# Plant diversity loss has limited effects on below-ground biomass and traits but alters community short-term root production in a species-rich grassland

Clara Gracia, Aleš Lisner, Markéta Applová, Thinles Chondol, Vojtěch

Dolejšek, Eva Janíková, Yogita Karpate, Marie Konečná, Athina

Papatheodoulou, Leyre Pedrós, et al.

► To cite this version:

Clara Gracia, Aleš Lisner, Markéta Applová, Thinles Chondol, Vojtěch Dolejšek, et al.. Plant diversity loss has limited effects on below-ground biomass and traits but alters community short-term root production in a species-rich grassland. Journal of Ecology, 2025, 10.1111/1365-2745.14488. hal-04923918

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# Plant diversity loss has limited effects on below-ground biomass and traits but alters community short-term root production in a species-rich grassland

Clara Gracia<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> ID

Aleš Lisner<fnref n="1" /> ID

Markéta Applová<fnref n="1" /> ID

Thinles Chondol<fnref n="1" />,<fnref n="4" /> ID

Vojtěch Dolejšek<fnref n="1" />

Eva Janíková<fnref n="1" /> ID

Yogita Karpate<fnref n="1" /> ID

Marie Konečná<fnref n="1" /> ID

Athina Papatheodoulou<fnref n="1" /> ID

Leyre Pedrós<fnref n="1" />,<fnref n="2" /> ID

Tereza Švancárová<fnref n="1" /> ID

Jan Lepš<fnref n="1" />,<fnref n="5" /> ID

Jules Segrestin<fnref n="1" /> ID

**Correspondence**

Clara Gracia

Email: claragraciamas@gmail.com

Jules Segrestin

Email: jsegrestin@gmail.com

**Funding information**

Grantová Agentura České Republiky, Grant/Award Number: 23-070875; Valencian Government, Grant/Award Number: AICO/2022/181

Handling Editor: Eric Allan

**Abstract**

1. Many biodiversity-ecosystem functioning (BEF) experiments have shown enhanced productivity with biodiversity, often explained by niche differentiation and complementarity effects. Yet, most of these results are based on artificial plant assemblages, with a major focus on above-ground productivity. Consequently, our comprehension of the BEF relationship in natural ecosystem and for below-ground functions remains largely unknown.

2. In this study, we simulated a long-term non-random species loss in a naturally diverse temperate meadow. We created a richness gradient spanning from 1 to more than 20 species per plot, removing the rare and sub-ordinate species from the vegetation. After 3 and 6 years of experimental manipulation, soil cores were collected to investigate the effects of species removal on below-ground biomass, vertical distribution of roots and root traits. We also used ingrowth cores to quantify the short-term root production of plant communities. We investigated the consistency of below- and above-ground responses to species removal.

3. Consistent with above-ground responses, species removal had little effect on root biomass and community root traits, except for a decrease in biomass in the uppermost soil layer (0-5 cm) found only after 6 years. Similarly, the vertical distribution of root biomass and traits was largely unaffected by diversity loss, suggesting little evidence for vertical niche differentiation. Nevertheless, species loss decreased the root production in spring (3 months), albeit this lower short-term root production was diluted after a year, when monocultures of dominants almost matched the annual root production of controls.

4. **Synthesis.** Our results from a non-random species removal experiment revealed minimal impact of a realistic diversity loss on root biomass and traits, but a decrease in short-term fine root production. These results challenge traditional

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Journal of Ecology* published by John Wiley & Sons Ltd on behalf of British Ecological Society.

<footnote n="1">¹Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic</footnote>

<footnote n="2">²Centro de Investigaciones sobre Desertificación, Consejo Superior de Investigaciones Científicas (CSIC-UV-GVA), Valencia, Spain</footnote>

<footnote n="3">³Faculty of Biological Sciences, University of Valencia, Valencia, Spain</footnote>

<footnote n="4">⁴Department of Functional Ecology, Institute of Botany, Czech Academy of Sciences, Třeboň, Czech Republic</footnote>

<footnote n="5">⁵Biology Centre, Institute of Entomology, Czech Academy of Sciences, České Budějovice, Czech Republic</footnote>
BEF theory and the niche differentiation hypothesis, suggesting that dominant species primarily determine below-ground biomass structure and characteristics. High diversity may only temporarily increase short-term root production.

KEYWORDS

below-ground productivity, biodiversity experiment, mass-ratio hypothesis, root production, root traits, species loss

# 1 | INTRODUCTION

The interplay between biodiversity and ecosystem functioning is a central and long-standing question in ecology. According to biodiversity-ecosystem functioning (BEF) experiments, frequently conducted on grassland communities, there is a general consensus about the positive association between biodiversity and productivity. The complementarity effect is one of the proposed explanations for the enhanced ecosystem functioning in diverse communities (Cardinale et al., 2007; Loreau & Hector, 2001; Tilman et al., 2001). This effect stems from the fact that, in more diverse communities, variations in species strategies are expected to optimize resource use, resulting in increased productivity. Although complementarity effects can arise from a wide range of mechanisms, in plants, they have often been linked with horizontal (von Felten & Schmid, 2008) or vertical niche differentiation (Li et al., 2018), especially below-ground, where the distribution of roots is expected to exhibit more variability in diverse plant communities (Brassard et al., 2013). The other proposed mechanism explaining the experimental BEF positive relationship is the selection effects, wherein the likelihood of a high-functioning species being present in an assemblage increases with the number of species (Cardinale et al., 2011).

In the last decades, there has been a debate on whether results from BEF experiments can provide insights into the effects of biodiversity loss on ecosystem functioning in natural communities (Doherty & Zedler, 2018; Lepš, 2004; Wardle, 2016). Most BEF experiments rely on random selections of species from a species pool, which often does not represent realistic communities on which species loss can be simulated. Although such experimental designs offer controlled simulations that can provide important mechanistic information (Eisenhauer et al., 2016), they can be misleading in some aspects as natural species loss does not behave as a random process (Schläpfer et al., 2005; Schmid et al., 2002). Indeed, the probability of a species to be lost from a community depends on multiple factors, but primarily on its relative abundance (Genung et al., 2020; Smith & Knapp, 2003; Spehn et al., 2005). Furthermore, most BEF experiments are based on evenly sown communities, where all species are initially sown with equal abundance, which fails to represent the environmental filters and biotic factors that govern assembly processes (Diaz et al., 2003). On the other hand, removal experiments in natural communities provide a promising alternative approach. Following a realistic scenario of species loss, it has been

shown that a few dominant species could compensate for the removal of rare and subordinate species and mitigate the effect of diversity loss on productivity (Lisner et al., 2023; Sasaki et al., 2017; Sasaki & Lauenroth, 2011; Smith & Knapp, 2003), further questioning the transferability of traditional BEF experiments to real-world communities.

Although most BEF experiments to date have focused on above-ground productivity (Cardinale et al., 2007), some studies have explored other ecosystem functions (Spehn et al., 2005; van der Plas, 2019), including below-ground productivity. However, the latter have mainly followed the same traditional experimental set-up with artificial communities (Mueller et al., 2013; Oram et al., 2018; Ravenek et al., 2014) consistently showing a positive relationship between species richness and root biomass. In natural communities, some observational studies found this relationship as well (Mommer et al., 2010; Zeng et al., 2020) but others contradict the idea of any effect of species richness on root biomass (Archambault et al., 2019; Meinen et al., 2009).

Whether the results obtained on the above-ground biomass would transfer to the below-ground biomass remains an open question since high species complementarity could be expected below-ground, due to the more symmetrical competition for resources (Lamb et al., 2009). Moreover, species above-ground biomass may not be correlated with below-ground biomass due to large variations in biomass allocation between species and years (Ottaviani et al., 2020). To address various knowledge gaps in BEF theory related to below-ground plant characteristics, our study examines the impact of a realistic plant diversity loss on below-ground functions. Employing a removal experiment on a semi-natural grassland, a gradient of species richness was established based on the observed dominance structure. This approach allows for the simulation of a more realistic scenario of species loss, integrating environmental and biotic factors inherent in natural communities. We compared above-ground and below-ground biomass along the diversity gradient after 3 and 6 years of species removal. Root characteristics were also determined in different soil layers. Finally, ingrowth cores were used to measure the spring (3 months) and annual root production in all plots. Comparing our results with the outcomes of classical BEF experiments using artificial communities will help us to test the reliability of previous work to assess the consequences of species loss on natural ecosystems. Our study aims to answer the following questions:
1. Does short-term root production and long-term standing root biomass decrease with species loss?

2. Is there any impact on the vertical distribution of roots? And on their traits?

We hypothesize that species loss may lead to a decrease in root biomass and alterations in fine root vertical distribution due to the loss of complementarity effects and reduced interspecific interactions. Regarding root traits, measured at the level of the community, we also expect a shift towards trait values associated with a lower acquisition rate and, more importantly, a lower trait variability with the loss of subordinate and rare species. Furthermore, we anticipate that plots with higher diversity will colonize more rapidly the available soil space and have higher root production in ingrowth cores, especially at short term.

## 2 | MATERIALS AND METHODS

### 2.1 | Study site

The study was conducted in a wet oligotrophic meadow located near České Budějovice, Czech Republic (48°57'10.8" N 14°35'34.8" E, 510 m a.s.l.). The site has a mean annual temperature of 8.3°C and a mean annual precipitation around 700 mm (Czech Hydrometeorological Institute, 1992–2022). It is a particularly species-rich meadow with an average species richness of c. 26 species per 0.25 m² (Lisner et al., 2023). The most dominant species is *Molinia caerulea* followed by *Danthonia decumbens* and *Betonica officinalis*. A complete list of species can be found in the data repository attached to Lisner et al. (2024). Traditionally, the meadow has been extensively managed with a single annual mowing, at the peak of the vegetation season, in mid-June. No special permits were required to conduct the experiment or collect the data.

### 2.2 | Experimental design

The manipulative experiment was established in 2016 and is described in detail in Lisner et al. (2023). A gradient of diversity loss was created by manually removing plant species based on their relative abundance in 30 1×1 m plots. Before the manipulation, the initial relative abundances of all species were determined in each plot by harvesting the above-ground biomass simultaneously to the annual mowing and hand-sorting each species biomass. Four levels of diversity loss were created where only the most abundant 1, 3, 6 and 12 species were kept (see Table S1 for targeted species composition). Additionally, we used two control treatments (naturally containing c. 26 species): the intact vegetation (C) and a disturbance control (D) to quantify the confounding effect of the disturbance caused by the experimental weeding. In

the intact plots, no species was removed, while in the disturbance plots, we randomly removed several individuals, aiming for an amount of biomass equivalent to the average biomass removed in removal treatments, but irrespective of the species identity. These six treatments were replicated five times in Latin-square-like design resulting in 30 plots (5 times × 6 treatments) (see Figure S1 for more details on the design). The plots were maintained twice a year—once in spring and once in late summer—by removing resprouting individuals, creeping stems from the surrounding vegetation and new seedlings. After 1 year (i.e. in spring 2017), the targeted species colonized most of the free space induced by the weeding. Consequently, the amount of biomass removed in each plot decreased significantly and contained only a small proportion of plants resprouting from below-ground organs. More information on the target species richness, effective species richness, dominance and removed biomass for each year can be found in Figure S2.

### 2.3 | Data collection: Standing roots

In 2019 and 2022, one soil core of 5 cm in diameter and 40 cm deep was collected in each plot. The sampling was conducted at the peak of the vegetation season, in mid-June. The samples were collected from at least 20 cm away from the edge of the plot to maximally reduce the presence of roots from plants growing in the surrounding plots and nearby vegetation. Each core was divided into five sections based on layer depth: 0–5; 5–10; 10–20; 20–30; and 30–40 cm (Figure 1) and stored in the freezer before further processing. During the following months, each layer was washed over a sieve of mesh size 0.5 mm with water, and plant biomass excluding litter was manually sorted into roots and other below-ground organs such as bulbs and rhizomes. Few roots of trees growing near the experiment could be identified and were excluded from the samples. All samples were oven-dried at 60°C for 48 h, and the dry weight was recorded. The same years, above-ground biomass was harvested at the peak of the vegetation season, in the inner 0.5 × 0.5 m area of each plot. The biomass was clipped close to the ground and sorted into species in the lab. Each species' dry mass was weighed after being oven-dried at 80°C for 48 h. For more detail, see Lisner et al. (2023).

### 2.4 | Data collection: Ingrowth cores

In 2022, we used ingrowth cores to estimate the short-term fine root production. The ingrowth cores consisted of plastic mesh cylinders of 3 cm in diameter, 15 cm in height and 5 mm in mesh size. One ingrowth core was established in each plot to measure the biomass of fine roots produced in spring (from 17 March to 21 June), while a second one was employed to measure the root production throughout the entire year (from 6 December 2021 to
<figure><img src="image_3.png" /><figcaption>FIGURE 1 In each 1×1 m plot of the experiment, two types of root data were collected close to the inner 0.5×0.5 m subplot. The standing root biomass (a) corresponds to the dry mass of fine roots in different soil layers of a core of 5 cm in diameter and 40 cm deep. Two ingrowth cores (b) of 3 cm in diameter and 15 cm deep were used to measure the biomass of fine roots produced in spring (after 3 months) and in a full year.</figcaption></figure>

4 January 2023). Following the drilling of soil cores of appropriate size, both ingrowth cores were installed on opposite sides of the 0.5×0.5 m inner subplot (Figure 1) and filled with root-free fresh soil. The soil was collected from the same site near the experiment in autumn 2021. It underwent thorough sieving through a 5-mm mesh to remove gravels, stones and unwanted plant material. It was then homogenized and oven-dried at 40°C for several days. Ingrowth cores were harvested by cutting all roots around the mesh with a knife before pulling the cylinder out of the ground. Post-harvest, ingrowth cores were washed with water, and fine roots were hand-sorted.

## 2.5 | Data collection: Root traits

Roots from each soil layer and from each ingrowth core were scanned using a flatbed scanner (Epson Perfection 4990 Photo) at a resolution of 600 dpi. We selected a representative subsample of roots from each sample (keeping a similar proportion of root types and diameters visually) that filled the scanned area without overlap. Subsamples ranged from approximately 14% to 100% depending on the sample size. The scanned images underwent analysis to measure the root length in 20 diameter classes of 0.1 mm, spanning from 0 to 2 mm, employing RhizoVision Explorer software v2.0.3 (Seethepalli & York, 2020) using algorithms described by Seethepalli et al. (2021). These data were utilized to calculate three community root traits for every layer and every ingrowth core: the community-specific root length (the total root length per unit dry mass) (as described in Freschet et al., 2021), community mean root diameter (the mean root diameter calculated from the root diameter distribution) and community root diameter variability (the variance in root diameter calculated from the root diameter distribution).

## 2.6 | Data analysis

The standing root biomass collected in each core was expressed per ground area in $g m^{-2}$. When analysing the vertical distribution of roots, the root biomass collected in each layer was expressed per unit soil volume in $mg cm^{-3}$. The same was applied to the biomass data collected in each ingrowth core. All biomass data, above-ground and below-ground, were log-transformed to avoid heteroscedasticity.

We used the effective number of species (diversity hereafter) as a plant diversity index. It was calculated for each plot and each year as the reciprocal of Simpson's diversity index, as:

$$Diversity = \frac{1}{\sum_{i=1}^{n} p_i^2},$$

where $p_i$ is the relative abundance of species i, calculated from the above-ground biomass of all species at the peak of the vegetation season, in mid-June. This index was preferred over the targeted species richness as it better captures variations in species abundances in each plot while adequately reflecting the experimental treatments (correlation between target species richness and effective number of species: $r_{2019}=0.81$; $r_{2022}=0.90$; Figure S2b).

Before conducting the main analyses, we tested whether the disturbance caused by the repeated species removal from the plots (removed biomass bi-annually; Figure S2d) had an impact on the different measures. We compared disturbance controls with intact vegetation controls using ANOVA models for each variable separately (total above-ground biomass (log scale), total below-ground biomass (log scale), root to shoot ratio, root density in each layer (log scale), root density in ingrowth cores after 3 and 12 months (log scale), community-specific root length (log scale), community mean root diameter (log scale), community root diameter variability in standing root cores and ingrowth cores). All tests showed no significant differences between the two types of controls (Figures S3 and
S4), indicating that the disturbance introduced by the experimental species removal had negligible effects. Therefore, in subsequent analyses, only the intact vegetation controls were kept.

Data analysis was conducted using linear mixed-effects models (LMM) in software R (ver. 4.3.0) with the *lme4* package (v1.1-26; Bates et al., 2015), and p-values were obtained with Satterthwaite's method with the *lmerTest* package (v.3.1-3; Kuznetsova et al., 2017). To test the effect of diversity on the total standing root biomass, a first LMM was fitted with diversity as the only predictor. The column and row identifiers of each plot were used as random factors to account for environmental gradients. A second LMM was fitted on the root biomass per layer with the same variables but including the interaction with soil layer, as a 5-level factor, to represent the root biomass vertical distribution. Since layers were collected from the same cores, the plot identity was included as a random factor in this model. To deepen our understanding of the effects on the different layers, individual LMM were fitted for each layer separately. Same method was used to test the effect of diversity on short-term root production in in-growth cores, fitting an LMM with diversity and its interaction with time, as a 2-level factor (3 and 12 months). As the interaction was not significant, it was eliminated from the final model. The R code required for the analysis is provided in the Supporting Information.

# 3 | RESULTS

## 3.1 | Standing root biomass and vertical distribution

On average, the standing root biomass was higher in 2019 (biomass±SD=801±262 g m⁻²) than in 2022 (522±163 g m⁻²) (paired t-test, t=5.3, df=24, p-value <0.001). At the same sampling dates, the above-ground biomass was lower in 2019 (biomass±SD=185±33 g m⁻²) than in 2022 (209±33 g m⁻²) (paired t-test, t=-3.5, df=24, p-value=0.002). It resulted in a higher root to shoot ratio in 2019 (ratio±SD=4.5±1.8) than in 2022 (ratio±SD=2.6±0.9) (paired t-test, t=5.7, df=24, p-value <0.001). These changes could not be related to the experimental treatments (all treatments had a similar response) but might rather be attributed to differences in climatic conditions between years. Indeed, 2019 was among the driest years in the last 20 years while 2022 was unusually wet (Meteoblue, 2024). Differences in water availability may have resulted in a redistribution of resources, shifting biomass allocation between above- and below-ground compartments (Mokany et al., 2006).

Species diversity had a positive effect on above-ground biomass in 2019 ($F_{1,16}=7.30$, p-value=0.016), but not in 2022 (Figure 2a,b). Note that 2019 is rather an exception as it is the only year out of 6 years of measurements with a significant diversity effect on above-ground biomass (see Lisner et al., 2023). In both years, species

<figure><img src="image_4.png" /></figure>

**FIGURE 2** Above-ground and below-ground biomass per m² and root to shoot ratio as a function of diversity for years 2019 (a, c, e) and 2022 (b, d, f). The solid line represents the only significant relationship with shaded confidence interval; corresponding statistical values are shown in each panel. Colours represent the targeted species richness of each plot. Diversity: effective number of species. C, controls.

removal had no significant effect on below-ground biomass, nor on the root to shoot ratio (Figure 2c-f).

When analysing the vertical distribution of root biomass, we found that the root density per soil volume decreased with depth both in 2019 and 2022 (2019-Layer: $F_{4,92}=78.7$, p-value <0.001; 2022-Layer: $F_{4,92}=111.6$, p-value <0.001) (Figure 3). We found no significant effect of plant diversity on the vertical distribution of roots in 2019 (interaction Diversity×Layer: $F_{4,92}=1.7$, p-value=0.149) (Figure 3). However, in 2022, plant diversity affected the vertical distribution of roots (interaction Diversity×Layer: $F_{4,92}=2.6$, p-value=0.042) and the root density in the topsoil layer (0–5 cm) was positively related to plant diversity (topsoil individual model: Diversity estimate=0.143, $F_{1,23}=7.1$, p-value=0.014). It resulted in a c. 40% decrease in root density in the 1-species plots compared to controls in this layer (Figure 3). We found no significant effect of plant diversity on the root density in deeper soil layers.

Community-level specific root length did not change between soil layers (Table 1; Figure S6a). However, roots tended to have a
smaller diameter (community mean root diameter) in the topsoil layer than in deeper layers (Table 1; Figure S6b). Finally, we found higher community root diameter variability in the topsoil layer than in deeper layers (Table 1; Figure S6c). None of the community-level traits were affected by plant diversity in any soil layer.

## 3.2 | Ingrowth cores and short-term root production

We found a significant positive effect of diversity on the short-term root production (Diversity estimate=0.149, $F_{1,19}=8.2$, p-value=0.010). Although the interaction factor was not significant in the initial model (interaction Diversity×Time: $F_{1,23}=3.1$, p-value=0.091), the diversity effect was much stronger for the spring root production than the annual root production (Figure 4).

The community-specific root length was higher after the 3-month growth period than after the 12-month period (Table 1;

<figure><img src="image_5.png" /><figcaption>FIGURE 3 Root biomass density in soil profiles as a function of diversity in 2019 (a) and 2022 (b). The solid line represents the only significant relationship for the 0–5 cm layer in 2022 with shaded confidence interval and the dashed lines represent non-significant relationships for the same year (see statistical values in the text). Diversity: effective number of species.</figcaption></figure>

Figure S7a). Yet it was not affected by species removal. Both mean root diameter and diameter variability remained constant over time and were unaffected by diversity loss (Table 1; Figure S7b,c).

## 4 | DISCUSSION

We conducted a long-term removal experiment (from 2016 to 2022) to simulate a realistic scenario of diversity loss in a semi-natural grassland. We explored the long-term effects of species removal on root biomass and traits and its relation with above-ground responses. According to previous results from BEF experiments and theories relating biodiversity to ecosystem function (e.g. the niche differentiation hypothesis), we expected a decrease in root biomass and changes on the root vertical distribution with species loss. However, standing root biomass remained largely unaffected and monocultures of a dominant species contained as much root biomass as high diversity plots, even after 6 years of species removal.

<figure><img src="image_6.png" /><figcaption>FIGURE 4 Short-term root production in spring (3 months) and in a year (12 months) depending on the diversity of the plot. The solid lines represent the significant relationships for each period of time with shaded confidence intervals. Diversity: effective number of species.</figcaption></figure>

<table><thead><tr><th rowspan="2"></th><th colspan="3">Specific root length</th><th colspan="3">Mean root diameter</th><th colspan="3">Root diameter variability</th></tr><tr><th>F</th><th>df</th><th>p</th><th>F</th><th>df</th><th>p</th><th>F</th><th>df</th><th>p</th></tr></thead><tbody><tr><td>Whole cores</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diversity</td><td>0.75</td><td>15</td><td>0.40</td><td>0.38</td><td>15</td><td>0.54</td><td>0.05</td><td>14</td><td>0.82</td></tr><tr><td>Soil layers</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diversity</td><td>0.69</td><td>14</td><td>0.41</td><td>2.25</td><td>15</td><td>0.15</td><td>0.20</td><td>14</td><td>0.66</td></tr><tr><td>Layer</td><td><strong>2.51</strong></td><td><strong>92</strong></td><td><strong>0.05</strong></td><td>1.73</td><td>92</td><td>0.15</td><td>1.60</td><td>92</td><td>0.18</td></tr><tr><td>Diversity×layer</td><td>1.05</td><td>92</td><td>0.38</td><td>0.49</td><td>92</td><td>0.73</td><td>1.43</td><td>92</td><td>0.23</td></tr><tr><td>Ingrowth cores</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diversity</td><td>0.00</td><td>18</td><td>0.98</td><td>0.13</td><td>23</td><td>0.72</td><td>0.00</td><td>18</td><td>0.97</td></tr><tr><td>Time</td><td><strong>15.6</strong></td><td><strong>23</strong></td><td><strong>&lt;0.01</strong></td><td>0.28</td><td>23</td><td>0.60</td><td>0.08</td><td>23</td><td>0.77</td></tr><tr><td>Diversity×time</td><td>1.91</td><td>23</td><td>0.18</td><td>0.40</td><td>23</td><td>0.53</td><td>0.01</td><td>23</td><td>0.92</td></tr></tbody></table>

Note: Bold values highlight *p*-values < 0.05.

<caption>TABLE 1 Community root trait responses to plant diversity loss in standing roots (whole cores and individual layers) and ingrowth cores (Time: 3 and 12 months). Diversity: Effective Number of Species. *F*, *df* and *p*: *F* value, degrees of freedom and *p*-value of type III ANOVA with Satterwhite's method, respectively.</caption>
Similarly, Yoshihara et al. (2020), in a removal experiment in arid grasslands, found no diversity effect in root biomass below the first 5cm of soil after 3 years of treatment. These results contrast with Ravenek et al. (2014), who reported that below-ground biomass in the Jena experiment depended on species richness and functional composition of the community. Wang et al. (2023) recently found a similar pattern, revealing a positive relationship between species richness and below-ground biomass using grass communities. Nonetheless, both studies are based on randomly assembled communities making it hard to distinguish the contribution of the selection effect (higher likelihood of finding a highly productive species in more diverse plots) and the complementarity effects on this positive effect of diversity (Oram et al., 2018). Indeed, the random set-up used in these studies involved monocultures of species that are unlikely to perform well alone, contributing to an artificially low biomass production of low diversity plots. In our study, monocultures of the most dominant species contained an equal or higher amount of root biomass than the average polyculture, not supporting the transgressive overyielding (Trenbath, 1974). This result is consistent with the lack of response found for above-ground biomass in the same experiment (Lisner et al., 2023). It shows that one single species can quickly compensate for the loss of more than 20 species (M. caerulea showed a 3-fold increase in above-ground productivity following the removal of other species) and be sufficient to maintain a high biomass at long-term both above- and below-ground.

Despite a lack of response of the total root biomass to species loss, we found a large decrease (~40%) in root density in the top 5-cm layer with species removal after 6 years. Similar results were found by Ravenek et al. (2014), where monocultures contained ~37% less root biomass than 16-species polycultures after 5 years. Hence, *M. caerulea* did not fully colonize the empty space produced by the removal of rare and subordinate species in the top layer. This might be explained by the fact that *M. caerulea* has the deepest rooting depth in the community (Kutschera et al., 1993) and might be able to uptake nutrients deeper than other species. Overall, it suggests that subordinate and rare species preferentially use free space in the upper soil layer that is not used by dominants, resulting in a higher root aggregation near the surface in high-diversity plots. One explanation could be that nutrient availability tends to decrease quickly with soil depth, making its uptake from deeper soil higher more costly (von Felten et al., 2009). The fact that this higher root density in the upper layer did not result in an overall overyielding suggests high interspecific competition in the topsoil layer (Yoshihara et al., 2020).

The lack of a diversity-loss effect on fine root traits, though unexpected, suggests small differences between species and aligns with the idea of a weak niche partitioning in our studied system. Diverse communities are expected to show high specific root length, which increases resource uptake efficiency for a given unit of biomass investment (Ostonen et al., 2007), high mean root diameter also related to efficiency (Peng & Chen, 2021) and a high root diameter variability, associated with a high soil exploration and resource partitioning. However, insignificant diversity effects have been

reported on community-specific root length (Bauhus et al., 2000; Gould et al., 2016; Siebenkäs & Roscher, 2016), as well as positive (Shu et al., 2018) and negative (Baxendale et al., 2014; Salahuddin Rewald et al., 2018). Similarly, contrasting results have been found on community mean root diameter (Peng & Chen, 2021). Furthermore, the lack of response in community root variability to species loss aligns with the findings of Herben et al. (2022, 2018), who showed that, contrary to pot experiments, root plasticity is usually low in natural communities (Belter & Cahill Jr, 2015; Gersani et al., 2001). Nevertheless, our study focused on traits related to resource uptake efficiency and it is possible that other root traits such as horizontal structure (von Felten & Schmid, 2008) or chemical composition (Bauhus et al., 2000) may change with diversity. Finally, the root-mycorrhizal association may respond to changes in plant diversity (Bergmann et al., 2020), warranting further investigation.

The most pronounced effect of species removal was found on the short-term root production. When comparing monocultures to controls, we found that species removal decreased by 47% the biomass produced in spring. The lower root production in spring found in low-diversity plot is likely due to the late phenology of the remaining dominant species. Indeed, *M. caerulea* is known to start its vegetative growth much later than other coexisting species (Doležal et al., 2019). Our results suggest that this late phenology also applies to below-ground growth. However, the annual root growth was only weakly affected by the species removal (~20% decreased), suggesting that, although late, dominants can produce a similar amount of root biomass during the rest of the year. This might be due to a higher growth rate compared to the species in naturally diverse controls. Another explanation could be that, despite differences in growth dynamics, 1 year of growth would be enough to saturate in-growth cores in all treatments. However, the root biomass produced in 1 year was still significantly lower than values measured in the standing root biomass (Figure S5).

One could argue that our results can be affected by the small size of the experimental plots used in our study. Indeed, plants from the surrounding vegetation may have colonized the empty space induced by the species removal, hence increasing the below-ground biomass in low-diversity plots. Although this phenomenon cannot be fully excluded, we harvested the below-ground biomass at least 20 cm from the edge of the plot to limit this effect. Moreover, a strong root colonization from the surrounding vegetation would have resulted in a high root to shoot ratio in monocultures, which was not found in our study. Nevertheless, only a detailed identification of the below-ground species composition, using molecular analyses, for example, would allow us to exclude completely the possibility of colonization from outside. Additionally, it could be argued that the low number of replicates in our experiment led to weak statistical tests, which may explain the low number of significant results. Nevertheless, the diversity effects observed in our study had comparable effect sizes to those reported in previous work. Specifically, in the Jena experiment, Ravenek et al. (2014) similarly reported an ~37% decrease in root biomass in the topsoil layer, closely matching our observed ~40% reduction, after 5 and 6 years, respectively. This suggests that
our experiment is robust enough to detect significant biodiversity effects, although some weaker effects may have been missed.

Our results indicate that the established diversity-ecosystem function relationships may not be as ubiquitous as previously thought when studied in manipulated real-world communities, especially in less explored below-ground characteristics. Nonetheless, we observed an increased short-term root production in diverse communities, likely driven by a phenological delay in monocultures composed by the dominant species *M. caerulea*. These results help us to identify important context dependencies on BEF relationships, allowing us to better understand how biodiversity will affect ecosystem functioning in a range of real-world scenarios. However, delving deeper into this topic would require a better identification of below-ground species composition. Therefore, the combination of a realistic species loss scenario approach with below-ground species identification techniques (Lamb et al., 2016) could shed light to the BEF relationship below-ground and poses a significant challenge for future research.

## AUTHOR CONTRIBUTIONS

Jan Lepš and Aleš Lisner conceived the long-term experiment; Jules Segrestin and Aleš Lisner conceived the ideas and designed the methodology; Jules Segrestin, Aleš Lisner, Eva Janíková, Markéta Applová, Marie Konečná and Tereza Švancárová conducted the experiment and collected the data, Clara Gracia analysed the data with the help of Thinles Chondol, Leyre Pedrós, Aleš Lisner and Jules Segrestin; Clara Gracia led the writing of the first draft with the help Yogita Karpate, Vojtěch Dolejšek, Athina Papatheodoulou, Jules Segrestin and Aleš Lisner. All authors contributed critically to the drafts and gave final approval for publication.

## ACKNOWLEDGEMENTS

This study was supported by the Czech Science Foundation (GAČR Project number: 23-07087S) and the Valencian Government (AICO/2022/181). We thank Blanka Divišová, Karolína Jackwerth and Natasha Sinkala for the technical assistance during the data collection. We also thank the numerous students involved in the long-term experiments. Open access publishing facilitated by Jihoceska Univerzita v Ceskych Budejovicich, as part of the Wiley - CzechELib agreement.

## CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

## PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/1365-2745.14488.

## DATA AVAILABILITY STATEMENT

Data and R code for analysis available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.p8cz8wb1j (Gracia et al., 2024). Above-ground species composition and abundance data from the Dryad Digital Repository: https://doi.org/10.5061/dryad.rxdwbrvj8 (Lisner et al., 2024).

## ORCID

Clara Gracia https://orcid.org/0009-0001-3855-4012

Aleš Lisner https://orcid.org/0000-0003-4881-7362

Markéta Applová https://orcid.org/0009-0002-1927-4215

Thinles Chondol https://orcid.org/0000-0003-1054-9980

Eva Janíková https://orcid.org/0000-0003-1649-9435

Yogita Karpate https://orcid.org/0000-0003-2545-9371

Marie Konečná https://orcid.org/0000-0002-9195-4576

Athina Papatheodoulou https://orcid.org/0000-0001-7198-5044

Leyre Pedrós https://orcid.org/0009-0009-4615-6485

Tereza Švancárová https://orcid.org/0009-0008-4028-7292

Jan Lepš https://orcid.org/0000-0002-4822-7429

Jules Segrestin https://orcid.org/0000-0001-7661-6061

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**Figure S1.** Latin-square-like experimental design used in our study. Thirty 1×1 m plots were arranged in 6 columns and 5 rows.

**Figure S2.** Community characteristics influenced by the experimental treatments over seven years (2016-2022).

**Figure S3.** No significant differences between intact and disturbance controls for biomass measurements in 2022: total aboveground biomass, total belowground biomass, root-to-shoot ratio, root density across different soil layers, and root density in ingrowth cores after 3 and 12 months.

**Figure S4.** No significant differences between intact and disturbance controls for community-level trait measurements: specific root length, community mean root diameter, and community root diameter variability in each soil layer of standing root cores and in ingrowth cores after 3 and 12 months.

**Figure S5.** Root density in ingrowth cores after 3 and 12 months compared with root density in standing root cores.

**Figure S6.** Community root traits in soil profiles as a function of diversity.

**Figure S7.** Community root traits in ingrowth cores as a function of diversity.

**Table S1.** Targeted species composition in each removal treatment. **Appendix S1.** R code attached to the manuscript: 'Plant diversity loss has limited effects on below-ground biomass and traits but alters community short-term root production in a species-rich grassland'.

**How to cite this article:** Gracia, C., Lisner, A., Applová, M., Chondol, T., Dolejšek, V., Janíková, E., Karpate, Y., Konečná, M., Papatheodoulou, A., Pedrós, L., Švancárová, T., Lepš, J., & Segrestin, J. (2025). Plant diversity loss has limited effects on below-ground biomass and traits but alters community short-term root production in a species-rich grassland. *Journal of Ecology*, 00, 1-10. https://doi.org/10.1111/1365-2745.14488