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Soil Biology & Biochemistry 73 (2014) 115e121

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Admixture of alder (Alnus formosana) litter can improve thedecomposition of eucalyptus (Eucalyptus grandis) litter

Fuzhong Wu a,c, Changhui Peng b,c, Wanqin Yang a,*, Jian Zhang a, Yu Han a, Tao Mao a

aKey Laboratory of Ecological Forestry Engineering, Institute of Ecology & Forestry, Sichuan Agricultural University, Chengdu 611130, Chinab Laboratory for Ecological Forecasting and Global Change, College of Forestry, Northwest A & F University, Yangling, Shaanxi 712100, ChinacDepartment of Biology Sciences, Institute of Environment Sciences, University of Quebec at Montreal, C.P. 8888, Succ. Centre-Ville, Montreal H3C 3P8,Canada

a r t i c l e i n f o

Article history:Received 7 October 2013Received in revised form18 February 2014Accepted 21 February 2014Available online 11 March 2014

Keywords:Alder litterDecomposition stageEucalyptus plantationLitter mixture decompositionMicrobial biomassNon-additive effects

* Corresponding author. Tel./fax: þ86 28 86290957E-mail address: scyangwq@163.com (W. Yang).

http://dx.doi.org/10.1016/j.soilbio.2014.02.0180038-0717/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The slow nutrient turnover of eucalyptus (Eucalyptus grandis) plantations has been well documented. Toexamine whether the admixture of alder (Alnus formosana) litter could improve the decomposition ofeucalyptus litter, a field litterbag experiment was conducted on a new eucalyptus plantation in south-western China. We investigated the mass loss rate from an alder and eucalyptus foliar litter mixtureevery half month from May 1st to October 1st, 2009. Five mixture proportions were examined: pureeucalyptus litter (10E), 70% eucalyptus litter mixed with 30% alder litter (7E:3A), 50% eucalyptus littermixed with 50% alder litter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter (3E:7A) and purealder litter (10A). Over 169 days of decomposition, approximately 79.22%, 70.23%, 62.82%, 49.95% and48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10E litter mixtures, respectively.Compared with pure eucalyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased 63.04%,44.54%, 29.29% and 2.80% of accumulated mass loss. The admixture of alder litter can significantlyimprove eucalyptus litter decomposition, and a small proportion of eucalyptus litter (3E:7A) may alsopromote alder litter decomposition. As the decomposition proceeded, the litter mixture displayed exactlyadditive effects in the initial stage and positive non-additive effects in the middle stage. However,negative non-additive effects were detected in the 7E:3A litter mixture in the later stage, althoughpositive non-additive effects were maintained throughout decomposition in the 5E:5A and 3E:7A mix-tures. Compared to pure eucalyptus litter, mixtures containing alder litter presented increased microbialbiomass carbon and bacterial DGGE (Denaturing Gradient Gel Electrophoresis) bands, but the littermixture decomposition relied more on microbial biomass than on microbial diversity. The results implythat alder litter can improve material cycling on eucalyptus plantations and that alder could be a po-tential species for mixed planting with eucalyptus.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The development of eucalyptus (normally Eucalyptus grandis)plantations has been increasing in the global commercial timberindustry (Forrester et al., 2006, 2013; Zhang et al., 2010; Leslieet al., 2012). Because of the plant’s short rotation (fast growth)and high consumption of water and soil nutrients, nutrient cyclingis one of the limitations to establishing sustainable eucalyptusplantation ecosystems (Lemma et al., 2007). Unfortunately, a thickleaf litter layer often accumulates on the floor of eucalyptusplantations, indicating a slow litter decomposition rate because of

.

low litter quality (Guo and Sims, 2001; Forrester et al., 2006).Several previous studies have reported that mixed-species plan-tations of eucalyptus with a dinitrogen (N2) fixation species havethe potential to increase productivity while maintaining soilfertility, enhancing soil organic carbon sequestration and acceler-ating nutrient cycling (Forrester et al., 2006, 2013; le Maire et al.,2013). It is therefore important to select N2 fixation species withreadily decomposable litter and high rates of nutrient cycling.However, both synergistic and antagonistic interactions (reviewfrom Gartner and Cardon, 2004) and even non-significant effects(Perez-Harguindeguy et al., 2008) have been observed in littermixture decomposition.

Climate, litter quality and the decomposer community areknown to be the main controllers of organic matter decomposition

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121116

(Couteaux et al., 1995). The non-additive effects on the decom-position of litter mixtures compared to that of monoculture littercan be mainly attributed to the changes of the chemical environ-ment and the physical alteration of the total litter surface wheredecomposition occurs (Hansen and Coleman, 1998; Kaneko andSalamanca, 1999; Hector et al., 2000). In general, higher-qualitylitter can stimulate decomposition in adjacent, more recalcitrantlitters, and conversely, leaf litter decomposition can be slowed byan admixture of lower-quality litter (Fyles and Fyles, 1993;McTiernan et al., 1997; Salamanca et al., 1998). The transfer ofnitrogen between the litters has been documented as a keymechanism in the interaction between decomposing litters(Berglund and Ågren, 2012; Berglund et al., 2013). Moreover, anincrease in microhabitats may also be correlated with increasedmass loss because of the creation of a more diverse and abundantdecomposer community (Hansen and Coleman, 1998; Gartner andCardon, 2004). Thus, chemical and physical changes in the leafmixture can influence decomposition rates both directly (physi-cally) and indirectly (through the decomposer community and itsactivities).

However, this superior chemical and physical diversity disap-pears as decomposition proceeds because the liable componentsare lost and the litter shape is destroyed after early rapid decom-position (Berg andMcClaugherty, 2008). The remaining substrate isrich in water-soluble defenses or inhibitory compounds (such aslignin and tannin), leading to co-limits with each other in the littermixture (Ostrofsky, 2007). Therefore, trends in the rates ofdecomposition and nutrient loss from mixtures of litter fromdifferent species are complex and inconsistent when compared tothose of monoculture litter. Moreover, litter in mixtures with N2fixation species does not necessarily decay faster than monoculturelitters of non-N2 fixation species (Rothe and Binkley, 2001; Binkleyet al., 2003). As a result, additional research is required on thedecomposition of litter mixtures of eucalyptus and N2 fixationspecies with the goal of providing effective information for mixed-species plantations of eucalyptus.

Alder (Alnus spp.) is an N2 fixation tree with a wide distributionfrom the boreal zone to the subtropical zone, and its leaf litter hasbeen well documented as possessing desirable decompositioncharacteristics (Chapman et al., 1988; Gartner and Cardon, 2004).The available information indicates that the admixture of alderlitter can improve the decomposition of other litter, such as that ofPopulus tremuloides (Taylor et al., 1989) and Pseudotsuga menziesii(Fyles and Fyles, 1993). Alder’s ability to improve the nutrientcycling of litter mixture decomposition makes it a candidate treespecies for mixed planting with eucalyptus, but little informationon this particular combination is available. Therefore, it is hy-pothesized that the admixture of alder litter can improve thedecomposition of eucalyptus litter, which is potentially beneficialfor eucalyptus plantations.

To test this hypothesis, a field litterbag experiment was con-ducted on a new eucalyptus plantation in southwestern China,where eucalyptus plantations cover more than 200 000 ha (Zhanget al., 2010, 2012). We measured the accumulated mass loss fromalder (Alnus formosana) and eucalyptus (E. grandis) foliar littermixtures every half month. Five mixed proportions were exam-ined: pure eucalyptus litter (10E), 70% eucalyptus litter mixed with30% alder litter (7E:3A), 50% eucalyptus litter mixed with 50% alderlitter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter(3E:7A) and pure alder litter (10A). The objectives were (1) todetermine whether the admixture of alder litter could improveeucalyptus litter decomposition and (2) to identify the aspects ofthe litter mixture decomposition. The results will be useful indetermining the practicality of alder as a candidate species formixed planting with eucalyptus.

2. Materials and methods

2.1. Study site

The studywas conducted in the Leshan region (E103�360, N29�370,413 m a.s.l) in western Sichuan Province, southwestern China. Theclimate is subtropical, with an annual mean temperature of 18.0 �Cand precipitation of 1137 mm. From a local weather station near thesample site, the monthly average air temperature was higher than20 �C fromMay to October 2009, with the highest average of 28.2 �Cin August. The majority of precipitation falls between June andAugust, whereas only 48.1mmand 74.3mm rainfall occurred inMayand October 2009, respectively. The soil is classified as ferralsol (SoilCensus Office, 1993), is derived from Pleistocene alluvium and has ayellow color, loamy texture and granular structure.

To avoid topological heterogeneity and the various effects of treeshadow, three new eucalyptus plantations of at least a 50 m� 50 mplot were established in the study site. The plantations wereplanted with E. grandis at a density of 2.5 m � 2.5 m betweenseedlings in March 2009. The soil had a pH of 4.95 and contained30.21 g kg�1 of soil organic carbon, 0.73 g kg�1 of soil nitrogen and0.67 g kg�1 of soil phosphorus.

2.2. Experimental design

Litter decomposition was studied using the common litterbagprocedure. In March 2009, fresh foliar litter of alder and eucalyptuswas collected from the floor of each pure plantation close to thesample plots. To avoid litter structure damage during oven drying,the fresh litter was air-dried for more than two weeks at roomtemperature. Five samples with approximately 20 g of air-driedlitter were oven-dried at 70 �C to determine the moisture content,which was 9.65 � 0.04% and 9.03 � 0.02% for air-dried alder litterandeucalyptus litter, respectively. A 20g sample (basedon theoven-dried mass) of the air-dried alder and eucalyptus litter mixture wasthen placed in a 20 cm � 20 cm nylon bag of a 1 mmmesh, and theedges of the bag were sealed. The following five mixtures wereplaced in the litterbags: 20 g pure eucalyptus litter (10E), 14 geucalyptus litter and 6 g alder litter (7E:3A), 10 g eucalyptus litterand 10 g alder litter (5E:5A), 6 g eucalyptus litter and 14 g alder litter(3E:7A), or 20 g pure alder litter (10A). The initial alder litter con-tained 437.09 � 12.51 g kg�1 organic carbon, 8.73 � 0.17 g kg�1 ni-trogen and 1.55 � 0.01 g kg�1 phosphorus; the initial eucalyptuslitter contained 513.74 � 7.57 g kg�1 organic carbon,8.59 � 0.02 g kg�1 nitrogen and 0.78 � 0.01 g kg�1 phosphorus.

The experiment began on April 15th, 2009. A total of 825 lit-terbags (five proportions � eleven sampling times � fivereplicates � three sampling plots) were placed on the floor of thethree sampled plots. To check the litter mixture decompositionprocesses, we sampled the litterbags every half month from May1st through the end of the growing season on October 1st, 2009.After four months of decomposition, the remaining litter in severallitterbags was insufficient to conduct the mass loss and microbialanalyses. We combined five litterbags into three on September 15thand October 1st, 2009. The retrieved litter was separated into twoparts after being completely mixed in each litterbag. One part wasstored in a refrigerator at 4 �C and prepared for the microbialbiomass and bacterial DGGE (Denaturing Gradient Gel Electro-phoresis) analyses, whereas the remaining part was oven-dried at70 �C for 48 h to determine the dry mass.

2.3. Microbial biomass analysis

The microbial biomass carbon (MBC) in the litter was deter-mined according to the differences between unfumigated and

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121 117

fumigated samples following extractionwith 0.5 mol L�1 K2SO4. Anefficiency factor (Kc ¼ 0.38) was used to correct for the incompleteextractability of the samples (Vance et al., 1987).

2.4. DNA extraction

Bacterial DNA was extracted from the litter samples using themethod described by Griffiths et al. (2000) in which litter sampleswere extracted with 0.5 g of glass beads (0.1 mm), 500 ml CTABextraction buffer, and 500 ml phenol-chloroform isoamyl alcohol(25:24:1) (pH 8.0) by beadbeating at full speed for 30 s with Mini-Beadbeater (Biospec Products, Bartlesville, OK, USA). Separation ofthe phases and precipitation of the nucleic acids was performed,and the separation was then treated using RNAase (Takara) ac-cording to the manufacturer’s instructions.

2.5. PCR-DGGE

The variable V3 region of the 16S rRNA gene sequence wasamplified by PCR (polymerase chain reaction) using the universalprimers 341f and 534r and the touchdown protocol (Muyzer et al.,1993). The extracted DNAwas amplified with a PCR mixture (50 ml)containing 37.55 ml of sterilizedMilli-Qwater, 5 ml of Mg-containingbuffer, 4 ml of deoxynucleoside triphosphate mixture, 1 ml of eachprimer (20 mM), 1 ml of the DNA solution, and 0.45 ml of HotStartVersion ExTaq DNA polymerase (Takara). PCR was performed with aBio-Rad iCycler thermocycler using the following protocol: 1 min at94 �C (denaturation), 1 min at 65 �C (annealing), and 3 min at 72 �C(elongation), with a 1 �C decrease in the annealing temperatureevery second cycle as a “touchdown” for 20 cycles, followed by 10cycles at an annealing temperature of 55 �C and a final cycle con-sisting of 10 min at 72 �C. After gel electrophoresis (1.5% [wt/vol]agarose gel) of 4 ml subsamples from the PCR product, the amountof amplified DNAwas quantified by comparing the band intensitiesto the standard curves obtained with a Low DNA Mass Ladder(Takara). The band intensities were measured with Quantity Oneanalysis software (Bio-Rad Laboratories, Hercules, CA).

Profiles of the amplified 16S rRNA gene sequences were pro-duced by DGGE using the CBS DGGE system (CBS Scientific, USA)(Muyzer et al., 1993). The PCR products were loaded onto a poly-acrylamide gel (8% [wt/vol] acrylamide in 1� TAE buffer with a 45e65% denaturant gradient (100% denaturant was 7 M urea and 40%[vol/vol] deionized formamide)). The wells were loaded with 25 mlof PCR product, and electrophoresis was conducted in the TAEbuffer at 100 V for 17 h at 60 �C. The DNA fragments were stained bysilver staining as described by Radojkovic and Ku�sic (2000). The gelwas destained in distilled water for 5 min. Images of the gels wereobtained after destaining using a Bio-Rad GS-800 CalibratedDensitometer (Bio-Rad Laboratories, Hercules, CA). The band pat-terns were analyzed using Quantity One software (Bio-Rad Labo-ratories, Hercules, CA) (Zhang and Jackson, 2008).

Each detected bandwas defined as an operational taxonomic unit(OTU), and the number of bands was defined as the genotypic rich-ness of each sample (Bell et al., 2005). The pixel intensity for eachband was detected by Quantity One software and was expressed asthe relative abundance (Pi) (Reche et al., 2005). The richness index(ShannoneWiener index, H0) (Trevors et al., 2010) was calculatedusing the relative abundance data based on the following equation:

H ¼ �Xs

i¼1

pi ln pi

where Pi ¼ ni/S, ni is the abundance of the ith OTU and S is the totalabundance of all OTUs in the sample.

2.6. Calculations

The accumulated mass loss (Ri) during litter decompositionbefore each stage were calculated using the following equation:

Rið%Þ ¼ 100� ðM0 �MiÞ=M0

where M0 is the dry mass of the initial litter and Mi is the dry massof the remaining litter in the bag at each stage after sampling.

The theoretical mass loss rates of the litter mixtures werecalculated using the following formula (Hoorens et al., 2003):

Theoretical mass loss rateð%Þ ¼ Ra � Pa þ Re � Pe;

where Ra and Re are the accumulated mass loss of pure alder andeucalyptus litter at each stage, respectively, and Pa and Pe are theinitial proportions of alder and eucalyptus litter in the mixture,respectively.

2.7. Statistical analysis

All of the variables, including themeasurements and calculationsamong the different littermixtures,were tested byone-wayanalysisof variance (ANOVA). When significant differences were detected,the LSD multiple range test was used to determine where the dif-ferences existed. Differenceswere considered significant at P< 0.05.Linear regressions were used to examine the relationships betweenthe accumulated mass loss and the microbial biomass and bacterialDGGE bands. All of the statistical analyseswere performed using theSPSS (Statistical Package for the Social Sciences) software package(Standard release version 16.0 for Windows, SPSS Inc., IL, USA).

3. Results

3.1. Mass loss

Over 169 days of decomposition, the accumulated mass loss wassignificantly different in the pattern 3E:7A > 10A > 5E:5A> 7E:3A > 10E. Approximately 79.22%, 70.23%, 62.82%, 49.95% and48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10Elitter mixtures, respectively (Fig. 1). Compared with pure euca-lyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased63.04%, 44.54%, 29.29% and 2.80% of accumulated mass loss. Fewdifferences in the accumulated mass loss were observed betweenthe different litter mixtures in the half month of decompositionbefore May 1st, and the 3E:7A litter mixture had a higher accu-mulated mass loss only in the last two months of the study (afterAugust 1st). The accumulated mass loss of pure eucalyptus litter(10E) was significantly lower than those of the other litter mixturesafter May 15th, but few differences were detected between theaccumulated mass loss of the 10E and 7E:3A litter mixtures afterSeptember 1st.

Additionally, the observed accumulated mass loss was close tothe theoretical values for each litter mixture on May 1st, but asignificant non-additive effect was detected over the course of thelitter mixture decomposition after May 1st. Although the observedaccumulated mass loss in the 7E:3A litter mixture after September1st was slightly lower than their theoretical values, the observedaccumulated mass loss in the other litter mixtures was higher thantheir theoretical values (Fig. 2).

3.2. Microbial biomass

Microbial biomass carbon (MBC) first increased and thendecreased as the litter mixture decomposition proceeded (Fig. 3),

a aa

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s lo

ss (

%)

10E 7E:3A 5E:5A 3E:7A 10A

Fig. 1. The accumulated mass loss in the litterbags during the decomposition of the E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3) and the different lettersdenote significant differences (P < 0.05) among different litter mixtures.

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121118

and its peak value did not appear at the same time for each littermixture. The highest MBC values during the decomposition of the10A and 7E:3A litter mixtures were observed on August 15th,whereas the highest values for the other mixtures were observedon September 15th. Few differences were observed among theMBCvalues of the different litter mixtures during the first two months(until June 1st) of decomposition. After this point, the MBC valuesof the pure alder litter (10A) decompositionwere higher than thoseof the other litter mixtures from June 1st to August 15th, and thevalues of the 3E:7A litter mixture were higher after September 1st.Conversely, the MBC values for the pure eucalyptus litter (10E)decomposition were the lowest among the other litter mixturesafter July 1st.

3.3. Bacterial community from PCR-DGGE

The PCR-DGGE bacterial analysis revealed that there were sig-nificant variations between the DGGE bands of the different littersamples during decomposition (Fig. 4). The DGGE bands showed anincrease followed by a decreasing tendency as the litter mixturedecomposition proceeded, with the exception of the 5A:5E littermixture, which showed the opposite tendency (Table 1). In com-parison with the DGGE bands for the pure eucalyptus litterdecomposition, the addition of alder litter increased the number ofDGGE bands, and the most DGGE bands were detected for the

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%)

Observed Theoretical

Fig. 2. The observed and theoretical values of the accumulated mass loss during the de

3E:7A litter mixture. The ShannoneWiener indices showed similarpatterns as the changes in DGGE bands.

4. Discussion

The hypothesis that the admixture of alder litter can improveeucalyptus litter decomposition was confirmed by this study. Boththe present study and previous results have demonstrated thatmixing eucalyptus litter with more readily decomposable litter canenhance the decomposition of eucalyptus litter (Briones andIneson, 1996). From a quantitative perspective, this study hypoth-esized that the litter mixture would decompose more slowly as theamount of slowly decomposing eucalyptus litter increases(Ostrofsky, 2007; Pérez-Suárez et al., 2012). Interestingly, our re-sults indicated that the presence of a certain amount of eucalyptuslitter actually enhanced the mass loss rate of the litter mixture(3E:7A) over that of the pure alder litter. Furthermore, a significantnon-additive effect was observed in this study, as the mass lossrates of the litter mixtures were higher than their theoretical valuesat most of the stages of decomposition. This result may beexplained as a synergistic effect (Gartner and Cardon, 2004) be-tween alder litter and eucalyptus litter, confirming that these twospecies facilitate each other’s litter decomposition.

Many previous studies have reported that the admixture ofhigher-quality litter can promote the decomposition of more

7E:3

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composition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).

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ry m

ass-1

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10E 7E:3A 5E:5A 3E:7A 10A

Fig. 3. The microbial biomass carbon (MBC) during the decomposition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).

Fig. 4. The silver-stained bands obtained by DGGE of bacteria during the decomposition of E. grandis and A. formosana litter mixtures.

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121 119

Table 1ShannoneWiener index (H) and the abundance of DGGE bands (S) during the decomposition of E. grandis and A. formosana litter mixtures.

Treatments Diversityindex

Sampling time

1-May 15-May 1-Jun 15-Jun 1-Jul 15-Jul 1-Aug 15-Aug 1-Sep 15-Sep 1-Oct

10E H 2.5875 2.7474 2.9745 3.0219 2.9705 3.0139 2.9245 2.9751 2.9759 2.9047 3.1636S 14 16 20 21 20 21 19 20 20 19 24

7E:3A H 2.4814 3.3088 3.1609 2.8392 3.6048 3.1887 3.2987 3.6621 3.4755 3.4436 3.4215S 12 28 24 18 38 25 28 40 33 32 33

5E:5A H 3.4453 3.5300 2.7516 2.7578 2.9809 3.1128 3.3390 3.3831 3.1169 3.2798 3.2387S 32 35 27 16 20 23 29 30 23 27 26

3E:7A H 3.4504 3.4821 3.4074 3.5550 3.4954 3.8154 3.8637 3.6211 3.5638 3.6503 3.3709S 33 34 31 36 34 47 50 39 37 40 31

10A H 3.4170 3.3547 3.4237 3.4552 3.5232 3.4300 3.5377 3.6465 3.5726 3.5455 3.5716S 31 29 31 32 34 31 35 40 37 35 33

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121120

recalcitrant litters, or vice versa (Fyles and Fyles, 1993; Briones andIneson, 1996; McTiernan et al., 1997), which is partly consistentwith the present study’s finding that an admixture of alder litteraccelerated the decomposition of eucalyptus litter in the presentstudy, but the addition of eucalyptus litter also stimulated alderlitter decomposition when the mixture proportion is considered.This result coincided with the mechanisms proposed by Ostrofsky(Ostrofsky, 2007), who declared that four principles control thedecomposition of mixed litter: (1) the presence of rapidly decom-posing leaves that favor the rapid colonization of decomposercommunities, which accelerate the mass loss of slowly processedrecalcitrant leaves; (2) the co-occurring reductions of the mass lossrates of rapidly decomposing leaves and slowly decomposingleaves caused by high concentrations of water-soluble defensive orinhibitory compounds (e.g., tannins and other phenolics); (3) amore persistent habitat for decomposing organisms provided bythe structural stability of the litter mixture beds to accelerate thedecomposition of slowly decomposing leaves by protecting theabrasion and leaching processes; (4) both facilitative and inhibitorymechanisms that occur simultaneously, as none of these effects aremutually exclusive, so that the observed accumulated mass losschanges in litter mixture decomposition represent the net effect ofmultiple processes (Ostrofsky, 2007; Pérez-Suárez et al., 2012).

However, the different litter mixtures did not follow the samepattern over the course of decomposition. In the initial decompo-sition stage, the litter mixture mass loss rate equaled the sum of thealder and eucalyptus litter mass loss rates alone, displaying acompletely additive effect, shown in Fig. 2. The initially intactshapes of both types of litter may limit their complete mixture,leading to few changes in nutrient transfer and decomposer habitat

(a)

R2 = 0.8013P =0.0000

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Accumulated mass loss (%)

MB

C (

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ass-1

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DG

GE

ban

d nu

mbe

r

Fig. 5. The linear relationships between the accumulated mass loss and microbial biomassbacteria (b).

(Hansen and Coleman, 1998; Hector et al., 2000; Berglund et al.,2013). We also observed that although the number of bacterialDGGE bands was higher in the litter mixtures (5E:5A and 3E:7Abefore May 15th) than in the pure alder and eucalyptus litters(Table 1), microbial biomass carbon did not significantly change(Fig. 3). As decomposition proceeded, increasing decomposercommunication and physical changes in the litter mixture structurecontributed to the mass loss of the litter mixture (Hansen andColeman, 1998; Hector et al., 2000), leading to the positive non-additive effects (obvious synergistic effects) observed in all of thelitter mixtures (Fig. 2) at this stage. However, after the rapid loss oflabile components, litter mixture decomposition can be slowed bythe decomposition of the remaining inhibitory compounds, such asphenolics and tannins (Fyles and Fyles, 1993; Salamanca et al.,1998). Less decomposable litter and more inhibitory compoundsremain in the litter mixture because of the reduction in labilecomponents. This observation is consistent with the 7E:3A littermixture, which displayed negative non-additive effects (antago-nistic interactions) after September 1st. The continuous synergisticeffects in the 5E:5A and 3E:7A litter mixtures may be explained bythe presence of relatively more labile components, which canmaintain the positive interactions for a longer time. These types oflitter mixtures may also exhibit antagonistic interactions in thelater decomposition stages. Therefore, our present results suggestthat the inconsistencies in previous observations (both negativeand positive non-additive effects that were reported in the reviewof Gartner and Cardon, 2004) of litter mixture decompositionsmight be attributed to the differences in the decomposition stagesand mixture proportions. Additional studies and longer decompo-sition experiments should be conducted to resolve these issues.

(b)

R2 = 0.2710P =0.007

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0 20 40 60 80 100Accumulated mass loss (%)

carbon (MBC) (a), and between the accumulated mass loss and DGGE band number of

F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121 121

Additionally, one of the essential promoters of litter mixturedecomposition is the increase of decomposer diversity because ofthe alteration of substrate diversity and microhabitat complexity(Kaneko and Salamanca, 1999; Wardle et al., 2006). In the currentstudy, we observed that the alder and eucalyptus mixture littershowed a higher number of bacterial DGGE bands as decomposi-tion proceeded compared to pure eucalyptus litter. However, ahigher number of bacterial DGGE bands were also detected in the3E:7A litter mixture than in the pure alder litter at most of thedecomposition stages, indicating obvious synergistic effects. Thisresult is in accordance with the conclusion of Gartner and Cardon(2004), who claimed that the interactions of litter from differentspecies in an ecosystem influenced the structure and activity of thedecomposer community, which is largely responsible for decom-position. In comparison with bacterial biodiversity, the microbialbiomass was more statistically correlated to the litter mixture massloss (Fig. 5). Microbial biomass refers to the biomass composed ofthe microorganisms on the suitable substrate, which representsbiological activity. In an incubation study, Thiessen et al. (2013) alsodocumented that organic matter decomposition mainly dependedon microbial biomass.

In conclusion, the admixture of alder litter can stimulate euca-lyptus litter decomposition, and a small amount of eucalyptus littermay also promote alder litter decomposition. Over the totalinvestigation period, the decomposition of each litter mixture dis-played positive non-additive effects. However, the effects differedover the decomposition process: exactly additive effects wereobserved in the initial stage, positive non-additive effects wereobserved in the middle stage, and negative non-additive effectswere observed in the later stage and would remain in progress asthe decomposition proceeded. Furthermore, litter mixturedecomposition relied more on microbial biomass than microbialdiversity. The results indicate that alder could be a potentialcandidate for mixed-species eucalyptus plantations because of itssynergistic effects on material cycling.

Acknowledgments

The authors are very grateful to their colleagues from EcologicalModelling and Carbon Science (ECO-MCS), Institute of EnvironmentSciences, University of Quebec at Montreal (UQAM) for providinghelpful suggestions in manuscript preparation. This research wasfinancially supported by the National Natural Science Foundation ofChina (31170423 & 31270498), the National Key Technologies R&Dof China (2011BAC09B05), and the Sichuan Youth Sci-tech Foun-dation (2012JQ0008 & 2012JQ0059).

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