Mass loss and nutrient dynamics during litter decomposition under three 
mixing treatments in a typical steppe in Inner Mongolia 
Yulian Tan1, 2, Jin Chen3, Liming Yan1,4, Jianhui Huang1*, Lixin Wang5,6, Shiping Chen1 
 
1State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese 
Academy of Sciences, Beijing 100093, China 
2Graduate University of Chinese Academy of Sciences, Beijing 100049, China 
3Ezhou Environmental Monitoring Station, Ezhou 436000, China 
4School of Life Sciences, Fudan University, Shanghai 200433, China 
5Department of Earth Sciences, Indiana University-Purdue University, Indianapolis (IUPUI), 
Indianapolis, Indiana 46202, USA 
6Water Research Center, School of Civil and Environmental Engineering, University of New 
South Wales, Sydney, NSW 2052, Australia 
 
*Corresponding author: 
Jianhui Huang, Ph.D. 
State Key Laboratory of Vegetation and Environmental Change 
Institute of Botany, Chinese Academy of Sciences 
No. 20 Nanxincun, Xiangshan, 
Beijing, 100093, China 
Tel: 86-10-62836279, Fax: 86-10-62599059 
Email: jhhuang@ibcas.ac.cn 
 
 
This is the author's manuscript of the article published in final edited form as:  
Tan, Y., Chen, J., Yan, L., Huang, J., Wang, L., & Chen, S. (2013). Mass loss and nutrient 
dynamics during litter decomposition under three mixing treatments in a typical steppe in Inner 
Mongolia. Plant and soil, 366(1-2), 107-118. 
http://dx.doi.org/10.1007/s11104-012-1401-6 
 
 1 
Abstract 
Background and Aims: Mixing effects during litter decomposition could occur 
between two or more different litter species because of the potential nutrient transfer. 
However, evidence of mixing effects is variable and the underlying mechanisms remain 
unclear. Using a three-year decomposition experiment, we aim to examine the effects of 
litter mixing and position on decomposition rates and nitrogen (N) and phosphorus (P) 
dynamics. 
Methods: We studied litter decomposition of Stipa krylovii (Sk) and Astragalus 
galactites (Ag), two dominant species with contrasting litter quality in a typical steppe 
of northern China in single decomposition and three mixing treatments. The three 
mixing treatments included thorough mixing (Sk-Ag), Ag over Sk (Ag/Sk), and Sk over 
Ag (Sk/Ag).  
Results: Both the Sk-Ag and the Sk/Ag mixture had negative mixing effects on the 
mass loss of the litter mixture, while the Ag/Sk mixture had a neutral mixing effect. The 
percent mass loss was higher when the litter species was placed at the top (25.0% and 
51.9% of mass remaining for Ag and Sk) than at the bottom (38.3% and 61.8% of mass 
remaining for Ag and Sk). The Sk/Ag mixture had negative effects on the release of N 
while all three mixing treatments had positive effects on the release of P.  
Conclusions: Our results indicate that: (1) mixing treatments can induce different 
mixing effects; (2) environmental factors likely play an important role in controlling the 
mixing effect; (3) litter-mixtures have different non-additive effects on N and P, which 
may further increase the heterogeneity of N and P availability as the two litter species 
may fall differentially in terms of space and time.  
Keywords: Litter decomposition, mixing effects, mass loss, nitrogen release, 
phosphorus release
 2 
Introduction 
In most terrestrial ecosystems, litter decomposition is a fundamental ecological 
process that provides soil nutrients for plant biological activity (Swift et al. 1979). 
Due to its importance in nutrient cycling, the dynamics of plant litter decomposition 
and the controlling factors have been the focus of a considerable number of studies. 
Previous studies have revealed that litter decomposition could be affected by a series 
of abiotic factors, such as precipitation (Austin and Vitousek 2000), UV-radiation 
(Austin and Vivanco 2006), water table depth (Moore et al. 2007), and soil nutrient 
availability (Dent et al. 2006; Hobbie and Vitousek 2000). In addition, biotic factors, 
such as litter quality (Berg and Ekbohm 1991), soil animals (Bradford et al. 2002), 
and soil microbes (Daniel and Anderson 1992) could have great impact on litter 
decomposition. In semiarid environments, litter decomposition is more complex and 
additional factors such as vegetation structure and soil moisture could also play 
important roles (e.g., Throop and Archer 2009; Wang et al. 2009). Among these 
factors, it is often believed that litter quality, i.e., nitrogen (N) content, C/N ratio and 
lignin/N ratio, is the most important factor in determining the rate of litter 
decomposition especially in a terrestrial ecosystem (Aerts 1997; Vivanco and Austin 
2006). 
Most of the previous studies, however, have only focused on the decomposition 
of single litter species, while litter layer in natural ecosystems is in fact a mixture of 
different plant species. Decomposition of one litter species is inevitably influenced by 
the presence of adjacent litter species, resulting in so-called mixing effects which may 
either facilitate or inhibit the rate of overall litter decomposition (Jonsson and Wardle 
2008; McTiernan et al. 1997; Wardle et al. 1997). Such mixing effects can also be 
regulated by environmental factors (Gartner and Cardon 2006; Madritch and 
 3 
Cardinale 2007) and litter quality of the component species, though the results are 
inconsistent among different studies (Hättenschwiler and Vitousek 2000; Hector et al. 
2000; Hobbie et al. 1999; Hoorens et al. 2003). Most studies compared the observed 
with the expected mass loss rate based on the decomposition rates of single 
component litter; few of these studies explored the underlying mechanisms inducing 
the mixing effects (Hättenschwiler et al. 2005; Wardle et al. 1997). Even fewer studies 
attempted to separate the component litter species from the litter mixture to 
investigate how the identity of the neighboring litter species affected decomposition 
interactively (Barantal et al. 2011; Hoorens et al. 2010; Wardle et al. 2003). 
In this study, we selected two dominant species, Stipa krylovii (Sk) and 
Astragalus galactites (Ag) with contrasting litter quality in terms of N content from a 
typical semiarid steppe ecosystem in Inner Mongolia, China to examine whether there 
were significant mixing effects on the decomposition of litter mixtures. In order to 
understand which factors determined the observed litter mixing effects, the two litter 
species in the litter mixture were mixed in three different treatments (thorough mixing, 
Sk on top of Ag, and Ag on top of Sk). We hypothesized that 1) the three mixing 
treatments would have different mixing effects as position could play an important 
role; 2) the high quality Ag litter would release nutrients while the low quality Sk 
litter would immobilize nutrients; and 3) the presence of Ag litter would facilitate the 
nutrient release whereas the Sk litter induce immobilization in the mixture.
 4 
Materials and methods 
Study site 
The experimental site was located at a temperate steppe in Duolun County, Inner 
Mongolia (42°02′N, 116°17′E, 1324 meters above sea level), China. This area is in a 
typical temperate zone characterized by a semiarid continental monsoon climate. 
Long-term mean annual precipitation is 385 mm, and mean annual temperature is 
2.1oC with monthly mean temperature ranging from -17.5oC in January to 18.9oC in 
July. Annual rainfall is 198.5 mm in 2007, 314.4 mm in 2008, and 172.6 mm in 2009, 
and small daily rainfall events (0-2 mm) account for 67%, 50%, and 68% of total 
rainfall from 2007 to 2009, respectively (Fig. 1). The sandy soil of the study site is 
classified as chestnut according to the Chinese classification, or Haplic Calcisols 
according to the Food and Agriculture Organization (FAO) classification. The surface 
10 cm soil layer is slightly alkaline (pH=7.2) and contains 20.4 g kg-1 total C, 1.63 g 
kg-1 total N with 10.35 mg kg-1 total available N, and 0.31 g kg-1 total phosphorus (P) 
(Liu et al. 2010). Mean soil bulk density is 1.31 g cm-3. 
Experimental design 
The selected species in this study, Stipa krylovii (Sk) and Astragalus galactites (Ag), 
are two dominant species in this typical steppe ecosystem in Inner Mongolia grassland. 
S. krylovii is a perennial bunchgrasses, and A. galactites is a perennial forb and an 
important legume species in the studied grassland system (Yang et al. 2011). The two 
litter species have quite different chemical and physical properties. The initial N 
concentration of the litter of Ag is about four times of that of Sk, and the initial P 
concentration of Ag is also significantly higher than that of Sk (Table 1). 
In late September 2006, the senescent leaf litters from the current year of the two 
species were collected, air-dried and placed in polyethylene litterbags (15×20cm, 
 5 
1mm mesh). Each bag was filled with 15g litter either from a single litter species (Ag 
or Sk), or a 1:1 mixture of the two litter species. Two litter species were either mixed 
thoroughly (Sk-Ag) or separated by a polyethylene sheet (0.20mm in mesh size) in the 
litterbags. For the two mixing treatments with a divider, either Sk or Ag was placed on 
top of the other (Sk/Ag and Ag/Sk). In total there were five treatments: two single 
litter treatments (Ag and Sk) and three litter mixing treatments (Sk-Ag, Sk/Ag and 
Ag/Sk). The initial litter weight (15g air-dried litter) in each bag was chosen based on 
the following considerations: (1) experimental duration (~3 years), (2) potential 
decomposition rate in this area, and (3) sufficient remaining mass for chemical 
analyses after decomposition. The choice of the litterbag size was constrained by the 
limited space among plant clumps in the studied community, i.e., it would be difficult 
to use litterbags larger than15cm x 20cm. 
On October 27 2006, 200 litterbags were deployed in five replicate plots, with 40 
in each plot. Litterbags were retrieved after 162, 252, 341, 620, 706, 911, 991, and 
1072 days, respectively, during the following three years. At each collection date, one 
set of litterbags, i.e., one litterbag of each single litter species and of each of the three 
mixing treatments from each replicate plot (25 total each time), was randomly 
collected, and then transported back to the laboratory. 
In the laboratory, five sub-samples of each type of the original litter were 
oven-dried at 70oC for 48 h before initial deployment to determine the ratio of 
air-dried versus oven-dried mass. This ratio was used to calculate the initial 
oven-dried mass of each litterbag from the air-dried mass. After the litterbags were 
collected, extraneous matter such as in-growth plant materials and small animals were 
removed from the decomposing litter, when the mixture of Sk/Ag and Ag/Sk were 
separated by species. The soil mixed with the litter sample was rinsed off in cold 
 6 
water, and the litter was then oven-dried at 70oC for 48 h to determine remaining dry 
mass.  
Chemical analyses 
To determine the initial litter chemistry, total C, N and P of the five original 
non-decomposed sub-samples were measured. Only N and P concentrations were 
examined for litter samples harvested during decomposition. All the litter samples 
were ground using a ball mill (Retsch MM 400, Retsch GmbH & Co KG, Haan, 
Germany), and passed through a No. 4 sieve. Total C content was determined using 
H2SO4-K2Cr2O7 oxidation method (Nelson and Sommers 1996), and total N 
concentrations with an Alpkem autoanalyzer (Kjektec System 1026 Distilling Unit, 
Sweden). After sub-samples were digested in the mixture of H2SO4 and H2O2, total P 
was measured using molybdenum blue colorimetric method at 880 nm. 
Statistical analyses 
The expected percent mass remaining of each litter mixture (Sk-Ag, Sk/Ag and Ag/Sk) 
was calculated as: 
Expected percent mass remaining (%) = [M1/( M1+M2)] × R1 + [M2/( M1+M2)] × R2  
where R1, R2 are the percent remaining mass (%) of the singly decomposed litter 
species 1 and 2, respectively, and M1 and M2 are the estimated initial dry mass of each 
litter species in the mixture (Hoorens et al. 2003). 
The expected percent N and P mass remaining in the litter mixtures were 
determined similarly based on N and P concentrations of each single litter species at 
each collection date. Any significant deviation from expected values indicates an 
interactive effect, either positive or negative, between the mixing litter species. 
Repeated measures ANOVA was used to test the significance of the overall 
differences between the observed and the expected percent mass remaining values and 
 7 
between different litter mixing treatments (i.e., Sk-Ag, Sk/Ag and Ag/Sk) across the 
whole decomposing period, where time was treated as within-subject factor. Repeated 
measures ANOVA was also used to test the differences of the mass loss and nutrient 
concentration of the same litter species placed at different positions (top or bottom) 
during the whole decomposing period. One-way ANOVA was used to test the nutrient 
concentration differences between the two positions in the litterbag at each collection 
date. The regressions between time and the ratios of the percent remaining mass of the 
top and bottom treatments of the same species were also conducted. The significant 
level of α= 0.05 was used. Data analyses were performed using SPSS (version 16.0). 
 
Results 
Mass loss of single-component litter and the mixtures 
After 1072 days, an average of 74.3% of the initial mass of the singly decomposed Ag 
litter was lost, compared with a 48.7% loss of the single Sk litter (Fig. 2a).  
Among the three types of litter mixing treatments, Sk-Ag had a significant 
negative mixing effect on mass loss (P=0.031). The amount of the observed percent 
mass remaining of the Sk-Ag treatment was 42.8% compared with the expected value 
of 41.0% at the end of this study. Sk/Ag also had a significant negative mixing effect 
on the mass loss (P<0.001), and the observed percent mass remaining was 45.1%, 
higher than the expected value of 41.0%. In contrast, Ag/Sk did not show significant 
mixing effect on the mass loss (P>0.05) (Fig. 2b, Table 2).  
Litter placed on the top, either Sk or Ag, decomposed much faster than that at the 
bottom. There were significant differences in the percent mass remaining between the 
top and the bottom positions for both Sk and Ag at all collection dates except the one 
at day 162 (Fig. 3a,b). The ratios between top and bottom percent remaining mass 
 8 
showed a significant exponential decrease with time for both Sk and Ag, and tended to 
level off at the end of this study (r2Sk=0.84, r2Ag=0.69, Fig. 3a,b).  
Nutrient concentrations of the component litter species and their nutrient release 
patterns 
N concentration of the litter species placed on the top was much higher than that of 
the same species placed at the bottom for both Sk and Ag (P=0.011 for Sk; P<0.001 
for Ag, Table 3, Fig. 4a,b) during decomposition. However, results of one-way 
ANOVA showed that significant difference in N concentrations of the two positions 
for the Sk litter was found only at the last collection date (Fig. 4a), while significant 
difference was found for the Ag litter retrieved at day 252, 341, 706 and 991 (Fig. 4b). 
N concentrations of Sk in Sk/Ag and Ag/Sk showed a small decrease at the beginning, 
increased continuously afterwards, and decreased again when it approached the end of 
the observation period (Fig. 4a). N concentrations of Ag in Ag/Sk and Sk/Ag showed 
remarkably large fluctuations during the decomposition period although there was an 
overall increasing trend after a dip at the beginning (Fig. 4b). Because the two 
component species could not be separated from each other in the thorough mixture 
Sk-Ag, we were unable to investigate separately the change of N and P concentrations 
of each decomposing litter species in the mixture. 
 Repeated measures ANOVA showed that there was no overall difference in P 
concentrations between the top and the bottom positions for both species (P=0.535 for 
Sk; P=0.163 for Ag, Table 3, Fig. 4c, d). In addition, there was no significant 
difference in P concentrations of the two positions for the Sk litter at any collection 
date (Fig. 4c, d). However, one-way ANOVA results showed that the P concentration 
of the Ag litter on the top was significantly higher than that of the Ag litter at the 
bottom collected at day 620, 706 and 911 (Fig. 4d). 
 9 
For both single decomposition and mixing treatments, there was a releasing phase 
for both N and P of the Ag litter. A leaching phase of N and P was found followed by 
immobilization and then a release phase for the Sk litter (Fig. 5a-d). When Sk 
decomposed with Ag, a stronger immobilization of N was found at both top and 
bottom positions (Fig. 5a, P=0.006). There was no significant difference in P release 
between the Sk litter decomposing singly and at the bottom position of the mixture, 
but the percent P remaining of the Sk litter at the upper position was significantly 
lower than the above two positions (Fig. 5b, P=0.002). For the Ag litter, the release of 
N was the fastest when Ag was placed above Sk. It became the slowest when Ag was 
placed below Sk (Fig. 5c, P<0.001). The release of P during the Ag litter 
decomposition showed similar pattern to that of N (Fig. 5d, P<0.001). 
Neither Sk-Ag nor Ag/Sk showed a significant mixing effect on N release (Table 
2, P>0.05). However, there was a negative effect of the Sk/Ag mixture on N release 
(Table 2, P<0.001), which was indicated by the higher observed percent N remaining 
value compared to the expected value over the whole decomposing period. Overall, N 
of the litter in the three mixtures was continuously released throughout the whole 
study period (Fig. 5e). All three mixtures had an overall significantly positive mixing 
effect on P release (Table 2). In addition, P was released throughout the study period 
with the exception of a large increase in the percent remaining after first year’s 
decomposition and a small increase for the last ~200 days of decomposition (Fig. 5f). 
 
Discussion 
Mass loss in the mixtures 
In natural terrestrial ecosystems, litter species in the litter layer usually decompose in 
mixtures rather than singly, and the decomposition of litter mixture may be either 
 10 
enhanced or retarded by the component litter species. Previous studies have indicated 
that there were mixing effects on the decomposition of different litter species in fen 
ecosystems (Hoorens et al. 2003), boreal forests (Nilsson et al. 1999), grasslands and 
agricultural systems (Wardle et al. 1997). In this study, consistent with our first 
hypothesis, there were different mixing effects for different mixing treatments. We 
found significant negative mixing effects on the mass loss of the Sk/Ag mixture, 
significant but weaker negative mixing effects on the mass loss of the Sk-Ag mixture, 
while no significant mixing effect on the mass loss of the Ag/Sk mixture (Table 2). 
We also found that the percent mass remaining was significantly different between the 
two mixing positions for both Sk and Ag after 162 days (Fig. 3). The ratios of percent 
mass remaining between the top and the bottom litter species, for both Sk and Ag, 
decreased exponentially with time but tended to level off later (Fig. 3 inset). The 
average difference of the eight collection dates showed that Sk in the Ag/Sk treatment 
was 5.4% higher in percent mass remaining than that of the single Sk, while Ag in the 
Ag/Sk treatment was 5.0% lower in percent mass remaining than that of the single Ag. 
This difference between the two component litter in the Ag/Sk treatment 
counterbalanced each other. However, Sk in the Sk/Ag treatment was 3.5% lower in 
percent mass remaining than the single Sk, while Ag in this mixture was 12.1% higher 
than the single Ag, resulting in a lower overall mass loss in the Sk/Ag mixture. 
Therefore, the difference in mixing effects on mass loss in different litter mixtures 
might be a result of the change in mixing positions. 
Studies have shown that litter quality and the environmental factors regulated 
mixing effects. Litter chemistry can influence the decomposition rates of the 
component litter species through the transfer of nutrients and secondary chemicals 
among different litter species. This movement of materials can simply be caused by 
 11 
leaching (Fyles and Fyles 1993; McArthur et al. 1994), and may also be mediated by 
the growth of fungi (McTiernan et al. 1997). Rainfall and soil moisture also play an 
important role in dryland decomposition (e.g., Wang et al. 2009; Yahdjian et al. 2006). 
The frequency of small rainfall events (less than 2 mm) accounted for more than 50% 
of the total rainfall in this semi-arid steppe area over the three years of observation, 
and may induce little effects on N leaching from the top litter to the bottom litter. 
Under this situation, the rain water might rarely reach the bottom litter species, and 
might hardly produce sufficient moisture conditions for the decomposition of the 
bottom litter species. Wardle et al. (2003) found that positive mixing effect was 
largely caused by low quality component litter due to its contribution to enhancing the 
moisture status in the litter layer. Their study results, supported by others, also 
suggested that the changes in environmental conditions affected the progress of 
decomposition and litter-mixing effects (Gartner and Cardon 2006; Jonsson and 
Wardle 2008; Madritch and Cardinale 2007). 
Mixing effect in this study could be affected by our specific litterbag design as 
the top and bottom layers of the two species were separated by a polyethylene barrier 
with 0.2 mm mesh size. This separation could lead to difference in moisture content 
of the top and the bottom litter as the upper litter might receive more water than the 
bottom litter when the amount of rainfall was small. At the same time, this was a full 
factorial experimental design, i.e., Ag/Sk treatment, Sk/Ag treatment, and thorough 
mixing (Sk-Ag). The same negative mixing effect was detected in both Sk/Ag and 
Sk-Ag treatments. Therefore, the effect of the experimental design on the mixing 
effects may not be significant. 
Our results showed that the litter placed on the top, which received more rainfall 
and solar radiation, decomposed much more quickly at the early period than the same 
 12 
litter species placed at the bottom. Berg and Laskowski (2006) also showed that early 
stage mass loss could be stimulated by climatic factors and the availability of nutrients 
such as N and P. Since there was no difference in chemical concentrations between the 
two positions at the beginning of the decomposition, we concluded that the 
environmental factors might induce significant differences in mass loss between the 
two positions since the very beginning of the decomposition. Besides, the overall 
higher decomposition rate of the component species on the top could be mainly due to 
the faster mass loss at the early stage, as the effect of position seemed to level off at 
the later stage (Fig. 3 inset). 
Nutrient dynamics during decomposition 
Studies have shown a typical triphasic pattern for nutrient release during litter 
decomposition. However, not all three phases will occur during decomposition. For 
example, only phase III will occur in the decomposition of litter species with high 
nutrient concentration (Berg and Laskowski 2006; Prescott 2005). Sometimes, only 
phase II and III occur during the decomposition of nutrient-poor litter species. 
The increase in N concentrations of the remaining litter during decomposition is 
common in most previous studies, and this has generally been interpreted as microbial 
immobilization. In this study, we also found that there was significant increase in N 
concentrations of both the Sk and the Ag litter (Fig. 4a,b). Fewer studies have shown 
that litter P concentrations also increase during litter decomposition as most studies 
only focused on N. Results from this study indicated that P concentrations of the Sk 
litter also increased throughout the decomposition (Fig. 4c). However, there was not 
much net immobilization as the remaining P did not exceed the initial litter P content 
(Fig. 5b). According to some of the previous studies, the increase in P concentrations 
and percent remaining have also been interpreted as microbial immobilization, 
 13 
especially when the availability of P is high due to fertilization (Liu et al. 2006; 
McGroddy et al. 2004) or high P content in the co-existing litter (e.g., Ag in this 
study). 
Our results showed that when Sk and Ag decomposed separately, the Ag litter 
generally released N and P over the three-year decomposition. The Sk litter, however, 
released N and P at the early stage of decomposition, followed by an immobilization 
period, and then released nutrients again (Fig. 5a-d). This generally agreed with our 
second hypothesis. Both litter species showed similar N and P release patterns 
regardless of their positions in the mixtures. When they were mixed, N was 
continuously released throughout the whole decomposition period regardless of the 
mixing treatments, which was similar to the pattern of individual Ag litter (Fig. 5c, e). 
The release pattern of P in the mixtures, on the other hand, was more similar to the 
pattern of individual Sk litter with short-term immobilization in the middle stage of 
decomposition (Fig. 5b, f). Therefore, the release of N from the mixtures during 
decomposition was primarily determined by the fast decomposing Ag litter while the 
release of P was largely determined by the slow decomposing Sk litter. In particular, 
when Ag litter was placed at the bottom of the litterbags, the decomposition of the 
Sk/Ag mixture was slowed down significantly, resulting in significant negative 
mixing effects on the N release. This also suggests that when the litter layer of a 
natural community has large amounts of Sk over Ag litter, high N retention will likely 
occur. 
Although P was immobilized in the individual Sk litter (Fig. 5c) and in the 
mixtures (Fig. 5f) at the middle stage of decomposition, the immobilization in the 
mixture was not as strong as in the Sk litter of the single decomposition treatment (Fig. 
5b). This indicates that the presence of the Ag litter during the Sk litter decomposition 
 14 
alleviated P limitation for microbial needs. Sk-Ag, Ag/Sk and Sk/Ag mixtures all had 
significant positive effects on P release (Table 2) although the mixing effects on mass 
loss were only found significantly negative for the Sk-Ag and the Sk/Ag mixtures, 
suggesting that the presence of Ag might stimulate P release of the Sk litter. This also 
partially confirmed our third hypothesis. Liu et al. (2006) found that the addition of P 
significantly accelerated the decomposition of Sk, indicating limitations of P in this 
area during litter decomposition. Therefore, mixing of different litter types may have 
different effects on the release of N and P. Litter mixing had different non-additive 
effects on N and P dynamics during litter decomposition, which was also found by 
Ball et al. (2009). This was possibly due to nutrient limitations or different capacities 
of N and P uptake by microbial communities (Hobbie and Vitousek 2000). 
In this typical semiarid steppe ecosystem, litter fall is a gradual process at the end 
of the growing season. Different litter species may be layered randomly either on the 
top or at the bottom position to form different types of litter mixtures, and this was 
generally neglected in most studies on the decomposition of litter mixtures. Gartner 
and Cardon (2004) proposed that the potential for interaction among different litter 
species may be determined by how leaves are mixed in litterbags (i.e., whether the 
leaves are layered in the order of the leaf fall or are thoroughly mixed). The results 
from this study clearly showed that there were different mixing effects with different 
mixing treatments. 
 
Conclusions 
Based on a three-year decomposition experiment, this study demonstrated that 
different mixing treatments of litter species had different mixing effects on the mass 
loss and the release of N. All three mixing treatments had positive effects on the 
 15 
release of P. We found that the release of N from the mixtures was primarily 
determined by the fast decomposing Ag litter while the release of P was largely 
determined by the slow decomposing Sk litter. The fast decomposition at the early 
stage more or less determines the overall rate of the mass loss. Based on these results, 
we think it is necessary to consider the litter placement in the litter layer when 
evaluating the decomposition of the mixtures and the cycling of nutrients. Our results 
also suggest that different mixing positions of litter species will result in different 
non-additive effects on the release of nutrients such as N and P, which may further 
increase the heterogeneity of the nutrient distribution pattern in the studied steppe 
ecosystem. 
 
Acknowledgements 
The authors would like to thank Ang Li and Jianyang Xia for their constructive 
comments on an earlier version of this manuscript. We are also extremely grateful for 
the two anonymous reviewers and the section editor Tim Moore for their constructive 
comments. This study was financially supported by the National Basic Research 
Program of China (2009CB421102) and the National Natural Science Foundation of 
China (41073056). We also thank the Duolun Restoration Ecology Research Station 
for permission to access the study site and for technical assistance. 
 
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Table 1 Initial litter chemistry for the two grassland plant species, Stipa krylovii (Sk) 
and Astragalus galactites (Ag), data are means ± SE (n=5). Different superscript 
letters in a column indicate significant difference between the two species. 
 
Species N (mg/g) P (mg/g) C (mg/g) N/P C/N C/P 
Sk 5.54 ± 0.29b 0.96 ± 0.03b 407.46 ± 6.23b 5.78 ± 0.28b 74.40 ± 4.42a 425.75 ±16.12a 
Ag 21.44 ±0.19a 1.59 ± 0.01a 438.19 ± 5.80a 13.46 ± 0.07a 20.44 ± 0.21b 275.20 ± 3.73b 
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Table 2 Summary of the repeated measures ANOVA results for the difference 
between the expected and observed values of percent mass remaining (%), percent N 
remaining (%) and percent P remaining (%) in the three mixtures. Sk, Stipa krylovii; 
Ag, Astragalus galactites. 
 
     Sk-Ag    Ag/Sk    Sk/Ag 
  F P F P F P 
Percent Mass remaining (%) 
 
 
Mixing (M) 6.8 0.031 0.1 0.739 76.6 <0.001 
Day (D) 1009.0 <0.001 867.2 <0.001 756.7 <0.001 
M×D 0.3 0.935 1.5 0.234 2.6 0.027 
Percent N remaining (%) 
 
 
Mixing (M) 0.0 0.992 0.3 0.576 78.8 <0.001 
Day (D) 38.9 <0.001 125.3 <0.001 98.6 <0.001 
M×D 0.5 0.666 0.8 0.598 1.9 0.090 
Percent P remaining (%) 
 
 
Mixing (M) 17.9 0.003 8.4 0.020 7.0 0.030 
Day (D) 34.5 <0.001 39.2 <0.001 38.8 <0.001 
M×D 6.7 <0.001 2.2 0.044 2.8 0.025 
Notes: Sk-Ag means a thorough mixing, Ag/Sk means Ag on the top, Sk/Ag means Sk on the 
top.
 23 
Table 3 Summary of the repeated measure ANOVA results for the difference between 
N and P concentration of the same species under different positions during 
decomposition. Sk, Stipa krylovii; Ag, Astragalus galactites. 
Species 
 N (mg/g) P (mg/g) 
 F P F P 
Ag 
 
 
Position (P) 137.217 <0.001 2.359 0.163 
Day (D) 34.581 <0.001 5.035 0.003 
P×D 2.55 0.042 0.543 0.706 
Sk 
 
 
Position (P) 10.705 0.011 0.42 0.535 
Day (D) 35.658 <0.001 23.087 <0.001 
P×D 0.99 0.448 0.541 0.787 
 24 
Figure legends 
Fig. 1 The frequency distribution of the daily precipitation amount and the monthly 
precipitation distribution (inset) during the three years (2007-2009). 
Fig. 2 Percent mass remaining (% of initial litter mass) in decomposition of Sk (Stipa 
krylovii) and Ag (Astragalus galactites) in single decomposition and mixtures during 
decomposition. a, decomposing singly; b, decomposing in mixtures 
Fig. 3 Percent mass remaining of the component species in the two mixing treatments. 
a, Sk from Sk/Ag (means Sk on the top) and Ag/Sk (means Ag on the top), 
respectively; b, Ag from Ag/Sk and Sk/Ag, respectively. Inset figure in each panel 
indicates the regressions between time and the ratios of the percent mass remaining of 
the top and bottom treatments. All the data are mean ± 1 SE, n =5. Sk, Stipa krylovii; 
Ag, Astragalus galactites. 
Fig. 4 The N and P concentration of the component species Sk and Ag in the litter 
mixture across the decomposition period (mg.g-1), a, N concentration of Sk; b, N 
concentration of Ag; c, P concentration of Sk; d, P concentration of Ag. Data are 
means ± 1 SE, n =5. * = P<0.05; ** = P<0.01; *** = P<0.001. Sk, Stipa krylovii; Ag, 
Astragalus galactites. 
Fig. 5 The nutrient dynamics (% of initial values) in the Sk and the Ag single 
decomposition and the three mixing treatments. a, b, Percent N and P remaining of 
the Sk litter decomposing in single decomposition and in mixtures with Ag; c, d, 
Percent N and P remaining of the Ag litter decomposing in single decomposition and 
mixtures with Sk. e, f, Percent N and P remaining of the observed and the expected 
values of the mixing litter. Values are mean ± 1 SE, n =5. Sk, Stipa krylovii; Ag, 
Astragalus galactites. 
 25 
Fig. 1 
 
 26 
Fig. 2 
 
 27 
Fig. 3 
 
 
 28 
Fig. 4 
 
 29 
Fig. 5 1 
 2 
 3 
 30