Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration and biochemical properties in a subtropical forest

Journal Pre-proof Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration and biochemical properties in a subtropical forest Yong PENG, Si-yi SONG, Zeng-yan LI, Shun LI, Guan-tao CHEN, Hong-ling HU, Jiulong XIE, Gang CHEN, Yin-long XIAO, Li LIU, Yi TANG, Li-hua TU PII:

S0038-0717(19)30358-X

DOI:

https://doi.org/10.1016/j.soilbio.2019.107694

Reference:

SBB 107694

To appear in:

Soil Biology and Biochemistry

Received Date: 4 August 2019 Revised Date:

7 December 2019

Accepted Date: 8 December 2019

Please cite this article as: PENG, Y., SONG, S.-y., LI, Z.-y., LI, S., CHEN, G.-t., HU, H.-l., XIE, J.-l., CHEN, G., XIAO, Y.-l., LIU, L., TANG, Y., TU, L.-h., Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration and biochemical properties in a subtropical forest, Soil Biology and Biochemistry, https://doi.org/10.1016/j.soilbio.2019.107694. 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 Elsevier Ltd. All rights reserved.

1

Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration

2

and biochemical properties in a subtropical forest

3 4 5 6

Figure 1 Location of study site in Ya’an, Sichuan province, China and the schematic design of N addition and aboveground litter manipulation.

1

CN LN HN

Cumulative litterfall (t ha-1)

200 Time effect (P < 0.001) Nitrogen effect (P = 0.921) Time x Nitrogen effect (P = 0.438)

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15 CN LN HN

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Figure 2 Seasonal variations in above-ground litter input in an evergreen broad-leaved forest in southwestern China from August 2015 to December 2017.

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11 12 13 Jun.-17 Jul.-17 Aug.-17 Sep.-17 Oct.-17 Nov.17 Dec.17

Jun.-17 Jul.-17 Aug.-17 Sep.-17 Oct.-17 Nov.17 Dec.17

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100

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4 Jan.-16

o

Soil temperature ( C) 22

Jan.-16

Soil moisture content (%)

10 24 (A) CNLLNLHNLCNL0 LNL0 HNL0 CNL+ LNL+ HNL+

10

8

6

Figure 3 Seasonal variations in soil temperature (A) and soil moisture content (B) in an evergreen

broad-leaved forest in southwestern China from January 2016 to December 2017.

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16 17 18 Feb.-16

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15 Jan.-16

-2 -1

Soil CO2 efflux (µmol CO2 m s )

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CNLLNLHNLCNL0 LNL0 HNL0 CNL+ LNL+ HNL+

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Figure 4 Seasonal variations in soil respiration in an evergreen broad-leaved forest in

southwestern China from January 2016 to December 2017.

4

-1 Cumulative C flux (t C ha )

25 a

CNLLNLHNLCNL0 LNL0 HNL0 CNL+ LNL+ HNL+

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ab bc bcd

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e

cd

ab bc a

bc

bc ab

bc cde

bcd

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ab b

bcd cd

c

c

bc

c

bcd

de

5

0

19 20 21

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2017

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Figure 5 Cumulative CO2 flux under different treatments.

5

Soil CO2 efflux (µmol CO2 m-2 s-1)

CNLLNLHNLCNL0 LNL0 HNL0 CNL+ LNL+ HNL+

(A)

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4

(B)

y = 0.562 e 0.095x P < 0.001 R2 = 0.759

4

2

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0

0 4

22 23

y = 0.653 x 0.223 P < 0.001 R2 = 0.149

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6

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10 12 14 16 o Soil temperature ( C)

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0

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400 600 -2 Litter input (g m )

800

Figure 6 Relationships between soil respiration and soil temperature (A) and litterfall (B).

6

Highlights •

The aboveground litter-input was added or reduced by only 50%

•

Nitrogen addition increased the topsoil TOC concentration when no litter changed

•

The increase of soil C content may be due to the inhabitation of SOM decomposition

•

Litterfall alteration reduced topsoil TOC concentration when N input increased

1

Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration

2

and biochemical properties in a subtropical forest

3

Yong PENGa, b, Si-yi SONGa, Zeng-yan LIa, Shun LIc, Guan-tao CHENd, Hong-ling HUa, Jiu-long

4

XIEa, Gang CHENa, Yin-long XIAOe, Li LIUf, Yi TANGg, Li-hua TUa,*

5 6

a

Key Laboratory of National Forestry & Grassland Administration on Forest Resources Conservation

7

and Ecological Safety in the Upper Reaches of the Yangtze River, College of Forestry, Sichuan

8

Agricultural University, Chengdu, Sichuan, 611130, China

9

b

The University of Tokyo Hokkaido Forest, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Furano, Hokkaido, 079-1564, Japan

10 11

c

Sichuan Academy of Forestry, Chengdu, Sichuan, 610084, China

12

d

Soil Science of Tropical and Subtropical Ecosystems, Faculty of Forest Sciences and Forest Ecology, University of Goettingen, Goettingen, 37077, Germany

13 14

e

College of Environmental Sciences, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China

15 16

f

Personnel Department, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China

17

g

College of Horticulture, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China.

18

*

Corresponding author: College of Forestry, Sichuan Agricultural University, No. 211 Huimin Road,

19

Wenjiang District, Chengdu, Sichuan, 611130, China.

20

E-mail address: [email protected]

21

Yong PENG, Si-yi SONG, Zeng-yan LI, and Shun LI contributed equally to this work.

22 23 24 1

25

Abstract:

26

Atmospheric nitrogen (N) deposition has rapidly increased in subtropical ecosystems and may have

27

altered the input of aboveground litter to soil, which substantially impacts soil carbon (C) and nutrient

28

cycling. But how the soil processes and properties respond to N deposition under uncertain fresh litter

29

input is poorly understood. In order to examine the responses of soil respiration and biochemical

30

properties to N addition and aboveground litter manipulation, a field N addition and litterfall

31

manipulation interaction experiment was performed in an evergreen broadleaf forest on the western

32

edge of the Sichuan Basin in China. Three levels of N addition, including an N control (CN, ambient N

33

input) and low N (LN, + 50 kg N ha

34

levels of litterfall manipulation, including intact litter input (L0, no litter alteration), litter reduction (L−,

35

reduced by 50%) and litter addition (L+, increased by 50%), were conducted monthly starting in

36

January 2014 and August 2015, respectively. Soil respiration was measured monthly from January

37

2016 to December 2017. Soil samples were collected four times, in October 2016 and January, April

38

and July 2017, to measure soil biochemical properties. The results showed that: (1) short-term N

39

addition did not significantly alter the aboveground litter input in this forest; (2) soil respiration

40

decreased with elevating N input and was associated with amount of litterfall input; (3) N addition

41

increased the total organic C (TOC) concentration in topsoil in subplots without litterfall alteration but

42

did not affect TOC in subplots with increased or decreased litter-input; (4) N addition decreased soil

43

pH and did not affect soil microbial biomass regardless of whether litterfall was altered or not; (5)

44

short-term litter manipulation did not affect any soil properties in the N control plots, but both litterfall

45

reduction and addition tended to reduce surface soil TOC concentration in the N-added plots; and (6)

46

both N addition and litterfall manipulation showed stronger effects on organic soil than on mineral soil.

47

These findings indicated that elevated N input increased the surface soil C content by reducing soil

48

respiration mainly via enhancing stabilization of soil organic matter rather than by reducing soil

49

microbial biomass, and that altered litterfall may mitigate the N-induced increase in soil C. Because of

50

temporal lag, long-term experimentation is needed to investigate the response of soil to altered litter

51

input under different N addition conditions.

52

Key words: Nitrogen addition; litter manipulation; soil respiration; soil carbon

−1

−1

year ) and high N (HN, + 150 kg N ha

−1

−1

year ), and three

53

2

54

Introduction

55

Over the past few decades, anthropogenic reactive nitrogen (N) inputs have substantially increased

56

and exceeded the N inputs through all-natural processes due to the application of N fertilizer and the

57

burning of fossil fuels (Davidson, 2009). Increased N deposition has been one of the major contributors

58

to global change and has altered terrestrial vegetation biomass (Devaraju et al., 2016; Yue et al., 2017)

59

and the input amount of aboveground litter to soil (Li et al., 2010a; Field et al., 2017). Soil, as the

60

largest carbon (C) pool in the terrestrial ecosystem, stores more organic C than vegetation and the

61

atmosphere and plays an important role in global and regional C and nutrient cycling as well as in C

62

feedback to global environmental changes (Schmidt et al., 2011; Lehmann and Kleber, 2015). Soil C

63

dynamics depend on the balance between C input and output and are regulated by many biotic and

64

abiotic factors, such as plant inputs, N availability, microbial transformations and stabilization of soil

65

organic matter (SOM) (Paul, 2016). Therefore, changes in N and aboveground litter inputs may affect

66

soil C dynamics, and the response of soil C dynamics to N addition and aboveground litter

67

manipulation may have considerable impacts on CO2 exchange between the biosphere and atmosphere.

68

Considerable attention has been devoted to the responses of soil C dynamics with elevated N inputs

69

to soil. Because ecosystem C dynamics are tightly coupled to N cycling (Ye et al., 2018; Wang et al.,

70

2019), the remarkable increases in atmospheric N deposition have considerably influenced processes of

71

natural ecosystems, such as plant productivity (Yan et al., 2014; Devaraju et al., 2016), litterfall (Li et

72

al., 2010a; Field et al., 2017) and litter decomposition (Whalen et al., 2018; Zhang et al., 2018), which

73

in turn impact soil C dynamics (Ye et al., 2018; Wang et al., 2019). Globally, anthropogenic N input has

74

led to an increase in soil C storage (Frey et al., 2014; Devaraju et al., 2016; Wang et al., 2017). With N

75

limiting in most terrestrial ecosystems (LeBauer and Treseder, 2008), N enrichment can generally

76

promote vegetation biomass (Yan et al., 2014; Devaraju et al., 2016; Wang et al., 2017) and thus

77

increase the C input to soil. In addition, elevated N inputs can reduce soil CO2 emission in many

78

ecosystems by reducing SOM decomposition rates (Bowden et al., 2004; Janssens et al., 2010; Fan et

79

al., 2014; Peng et al., 2018; Zhou et al., 2018; Wang et al., 2018). However, a neutral or negative effect

80

of N addition on soil C dynamics was also observed in some ecosystems (Lu et al., 2011; Reid et al.,

81

2012; Forstner et al., 2018). For instance, Forstner et al. (2018) reported that enhanced N input

82

decreased SOC pools in the mineral topsoil in two temperate coniferous forests, which probably 3

83

resulted from decreased belowground C input and/or increased C output due to accelerated

84

decomposition.

85

Plant litter is an inherent part of C and nutrient cycling and also a buffer or protective layer on the

86

surface soil of soil C, and variation in litter input can thus affect the soil C dynamics directly and

87

indirectly (Sayer, 2005). A great number of litter manipulation experiments have been conducted to test

88

the effect of alterations in litter input on belowground C dynamics (Sayer et al., 2011; Leff et al., 2012;

89

Lajtha et al., 2014a, 2014b, 2018; Han et al., 2015; Pisani et al., 2016; Wu et al., 2017; Cusack et al.,

90

2018). Numerous studies demonstrated that soil respiration, microbial biomass, and soil C content are

91

associated with the amount of litterfall (Xu et al., 2013a; Lajtha et al., 2014a; Chen and Chen, 2018;

92

Cusack et al., 2018). Generally, litter exclusion leads to reduced surface soil C storage due to a direct

93

reduction in the input of C to soil (Bowden et al., 2014; Lajtha et al., 2014a, 2014b, 2018; Cusack et al.,

94

2018). However, the addition of litter does not always lead to increased soil C content. In many

95

ecosystems, litter addition does not affect or even reduces soil C content (Bowden et al., 2014; Pisani et

96

al., 2016; Lajtha et al., 2014b, 2018). The neutral or negative effect has been considered due to the

97

accelerated native SOM degradation caused by increased litter input, i.e., a priming effect (Kuzyakov et

98

al., 2000; Bowden et al., 2014; Lajtha et al., 2018). It is noteworthy that aboveground litters were either

99

completely excluded or doubled in most litter manipulation experiments (Sayer et al., 2011; Bowden et

100

al., 2014; Pisani et al., 2016; Gao et al., 2018; Cusack et al., 2018; Rodtassana and Tanner, 2018).

101

However, complete removal or doubling of litterfall can only explore the role of litter in an ecosystem

102

and cannot properly simulate the impact of an increase or decrease of litterfall with changes in net

103

primary productivity (NPP) on belowground physicochemical and biological processes. First, the

104

magnitude of alteration of litterfall due to the global change in natural ecosystems is much smaller than

105

100%. For instance, the increase in litterfall induced by increased N input to date is less than 20%

106

across all terrestrial ecosystems and is only ~10% on average in forest ecosystems (Liu and Greaver,

107

2010; Yue et al., 2016). Magill et al. (2004) reported that long-term N application in an N-saturated

108

forest led to forest decline, thereby reducing litterfall. Predictably, this reduction of litter input is

109

unlikely to reach 100% and shows little effect on the fauna and microflora in the original litter layer. In

110

addition, the removal of aboveground litter completely removes the animal and microbial communities

111

in the original litter layer and also impacts the temperature and moisture of the soil surface (Sayer,

112

2005; Xu et al., 2013a). Therefore, complete removal of litter is not suitable for simulating the litter 4

113

reduction scenario. To be more in line with natural conditions, the original litter layer should be present

114

during a litterfall manipulation experiment. However, to our knowledge, no such study has been

115

published to date. Klotzbücher et al. (2012) looked like did a similar experiment in which the effect of

116

80% litter input alteration on dissolved organic matter was studied in a Norway spruce (Picea abies)

117

stand, but they also completely removed or doubled the aboveground litter input during the snow-free

118

period (approximately 80% of the year) and did not conduct any litter manipulations during

119

winter-time.

120

Here, the effects of alteration in aboveground litter input on the belowground physicochemical and

121

biological processes were investigated by periodically increasing or decreasing 50% of recent litterfall

122

without changing the original litter layer.

123

The Rainy Zone of Western China, which is a large and complex ecotone on the western edge of the

124

Sichuan Basin, ranges 400–450 km from north to south and 50–70 km from east to west, with an area

125

of approximately 25,000 km2 (Zhuang and Gao, 2002). This region is located in a mid-subtropical

126

evergreen broad-leaved forest zone with a humid and monsoon-influenced climate and is an important

127

habitat for the giant panda (Ailuropoda melanoleuca). Due to the influence of the steep terrain on the

128

eastern side of the Tibetan Plateau and the monsoon climate, warm moist air from the Sichuan Basin

129

readily reaches supersaturation and forms abundant rainfall on the western edge of the basin (Zhuang

130

and Gao, 2002; Li et al., 2010b), and thus, atmospheric N deposition in this area is mainly via wet

131

deposition (Yang et al., 2018). The mean annual atmospheric N wet deposition in the center of this

132

zone (Ya’an city) was approximately 95 kg N ha−1 (Xu et al., 2013b), which is much greater than the

133

average level of China (21.1 kg N ha−1) (Liu et al., 2013). Previous N addition experiments conducted

134

in this area demonstrated that elevated N input lessened the soil C emission of two natural evergreen

135

broadleaf forests by reducing fine root biomass, soil microbial biomass and litter decomposition (Zhou

136

et al., 2017, 2018; Peng et al., 2017, 2018). This negative response of soil C efflux might increase soil

137

C sequestration in these evergreen broad-leaved forests. However, the responses of aboveground plant

138

input to N addition and of soil biochemical processes to altered litterfall under different N-addition

139

conditions remain unclear.

140

Thus, we performed a field N addition and aboveground litter manipulation experiment in a

141

subtropical evergreen broadleaf forest in the Rainy Zone of Western China to investigate the response

142

of soil biochemical properties to N addition and/or aboveground litter alteration. We hypothesized that: 5

143

(1) N addition would enhance soil C content by reducing soil respiration; (2) litterfall reduction would

144

decrease the soil respiration rate because of reduced substrate, while litterfall addition would stimulate

145

soil CO2 emission due to enhanced substrate availability and by a priming effect; and (3) a combination

146

of N and litterfall addition would lead to a minor change in soil respiration, while combined N addition

147

and litterfall reduction would cause further reduction of soil respiration.

148

Materials and methods

149

Site description

150

The field experiment was conducted in an evergreen broadleaf secondary forest in Bifeng Valley

151

Ecological Scenic Area (30º 04′ N, 103º 00′ E, 1,030 m a.s.l.), a national park in Ya’an, Sichuan

152

Province, China (Fig. 1 A, B), which is located in the centre of the Rainy Zone of Western China. The

153

annual average precipitation and temperature are 1750 mm (with 215 annual rainy days, Li et al.,

154

2010b) and 13.9 ºC, respectively. The soil in this study site is classified as a Ferralsol (according to

155

World Reference Base for Soil Resources 2014), with old alluvial yellow loam, with a granular

156

structure, > 60 cm depth. The dark humus layer of the soil here is loam, around 10 cm thick, with little

157

stones. The soil beneath the humus layer is a yellow iron-aluminum accumulation layer of about 50 cm,

158

with a strong viscosity. The nature of bedrock is granite. The litter stock in this forest was 20.6 ± 3.4 t

159

ha . The forest canopy mainly included Castanopsis fargesii, Schima superba, Symplocos botryantha,

160

S. setchuensis, Quercus fabri and Cunninghamia lanceolata, with average tree density and

161

breast-height diameter of 1,631 ± 130 stems ha

162

were primarily Daphniphyllum macropodum, Toxicodendron succedaneum, and Smilax china.

163

Common herbs included Murdannia triquetra, Sarcopyramis bodinieri and ferns.

164

Experimental design

−1

−1

and 11.6 ± 0.7 cm, respectively. The understory trees

165

In November 2013, twelve 20 m × 20 m experimental plots were established as a randomized block

166

design, at intervals of > 20 m (Fig. 1 C). These plots were randomly divided into three N addition

167

treatments, i.e., control N treatment (CN, ambient N deposition), low N (LN, + 50 kg N ha

168

and high N (HN, + 150 kg N ha

169

treatments, LN and HN, were applied to simulate scenarios of N wet deposition roughly increased by

170

50% and 150% in this region, respectively. Nitrogen was added in the form of ammonium nitrate

−1

−1

−1

year )

−1

year ), with four replicates for each treatment. The two N addition

6

171

(NH4NO3) to simulate increased N deposition because atmospheric N deposition is mainly composed

172

of ammonium (NH4 ) and nitrate (NO3 ). NH4NO3 dissolved in 10 L water was added to the soil

173

surface monthly using a backpack sprayer starting in January 2014. Control plots received 10 L of

174

water without NH4NO3. The amount of external water added to each plot when N is applied every year

175

is only equivalent to an increase of 0.3 mm (0.017%) annual precipitation. Thus, the influence of

176

additional water can be ignored.

177

+

At the end of July 2015, nine 2 m

−

× 2 m subplots were randomly established in each N treatment

178

plot and divided into three litter treatments with tree replicated for each treatment (Fig. 1 D): litter

179

reduction (L−, reduced by 50%), litter addition (L+, increased by 50%), and intact litter input (L0, no

180

litter alteration). For the L− treatment, a 2 m × 2 m nylon mesh (size is 1 mm) litter trap was set above

181

each L− subplot to prevent fresh litter input at the end of July 2015 (Fig. 1 E). Beginning in August

182

2015, the litter materials in each litter trap were weighed and recorded on-site monthly and divided into

183

two portions; then, one portion was evenly added to the L− subplot, and the other portion was added to

184

an adjacent L+ subplot. For the L0 subplot, the natural aboveground litter input was maintained. In

185

addition, to calculate the litter water content, litter materials of each plot were collected from a 0.5 m ×

186

0.5 m litter trap which was randomly set in each N treatment plot and brought back to the laboratory.

187

Litter manipulation occurred before N application in every month.

188

Soil respiration measurement

189

In December 2015, a PVC soil respiration (RS) sampling collar (20 cm inside diameter and 8 cm

190

height) was inserted in each subplot for RS measurement. PVC collars were inserted into the soil

191

surface at approximately 6 cm depth and left in situ throughout the study period. A Li-8100 automated

192

soil CO2 efflux system (LI-COR Inc., Lincoln, NE, USA) was used to measure RS once monthly from

193

January 2016 to December 2017. All RS measurements included both litter layers and mineral soil and

194

occurred between 10:00 am and 15:00 pm (local time) on a rainless day before litter manipulation and

195

N addition (Peng et al., 2018). Our previous measurements showed that the soil CO2 fluxes measured

196

within same period approximately equal to the average soil respiration rates of the diel cycle. The

197

average RS rate of three subplots under same treatment in a plot was assumed to represent the diurnal

198

mean of the treatment in the plot, and the total quantity of CO2 emission on that day was multiplied by

199

the number of days of the sampling month to estimate the total CO2 emission of that month (Fan et al., 7

−2

−1

−1

200

2014). The rates of RS and CO2 emission were expressed as µmol CO2 m

201

respectively. The soil temperature at 10 cm below the soil surface was measured for each subplot with

202

a temperature probe at the same time when RS was measured, while the soil moisture content in the

203

upper 5 cm of the soil horizon was measured only for L0 subplots (i.e., soil moisture contents in L−

204

and L+ subplots were not measured) by the gravimetric method with oven drying (dried at 105 °C for

205

24 h).

206

Soil sampling

s

and t C ha ,

207

Soil samples were collected four times: in October 2016 and in January, April and July 2017.

208

Because both N addition and litter manipulation occurred at the surface of the soil and were carried out

209

for only 3-4 years and 1-2 years, respectively, which is unlikely to impact deep soil, thus we only

210

sampled the first 20 cm soil. At each time, within each subplot, the plant litter was removed, and a soil

211

core of the top 20 cm soil horizon was randomly obtained using a soil auger and divided into the

212

organic horizon (including the O and A horizons, with dark color and thickness of approximately 10 cm)

213

and mineral layer (the soil beneath the organic horizon, with thickness of approximately 10 cm) based

214

on soil color. Soil cores of the same soil layer from three subplots under the same litter treatment were

215

mixed as a soil sample. Therefore, there were three different soil samples collected from each N

216

treatment plot for each soil layer, and in total, thirty-six soil samples for each soil layer were obtained

217

for each sampling time.

218

Soil chemistry analysis

219

Fresh soil samples were brought back to the laboratory and immediately ground and sieved through a

220

2-mm mesh after carefully removing the visible roots using tweezers. Then, these samples were divided

221

into two parts. One part was stored at 4 ºC for measuring ammonium (NH4 ), nitrate (NO3 ), microbial

222

biomass carbon (MBC), microbial biomass nitrogen (MBN) and extractable dissolved organic carbon

223

(EDOC) within one week; the other part was air-dried and used for measuring total organic carbon

224

(TOC), readily oxidizable carbon (ROC) and total nitrogen (TN) after being ground and sieved through

225

a 0.25-mm mesh and for measuring soil pH after being ground and sieved at 2 mm.

+

+

−

−

226

Soil NH4 and NO3 were extracted by a 2 M KCl solution (Maynard et al., 2007), and measured

227

with a colorimetric method based on the color reaction between NH4 and a weakly alkaline mixture of

228

Na salicylate and a chlorine source in the presence of Na nitroprusside (Baethgen and Alley, 1989) and

+

8

229

a two-wavelength ultraviolet spectrum approach (Edwards et al., 2001), respectively. The soil MBC

230

and MBN were measured with the 24-h chloroform fumigation extraction technique (Wu et al., 1990;

231

Brookes et al., 1985) and a total C/N analyzer (Shimadzu model TOC-VcPH +TNM-1, Kyoto, Japan).

232

Soil MBC and MBN were calculated as the differences in 0.5 M K2SO4-extractable C or N between

233

fumigated and unfumigated soils, which were divided by 0.45 (Wu et al., 1990) and 0.54 (Brookes et

234

al., 1985), respectively. The K2SO4-extractable C in unfumigated soils was calculated as EDOC. Soil

235

TOC and ROC were measured by the dichromate digestion method (Soon and Abboud, 1991) and

236

KMnO4 oxidation method (Blair et al., 1995), respectively. The soil TN concentration was determined

237

by the Kjeldahl method (Rutherford et al., 2007). Soil pH was determined using a glass electrode in

238

aqueous extracts with a ratio of 1:2.5 (w/v).

239

Statistical analysis

240

Average values of all parameters from each plot under the same treatment from each sampling date

241

were used for data analysis. All parameters were first tested for normal distribution and homogeneity of

242

variances using Shapiro-Wilk’s test and Levene’s test, respectively. Parameters with nonnormal or

243

unequal variances were transformed using the Box-Cox method. One-way repeated-measures analysis

244

of variance (ANOVA) with a post hoc LSD test was used to detect the effects of N addition

245

(between-subjects factor), sampling time (within-subjects factor), and their interaction on litter input.

246

One-way ANOVA with a post hoc LSD test was used to determine the effect of N addition on litterfall

247

for 2015, 2016, 2017 and the whole study period, separately. Two-way repeated-measures ANOVA

248

with a post hoc LSD test was used to determine the effects of N addition and litter manipulation (two

249

between-subjects factors), sampling time (within-subjects factor), and their interactions on RS, soil

250

temperature, soil moisture and soil properties (TOC, ROC, EDOC, TN, NH4+, NO3−, MBC, MBN, C/N

251

and pH). For RS, separate analyses were conducted for 2016, 2017 and whole study period. For soil

252

biochemical characteristics, analyses were conducted for each soil layer separately. Two-way ANOVA

253

was used to detect the effects of N addition and litter manipulation on soil cumulative CO2 efflux for

254

2016, 2017 and whole study period. Nonlinear regression was used to determine the correlations

255

between RS rates and soil temperature, litter inputs or soil moisture. All the above analyses were

256

conducted using IBM SPSS statistics 20.0 for Windows (IBM Crop., Armonk, New York, USA), and

257

statistically significant differences were determined at P < 0.05. 9

258

Results

259

Aboveground litter input

260

Aboveground litter input in this forest displayed significant variation throughout the research period

261

(Fig. 2). The annual mass of litterfall was 5.7 ± 0.7 t ha−1 and 4.5 ± 0.5 t ha−1 in 2016 and 2017,

262

respectively. Generally, there were two litter input peaks (May to July and October to December) each

263

year. The proportion of litterfall in these months reached 66%. The lowest litterfall was observed in

264

January, only 1% of the annual amount. According to the repeated measures ANOVA, N additions did

265

not alter the aboveground litter input. In the whole study period (August 2015 to December 2017), the

266

total amount of added or reduced litterfall in L+ or L− subplots was 6.5 t ha−1.

267

Soil temperature and moisture

268

In this forest, soil temperature varied greatly throughout the study period (Fig. 3A). The annual

269

average temperature was 12.9 °C, with the highest value (19.6 °C) in August and the lowest value

270

(6.1 °C) in January. However, the soil water content remained relatively stable over the study period,

271

ranging from 43.1% to 67.6% (Fig. 3B). The annual mean soil moisture content was 56.2% and 55.1%

272

in 2016 and 2017, respectively. Repeated measures ANOVA showed that neither N addition nor

273

litterfall alteration significantly affected the soil temperature; there was also no significant N effect

274

observed on the soil water content (Table 1). Because the soil moisture contents in the L− and L+

275

subplots were not measured, the influence of litter input manipulation on soil moisture was not

276

analyzed.

277

Soil respiration and microbial biomass

278

Similar to soil temperature, soil respiration showed significant seasonal dynamics, with the lowest

279

value (1.07 µmol CO2 m−2 s−1) in January and the highest value (4.58 µmol CO2 m−2 s−1) in July (Fig. 4).

280

Annual cumulative CO2 fluxes in this forest were 8.2 and 11.3 t C ha−1 yr−1 in 2016 and 2017,

281

respectively (Fig. 5). The seasonality of soil respiration was mainly explained by soil temperature and

282

aboveground litter input. In this forest, soil respiration was significantly positively exponentially

283

related to soil temperature (P < 0.001, R2 = 0.759; Fig. 6 A) and positively related to litterfall via a

284

power function (P < 0.001, R2 = 0.149; Fig. 6 B). However, the correlation between soil respiration and

285

soil moisture was insignificant because soil moisture content varied slightly across the study period. 10

286

Two-way repeated-measures ANOVA revealed that both N addition and litter input alteration

287

significantly altered soil respiration (N effect, P < 0.001; litter effect, P = 0.003), but no significant

288

interactive effects among sampling time, N addition and litter alteration were observed (Table 2).

289

Nitrogen addition significantly reduced soil respiration in subplots with intact litter input in the whole

290

study period by 14.8%−29.3% (P = 0.006; Fig. 5) but insignificantly in subplots with litter reduction or

291

addition (P = 0.161 and P = 0.082, respectively; Fig. 5). Soil CO2 efflux significantly increased with

292

increasing aboveground litter input. The combined L− and N addition treatments significantly (P <

293

0.05) decreased soil respiration and showed a stronger effect than L− or N addition alone; however, the

294

combination of L+ and N addition did not affect soil CO2 efflux. Litter reduction and addition likely

295

promoted and alleviated the negative effect of N addition on soil CO2 emission, respectively.

296

The soil MBC and MBN in the organic horizon and mineral horizon varied significantly in different

297

seasons (P < 0.001; Table 3). The soil MBC and MBN in the organic horizon were greater than those

298

in the mineral horizon (P < 0.001). Added N did not change the soil MBC and MBN in both layers

299

except for a marginally decreased MBN of the organic horizon by 16%−24% (P = 0.053). Litter input

300

alteration did not affect the soil MBC and MBN in both layers. No significant interactive effects among

301

sampling time, N addition and litter manipulation on the soil MBC and MBN were observed.

302

Soil carbon

303

The soil TOC in the organic horizon and ROC and EDOC in both layers displayed significant

304

seasonal variations (P < 0.001; Table 3). The soil TOC and ROC concentrations in the organic horizon

305

were much higher than those in the mineral horizon (P < 0.001). However, there was no significant

306

difference in soil EDOC concentrations between the two soil layers. Two-way repeated-measures

307

ANOVA revealed that both N addition and litterfall manipulations significantly affected soil TOC

308

concentrations in the organic soil layer (P < 0.01). Nitrogen addition increased the soil TOC

309

concentration in the organic soil layer in the litter control subplots but not in the subplots with litter

310

reduction or addition. In the N-added subplots, both L– and L+ decreased soil TOC concentration in

311

organic soil, but no significant effect was found in N controls. Nitrogen addition was associated with a

312

significant increase in soil ROC concentration in the organic horizon (P = 0.008). The soil ROC

313

concentration in the organic soil in subplots without litterfall alteration significantly increased with N

314

addition but did not change in the subplots with litterfall reduction or addition. However, litterfall 11

315

alteration was not associated with soil ROC in the organic soil layer. Neither N addition nor litterfall

316

manipulation influenced TOC and ROC in the mineral soil layer or EDOC in both soil layers. There

317

were no interactive effects among sampling time, N addition and/or litter manipulation observed.

318

Soil nitrogen

319

The soil TN, NO3− and NH4+ concentrations in both the organic horizon and mineral horizon varied

320

significantly throughout the study period (P < 0.001; Table 3). Neither N addition nor litter input

321

manipulation altered soil TN in both soil layers. Nitrogen addition significantly increased the average

322

NO3− concentrations in the organic horizon and mineral horizon by 28.6%−68.9% and 18.5%−103.8%

323

and increased the average NH4+ concentrations by 37.6%−217.9% and 10.0%−108.1%, respectively (P

324

< 0.001). Nitrogen addition showed a significant interactive effect with sampling time on NO3−

325

concentrations in the organic horizon and NH4+ concentrations in both soil layers (P < 0.001). The

326

positive responses of soil NO3− and NH4+ concentrations to N addition were observed in all

327

litter-manipulation treatments and both soil layers (P < 0.005). Combined with N addition, L−

328

significantly increased NH4+ concentrations in the organic horizon (P < 0.05) but did not affect the

329

NH4+ concentration in mineral soil or NO3− concentrations in both soil layers. There was also no

330

interactive effect between N addition and litter manipulation on soil TN, NO3−, and NH4+

331

concentrations.

332

Soil C: N ratio and pH

333

The soil C:N ratio varied significantly throughout the study period (P < 0.003, Table 3). In the

334

organic horizon, N addition significantly increased the soil C:N ratio (P = 0.004). Both L− and L+

335

tended to reduce the soil C:N ratio in the organic horizon, which reached a significant level when

336

combined with LN treatment (P < 0.05). Neither N treatments nor litter manipulations affected the soil

337

C:N ratio in the mineral horizon. There was no interactive effect between N addition and litter input

338

alteration on the soil C:N ratio.

339

The soil pH varied significantly throughout the study period (P < 0.001, Table 3). Nitrogen addition

340

was associated with a significant decrease in soil pH in both soil layers (P < 0.001). Litter manipulation

341

did not change soil pH in either layer. There was no interactive effect between N addition and litter

342

input alteration on soil pH.

12

343

Discussion

344

Effect of nitrogen additions on aboveground litter input

345

As the major pathway to return C and nutrients to the soil from vegetation, litterfall reflects the net

346

primary productivity of forest ecosystems (Malhi et al., 2011). Because most forest ecosystems are

347

N-limited (LeBauer and Treseder, 2008), N addition is believed to potentially alleviate this limitation,

348

thereby promoting net primary productivity (NPP) (Law, 2013; Yan et al., 2014) and, in turn, increasing

349

the amount of aboveground litter (Magill et al., 2000; Li et al., 2010a; Field et al., 2017). By

350

synthesizing studies in 32 forest ecosystems around the world, Yue et al. (2016) reported that global

351

forest litterfall increased by an average of 10.8% with the recorded increase of N input and that the

352

effects of high N addition levels were more pronounced compared with the effects of low N addition

353

levels. However, our results showed that 3−4 years of N application had no significant influence on

354

litterfall, consistent with some previous studies (Mo et al., 2008; Cusack et al., 2011; Peng et al., 2017),

355

probably because 3-4 years of N addition do not supply an extra input of N enough to result in

356

significant increases in plant production. This pattern may occur because this forest is N-rich, as the

357

atmospheric N-deposition rate in this region is very high (95 kg N ha−1 yr−1, Xu et al. 2013b) and thus

358

led to a high N concentration (41.9 g kg−1 and 9.5 g kg−1 for the organic and mineral horizon,

359

respectively) in soil in this forest. Similarly, the N concentrations in soils in the above-cited studies

360

(Mo et al., 2008; Cusack et al., 2011; Peng et al., 2017) are also very high. Therefore, the possibility

361

that these forests have become N-saturated may be another reason why N addition did not affect

362

litterfall. Lu et al. (2018) reported that even 10 years of N addition did not significantly alter litterfall

363

production in an N-rich tropical forest. However, in a temperate forest, litterfall response to N addition

364

changed from increasing early in the chronic N addition to decreasing over time when the soil became

365

N-saturated (Magill et al. 2004). It is likely that the direction and extent of litterfall responses to N

366

addition differ among different forest ecosystems and are determined by the amount and duration of N

367

addition.

368

Effect of nitrogen addition on soil respiration and biochemical properties

369

In this forest, we observed that four years of N addition significantly reduced soil respiration,

370

which is consistent with many other studies (Mo et al., 2008; Maaroufi et al., 2015; Zhou et al., 2018;

371

Peng et al., 2018). Soil CO2 efflux is mainly from two sources: root respiration (rhizospheric 13

372

respiration) and microbial decomposition of SOM (heterotrophic respiration), and the latter is

373

considered the basal respiration (Kuzyakov, 2006). In another similar forest in the same region, it was

374

reported that SOM-derived CO2 fluxes accounted for 75% of the total soil CO2 emission and that N

375

addition reduced both rhizospheric and heterotrophic respiration (Peng et al., 2017). The N-induced

376

reduction in soil respiration suggests that SOM decomposition was inhibited by N addition. On one

377

hand, increased N input may stabilize SOM and enhance its resistance to microbial decomposition by

378

incorporating part of the inorganic N into SOM, forming some recalcitrant compounds, such as indoles

379

and pyrroles (Thorn and Mikita, 1992; Riggs et al., 2015). In their work Berg and Matzner (1997) also

380

proposed that N can regulate the accumulation of SOM, as N-containing compounds can polymerize

381

with aromatic substances in the soil to form recalcitrant organic matter. On the other hand, many

382

previous studies demonstrated that N addition negatively affected soil microbial biomass (Treseder,

383

2008; Frey et al., 2014; Peng et al., 2017), which would directly lead to a decrease in SOM degradation.

384

However, in this study, the soil MBC and MBN did not significantly respond to N additions, which

385

may imply that N application had little effect on soil microbial biomass in this forest. Therefore, the

386

SOM decomposition rate in this study may be predicted to decline mainly due to the enhanced

387

stabilization of SOM. In this region, a negative response of root biomass to N addition was also

388

observed in other similar forests (Peng et al., 2017; Zhou et al., 2018). Thus, we hypothesize that a

389

similar response of root biomass would also occur in this forest, which thereby contributes some extent

390

to the decline of soil CO2 efflux.

391

In the present study, the soil TOC concentration in the organic horizon in subplots without litter

392

manipulations significantly increased as N input was elevated. This result may indicate that N addition

393

results in an increase in soil C in topsoil. Globally, increased N deposition is believed to promote

394

terrestrial ecosystems C sequestration because N is considered a limiting nutrient for terrestrial

395

ecosystems (Wang and Houlton, 2009; Norby et al., 2010). Numerous previous studies have

396

demonstrated that N addition resulted in an increase in soil C sequestration in different types of forest

397

(Field et al., 2017; Yue et al., 2017; Forstner et al., 2018; Yan et al., 2018). Wang et al. (2017) assessed

398

that the globally averaged increase in forest C storage due to atmospheric N deposition for 1997-2013

399

was 0.27 ± 0.13 Pg C yr−1, and 29% (0.079 ± 0.034 Pg C yr−1) of that C was stored in forest soil. Two

400

possible reasons can explain this increase in forest soil C storage. First, elevated N deposition increases

401

N availability and then mitigates N limitation of a forest, which in turn boosts forest productivity and, 14

402

subsequently, increases the inputs of organic matter from plant litter (Li et al., 2010a; Field et al., 2017).

403

Second, N addition may suppress SOM decomposition (Riggs et al., 2015; Zang et al., 2016) and thus

404

increase soil C storage. However, in this forest, no significant change was observed in aboveground

405

litter mass after N application; thus, we speculated that the N-induced increase in TOC concentration in

406

organic soil was mainly due to suppressed SOM decomposition rather than boosted forest productivity

407

(Frey et al., 2014). Although a slight increase in ROC concentration in organic soil was observed in this

408

forest, the proportion of ROC in soil TOC is so small (only ~0.03%) that the influence of changes in

409

ROC may be negligible.

410

Surprisingly, we observed that the soil TN concentration in this forest was 41.9 g kg−1 in organic

411

soil, which is much greater than that in other (sub-)tropical forests (Mo et al., 2008; Cusack et al.,

412

2011). One important cause may be the high ambient atmospheric N deposition rate (95 kg N ha−1 yr−1,

413

Xu et al., 2013b), which would lead to accumulation of N in topsoil in this region. In this forest, the LN

414

treatment showed little effect on the soil inorganic N (NH4+ and NO3−) concentration, while the HN

415

treatment considerably increased the soil inorganic N concentration in both soil layers, but no changes

416

were observed in soil TN concentration. There is no doubt that soil NH4+ and NO3− concentrations

417

would increase after a large amount of N input, as NH4NO3 was directly added to the soil surface when

418

performing the N application. Moreover, heightened soil inorganic N concentration is considered to

419

potentially stimulate the process of mineral N production (N mineralization and nitrification) and

420

restrain microbial N immobilization (Baldos et al., 2015), which in turn would increase soil inorganic

421

N concentration. As a result of stimulated nitrification, more protons (H+) may be accumulated in the

422

soil in N-added plots, leading to stronger soil acidification (Peng et al., 2017). In our study, a reduction

423

in soil pH in both organic and mineral horizons was observed. This pattern is corroborated by many

424

studies by our group or others

425

to the high atmospheric N deposition, soils in this region likely show greater N concentration and lower

426

pH compared with other subtropical forests.

(Peng et al., 2017, 2018; Zhou et al., 2018; Forstner et al., 2018). Due

427

In this study, the C pool in mineral soil did not respond to N addition, whereas the soil TOC and

428

ROC concentrations and the C:N ratio in the organic horizon were significantly associated with

429

elevated N input. Therefore, it can be suggested that organic soil is more sensitive to N addition than

430

mineral soil. Previous studies also revealed that simulated N deposition showed a stronger effect on the

431

organic layer C pool than on mineral soil (Frey et al,. 2014; Tonitto et al., 2014; Maaroufi et al., 2015). 15

432

For example, Frey et al. (2014) reported that N addition (50 kg N ha−1 yr−1) significantly increased the

433

SOC in organic soil in both hardwood and pine stands but did not affect that in mineral soil.

434

Effect of aboveground litter alteration on soil respiration and biochemical

435

properties

436

Although soil C is mostly composed of root-derived C (Rasse et al., 2005), litterfall is also well

437

known as an important source of carbon and nutrients returning from vegetation to topsoil ( Rasse et al.,

438

2005; Jia et al., 2018); thus, changes in litterfall input would impact topsoil substrate and nutrient

439

availability and ultimately affect microbial community and carbon dynamics in topsoil (Xu et al.,

440

2013a; Lajtha et al., 2014a, 2014b, 2018; Wu et al., 2017; Cusack et al., 2018). In general, soil

441

respiration, soil microbial biomass, and soil total C and N are considered to be positively correlated

442

with the amount of litterfall input (Xu et al., 2013a). For instance, many previous litter manipulation

443

experiments and meta-analyses reported that aboveground litter removal and addition respectively

444

decreased and increased soil respiration to varying degrees (Sayer et al., 2011; Leff et al., 2012; Xu et

445

al., 2013a; Han et al., 2015; Wu et al., 2017; Chen and Chen, 2018). In this study, the soil respiration

446

rate increased with the increase of aboveground litter input, showing a positive power correlation with

447

litterfall, although the difference in soil respiration between the litter control and L− or L+ did not

448

reach a significant level. These minor changes are likely because only 50% of the litterfall was

449

removed from L− subplots and added to L+ subplots, which is a smaller alteration in litterfall

450

compared to most other experiments conducted in different forests (Sayer et al., 2011; Leff et al., 2012;

451

Han et al., 2015; Bréchet et al., 2018). However, due to the large difference in the amount of litterfall

452

input between L− and L+, a significant difference in the cumulative CO2 effluxes between them was

453

observed. In this study, the effect of litterfall alteration on soil respiration is most likely due to the

454

direct change of substrate-input but not caused by other biotic (e.g., microbial biomass) and abiotic

455

(e.g., temperature and moisture) factors, because those factors were not affected by altered litter-input.

456

During the decomposition process, plant litter releases most C into air, but a few organic C into

457

the surface soil, and most models assumed a direct relationship between litter input and soil C storage

458

(Gottschalk et al., 2012). Therefore, it was generally considered that surface soil C content would be

459

associated with the amount of litter input (Lajtha et al., 2018). Many studies did demonstrate that

460

long-term litter removal reduced soil C content in topsoil and litter addition enhanced the C content 16

461

(Lajtha et al., 2014a; Cusack et al., 2018). Litter removal likely leads to a decline in soil C in most

462

forests, while litter addition might not always positively affect soil C and showed little and even

463

negative effects on soil C in some ecosystems (Bowden et al., 2014; Lajtha et al., 2014b, 2018). This

464

minor change in soil C content after increased litter input can be mainly explained by the priming effect

465

because the enhanced soil C derived from increased plant litter may be offset by the accelerated

466

decomposition of native SOM (Sayer et al., 2011; Xu et al., 2013a). In this study, although we did not

467

observe that litter addition changed soil C concentration, there was no evidence that the priming effect

468

played an important role in this process because the soil CO2 efflux in the L+ subplots did not

469

significantly differ from that in the litter control. In addition, there were also no differences in soil C

470

between the L− and litter control subplots and in other soil properties among litter manipulations.

471

Therefore, we attributed these unaffected results to the smaller alteration of litterfall input (only 50%)

472

and shorter experiment duration (< 2.5 years) compared to other studies, in which litters were doubled

473

or entirely excluded continually for 10 or more years (Lajtha et al., 2014a; Cusack et al., 2018). Other

474

short-term experiments also reported a similar result: minor changes were observed in soil C and N

475

contents after less than 5 years of litter treatment (Holub et al., 2005; Zhao et al., 2017). Because the

476

effect of litter manipulation on soil may have a significant temporal lag (Lajtha et al., 2014b),

477

long-term observation (decadal-scale) is necessary to investigate the response of soil to altered litter

478

input (Sayer, 2005; Lajtha et al., 2018), especially in experiments with small proportions of litter

479

manipulation.

480

Effect of aboveground litter manipulation on soil respiration and biochemical

481

properties under different nitrogen-addition conditions

482

Our data indicated that, under each N-addition condition, soil respiration increased with increasing

483

aboveground litter input. Combined N addition and litter reduction significantly decreased soil

484

respiration and showed a stronger effect than individual N addition or litterfall reduction. This pattern

485

occurs because both N addition and litterfall reduction can cause a decrease in soil CO2 efflux, as

486

discussed before (Leff et al., 2012; Han et al., 2015; Wu et al., 2017; Peng et al., 2018; Zhou et al.,

487

2018). However, the soil C content in subplots treated with a combination of N addition and litter

488

reduction showed no significant change compared to subplots without N addition and litter alteration,

489

which might suggest that an N-induced increase in soil C content may be partly offset by decreased 17

490

litter input. Conversely, combined N and litter addition did not significantly affect soil CO2 effluxes,

491

which indicated that litter addition likely mitigated the negative response of soil respiration to N

492

addition because soil respiration is positively associated with litterfall (Sayer et al., 2011; Leff et al.,

493

2012; Han et al., 2015; Wu et al., 2017).

494

Surprisingly, no changes were observed in soil TOC concentration among litterfall manipulations

495

in N control plots, but both the L− and L+ treatments were found to significantly reduce soil TOC in

496

the organic horizon in N-added plots, which may suggest that topsoil C content is sensitive to

497

alterations of aboveground litter input when N input is elevated. This relation would have important

498

implications on the response of soil C storage to global change. In the HN treatment plots, L−

499

significantly reduced the soil TOC concentration in organic soil by 10.7% relative to L0. Firstly,

500

reduced litterfall directly results in a reduction in aboveground C input. In addition, previous studies

501

have noted that reduced aboveground litter input can potentially lead to a decrease in root biomass and

502

production, which is an important soil C source (Rodtassana and Tanner, 2018). Therefore, it can be

503

surmised that a reduction in aboveground litter in HN plots may lead to a decreased soil C content by

504

reducing aboveground C input directly and by reducing belowground C input indirectly. Unexpectedly,

505

the L+ treatment was observed to reduce the surface soil TOC by ~13% compared with L0 in N-added

506

plots. Many studies demonstrated a neutral or even negative effect of litter addition on soil C content as

507

a result of higher rates of SOM degradation due to a priming effect (Sayer et al., 2011; Leff et al., 2012;

508

Zhang et al., 2017; Pisani et al., 2016). However, in this study, the soil CO2 efflux in L+ did not

509

significantly differ from L0 in N-added plots, which did not support the priming effect; thus, the

510

potential mechanism needs further research.

511

Our results showed that L− significantly increased soil NH4+ concentration in topsoil in N-added

512

plots but not in plots without N addition, while L+ did not affect that factor in plots either with or

513

without N addition. Similarly, Holub et al. (2005) also reported that soil NH4+ concentration under high

514

N deposition was greater in plots with no litter input compared to intact litter plots but did not change

515

in doubled litter plots or under low N deposition. Because the litter layer acts as a kind of buffer and

516

barrier which regulates nutrients the underlying horizon will receive from precipitation, part of the

517

NH4+ added to the surface of the litter cannot reach mineral soil but is retained in the litter layer.

518

Therefore, it is logical that the smaller the amount of litter above the soil, the more the proportion of

519

the added NH4+ that will reach the soil. Additionally, as C:N ratio in litter is generally higher than that 18

520

in microbes, a litter net N immobilization commonly occurs in terrestrial ecosystems (Holub et al.,

521

2005; Parton et al., 2007). In this study, because of decreased litter input, immobilization of added

522

NH4+ was also decreased; thus, more NH4+ was retained in the soil of L− subplots compared to litter

523

intact subplots. However, the little change in soil NH4+ concentration in L+ subplots was probably due

524

to the limitation of microbes in the topsoil and litter layer.

525

Conclusion

526

Our results highlight that elevated N input significantly increased the surface soil C content by

527

suppressing soil respiration mainly due to enhanced stabilization of SOM rather than decreased soil

528

microbial biomass. Due to the smaller alteration and shorter experimental duration, the aboveground

529

litter manipulation did not affect any soil properties characterized in this study, but the soil CO2

530

emission was significantly associated with the amount of litter input. Because of temporal lag, a

531

long-term litter manipulation experiment is necessary to investigate the response of soil to altered litter

532

input. Aboveground litter reduction likely promotes a negative effect on soil respiration due to N

533

addition, while litter addition tended to mitigate the change in soil respiration. Under N addition, litter

534

reduction tended to decrease the surface soil C content by reducing the aboveground C input directly

535

and the belowground C input indirectly. Aboveground litter addition also tended to reduce the topsoil C

536

content when the N input is enhanced, but the potential mechanism still needs further exploration

537

because no evidence was observed to support a priming effect. Both N and aboveground litter

538

manipulations showed a stronger effect on the organic soil layer than on the mineral soil layer. Finally,

539

because the soil in this forest shows very high N content and is probably already N-saturated, as a

540

result of the high ambient atmospheric N deposition rate here, some of our results would not apply for

541

forests not (or less) affected by this situation.

542 543 544 545 546

Acknowledgement This project was supported by the National Natural Science Foundation of China (No. 31300522) and Major Project of Education Department in Sichuan Province (No. 17ZA0310).

547

19

548

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549

Baethgen, W.E., Alley, M.M., 1989. A manual colorimetric procedure for measuring ammonium

550

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Blair, G.J., Lefroy, R.D.B., Lisle, L., 1995. Soil carbon fractions based on their degree of oxidation,

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Bowden, R.D., Davidson, E., Savage, K., Arabia, C., Steudler, P., 2004. Chronic nitrogen additions

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Bowden, R.D., Deem, L., Plante, A.F., Peltre, C., Nadelhoffer, K., Lajtha, K., 2014. Litter input

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Bréchet, L.M., Lopez-Sangil, L., George, C., Birkett, A.J., Baxendale, C., Trujillo, B.C., Sayer, E.J.,

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woodland and tropical forest. Ecology and Evolution 8, 3787 – 3796.

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Figure legends:

788 789

Figure 1 Location of study site in Ya’an, Sichuan province, China and the schematic design of N

790

addition and aboveground litter manipulation. CN, LN and HN indicate N control (ambient N

791

input), low level (+ 50 kg N ha−1 year−1) and high level (+ 150 kg N ha−1 year−1) of N addition,

792

respectively. L0, L− and L+ indicate natural litter input, aboveground litter reduction (reduced by 50%)

793

and addition (increased by 50%), respectively.

794 795

Figure 2 Seasonal variations in above-ground litter input in an evergreen broad-leaved forest in

796

southwestern China from August 2015 to December 2017. Histograms indicate cumulative litter

797

input in each year (litter traps were set in the end of July 2015, thus only last five months were included

798

in 2015) and total input of all the months. Values are means, n=4. Error bars were not given to improve

799

the clearness of the graph. The results of repeated measures ANOVA are shown. CN: N control

800

(ambient N input); LN: low level of N addition (+ 50 kg N ha−1 year−1); HN: high level of N addition

801

(+ 150 kg N ha−1 year−1).

802 803

Figure 3 Seasonal variations in soil temperature (A) and soil moisture content (B) in an evergreen

804

broad-leaved forest in southwestern China from January 2016 to December 2017. Values are

805

means, n=4. Error bars were not given to improve the clearness of the graph. CNL−: Nitrogen control

806

with litter reduction; LNL−: Low N addition with litter reduction; HNL−: High N addition with

807

litter reduction; CNL0: Nitrogen control with intact litter input; LNL0: Low N addition with

808

intact litter input; HNL0: High N addition with intact litter input; CNL+: Nitrogen control with

809

litter addition; LNL+: Low N addition with litter addition; HNL+: High N addition with litter

810

addition.

811 812

Figure 4 Seasonal variations in soil respiration in an evergreen broad-leaved forest in

813

southwestern China from January 2016 to December 2017. Values are means, n=4. Error bars

814

were not given to improve the clearness of the graph. CNL−: Nitrogen control with litter

815

reduction; LNL−: Low N addition with litter reduction; HNL−: High N addition with litter 28

816

reduction; CNL0: Nitrogen control with intact litter input; LNL0: Low N addition with intact

817

litter input; HNL0: High N addition with intact litter input; CNL+: Nitrogen control with litter

818

addition; LNL+: Low N addition with litter addition; HNL+: High N addition with litter

819

addition.

820 821

Figure 5 Cumulative CO2 flux under different treatments. CNL−: Nitrogen control with litter

822

reduction; LNL−: Low N addition with litter reduction; HNL−: High N addition with litter

823

reduction; CNL0: Nitrogen control with intact litter input; LNL0: Low N addition with intact

824

litter input; HNL0: High N addition with intact litter input; CNL+: Nitrogen control with litter

825

addition; LNL+: Low N addition with litter addition; HNL+: High N addition with litter addition.

826

Error bars indicate ± 1SE, n=4. Different letters indicate significant difference among different N

827

additions and litter manipulations within each year.

828 829

Figure 6 Relationships between soil respiration and soil temperature (A) and litterfall (B). Values

830

are means, n=4. Error bars were not given to improve the clearness of the graph. CNL−: Nitrogen

831

control with litter reduction; LNL−: Low N addition with litter reduction; HNL−: High N

832

addition with litter reduction; CNL0: Nitrogen control with intact litter input; LNL0: Low N

833

addition with intact litter input; HNL0: High N addition with intact litter input; CNL+: Nitrogen

834

control with litter addition; LNL+: Low N addition with litter addition; HNL+: High N addition

835

with litter addition.

29

1

Influences of nitrogen addition and aboveground litter-input manipulations on soil respiration

2

and biochemical properties in a subtropical forest

3 4 5 Table 1 Results of repeated measures ANOVA concerning the effects of sampling time, nitrogen treatment, litter manipulation, and their interactions on soil temperature and soil moisture in an evergreen broad-leaved forest in southwestern China from January 2016 to December 2017 Temperature (ºC)

Factors

Moisture (%)

df

F-value

P-value

df

F-value

P-value

Time (T)

23

8507.256

< 0.001

23

10.863

< 0.001

Nitrogen (N)

2

2.056

0.148

2

1.303

0.319

Litter (L)

2

0.944

0.402

−

−

−

N×L

4

0.327

0.857

−

−

−

T×N

46

0.724

0.747

46

0.963

0.485

T×L

46

0.607

0.855

−

−

−

T×N×L

92

0.635

0.921

−

−

−

Soil moistures for subplots with litter manipulations were not measured, thus effect of litter alteration on soil moisture was not shown.

6 7 8 Table 2 Results of two-way repeated measures ANOVA concerning the effects of sampling time, nitrogen treatment, litter manipulation, and their interactions on soil respiration in an evergreen broad-leaved forest in southwestern China from January 2016 to December 2017 Factors

2016

2017

Whole study period

df

F-value

P-value

df

F-value

P-value

df

F-value

P-value

Time (T)

11

175.214

< 0.001

11

166.122

< 0.001

23

166.315

< 0.001

Nitrogen (N)

2

12.038

< 0.001

2

7.641

0.002

2

10.854

< 0.001

Litter (L)

2

7.078

0.003

2

4.969

0.015

2

6.765

0.004

N×L

4

0.957

0.447

4

0.755

0.564

4

0.891

0.483

T×N

22

1.568

0.109

22

2.062

0.063

46

1.788

0.065

T×L

22

1.679

0.079

22

1.227

0.299

46

1.290

0.239

T×N×L

44

0.868

0.642

44

0.787

0.668

92

0.779

0.740

9

1

Table 3 Averaged soil properties for four times and P-value of two-way repeated ANOVA focus on effects of sampling time, N addition, litter manipulation, and their interactions on soil chemistry TOC (g kg−1)

ROC (mg kg−1)

EDOC (mg kg−1)

TN (g kg−1)

NO3− (mg kg−1)

Factors

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

CNL−

174.8 ± 5.1 bc

30.5 ± 2.0

53.1 ± 1.7 ab

9.4 ± 1.0

0.34 ± 0.03

0.37 ± 0.06

47.1 ± 0.9

10.3 ± 0.6

46.4 ± 1.2 e

27.9 ± 7.0 c

CNL0

167.6 ± 6.1 bc

28.3 ± 1.7

49.8 ± 2.0 b

9.0 ± 0.8

0.32 ± 0.03

0.40 ± 0.04

41.9 ± 1.4

9.5 ± 0.7

50.8 ± 4.5 e

24.8 ± 3.8 c

CNL+

160.7 ± 2.1 c

28.8 ± 1.5

52.0 ± 2.2 ab

9.6 ± 0.9

0.33 ± 0.01

0.35 ± 0.06

43.3 ± 1.5

10.0 ± 0.6

50.4 ± 3.2 e

26.5 ± 2.1 c

LNL−

161.4 ± 12.8 bc

29.9 ± 3.5

52.1 ± 3.4 ab

9.6 ± 1.4

0.33 ± 0.05

0.32 ± 0.03

44.8 ± 3.1

10.8 ± 0.8

69.1 ± 9.5 bcd

35.0 ± 3.1 bc

LNL0

179.5 ± 7.0 b

28.8 ± 1.3

57.8 ± 2.4 a

9.3 ± 0.8

0.33 ± 0.01

0.35 ± 0.05

45.1 ± 2.1

9.8 ± 0.2

57.7 ± 4.4 de

27.1 ± 2.7 c

LNL+

155.6 ± 9.5 c

28.6 ± 2.2

52.9 ± 1.3 ab

10.5 ± 1.3

0.30 ± 0.04

0.32 ± 0.03

44.1 ± 0.8

10.0 ± 0.4

63.0 ± 5.2 cde

31.8 ± 5.5 c

HNL−

177.0 ± 3.5 bc

29.5 ± 3.5

59.5 ± 1.8 a

9.4 ± 0.7

0.33 ± 0.04

0.40 ± 0.05

44.3 ± 0.8

10.1 ± 0.8

86.5 ± 8.9 a

53.6 ± 9.3 a

HNL0

198.2 ± 4.1 a

29.5 ± 3.4

57.9 ± 4.2 a

9.5 ± 1.3

0.33 ± 0.04

0.40 ± 0.05

45.1 ± 1.1

9.7 ± 0.6

84.1 ± 6.5 ab

56.0 ± 8.7 a

HNL+

173.1 ± 4.7 bc

26.1 ± 2.2

59.0 ± 3.1 a

8.9 ± 0.7

0.35 ± 0.06

0.38 ± 0.03

44.9 ± 1.0

9.2 ± 0.6

78.7 ± 4.5 abc

51.9±5.5ab

Time (T)

< 0.001

0.567

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

Nitrogen (N)

0.009

0.905

0.008

0.775

0.865

0.319

0.863

0.552

< 0.001

< 0.001

Litter (L)

0.009

0.601

0.971

0.904

0.959

0.727

0.506

0.315

0.745

0.821

N×L

0.283

0.951

0.456

0.915

0.983

0.996

0.347

0.912

0.639

0.918

T×N

0.376

0.504

0.242

0.511

0.575

0.413

0.662

0.226

< 0.001

0.277

T×L

0.499

0.949

0.614

0.123

0.935

0.792

0.174

0.223

0.556

0.792

T×N×L

0.610

0.919

0.855

0.728

0.966

0.986

0.255

0.946

0.520

0.657

2

(Continued) NH4+ (mg kg−1)

MBC (mg kg−1)

MBN (mg kg−1)

C : N ratio

pH

Factors

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

Organic horizon

Mineral horizon

CNL−

20.1 ± 4 4 d

12.4 ± 2.4 c

1.46 ± 0.24

0.33 ± 0.03

0.27 ± 0.03

0.067 ± 0.012

3.70 ± 0.11 bc

2.98 ± 0.12

3.45 ± 0.03 abc

3.91 ± 0.05 a

CNL0

19.0 ± 4.4 d

15.0 ± 1.3 c

1.32 ± 0.18

0.32 ± 0.06

0.25 ± 0.03

0.077 ± 0.010

3.99 ± 0.11 b

3.04 ± 0.07

3.52 ± 0.03 a

3.90 ±0.03 a

CNL+

19.2 ± 3.8 d

12.8 ± 1.8 c

1.12 ± 0.26

0.37 ± 0.04

0.24 ± 0.03

0.070 ± 0.00

3.70 ± 0.15 bc

2.89 ± 0.12

3.52 ± 0.03 a

3.91 ± 0.04 a

0.37 ± 0.05

0.24 ± 0.02

0.064 ± 0.01

LNL−

42.4 ± 4.9 c

15.6 ± 2.3 c

1.11 ± 0.09

3.58 ± 0.06 c

2.73 ± 0.14

3.40 ± 0.04 bcd

3.84 ± 0.02 ab

LNL0

19.9 ± 4.4 d

15.0 ± 1.2 c

0.99 ± 0.20

0.33 ± 0.03

0.21 ± 0.03

0.089 ± 0.02

4.00 ± 0.12 b

2.96 ± 0.09

3.47 ± 0.05 ab

3.86 ± 0.04 a

LNL+

17.9 ± 3.7 d

13.7 ± 1.3 c

0.98 ± 0.14

0.34 ± 0.03

0.19 ± 0.02

0.069 ± 0.01

3.52 ± 0.16 c

2.84 ± 0.10

3.45 ± 0.02 abc

3.87 ± 0.04 a

HNL−

69.6 ± 9.9 a

27.2 ± 3.2 ab

1.08 ± 0.22

0.31 ± 0.05

0.19 ± 0.03

0.050 ± 0.01

4.02 ± 0.17 b

2.92 ± 0.09

3.35 ± 0.03 d

3.74 ± 0.03 c

HNL0

53.4 ± 3.6 bc

25.1 ± 1.4 b

0.97 ± 0.07

0.29 ± 0.03

0.19 ± 0.03

0.063 ± 0.00

4.43 ± 0.13 a

3.33 ± 0.47

3.36 ± 0.03 cd

3.71 ± 0.03 c

HNL+

62.3 ± 6.8 ab

31.6 ± 1.5 a

0.95 ± 0.19

0.35 ± 0.07

0.20 ± 0.02

0.060 ± 0.01

3.85 ± 0.19 bc

2.86 ± 0.10

3.37 ± 0.04 cd

3.75 ± 0.03 bc

Time (T)

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

< 0.001

0.002

< 0.001

< 0.001

Nitrogen (N)

< 0.001

< 0.001

0.112

0.704

0.053

0.119

0.004

0.434

< 0.001

< 0.001

Litter (L)

0.013

0.773

0.432

0.543

0.621

0.168

0.001

0.202

0.146

0.718

N×L

0.177

0.178

0.970

0.950

0.962

0.918

0.878

0.820

0.900

0.972

T×N

< 0.001

<0.001

0.454

0.397

0.576

0.753

0.134

0.521

0.075

0.016

T×L

< 0.001

0.009

0.951

0.596

0.751

0.296

0.008

0.061

0.309

0.558

T×N×L

0.175

0.525

0.999

0.983

0.851

0.645

0.623

0.517

0.655

0.999

CNL−: Nitrogen control with litter reduction; LNL−: Low N addition with litter reduction; HNL−: High N addition with litter reduction; CNL0: Nitrogen control with intact litter input; LNL0: Low N addition with intact litter input; HNL0: High N addition with intact litter input; CNL+: Nitrogen control with litter addition; LNL+: Low N addition with litter addition; HNL+: High N addition with litter addition. Different letters indicate significant difference among treatments (P < 0.05)

3