# Leaf isotopes reveal tree diversity effects on the functional responses to the pan-European 2018 summer drought

Xin Jing, Christel Baum, Bastien Castagneyrol, Nico Eisenhauer, Olga

Ferlian, Tobias Gebauer, Peter Hajek, Hervé Jactel, Bart Muys, Charles A Nock, et al.

► To cite this version:

Xin Jing, Christel Baum, Bastien Castagneyrol, Nico Eisenhauer, Olga Ferlian, et al. Leaf isotopes reveal tree diversity effects on the functional responses to the pan-European 2018 summer drought. New Phytologist, 2024, pp.1-17. 10.1111/nph.19931. hal-04642132

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# Leaf isotopes reveal tree diversity effects on the functional responses to the pan-European 2018 summer drought

Xin Jing<fnref n="1" />,<fnref n="2" /> ID, Christel Baum³ ID, Bastien Castagneyrol⁴ ID, Nico Eisenhauer⁵,⁶ ID, Olga Ferlian⁵,⁶ ID, Tobias Gebauer⁷ ID, Peter Hajek⁷ ID, Hervé Jactel⁴ ID, Bart Muys<fnref n="2" /> ID, Charles A. Nock⁷,⁸ ID, Quentin Ponette⁹ ID, Laura Rose⁷,¹⁰ ID, Matthias Saurer¹¹ ID, Michael Scherer-Lorenzen⁷ ID, Kris Verheyen¹² ID and Koenraad Van Meerbeek<fnref n="2" />,¹³ ID

## Summary

* Recent droughts have strongly impacted forest ecosystems and are projected to increase in frequency, intensity, and duration in the future together with continued warming. While evidence suggests that tree diversity can regulate drought impacts in natural forests, few studies examine whether mixed tree plantations are more resistant to the impacts of severe droughts.

* Using natural variations in leaf carbon (C) and nitrogen (N) isotopic ratios, that is δ¹³C and δ¹⁵N, as proxies for drought response, we analyzed the effects of tree species richness on the functional responses of tree plantations to the pan-European 2018 summer drought in seven European tree diversity experiments.

* We found that leaf δ¹³C decreased with increasing tree species richness, indicating less drought stress. This effect was not related to drought intensity, nor desiccation tolerance of the tree species. Leaf δ¹⁵N increased with drought intensity, indicating a shift toward more open N cycling as water availability diminishes. Additionally, drought intensity was observed to alter the influence of tree species richness on leaf δ¹⁵N from weakly negative under low drought intensity to weakly positive under high drought intensity.

* Overall, our findings suggest that dual leaf isotope analysis helps understand the interaction between drought, nutrients, and species richness.

Author for correspondence:
Xin Jing

Email: jingx@lzu.edu.cn

Received: 19 February 2024

Accepted: 5 June 2024

New Phytologist (2024)

doi: 10.1111/nph.19931

**Key words:** biodiversity, climate change, drought resistance, ecosystem functioning, species richness, stable isotope, TreeDivNet, tree plantations.

## Introduction

Drought occurrences have seen an upward trend alongside climate change and are anticipated to further amplify in terms of frequency, intensity, and duration in the future (IPCC, 2021). Europe has experienced multiple severe drought events since the start of the twenty-first century (Stahl et al., 2016), and warm-season droughts with prolonged soil water scarcity are recognized as the dominant drought type in Europe (Markonis et al., 2021). During the summer of 2018, large parts of the European continent faced a record-breaking severe drought and heat wave (Schuldt et al., 2020). Importantly, during the 2018 summer drought, the air temperature was 3.3°C warmer than the

long-term average between 1961 and 1990 (Schuldt et al., 2020). Due to the likelihood that drought and heat wave will occur more frequently as human-caused climate change progresses, there is an urgent need to assess how ecosystems respond to warm-season droughts, which continues to be a primary goal of ecological research (Allen et al., 2010; Doblas-Miranda et al., 2015; Keenan, 2015; Markonis et al., 2021).

Drought impacts on natural forests have been well documented, such as early leaf senescence, canopy dieback, growth reduction, and tree mortality (Schuldt et al., 2020; Schnabel et al., 2022). To better understand how to mitigate drought impacts, and to help adapt management practices to the novel drought conditions, a better understanding of the processes involved is

<footnote n="1">¹State Key Laboratory of Herbage Improvement and Grassland Agro-Ecosystems, and College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, China;</footnote>

<footnote n="2">²Department of Earth and Environmental Sciences, KU Leuven, Leuven, 3001, Belgium; ³Soil Science, Faculty of Agricultural and Environmental Sciences, University of Rostock, Justus-von-Liebig-Weg 6, Rostock, 18059, Germany; ⁴BIOGECO, INRAE, University of Bordeaux, Cestas, 33610, France; ⁵German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Puschstrasse 4, Leipzig, 04103, Germany; ⁶Institute of Biology, Leipzig University, Puschstrasse 4, Leipzig, 04103, Germany; ⁷Geobotany, Faculty of Biology, University of Freiburg, Schätzlestr. 1, Freiburg, 79104, Germany; ⁸Renewable Resources, Faculty of Agriculture, Life and Environmental Sciences, University of Alberta, Edmonton, AB, T6G 2R3, Canada; ⁹Earth and Life Institute, Université catholique de Louvain, Croix du Sud 2 - box L7.05.24, Louvain-la-Neuve, 1348, Belgium; ¹⁰Ministry of Environment, Climate, Energy and Agriculture (BUKEA), Hamburg, 21109, Germany; ¹¹Forest Dynamics, Swiss Federal Research Institute WSL Birmensdorf, Zuercherstrasse 111, Birmensdorf, 8903, Switzerland; ¹²Forest & Nature Lab, Campus Gontrode, Department of Environment, Ghent University, Melle-Gontrode, 9090, Belgium; ¹³KU Leuven Plant Institute, KU Leuven, Leuven, 3001, Belgium</footnote>
required. For example, research has theoretically and experimen-
tally demonstrated that biodiversity not only promotes tree pro-
ductivity (Scherer-Lorenzen, 2014; Ammer, 2019; van der
Plas, 2019) but can also enhance the stability of natural forests in
response to droughts (Grossiord, 2020; Schnabel et al., 2021;
Hatton et al., 2024). Two nonmutually exclusive processes
potentially contribute to the mitigating effects of tree diversity on
the functional responses to droughts (Kunert & Mercado, 2015;
Grossiord, 2020; Trogisch et al., 2021): selection and comple-
mentarity effects. Selection effects can arise from: a stochastic
component, suggesting that as species richness increases, there is
a greater probability of desiccation-tolerant species being
incorporated into the community (Huston, 1997); while the
functional component of selection effects suggests that
desiccation-tolerant species can become dominant, thereby
increasing drought resistance of the community. Conversely,
complementarity effects may stem from: niche differentiation,
leading to not only reduced resource competition for water in
space and time but also more efficient use of resources that are
often not evenly distributed across the vertical stratification of
roots and canopy; as well as from acilitation, wherein one species
experiences positive impacts from the presence of other species,
for example, due to hydraulic redistribution (Wright et al., 2017;
Grossiord, 2020). Although these processes theoretically suggest
positive effects of tree species diversity on forest drought
responses, empirical evidence is not conclusive, and only 42% of
the empirical studies in a recent review showed positive effects
of tree species diversity in mediating drought impacts on forests
(Grossiord, 2020). Compared with monocultures, mixed forests
are typically more productive and require more water, which
could cause the water supply in the soil to run out sooner and
leave them more susceptible to severe droughts (Grossiord
et al., 2014b; Forrester, 2015). These research findings hold con-
siderable significance, given the growing recognition of forest
planting as a pivotal nature-based solution to counteract climate
change, rehabilitate degraded land, and sustain biodiversity and
ecosystem functioning (Messier et al., 2021).

Nonetheless, it is important to acknowledge that transferring
research insights, derived from natural forests, directly into tree
plantations might not be straightforward. Plantations typically
comprise stands of uniform age, exhibiting reduced structural
and genetic diversity. Over time, the impact on biodiversity
may evolve and strengthen (Meyer et al., 2016; Huang
et al., 2018; Wagg et al., 2022), while susceptibility to drought
can also vary with plantation age (Socha et al., 2023). Moreover,
differences in drought vulnerability between naturally regener-
ated and planted forests have been documented (Zadworny
et al., 2014). Hence, there is still debate over whether mixed tree
plantations are more drought-resistant than monocultures in
forest plantations (Kunert & Mercado, 2015; Hajek
et al., 2022). Two mechanisms are crucial to define drought
resistance, that is desiccation delay and desiccation tolerance
(Tyree et al., 2003). The former is associated with plant traits
such as deep rooting depth and early stomatal closure, which
can improve access to water while decreasing water loss. Desic-
cation tolerance is associated with plant traits such as leaf

relative water content and water potentials, which enable plants
to endure desiccation. However, prior research has rarely eluci-
dated the mechanisms behind the drought resistance of tree
plantations. Therefore, a better mechanistic understanding of
the drought resistance in tree plantations is required to improve
the design and management of forest ecosystems in response to
droughts (Messier et al., 2021).

Across a broad spectrum of species and environmental condi-
tions, the carbon (C) isotopic ratio (δ¹³C) in the aboveground
tissues is recognized as a reliable indicator to assess drought
impacts on plants, because δ¹³C is associated with plant water
availability (Grossiord et al., 2014b; Bonal et al., 2017; Jucker
et al., 2017). For instance, under drought conditions, C₃ plants
may close their stomata to prevent desiccation (i.e. desiccation
delay). Consequently, the intercellular concentrations of CO₂
will decrease (Grossiord et al., 2014b; Jucker et al., 2017), which
results in a greater amount of ¹³C being fixed, thereby increasing
δ¹³C in plant tissues (Farquhar et al., 1982, 1989; Arndt &
Wanek, 2002; Peuke et al., 2006). With tree diversity mitigating
drought impacts due to complementarity or selection effects,
increases in δ¹³C will be lower in species-rich forests than those
in monospecific stands. As a result, a negative relationship
between tree species richness and δ¹³C should be observed during
droughts (Grossiord et al., 2014b; Forrester et al., 2016; Jucker
et al., 2017). However, earlier work in mature European forests
on plant δ¹³C using an approach of dendrochronological analysis
has demonstrated inconsistent results, including positive, neutral,
or negative effects of tree diversity on desiccation tolerance
(Grossiord et al., 2014b; Forrester et al., 2016; Jucker
et al., 2017). These studies were performed in natural forest
ecosystems, where confounding factors may override potential
tree diversity effects. Within the present study, we propose to use
observations from tree diversity experiments across a wide range
of climatic and environmental conditions using the natural C iso-
topic ratio. Moreover, such controlled tree diversity experiments
provide a unique opportunity to investigate the mechanisms
underlying tree diversity effects on the functional response of tree
plantations to droughts.

The interaction of droughts with nutrient availability and
uptake is important for understanding drought impacts on trees
(Gessler et al., 2017). However, patterns of nitrogen (N) isotopic
ratio (δ¹⁵N) in leaves are often highly variable, since it is affected
by many factors, such as changes in N availability, soil microbial
activities, distribution of N assimilation between roots and
shoots, and N metabolism within the plants (Robinson, 2001).
Unlike δ¹³C in plant aboveground tissues, the response of leaf
δ¹⁵N to droughts is not well understood. Previous literature has
shown that plant δ¹⁵N increases with a decline in mean annual
precipitation (MAP) (Handley et al., 1999; Scherer-Lorenzen
et al., 2003; Craine et al., 2009; Ma et al., 2012). This implies
that N cycling in water-limited systems is more open and typi-
cally results in higher ¹⁴N losses, but a higher ¹⁵N retention (Ma
et al., 2012). Consequently, there is an enrichment of the soil
δ¹⁵N pool, which results in a subsequent increase in plant δ¹⁵N
under droughts (but see Peuke et al., 2006; Dudley et al., 2023).
These studies, however, did not investigate the effects of tree
<table><caption>Table 1 Site characteristics, experiment design, and sampling information of the seven experiments of the tree diversity network (TreeDivNet).</caption><thead><tr><th></th><th>ECOLINK</th><th>MyDiv</th><th>FORBIO</th><th>FORBIO</th><th>FORBIO</th><th>IDENT</th><th>ORPHEE</th></tr></thead><tbody><tr><td><strong>Site characteristics</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Location</td><td>Rostock</td><td>Bad Lauchstädt</td><td>Hechtel-Eksel</td><td>Zedelgem</td><td>Gedinne</td><td>Freiburg</td><td>Pierroton</td></tr><tr><td>Country</td><td>Germany</td><td>Germany</td><td>Belgium</td><td>Belgium</td><td>Belgium</td><td>Germany</td><td>France</td></tr><tr><td>Latitude/</td><td>54°N, 12°E</td><td>51°N, 12°E</td><td>51°N, 5°E</td><td>51°N, 3°E</td><td>50°N, 5°E</td><td>48°N, 8°E</td><td>45°N, -1°E</td></tr><tr><td>Longitude</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Elevation (m asl)</td><td>13</td><td>118</td><td>55-56</td><td>10-16</td><td>367-376, 421-426</td><td>278</td><td>60</td></tr><tr><td>Mean annual temperature (°C)</td><td>8.4</td><td>8.8</td><td>10.2</td><td>10.9</td><td>8.3</td><td>10.4</td><td>13.8</td></tr><tr><td>Mean annual precipitation (mm)</td><td>591</td><td>484</td><td>887</td><td>850</td><td>1336</td><td>887</td><td>944</td></tr><tr><td>Former land use</td><td>Agriculture</td><td>Agriculture</td><td>Heathland converted to pine plantation in 1908</td><td>Agriculture: crops and grass</td><td>Forest: broadleaved until 1920, spruce plantations until 2005</td><td>Military training area and barracks until 1945, then grassland with sheep grazing</td><td>Forest: Pinus pinaster plantation</td></tr><tr><td>Soil type</td><td>Stagnic cambisol</td><td>Haplic Chernozem developed from Loess</td><td>Dry sandy soil with gravel substrate</td><td>Relatively dry sandy soil to moderately wet loamy sand soil</td><td>Moderately dry stony loam soils</td><td>Cambisol, loam</td><td>Sandy podzol on sandstone bedrock</td></tr><tr><td><strong>Experiment information</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Planting year</td><td>2014</td><td>2015</td><td>2012</td><td>2009, 2010</td><td>2010</td><td>2013</td><td>2008</td></tr><tr><td>Experiment design</td><td>Randomized block</td><td>Randomized block</td><td>Randomized block</td><td>Randomized block</td><td>Randomized block</td><td>Randomized block</td><td>Randomized block</td></tr><tr><td>Number of blocks</td><td>3</td><td>2</td><td>2</td><td>2</td><td>2</td><td>4</td><td>8</td></tr><tr><td>Number of plots</td><td>9</td><td>80</td><td>41</td><td>42</td><td>44</td><td>415</td><td>256</td></tr><tr><td>Plot size (m²)</td><td>92</td><td>121</td><td>1296</td><td>1764</td><td>1575, 1764</td><td>13</td><td>400</td></tr><tr><td>Number of trees planted</td><td>1296</td><td>11 200</td><td>23 040</td><td>32 810</td><td>33 404</td><td>19 992</td><td>25 600</td></tr><tr><td>Level of genetic diversity</td><td>1, 2</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Level of species richness</td><td>—</td><td>1, 2, 4</td><td>1, 2, 3, 4</td><td>1, 2, 3, 4</td><td>1, 2, 3, 4</td><td>1, 2, 4, 6</td><td>1, 2, 3, 4, 5</td></tr><tr><td>Size of species pool</td><td>2</td><td>10</td><td>5</td><td>5</td><td>5</td><td>12</td><td>5</td></tr></tbody></table>
<table><caption>Table 1 (Continued)</caption><thead><tr><th></th><th>ECOLINK</th><th>MyDiv</th><th>FORBIO</th><th>FORBIO</th><th>FORBIO</th><th>IDENT</th><th>ORPHEE</th></tr></thead><tbody><tr><td>Species pool</td><td>Salix varieties: Salix dasyclados var. 'Loden', Salix schwerinii × Salix viminalis var. 'Tora'</td><td>Acer pseudoplatanus, Aesculus hippocastanum, Fraxinus excelsior, Prunus avium, Sorbus aucuparia, Betula pendula, Carpinus betulus, Fagus sylvatica, Quercus petraea, Tilia platyphylos</td><td>Betula pendula, Fagus sylvatica, Pinus Sylvestris, Quercus robur, Tilia cordata</td><td>Betula pendula, Larix kaempferi, Pinus Sylvestris, Pseudotsuga menziesii, Quercus petraea</td><td>Acer pseudoplatanus, Fagus sylvatica, Larix eurolepis, Pseudotsuga menziesii, Quercus petraea</td><td>Acer platanoides, Acer saccharum, Betula papyrifera, Betula pendula, Larix decidua, Larix laricina, Picea abies, Picea pungens var. glauca, Pinus strobus, Pinus sylvestris, Quercus robur, Quercus rubra</td><td>Betula pendula, Pinus pinaster, Quercus pyrenaica, Quercus robur, Quercus ilex</td></tr><tr><td>Sampling information</td><td>2018/06</td><td>2018/10</td><td>2018/08</td><td>2018/10</td><td>2018/10</td><td>2018/08</td><td>2018/10</td></tr><tr><td>Sampling year/ month</td><td>3</td><td>2</td><td>2</td><td>2</td><td>2</td><td>4</td><td>3</td></tr><tr><td>Number of blocks sampled</td><td>9</td><td>79</td><td>40</td><td>42</td><td>43</td><td>193</td><td>24</td></tr><tr><td>Number of plots sampled</td><td>12</td><td>197</td><td>99</td><td>102</td><td>99</td><td>470</td><td>44</td></tr><tr><td>Number of samples</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mean δ<sup>13</sup>C ± SE (%)</td><td>-27.1 ± 0.12de</td><td>-27.6 ± 0.08e</td><td>-28.2 ± 0.07 cd</td><td>-30.5 ± 0.09a</td><td>-28.2 ± 0.12 cd</td><td>-29.1 ± 0.08b</td><td>-28.5 ± 0.14c</td></tr><tr><td>Mean δ<sup>15</sup>N ± SE (%)</td><td>-0.55 ± 0.26d</td><td>0.22 ± 0.10d</td><td>-5.77 ± 0.14a</td><td>0.89 ± 0.15e</td><td>-4.86 ± 0.13b</td><td>1.52 ± 0.06f</td><td>-2.48 ± 0.07c</td></tr></tbody></table>

Bold indicates that leaf samples of the tree species were not collected. Different letters indicate significant differences in leaf C and N isotopic ratios among research locations at *P* < 0.05 (the Tukey Honest Significant Differences method was used). The seven experiments are ECOLINK in Rostock, MyDiv in Bad Lauchstädt, FORBIO in Hechtel-Eksel, FORBIO in Zedelgem, FORBIO in Gedinne, IDENT in Freiburg, and ORPHEE in Pierroton (see more details in *Materials and Methods* section). The research location is sorted from north to south.
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Conceptual framework illustrating how the relationships between tree species richness (SR) and leaf C and N isotopic ratios are expected to change with climatic conditions. (a) In dry years, drought increases δ¹³C, while species-rich plantations (High SR) are expected to have lower water stress and lower ¹³C being fixed, and thus have lower ¹³C than species-poor (Low SR) plantations. Box plots showcase the distribution of δ¹³C in wet years (grey) and in dry years (light green represents Low SR and dark green represents High SR). (b) We expect a negative relationship between tree species richness and δ¹³C in dry years (H1a), and this negative relationship might become stronger under more intense drought conditions (H1b). Dark blue lines represent high-intensity droughts and light blue lines represent low-intensity droughts. (c) We expect that species-rich plantations may have lower δ¹⁵N (H2a). (d) Droughts are expected to increase leaf δ¹⁵N, but there are no *a priori* expectations on differences in this increase between diversity levels (H2b).</figcaption></figure>

ecosystem functions. At the Rostock site, two Salix varieties (Salix dasyclados var. 'Loden' and S. schwerinii × S. viminalis var. 'Tora') were planted alone or in a mixture in nine plots (each 92 m² in size) in three replicate blocks. The MyDiv experiment investigates the significance of mycorrhizas in tree diversity-ecosystem functioning relationships. Ten tree species (five AM species and five ECM species) were planted as monocultures, two- and four-species polycultures in 80 plots (each 121 m² in size) distributed over two blocks. The FORBIO experiment examines the effects of tree species diversity on ecosystem functioning at three sites in Hechtel-Eksel, Zedelgem, and Gedinne, Belgium, with contrasting environmental conditions. Five tree species were planted in 127 plots (41, 42, and 44 plots per site respectively, between 1296 and 1764 m² in size) with one up to four tree species per plot. The experiment IDENT Freiburg is part of the International Diversity Experiment Network with Trees (IDENT), to study tree diversity effects at early stages after planting by using high-density planting (Wein et al., 2016).

Twelve tree species were planted in 415 plots (each 13 m² in size) in four blocks with monocultures, two-species, four-species, and six-species polycultures, from which the nonfertilized ones were used here (total of 193 plots). Finally, the ORPHEE experiment in the southwest of France is established to study the effects of climate change on tree diversity and ecosystem functioning relationships. Five tree species were planted in 256 plots (each 400 m² in size) in eight blocks with monocultures, two- and five-species polycultures.

## Leaf sampling and C and N stable isotope measurement

Leaves were collected between June and October 2018 from each tree species per plot following the protocol of Perez-Harguindeguy et al. (2013). In total, 28 different European tree species were sampled (Tables 1, S1). Since initial signs of stress recovery in trees are typically detectable through the regeneration of leaves (Dobbertin, 2005), newly formed leaves (i.e. deciduous and
evergreen species) and needles (i.e. coniferous species) during the drought were collected to detect the signal of drought. That is, the collected leaves and needles were the youngest ones, unless it was clear that they were formed after the drought. Specifically, leaves were collected using the following criteria: (1) leaves were fully expanded sun leaves or as close to full sun leaves as possible, that is between 2/3 and the top of the living crown; (2) leaves were collected from each of three trees per species in each plot; and (3) a minimum of five leaves and 4 g of fresh leaf/needle materials were collected and pooled into one sample per species per plot. Leaf samples were stored in paper envelopes and oven-dried at 60°C for 48 h in the laboratory to constant mass immediately after collecting and then stored under dry conditions until stable isotope composition analysis.

To detect the effects of tree species richness on tree responses to extreme drought, we measured leaf C and N stable isotopes during the 2018 summer European drought. We focused on leaf isotope composition, rather than plant growth for three reasons. First, isotope composition does not directly connect to plant growth (i.e. C sequestration and allocation to wood) in response to drought (Jucker et al., 2017). Second, leaf isotope composition, that is δ¹³C, is defined by only few and specific leaf-level physiological changes, in particular stomatal conductance, while growth is determined by many more factors, in particular sink limitation during growth (no growth due to hydraulic constraints). This gives then raise to different information contained in the two variables, that is leaf isotope composition and plant growth, with their relationship extremely variable (Timofeeva et al., 2017). Third, we wanted to shed more light on the mechanisms driving species diversity effects, and leaf isotope composition gives us more information on what happens in the plant. Leaf samples were first grounded with a ball mill to a fine powder. Next, leaf δ¹³C and δ¹⁵N were analyzed at the Stable Isotope Facility of KU Leuven, Leuven, Belgium (except for leaf samples from IDENT Freiburg, which were measured at the Swiss Federal Institute for Forest, Snow and Landscape Research, Forest Dynamics, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland). All samples were combusted in an elemental analyzer at 1020°C and analyzed using a continuous-flow isotope ratio mass spectrometer (Sercon Ltd, Cheshire, UK). Leaf δ¹³C and δ¹⁵N were calculated using the following equations:

$$ \delta^{13}\text{C} = \left( \frac{\left( \frac{^{13}\text{C}}{^{12}\text{C}} \right)_{\text{leaves}}}{\left( \frac{^{13}\text{C}}{^{12}\text{C}} \right)_{\text{VPDB}}} - 1 \right) \times 1000 \qquad \text{Eqn 1} $$

where δ¹³C was expressed as the C isotopic ratio (¹³C : ¹²C) of the leaf samples and of the Vienna Pee Dee Belemnite standard (VPDB, ‰).

$$ \delta^{15}\text{N} = \left( \frac{\left( \frac{^{15}\text{N}}{^{14}\text{N}} \right)_{\text{leaves}}}{\left( \frac{^{15}\text{N}}{^{14}\text{N}} \right)_{\text{AIR}}} - 1 \right) \times 1000 \qquad \text{Eqn 2} $$

where δ¹⁵N was expressed as the N isotopic ratio (¹⁵N : ¹⁴N) of leaf samples and of the International Atomic Energy

Agency standard air (AIR, ‰). The precision for the repeated analysis of standard materials was 0.1‰ for δ¹³C and 0.3‰ for δ¹⁵N.

## Tree trait data

To better understand how tree species richness affects leaf C and N isotopic ratios under drought conditions, we compiled data on desiccation tolerance and mycorrhizal type for each tree species. Desiccation tolerance data were compiled from the study of Niinemets & Valladares (2006) based on the rankings of site characteristics and plant physiological potentials for conifers, deciduous and evergreen broad-leaf species. Specifically, the duration of the dry period, the ratio of precipitation to potential evapotranspiration (PET), and the total annual precipitation were among the site characteristics. The physiological potential of plants was the lowest amount of soil water that may be sustained over an extended period without causing more than 50% of leaf damage or plant dieback. Desiccation tolerance was categorized into five categories: (1) very intolerant; (2) intolerant; (3) moderately tolerant; (4) tolerant; and (5) very tolerant. Of the 28 tree species that were examined, four species (Larix eurolepis A., Pinus pinaster Ait., S. dasyclados var. 'Loden', and S. schwerinii × S. viminalis var. 'Tora') lack desiccation tolerance data and were subsequently excluded from this analysis (Table S2). We classified the 28 tree species into two groups of mycorrhizal types based on the literature (Harley & Harley, 1987). These mycorrhizal types were AM and ECM. Since the roots of Salix varieties may be colonized by AM and ECM fungi, as well as dark-septate endophytes (Baum et al., 2018; Huang et al., 2018), we subsequently excluded S. dasyclados var. 'Loden' and S. schwerinii × S. viminalis var. 'Tora' from this analysis (Table S3).

## Climate data

To test whether climate mediates the effects of tree species richness on leaf C and N isotopic ratios, we compiled climate data for each of the seven study sites. These climate data included the Standardized Precipitation Evapotranspiration Index (SPEI; unitless), MAT (°C), MAP (mm yr⁻¹), and PET (mm). SPEI data were compiled from the SPEI Global Drought Monitor database (Vicente-Serrano et al., 2010). The SPEI data were derived based on monthly precipitation and PET at a spatial resolution of 0.5 degrees between 1950 and 2019. We only used the SPEI data throughout the sampling month for statistical analysis, while the SPEI data between 1950 and 2018 were used to visualize and inspect the temporal trends of SPEI (See Figs S2–S4 for more details). MAT, MAP, and PET data were compiled from the gridded agro-meteorological database in Europe, that is AGRI4-CAST (http://mars.jrc.ec.europa.eu/mars). The database contains interpolated climate data derived from meteorological observations, remote sensing imagery, and agro-meteorological modeling at a 25 km × 25 km spatial resolution in the form of a daily basis between 1979 and 2020. After an inspection of the correlations between climate variables, we selected MAP and PET to calculate a MAP : PET ratio that was used to define long-term water
availability in a region as in Jucker *et al.* (2016) for further data analysis.

## Soil characteristics data

To test whether soil characteristics mediate the effects of tree species richness on leaf C and N isotopic ratios, we compiled soil characteristics data of each research site from the Land Use and Cover Area frame Statistical Survey (LUCAS) database (Ballabio *et al.*, 2016). These soil characteristics data included soil available water capacity (vol %), bulk density (T m⁻³), topsoil sand proportion (%), topsoil clay proportion (%), and topsoil silt proportion (%) (see Table S4 for details). The LUCAS database was derived using the model of multivariate additive regression splines based on c. 20 000 topsoil samples in Europe at 500 m × 500 m spatial resolution. After an inspection of the correlations between these soil properties, we selected soil sand content that was used to estimate soil nutrient and water retention potential as in Ratcliffe *et al.* (2017) for further data analysis.

## Statistical analyses

In this study, we developed a two-step analytical approach. First, we examined the general relationship between the isotope composition of leaves across a diversity gradient within specific sites. During severe drought conditions, leaf isotope composition changes because stomata close to avoid desiccation, with the rate of closure increasing with greater exposure to drought. Given consistent climatic and soil conditions within each site, this set-up allows us to identify tree diversity effects on the need for desiccation avoidance and stomatal closure. Two key mechanisms potentially underlie the diversity effects: complementarity effects, driven by niche complementarity and facilitation, mitigate drought stress experienced by individual trees, alongside selection effects that promote the presence and dominance of drought-resistant species as tree diversity increases. However, given that all tree species have an equal chance of presence across diversity gradients, and we sampled leaves from all species present in each plot, our analyses cannot identify selection effects, and the observed diversity effects can be attributed to complementarity effects alone. In a subsequent analysis, we examined whether the species-specific impacts of tree diversity on leaf isotope composition were influenced by their desiccation tolerance (Niinemets & Valladares, 2006). *Pinus sylvestris* L. was an extreme outlier in this analysis, inducing a marginally significant effect (Fig. S5). We therefore report the results excluding this species. Given the acknowledged influence of mycorrhizas on plant drought resistance, we also investigated the correlation between species-specific effects and the mycorrhizal type of the studied trees.

To examine the hypothesized effects of tree species richness on leaf C and N isotopic ratios in the summer drought of 2018, and how these tree diversity effects rely on drought conditions, we separately fitted the linear mixed-effects models for leaf δ¹³C and δ¹⁵N. Fixed effects included tree species richness (log₂ transformed), drought intensity (i.e. SPEI) throughout the

sampling month, long-term water availability (MAP : PET ratio) of a site, and soil sand content. To evaluate whether drought intensity influences the effects of tree species richness on leaf isotopes, an interaction term between tree species richness and drought intensity was included. Fixed factors were centered to allow the main effects (tree species richness and drought condition variables) to be biologically explainable (Schielzeth, 2010). Random effects included in the linear mixed-effects models were a block factor nested within a site factor (locality), a species identity factor, and an interaction factor between species identity and species richness. A visual inspection of residual plots was conducted to identify any deviations from a linear form. For the estimated coefficients of fixed factors, we used the bootstrap sample to calculate the 95% confidence intervals. We conducted 500 repetitions of bootstrap samplings for each linear mixed-effects model. A pseudo-R² was estimated to determine the marginal coefficient of determination (variance explained by the fixed factors) and the conditional coefficient of determination (variance explained by both the fixed and random factors; Nakagawa *et al.*, 2017). We conducted a sensitivity analysis of linear mixed-effects models by removing the Zedelgem site owing to the lower values of the leaf δ¹³C, specifically -30.5 ± 0.09‰ (Table S5).

To determine species-specific effects of tree species richness on leaf C and N isotopic ratios in the summer drought of 2018, we conducted general linear models following Hector *et al.* (1999). For each of the 28 tree species (Table S1), leaf δ¹³C and δ¹⁵N were separately regressed on tree species richness (log₂ transformed). If the estimated coefficients (slopes) were positive, the leaf δ¹³C and δ¹⁵N values were higher in monocultures. Instead, if the estimated coefficients (slopes) were negative, the leaf δ¹³C and δ¹⁵N values were higher in monocultures. Experimental sites (i.e. locality) and blocks were also included before the tree species richness term was entered into the general linear models. We, therefore, calculated the total sums of squares explained by tree species richness by accounting for the influences of experimental sites and blocks.

To determine whether tree traits mediate species-specific effects of tree species richness on leaf C and N isotopic ratios, we assessed the associations between the estimated coefficients of general linear models (i.e. the effects of tree species richness on leaf isotopes) and desiccation tolerance and mycorrhizal type, respectively. We computed Pearson correlation coefficients to investigate the relationships between estimated coefficients and desiccation tolerance. A permutation test on correlation was further used to assess the significance of correlation coefficients. We conducted general linear models to assess the effects of mycorrhizal type on the estimated coefficients. A permutation t-test was further used to assess the significance of mycorrhizal type effects (Figs S6, S7). All permutation tests were conducted with 9999 simulations.

All statistical analyses were performed in R (v.4.0.3) (R Development Core Team, 2019). We used LME4 package (Bates *et al.*, 2015) for linear mixed-effects models and the TIDYVERSE package (Wickham *et al.*, 2019) for data preprocessing and visualization.
Results

Effects of tree species richness on leaf δ¹³C and δ¹⁵N

A total of 1023 leaf samples were analyzed from the seven Eur-
opean tree diversity experiments (Table 1). We found that leaf
δ¹³C decreased significantly with tree species richness (Fig. 2a);
the estimated coefficient/slope was −0.19 with 95% confidence
interval [−0.33, −0.05] (Table 2). The predicted mean leaf δ¹³C
ranged from −28.1‰ (95% confidence interval [−29.2‰,
−27.1‰]) in monocultures to −28.6‰ (95% confidence inter-
val [−29.7‰, −27.6‰]) in mixtures of six tree species
(Fig. 2a). The effects of tree species richness on leaf δ¹³C were
independent of SPEI, MAP : PET, and soil sand content (all
interaction terms were not significant in the linear mixed-effects
model, Table 2). Among the random effects assessed, the variabil-
ity in leaf δ¹³C was mainly explained by site (38.0%) and species
identity (31.5%; Table 2).

Leaf δ¹⁵N and tree species richness relationships varied with drought intensity, as represented by the interaction between tree species richness and SPEI in the sampling months (the estimated effects of interaction = -0.22, 95% confidence interval [-0.32, -0.12]; Table 2). Specifically, the predicted mean of leaf δ¹⁵N increased with tree species richness under high drought intensity, while it decreased with tree species richness under low drought intensity (Fig. 2b). However, the main effects of tree species richness were negligible (the estimated effects of tree species richness = 0.02, 95% confidence interval [-0.08, 0.13] and the

estimated main effects of SPEI = -2.68, 95% confidence inter-
val [-8.68, 3.31]; Table 2). Among the random effects, the
variability in leaf δ¹⁵N was mainly explained by the site (84.4%),
followed by species identity (7.7%; Table 2).

Species-specific effects of tree species richness on leaf δ¹³C and δ¹⁵N

The effects of tree species richness on leaf δ¹³C varied among species (Fig. 3a,b). After adjusting for the influences of research sites and experimental blocks, nine tree species (32%) out of 28 showed significantly negative effects of tree species richness on leaf δ¹³C (i.e. no overlap between zero and the 95% confidence interval; Fig. 3a; Table S6). *Quercus robur* L. had the largest negative effect of tree species richness, followed by three coniferous tree species *Pinus strobus* L., *Larix laricina* (Du Roi) K., and *P. sylvestris* L. *eurolepis* was the only species with significantly positive effects of tree species richness (Fig. 3a). Variance in leaf δ¹³C explained by tree species richness varied between 0 and 30.1% (Fig. 3b; Table S6).

The effects of tree species richness on leaf δ¹⁵N varied consid-
erably among species (Fig. 3c,d). After adjusting for differences
in research sites and experimental blocks, *Fagus sylvatica* L. and
*Pseudotsuga menziesii* (Mirb.) F. were the only two species, com-
prising only 7% of the total 28 species evaluated, which exhibited
significantly negative effects of tree species richness, while *Picea
abies*, *P. strobus*, and *Q. robur* had significantly positive effects of
tree species richness, comprising 11% of the species studied
(Fig. 3c,d; Table S7).

<figure><img src="image_4.png" /><figcaption>**Fig. 2** Effects of tree species richness on leaf δ¹³C (a) and δ¹⁵N (b) in the summer drought of 2018 across the seven European tree diversity experiments. (a) Tree species richness significantly decreases leaf δ¹³C, while the effect does not vary with drought intensity (i.e. no interaction). The line represents the estimated coefficient, and the band represents the 95% confidence intervals of the linear mixed-effects models. (b) The effect of tree species richness on leaf δ¹⁵N varies with drought intensity. The line represents the estimated coefficients. Dark lines represent high-intensity droughts and light lines represent low-intensity droughts. The points in (a, b) are jittered for visualization and represent the predicted values of leaf δ¹³C and δ¹⁵N using the linear mixed-effects models. Drought intensity is represented by the Standardized Precipitation Evapotranspiration Index (SPEI) which is derived from the Global SPEI database for the given sampling month at each tree diversity experiment site (Materials and Methods section). See Table 2 for detailed test statistics and supporting Information Figs S2–S4 for the trend of SPEI at each tree diversity experiment site.</figcaption></figure>
<table><caption>**Table 2** Effects of tree species richness on leaf carbon (C) and nitrogen (N) isotopic ratios in the summer drought of 2018.</caption><thead><tr><th></th><th>δ<sup>13</sup>C</th><th>δ<sup>15</sup>N</th></tr></thead><tbody><tr><td><strong>Fixed effects</strong></td><td></td><td></td></tr><tr><td>Intercept</td><td>-28.4 [-30.1, -26.8]<sup>†</sup></td><td>-0.65 [-5.06, 3.76]</td></tr><tr><td>Species richness<br>(log<sub>2</sub> scale)</td><td>-0.19 [-0.33, -0.05]</td><td>0.02 [-0.08, 0.13]</td></tr><tr><td>SPEI</td><td>0.14 [-2.14, 2.43]</td><td>-2.68 [-8.68, 3.31]</td></tr><tr><td>PPET</td><td>-1.65 [-11.5, 8.24]</td><td>-11.4 [-36.9, 14.2]</td></tr><tr><td>Sand</td><td>-0.01 [-0.07, 0.05]</td><td>-0.01 [-0.17, 0.14]</td></tr><tr><td>Richness × SPEI</td><td>0.06 [-0.05, 0.17]</td><td>-0.22 [-0.32, -0.12]</td></tr><tr><td><strong>Variance components (VC%)</strong></td><td></td><td></td></tr><tr><td>Site</td><td>1.15 (38.0)</td><td>8.99 (84.4)</td></tr><tr><td>Block</td><td>0.02 (0.6)</td><td>0.02 (0.2)</td></tr><tr><td>Species identity<br>(Identity)</td><td>0.96 (31.5)</td><td>0.81 (7.7)</td></tr><tr><td>Richness × Identity</td><td>0.07 (2.4)</td><td>0.02 (0.2)</td></tr><tr><td>Residuals</td><td>0.84 (27.5)</td><td>0.80 (7.5)</td></tr><tr><td><strong>Pseudo-R<sup>2</sup>‡</strong></td><td></td><td></td></tr><tr><td>Marginal</td><td>0.07</td><td>0.21</td></tr><tr><td>Conditional</td><td>0.74</td><td>0.94</td></tr></tbody></table>

Fixed effects, variance components of random factors, and pseudo-R² are shown for the linear mixed-effects models of leaf δ¹³C and δ¹⁵N, respectively. Significant fixed terms are shown in bold. Insignificant interaction terms are excluded from the linear mixed-effects models. SPEI denotes the Standardized Precipitation Evapotranspiration Index, PPET denotes MAP: PET ratio, and sand denotes sand content. MAP, mean annual precipitation; PET, potential evapotranspiration.

†Estimated coefficients of the linear mixed-effects models with 95% confidence interval.

‡Marginal R² represents model variation explained by the fixed factors, and conditional R² represents model variation explained by the fixed and random factors.

## Associations between species-specific effects of tree species richness on leaf isotopes and desiccation tolerance and mycorrhizal type

We found that desiccation tolerance did not mediate the species-specific effects of tree species richness on leaf δ¹³C (Fig. 4a; Pearson correlation coefficient *r* = -0.28, *P*-value = 0.211, *P*-value of the permutation test = 0.208). Similarly, species-specific effects of tree species richness on leaf δ¹⁵N were not significantly associated with desiccation tolerance (Fig. 4b; *r* = -0.38, *P*-value = 0.078, *P*-value of the permutation test = 0.078). Species-specific effects of tree species richness on leaf δ¹³C and δ¹⁵N were not mediated by mycorrhizal type (Figs 4c,d, S6, S7).

## Discussion

The seven research sites of tree diversity experiments experienced an extreme natural drought event in 2018. This occurrence offers a rare chance to evaluate whether and how the impacts of extreme drought on leaf C and N isotopic ratios can be mediated by tree species richness. We show a generally negative relationship between tree species richness and leaf δ¹³C across the seven tree diversity experiments (Fig. 2). We further show that the effects of tree species richness on leaf δ¹³C vary depending on the tree species examined but found no evidence that they are modulated by

their desiccation tolerance (Figs 3, 4). Although an overall relationship between tree species richness and leaf δ¹⁵N did not become evident, our work demonstrates that this relationship is modulated by drought intensity. The relationship varies from weakly negative under low-intensity droughts to weakly positive under high-intensity droughts (Fig. 2).

## Effects of tree species richness on leaf C isotopic ratio under drought conditions

Increases in C isotope values are frequently observed during droughts (Sarris *et al.*, 2013; Jucker *et al.*, 2017; Siegwolf *et al.*, 2023). Our work shows a significantly negative relationship between tree species richness and leaf δ¹³C under drought conditions (Fig. 2), indicating that the drought-induced increase in leaf δ¹³C is smaller in species-rich tree plantations than in species-poor tree plantations. This result is in line with our hypothesis H1a and is supported by previous research that often found negative effects of tree species richness on C isotopic ratio using stable C isotope values from tree rings rather than leaves (Grossiord *et al.*, 2014b; Forrester *et al.*, 2016). As species-rich forests and tree plantations usually show better resource partitioning and facilitation (e.g. hydraulic lift of deeper soil water by tree species with deeper rooting depth) than species-poor ones (Grossiord, 2020), this reduces water stress and, consequently, lowers the rise in δ¹³C values during drought events (but see the exceptions in Grossiord *et al.*, 2014b; Forrester *et al.*, 2016). Importantly, we found that tree species richness decreased leaf δ¹³C by 0.5‰ from monocultures to six tree species plots. Grossiord *et al.* (2014b) reported that, on average across a wide range of European forests, droughts increased tree ring δ¹³C from 0.55‰ to 1.11‰ (i.e. the differences in tree ring δ¹³C between a dry year and a wet year). Our result, therefore, suggests that tree species richness may completely or partially mitigate the impacts of drought on δ¹³C. These findings highlight that biodiversity is an important ecosystem property that can promote forest drought resistance (Sousa-Silva *et al.*, 2018; Grossiord, 2020).

When delving into the species-specific relationships, we found considerable variation in the slopes between tree diversity and δ¹³C. Only nine of the 28 tree species (c. 32%) have significantly lower leaf δ¹³C values in species-rich tree plantations than in species-poor ones (Fig. 3). We even found a positive slope for *L. eurolepis*, indicating that this species is more water-stressed in species-rich tree plantations than in species-poor ones. *L. eurolepis* is among the fastest-growing tree species in the species pool. Its higher observed drought stress when mixed with other tree species could be related to the corresponding reduced belowground competition due to facilitation and resource partitioning among species (Grossiord, 2020), which in turn might have resulted in a proportionally lower investment in the root system. However, *L. eurolepis* has only been planted in one experiment (FORBIO-Gedinne in Belgium; Table S1), so results must be interpreted with caution. Contrary to our expectations under the presence of complementarity effects, the species-specific effects of tree species richness on leaf δ¹³C did not tend to change with species desiccation tolerance (Fig. 4). Although our
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Species-specific effects of tree species richness on leaf δ¹³C (a, b) and δ¹⁵N (c, d) in the summer drought of 2018 across the seven European tree diversity experiments. Species-specific effects of tree species richness are indicated by the estimated coefficients/slopes of general linear models. Tree species are ranked by the estimated coefficients/slopes of leaf δ¹³C (a) or δ¹⁵N (c) that show the magnitude of species-specific effects of tree species richness on leaf δ¹³C and δ¹⁵N. Black points represent significantly positive coefficients, red points, significantly negative coefficients, and grey points, nonsignificant coefficients. Thick error bars represent the estimated coefficients ±1 SE, and thin error bars represent the estimated coefficients ±2 SE (i.e. 95% CI). The thin bar does not overlap zero which is considered a significant coefficient. The bars and numbers in (b, d) represent the total variance (sums of squares) explained by tree species richness after adjusting for differences in experimental sites and blocks. See Supporting Information Tables S6 and S7 for detailed test statistics.</figcaption></figure>

methodology may not have been capable of detecting selection
effects, this outcome implies that the presence of such selection
effects associated with drought resistance is improbable. This

might be driven by the maladaptation of the studied forest plan-
tations to drought events as rare and extreme as the 2018
pan-European drought.
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Species-specific effects of tree species richness on leaf δ¹³C (a, c) and δ¹⁵N (b, d) as a function of desiccation tolerance (a, b) and mycorrhizal type (c, d). Species-specific effects of tree species richness are indicated by the estimated coefficients/slopes of general linear models. (a, b) Associations between species-specific effects of tree species richness and desiccation tolerance. Pearson correlation coefficients and the associated *P*-values are shown. Dashed fitted line represents a nonsignificant association. Thick error bars represent the estimated coefficients ±1 SE, and thin error bars represent the estimated coefficients ±2 SE. (c, d) Associations between species-specific effects of tree species richness and mycorrhizal type. The *t*-values of general linear models and the associated *P*-values are shown. AM represents arbuscular mycorrhiza and ECM represents ectomycorrhiza. Black points represent significantly positive coefficients, red points significantly negative coefficients, and grey points nonsignificant coefficients.</figcaption></figure>

Environmental conditions, especially climatic conditions, are
known to modulate biodiversity and ecosystem functioning rela-
tionships (Jucker *et al.*, 2016; Ratcliffe *et al.*, 2017; Fei
*et al.*, 2018; Eisenhauer *et al.*, 2019; Jing *et al.*, 2022). Indeed,

context dependency in tree diversity effects on δ¹³C is regularly
observed in the literature. For example, Grossiord *et al.* (2014b)
observed a negative relationship between tree species diversity
and tree ring δ¹³C in two major European forest types (i.e.
temperate beech and thermophilous deciduous), a positive one in
boreal forests (Grossiord et al., 2014a), while three other forest
types (hemi-boreal, mountainous beech, and Mediterranean) did
not show significant tree species diversity effects. Similarly, For-
rester et al. (2016) observed two negative, one positive, and three
neutral effects of tree diversity on tree ring δ¹³C. However,
unlike these previous studies, our work focuses on leaf δ¹³C,
while the others studied δ¹³C in woody tissues (Arndt &
Wanek, 2002; Grossiord et al., 2014b; Forrester et al., 2016).
Leaf δ¹³C is highly associated with the processes of stomatal regu-
lation of stable C isotope discrimination during C fixation (Far-
quhar et al., 1982, 1989) and can be immediately affected by
droughts (Arndt & Wanek, 2002; Siegwolf et al., 2023). On the
contrary, it may take time to incorporate photosynthetically fixed
δ¹³C into woody tissues with multiple C allocation processes
involved (Arndt & Wanek, 2002). Additionally, most tree ring
growth occurs during the spring, and therefore, the subsequent
tree ring stable C isotope signal may not accurately represent a
period of dry and hot summer climate with very little tree ring
growth (Sarris et al., 2013; Jucker et al., 2017; Siegwolf
et al., 2023). Our work shows that the negative effects of tree spe-
cies richness on leaf δ¹³C are present across the seven tree diver-
sity experiments, irrespective of the variation in drought intensity
during the sampling months across sites, as well as the long-term
water availability and soil water retention capacity at the study
sites (Table 2). These results do not support our hypothesis
that higher drought intensity increases the effects of tree species
richness on leaf δ¹³C (i.e. stress gradient hypothesis; H1b).
This underscores the likelihood that the mechanisms driving
these effects may differ significantly between sites (Fig. S8;
Tables S8, S9; Grossiord et al., 2014b). Future research could
aim to more precisely discern the relative significance of resource
partitioning and facilitation, among other mechanisms not
addressed in this study, including competition, species
dominance, neighbor identity, and environmental filtering
(Grossiord, 2020).

Effects of tree species richness on leaf N isotopic ratio under drought conditions

The overall effect of tree species richness on leaf δ¹⁵N under the 2018 summer drought is not significant. This result does not support our hypothesis that there is a negative relationship between tree species richness and leaf δ¹⁵N (H2a), driven by lower soil N losses in high-diversity plots (Zak *et al.*, 2003; Lang *et al.*, 2013; Chen *et al.*, 2020). Zooming in on each of the 28 tree species examined, we found no tree species richness effects on leaf δ¹⁵N for 82% of tree species (Fig. 3). Species-specific results were not found to be mediated by the type of mycorrhizal association (Fig. 4).

We found that experimental sites accounted for 84.2% of the
variability in leaf δ¹⁵N (Table 2), implying that the relationship
between tree species richness and leaf δ¹⁵N is highly context-
dependent. Such strong context-dependency effects on biodiversity–
ecosystem functioning relationships have been reported in forest
ecosystems before (Jucker et al., 2016; Ratcliffe et al., 2017; Fei

et al., 2018; Mori, 2018). Moreover, although the main effect of tree species richness on leaf δ¹⁵N was not significant, we highlight the role of drought intensity in shaping context dependency. Specifically, we observed a significantly negative interaction between species richness and drought intensity. The marginal effects plot, incorporating both main and interaction effects (Fig. 2b), illustrates that leaf δ¹⁵N increases with drought intensity, partially aligning with our hypothesis H2b that droughts elevate leaf δ¹⁵N and indicating a transition toward more open N cycling as water availability declines. Furthermore, we demonstrate that severe droughts may counterbalance the adverse effects of tree species richness, resulting in a weakened negative association between tree species richness and leaf δ¹⁵N under heightened drought intensity. We found a slightly negative relationship between tree species richness and leaf δ¹⁵N under low drought intensity, while under higher drought intensity the relationship becomes slightly positive. Hence, not only the magnitude of tree species richness effects on leaf δ¹⁵N changes with drought intensity, but also the direction of tree diversity effects. Future research is necessary to further reveal the mechanisms driving the interaction between drought intensity and species richness, offering insights into N cycling under extreme droughts.

Conclusion

Considering the increasing frequency of droughts due to climate
change (IPCC, 2021), and the need to design resilient tree plant-
tations capable of providing diverse ecosystem services even dur-
ing extreme drought conditions (Messier et al., 2021),
understanding how tree diversity influences ecosystem responses
to severe droughts could enable mitigation of their impacts on
tree plantations. In our study, we present clear evidence that
drought directly contributes to an increase in leaf δ¹³C, while
tree species richness decreases leaf δ¹³C. The negative relation-
ship between tree species richness and leaf δ¹³C is likely driven
by complementarity effects. Furthermore, we found that the
species-specific effects of tree species richness on leaf δ¹³C did
not vary with species desiccation tolerance, suggesting that the
presence of drought-related selection effects in the studied forest
plantations is improbable. Unlike leaf C isotopic ratio, the
effect of tree species richness on leaf δ¹⁵N depends on drought
intensity, with an increasingly positive effect observed under
high drought intensity. Thus, our findings suggest that dual leaf
isotope analysis aids in comprehending the relationship among
species richness, nutrients, and drought. Our work holds signifi-
cant relevance for the management of tree plantations, with
more diverse plantations being more resilient – and thus eco-
nomically more attractive – under ongoing climate change with
more frequent droughts. However, given the considerable varia-
tion in the observed impact of tree species richness on leaf δ¹³C
across different sites, further research is required to clarify the
relative significance of resource partitioning and facilitation,
among other mechanisms not addressed in this study. It would
also be of interest for future research to broaden the framework
for better understanding how tree species interact with each
other in response to extreme climate events across varying envir-
onmental gradients.
Acknowledgements

The authors thank site managers, field technicians, and student
 helpers for establishing and managing the TreeDivNet experi-
 ments and for collecting the leaf samples. We thank Marijn Bau-
 ters and Pascal Boeckx for the discussion about the
 interpretation of the results of the nitrogen isotope. We thank
 Margaux Casier for the help in compiling data on desiccation
tolerance. We thank the team of the Stable Isotope Research
 Centre (SIRC) at WSL Birmensdorf for support in the labora-
tory. The research was supported by the Internal Funds of the
 KU Leuven (XJ). CB acknowledges funding from the German
 Research Foundation (DFG, project no.: BA 1494/9-1). NE and
 OF acknowledge the support from the German Centre for Inte-
grative Biodiversity Research (iDiv) Halle-Jena-Leipzig funded
 by the German Research Foundation (DFG- FZT 118,
 202548816) and funding by the DFG (Ei 862/29-1 and Ei
 862/31-1). LR, PH, CAN, and MSL acknowledge funding by
 the German Research Foundation (DFG 384026712 to LR;
 DFG 316733524 to CAN) and by the University of Freiburg
 (Innovationsfonds Forschung to MSL). The maintenance of the
 FORBIO-Gedinne site is partly supported by the 5-yr research
 program ‘Accord-cadre de recherches et de vulgarisation forest-
ières’ funded by the Walloon Forest Service (SPW-DNF). The
 research presented here contributes to the Global Network of
 Tree Diversity Experiments (TreeDivNet; https://treed.ivnet.
ugent.be/).

Competing interests

None declared.

Author contributions

KVM, BM, and KV conceived of the study. XJ and KVM led the
analyses and writing of the manuscript. All the authors contribu-
ted substantially to the result interpretation and manuscript revi-
sion.

ORCID

Christel Baum https://orcid.org/0000-0003-1179-2149

Bastien Castagneyrol https://orcid.org/0000-0001-8795-7806

Nico Eisenhauer https://orcid.org/0000-0002-0371-6720

Olga Ferlian https://orcid.org/0000-0002-2536-7592

Tobias Gebauer https://orcid.org/0000-0003-2751-4071

Peter Hajek https://orcid.org/0000-0001-5268-8917

Hervé Jactel https://orcid.org/0000-0002-8106-5310

Xin Jing https://orcid.org/0000-0002-7146-7180

Bart Muys https://orcid.org/0000-0001-9421-527X

Charles A. Nock https://orcid.org/0000-0002-3483-0390

Quentin Ponette https://orcid.org/0000-0002-2726-7392

Laura Rose https://orcid.org/0000-0003-4523-4145

Matthias Saurer https://orcid.org/0000-0002-3954-3534

Michael Scherer-Lorenzen https://orcid.org/0000-0001-9566-590X

Koenraad Van Meerbeek https://orcid.org/0000-0002-9260-3815

Kris Verheyen https://orcid.org/0000-0002-2067-9108

Data availability

The data for replication of the results in this work are openly
available in Tables S2, S3, S6, S7, and S10.

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Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Hypothesis on the relationship between desiccation tolerance of tree species and their species-specific relationship between species richness and leaf δ¹³C.

**Fig. S2** Pattern of the Standardized Precipitation Evapotranspiration Index (SPEI) at the tree diversity experimental sites between 1950 and 2019.

**Fig. S3** Changes in the Standardized Precipitation Evapotranspiration Index (SPEI) in 2018 across the studied regions.

**Fig. S4** Trends of the Standardized Precipitation Evapotranspiration Index (SPEI) in 2008 across the tree diversity experiments.
**Fig. S5** A sensitivity analysis on the species-specific effects of tree species richness on leaf δ¹³C and leaf δ¹⁵N as a function of desiccation tolerance when including *Pinus sylvestris*.

**Fig. S6** Permutation *t*-test for the associations of species-specific effects of tree species richness on leaf δ¹³C as a function of mycorrhizal type.

**Fig. S7** Permutation *t*-test for the associations of species-specific effects of tree species richness on leaf δ¹⁵N as a function of mycorrhizal type.

**Fig. S8** The effects of tree species richness on leaf δ¹³C vary with sites.

**Table S1** Number of leaf samples measured in the laboratory per species per location.

**Table S2** Desiccation tolerance of the tree species examined in this study.

**Table S3** Mycorrhizal types of the tree species examined in this study.

**Table S4** Soil characteristics of the research site.

**Table S5** Sensitivity analysis for the effects of tree species richness on leaf C and N isotopic ratios in the summer drought of 2018 by removing the Zedelgem site.

**Table S6** Species-specific relationships between tree species richness and leaf δ¹³C in the summer drought of 2018.

**Table S7** Species-specific relationships between tree species richness and leaf δ¹⁵N in the summer drought of 2018.

**Table S8** Effects of tree species richness on leaf δ¹³C across sites.

**Table S9** Effects of tree species richness on leaf δ¹³C across sites and species.

**Table S10** Leaf isotope data for replication of the results in this work.

Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.