Data from: Plant diversity loss has limited effects on belowground biomass and traits but alters community short-term root production in a species-rich grassland
Creators: name: Gracia, Clara
name: Lisner, Aleš
name: Applova, Marketa
name: Chondol, Thinles
name: Dolejšek, Vojtěch
name: Janíková, Eva
name: Karpate, Yogita
name: Konečná, Marie
name: Papatheodoulou, Athina
name: Pedrós, Leyre
name: Švancárová, Tereza
name: Lepš, Jan
name: Segrestin, Jules
Publisher: name: Zenodo
Year: 2025
Subjects: biodiversity experiment
Belowground productivity
mass-ratio hypothesis
root traits
root production
species loss

description: 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 aboveground productivity. Consequently, our comprehension of the BEF relationship in natural ecosystem and for belowground functions remains largely unknown.

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 belowground 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 aboveground responses to species removal.

Consistent with aboveground responses, species removal had little effects on root biomass and community root traits, except for a decrease in biomass in the uppermost soil layer (0-5cm) 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.

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 BEF theory and the niche differentiation hypothesis, suggesting that dominant species primarily determine belowground biomass structure and characteristics. High diversity may only temporarily increase short-term root production.
description: Funding provided by: Czech Science FoundationROR ID: https://ror.org/01pv73b02Award Number: GAČR Project number: 23-07087S
description: 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. 29 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.

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 aboveground 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 ca. 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 6 treatments were replicated five times in Latin-square-like design resulting in 30 plots (5 times x 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 one 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 belowground organs. More information on the target species richness, effective species richness, dominance, and removed biomass for each year can be found in Figure S2.

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 belowground 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 60ºC for 48 hours, and the dry weight was recorded. The same years, aboveground biomass was harvested at the peak of the vegetation season, in the inner 0.5 x 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 hours. For more detail, see Lisner et al. (2023).

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 4 January 2023). Following the drilling of soil cores of appropriate size, both ingrowth cores were installed on opposite sides of the 0.5 x 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.

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 and 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).

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, aboveground and belowground, 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 = 1/(sum(pi2)

where pi is the relative abundance of species i, calculated from the aboveground 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: r2019 = 0.81; r2022 = 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 S2.d) had an impact on the different measures. We compared disturbance controls with intact vegetation controls using ANOVA models for each variable separately (total aboveground biomass (log scale), total belowground 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 (Figure 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 (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 ingrowth cores, fitting an LMM with diversity and its interaction with time, as a 2-level factor (3 months and 12 months). As the interaction was not significant, it was eliminated from the final model. R code necessary for conducting the analysis can be found in Supporting Information.