass and reduced soil temperature (both directly and indirectly via biomass), but neither pathway translated into higher SOC. Under drought, plant diversity increased biomass, which in turn positively influenced SOC, a pathway absent under control conditions. Aboveground processes alone were insufficient to explain SOC variation under control conditions but became important during drought. This indicates that under environmental stress, aboveground productivity becomes a key short-term determinant of soil C dynamics. Over longer time scales, consistent aboveground inputs should accumulate, potentially leading to more pronounced increases in SOC (Bardgett and van der Putten 2014). Belowground processes are also fundamental to how plant diversity influences SOC. Root-derived C contributes disproportionately to SOC formation through direct soil entry and coupling with microbial and mineral interactions (De Deyn et al. 2008; Lange et al. 2015; Kravchenko et al. 2021), with plant diversity influencing these rhizosphere processes (Bardgett et al. 2014). Future studies should therefore integrate belowground processes and repeated drought events to determine whether plant diversity can sustain soil C storage under increasingly variable climatic conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study shows that diversity-driven SOC gains are maintained under short-term drought. After seven years, mixtures maintained approximately 10% higher SOC than monocultures regardless of water availability, and, under drought, this was driven indirectly by sustained aboveground biomass production. This suggests that the role of productivity in maintaining soil C becomes particularly important when ecosystems face stress. As drought events intensify and become more frequent with climate change, maintaining diverse plant communities may be critical for preserving soil C stocks and the ecosystem services they support. Our findings highlight the importance of long-term experiments that integrate multiple stressors and examine both above- and belowground C pathways, particularly as the mechanisms driving SOC accumulation under ambient conditions remain unclear.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSOC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003esoil organic carbon\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003estructural equation model\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003ecarbon\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlya Kingsland-Mengi\u003c/strong\u003e: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJulia Mayr:\u003c/strong\u003e conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eYuheng Chen:\u003c/strong\u003e conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMink R. Verschoor:\u003c/strong\u003e conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eDiana Hoekstra:\u0026nbsp;\u003c/strong\u003edata curation, methodology. \u003cstrong\u003eNehizena Osagie:\u003c/strong\u003e data curation, methodology. \u003cstrong\u003eRola E. Johannes:\u003c/strong\u003e methodology, project administration, resources. \u003cstrong\u003eBetty P. Verduyn:\u0026nbsp;\u003c/strong\u003emethodology, project administration, resources. \u003cstrong\u003ePeter Veenhuizen:\u003c/strong\u003e methodology, project administration, resources. \u003cstrong\u003eJanna M. Barel:\u003c/strong\u003e methodology, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eYann Hautier:\u003c/strong\u003e conceptualization, funding acquisition, investigation, methodology, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eGeorge A. Kowalchuk:\u003c/strong\u003e conceptualization, funding acquisition, investigation, methodology, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eKathryn E. Barry:\u003c/strong\u003e conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, resources, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eWe are deeply grateful to all the field workers of the BIOCLIVE whose efforts made this experiment and its extensive measurements possible. The BIOCLIVE experiment is supported by the Ecology \u0026amp; Biodiversity Group, the Department of Biology of Utrecht University, and the Utrecht University Fund. We extend our sincere thanks to Ali Miedema and Laurens Gaarenstroom for their invaluable contributions. We are also grateful for the financial support from the trusts and foundations associated with the Utrecht University Fund: K.F. Hein Fonds, WNF INNO-Fonds, Ars Donandi, Stichting Thurkowfonds, and the M.A.O.C. Gravin van Bylandt Stichting. Alya Kingsland-Mengi was funded by the Startersbeurs Barry 2022 Grant (BB.000725.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe data and code that support the findings of this study are openly available in Zenodo at https://zenodo.org/records/19056618.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmelung, W., Bossio, D., de Vries, W., K\u0026ouml;gel-Knabner, I., Lehmann, J., Amundson, R., Bol, R., Collins, C., Lal, R., Leifeld, J., Minasny, B., Pan, G., Paustian, K., Rumpel, C., Sanderman, J., van Groenigen, J. W., Mooney, S., van Wesemael, B., Wander, M., \u0026amp; Chabbi, A. (2020). 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Grassland Responses to Three Years of Elevated Temperature, Co2, Precipitation, and N Deposition. \u003cem\u003eEcological Monographs\u003c/em\u003e, \u003cem\u003e73\u003c/em\u003e(4), 585\u0026ndash;604. https://doi.org/10.1890/02-4053\u003c/li\u003e\n\u003cli\u003eZhou, R., Wang, J., Wang, Q., Liu, N., Ye, C., Shi, J., Liu, M., Gao, Z., Chu, H., Zhang, Z., Niu, B., Wang, S., Zhang, R., Tian, D., \u0026amp; Niu, S. (2026). Decadal extreme drought reduces alpine subsoil carbon stocks. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, \u003cem\u003e123\u003c/em\u003e(8), e2517468123. https://doi.org/10.1073/pnas.2517468123\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Plant diversity, Soil organic carbon, Soil microclimate, Aboveground biomass, Drought stress","lastPublishedDoi":"10.21203/rs.3.rs-9141925/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9141925/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background and Aims\n\nSoil organic carbon (SOC) plays a central role in global carbon cycling and is a key component of nature-based climate solutions. However, climate change and biodiversity loss are interrelated threats that may jointly undermine SOC. While plant diversity is known to enhance ecosystem functioning, its potential to buffer SOC under climate extremes such as drought, remains poorly understood. This study investigates whether plants growing in mixtures can mitigate SOC losses under drought by maintaining biomass production and moderating soil microclimate conditions.\n\nMethods\n\nThis study was conducted within a large-scale factorial grassland biodiversity and climate variability experiment (UU-BioCliVE) at Utrecht University, manipulating planted species richness (1, 4, 8, 12 species) and precipitation to impose drought. We used structural equation modeling (SEM) to understand how plant diversity alters and/or maintains SOC via aboveground biomass, soil temperature, and soil moisture under drought conditions.\n\nResults\n\nSOC was higher in soils under plant mixtures than under monocultures across both control and drought treatments. Under drought, SEM showed a positive association between aboveground biomass and SOC in mixtures, while soil microclimate variables were not related to SOC. Under control conditions, mixtures increased biomass and reduced soil temperature, but neither factor explained SOC.\n\nConclusion\n\nOur findings show that plant diversity supports SOC retention under short-term drought by sustaining biomass production. This underscores the importance of conserving plant diversity to sustain carbon storage under future drought stress.","manuscriptTitle":"Higher soil organic carbon in diverse plant communities linked to sustained biomass production during short-term drought","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 10:21:29","doi":"10.21203/rs.3.rs-9141925/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-07T02:45:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-03-27T04:17:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T07:57:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-03-16T17:40:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"736ee08b-3164-426f-a0d6-caec3f01dd82","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T10:21:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 10:21:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9141925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9141925","identity":"rs-9141925","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}