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RESEARCH ARTICLE

# The afterlife effects of leaf and root litter traits on soil N cycling

Janna Wambsganss<fnref n="1" />,²,³ ID

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Raoul Huys<fnref n="1" /> ID

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Hättenschwiler Stephan⁴

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Vincent Poirier⁵

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Alison D. Munson⁶ ID

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Grégoire T. Freschet<fnref n="1" /> ID

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**Correspondence**

Janna Wambsganss

Email: janna.wambsganss@gmail.com

**Funding information**

Mediterranean Center for Environment and Biodiversity (CEMEB), Grant/Award Number: ANR-10-LABX-0004; Towards a Unified theory of biotic Interactions (TULIP), Grant/Award Number: ANR-10-LABX-0041; EC2CO grant 'MULTIVERS'

Handling Editor: Natalia Pérez-Harguindeguy

**Abstract**

1. Litter nitrogen (N) release during decomposition crucially influences ecosystem N cycling and the amount of N available to plants and other soil organisms. However, the role of initial litter traits in affecting patterns of temporal N release from litters and determining the fate of N in the soil is still poorly understood.

2. Here, we measured litter N release during the 12-month decomposition of ¹⁵N-labelled leaf and root litters of 12 common Mediterranean herbaceous species. We further traced the fate of N during decomposition of the litters into the soil and measured the inorganic N content in the soil solution as well as total N in the particulate (POM) and mineral-associated organic matter (MAOM) fractions at the end of the incubation.

3. While most litters exhibited a very rapid initial N release, they were also quite variable in their N dynamics. As such they differed in the quantity and temporal patterns of N release, the accumulation of inorganic N in the soil solution, as well as in the incorporation of N in stable soil organic matter (SOM) fractions during decomposition. This variability was driven by differences in initial litter chemistry, particularly N, but also P and Mn, and litter C leachate concentrations. These and other traits (including water-soluble compounds and lignin concentrations) explained up to 81% of the variance in N release rate, 48% for N accumulation in the soil solution, 57% for N entering the soil POM and 55% for MAOM fraction.

4. Synthesis. We identified litter traits and litter types (root versus leaf) with afterlife effects on N cycling in soils and important implications for ecosystem functioning. Highly decomposable litters (typically high N; including most leaf litters) generally support a fast N release through mineralization, thereby the immediate supply of N to plants, but also potentially higher losses from the system, and long-term storage as MAOM-N, likely within microbial resynthesis products. In

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<footnote n="1">¹Station d'Ecologie Théorique et Expérimentale, CNRS, Moulis, France; ²Chair of Silviculture, Faculty of Environment and Natural Resources, University of Freiburg, Freiburg, Germany; ³Research Institute for Forest Ecology and Forestry (FAWF), Trippstadt, Germany; ⁴Université Montpellier, CNRS, EPHE, IRD, Montpellier, France; ⁵Unité de Recherche et Développement en Agriculture et Agroalimentaire (URDAAT), Université du Québec en Abitibi-Témiscamingue (UQAT), Notre-Dame-du Nord, Canada and ⁶Centre d'Étude de la Forêt, Université Laval, Québec, Canada</footnote>

<footnote>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.</footnote>

<footnote>© 2025 The Author(s). *Journal of Ecology* published by John Wiley & Sons Ltd on behalf of British Ecological Society.</footnote>
contrast, poorly decomposable litters (typically low N; including most root litters) promote longer and weaker N release and foster N retention via lower N losses and increased POM-N formation.

KEYWORDS

inorganic nitrogen, litter chemistry, MAOM, nitrogen release, POM, temporal N dynamics, Weibull residence time model

# 1 | INTRODUCTION

Nitrogen (N) is the dominant element in the Earth's atmosphere, and an important constituent of the biosphere, yet it often limits ecosystem productivity owing to its near-absence in bedrock (LeBauer & Treseder, 2008). The availability of N to plants is mainly controlled by organic matter decomposition, during which organic N is immobilized as it becomes incorporated in microbial biomass, or mineralized, that is converted into inorganic forms (Figure 1). The inorganic N content in the soil solution is strongly determined by the rate of net N mineralization (Li et al., 2019; Schimel & Bennett, 2004), that is when mineralized N exceeds microbial demand. It is also important for ecosystem productivity as inorganic N is the predominant source of N for plants.

The rate and temporal dynamics of N mineralization and release in the soil solution, and its incorporation into soil fractions are of fundamental importance for plant growth and ecosystem productivity. Yet, our knowledge of how litter decomposition drives N dynamics and affects the fate of N in the soil is still rather limited.

Nitrogen dynamics during decomposition in soils are affected by various abiotic (e.g. soil properties; Villarino et al., 2023) and biotic (e.g. rhizodeposition; Berenstecher et al., 2023; Jilling et al., 2018) parameters. Yet, multiple studies have shown that initial litter chemistry may be the predominant influence on N dynamics during decomposition and eventually on the quantity of inorganic N in the soil solution (Chen et al., 2015; Orwin et al., 2010; Trinsoutrot, Recous, Bentz, et al., 2000; Trinsoutrot, Recous, Mary, et al., 2000; van Huysen et al., 2013). As such, ample evidence exists that the initial litter N concentration and the C/N ratio strongly affect litter net N release, that is the loss of N measured by changes in litter N concentration (Kriiska et al., 2021; Manzoni et al., 2008; Moore et al., 2011; Parton et al., 2007; Pei et al., 2019; van Huysen et al., 2013). Litters with a relatively high C/N ratio, which may reinforce microbial N limitation, often lead to net N immobilization during the initial stages of decomposition, contrary to litters with a relatively low C/N ratio that favour net N mineralization (Trinsoutrot, Recous, Bentz, et al., 2000). Yet, the litter C/N is not always the main determinant of the net release of inorganic N into the soil solution (Chen et al., 2014).

<figure><img src="image_9.png" /><figcaption>FIGURE 1 Schematic overview of pools and fluxes related to N dynamics during decomposition. Inspired by Geisseler et al. (2010), Averill and Waring (2018), Cotrufo et al. (2021), Jilling et al. (2018) and Zhang et al. (2021).</figcaption></figure>
In particular, it has also been observed that high initial litter cellulose concentrations may promote net N mineralization (Chen et al., 2015), while high initial litter lignin (van Huysen et al., 2013) and polyphenol concentrations (Fernández-Alonso et al., 2018; Oglesby & Fownes, 1992; Trinsoutrot, Recous, Bentz, et al., 2000) appear to retard N mineralization. Tannins are known to form complexes with proteins and thereby inhibit N mineralization (Schimel et al., 1998) and nitrification (Baldwin et al., 1983). Large amounts of phenolic compounds have also been shown to accelerate bulk litter mass loss when C availability rather than N availability primarily limits microbial decomposers (Hättenschwiler & Jørgensen, 2010). Other litter traits, such as calcium (Ca) (Hobbie, 2015), manganese (Mn) and magnesium (Mg) concentrations (Vivanco & Austin, 2019) could also play an important role in decomposition and N cycling. Ca and Mg are important base cations that may accelerate initial decomposition (Aponte et al., 2013; García-Palacios et al., 2016), potentially leading to faster N release from litter. Mn may be significant for the degradation of lignin (Keiluweit et al., 2015) with positive consequences for the release of N from formerly shielded N compounds.

During organic matter decomposition, N contained in free particulate organic matter (POM, which is a heterogeneous pool of small litter fragments, litter decomposition by-products and soil OM not associated with minerals) can be incorporated into soil aggregates (i.e. aggregated POM), which typically confers only a moderate protection from microbial degradation (Lavallee et al., 2020; von Lützow et al., 2006). Stronger protection is provided when both organic and inorganic N are immobilized by being sorbed onto minerals or by forming interactions with organo-mineral complexes, thereby becoming part of the mineral-associated OM (MAOM) fraction (Hatton et al., 2012; Hatton, Remusat, et al., 2015; Poirier et al., 2014; von Lützow et al., 2006). In contrast to POM, which typically represents a more labile fraction with a shorter residence time, MAOM is more stable, serving as a key component in nutrient storage and slow-release processes over time (Lavallee et al., 2020).

Litter quality may also be an important control of the N stabilized in MAOM (Angst et al., 2021; Córdova et al., 2018). According to the *Microbial Efficiency-Matrix Stabilization* (MEMS) framework (Cotrufo et al., 2013), a higher initial litter quality (e.g. a higher N concentration and labile C compounds content) may promote the stabilization of SOM via the microbial pathway (Angst et al., 2019; Cyle et al., 2016; Hatton, Castanha, et al., 2015; Liang et al., 2019) through a higher production of microbial-derived products, which may eventually be stabilized as MAOM (Cotrufo et al., 2013). However, opposite effects of litter quality on the stabilization of SOM have also been reported. As such, several studies found that less MAOM was stabilized by high-quality litters. Suggested underlying mechanisms included soil C saturation (Castellano et al., 2015; Huys et al., 2022), higher C mineralization rates of high-quality litters (Córdova et al., 2018), or the role of fungi, whose growth may be promoted by low-quality litters, in MAOM formation (Zheng et al., 2021). Another study observed that the addition of low C/N (i.e. high quality) material induced a positive priming effect, and thus stimulated the breakdown of SOM (Mason-Jones et al., 2018). These observations question

the generality of the MEMS framework. While the vast majority of studies related to MAOM and POM formation during decomposition have focused on C, understanding the determinant of N stabilized in MAOM-N and in POM-N has largely been understudied despite its crucial importance for our understanding of the N cycle (but see Jilling et al., 2018).

Most of these previous studies focused on leaf litter only, although fine-root litter represents another major source of N to the soil (Freschet et al., 2013) and may more strongly affect SOM dynamics (Kyaschenko et al., 2019). Leaf and fine-root litter can greatly differ in their initial chemical composition, that is such as in initial N concentrations (e.g. Song et al., 2021), and fine-root litter is more intimately in contact with the soil matrix. Consequently, litter N release patterns associated with their decomposition (Fornara et al., 2009; Moretto et al., 2001; Parton et al., 2007) and N availability to plants (Bird et al., 2008; Hatton, Castanha, et al., 2015) may also vary between root and leaf litter types.

In contrast to C dynamics, which follow the same pattern as litter mass loss during decomposition, N dynamics can differ considerably (Lindahl et al., 2007; Xiong et al., 2013). Typically, N release patterns are characterized by three phases: an initial phase dominated by N leaching, followed by a phase of immobilization where N concentration in the decomposing litter increases, and finally, a phase of continuous release of N (Ball et al., 2009). These phases and the timing of N release can vary greatly depending on initial litter quality (Parton et al., 2007). Manzoni et al. (2010) suggested that the initial C/N ratio is among the most important determinant of the N release patterns. The relative importance of initial litter traits for N dynamics can vary during the different decomposition stages. For example, higher initial litter N concentrations may accelerate decomposition and N release during early-stage decomposition, while it can have the opposite effect (Hobbie, 2015) or no effect during later stages, when lignin concentration may be the primary control (Oglesby & Fownes, 1992; Pei et al., 2019). Most studies have focused on the early stages of litter decomposition and, more importantly, have not quantified the fate of the N released from litter in the soil. Furthermore, it remains unclear whether and how leaf and fine-root litter differ in N release dynamics under the influence of different litter traits.

With this study, we compared litter N release dynamics and the fate of N within the soil between leaf and fine-root litter from 12 plant species using a stable isotope approach. Indeed, the labelling of litter with the stable isotope ¹⁵N allows more detailed insight into N transformation processes during decomposition (Zeller et al., 2000, 2001) and how it impacts N availability in the soil solution (Schimel & Bennett, 2004).

We hypothesized that:

1. During leaf and fine-root litter decomposition, litter N release and the availability of soil inorganic N increase with increasing initial litter N and decreasing initial litter C/N.

2. Beyond initial N and C/N, high concentrations in other litter nutrients such as Ca, Mg and Mn positively affect litter N release and soil inorganic N accumulation during early stages of
decomposition. In contrast, higher initial concentrations of lignin negatively affect litter N release and soil inorganic N accumulation during later stages of decomposition.

3. High litter N release is linked to less N in the POM pool and more N stabilized in the MAOM pool.

4. The overall distinct trait syndrome of leaf vs. fine-root litter results in different N dynamics and fates during decomposition. Leaf litter generally releases N faster, contributes more N to the soil solution during early-stage decomposition and leads to a higher proportion of N in the MAOM-N soil fraction compared to fine-root litter.

## 2 | MATERIALS AND METHODS

We selected 12 herbaceous species (Table 1) based on their diversity in life history, taxonomic groups and functional traits (above- and below-ground), representative of southern France's Mediterranean old-field succession. We produced ¹⁵N-labelled plant litter material that we used to measure litter N decomposition and to set up the litter incubation experiment (Huys et al., 2022).

### 2.1 | Preparation of ¹⁵N labelled plant material

Seeds from the 12 species were collected (August 2014) and germinated for 3 weeks (September 2014). Subsequently, seedlings were grown in large pots (with N-poor soil; soil characteristics are further described below, as it was the same soil used for the litter incubation) at a plant density of 200 plants m⁻² for five months in a greenhouse. Every month, a ¹⁵N solution (50/50 nitrate/ammonium in the form of 10 at% ¹⁵N Ca(¹⁵NO₃)₂Na and ¹⁵NH₄Cl in increasing amounts from 2 to 8 g N m⁻² up to a total of 18 g N m⁻² over time) was added. Plants were harvested after irrigation had gradually been reduced in the last month to reproduce typical conditions of Mediterranean climate summer drought, which induces plant senescence. Harvested plants were then sorted into leaf, stem, fine roots (i.e. the first three most distal root orders; Freschet et al., 2021) and coarse roots (i.e. higher root orders). Roots were gently cleaned with water before being sorted and all litter samples were air-dried after sorting. After careful homogenization of each litter, three subsamples were taken per litter type (leaf and root) and species and ground, and ¹⁵N concentrations were measured on all three subsamples for each tissue and species with a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). The average enrichment of leaf litter was 6.49 at% ¹⁵N and that of fine-root litter was 6.32 at% ¹⁵N.

### 2.2 | Initial litter trait measurement

In total, 14 chemical traits were measured on two replicate subsamples of each of the 12 leaf and fine-root litter species. Replicate

values were later averaged to obtain one trait value per litter type and species. These included concentrations of C, N, P, Mg, Ca and Mn, concentrations of water-soluble compounds (WSC), cellulose, hemicellulose, lignin, condensed tannins and the concentrations of C, N and P in litter leachates.

Plasma emission spectrometry (ICP-MS, Thermo Scientific iCAP Q, Thermo Fisher Scientific GmbH, Germany) was used to measure P, Ca and Mn after acid mineralization. The van Soest method (van Soest, 1963) was used to measure concentrations of WSC, hemicellulose, cellulose and lignin with a Fibersac 24 fibre analyser (Ankom, Macedon, NJ, USA). The acid butanol method (Waterman & Mole, 1994) was used to measure condensed tannins. Soluble C, N and P concentrations were quantified on litter leachates. This was done after the extraction of 0.5 g of litter (air-dried and cut into 1 cm long pieces) in 30 mL of distilled water for 30 min on an end-overend shaker (20 rpm at 20°C) and then filtered (0.45 µm). An automated TOC-TN analyser (Shimadzu, TOC-Vcph, Japan) was used to quantify the total organic C and N contents in the extracts. Total P content was measured colorimetrically after digestion with sulfuric acid and hydrogen peroxide (35 min at 100°C and 2 h at 360°C) using the molybdenum blue method (Grimshaw et al., 1989), by an autoanalyser (Evolution II, Alliance Instrument, Frépillon, France). Total amounts of litter C, N and P leached were expressed per mass of initial litter (mg g⁻¹).

### 2.3 | Litter incubation

Prior to incubation, leaf and fine-root litter material of each species were cut into pieces of 1 cm² and 1 cm length, respectively, and homogenized. For the incubation, we used a naturally decarbonated soil of a sandy-loam texture (9% clay, 26% silt, 65% sand), with a pH of 7 and relatively low C and N concentrations (15 and 1.1 g kg⁻¹, respectively), P availability (0.035 g kg⁻¹ Olsen-P) and cation exchange capacity (7.75 cmol kg⁻¹). The soil, excavated in Villefort (France; 44°43' N, 3°92' E), was a brunisol developed on a schist parent rock. Before incubation, soil was sieved (at 5 mm) and moistened with the purpose to induce germination of the seed bank. Germinated seeds were manually removed after 2 weeks, and soil was air-dried (to constant mass), again sieved (at 2 mm) and homogenized. Soil residual humidity (oven-dried at 105°C, soil water content 0.014 g g⁻¹) and soil field capacity (0.396 g g⁻¹) were quantified on soil subsamples.

For each of the 12 species and two litter types (leaf and fine root), 12 litter samples of 0.4 g were prepared and subsequently mixed with 50 g of air-dried soil, corresponding to three replicates each during four sequential harvests. Mean values of these three replicates were used for all statistical analyses. Half of the soil-litter mixture was transferred to a microcosm (80 mL), and a polyester mixed-bed ionic resin capsule (PST1; Unibest, Bozeman, Montana, USA) was then placed in the centre of the microcosm before the other half of the soil-litter mixture was added. The resin capsule was used to determine the amount of inorganic N released in the soil solution. While the amount of N captured in the resin capsules
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<table><caption>TABLE 1 Initial trait values of leaves and root litter species.</caption><thead><tr><th>Species (family)</th><th>Life form</th><th>Litter type</th><th>Cellulose (mgg<sup>-1</sup>)</th><th>Hemicellulose (mgg<sup>-1</sup>)</th><th>Lignin (mgg<sup>-1</sup>)</th><th>Tannins (mgg<sup>-1</sup>)</th><th>C (mgg<sup>-1</sup>)</th><th>N (mgg<sup>-1</sup>)</th><th>Mg (mgg<sup>-1</sup>)</th><th>Ca (mgg<sup>-1</sup>)</th><th>Mn (mgg<sup>-1</sup>)</th><th>C/N</th><th>WSC (mgg<sup>-1</sup>)</th><th>C<sub>L</sub> (mgg<sup>-1</sup>)</th><th>P<sub>L</sub> (mgg<sup>-1</sup>)</th><th>N<sub>L</sub> (mgg<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Bituminaria bituminosa (Fabaceae)</td><td>Legume</td><td>Leaf</td><td>146.75</td><td>180.07</td><td>77.35</td><td>0.46</td><td>413.17</td><td>30.10</td><td>13.30</td><td>36.48</td><td>0.10</td><td>14</td><td>595.83</td><td>102.17</td><td>0.63</td><td>7.78</td></tr><tr><td></td><td></td><td>Root</td><td>206.02</td><td>282.19</td><td>106.48</td><td>0.12</td><td>377.20</td><td>32.91</td><td>2.69</td><td>13.64</td><td>0.10</td><td>11</td><td>405.32</td><td>38.28</td><td>1.03</td><td>4.71</td></tr><tr><td>Brachypodium phoenicoides (Poaceae)</td><td>Graminoid</td><td>Leaf</td><td>289.00</td><td>350.06</td><td>41.70</td><td>0.55</td><td>432.48</td><td>21.88</td><td>3.18</td><td>5.94</td><td>0.14</td><td>20</td><td>319.24</td><td>43.15</td><td>0.52</td><td>6.21</td></tr><tr><td></td><td></td><td>Root</td><td>301.36</td><td>328.05</td><td>119.48</td><td>0.19</td><td>435.05</td><td>16.57</td><td>1.33</td><td>5.32</td><td>0.31</td><td>26</td><td>251.11</td><td>25.91</td><td>0.89</td><td>4.39</td></tr><tr><td>Bromus erectus (Poaceae)</td><td>Graminoid</td><td>Leaf</td><td>284.01</td><td>258.88</td><td>31.67</td><td>0.35</td><td>411.59</td><td>29.59</td><td>4.73</td><td>8.03</td><td>0.13</td><td>14</td><td>425.44</td><td>68.89</td><td>0.93</td><td>10.58</td></tr><tr><td></td><td></td><td>Root</td><td>301.19</td><td>360.29</td><td>102.84</td><td>0.06</td><td>419.14</td><td>19.15</td><td>1.07</td><td>4.42</td><td>0.18</td><td>22</td><td>235.69</td><td>26.44</td><td>0.40</td><td>3.83</td></tr><tr><td>Bromus madritensis (Poaceae)</td><td>Graminoid</td><td>Leaf</td><td>249.36</td><td>273.78</td><td>32.73</td><td>0.45</td><td>409.52</td><td>34.41</td><td>5.39</td><td>17.69</td><td>0.18</td><td>12</td><td>444.13</td><td>50.09</td><td>1.03</td><td>10.88</td></tr><tr><td></td><td></td><td>Root</td><td>293.35</td><td>404.39</td><td>81.89</td><td>0.06</td><td>407.42</td><td>17.43</td><td>1.66</td><td>5.81</td><td>0.23</td><td>23</td><td>220.38</td><td>19.42</td><td>0.90</td><td>2.89</td></tr><tr><td>Clinopodium nepeta (Lamiaceae)</td><td>Forb</td><td>Leaf</td><td>94.91</td><td>127.47</td><td>76.86</td><td>0.47</td><td>429.62</td><td>36.62</td><td>8.13</td><td>21.90</td><td>0.10</td><td>12</td><td>700.76</td><td>96.51</td><td>0.84</td><td>8.20</td></tr><tr><td></td><td></td><td>Root</td><td>240.39</td><td>211.39</td><td>189.46</td><td>0.06</td><td>421.72</td><td>21.69</td><td>4.34</td><td>11.77</td><td>0.17</td><td>19</td><td>358.76</td><td>29.73</td><td>0.41</td><td>2.04</td></tr><tr><td>Crepis foetida (Asteraceae)</td><td>Forb</td><td>Leaf</td><td>120.01</td><td>168.01</td><td>96.35</td><td>0.39</td><td>379.18</td><td>35.29</td><td>14.34</td><td>31.04</td><td>0.09</td><td>11</td><td>615.62</td><td>78.81</td><td>1.21</td><td>11.50</td></tr><tr><td></td><td></td><td>Root</td><td>190.57</td><td>208.93</td><td>172.73</td><td>0.18</td><td>400.40</td><td>26.94</td><td>3.67</td><td>14.54</td><td>0.18</td><td>15</td><td>427.76</td><td>33.58</td><td>1.11</td><td>3.70</td></tr><tr><td>Dactylis glomerata (Poaceae)</td><td>Graminoid</td><td>Leaf</td><td>267.01</td><td>270.05</td><td>36.58</td><td>0.47</td><td>410.29</td><td>28.48</td><td>5.35</td><td>8.63</td><td>0.17</td><td>14</td><td>426.36</td><td>69.52</td><td>0.68</td><td>10.36</td></tr><tr><td></td><td></td><td>Root</td><td>296.05</td><td>352.46</td><td>110.26</td><td>0.18</td><td>432.67</td><td>14.45</td><td>0.99</td><td>4.45</td><td>0.15</td><td>30</td><td>241.23</td><td>20.49</td><td>0.55</td><td>2.52</td></tr><tr><td>Daucus carota (Apiaceae)</td><td>Forb</td><td>Leaf</td><td>153.69</td><td>233.84</td><td>64.29</td><td>0.54</td><td>397.66</td><td>25.56</td><td>12.96</td><td>38.71</td><td>0.10</td><td>16</td><td>548.19</td><td>84.68</td><td>0.59</td><td>8.06</td></tr><tr><td></td><td></td><td>Root</td><td>197.55</td><td>212.76</td><td>154.24</td><td>0.07</td><td>408.59</td><td>21.93</td><td>3.51</td><td>10.32</td><td>0.13</td><td>19</td><td>435.44</td><td>57.15</td><td>0.77</td><td>5.80</td></tr><tr><td>Medicago minima (Fabaceae)</td><td>Legume</td><td>Leaf</td><td>170.75</td><td>243.00</td><td>56.40</td><td>0.30</td><td>401.28</td><td>33.94</td><td>5.59</td><td>36.73</td><td>0.12</td><td>12</td><td>529.86</td><td>109.69</td><td>0.76</td><td>10.17</td></tr><tr><td></td><td></td><td>Root</td><td>224.63</td><td>250.70</td><td>130.45</td><td>0.12</td><td>389.44</td><td>29.98</td><td>3.54</td><td>13.88</td><td>0.37</td><td>13</td><td>394.23</td><td>30.68</td><td>0.85</td><td>3.82</td></tr><tr><td>Picris hieracioides (Asteraceae)</td><td>Forb</td><td>Leaf</td><td>160.58</td><td>178.11</td><td>43.13</td><td>0.72</td><td>385.05</td><td>37.02</td><td>13.28</td><td>31.86</td><td>0.18</td><td>10</td><td>618.18</td><td>79.68</td><td>1.23</td><td>10.58</td></tr><tr><td></td><td></td><td>Root</td><td>193.43</td><td>179.44</td><td>154.78</td><td>0.30</td><td>412.55</td><td>26.28</td><td>4.07</td><td>12.08</td><td>0.40</td><td>16</td><td>472.34</td><td>42.87</td><td>0.78</td><td>4.87</td></tr><tr><td>Tordylium maximum (Apiaceae)</td><td>Forb</td><td>Leaf</td><td>159.52</td><td>113.60</td><td>38.47</td><td>0.27</td><td>362.02</td><td>41.78</td><td>12.07</td><td>52.24</td><td>0.19</td><td>9</td><td>688.42</td><td>104.76</td><td>0.72</td><td>12.24</td></tr><tr><td></td><td></td><td>Root</td><td>214.78</td><td>252.65</td><td>130.30</td><td>0.07</td><td>341.29</td><td>25.07</td><td>4.37</td><td>17.09</td><td>0.64</td><td>14</td><td>402.27</td><td>23.69</td><td>0.92</td><td>3.41</td></tr><tr><td>Trifolium angustifolium (Fabaceae)</td><td>Legume</td><td>Leaf</td><td>172.75</td><td>239.03</td><td>44.82</td><td>0.51</td><td>403.11</td><td>31.74</td><td>7.00</td><td>43.86</td><td>0.12</td><td>13</td><td>543.39</td><td>116.56</td><td>1.09</td><td>7.11</td></tr><tr><td></td><td></td><td>Root</td><td>242.60</td><td>268.36</td><td>136.46</td><td>0.03</td><td>390.78</td><td>30.04</td><td>3.15</td><td>11.24</td><td>0.17</td><td>13</td><td>352.57</td><td>17.75</td><td>0.69</td><td>2.18</td></tr></tbody></table>

Note: Please note that the C/N values are lower than those of (unfertilized) plants grown in the field (Birouste et al., 2012).

Abbreviations: C<sub>L</sub>, C leachates; N<sub>L</sub>, N leachates; P<sub>L</sub>, Pleachates; WSC, water-soluble compounds.
<figure><img src="image_11.png" /></figure>

provides an estimate of plant-available N, it may not fully reflect the total N available under natural conditions (Schimel & Bennett, 2004). Soil field capacity was raised to 80% with distilled water at the beginning of the experiment. By using pierced lids, gas exchange was ensured after microcosms were closed. Microcosms were incubated at 25°C in the dark in three climatic chambers (i.e. one per replicate microcosm).

A total of 303 microcosms were prepared, including 15 microcosms of bare control soil. Three replicates of bare control soil were harvested 24h after the start of the experiment to determine the initial soil conditions after rewetting. Three replicates of each type of microcosm were harvested after 10, 38, 156 and 367 days each.

## 2.4 | Litter and soil N measurements

Following the harvest, the microcosm content was spread on plates to allow for rapid air-drying at 25°C in a well-ventilated room. Subsamples of the soil-litter fragments mixture were analysed for C and N concentrations and ¹⁵N at% on a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Resin capsules were brushed free of soil, then further cleaned under a gentle flow of distilled water. The mineral N (NO₃⁻, NH₄⁺) that had accumulated over time in the ionic resin capsules was desorbed with 1 mol L⁻¹ KCl and determined colorimetrically with an autoanalyser (Futura II, Alliance Instrument, Frépillon, France). Nitrates were quantified by the sulfanilic acid method after reduction to nitrites in a cadmium column. Ammonium was determined by the indophenol blue reaction.

Litter fragments remaining in the soil were manually sorted from a 10g subsample and subsequently ground with a ball mill. Litter mass remaining (g) was derived from these fragments and their C and N concentrations were measured with an elemental analyser (CHN model EA1108; Carlo Erba Instruments, Milan, Italy).

## 2.5 | Soil fraction analyses

From the soil collected after 367 days, we fractionated subsamples following the procedure described in Golchin et al. (1994). We separated soil subsamples (6g) into the particulate organic matter fraction (POM; <1.6 g cm⁻³) and the mineral-associated organic matter fraction (MAOM; >1.6 g cm⁻³). A two-step procedure was used to separate the POM from the MAOM fraction. At first, soil samples were suspended in 25 mL sodium iodide (NaI) solution of 1.6 g cm⁻³ density within 50 mL polypropylene centrifugation tubes (Sarstedt AG & Co, Nümbrecht, Germany).

The free light fraction was extracted using a filtration unit (200 mL distilled water on a 0.45 µm filter) after gently shaking the tubes (20 times) and subsequently centrifuging them for 35 min at 8000 RPM. To liberate the light fraction occluded in aggregates, aggregates of the remaining soil were destroyed by shaking the samples for 1 h on a reciprocal shaker and centrifuging them for 35 min

at 8000 RPM. Filtration was used again to recover the light fraction. The residual soil was resuspended with distilled water to rinse the remaining dense fraction (MAOM). For that, tubes were vigorously shaken (1 min) and centrifuged for 15 min at 8000 RPM. An aspiration unit was used to remove water. This rinsing procedure was done three times. After drying (60°C), the mass and ¹⁵N concentrations of the POM and MAOM fractions were determined, and litter-derived N concentrations were calculated using the same equation as for the total soil (see calculations below).

## 2.6 | Calculations

The amount of N in the initial litter was quantified as the initial litter N concentration multiplied by the initial litter mass (0.4 g). The amount of N remaining in the litter (hereafter 'litter N remaining' (g)) at time $t_n$ was calculated as the litter mass remaining multiplied by its N concentration at harvest $t_n$ both estimated from manual sorting of litter fragments, as described above.

Average daily litter N release (g day⁻¹) was calculated for each period between harvests by dividing the amount of N released from the litter during the respective period by the number of days of that period (i.e. 10, 28, 118 and 211 days, respectively).

The amount of inorganic N that had accumulated in the resin capsules (hereafter 'total N in resin capsules' (g)) at time $t_n$ was calculated by summing the amounts of NO₃⁻ and NH₄⁺ extracted from the capsules (¹⁵N concentrations were not measured). The values were corrected by subtracting the resin N content in the bare (control) soil (taken 24h after the start of the experiment and soil rewetting) representing the initial flush of N due to the rewetting of the soil. To specifically determine the N accumulation in the resin capsules due to the presence of litter, as compared to the bare control soil (hereafter 'litter-induced N in resin capsules (g)), the amount of resin N in the bare control soil at $t_n$ was subtracted from the total resin N at $t_n$, at each harvest step. Average daily N accumulation in resin capsules (µg day⁻¹) was calculated for each harvesting period by dividing the total amount of N in resin capsules for a given point of incubation time by the number of incubation days until that point (i.e. 10, 38, 156, 376).

The 'litter-derived soil N' (g) refers to the litter-derived N that entered the MAOM and POM (including remaining litter fragments) fractions of the soil and microbial biomass N, excluding litter N that entered resin capsules or was volatilized. At time $t_n$, it was assessed using the isotopic mixing model:

$$ \text{Litter - derived N (g)} = (\delta_{\text{treat}} - \delta_{\text{cont}}) / (\delta_{\text{litter}} - \delta_{\text{cont}}) \times [\text{microcosm N}] \times M \quad (1) $$

where $\delta_{\text{treat}}$ and $\delta_{\text{cont}}$ represent the ¹⁵N of the microcosm (soil and remaining litter mix) and of the control soil, respectively, at harvest $t_n$. $\delta_{\text{litter}}$ equals the initial litter ¹⁵N. [microcosm N] equals the N concentration in the microcosm (soil and remaining litter mix) at harvest $t_n$ (g g⁻¹) and M represents the initial dry mass (50 g) of soil in the microcosm. The 'litter-derived POM-N and MAOM-N'
(g) were calculated using the same equation, but with $δ_{treat}$ representing the $δ^{15}$N of the POM and MAOM in the microcosm, respectively. Owing to the near-complete decomposition of all litter fragments in mesocosms analysed for POM and MAOM (final harvest at day 367), litter fragments remaining in the soil, typically below $2\text{mm}^2$, were considered as POM. All focal variables were expressed in absolute and in standardized (per initial litter N content) terms.

## 2.7 | Data analysis

All statistical analyses were performed using R version 3.5.1. (R Core Team, 2018), with differences considered significant at $p=0.05$. Wilcoxon-singed-rank tests or t-tests were used to test for differences in N pools among the two litter types (i.e. leaves vs. roots).

### 2.7.1 | Fitting of the temporal dynamics

We fitted different models to better describe the course of decomposition (i.e. litter N release and resin N accumulation dynamics) and to extract model parameters that can be used as response variables in further analyses.

We used a Weibull residence model to fit the course of litter N release via decomposition:

$$f = e^{-(t/T)^{\alpha}} \quad (2)$$

This model has been suggested to be more suitable for heterogeneous litter data than previous models used including single-exponential models (Cornwell & Weedon, 2014; Gill et al., 2021; Rosenfield et al., 2020). As such, it is associated with higher flexibility regarding the shape ($α$) and scale ($β$) of the decomposition patterns (Cornwell & Weedon, 2014). In line with Cornwell and Weedon (2014), an optimization algorithm was used for fitting the model parameters $α$ and $β$. In this study, we present $1/β$ values rather than $β$ so that increasing $1/β$ correspond to increasing rate of N release. Weibull's $α$ values were generally $\ll 1$, affirming the absence of a 'lag' phase (Cornwell & Weedon, 2014), that is a retarded initial decomposition phase. We, therefore, did not further include it in our analyses.

We additionally fitted conventional single negative exponential models and compared them to the Weibull fits using Akaike Information Criterion (AIC). In all 24 cases, except for one (i.e. leaf litter of *T. maximum*), the Weibull models yielded better fits than the single negative exponential model (indicated by lower AIC values, Table S1). We derived half-life values (i.e. $T_{0.5}$: time (days) until 50% of the N is released from the litter) from Weibull models for better interpretation of the decomposition dynamics (Freschet et al., 2012):

$$t_{0.5} = \beta \left( \ln \left( \frac{1}{0.5} \right) \right)^{\frac{1}{\alpha}} \quad (3)$$

To model the accumulation of N in resin capsules, we fitted an equation derived from the Michaelis-Menten equation, where incubation time (T) replaced substrate concentration and N accumulation replaced reaction rate:

$$f = \frac{A_{\max} \times T}{K_{A} + T} \quad (4)$$

and the two parameters $A_{max}$ and $K_A$ represent the maximum N accumulation in the resin capsules and the time at which half of the N has been accumulated, respectively. $K_A$ can be further interpreted here as the steepness of the slope of the N accumulation (a higher $K_A$ indicating a flatter curve and thereby a slower N accumulation; a lower $K_A$ indicating a steeper curve and thereby a faster N accumulation).

### 2.7.2 | Testing the effect of initial litter traits

To address our overall aim of testing how initial litter traits affect the N dynamics during decomposition, we did a literature review and mainly selected predictor variables that were previously reported to be important for N dynamics. These included initial litter N and C/N (Cissé et al., 2021; Moore et al., 2011; Parton et al., 2007; Prescott, 2005), P (Cissé et al., 2021; Prescott, 2005), cellulose (Chen et al., 2015), lignin (van Huysen et al., 2013), tannins (Schimel et al., 1998), Ca and Mn (Hobbie, 2015). We also included WSC (Pei et al., 2019) and leachates N, P and C as predictors because of their potential influence on the limitation of microbial activities (Manzoni et al., 2010). In addition, initial litter hemicellulose and Mg were also tested owing to their overall role in litter decomposition (Makkonen et al., 2012).

Model selection and averaging were used to investigate which initial traits best predict the response variables using linear regressions (variables describing temporal dynamics, i.e. Weibull $1/β$, $A_{max}$ and $K_A$, and variables reflecting the fate of litter N, that is litter-derived MAOM-N, POM-N and total resin N). In case model assumptions such as normality of the residuals were not fulfilled, response variables were transformed. All predictor variables were scaled using the 'scale' function, which centres the variables around their means. To avoid model overfitting, we used a hierarchical modelling approach, that is we fitted three separate models, each with a different set of predictor variables, and subsequently applied model selection using 'dredge' and averaging using 'mod.avg', with $ΔAIC=4$ (R package *MuMln*, Barton, 2019, see Tables S2-S4). All predictors retained in the respective most parsimonious models were then included in final synthesis models, for which model selection and averaging were applied again. Collinearity among predictors was assessed using variance inflation factors (vif), considering $vif > 5$ as threshold for collinearity (R package *car*, Fox & Weisberg, 2011). As initial litter N and initial litter C/N were highly correlated, we ran the models including either N or C/N. The results (model estimates and significance levels) did not change significantly, and therefore, we only report the results including initial litter N.
As litter type (i.e. leaf vs. root litter) covaried with most of the initial traits, we generally did not include it as a predictor in the models. Yet, we still tested whether including 'litter type' as a factor in the models led to significant changes in the model outputs (for approximately two-thirds of the models there was no effect, while the results of none of the synthesis models were changed by the addition of 'litter type' as a factor).

# 3 | RESULTS

## 3.1 | Temporal N dynamics

### 3.1.1 | N release from litter

Generally, N was lost quite rapidly during the first 10 days of the incubation across all litter species and types (Figures 2a and 3b; Figure S1b). Likewise, litter C showed a similar pattern (Figure 3a). By the end of the 367 days of the incubation period, the total litter C and N proportions remaining were on average 23.5±0.7% and 3.7±0.8% across all species and both tissue types, respectively.

The Weibull residential model fitted the course of litter N remaining very well (across all litter types, $R^2=0.96$, Figure 2a), and typically better (with overall lower AIC) than the commonly used single negative exponential model (Table S1). Weibull's $1/β$ values were on average 1929±702 across all litters. Accordingly, half-life values ($T_{0.5}$) derived from the Weibull model were on average 3.9±1.4 days and predicted an extremely rapid loss of litter N within the first few days of incubation for most litters.

Across species, patterns of litter N release from root litters appeared to be generally slower than that of leaf litters over the course of the incubation period (Figure 2a; Figure S2). Within

the first 10 days of incubation, average daily litter N release rates were also significantly higher (ca. 60%) for leaf than for root litters (Figure S3).

### 3.1.2 | Total and litter-induced N accumulation in resin capsules

In general, the rate of total inorganic N accumulation in the resin capsules was relatively high during the initial decomposition phase. It declined over time. Across litters, the modified Michaelis-Menten function described the overall temporal pattern of the total N in resin capsules well ($R^2=0.97$, Figure 2b).

The median $A_{max}$ across all litters was 0.014±0.005 g, whereas the median $K_A$ was 274±300 days. This indicates that after on average 274 days 50% of the N had accumulated in the resin capsules. During the earlier stages of decomposition, the accumulation rate in resin capsules was faster (approximately two times) for leaf than for root litters (Figures S4 and S5). This was indicated by lower overall $K_A$ values for leaf litter (196 vs. 394 days in leaf and root litters, respectively, $p=0.06$, Figure 2).

### 3.1.3 | Litter-derived soil N

Litter-derived soil N sharply increased within the first 10 days of the incubation for most litters (Figure 3c). Afterwards, it appeared to steadily decline for leaf litters while it remained fairly constant for root litters over time. The total amount of litter-derived soil N did not significantly differ between leaf and root litters at any of the harvests. Yet, litter-derived soil N relative to the initial litter N was significantly higher (ca. 44%) for root than leaf litters during the

<figure><img src="image_12.png" /><figcaption>**FIGURE 2** Temporal dynamics of (a) litter N remaining (% of initial) and (b) N accumulation in resin capsules (g) by litter type (superscript letters behind values indicate statistically significant differences between root and leaf litters using t-tests). Error bars represent SEs. $R^2$ values, which represent the goodness of fit, were 0.96 for all four models. Curves represent fitted (a) Weibull distributions where $α$ describes acceleration (when $α>1$) or deceleration (when $α<1$) of a decomposition rate $1/β$ (note that a high $1/β$ indicates a high rate) along time; and (b) a modified Michaelis-Menten function, where $A_{max}$ represents the maximum accumulation of N in resin capsules and $K_A$ the time needed to reach half of maximum N accumulation. Note that the high values for $K_A$ and $A_{max}$ are linked to the fact that the N resin accumulation values are derived from both litter N and soil N (i.e. not only litter-induced N in resins) and that the accumulation of N is expected to continue after the litters have fully decomposed.</figcaption></figure>
<figure><img src="image_13.png" /></figure>

FIGURE 3 Species-specific patterns of (a) litter C remaining (g C g⁻¹ initial litter C), (b) litter N remaining (g N g⁻¹ initial litter N), (c) litter-derived soil N (g N g⁻¹ initial litter N) and (d) litter-induced N in resin capsules (i.e. the difference between each litter+soil microcosm and the bare soil control, in g N g⁻¹ initial litter N) by litter type. (Recall, in (d) the displayed N accumulation in the capsules under litter addition is the one measured minus the accumulation in the resin capsules in the control soil (see Section 2)).

later stages of the decomposition (i.e. 156 and 367 days, *p* < 0.05 and *p* < 0.001, respectively, Table S6).

## 3.2 | Fate of N after 376 days of incubation

Overall, the proportion of unrecovered litter N in the experiment was rather high, with nearly 49.3±3.5% for leaf and 42.8±3.7% for root litters (Figure 4).

### 3.2.1 | Total and litter-induced N in resin capsules at the end of the incubation

The total and litter-induced amount of N accumulated in the resin capsules at the end of the incubation was on average 0.008±0.0003 and 0.001±0.0003 g, respectively. Across all litters, the total and litter-induced amounts of N in the resin capsules after 367 days of incubation were higher (*p* < 0.01) for leaf than for root litters (Figure S6). Litter-induced N was more than four times higher for leaf than for root litters. The litter-induced N in resin capsules also differed significantly between leaf and root litters when expressed in % of initial litter N (Figure 4; Figure S7).

### 3.2.2 | Litter-derived soil N (MAOM and POM) at the end of the incubation

After 367 days of incubation, on average 82.4±1.9% of the total litter-derived soil N pool was MAOM-N, while 17.6±1.9% was

<figure><img src="image_14.png" /></figure>

FIGURE 4 Percentage of N of added litter N (%) in the different pools after 367 days of incubation. Error bars represent SE. Asterisks indicate significant differences between the two litter types (\*\* *p* < 0.05, \*\*\* *p* < 0.01) using t-tests.

POM-N. When expressed in % of the initial litter N (which was higher for leaf than for root litter), both litter-derived POM-N and MAOM-N were significantly higher for root compared to leaf litters (*p* < 0.01, Figure 4; Figure S8). In terms of absolute values, only litter-derived POM-N was significantly higher for root compared to leaf litters (*p* < 0.05), while MAOM-N values did not differ between the two litter types (Figure S6c).

## 3.3 | Influence of initial traits on N dynamics

### 3.3.1 | Temporal N dynamics

The averaged (synthesis) model across all litter types showed that initial litterWSC and leachate C concentrations were the most
<figure><img src="image_15.png" /></figure>

FIGURE 5 Significant relationships of initial traits with different variables related to N dynamics, that is (a, b) the rate of decrease in litter N remaining (represented by Weibull 1/β, with a higher 1/β meaning a higher rate), (c) the rate of N accumulation in resin capsules ($K_A$, which represents the time needed to reach half of maximum N accumulation) as well as (d) total resin N. Green and brown data points represent leaf and root litter samples, respectively. The solid lines represent linear regressions for the two variables of interest (across both leaf and fine-root litter samples), including a 95% confidence interval (shaded grey area). R²-values represent Pearson's correlation coefficients. Note that Weibull 1/β and $K_A$ were transformed to comply with assumption of normality using orderNorm and boxcox transformations, respectively.

important predictors of the rate of decrease in litter N, represented by Weibull 1/β (both *p* < 0.01, adj. $R^2$=0.81; Figure 5a,b; Table S5), whereas the rate of N accumulation in resin capsules (represented by $K_A$) and the maximum N accumulation in resin capsules (represented by $A_{max}$) were mainly affected by initial litter N and leachate P concentrations (both *p* < 0.01, adj. $R^2$=0.50 and $R^2$=0.29), respectively.

Different predictors influenced average daily litter N release and resin N accumulation during different phases of the incubation (Table S5). For example, initial litter cellulose and litter N concentrations negatively and positively affected average daily litter N release during the first 10 days of the incubation, respectively (both *p* < 0.01), while initial litter hemicellulose (*p* < 0.05) and litter N (*p* < 0.1) positively and negatively influenced N release for the period of 156–367 days of the incubation, respectively. Similarly, initial litter cellulose and N concentrations had a positive effect on average daily resin N accumulation during the first 10 days of the incubation (both *p* < 0.01), while initial litter cellulose (*p* < 0.01), lignin (*p* < 0.05) and tannin (*p* < 0.01) concentrations negatively affected resin N accumulation during 38–156 days of the incubation (Table S5).

### 3.3.2 | Fate of N after 376 days of incubation

Initial litter lignin and P concentrations were the most important predictors of litter-derived POM-N across both litter types (*p* < 0.01 and *p* < 0.05, respectively, adj. $R^2$=0.48, Figure 6c,d), whereas initial litter C, N and Mn concentrations best predicted litter-derived MAOM-N (*p* < 0.1, *p* < 0.01 and *p* < 0.01, respectively, adj. $R^2$=0.36,

Table S5, Figure 6a,b) at the end of the incubation (see also Table S5 for linear regression results). A separate analysis for leaf and root litters, respectively, showed that initial litter Mn significantly affected litter-derived MAOM-N of root but not leaf litters (Figure 6b). Total (and litter-induced N, not shown) that had accumulated in resin capsules were mainly affected by leachate C concentrations (*p* < 0.01, adj. $R^2$=0.48, Figure 5d).

The relationships between Weibull 1/β and overall (both leaf and root) litter-derived POM-N and MAOM-N, respectively, were not significant ($R^2$=0.001, *p* > 0.05 and $R^2$=0.04, *p* > 0.05, respectively). When tested separately for leaf and for root litters, only Weibull 1/β and POM-N for root litters were significantly correlated ($R^2$=0.44, *p* < 0.05).

## 4 | DISCUSSION

The overall objective of this study was to assess the role of litter traits in the temporal dynamics of the release of litter N and its fate within the soil. Initial litter quality, which varied substantially across litters, influenced the temporal patterns of organic N release from litter and its transfer into the inorganic or SOM pools. More specifically, highly decomposable litter, mainly characterized by high initial litter N favoured a rapid N release, a fast accumulation of inorganic N in the soil solution as well as the formation of MAOM-N, as compared to poorly decomposable litter (Figure 7). This dichotomy between highly and poorly decomposable litters appears partly consistent across leaf versus root litter, of typically high versus low
<figure><img src="image_16.png" /></figure>

FIGURE 6 Significant relationships of initial traits with (a, b) litter-derived N in mineral-associated organic matter fractions (MAOM-N, g) and (c, d) litter-derived N in particulate organic matter fractions (POM-N, g) at the end of the incubation period across all 24 litter types. Green and brown data points represent leaf and root litter samples, respectively. The solid lines represent linear regressions for the two variables of interest (across both leaf and fine-root litter samples), including a 95% confidence interval (shaded grey area). R²-values represent Pearson's correlation coefficients. Note that for (b), the regression line and R²-value only relates to the fine-root litter samples.

<figure><img src="image_17.png" /></figure>

FIGURE 7 Conceptual summary of the soil N cycling dynamics including its afterlife effects determined by initial litter traits. The size of the boxes and thickness of the arrows highlight differences between the left and right side of the figure in the size of the pool and strength of the flux, respectively. Note that where several fluxes (arrows) lead to the same pool (box) the relative importance of each flux is unknown as all measures were made on pools rather than fluxes. The effects of the respective initial litter traits are indicated by the triangular shaped boxes in the centre of the figure (the direction of the boxes indicates the direction of the effect and the colour of the boxes indicates for which pool and flux the effects were found). POM, particulate organic matter; MAOM, mineral-associated organic matter; WSC, water-soluble compounds; DIN, dissolved inorganic N; min., mineralization rate.

decomposability, respectively. Initial litter traits therefore determine the overall rate of litter N cycling in the soil, with important consequences for ecosystem functioning. Specifically, the rapid N release and accumulation of inorganic N observed for highly decomposable

litters (Figure 7) likely favours higher, but shorter peaks of N availability for plant uptake and increase the potential for soil N loss via leaching. In contrast, the slower N release associated with less decomposable litters may support weaker, but longer peaks of N
availability for plant uptake and decreases the potential for soil N loss.

## 4.1 | Initial litter N strongly determines the fate of decomposing litter N

In line with our first hypothesis, we found that initial litter N concentrations significantly affected the N dynamics during decomposition. Particularly, litter N increased the amount and rate of litter N release (Table S3), the accumulation of inorganic N in the soil solution (Figure 5c) as well as the total amount of MAOM-N at the end of our experiment (Figure 6a). These results support previous findings with N-rich litters (with low C/N) that generally showed a fast N release (Fornara et al., 2009; Manzoni et al., 2008; Moore et al., 2011; Parton et al., 2007; Seneviratne, 2000) and accumulation of inorganic N in the soil solution (Khalil et al., 2005; Mooshammer et al., 2014; Schleuss et al., 2021). The accumulation of inorganic N in the soil solution is probably the result of net N mineralization (Manzoni et al., 2008). This strongly depends on microbial nutrient demand relative to C demand, as well as on the availability of these resources in their substrate. $NH_4^+$ may be released as excess N by microorganisms when N availability is relatively high compared to C availability and with a switch from microbial N to C (and energy) limitation (Mooshammer et al., 2014). More specifically, when N is relatively available, microbial N use efficiency decreases and less N is converted into microbial biomass, allowing for a higher proportion of organic N taken up to be mineralized and released as $NH_4^+$ (Mooshammer et al., 2014). Mooshammer et al. (2014) proposed a threshold for a switch from primarily N to C limitation at a C/N ratio of 20 (Mooshammer et al., 2014). With a range of 9 to 26, the C/N ratios of the litters used in our study were close to or below this threshold, which may have led to a relatively high release of mineral N.

While the presence of litter in soil tended to increase the mineral N pool captured by resin capsules for most of the litters we used, the majority of inorganic N in the resin capsules was of soil origin. Part of the increase in the mineral N pool due to litter input may be attributed to desorption (and/or micro-aggregate destabilization) processes that could have led to the degradation of SOM-N present prior to the start of the experiment, such as N incorporated into native MAOM or N adsorbed on clay particles (Poirier et al., 2014). Possible causes for this could have been a stimulation of microbial activity and N-mining following litter additions (Jilling et al., 2021), as MAOM-N may be an important N source for plants and microbes (Jilling et al., 2018).

Although, the litter N release rate was not directly related with N in either of the POM or MAOM pools (hypothesis 3), the positive effect of initial litter N concentration on the total amount of MAOM-N at the end of our experiment is consistent with the MEMS framework (Cotrufo et al., 2013). It is also in line with the findings of others (Angst et al., 2019; Garcia-Pausas et al., 2012), who showed that OM rich in N was associated with the formation of more stable SOM than OM that was poorer in N. This was not the case for the litter-derived MAOM-C in the same dataset, though (Huys et al., 2022). More labile and rapidly decomposing litter, characterized by higher

initial litter N is more rapidly degraded by microbes, therefore, enhancing the accumulation of microbially derived N-containing products, which may then be stabilized in the soil matrix and form MAOM (Cotrufo et al., 2013). However, it is also likely that some of the positive influence of litter N on MAOM-N is due to sorption of inorganic N on mineral particles (Poirier et al., 2014, 2020). Yet, our experiment does not allow us to distinguish between these two possible pathways leading to higher MAOM-N.

## 4.2 | Nutrients and easily accessible compounds co-determine litter N afterlife effects

Our statistical modelling results suggest that for some litter N afterlife effects, the initial litter N has less predictive value than certain other initial litter traits (Table S5). In partial support of our second hypothesis, initial litter total WSC concentration was the most important predictor of the rate of litter N release patterns. This underscores the notion that litters associated with high WSC concentrations tend to lose soluble nutrients, such as N, more rapidly during the initial stages of decomposition (Berg & McClougherty, 2020; Pei et al., 2019; Trinsoutrot, Recous, Bentz, et al., 2000). In contrast to what we hypothesized, the concentration of the structural compounds such as lignin, but also hemicellulose, and cellulose had a limited influence on N dynamics during the later stages of the incubation but rather played a role in the early stages. As such, higher litter cellulose delayed litter N release during the first 10 days of incubation, whereas higher litter hemicellulose tended to accelerate N release during later stages. Generally, cellulose and hemicellulose are relatively easily accessible C sources (Berg & McClougherty, 2020) and may stimulate DON production through increased microbial substrate use efficiency (Moorhead et al., 2013). Lignin was negatively correlated with inorganic N accumulation in the soil solution during the intermediate phase of the incubation. However, lignin was not associated with any change in daily N release rates, affirming another study (Seneviratne, 2000), which showed that lignin was not a reliable predictor of N release in the soil. In addition, the negative correlation of tannins with inorganic N accumulation could hint at complexation of tannins with compounds containing N, such as proteins (Kraus et al., 2003).

We further found that—besides initial litter N—initial litter C positively influenced the build-up of MAOM-N. While there are only a few studies that have focused on MAOM-N in this regard, this result is comparable with a study showing that, somewhat surprisingly, the presence of some complex compounds such as the lignin monomer syringol can be related to a higher microbial C use efficiency (and higher fungal abundance) as well as to the accumulation of SOC in soil (Kallenbach et al., 2016).

Another important predictor of increasing litter-derived MAOM-N was increasing initial litter Mn. The importance of Mn for the stabilization of organic compounds as MAOM has recently been highlighted (Li et al., 2021; Poirier et al., 2018). As Mn plays a key role in one of the major lignin degradation pathways (Berg & McClougherty, 2020; Li et al., 2021), it is especially important during later stages of the
<figure><img src="image_18.png" /></figure>

decomposition process potentially increasing DOM production (Kalbitz et al., 2006; Trum et al., 2015), which may then enhance stabilization of OM as MAOM rather than POM (Kalbitz & Kaiser, 2008).

This Mn-driven increase in MAOM-N via lignin breakdown in later decomposition stages was accompanied by an increase of POM-N accumulation at the end of the incubation period with higher initial litter lignin concentration. The recalcitrance of lignin may shield part of the litter N (von Lützow et al., 2006), which is then directed along with lignin degradation by-products to the formation of POM (Huys et al., 2022). The positive effect of lignin concentrations on POM-N could further point to the role of aggregate formation by hyphae or mucilage from fungi (both being characterized by lower C/N than SOM; Lucas et al., 2014; Miller & Jastrow, 1990; Tisdall & Oades, 1982) involved in the degradation of lignin. In this case, POM-N could be physically protected from microbial degradation for decades (von Lützow et al., 2008).

## 4.3 | Consequences for ecosystem functioning of litter quality

Generally, our results suggest contrasting afterlife effects and thereby consequences for ecosystem functioning of highly (N-rich) and poorly (N-poor) decomposable litters. In particular, highly decomposable litters displayed a quite rapid litter N release and shorter, but higher peaks of litter-derived inorganic N accumulation in the soil solution. Overall, this indicates a fast N cycling, with a high amount of litter N readily available for plant uptake for these litters. However, it remains to be tested whether this N input is beneficial to plant N uptake. In biomes where plants shed most litter at the end of the growing season, it is unclear whether this fast release would be immediately taken up by plants or would simply exceed plants' demand. Yet, the apparently relatively large mineral N pool derived from highly decomposable litter may also be lost from the system via leaching of $NO_3^-$ (Neff III et al., 2003) or denitrification. The potential for N leaching may especially be high in sandy soils (van Kessel et al., 2009), for which the stabilization through organo-mineral interactions is limited (Castellano et al., 2015). Moreover, N leaching may be promoted in temperate and cold climates, where, in autumn and winter, rain events coincide with leaf and root senescence and a low N demand by plants and soil organisms (Thorup-Kristensen et al., 2003). In contrast, poorly decomposable litters related to a slower N cycling (i.e. a slower litter N release and longer but longer peak of inorganic N accumulation in the soil solution) can be associated with a greater N retention and reduced potential for N leaching.

We acknowledge that our study under controlled conditions excluded a series of environmental and biotic factors that could additionally affect the measured processes under natural conditions. In particular, to make inferences related to ecosystem-level processes and functions, such as N leaching or ecosystem productivity, it is necessary to study how temporal patterns of plant N demand coincide with the patterns of available N in the soil. The amount of N accumulated in resin capsules is only a rough indicator of N

leaching potential or of the total amount of N available to plants and potentially taken up by them under natural conditions (Schimel & Bennett, 2004). Plants together with their mycorrhizal associates actively forage for soil N and compete with heterotrophic microbes (Kuzyakov & Xu, 2013), a complex and dynamic process that is difficult to predict from a lab study without plants. Plants may also substantially influence (enhance or suppress) microbial decomposition processes, for example through the release of exudates that can result in increased mineralization of N (Kuzyakov & Xu, 2013) and increased incorporation in MAOM (Jilling et al., 2021). We note that the relatively high unrecovered proportion of N could hint at high denitrification rates due to favourable moisture conditions and high N availability in the soils, highlighting again the need to perform such experiments in soils with growing plants. Finally, the low clay content and relatively low cation exchange capacity (CEC) of the soil used in this study likely constrain the formation and stabilization potential of MAOM-N. Soils with higher clay content could promote MAOM formation, thereby enhancing N retention and reducing potential N losses via leaching. These interactions between soil texture, CEC and N dynamics call for further studies encompassing longer temporal scales and a wider range of soil types.

## 4.4 | Contrasting afterlife effects of leaf versus root litters

In general agreement with our hypothesis that N dynamics differ between leaf and root litters, leaf litters were associated with accelerated initial litter N release and an overall greater inorganic N built-up in resins compared to root litters. A faster initial N release of foliar compared to root litter was also observed by others (Hicks Pries et al., 2017) and is in our study likely related to overall higher concentrations in initial litter N, WSC, Mg and Ca in leaf compared to root tissues. However, other studies reported opposite findings (Fornara et al., 2009; Moretto et al., 2001; Parton et al., 2007), which may reflect non-consistent differences in these leaf and root chemical trait values among plant species.

According to the MEMS framework (Cotrufo et al., 2013), which was supported by the findings of some studies (Bird et al., 2008; Garcia-Pausas et al., 2012; Hatton, Castanha, et al., 2015), we hypothesized that the more labile leaf litter would result in relatively greater incorporation of litter N into the stable MAOM pool. Our results do not support this hypothesis, as only the accumulation of POM-N (absolute values) significantly differed between the two litter types (Figure S6c). Hence, they agree with the findings of Hicks Pries et al. (2017), who also reported no litter type effect in association with MAOM, but an effect on the POM fraction. One explanation for the lack of a greater MAOM-N accumulation associated with leaf litters could be their faster and massive N release early in the decomposition process. This rapid release may exceed the microbial community's needs leading to higher mineral N availability in the soil solution. As a result, more N could be adsorbed onto resin capsules and lost through denitrification. In situ, as opposed to our closed
<figure><img src="image_19.png" /></figure>

microcosm, this may further lead to substantial N loss through leaching. However, it is also possible that the type of N-containing compounds in leaf versus root litter plays a key role in determining whether N from these litters is incorporated into MAOM-N or remains in other soil pools.

Overall, our results showed that leaf and root litters distinctly affect N dynamics and overall soil N availability, indicating a different functional role of these two litter types, at least for the herbaceous species studied here. While root litters appear to be associated with greater soil N retention potential, leaf litters may increase the risk for N loss from the system.

## 5 | CONCLUSIONS

As one of the rare studies integrating different variables characterizing the N cycle simultaneously, our results provide a more complete picture of N dynamics during litter decomposition under controlled conditions than the bulk of litter N loss studies. We identified initial litter traits and litter types (root vs. leaf) that determine the direction of litter afterlife effects on N cycling in soils, that is towards faster N cycling and potential N loss or towards slower N cycling and N retention (Figure 7). Specifically, litter traits such as initial N may be good proxies for the fate of litter-derived N in the soil, that is its availability in the soil solution, its medium- to longer-term stabilization or its tendency to being lost from the system. However, the presence of living plants may modify the responses documented in our study, and N dynamics will also depend on the soil type and other environmental factors (Cornwell & Weedon, 2014). Further, we note that differences between leaf and root litters may even be stronger under natural conditions when leaf litter decomposes mostly on the soil surface and fine-root litter within the soil matrix, and leaf and root litter turnover follow different temporal dynamics. Nonetheless, our findings open promising perspectives for further testing these concepts in situ, whether in semi-natural ecosystems or in human-managed ecosystems, where such knowledge may allow acting on the N cycle by favouring the input of specific litter types and traits (Cornwell & Weedon, 2014).

### AUTHOR CONTRIBUTIONS

Grégoire T. Freschet conceived the experiment, with input from Hättenschwiler Stephan. Grégoire T. Freschet, Hättenschwiler Stephan and Vincent Poirier performed the experiment and ran the analyses. Janna Wambsganss, Raoul Huys and Grégoire T. Freschet analysed the data. Janna Wambsganss wrote the first draft of the paper. All authors contributed to the writing of the paper.

### ACKNOWLEDGEMENTS

This study was supported by the EC2CO grant 'MULTIVERS' to G.T.F. and the 'Laboratoire d'Excellence' TULIP (ANR-10-LABX-0041). This experiment was further supported by the CEFE teams 'terrain d'experiences' and 'plateforme d'analyses chimiques' (LabEx CeMEB, an ANR 'Investissements d'avenir' program

ANR-10-LABX-0004). We are grateful to Malo Bourget, Catherine Roumet, Nathalie Fromin, Katherine Urban-Mead, Yamei Chen, Lin Chao, David Degueldre, Thierry Matthieu, Pauline Durbin, Bruno Buatois, Nicolas Barthes and Raphaëlle Leclerc for their help at various steps of the experiment. Open Access funding enabled and organized by Projekt DEAL.

### CONFLICT OF INTEREST STATEMENT

Grégoire T. Freschet is an Associate Editor of *Journal of Ecology*, but took no part in the peer review and decision-making processes for this paper.

### PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/1365-2745.70011.

### DATA AVAILABILITY STATEMENT

Data are available at the Dryad Repository: https://doi.org/10.5061/dryad.msbcc2g89 (Wambsganss et al., 2025).

### ORCID

Janna Wambsganss https://orcid.org/0000-0002-2968-558X

Raoul Huys https://orcid.org/0000-0002-7499-674X

Alison D. Munson https://orcid.org/0000-0001-6013-7998

Grégoire T. Freschet https://orcid.org/0000-0002-8830-3860

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## SUPPORTING INFORMATION

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

**Table S1.** Comparison of Akaike Information Criterion (AIC) values for model fits (i.e. Weibull and single-negative exponential model) for the course of litter N release via decomposition (see Section 2).

**Table S2.** Linear regression results after model selection and averaging testing which initial litter traits (here: Cellulose, Hemicellulose, Lignin, and Tannins) best predict the response variables.

**Table S3.** Linear regression results after model selection and averaging testing which initial litter traits (here: Litter N (Nitrogen), C (Carbon), P (Phosphorus), Ca (Calcium), Mg (Magnesium), Mn (Manganese)) best predict the response variables.

**Table S4.** Linear regression results after model selection and averaging testing which initial litter traits (here: WSC (water-soluble compounds), N<sub>L</sub> (N leachates), P<sub>L</sub> (P leachates), and C<sub>L</sub> (C leachates)) best predict the response variables.

**Table S5.** Linear regression results after model selection and averaging results of the synthesis models (which included the significant predictors of the models reported in Tables S2–S4).

**Table S6.** Mean values (±SE) of N pools and rates and test statistics.

**Figure S1.** Species-specific patterns of (a) C remaining (g), (b) litter N remaining (g), (c) litter-derived soil N (g), and (d) litter-induced N in resin capsules (g N capsule⁻¹) by litter type.

**Figure S2.** Proportion of litter N remaining (% of initial) by species and litter type, i.e., leaves (green) and roots (brown) throughout the decomposition period of 367 days.

**Figure S3.** Average daily litter N release rate (mg N resin day⁻¹) by leaf and root litter type during the different incubation intervals. Error bars represent SE.

**Figure S4.** Total N in resin capsules by species and litter type over the course of the incubation period.

**Figure S5.** Average daily resin N accumulation rates (µg day⁻¹) by leaf and root litter type during the different incubation intervals. Error bars represent SE.

**Figure S6.** (a) Total N in resin capsules (g), (b) litter-induced N in resin capsules (g capsule⁻¹), and (c) mean N in soil fractions, i.e. mineral-associated organic matter (MAOM) and particulate organic matter (POM) fractions (g) by litter type after 367 days of incubation. Error bars represent SE.

**Figure S7.** Total N in resin capsules relative to initial litter N after 367 days of incubation for all 24 litters and by plant functional type.

**Figure S8.** Species-specific patterns of litter-derived MAOM-N and POM-N (gg⁻¹ initial litter N) after 367 days of incubation for all 24 litter types and by plant functional type.

**How to cite this article:** Wambsganss, J., Huys, R., Stephan, H., Poirier, V., Munson, A. D., & Freschet, G. T. (2025). The afterlife effects of leaf and root litter traits on soil N cycling. *Journal of Ecology*, 100, 1–18. https://doi.org/10.1111/1365-2745.70011