Root litter diversity and functional identity regulate soil carbon and nitrogen cycling in a typical steppe

Journal Pre-proof Root litter diversity and functional identity regulate soil carbon and nitrogen cycling in a typical steppe Jing Man, Bo Tang, Wen Xing, Yang Wang, Xuezhen Zhao, Yongfei Bai PII:

S0038-0717(19)30352-9

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https://doi.org/10.1016/j.soilbio.2019.107688

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Soil Biology and Biochemistry

Received Date: 19 July 2019 Revised Date:

21 October 2019

Accepted Date: 24 November 2019

Please cite this article as: Man, J., Tang, B., Xing, W., Wang, Y., Zhao, X., Bai, Y., Root litter diversity and functional identity regulate soil carbon and nitrogen cycling in a typical steppe, Soil Biology and Biochemistry (2019), doi: https://doi.org/10.1016/j.soilbio.2019.107688. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Root litter diversity and functional identity regulate soil carbon and

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nitrogen cycling in a typical steppe

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Jing Man1,2†, Bo Tang1,2†, Wen Xing1,2, Yang Wang1, Xuezhen Zhao1,2, Yongfei Bai1,2*

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1

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Chinese Academy of Sciences, Beijing 100093, China

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2

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19A Yuquan Road, Beijing 100049, China

State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,

Colleage of Resources and Environment, University of Chinese Academy of Sciences, No.

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* Correspondence address:

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Yongfei Bai

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State Key Laboratory of Vegetation and Environmental Change,

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Institute of Botany, the Chinese Academy of Sciences

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20 Nanxincun, Xiangshan 100093

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Beijing, P.R. China

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Tel: (+86)-10-6283-6272

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Fax: (+86)-10-8259-5771

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Email: [email protected]

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† These authors contribute equally to this work

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Abstract

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Root litter decomposition is the dominant source of soil organic carbon (C) and nitrogen (N)

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in grasslands. Few studies, however, have explored the effect of root litter diversity on soil C

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and N cycling. This study investigated the effects of species diversity and functional traits of

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root litter on soil CO2 and N2O release, net ammonification, net nitrification, and net N

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mineralization based on a 56-day incubation of grassland soils with root litter mixtures

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containing one, two, or four native plant species. The increasing species richness of root litter

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decreased the cumulative CO2 and N2O release in the soil, but enhanced the net

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ammonification, nitrate immobilization, and N mineralization. Root litter diversity has a

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predominant non-additive antagonistic effect on the release of soil CO2 and N2O, and a

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synergistic effect on the net ammonification, nitrate immobilization, and N mineralization in

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the soil. The functional identity rather than functional diversity of root traits explains most of

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the variation in soil C and N cycling. A high C: N ratio and low concentrations of N, P, K,

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and Di-O-alkyl-C (characteristic of celluloses) were found to be key to the antagonistic

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effects associated with cumulative release of CO2 from the soil. For net N ammonification

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and mineralization, the synergistic effect was principally induced by the high levels of

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carbohydrate-C and N and the low C: N ratios in root litter mixtures. Our study highlights the

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role and mechanisms of increased root litter diversity in decreasing soil CO2 and N2O release

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and in increasing the net N mineralization via non-additive antagonistic and synergistic

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effects of dominant root traits.

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Keywords: root litter diversity; soil C and N cycling; non-additive effect; root chemical traits;

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functional root traits; functional identity. 2

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1. Introduction

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Decomposition of both leaf and root litter plays a critical role in global carbon (C)

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cycle and provides nutrients for plants to maintain the ecosystem via primary production,

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especially of nitrogen (N), a limiting element in most terrestrial ecosystems (Parton et al.,

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2007; Gessner et al., 2010). However, despite the increased interest in leaf litter

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decomposition, the effect of root litter decomposition and its quality on soil C and N cycling

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has been frequently overlooked (Zhang et al., 2008; Freschet et al., 2013). In grasslands, the

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plant biomass allocated to roots is more than three times higher than that allocated to

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aboveground tissues, and root litter accounts for 33% of the annual litter generated (Jackson

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et al., 1996; Freschet et al., 2013). Moreover, root-derived C is sequestered in soil more

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efficiently than in leaves, thus affecting the global C budget (Rasse et al., 2005; Schmidt et al.,

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2011; Sokol and Bradford, 2018). Although few studies have analyzed the decomposition of

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fine roots, almost all of these studies focused on single root decomposition per se, that is loss

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of litter mass (Gill and Burke, 2002; Vivanco and Austin, 2006; Smith et al., 2014). However,

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plant roots in natural grasslands are frequently intermingled and decomposed in mixtures.

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Thus, a better understanding of root litter diversity and its effect on soil C and N cycling is

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imperative, particularly in grassland ecosystems.

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Studies investigating the decomposition of diverse leaf litter reported that the effects

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of species litter diversity on decomposition cannot be predicted from the data based on single

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species within the litter mixtures, suggesting a non-additive effect involving multiple

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decomposing species (Wardle et al., 1997; Hättenschwiler et al., 2005; Lecerf et al., 2011).

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Mixed leaf litter generally has a synergistic effect on decomposition rates and nutrient release

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(Gartner and Cardon, 2004; Lecerf et al., 2011; Chen et al., 2017b; Zhou et al., 2019). By 3

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contrast, sporadic studies analyzing the decomposition of root litter mixtures failed to explain

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the general pattern and even yielded conflicting results (Cong et al., 2015; Guerrero-Ramírez

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et al., 2016; Prieto et al., 2017). For example, Cong et al. (2015) found that decomposition of

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mixed grass roots didn’t show a significant non-additive effect on soil CO2 release. Prieto et

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al. (2017) detected both inhibitory and additive effects on mass loss during the decomposition

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of mixed grass root litter in a Mediterranean ecosystem. Moreover, studies focused on the

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effects of mixed root litter decomposition on the ecosystem mostly from the perspective of C

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loss, with little insight into soil N transformation. A previous study suggested that the

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decomposition of fine roots promoted soil N mineralization (Fornara et al., 2009). Therefore,

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further studies are needed to simultaneously evaluate the effect of diverse root litter species

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decomposition on soil C and N cycling.

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The mechanisms underlying the non-additive effect of diverse mixtures of leaf and

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root litter are still disputed, because of inconsistent effects (synergism and/or antagonism) in

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different studies investigating litter composition under varying experimental durations

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(Srivastava et al., 2009; Lecerf et al., 2011). An increasing number of studies, however, have

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confirmed that the composition of the litter species is the most fundamental factor underlying

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this effect, and more deeply, the chemistry of the composite litter rather than species richness

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under specific environmental conditions (Hättenschwiler et al., 2005; Handa et al., 2014;

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Santonja et al., 2017). This finding might be more convincing for root litter decay since roots

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usually decompose in the soil, which has a buffer effect on extreme climate. Thus, under

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specific environments, root litter quality might be more important than environmental factors

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(e.g. climatic fluctuations) in controlling root decomposition (Silver and Miya, 2001; 4

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Bradford et al., 2016). High N and phosphorus (P) content but poor lignin levels and a low

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C:N ratio in the roots accelerate root decomposition and decay, and vice versa (Silver and

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Miya, 2001; Smith et al., 2014). Although these traits have been reported to influence

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decomposition of single root species, few studies have described their effects in non-additive

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soil C and N dynamics at the community level (i.e. integrate traits of individual species into a

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community-level feature, which is defined as “functional parameter” of a plant community)

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(Violle et al., 2007).

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Functional traits are defined as any traits (e.g. biochemical, morphological and

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ecophysiological traits) of an organism that influence individual performance and ecosystem

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processes (Petchey and Gaston, 2006; Violle et al., 2007; de Bello et al., 2010). Functional

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traits diversity, which includes trait-based functional diversity and functional identity, has

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been widely used to explain and predict the effects of species composition on primary

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productivity and litter decomposition in the ecosystem (Petchey and Gaston, 2006; Meier and

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Bowman, 2008). For example, recent studies have reported that dissimilarity of functional

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traits within mixed leaf litter (i.e., functional diversity, FD) partly accounted for the

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non-additive effects on litter mass loss or belowground soil processes (Lecerf et al., 2011;

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Chen et al., 2017b). Interactions between litter species with diverse chemical traits were

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driven by the actions of soil microorganisms, and chemically diverse litter mixtures would

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provide a rich source of nutrients for microbial communities, thereby enhancing or inhibiting

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the decomposition processes (Meier and Bowman, 2008; Lecerf et al., 2011; Handa et al.,

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2014). In contrast, functional identity (FI), which is another dimension for measuring

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functional trait diversity, proposes that functional traits of the dominant species in a 5

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community determine the processes in the ecosystem (Grime, 1998; Tobner et al., 2016). This

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approach has rarely been studied although it also accounted for non-additive cycling effect of

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soil C and N in mixtures of leaf litter (Meier and Bowman, 2010). In fact, both FD and FI are

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not mutually exclusive in explaining and predicting the ecosystem processes; however, their

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effects on decomposition may not be equally significant (Mokany et al., 2008).Notably,

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almost all studies investigating the effects of FD or FI on the decomposition of litter mixtures

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have focused on aboveground traits (Laliberte, 2017). Few studies have simultaneously

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compared the relative contributions of FD and FI of root chemistry to soil C and N cycling.

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In this study, we tried to elucidate the effects of species diversity of root litter and FD

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and FI of root chemical traits on soil C cycling and N transformation. To isolate the effects of

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root litter diversity from other limiting factors such as temperature and moisture, we

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conducted a 56-day laboratory experiment incubating grassland soils with root litter mixtures

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containing one, two, or four native plant species. Specifically, we addressed two questions: 1)

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How does species diversity of root litter affect the cumulative release of soil CO2 and N2O,

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net ammonification, net nitrification, and net N mineralization in the Inner Mongolia

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grassland? 2) How do the effects of FD and FI of root differ in controlling the release of soil

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CO2 and N2O, and N mineralization? We hypothesized that high species diversity of root

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litter has a negative effect on cumulative CO2 release in the soil because it increases the

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probability of including species with hardly decomposable roots (e.g. high C:N ratio and/or

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high contents of lignin, tannins or polyphenols), which reduces the overall decomposition in

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root mixtures by inhibiting microbial activities, producing a non-additive antagonistic effect

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on cumulative CO2 in the soil (Prieto et al., 2017). We further hypothesized that high species 6

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diversity of root has a positive effect on soil N mineralization. Previous studies have provided

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evidence that niche complementarity may play a more important role in affecting the litter N

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dynamics than C losses, because more N and N-related compounds in litter mixtures may

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elevate the nutrient availability for microbial communities (Hättenschwiler et al., 2005;

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Garcia-Palacios et al., 2017), thereby resulting in a non-additive synergistic effect on soil N

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mineralization. Finally, we state that root FD and FI are two key variables explaining the

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varying degrees of non-additive synergistic or antagonistic effects of root mixtures on the

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release of soil CO2, and N mineralization.

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2. Materials and Methods

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2.1. Study site, and root and soil sampling

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Our study was conducted at the Inner Mongolia Grassland Ecosystem Research

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Station (IMGERS) of the Chinese Academy of Sciences, which is located in the Xilin River

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Basin, Inner Mongolia Autonomous Region, China (116°42′ E, 43°38′ N) (Bai et al., 2010).

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The altitude of the study site was about 1260 m a.s.l. The mean annual temperature of our

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study site was 0.3ºC, with the mean monthly temperatures ranging from -21.6ºC in January to

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19.0ºC in July. The mean annual precipitation was 346 mm, with 60 to 80% occurring during

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the growing season (May to August). The soil was considered as dark chestnut type

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(Kastanozems according to IUSS Working Group WRB, 2015).

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The plant roots and soil samples were collected within the permanent research plot of

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IMGERS, which was an overgrazed area before 2013 and was fenced against grazing in June

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2013. Leymus chinensis Trin. Tzvel (perennial rhizomatous grass) and Stipa grandis P. Smirn

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(perennial C3 bunchgrass) are two dominant species, and Carex korshinskyi Kom (perennial 7

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sedge) and Cleistogenes squarrosa Trin Keng (perennial C4 bunchgrass) are two common

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species in the study area, together accounting for more than 80% of the total aboveground

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biomass. The roots of L. chinensis, S. grandis, C. korshinskyi, and C. squarrosa were

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sampled by randomly excavating several 20 × 20 × 30 cm soil cores within a 1ha area in

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September 2016. The collected soil cores were transported to the laboratory, and the roots

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were separated according to species based on their morphological characteristics. The roots

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of the same species were pooled together and washed under running water. All of the

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collected roots were air-dried for three weeks, and cut into lengths of 1cm and stored at room

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temperature. To ensure homogeneity, we did not distinguish between living and dead roots in

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the absence of significant differences in N and P concentrations (Aerts, 1990; Aerts et al.,

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1992). Soils at a depth of 0 to 10 cm, where most of the fine roots distributed, were collected

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in early June 2017 from the same locations within the study site. Roots and rocks were

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removed by hand. Each soil sample was sieved through a 1mm screen, homogenized, kept at

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the moisture content, and stored at 4ºC before mixing with the root litter.

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2.2. Experimental design To explore the impact of species richness of root litter and functional traits diversity

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(i.e. functional diversity and functional identity) of root litter on soil C and N cycling, we

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incubated grassland soil samples with root litter mixtures containing one, two, or four native

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dominant and common species. The experimental approach was used to distinguish the

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effects of root litter biodiversity from other confounding environmental factors, such as soil

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temperature and moisture, which influence litter decomposition. Before the experiment, the

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root litters of four plant species were sterilized by autoclaving twice consecutively (at 121ºC 8

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for 20 min) to ensure maximum levels of soil decomposer communities (Fanin and Bertrand,

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2016). There were 11 treatments and five replicates for each treatment in the experiment. The

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experiment was set up as a randomized block design with five blocks as replicates. The 11

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treatments included soils with root litter mixtures containing one (four treatments), two (six

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treatments), and four (one treatment) native dominant and common plant species. For the

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treatment of each single species, 50 g of dry-weight soil were mixed thoroughly with 2 g root

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litter derived from each of the four species. For the treatment of two species, 50 g of

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dry-weight soil were mixed thoroughly with 2 g of root litter derived from two species, each

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species accounting for 50%. For the treatment of four species, 50 g of dry-weight soil were

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mixed thoroughly with 2 g root litter obtained from four species, each species accounting for

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25%. All mixtures of root litters were incubated in 250 mL flasks. To destructively sample

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twice, we constructed two identical sets of soil-root litter mixtures, each set including 55

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flasks (11 treatments × 5 replicates). One set was sampled on day 28 to measure the soil

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ammonium (NH4+-N) and nitrate (NO3--N) levels, and the other was used to measure soil

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CO2 and N2O released during the experiment and harvested to measure NH4+-N and NO3--N

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on day 56. A total of 110 soil microcosms were adjusted to 60% water-filled pore space

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(WFP), covered with semi-permeable membrane (the membrane was perforated which can

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reduce water evaporation from the soil while allow gas to diffuse), and transferred into a

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growth chamber for dark incubation (Linn and Doran, 1984; Mi et al., 2014). The

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temperature in the growth chamber was set to 23ºC during the daytime (12h) and 15ºC at

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night (12 h) to mimic diurnal variation in field temperature, because a constant temperature

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(i.e. 25ºC, the optimum temperature for microbial respiration) may overestimate the actual 9

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microbial C mineralization (Linn and Doran, 1984; Ci et al., 2015). The changing diurnal and

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nocturnal temperatures corresponded to the temperature difference between day and night in

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the peak growing season (July to August), which was around 8ºC at our study site. Soil

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moisture was maintained around 60% WFP by weighing each flask every four days and

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re-adjusting the mass by adding sterile distilled water when necessary.

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2.3. Measurement of initial root chemical composition The sterilized roots were used to measure the initial chemical traits. Root C and N

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concentrations were measured using an elemental analyzer (Vario EL III, Elementar,

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Germany). The concentrations of root P, aluminum (Al), calcium (Ca), magnesium (Mg), and

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potassium (K) were determined via inductively coupled plasma-optical emission

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spectrometry (ICP-OES; ICAP 6300, Thermo Scientific, USA) after microwave digestion in

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concentrated sulfuric acid.

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We measured some other root chemical compounds, such as cutins, celluloses, lignin

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and tannin via solid-state 13C-cross-polarization/total sideband suppression (CP/TOSS)

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nuclear magnetic resonance (NMR). This method has been used successfully to characterise

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the organic C components in litter. Compared with conventional wet-chemical analyses,

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NMR classified the litter organic components into different C fractions that have biological

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relevance, which can better assess the effect of litter chemical compositions on soil processes

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(Wang et al., 2004; Ono et al., 2010). The NMR spectra were obtained using a Bruker

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AVANCE-III 400 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) at a

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frequency of 100.64 MHz. Root samples were packed in a 4-mm diameter cylindrical

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zirconia rotor with Kel-F endcaps. The magnetic angle spinning rate was 5000 Hz, and the 10

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contact time was 1 ms with a 1 s recycle delay. For each sample, the acquisition time was 13

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ms, and approximately 3500 scans were collected. Chemical shifts were externally referenced

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to glycine at 176.03 ppm. All spectral processing was completed using Bruker TopSpin 2.1

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software. Eight common chemical shifts, i.e. 0–45, 45–60, 60–90, 90–110, 110–145, 145–165,

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165–190, and 190–210 ppm, were assigned to alkyl-C, N-alkyl-C, carbohydrate-C,

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Di-O-alkyl-C, aryl-C, O-aryl-C, carbonyl-C, and ketone-C, respectively (Mathers et al.,

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2007). The relative content (%) of each C form was calculated by integrating the signal

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intensities in the respective chemical shift region and expressed as percentages of the total

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area (0–210 ppm) (Table S1). Specifically, alkyl-C was associated with waxes, cutins,

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suberins and lipids; N-alkyl-C was found in amino acids; carbohydrate-C was mainly

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associated with sugars and polysaccharides; Di-O-alkyl-C was mainly found in celluloses;

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aryl-C and O-aryl-C were associated with phenols, polyphenols, lignin and tannin; and

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carboxyl-C and ketone-C were associated with organic acids (Mathers et al., 2007; Bonanomi

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et al., 2011).

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Based on root chemical traits measured above, FI was measured using community

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weighted means (CWM) (Garnier et al., 2004). FD was measured via Rao’s quadratic entropy

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(Rao’s Q) (Botta-Dukat, 2005). For each trait, FI and FD of the litter mixtures were

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calculated as follows: × traiti,

CWM = ∑

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(1)

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where pi is the relative abundance of the species i in the litter mixture and traiti denotes the

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trait value of species i.

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Raoi = ∑

∑

× 11

,

(2)

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where pi and pj represent the relative abundance of the species i and j in the litter mixture,

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respectively, and dij denotes the trait dissimilarity coefficient based on the Euclidean distance

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between species i and j in the multivariate trait space of the litter mixture. All litter trait

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values were normalized using z-score standardization before calculating these two indices.

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2.4. Soil cumulative CO2 and N2O release We measured the soil CO2 and N2O concentrations at 15ºC and 23ºC, respectively, on

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days 2, 3, 6, 7, 14, 21, 28, 42, and 56. All 55 microcosms were sealed with airtight butyl

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rubber stoppers, which had one inlet and one outlet pinhole. We flushed room air into each

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flask for about 3 min. Three empty flasks were also prepared similarly for calibration. All

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flasks were sealed with parafilm and incubated for 3 h to generate cumulative CO2 and N2O

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in headspace. Later, the headspace gas was mixed 3 to 5 times using gastight syringes with

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three-way stopcocks. A 30 mL sample of thoroughly mixed headspace gas was analyzed with

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a gas chromatograph (Agilent 7890A, Santa Clara, CA). After each measurement of CO2 and

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N2O concentrations, all 55 flasks were covered with semi-permeable membrane again. CO2

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and N2O release rates (µgCO2-C g-1 soil d-1 and µgN2O-N kg-1 soil d-1) at 15ºC and 23ºC were

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calculated, respectively, on each sampling day according to the concentrations of CO2 and

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N2O, the Ideal Gas Law, headspace volume of flask and dry mass of soil in the flask. The

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final CO2 or N2O release rate was expressed as the average values at 15ºC and 23ºC. Finally,

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we calculated the cumulative CO2 and N2O release (µgCO2-C g-1 soil and µgN2O-N kg-1 soil)

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via linear interpolation based on CO2 and N2O release rates measured on days 2, 3, 6, 7, 14,

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21, 28, 42, and 56.

268 12

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2.5. Soil net N transformation Contents of soil NH4+-N and NO3--N were extracted with 12.5 mL of 2M KCl from

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2.5 g fresh soil before and after incubation. The solution was shaken for 1 h and then filtered

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using a filter paper (Whatman no.1, 9 cm in diameter), and NH4+-N and NO3--N

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concentrations were measured using a flow injection autoanalyzer (SEAL Analytical,

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Germany). Soil net ammonification and nitrification levels on days 28 and 56 were calculated

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by subtracting the initial content of NH4+-N and NO3--N in the soil from those in the

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incubated soil. Soil net N mineralization on days 28 and 56 was calculated by subtracting the

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initial concentrations of inorganic N (sum of NH4+-N and NO3--N) in the soil from those in

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the incubated soil.

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Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) contents in

280

the soil were measured by the chloroform fumigation extraction method (Vance et al., 1987).

281

The extractable C and N in the fumigated and un-fumigated soil samples extracted by 0.5M

282

K2SO4 were measured via a total C/N analyzer (Multi-N/C 2100, Analytic Jena AG,

283

Germany). Efficiency factors (Ken) of 0.45 for C (Joergensen, 1996) and 0.54 for N

284

(Joergensen and Mueller, 1996) were used to calculated the MBC and MBN respectively.

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2.6. Data analyses

287

We used one-way ANOVA followed by Duncan multiple comparison to test the

288

effects of species richness of root litter (i.e. one, two, and four species) on seven soil response

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variables (i.e. cumulative CO2, N2O release, net ammonification, net nitrification, net N

290

mineralization, MBC and MBN) on days 28 and 56. The same procedure was conducted to

291

determine the effects of root litters containing single and mixed species on seven soil 13

292 293

variables on days 28 and 56. Based on the values obtained from single species litter, we calculated the expected

294

values (E) of each soil variable in two- and four-species mixtures according to the following

295

equation (Wardle et al., 1997): E=∑

296

/ ,

(3)

297

where Ri is the soil response variable when only species i was included, and S denotes the

298

number of species in the litter mixtures. For each two- and four-species mixture, we

299

determined the difference between the observed values (O) and E via paired t-test for

300

non-additive effects in each response variable. For each response variable, a significant

301

difference between O and E (P < 0.05) indicated a non-additive effect; otherwise, an additive

302

effect was inferred. The direction and magnitude of non-additive effect (or litter mixture

303

effect) were calculated as following (Wardle et al., 1997):

304

Litter mixture effect = ( − )⁄ ,

305

wherein, positive (O-E) values indicate synergistic effects, and negative (O-E) values

306

indicate antagonistic effects.

307

(4)

For soil variables that showed a non-additive effect, we used a general linear model to

308

determine the impact of each root species. We determined the relative contributions of FI

309

(CWM) and FD (Rao’Q) to the litter effect of each soil variable using the variation

310

partitioning analyses with the calc.relimp function in the ‘relaimpo’ package. Given the high

311

number of litter traits measured, and different soil variables affected by different litter traits,

312

we performed Pearson correlations between each soil response variable on day 56 (in single

313

or mixed species) and 16 litter traits with preselected traits for calculation of CWM and 14

314

Rao’Q. Root traits that significantly correlated with soil response variables in single- or

315

multiple- species mixtures (P < 0.05) were both retained (Table S2 and S3). Next, we

316

performed a principal component analysis (PCA) using CWMs and Rao’Q values of all traits

317

selected for each soil variable. CWMs and Rao’Q values were standardized using z-scores

318

prior to PCA. For each PCA, the first two PC axes (PC1 and PC2) were retained to capture

319

the variance in CWM and Rao’Q values (Fig S3 and S4). The relative contributions of CWM

320

and Rao’Q were tested by modeling the non-additive effects of each soil variable as a

321

function of PC scores (both CWMs and Rao’Q) via variation partitioning analyses, because

322

each soil variable corresponded to a pair of PCAs (i.e. CWM and Rao’Q). For cumulative

323

soil CO2, we used CWM1CO2 and CWM2CO2 to represent the first two PC axes associated with

324

CWMs, Rao1CO2; and Rao2CO2 to represent the first two PC axes generated by Rao’Q.

325

Similarly, we defined the other four soil variables. Specifically, CWM1N2O, CWM2N2O,

326

Rao1N2O, and Rao2N2O were used for cumulative soil N2O. CWM1amf, CWM2amf, Rao1amf,

327

and Rao2amf were used for net ammonification. CWM1nif, CWM2nif, Rao1nif, and Rao2nif

328

were used for net nitrification. CWM1Nmin, CWM2Nmin, Rao1Nmin, and Rao2Nmin were used for

329

net N mineralization. All data were tested for normality and homogeneity. One-way ANOVA,

330

paired t-test, and Pearson correlations were performed using SPSS software (version 16.0;

331

SPSS Inc., Chicago, IL, USA). General linear model, PCA, and variation partitioning were

332

conducted with R 3.5.1 (R Development Core Team 2018).

333 334

3. Results

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3.1. Effects of root species diversity on soil cumulative CO2 and N2O releases

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Species richness of root litter showed a significant negative effect on cumulative soil 15

337

CO2 and N2O release after 28 and 56 days of incubation (Fig. 1a, b). The average cumulative

338

soil CO2 and N2O release declined substantially in two- and four-species root litters

339

compared with single-species root litters. Within the single-species root litter, the effect of

340

plant species on cumulative soil CO2 and N2O release differed substantially after 28 and 56

341

days of incubation (Fig. S1a, c and Fig. 2a, c). Soils with root litter containing Leymus

342

chinensis (Lc) had the highest cumulative CO2 release but the lowest cumulative N2O release,

343

and soils with a root litter of Stipa grandis (Sg) exhibited the lowest cumulative CO2 release

344

and a low cumulative N2O release. Soils containing the root litter of Cleistogenes squarrosa

345

(Cs) showed the highest cumulative N2O release, and soils with a root litter of Carex

346

korshinskyi (Ck) exhibited the second highest release of cumulative CO2 and N2O (Fig. S1a,

347

c and Fig. 2a, c).

348

The cumulative soil CO2 and N2O release from two- and four-species root litters also

349

differed after 28 and 56 days of incubation (Fig. S1b, d and Fig. 2b, d). As expected, root

350

litter mixtures containing the single species S. grandis generally exhibited a lower cumulative

351

release of soil CO2 compared with root litter mixtures containing L. chinensis (Fig. S1b and

352

Fig. 2b). Similarly, root litter mixtures containing C. korshinskyi and C. squarrosa generally

353

showed higher cumulative soil N2O release compared with root litter mixtures containing L.

354

chinensis and S. grandis (Fig. S1d and Fig. 2d). Interestingly, most mixtures showed a

355

significantly lower release of observed cumulative CO2 and N2O compared with the values

356

expected from single-species root litter treatments, suggesting a non-additive antagonistic

357

effect (Fig. S1b, d and Fig. 2b, d). On average, the observed cumulative release of soil CO2

358

was 13.2% lower than expected after 28 days, and 12.6% lower than expected after 56 days 16

359

of incubation (Fig. 3a). For cumulative soil N2O release, the average non-additive

360

antagonistic effect was -30.3% after 56 days of incubation (Fig. 3b).

361 362

3.2. Effects of root species diversity on soil net ammonification, net nitrification and net N

363

mineralization

364

Species richness of root litter showed a significant positive effect on net

365

ammonification and net N mineralization in the soil after 28 and 56 days of incubation,

366

respectively (Fig.1 c, e). The average levels of net ammonification and N mineralization in the

367

soil increased in two- and four-species root litters compared with single-species root litters.

368

However, for the net NO3--N immobilization, the species richness of root litter showed a

369

negative effect after 28 days of incubation, and a positive effect after 56 days of incubation

370

(Fig. 1d). Within single-species root litter treatments, soils with root litters of L. chinensis or

371

C. korshinskyi showed greater net ammonification and net N mineralization compared with

372

soils containing root litters of C. squarrosa or S. grandis; however, they showed a lower net

373

NO3--N immobilization than soils with root litters of C. squarrosa or S. grandis (Fig. S2a, c,

374

e; Fig. 4a, c, e).

375

For two- and four-species root litter mixtures, the net values of soil ammonification,

376

NO3--N, and N mineralization varied with the species combinations after 28 and 56 days of

377

incubation (Fig. S2b, d, f; Fig. 4b, d, f). Root litter mixtures containing L. chinensis generally

378

showed higher soil net ammonification and net N mineralization compared with root litter

379

mixtures containing C. korshinskyi (Fig. S2b, f; Fig. 4b, f). Soil net NO3--N immobilization

380

was the highest in root litter mixtures containing L. chinensis and C. korshinskyi, and C.

381

squarrosa and C. korshinskyi, and the lowest in root litter mixtures of L. chinensis and C. 17

382

squarrosa, and S. grandis and C. korshinskyi after 28 and 56 days of incubation, respectively

383

(Fig. S2d; Fig. 4d). Accordingly, for most root litter mixtures, the observed soil net

384

ammonification and net N mineralization were significantly higher than the values expected

385

from single-species root litter treatments, suggesting a non-additive synergistic effect (Fig.

386

S2b, f; Fig. 4b, f). A predominant non-additive antagonistic effect on soil net NO3--N

387

immobilization was detected after 28 days of incubation, while a non-additive synergistic

388

effect was dominant after 56 days of incubation (Fig. S2d; Fig. 4d). On average, the root litter

389

mixtures showed increased soil net ammonification, net NO3--N immobilization and net N

390

mineralization by 34.6%, 16.9%, and 127.1%, respectively, after 56 days of incubation (Fig.

391

3c-e).

392 393 394

3.3. Effects of root species diversity on soil MBC and MBN Species richness of root litter had no significant effect on soil MBC and MBN after 28

395

and 56 days of incubation, although the average soil MBC and MBN tended to decline from

396

single-species root litter to two- and to four-species root litter mixtures after 56 days of

397

incubation (Fig. S3). Within single-species root litter, soil with root litter of L.chinensis

398

showed highest MBC and MBN, while soil with root litter of S.grandis and C.squarrosa

399

exhibited the lowest MBC and MBN, respectively, after 56 days of incubation (Fig. S4a, c).

400

Among two- and four-species root litter mixtures, soil MBC and MBN were the

401

highest in root litter mixtures containing L. chinensis and C. korshinskyi, while both were the

402

lowest in root litter mixtures of S. grandis and C.squarrosa after 56 days of incubation (Fig.

403

S4b, d). For most root litter mixtures, there was no significant difference between observed

404

and expected values (i.e. additive effects), except for two-species root mixtures containing S. 18

405

grandis and C.squarrosa and four-species root mixtures showed an antagonistic effect on soil

406

MBC and MBN (Fig. S4b, d).

407 408

3.4. Non-additive soil C and N cycling related to root species

409

The presence or absence of root species had a significant effect on non-additive soil C

410

and N cycling after 28 and 56 days of incubation (Table S4). Specifically, root litter mixtures

411

containing L. chinensis or C. korshinskyi had a lower antagonistic effect on soil cumulative

412

CO2 compared with root litters without these species. Root litters containing S. grandis

413

showed a higher antagonistic effect compared with those lacking S. grandis (Fig. 5a). Root

414

litters containing L. chinensis had a lower antagonistic effect on soil cumulative N2O release

415

than those without L. chinensis, while the presence of C. squarrosa in root mixtures had a

416

greater inhibitory effect on soil cumulative N2O than those without C. squarrosa (Fig. 5b).

417

Root litters containing L. chinensis induced higher levels of soil net ammonification,

418

nitrification and N mineralization than expected, while root litters containing C. korshinskyi

419

inhibited net ammonification, nitrification and N mineralization than expected (Fig. 5c, d, e).

420 421 422

3.4. Relative contributions of FI and FD to non-additive soil C and N cycling Variation partitioning analyses showed that both CWM and Rao’Q of root traits had a

423

significant or a marginally significant effect on non-additive soil C and N cycling after 56

424

days of incubation. However, compared with Rao’Q, CWM accounted for a large proportion

425

of variance in non-additive soil C and N cycling (Table 1; Table 2). Specifically, CWM1CO2

426

was negatively correlated with non-additive soil cumulative CO2, and the association of the

427

chemical traits with CWM1CO2 suggested that the high C:N ratio in root mixtures increased 19

428

the antagonistic effect on cumulative soil CO2, and the high concentrations of N, P, K and

429

Di-O-alkyl-C in root litter mixtures reduced the antagonistic effect on cumulative CO2.

430

Rao2CO2 had a negative, but marginally significant effect on non-additive cumulative soil

431

CO2 (Table 1; Fig. S3a, b). CWM1N2O had a positive, but marginally significant effect on

432

non-additive cumulative N2O (Table 1). The high contents of aryl-C in root mixtures resulted

433

in a synergistic effect on net ammonification, while the high Mg and alkyl-C content in root

434

mixtures generated antagonistic effects against net ammonification (associated with

435

CWM1amf, Table 2; Fig. S4a). Rao1amf had a smaller, but significantly positive effect on

436

non-additive net ammonification, indicating increased synergistic effects with increasing

437

variation in N and K content within root mixtures, and decreased synergism following

438

increasing variation in alkyl-C level (Table 2; Fig. S4b). CWM1nif was positively correlated

439

with non-additive net nitrification (Table 2). The association between chemical traits and

440

CWM1nif suggested that the high C:N ratio in the root mixtures increased the synergistic

441

effects on net nitrification, while high N, Mg, K and Di-O-alkyl-C levels in root mixtures

442

promoted synergistic effects on net NO3--N immobilization (Fig. S4c). In contrast, both Rao1

443

and Rao2 had no significant effect on non-additive net nitrification (Table 2). Root mixtures

444

containing high C:N ratios and carbohydrate-C levels exhibited greater net N mineralization

445

than expected, while higher levels of N, Mg, K, Di-O-alkyl-C, O-aryl-C, and alkyl-C

446

increased the antagonistic effects on net N mineralization (associated with CWM1Nmin and

447

CWM2Nmin; Table 2; Fig. S4e). Rao1Nmin had a negative, but marginally significant effect on

448

non-additive net N mineralization (Table 2).

449 450

4. Discussion 20

451

4.1. Effects of root litter diversity on cumulative CO2 release and net N transformation in the

452

soil

453

Our study suggests that high species diversity of root litter reduces the cumulative

454

release of soil CO2 and N2O and increases the net N mineralization of the soil. The findings

455

indicate that the diversity of root litter potentially increases soil C storage, elevates available

456

plant N, and simultaneously reduces soil greenhouse gas emissions. In line with our initial

457

hypothesis, the cumulative release of soil CO2 decreased in root mixtures with high species

458

richness, and almost all root mixtures carried lower levels of cumulative soil CO2 compared

459

with the expected values calculated from the constituent single species, suggesting a

460

non-additive antagonistic effect. This result confirms previous studies indicating that the

461

decomposition of community roots was negatively affected by species richness, and inhibited

462

the mass loss in root mixtures compared with the respective single species (Chen et al., 2017a;

463

Prieto et al., 2017). Based on the decomposition of mixed root litter, our results suggest that

464

the non-additive antagonistic effect on cumulative soil CO2 release in root mixtures is likely

465

resulted from the differences in species composition of root litter. This means that root

466

mixtures containing species with hardly decomposable roots (e.g. high C:N ratio or high

467

contents of lignin, tannins or polyphenols) increase the antagonistic effect on cumulative soil

468

CO2. Indeed, we found that root litter mixtures containing S. grandis had lower cumulative

469

CO2 release but higher antagonistic effect than litter mixtures containing L.chinensis; because

470

S. grandis is less decomposable than L. chinensis due to its higher C:N ratio and lower N, P

471

and Di-O-alkyl-C (characteristic of celluloses) contents, which decreased microbial biomass

472

C and N and inhibited microbial activities (Gessner et al., 2010; Prieto et al., 2016). This 21

473

suggests that species identity may play a more important role than species richness in

474

controlling the non-additive antagonistic effect on cumulative soil CO2 release in root

475

mixtures. But contrary to the findings based on leaf litter mixtures suggesting a

476

predominantly synergistic effect (Gartner and Cardon, 2004; Lecerf et al., 2011). The

477

non-additive effects associated with the interaction between leaf and root litter may primarily

478

arise from the differences in litter quality. Root litter contains higher levels of recalcitrant C,

479

such as lignin, tannin, and suberins, which are resistant to most decomposing microbes

480

(Angst et al., 2016; Dong et al., 2016). Besides, compared with the current experimental

481

design, most of the results based on mixed leaf litters were obtained using “litter bags” in the

482

field, which permit the entrance of soil fauna leading to increased decomposition

483

(Hättenschwiler and Gasser, 2005). Our findings indicate that it is not accurate to predict the

484

diverse effects of root litter decomposition on soil C cycling based on the results of

485

decomposition of individual species. The results are also corroborated by studies conducted

486

in grasslands that reported the sequestration of C derived from root decay in soil more

487

efficiently under higher levels of root species diversity compared with aboveground litter

488

(Jackson et al., 1996; Fornara et al., 2009; Chen et al., 2017a).

489

The net ammonification, net NO3--N immobilization and net N mineralization in the

490

soil increased with increasing species richness of root litter after 28 and 56 days of incubation,

491

except for the net NO3--N immobilization after 28 days. This finding partially supports our

492

second hypothesis. A predominant non-additive synergistic effect was detected concurrently,

493

while the cumulative N2O release showed an opposite trend. The increased net

494

ammonification may be attributed to the higher concentrations of organic C and N, and a 22

495

richer array of chemical compounds (e.g. carbohydrate-C and aryl-C) in the root litter

496

mixtures with higher species diversity, which facilitate the transformation of organic N to

497

NH4+-N (Hosokawa et al., 2017). The net decrease in nitrification (i.e. increased net NO3--N

498

immobilization) with increasing species diversity of root litter after 56 days of incubation,

499

probably due to the high species diversity of root litter may promote the activity of nitrifiers

500

and thereby increased the immobilization of NO3--N (Laughlin et al., 2009). The net decrease

501

in nitrification with increasing species diversity of root litter may have contributed to the

502

decrease in cumulative soil N2O release because NO3--N is the substrate for the production of

503

N2O (Kuypers et al., 2018). Overall, the increase in net mineralization with increasing species

504

diversity of root litter is mainly attributed to net ammonification, whose magnitude was much

505

greater than that of net nitrification. The effects of root species diversity on soil N

506

transformation were also mediated by root species identity. For example, root litter mixtures

507

containing L.chinensis exhibited a synergistic effect, while root litter mixtures containing

508

C.korshinskyi showed an antagonistic effect. The synergistic effect might be resulted from the

509

presence of root species (e.g. L.chinensis) with high N and Di-O-alkyl-C in litter mixtures,

510

because these traits have been reported positively affect soil N mineralization (Fornara et al.,

511

2009; Meier and Bowman, 2010).

512

Our results showed that root litter diversity had contrasting effects on soil C cycling

513

and N transformation, suggesting that the underlying mechanisms may substantially differ.

514

Unlike the soil C cycling, which involves all living microorganisms and their activities are

515

mainly influenced by resource availability, soil N transformation is usually driven by

516

particular groups of microbes. Hence, soil N transformation process is not only affected by 23

517

resource availability, but also highly depends on microbial communities (Monson et al., 2006;

518

Delgado-Baquerizo et al., 2016). Thus, further studies of soil microbial composition are

519

needed to explain the different responses of soil C cycling and N transformation to species

520

diversity of root litter. Our results provide robust evidence that the high diversity of root litter

521

(i.e. via species diversity and identity effects) not only increases soil C sequestration by

522

decreasing CO2 release and elevates plant available N by increasing net N mineralization, but

523

also reduces greenhouse gas emissions by inhibiting soil CO2 and N2O releases.

524 525

4.2. The relative contributions of functional identity and functional diversity to soil C and N

526

cycling

527

Several studies have shown that litter chemical traits represent the ultimate cause of

528

non-additive synergistic or antagonistic effects (Hättenschwiler et al., 2005; Srivastava et al.,

529

2009; Chen et al., 2018). However, the relative contributions of functional diversity and

530

functional identity of root traits to soil C and N cycling are not well established. In line with

531

our hypothesis, we found that both the community-weighted mean traits (i.e. functional

532

identity) and litter trait diversity measured as Rao’Q (i.e. functional diversity) are important

533

drivers of soil C cycling and N transformation. Community-weighted mean traits accounted

534

for a higher variation in soil C cycling and N transformation than Rao’Q, indicating that the

535

functional identity was more important than functional diversity in predicting the effects of

536

species diversity of root litter on soil C and N dynamics. Our results concur with findings

537

derived from grasslands, temperate and subtropical forests (Mokany et al., 2008; Meier and

538

Bowman, 2010; Tobner et al., 2016). Furthermore, our results validate that species

539

composition (species identity) is more important than species richness, since functional 24

540

identity implies the overwhelming influence of species on ecosystem processes, while

541

diversity indicates species richness (Mokany et al., 2008). However, the low explanatory

542

power of functional diversity may be attributed to two factors. First, our results are based

543

only on brief incubation. Previous studies indicated that functional diversity resulted in

544

higher variance with the advance of time (Cardinale et al., 2007; Tobner et al., 2016). Second,

545

although a large number of litter traits have been used to calculate functional diversity,

546

important morphological traits (e.g. specific root length) were not included in our analysis

547

(Smith et al., 2014), which can affect root decomposition and may have partially affected our

548

results.

549

The dominant root traits determining the direction of non-additive effect differ with

550

soil processes. Specifically, for cumulative soil CO2, high C:N ratio and low concentrations

551

of N, P, K and Di-O-alkyl-C (characteristic of celluloses) were the key traits resulting in

552

antagonistic effects. Although these traits have been reported to affect root litter

553

decomposition of single species (Silver and Miya, 2001; Vivanco and Austin, 2006; Smith et

554

al., 2014), our results provide new insight into traits affecting diverse root decomposition at

555

the community level, and explain the significantly increased antagonistic effect due to the

556

presence of S. grandis within litter mixtures, because of the higher C: N ratio, and lower N, P,

557

K and Di-O-alkyl-C content compared with other species, such as L. chinensis. Based on

558

findings obtained from mixed leaf litters, the presence of species with hardly decomposable

559

roots attenuates the decomposition of other species in root mixtures, thus inducing an

560

antagonistic effect (Liu et al., 2009; Gessner et al., 2010). For net ammonification and net N

561

mineralization in root mixtures, the synergistic effect was mainly attributed to high levels of 25

562

carbohydrate-C concentration, which was mainly associated with sugars and polysaccharides.

563

The synergistic effect of net NO3--N immobilization was mainly ascribed to the high levels of

564

N, Mg, and Di-O-alkyl-C, and the low C: N ratios in root mixtures. These results are in

565

agreement with studies focused on leaf litter decomposition, which reported that high N,

566

sugar concentrations, and/or low C: N ratios in leaf mixtures promoted soil nitrification and

567

N mineralization (Meier and Bowman, 2008; Laughlin, 2011).

568

Our study provides a comprehensive analysis of root traits driving soil N

569

transformation at the community level. Our findings confirmed that the root chemistry

570

compared with leaf chemistry also resulted in a non-additive synergistic effect on net N

571

transformations in the soil. Although the synergistic effect was predominantly seen in the net

572

N transformation, we also detected an antagonistic effect in a few root mixtures. For example,

573

mixtures of L. chinensis with C. korshinskyi and C. korshinskyi with C. squarrosa had an

574

antagonistic effect on net ammonification, net nitrification and net N mineralization. Related

575

chemical traits included high Mg, alkyl-C (associated with waxes, cutins and suberins), N

576

and O-aryl-C concentrations, because the high concentrations of tannin and phenols

577

(measured as O-aryl-C) in the root mixtures inhibited net N mineralization. The underlying

578

mechanisms may involve specific components such as tannins and phenols, which inhibit

579

microbial activity and result in non-additive effects on decomposition (Hättenschwiler et al.,

580

2005). Furthermore, we detected a few recalcitrant sources of C such as waxes, cutins and

581

suberins (measured as Alkyl-C) in root mixtures, which also induced non-additive

582

antagonistic effects on the net N mineralization of the soil. Interestingly, previous studies

583

demonstrated that species with high N or O-aryl-C content promoted N mineralization (Meier 26

584

and Bowman, 2008; Fornara et al., 2009; Garcia-Palacios et al., 2017). In the current study,

585

however, we found that mixtures of L. chinensis and C. korshinskyi (both carrying high N and

586

O-aryl-C content) induced an antagonistic effect, probably due to the formation of tannin-N

587

complexes that inhibit microbial growth, suggesting that the chemical interactions between

588

litter components also contribute to non-additive effects (Meier and Bowman, 2008).

589 590 591

5. Conclusions Our results based on a 56-day laboratory incubation experiment demonstrate that

592

species and functional diversity of root litter had significant non-additive effects on soil C

593

and N cycling in the steppe ecosystem. Root litters with high species diversity can potentially

594

promote soil C storage and enhance plant N availability. A predominantly antagonistic effect

595

of root litter diversity on the cumulative release of soil CO2 and N2O was found, and a

596

synergistic effect on net N transformation was predominantly seen in root litter mixtures. The

597

pattern of non-additive effects on soil C and N cycling was mainly determined by the

598

dominant traits within root mixtures. Our study also provides evidence suggesting that

599

species identity (e.g. specific root chemical traits) was more important than species richness

600

in influencing soil C and N cycling in specific cases. A high C:N ratio and low levels of N, P,

601

K, and Di-O-alkyl-C within root mixtures inhibit the cumulative release of soil CO2. In

602

contrast, the root traits that affect non-additive net N transformations such as Aryl-C, Alkyl-C,

603

Di-O-alkyl-C, N, and Mg levels, show greater variation within root litter mixtures.

604 605 606

Acknowledgements We thank the staff at the Inner Mongolia Grassland Ecosystem Research Station

27

607

(IMGERS), Chinese Academy of Sciences for their help with field sampling. This study was

608

supported by grants from the National Natural Science Foundation of China (31630010,

609

31320103916).

610 611 612

Author contributions YB conceived the study; JM and BT conducted the field sampling and laboratory

613

analysis; JM, BT, WX, YW, XZ, and YB analyzed the data and contributed to the manuscript

614

writing; JM and BT contribute equally to this work.

615 616

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811

34

812

Tables

813 814

Table 1 Variation partitioning based on the results of principal component analysis (PCA) of

815

the effects of community-weighted means (CWM) and functional diversity (Rao’Q) on

816

cumulative CO2 and N2O release of litter mixtures after 56 days of incubation Cumulative CO2 PC axes

Cumulative N2O

Estimate

P

R2

CWM1CO2

-0.043

0.001

0.300

CWM2CO2

-0.005

0.666

Rao1CO2

-0.010

Rao2CO2

-0.021

PC axes Estimate

P

R2

CWM1N2O

0.146

0.073

0.102

0.003

CWM2N2O

0.060

0.521

0.011

0.360

0.011

Rao1N2O

0.047

0.566

0.008

0.071

0.087

Rao2N2O

0.003

0.976

0.007

817 818

35

819

Table 2 Variation partitioning based on the results of principal component analysis (PCA) of the effects of community-weighted means (CWM)

820

and functional diversity (Rao’Q) on net ammonification, net nitrification, and net N mineralization of litter mixtures after 56 days of incubation

821

Net ammonification PC axes

Net nitrification

Estimate

P

R2

CWM1amf

0.492

<0.001

0.393

CWM2amf

0.132

0.181

Rao1amf

0.290

Rao2amf

0.081

PC axes

Net N mineralization

Estimate

P

R2

CWM1nif

0.333

<0.001

0.467

0.017

CWM2nif

-0.113

0.077

0.005

0.116

Rao1nif

-0.039

0.412

0.040

Rao2nif

-0.009

36

PC axes Estimate

P

R2

CWM1Nmin

2.812

0.003

0.216

0.055

CWM2Nmin

-2.294

0.002

0.181

0.539

0.024

Rao1Nmin

-1.300

0.074

0.091

0.879

0.003

Rao2Nmin

0.435

0.610

0.029

822

Figure Legends

823 824

Fig. 1 Effects of species richness of root litter on cumulative release of soil CO2 and N2O, net

825

ammonification, net nitrification, and net N mineralization after 28 and 56 days of incubation

826

(a-e). Values represent mean ± SE (n = 5). Different lowercase letters on each sampling date

827

indicate significant differences between root litters with different levels of species (P < 0.05).

828 829

Fig. 2 Effects of species composition of root litter on cumulative soil CO2 (a-b) and N2O (c-d)

830

release in single species (a, c) and combinations of species (b, d) after 56 days of incubation.

831

Values represent mean ± SE (n = 5). Different lowercase letters represent cumulative release

832

of CO2 and N2O indicating significant differences between the observed values (P < 0.05).

833

Significant differences between observed and expected values are indicated with the

834

respective symbols: + for P < 0.1, * for P < 0.05, ** for P < 0.01, *** for P < 0.001 and ns

835

for non-significant. Species abbreviations: Lc, Leymus chinensis; Ck, Carex korshinskyi; Cs,

836

Cleistogenes squarrosa; and Sg, Stipa grandis. Letter combinations correspond to species

837

combinations in root litter mixtures.

838 839

Fig. 3 Effect of litter mixtures on cumulative release of soil CO2 and N2O, net

840

ammonification, net nitrification, and net N mineralization after 28 and 56 days of incubation

841

(a-e). Values represent mean ± SE (n = 5). Significant differences between effects of litter

842

mixtures are indicated with the respective symbols: + for P < 0.1, * for P < 0.05, ** for P <

843

0.01, *** for P < 0.001 and ns for non-significant. Positive deviation from 0 indicates a 37

844

synergistic effect and negative deviation from 0 indicates an antagonistic effect.

845

Abbreviations: O, observed values; E, expected values. Letter combinations correspond to

846

species combinations in root litter mixtures.

847 848

Fig. 4 Effects of species composition of root litter on soil net ammonification (a-b), net

849

nitrification (c-d) and net N mineralization (e-f) in a single species and in a combination of

850

species after 56 days of incubation. Values represent mean ± SE (n = 5). Different lowercase

851

letters for net ammonification (nitrification and N mineralization) indicate a significant

852

difference between the observed values (P < 0.05). Significant differences between observed

853

and expected values are indicated with the respective symbols: + for P < 0.1, * for P < 0.05,

854

** for P < 0.01, *** for P < 0.001 and ns for non-significant. Species abbreviations: Lc,

855

Leymus chinensis; Ck, Carex korshinskyi; Cs, Cleistogenes squarrosa; Sg, Stipa grandis.

856

Letter combinations correspond to species combinations in root litter mixtures.

857 858

Fig. 5 Effects of root species on litter mixture in terms of cumulative CO2 (a), cumulative

859

N2O (b), net ammonification (c), net nitrification (d), and net N mineralization (e) after 56

860

days of incubation. Values shown in the figure represent mean ± SE (n = 15). Significant

861

effects of individual root species are represented as follows: +, P < 0.1; *, P < 0.05; **, P <

862

0.01; ***, P < 0.001; ns, non-significant.

863

38

864

Figures

865

866 867 868

Fig. 1

869

39

870

871 872 873

Fig. 2

874

40

875

876 877 878

Fig. 3

879

41

880

881 882 883

Fig. 4

884 42

885

886 887 888

Fig. 5

889

43

Highlights

High root litter diversity decreases cumulative CO2 and N2O release in the soil. Great root litter diversity increases soil net N ammonification and mineralization. The decreasing soil CO2 release is caused by antagonistic effects of root traits. Root litter diversity regulates soil N cycling via synergistic effects of root traits. Functional identity explains more than functional diversity in soil C and N cycles.

October 21, 2019 Editor Soil Biology and Biochemistry

Dear Editor: The authors declare there are no competing interests in the submitted manuscript entitled, “Root litter diversity and functional identity regulate soil carbon and nitrogen cycling in a typical steppe (SBB15414)”.

Sincerely yours,

Yongfei Bai on behalf of all co-authors Professor of Ecology