# In situ and laboratory non-additive litter mixture effect on C dynamics of Sphagnum rubellum and Molinia caerulea litters

Sébastien Gogo, Fatima Laggoun-Défarge, Fatima Merzouki, Stéphane

Mounier, Audrey Guirimand-Dufour, Nevila Jozja, Arnaud Huguet, Frédéric

Delarue, Christian Défarge

► To cite this version:

Sébastien Gogo, Fatima Laggoun-Défarge, Fatima Merzouki, Stéphane Mounier, Audrey Guirimand-Dufour, et al.. In situ and laboratory non-additive litter mixture effect on C dynamics of Sphagnum rubellum and Molinia caerulea litters. Journal of Soils and Sediments, 2016, 16 (1), pp.13-27. 10.1007/s11368-015-1178-3. insu-01164205

HAL Id: insu-01164205

https://insu.hal.science/insu-01164205

Submitted on 16 Jun 2015

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
3 *In situ* and laboratory non-additive litter mixture effect on C dynamics of *Sphagnum rubellum* and *Molinia caerulea* litters
4
26

*Purpose*. The accumulation of carbon in peatlands originates from the slow rate of *Sphagnum* litter decomposition. Vegetation shifts can alter the *Sphagnum* decomposition rate through a litter mixture effect. This is rarely studied in peatlands. In a site colonized by vascular plants, we examined the effect of mixing litters of *Sphagnum* species with those of *Molina caerulea* and *Betula* spp on litter C dynamics. We tested that water content and pH may explain the potential non-additive litter mixing effect.

27

28

29

30

31

32

*Materials and methods*. Litter bags with *S. cuspidatum* or *S. rubellum* and *M. caerulea* or *Betula* spp were placed in situ, in a *Sphagnum* decomposing environment and retrieved after one year of incubation. In the laboratory, the specific interaction between *S. rubellum* and *M. caerulea* was investigated. Solid, soluble and gaseous forms of C were studied in addition to the fluorescence of the dissolved organic matter (FDOM).

33

34

35

36

37

*Results and discussion*. Laboratory and field experiments showed that there is a non-additive effect of mixing *S. rubellum* and *M. caerulea* litter on C dynamics. The analyses of FDOM suggested a relatively higher living biomass in the laboratory measured mixture than in the expected one. The in situ *S. rubellum* moist environment could stimulate the decomposition of *M. caerulea* that experiences much drier conditions in its native environment. In the laboratory experiment, *M. caerulea* were kept moist, and no significant difference in water content between the measured and expected mixture was found. Also, pH decreased in the measured mixture, ruling out any direct effect on microbial activity.

38

39

40

41

42

43

44

45

*Conclusions*. The non-additive mixture effect observed in the laboratory may be triggered by an increase of the microbial biomass. This increase was not explained by direct moisture or a pH effect. The suggested hypothesis that a lower pH could affect the availability of labile organic substrate through increased OM hydrolysis and thus stimulate microbial growth has to be further studied.

46

47

48

49

50

**Keywords** Aromaticity • Fluorescence • Litter-mixture effect • Peatland invading species • Water extractable organic matter

51

52

53

54
# 1 Introduction

Temperature and water regime perturbations are provoking shifts in the limits of plant distribution and changes in plant abundances (Weltzin et al. 2003; IPCC 2007; Limpens et al. 2008). These changes will modify the functioning of ecosystems through the occurrences of new interactions (as some plant distribution limits move northward), or the strengthening of previously weak interactions (increased abundances of previously sparse species; Cornelissen et al. 2001). Among ecosystem processes, litter decomposition will be affected by vegetation changes especially because of litter mixture effects (Gartner and Cardon 2004). Litter decomposition is an important component in the functioning of ecosystems as it influences the carbon (C) and nutrient cycles (Berg and McClaugherty 2003). At the local scale, litter decomposition influences primary productivity through the mineralization of nutrients, and at the global scale, it affects the C cycle through soil organic matter (SOM) stabilization and accumulation (Limpens et al. 2008; Bardgett and Wardle 2010). When a vegetation change occurs, new litter mixture effects are expected to occur. These effects can be detected by comparing the measured values of two or more litters in mixture to theoretical values calculated from the same litters incubated in monoculture (Gartner and Cardon 2004). An additive effect is identified when there is no difference between the measured and expected decomposition rate. When a difference is observed, the litter mixture effect is non-additive. It can be synergistic, i.e. stimulation of the decomposition, or antagonistic, i.e. inhibition of the decomposition. Hoorens et al. (2010) showed that the litter mixture effect may be additive at the plant functional level, because these opposite interactions may take place at the same time. However, such an "averaging" effect may be limited in low biodiversity ecosystems. Identifying such a litter mixture effect in ecosystems and understanding the mechanisms behind it is essential to specify which vegetation change can enhance (increased SOM mineralization) or buffer (increased SOM accumulation) greenhouse gas emissions and/or dissolved organic carbon export.

To better understand soil C dynamics, Cotrufo et al. (2010) suggested that simultaneous measurements of (i) CO₂ production, (ii) remaining mass and (iii) water extractable organic carbon (WEOC) should be undertaken. As suggested by Schimel and Weintraub (2003), this latter compartment is thought to be the intermediate pool between remaining mass (solid C) and CO₂ production (gaseous C). Following the suggestions of Cotrufo et al. (2010) and after adapting the model of Schimel and Weintraub (2003) to make it experimentally testable, Gogo
86

et al. (2014) showed in laboratory incubation of peatland plant litters that the WEOC compartment is accurately accounted for when it is assumed that it results from the balance between solid OM decomposition and soluble C respiration, and is quantitatively the most sensitive pool to changes in solid OM decomposition and respiration rates. In the context of the litter mixture effect, quantities as well as the chemical characteristics of WEOC are probably affected. Creamer et al. (2015) showed that litter WEOC is more sensitive to temperature change than WEOC from the mineral soil. This fraction may also be sensitive to other modified conditions such as litter mixture with invading species.

87

88

89

90

91

92

93

94

Peatlands contain up to a third of the world soil C (Gorham 1991). Their accumulation capacity originates from the particularly low decomposition rates of *Sphagnum* species (Clymo 1983). Any modification in their decomposition rate, through the litter mixing effect caused by vegetation change, would thus undermine the regulatory role of peatlands on the global C cycle (Limpens et al. 2008). Hoorens et al. (2002) showed that an increased atmospheric CO₂ concentration can lead to an excess microbial respiration in *Sphagnum recurvum* and *Carex rostrata* mixtures. In spite of the importance of such a result and the unfolding vegetation change in peatlands, very few studies have actually addressed the issue of a litter mixing effect in such ecosystems.

95

96

97

98

99

100

101

102

103

The two aims of this work are (i) to determine the occurrence of a mixture effect of *Sphagnum* and vascular plant litters and (ii) to identify the mechanisms behind such an effect. For this, two experiments were undertaken: a field experiment to test the effect of mixing *Sphagnum rubellum* and *cuspidatum* with *Betula* spp and *Molinia caerulea* on litter remaining mass, and a laboratory incubation experiment testing the effect of mixing *Sphagnum rubellum* and *Molinia caerulea* on solid, water extractable and gaseous C from these litters. In the latter experiment, the model developed in Gogo et al. (2014) was implemented to estimate overall exo-enzyme activity and respiration rate. Litter mixture effect can result from nutrient transfer between litters, influences caused by specific compounds released by the litters, changes in microclimatic conditions and modification of trophic levels interactions (Gartner and Cardon 2004; Hättenschwiler et al. 2005). Nutrient content of *Sphagnum rubellum* and *Molinia caerulea* is very low (Gogo et al. 2011). Thus, nutrient transfer may not be a pertinent cause of possible litter mixture effect. Instead, physical changes may occur. *Sphagnum* can affect the pH significantly and these species can retain up to 20 times its weight in water (Rydin and Jeglum 2013). Both

104

105

106

107

108

109

110

111

112

113

114

115

116
parameters are known to have an effect on OM decomposition (Bergman et al. 1999; Prescott 2010). To explain potential litter mixture effects in the laboratory experiment, the following two hypotheses were tested: compared to the theoretical expected values, mixing the two litters increases (a) litter water content or (b) pH of the water extract. To test these hypotheses, the water content of all litters was assessed as well as the pH of the water extract. To analyse in greater detail the water extract characteristics, the UV absorption (aromaticity) and the fluorescence properties (differentiation of pools of OM) of the soluble OM were investigated. Fluorescence analyses of DOM gave excitation-emission matrices (EEMs) that were treated using PARAFAC modelling.

## 2 Materials and methods

### 2.1 Sampling site

All litters for *in situ* and lab experiments were collected in La Guette peatland (Sologne, France), a site of the French Peatland Monitoring Service: Service National d'Observation Tourbières. It is located in Neuvy-sur-Barangeon (Cher) in the south-eastern part of the French Centre Region (altitude: 154m, N: 47°19', E: 2°16', Gogo et al. 2 011). La Guette peatland is composed of vegetation patches ranging from a transitional fen to wet heathland dominated by *Molinia caerulea*, *Erica teralix* and *Calluna vulgaris*. The dominant *Sphagnum* species are *Sphagnum cuspidatum* and *Sphagnum rubellum*. In recent decades, the abundance of vascular species already present in the ecosystem, *Molinia caerulea* and *Betula* spp., increased dramatically. Trees covered 15% of the peatland surface in 1980, 50% in 2000 and 69% in 2009. The reasons for such an abrupt increase may be hydraulic works and pine plantation in the surroundings of the site, as well as nutrient inputs.

### 2.2 Coupling field and laboratory experiments

Studying the effect of mixing litters with *Sphagnum* species is not as straightforward as with vascular plants. In the field, the incubation environment and the *Sphagnum* litter to decompose in the bag are the same material: *Sphagnum* litter. Whether in mixture with *Sphagnum* or in monoculture, the other litter composing the mixture experiences the same incubation conditions. To avoid this issue, Hoorens et al. (2003) and Hoorens et al. (2010) decided to incubate the litter bags in another environment than the natural one, with similar abiotic conditions. This
allows a fair contribution of both litters, but as the incubation conditions are not strictly the same as the natural ones, the results are difficult to transpose to the true environment.

Gartner and Cardon (2004) suggested that the remaining mass of each litter incubated in mixture should be weighed. Thus, following this recommendation, it was initially planned to separate the litter in mixture samples. However, preliminary tests showed that separating the *Sphagnum* litter which can be composed of very small leaves is a difficult and time consuming task that can lead to biased results as it is difficult to be sure that all the *Sphagnum* leaves have been separated,

In this study, it was decided to implement the field experiment in *Sphagnum* hummocks and lawns to test only the effect of the vascular plant litter on *Sphagnum* litter decomposition.

Vascular plant litters were also incubated in their native environment. The field experiments were further complemented with a laboratory incubation experiment, where all the litters were incubated in the very same conditions. It has been shown that the initial laboratory respiration rate could give a fair representation of long-term litter decomposability (Aerts and de Caluwe et al. 1997). In addition to respiration, the litter remaining mass and WEOC quantity and characteristics were also measured.

## 2.3 Field experiment

Litter bags (0.5 mm mesh) were filled with air-dried litter (about 750 mg) of different species: *Sphagnum cuspidatum*, *Sphagnum rubellum*, and leaves of *Betula* spp and *Molinia caerulea*.

These samples are referred to as "monoculture". The mixture samples were composed of 50% of *Sphagnum* litter (either *S. cuspidatum* or *S. rubellum*) with 50% of a vascular plant litter (either *M. caerulea* or *Betula* spp). The litter bags (n=4) were placed in situ in March 2009. In *S. rubellum* hummocks were placed: *S. rubellum*, *M. caerulea* and *Betula* spp litters in monoculture and in mixture (3 monocultures and 2 mixtures: *S. rub* + *M. cae* and *S. rub* + *Bet. spp*). In *S. cuspidatum* lawns were placed: *S. cuspidatum*, *M. caerulea* and *Betula* spp in monoculture and in mixture (3 monocultures and 2 mixtures: *S. cus* + *M. cae* and *S. cus* + *Bet. spp*).

In addition, the same protocol was followed to prepare *M. caerulea* and *Betula* spp. litter bags in monoculture that were placed in their native decomposition environment. Litter bags of *M. caerulea* were placed in the litter of *M. caerulea* in tussocks. The litter bags of *Betula* spp were
placed in the litter of *Betula* spp. Both experiments were located in the same area and undertaken at the same time.

The litters were collected in March 2010 after one year of incubation. In the laboratory, the roots were retrieved from the litter bags with tweezers and the litters were gently washed with deionised water. The litter bags were dried at 50°C for 2 days and weighed and the proportion of remaining mass was calculated (100 x remaining dry mass / initial dry mass).

There was an attempt to separate the litters, but this raised some issues. First, as the samples were extracted with water, some materials were found on the filter and some of these materials were so small that it was impossible to pool them with one or another litter. Furthermore, most of the vascular plant litter remained in one piece, which was absolutely not the case for *Sphagnum* litter, which scattered. This made the recovering of *Sphagnum* litter difficult to undertake without introducing an error in the estimation of the mass remaining, and thus a possible error in the detection of a mixture effect. It was preferred not to separate the litter to have a better guarantee on the whole mixture effect.

## 2.4 Laboratory experiment

As the field experiment showed significant differences between measured and expected values in the *S. rubellum* and *M. caerulea* mixture, the laboratory experiment focused on these 2 litters. The litters were collected in April 2011 and air dried before incubation, until they reached constant weight Surface peat water was collected in the same site. There was enough free water to collect it with a beaker and poor it into a flask. Then the water was filtered at 100 µm to get rid of the coarsest particles, without excluding microbial communities.

### 2.4.1 Sample preparation and incubation

The day after the water was collected from the peatland, air dried *S. rubellum* and *M. caerulea* litter samples were weighed (about 250 mg) and inoculated with 20 ml peat water and left overnight. Then, the excess water of the litters was removed with a tissue and the litters were placed in a 50 ml tube, which contained 5 ml of a potassium sulfate saturated solution (120 mg l⁻¹) to maintain moist conditions (Aerts and de Caluwe 1997). Glass marbles were also placed at the bottom of the tube and covered with a PVC filter (0.5 mm mesh) to avoid contact of the litter with the potassium sulfate solution. A total of 126 samples were prepared: 3 litter types (S.
209 *rubellum*, *M. caerulea* and the mixture of the two species, 50% each), 42 samples per litter type,
210 and incubated in controlled conditions. An insulated container (0.3 m x 1 m x 1 m) was built with
211 pieces of PVC covered with aluminum foil to ensure constant environmental conditions during
212 incubation. To obtain a water-saturated air, the atmosphere of the container was filled with air
213 that was pumped through warm water. Sets of samples (*S. rubellum*, *M. caerulea* and the
214 mixture) were randomly put in racks that were placed in the container. The position of the racks
215 within the container was changed every week. Temperature and humidity were monitored and
216 showed that the experimental setup ensured a constant water-saturated atmosphere and an air
217 temperature of 22.5 ± 0.3 °C (n = 32).

218 A total of 126 samples allowed a kinetic study of 7 dates with 6 samples of each litter type at
219 each date. To calculate expected mixture results from additive effect (50% of each litter type),
220 groups were composed of one sample of each type of litter (total of 3 samples per group: 1 *M.*
221 *caerulea* alone, 1 *S. rubellum* alone, 1 mixture of both). At each date, 6 groups were randomly
222 retrieved after 2, 6, 12, 20, 33, 47 and 64 days of incubation. Immediately after the sample had
223 been taken out, the CO₂ production was measured. Then the water extraction of litters was
224 undertaken and finally the sample was dried at 50°C during 2 days before weighing.
225

226
2.4.2 CO₂ production

227 The tubes were placed in a chamber during 20 to 30 minutes. The cover of the chamber was
228 fitted with a mounting flange, which could receive a Vaisala CO₂ infra-red probe (GMP343). The
229 CO₂ concentrations were monitored and recorded directly on a computer. The CO₂ production
230 was calculated from the slope of the increasing concentration with time.
231

232
2.4.3 Water Extractable Organic Carbon (WEOC)

233 After the CO₂ production measurement, the litter was taken out of the tube and placed in an
234 aluminum cup at room temperature. The litter was rinsed twice with 15 ml and once with 10 ml
235 deionised water and the extract was filtered (0.45 µm). The filter was weighed before filtering
236 and was dried 48 h at 50 °C after filtering. The difference in filter mass was added to the mass
237 of litter left in the aluminum cup (litter also dried at 50 °C during 48 h and weighed) to obtain the
238 whole remaining mass. A filtrate subsample was dedicated to dissolved organic carbon (DOC)
239 analysis (Shimadzu TOC 5000), which allowed the calculation of the WEOC (in g C g⁻¹ initial
240 litter dry weight). Another filtrate subsample was dedicated to UV absorption and fluorescence spectroscopy analyses. Before injection into the Shimadzu TOC 5000, the samples were acidified and bubbled to eliminate dissolved inorganic C, leaving only the DOC to be analyzed.
241
242
243 Phtalate was used for calibration and each sample was replicated until the coefficient of variation was less than 10% (maximum replication = 5). This procedure was undertaken with each sample (n = 6) from each treatment. Just after extraction, the pH of the extracts was measured on subsamples.
244
245
246
247

248
2.4.4 Calculation of remaining mass

For both field and laboratory experiments, i.e. laboratory and field incubations, the litters were
249
250 air-dried until reaching a constant weight. Then, a set of air-dried samples of each litter was
dried at 50 °C during 48 h and weighed. This allows the determination of the air to oven dried
251
252 ratio. This ratio was used to calculate the initial oven dried mass of all incubated air-dried litters.
253 The sum of the litter left after extraction and the litter on the filter gave the remaining mass. This
254 percentage was calculated by dividing the remaining mass at any time by the initial mass of the
255 litter and multiplied by 100. The mass of wet samples was also measured and the water content
256 in g of water per g of remaining mass was calculated.
257

258
2.4.5 SUVA₂₉₀

The water samples were neutralized with NaOH before measurement as recommended by
259
Weishaar et al 2003). Most of the samples were in the range of 10 to 20 mg L⁻¹, with no
260
samples higher than 31 mg L⁻¹ or lower than 7 mg L⁻¹. UV absorption at 280 nm of all water
261
samples was acquired with a Hitachi U-1900 absorbance spectrophometer using a 1 cm path-
262
length quartz cell (6030-UV Hellma Analytics). The zero absorbance adjustment was carried out
263
with deionised water before each batch of measurements. Absorbance spectra was measured
264
between 1100 nm and 200 nm. The absorbance at 280 nm was divided by the DOC content of
265
the sample to give the Specific UV Absorption at 280 nm (SUVA₂₉₀ in L cm⁻¹ mg⁻¹). The SUVA₂₉₀
266
informs on the aromaticity of the DOC (Chin et al 1994).
267
268

268
269
2.4.6 Natural dissolved organic matter fluorescence (FDOM) measurement and signal
270 processing
271 Fluorescence properties of DOM have been used to study the mixing of fresh and salt waters in estuaries (Coble 1996; Parlanti et al. 2000, Huguet et al. 2009), as well as humic substances in soil and peat water (Zsolnay et al. 1999; Alberts and Takács 2004; Zaccone et al. 2009). In this study, FDOM was measured to assess whether this technique could differentiate between sources of DOM in decomposition experiments.

276 After UV-vis analysis, the same sample was used to characterize FDOM. Excitation-Emission Matrices (EEMs) were acquired with a Hitachi F-7000 fluorescence spectrophotometer equipped with FL solutions software, using 10 x 10 mm quartz cell (1000-OS Hellma Analytics). For excitation and emission monochromators, slit widths were set at 2.5 nm. EEMs were recorded using a scan speed of 1200 nm/min, at 950 V, over ranges of excitation and emission wavelengths of 250-500 and 300-600 nm, respectively. Excitation and emission wavelengths were stepped by 10 and 1 nm respectively and no inner effect correction was necessary. After subtraction of the blank (deionised water), the EEMs were treated by CP/PARAFAC algorithm to extract independent fluorescence contribution.

285 PARAFAC analysis identified four groups of components in the water extracted organic matter (WEOM) from incubated litter (Table 1) with a CORCONDIA of 68.9%. Three ratios were calculated using the C2 fluorophores corresponding to α or C (Table 1) as the denominator. The ranges of excitation-emission of the four groups were compared to published data (Table 1). The C2 and C3 components were related to humic substances (Table 1). It is assumed that C3/C2 ratio corresponds to α'/α ratio. As both humic substances have α' and α components, it is not possible to use this ratio to distinguish humic substances. The component α is composed of more humified and older compounds than those found in the component α'. An increase in C3/C2 ratio could indicate an increasing proportion of recent material. C1 Em/Ex couples were similar to those found in extracts of IHSS standard humic substances (Alberts and Takács 2004) and marine samples corresponding to marine humic-like substances (Coble 1996; Parlanti et al. 2000). The C1/C2 ratio was also calculated and the correlation between the C1/C2 and C3/C2 ratios was significant (R = 0.87, P<0.0001). This suggests that, like the C3/C2 ratio, the C1/C2 ratio cannot be used to distinguish humic substances.

299 The C4 component is related to protein-like compounds (Table 1). It is assumed that the C4/C2 ratio corresponds to the γ/α ratio used to assess the relative variations of protein-like substances compared to humic substances (Sierra et al. 2005). Zsolnay et al. (1999) observed
a relative increase in the γ fluorophores (C4 component in this study) compared to the α fluorophores (C2 component in this study) in soil water extract after fumigation. In the microbial biomass estimation, fumigation is used to achieve lysis of microbial cells, so cellular constituents are released into the soil solution and can be readily extracted with water and measured (Brookes et al. 1985). Low values of the C4/C2 ratio could indicate that protein-like substances are in living microbial cells and not extractable, whereas high values could indicate that microbial cell death has released protein-like compounds in the soil that are extractable. To corhoborate this assumption, C4/C2 was related to proxies of microbial activities (catalysis activities and respiration).

311

## 2.5. Model description and parameters estimation

As the experimental design was similar to that in Gogo et al. (2014), the data were calculated to allow their use as inputs to the Gogo et al. (2014) model. This model is based on Schimel and Weintraub (2003). It assumes that solid organic matter is catalysed by exo-enzymes into soluble organic matter. Then the soluble matter is absorbed by the microbial and used as an energy source for different microbial function (enzymes production, maintenance, growth) and released into the environment in the form of CO₂ (Schimel and Weintraud 2003). Gogo et al. (2014) simplified this model to make it experimentally testable. It consist of a three compartment: (i) the “L” compartment corresponding to the fraction of C contained in the litter (solid fraction), (ii) the “W” compartment corresponding to fraction of C contained in the WEOC (dissolved fraction) and (iii) the “G” compartment corresponding to fraction of C contained in the cumulative CO₂ released by microbial respiration (gaseous fraction). The flux between the L and W compartment corresponds to the exo-enzymes catalysis rate. The flux between the W and G compartment correspond to the respiration. Equations were written to give account of the simultaneous change in time of the state variable (L, W, and G) and the reaction rates. At any time of the course of the experiment the sum of all these three fractions is equal to 1. The three fractions corresponding to the three compartments were experimentally measured. The two reaction rates (catalysis or “c” and respiration or “r”) were tuned simultaneously to fit the model to the three experimentally assessed compartments.

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

Gogo et al (2014) showed that allowing the reaction rate to change in the course of the experiment improved the goodness of fit. The reaction rates were allowed to follow a negative
333 exponential decrease with time with the following parameters to describe the shape of the curve: "a + b" is the initial reaction rate, "a" is the final rate, "m" rate of decay with time of the reaction rate (Rovira and Rovira 2010).
334
335

336

## 2.6 Statistical analyses

338 Paired *t*-tests and repeated measures ANOVA (with litter type or mixture type as within effect) were conducted with Statistica (2008) to test for differences in response variables between *Sphagnum* spp and vascular plant litters and for differences between measured and calculated values of the mixture. Correlations were calculated on log transformed data when necessary to assess the relationship between two response variables, using Statistica (2008). Model II regression (ranged major axis; Legendre and Legendre 1998) was performed on log transformed data when necessary to calculate equation coefficients.
339
340
341
342
343
344
345

346

# 3 Results

## 3.1 Field experiment

348 *S. rubellum* litter decomposed significantly faster in the presence of *Molinia caerulea* than without (*P* < 0.02; Table 2). This was the only significant non-additive litter mixture effect observed. On the whole, in the field conditions and over a year, *Molinia caerulea* and *Betula* spp decomposed faster than *Sphagnum* species (Table 2). *M. caerulea*, but not *Betula* spp., decomposed faster in the *Sphagnum* environments than in its native one (Table 3). As a mixture effect was found only with *S. rubellum* and *M. caerulea* and as *Sphagnum* environments affected only *Molinia caerulea*, the laboratory experiment focused on this species.
349
350
351
352
353
354
355

## 3.2 Laboratory experiment

### 3.2.1 *C* dynamics

358 *S. rubellum* litter mass decreased faster, contained more WEOC and tended to produce more CO₂ than *M. caerulea* litter (Fig. 1a, c, e; Table 4). The measured remaining mass of the *S. rubellum* and *M. caerulea* mixture was significantly lower than expected from calculations (*P* < 0.002; Fig. 1b; Table 4). The non-additive effect of the mixture was also observed on the WEOC and the CO₂ production, with higher measured values than expected (Fig. 1d, f; Table 4).
359
360
361
362
Applying the model of Gogo et al. (2014) gave a good account of the observations with RMSE in the same range as Gogo et al. (2014): between 0.16 and 17.8 in this study (Table 5) and between 0.23 and 14.5 in (Gogo et al. 2014) for *M. caerulea*. Initial exo-enzyme activity and respiration rate in the measured mixture were in the expected range (no more than 10% of difference; Table 5; Fig. 2b, d). However, the rates at which the activities decreased afterward (the "m" parameters in Table 6) were much lower than expected. This implies that both activities in the measured mixture were higher than expected (Fig. 2). The difference was more pronounced for exo-enzyme activities (75% difference) than for respiration (46% difference; Table 6).

Contrary to the stated hypothesis, the water content in the measured mixture was not different than the calculated one (Table 4; Fig. 3 a and b). The pH was different and significantly lower in the measured mixture than in the calculated one (Table 4; Fig. 3 c and d).

### 3.2.3 Specific Ultraviolet Absorption at 280 nm

In all litters, SUVA<sub>280</sub> decreased with time (Fig. 4). On the whole WEOC from *Molinia caerulea* litter was more aromatic than the *Sphagnum rubellum* WEOC (*P* < 0.0001; Fig. 4ab). Also, the WEOC measured in the mixture was found to be more aromatic than expected (Fig. 4c).

### 3.2.4 Fluorescent dissolved organic matter

Both C1/C2 and C3/C2 ratios increased with time (Fig. 5a, b, c, d), showing an increased contribution of more recent humic substances. The C1/C2 ratio in *M. caerulea* WEOM was significantly higher than in *S. rubellum* (*t* = 6.86, *P* < 0.0001, *n* = 42; Fig. 5a), but there was no difference between measured and calculated mixtures (*t* = 0.035, *P* = 0.97, *n* = 42; Fig. 5b). In contrast, the C3/C2 ratio was significantly lower in the measured than in the calculated mixture (*t* = 3, *P* < 0.005, *n* = 42; Fig. 5d). However, this ratio in the single litter WEOM was not different (*t* = 0.82, *P* = 0.42, *n* = 42; Fig. 5c).

The C4/C2 ratio in all litter types increased with time, showing an increased contribution of protein-like compounds compared to humic substances (Fig. 5e, f; the same pattern is observed when C3 or C1 substitute C2). This increase was particularly high for the *M. caerulea* litter compared to the *S. rubellum* litter (*t* = 8.93, *P* < 0.0001, *n* = 42; Fig. 5e). However, as well as C1/C2, there was no significant difference between measured and calculated mixtures (*t* =
0.055, P = 0.96, n = 42; Fig. 5f). In our study, the C4/C2 ratios were always negatively correlated to the modelled exo-enzyme activity and respiration rates (all R < -0.89, all P < 0.01; Fig 6). Also, the relationship was in a chronological order: as time passes, the microbial activity decreased, whereas the relative fluorescence of protein-like compounds increased.

398

## 4 Discussion

### 4.1 Laboratory synergistic effect of litter mixture on OM dynamics

In the laboratory experiment, mixing *M. caerulea* and *S. rubellum* litters increased litter mass loss (decomposition) and CO₂ production (microbial respiration) compared to what can be expected in an additive scenario (Fig. 1; Table 4). Krab et al. (2013) already demonstrated a *Sphagnum*-vascular plant mixture effect on mass loss in the field, but with *S. fuscum* and *Betula pubescens*. To our knowledge, such a non-additive effect of mixing *M. caerulea* and *S.rubellum* litters on microbial activity has never been shown before. The mixture effect between *M. caerulea* and *S.rubellum* litters could result from a higher exo-enzyme activity and respiration rate in measured than in calculated mixtures during all or most of the incubation time respectively (Fig. 2b, c).

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

WEOC content was also increased by the mixture. The WEOC compartment receives input from litter exo-enzyme catalysis (provoking litter mass loss), and is consumed by the microbial biomass (resulting in output by respiration in the form of CO₂; Schimel and Weintraub 2003; Gogo et al. 2014). As such, WEOC is sensitive to any change in input and output rates (Gogo et al. 2014). The build-up of WEOC built up in the measured mixture, compared to the calculated mixture, can only result from a relatively higher input than output rate. This suggests that in the first stage of decomposition, mixing *S. rubellum* and *M. caerulea* increased exo-enzyme activity more than respiration, compared to what could be expected. The model showed that both rates decreased with time in all types of litter. Thus, the higher WEOC content in the measured mixture could only be caused by an output rate that decreased faster than the input rate (Table 6).

421

### 4.2. Increase of aromatic compounds in the WEOC

The higher WEOC in the measured mixture was simultaneous to a significant increase of WEOC aromaticity (Fig. 4). Aromaticity variation can be due to (i) input/output of aromatic

423

424
compounds in the WEOC compartment and/or (ii) input/output of non-aromatic compounds (e.g. carbohydrates), mechanically decreasing/increasing the SUVA₂₉₀.

Degradation of lignin, tannin and/or polyphenols of the litters can be a significant source of soluble aromatic compounds into the WEOC. Dignac et al. (2005) showed in a cropland soil that lignin turnover could be faster than the whole soil organic carbon turnover. In the present study, it was shown that decomposition rate of the solid phase in the measured mixture was faster than expected, with thus the potential to draw more aromatic compound into the WEOC than expected. Furthermore, aromatic compounds such as tannin are more difficult to metabolise by the microorganisms, than carbohydrates monomers (Davidson and Janssens 2006). In the present study, it was shown that respiration rate was faster in the measured mixture than in the expected one, leaving into the WEOC probably more aromatic compounds than carbohydrates.

The increased of WEOC aromaticity in the measured mixture is thus coherent with the combine effect of increased rate of solid matter decomposition and WEOC respiration.

438

439 4.3 Contribution of fluorescence analysis to the study of litter C dynamics

Among the fluorescent components highlighted by the PARAFAC analysis, C4 is the only one that is not attributed to humic substances, but to tyrosine and tryptophane containing materials (i.e. proteins; Coble 1996; Parlanti et al. 2000). These compounds contain nitrogen, which is one of the most limiting nutrients in soils. The occurrence of such fluorophores may be related to a release of exo-cellular enzymes. However, if a high contribution of protein-like fluorophores were to be related to an increased release of exo-enzymes, an increased decomposition rate would be expected when the C4/C2 ratio is high. This was not the case. First, the *S. rubellum* litter decomposed faster than the *M. caerulea* litter, whereas its C4/C2 was much lower (Figs. 1a and e and 5e). Second, as time passes, the microbial activity decreased in all samples, whereas the γ fluorophores contribution increased. The occurrence of such fluorophores may also be related to the release into the solution of proteins associated to the decomposing tissues. If this were the case, increased contribution of the γ fluorophores would be associated to an increased mass loss. However, the exact opposite was observed: *M. caerulea*, which decomposition was slow, had a WEOC with the most important γ fluorophore contribution. Furthermore, because of the translocation process (Taylor et al. 2001) *M. caerulea* litter has a very low N content: more than 2 times less than *S. rubellum*, with 1.0 mg g⁻¹ and 2.2 mg g⁻¹

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455
respectively (Gogo et al 2010). Such a high input of protein-like compounds from such a slow decomposing N poor litter may not be realistic.

In peat, the microbial biomass concentrates a significant fraction of the soil total N (peat C/N from 16 to 36, microbial biomass C/N from 6 to 14; Francez et al. 2000). Parlanti et al. (2000) showed in a macro-algae degradation experiment that the y/α ratio increased in the first 15 days of the incubation corresponding to degradation products of the organisms, probably originating from cell membranes. Zsolnay et al. (1999) also showed that such fluorophores occurred when the microbial cell were lysed, leaving in solution the protein rich cytoplasmic content. As suggested above, protein amount in soil can increase because of exo-enzymes release. However, the expected increase of OM degradation in such case was not observed: microbial activity decreased as the protein-like fluorophores contribution increased. What could happen is that as the microbial biomass dies off, the cell contents are released in the media without being taken up again, provoking a build-up of proteinaceous compounds in the WEOM. Thus, it suggests that the relative increase in the C4/C2 ratio in time could reflect an increasing solubilisation of microbial cell products after their death. The C4/C2 ratio in the context of litter laboratory incubation can be used as an index of the microbial dead/living biomass ratio.

As solid OM composition differs between *Sphagnum* species and *M. caerulea* litter (Gogo et al. 2011; Gogo et al. 2014), it was expected that the fluorescence of the WEOM produced by solid OM degradation would differ as well. Such a difference would allow identification of the relative contribution of each litter in the mixture, making it possible to identify which litter contributes more to the input into the WEOM. However, the results show that when a difference was found between single litters, there was no non-additive effect in the mixture (C1/C2 and C4/C2, Fig. 5a, b, e and f). In contrast, a non-additive effect was detected in the mixture where no difference was found between single litters, (C3/C2, Fig. 5c, d). Thus, in the present case, fluorescence analysis is not a pertinent tool to study chemical changes linked to mixture.

Most fluorescent materials (C1, C2, C3) are humic substances (Coble 1996; Parlanti et al. 2000; Alberts and Takács 2004). They are not the direct product of litter exo-enzymatic attack. They were certainly present in the water used to inoculate the litter. Humic substances are known to be recalcitrant to decay (Jenkinson and Rayner 1977). This may explain why the non-additive effect observed on mass loss, WEOC content and respiration was not reflected in these compounds.
## 4.4 Mechanisms of in situ and laboratory litter mixture effect

As in the laboratory, there was an in situ synergistic effect of mixing *S. rubellum* and *M. caerulea* litters on OM decomposition after one year of incubation (Table 2). This effect of mixing *M. caerulea* with a *Sphagnum* moss on OM decomposition has never been evidenced before. Because the *M. caerulea* decomposing environment is composed of *Sphagnum* litter, it was not possible to test the effect of *Sphagnum* litter on *M. caerulea* litter decomposition. It can however be concluded that the in situ mixture effect observed originated from an effect of *M. caerulea* on the rate of decay of *S. rubellum*, which is an additional information compared to the laboratory incubation.

The effect of the *Sphagnum* environment on *M. caerulea* can be approached by comparing the decomposition rate obtained in *Sphagnum* and in the native tussock environment. *M. caerulea* litter in its in situ native environment decomposed at a much slower rate than in both *S. rubellum* and *S. cuspidatum* environments (Table 3). Van Vuuren and van der Eerden (1993) also found low decomposition rate of *M. caerulea* in its native environment in a heathland (80% of remaining mass after 11months of incubation). The decomposition of *M. caerulea* litter increased by 17 - 21% in a *Sphagnum* environment compared to their native environment (Table 3). The non-additive litter mixture effect may result from changes in physical properties caused by one litter (Gartner and Cardon 2004). *M. caerulea* is a tufted plant forming tussocks and because of this microtopography most of the litter experiences dry conditions in the first year that limit decomposition. Moisture in the range of 60-75% (wet weight basis) does not limit decay, whereas its variations could influence mass loss in the range of 75-80% and more extensively in the range of 30-60% (Prescott 2010). Microbial activity decreases as the conditions become drier and wetter. A change toward an increase of moisture compared to its native environment would stimulate *M. caerulea* litter decomposition. Through their morphological characteristics, *Sphagnum* mosses are able to retain up to 15 to 20 times their dry weight in water (Rydin and Jeglum 2013, in accordance with Fig. 3), which maintains moist conditions over dry periods. Thus, in field conditions, when *M. caerulea* litter decomposes in a *Sphagnum* environment, it spends more time in favourable conditions for decomposition than in its native environment.
Water content seems to explain the differences observed. Although *M. caerulea* did decompose faster than in its native environment, the decomposition rate in *Sphagnum* environment was low (about 70% of mass remaining after one year; Table 3). Hoorens et al. (2003) reported a faster decomposition rate of *M. caerulea* litter in a *Polytrichum commune* decomposing environment (59%). Once a limiting factor was alleviated, other factor influencing OM decomposition may take place. *Sphagnan* a carbohydrate polymer specific to *Sphagnum* environment is thought to preserve OM through different mechanisms (Hájek et al. 2011). A longer experiment and/or comparison of *M. caerulea* litter decomposition in other environments (*M. caerulea* can grow in a wide array of environments) would help to assess the effect of *Sphagnum* environment on *M. caerulea* decomposition.

As mentioned above, *M. caerulea* is a tufted plant forming tussocks, which implies dry conditions in the first year that limit decomposition. From the in situ experiment, it was suggested that litter moisture is an important factor controlling the effect of the *Sphagnum* environment on the decomposition of *M. caerulea*. In the laboratory conditions, all the litters were subjected to the same conditions. The *M. caerulea* litters in both treatments (in monoculture and in mixture) were moist (unlike native field conditions). A significant correlation was found between microbial activity and water content for all litters (Fig. 6), which supports the role of water content on microbial activity. However, no significant difference in litter water content was found between measured and calculated mixtures (Table 4; Fig. 3a and b). This suggests that additional mechanisms are at work to explain the observed non-additive effect of *S. rubellum* on *M. caerulea* in the laboratory and of *M. caerulea* on *S. rubellum* in the field.

Microbial activity can be very sensitive to changes in pH. The optimum pH for CO₂ production in peat is above the pH usually measured in peatlands (Bergman et al. 1999). In our study, although the pH in the mixture extract decreased compared to an additive scenario, microbial activity was enhanced by mixing the two litters. This rules out any direct role of this factor on the mechanism that could explain the synergistic effects observed. However, increase of acidity could have an indirect effect on labile carbon supply to microorganisms. Low pH could accelerate litter OM hydrolysis, releasing both lignin (as suggested above) and carbohydrate monomers. The latter could have stimulated microbial activity, which in turn could have increased litter degradation. Such an hypothesis has to be further explored.

547
549 Hoorens et al. (2010) showed that the litter mixture effect may be additive at the plant functional
550 level, implying that the decomposition rate at the ecosystem level could be calculated from the
551 abundant literature on individual litter decomposition rates. While this may be true for
552 ecosystems supporting enough biodiversity, it is to stress that biodiversity is dramatically
decreased in peatlands invaded by *Molinia caerulea*. In the study site, with the exception of
553 some wetter areas dominated by *Rynchospora alba*, *M. caerulea* together with very sparse
554 *Eriophorum angustifolium* dominates the herbaceous strata and covers almost all the site. In
555 some wetter places, where *S. rubellum* can develop, *M. caerulea* is the only herbaceous plant.
556 *M. caerulea* cover increases because of disturbances. In such a situation, the negative and
557 positive non-additive interaction may not cancel out as suggested by Hoorens et al. (2010),
558 leaving only a positive non-additive effect. The ecosystem level decomposition rate estimation
559 using individual litter data in ecosystems where *M. caerulea* dominates should therefore not be
560 used in modelling. Furthermore, the occurrence of a mixture effect between different *Sphagnum*
561 species and different invading vascular plants (Krab et al. 2013; this study) clearly highlights the
562 need to take peatland vegetation change into account when simulating future C stock and
563 fluxes.
564

565 One must be careful when transposing results obtained in the laboratory to the field. That is why
566 here laboratory experiment was coupled to in situ incubation. Both experiment showed an
567 acceleration of decomposition in litter in mixture. However, the laboratory experiment was much
568 shorter than the field experiment and mixture effect observed in both experiment may not
569 originate from the same process.

570 One aim of this study was to elucidate the mechanisms behind the mixture effect. However,
571 direct effect of the variations of the factors studied (water content and pH) could not explain the
572 observation. As an alternative, the results of the present study suggest that the interaction
573 between labile OM and microbial biomass activity may be the key interaction to focus on in
574 further research. This is crucial because such a synergistic effect can jeopardize the C storing
575 capacity of *Sphagnum* peatlands in the context of vegetation change. Considering the vast
576 areas covered by *Sphagnum* species, especially in Siberia and Canada and the increasing
577 abundance in Northern peatlands of *M. caerulea* and other colonising vascular plant species
578 (e.g. *Betula* spp, *Pinus* spp, *Phragmites australis*), the litter mixture effect of invading species
579 litter with *Sphagnum* spp litters definitely requires more attention. For example, other mixing
580 experiment should be undertaken with *Sphagnum* species and other vascular plant to assess to
581 which extent the mixture effect is occurring in these ecosystems.

582

583 Acknowledgements

584 The salary of S. Gogo was financially supported by the Conseil Général du Loiret, the CPER
585 (Contrat Plan Etat-Région Centre) project Spatioflux n°734-30805 and the Labex VOLTAIRE.

586 This paper is a contribution of the Labex VOLTAIRE (ANR-10-LABX-100-01). CETRAHE was
587 supported by the DRRT (Délégation Régionale à la Recherche et à la Technologie Centre) and
588 the Région Centre, through the “Cellules R&D” program of CPER. This study was undertaken in
589 the framework of the Service National d'Observation Tourbières (French Peatland Observatory),
590 accredited by the INSU- CNRS.

591

592 References

593 Aerts R, de Caluwe H (1997) Initial litter respiration as indicator for long-term leaf litter
594 decomposition of *Carex* species. Oikos 80:353-361

595 Alberts JJ, Takács M. (2004) Total luminescence spectra of IHSS standard and reference fulvic
596 acids, humic acids and natural organic matter: comparison of aquatic and terrestrial source
597 terms. Org Geochem 35:243-256

598 Bardgett RD, Wardle DA (2010) Aboveground-belowground linkages - Biotic interactions,
599 Ecosystem processes and global change. Oxford University Press, Oxford

600 Berg B, McClaugherty C (2003) Plant litter, decomposition, humus formation, carbon
601 sequestration. Springer-Verlag, Berlin

602 Bergman I, Lundberg P, Nilsson M (1999) Microbial carbon mineralisation in an acid surface
603 peat: effects of environmental factors in laboratory incubations. Soil Biol Biochem 30:729-
604 751

605 Brookes PC, Landman A, Prudent G, Jenkinson DS (1985) Chloroform fumigation and the
606 release of soil nitrogen: a rapid direct extraction method to measure microbial biomass
607 nitrogen in soil. Soil Biol Biochem 17:837-842

608 Chin Y-P, Alken G, O'Loughlin E (1994) Molecular weight, polydispersity and spectroscopic
609 properties of aquatic humic substances. Environ Sci Technol 28:1853-1858
610
Clymo RS (1983) Peat. In: Gore AJP (ed), Ecosystems of the World, vol. 4A. Mires: Swamp,
611
Bog, Fen and Moor. Elsevier, Amsterdam pp 159-224

612
Coble PG (1996) Characterization of marine and terrestrial DOM in seawater using excitation-
613
emission matrix spectroscopy. Mar Chem 52:325-346

614
Cornelissen JHC, Callaghan TV, Alatalo JM et al. (2001) Global change and arctic ecosystems :
615
is lichen decline a function of increases in vascular plant biomass? J Ecol 89:984-994

616
Cotrufo MF, Del Galdo I, Piermatteo D (2010) Litter decomposition: concepts, methods and
617
future perspectives. In Kutsch WL, Bahn, MA, Heinemeyer A (eds), Soil Carbon dynamics.
618
Cambridge University Press, Cambridge, pp 76-90

619
Creamer CA, de Menezes AB, Krull ES, Sanderman J, Newton-Walters R, Farell M (2015).
620
Microbial community structure mediates response of soil C decomposition to litter addition
621
and warming. Soil Biol Biochem 80:175-188

622
Davidson EA, Janssens IA (2006) Temperature sensitivity of soil carbon decomposition and
623
feedbacks to climate change. Nature 440:165-173

624
Dignac M-F, Bahri H, Rumpel C, Rasse DP, Bardoux G, Balesdent J, Girardin C, Chenu C,
625
Mariotti A (2005) Carbon-13 natural abundance as a tool to study the dynamics of lignin
626
monomers in soil: an appraisal at the Closeaux experimental field (France). Geoderma
627
128:3-17

628
Francez A-J, Gogo S, Josselin N (2000) Distribution of potential CO2 and CH4 productions,
629
denitrification and microbial biomass C and N in the profile of a restored peatland in Brittany
630
(France). Eur J Soil Biol 36:161-168

631
Gartner TB, Cardon ZG (2004) Decomposition dynamics in mixed-species leaf litter. Oikos
632
104:230-246

633
Gogo S, Laggoun-Défarge F, Delarue F, Lottier N (2011). Invasion of a *Sphagnum*-peatland by
634
*Betula* spp and *Molinia caerulea* impacts on organic matter biochemistry. Implications for
635
carbon and nutrient cycling. Biogeochemistry 11:545-551

636
Gogo S, Francez A-J, Laggoun-Défarge F, Gouélibo N, Delarue F, Lottier N (2014)
637
Simultaneous estimation of actual litter enzymatic catalysis and respiration rates with a
638
simple model of C dynamics in *Sphagnum* dominated peatlands. Ecosystems 17:302-316

639
Gorham E (1991) Northern peatlands: role in the carbon cycle and probable responses to
640
climatic warming. Ecol Appl 1:182-195
641 Hájek T, Balance S, Limpens J, Zijlstra M, Verhoeven JTA (2011) Cell-wall polysaccharides play an important role in decay resistance of Sphagnum and actively depressed decomposition in vitro. Biogeochemistry 103:45-57

644 Hättenschwiler S, Tiunov A, Scheu S (2005) Biodiversity and litter decomposition in terrestrial ecosystems. Annu Rev Ecol Evol Syst 36:191-218

646 Hoorens B, Aerts R, Stroetenga M (2002) Litter quality and interactive effects in litter mixtures: more negative interactions under elevated CO₂? J Ecol 90:1009-1016

648 Hoorens B, Aerts R, Stroetenga M (2003) Does litter chemistry explain litter mixture effect on decomposition? Oecologia 442:578-586

650 Hoorens B, Stroetenga M, Aerts R (2010) Litter mixture interactions at the level of plant functional types are additives. Ecosystems 13:90-98

652 Huguet A, Vacher L, Relaxans S, Saubusse S, Froidefond JM, Parlanti E (2009) Properties of fluorescent dissolved organic matter in the Gironde estuary. Org Geochem 40:706-719

654 IPCC, 2007. Climate Change 2007: synthesis report. Contribution of working groups I, II and III to the fourth assessment report of the Intergovernmental Panel on Climate Change [Core writing team, Pachauri RK, Reisinger A (eds)]. IPCC, Geneva, Switzerland, 104 pp

657 Jenkinson DS, Rayner JH (1977) The turnover of soil organic matter in some of the Rothamsted classical experiments. Soil Sci 123:298-305

659 Krab EJ, Berg MP, Aerts R, van Logtestjin RSP, Cornelissen JHC (2013). Vascular plant litter input in subarctic peat bogs changes Collembola diets and decomposition patterns. Soil Biol Biochem 63:106-115

662 Legendre P, Legendre L (1998) Numerical Ecology. Elsevier Science B.V., Amsterdam

663 Limpens J, Berendse F, Blodeau C, Canadell JG, Freeman C, Holden J, Roulet N, Rydin H, Schaepman-Strub G (2008) Peatlands and the carbon cycle: from local processes to global implications - a synthesis. Biogeosciences 5:325-329

666 Parlanti E, Wörz K, Geoffroy L, Lamotte M (2000) Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs. Org Geochem 31:1765-1781

669 Prescott CE (2010) Litter decomposition: what controls it and how can we alter it to sequester more carbon in forest soils? Biogeochemistry 101:133-149
671 Rovira P, Rovira R (2010) Fitting decomposition datasets to mathematical curves: towards a generalised exponential approach. Geoderma 155:329-343

672
Rydin H, Jeglum JK (2013) The biology of peatlands. Oxford University Press, Oxford

673
Schimel JP, Weintraub MN (2003) The implications of exoenzyme activity on microbial carbon and nitrogen limitation in soil: a theoretical model. Soil Biol Biochem 35:549-563

676 Sierra MMD, Giovanela M, Parlanti E, Soriano-Sierra EJ (2005) Fluorescence fingerprint of fulvic and humic acids from varied origins as viewed by single-scan and excitation/emission matrix techniques. Chemosphere 58:715-733

679 Statsoft Inc (2008) STATISTICA for Windows Version 8.0. Tulsa Oklahoma, Statsoft, Inc.

680 Taylor K, Rowland AP, Jones HE (2001) *Molinia caerulea* (L.) Moench. J Ecol 89:126-144

681 Van Vuuren MMI, Berendse F (1993) Changes in soil organic matter and net nitrogen mineralization in heathland soils, after removal, addition or replacement of litter from Erica tetralix or Molinia caerulea. Biol Fertil Soils 15:268-274

683
Weishaar JL, Aiken GR, Bergamaschi BA, Fram MS, Fujii R, Mopper K (2003) Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon. Environ Sci Technol 37:4702-4708

684
Weltzin JF, Bridgham SD, Pastor J, Chen J, Harth C (2003) Potential effects of warming and drying on peatland plant community composition. Glob Change Biol 9:141-151

687
Zaccone C, D'Orazio V, Shotyk W, Miano TM (2009) Chemical and spectroscopic investigation of porewater and aqueous extracts of corresponding peat samples throughout a bog core (Jura Mountains, Switzerland). J. Soils Sediments 9:443-456

688
Weltzin JF, Bridgham SD, Pastor J, Chen J, Harth C (2003) Potential effects of warming and drying on peatland plant community composition. Glob Change Biol 9:141-151

689
Zaccone C, D'Orazio V, Shotyk W, Miano TM (2009) Chemical and spectroscopic investigation of porewater and aqueous extracts of corresponding peat samples throughout a bog core (Jura Mountains, Switzerland). J. Soils Sediments 9:443-456

691
(Jura Mountains, Switzerland). J. Soils Sediments 9:443-456
692
Table 1. Major components found by a PARAFAC analysis of the fluorescence excitation-emission matrices of the water extractable samples, and the assumed corresponding origin of the substances of each component from different literature sources. M and β were usually attributed to marine humic substances, but were also found in extract of humic substances standard (Alberts and Takács, 2004).

<table><thead><tr><th>PARAFAC<br>Components</th><th>Coble et al.<br>(1996)</th><th>Parlanti et al.<br>(2001)</th><th>Excitation<br>max. (mm)</th><th>Emission<br>max. (mm)</th><th>Compound type</th></tr></thead><tbody><tr><td>C1</td><td>M</td><td>β</td><td>300-340</td><td>400-440</td><td>Humic substances</td></tr><tr><td>C2</td><td>C</td><td>α</td><td>330-370</td><td>420-480</td><td>humic substances</td></tr><tr><td>C3</td><td>A</td><td>α'</td><td>250-270</td><td>380-480</td><td>humic substances + recent materials</td></tr><tr><td>C4</td><td>B-T</td><td>γ</td><td>270-280</td><td>300-340</td><td>Protein-like material</td></tr></tbody></table>
Table 2. Mean (± 1 s.e., n = 4) remaining mass (%) after one year *in situ* incubation of *Sphagnum* and vascular plant litters, in monoculture and in mixture (Mix). Significant difference between *Sphagnum* and the vascular plant litters decomposition are in italics and significant difference between measured and calculated decomposition in mixture are in bold italics.

<table><thead><tr><th>Incubation site</th><th>Litter</th><th>Mass remaining ± s.e.</th><th>P</th></tr></thead><tbody><tr><td rowspan="8">S. rubellum hummocks</td><td>S. rubellum</td><td>86.9 ± 0.95</td><td rowspan="2">0.007</td></tr><tr><td>M. caerulea</td><td>68.5 ± 2.56</td></tr><tr><td>Mix measured</td><td>71.1 ± 1.74</td><td rowspan="2">0.02</td></tr><tr><td>Mix calculated</td><td>77.6 ± 1.39</td></tr><tr><td>S. rubellum</td><td>86.9 ± 0.95</td><td rowspan="2">0.01</td></tr><tr><td>Betula spp.</td><td>59.6 ± 5.36</td></tr><tr><td>Mix measured</td><td>74.0 ± 1.45</td><td rowspan="2">0.88</td></tr><tr><td>Mix calculated</td><td>72.9 ± 2.19</td></tr><tr><td rowspan="8">S. cuspidatum lawns</td><td>S. cuspidatum</td><td>83.1 ± 1.66</td><td rowspan="2">0.05</td></tr><tr><td>M. caerulea</td><td>71.6 ± 2.74</td></tr><tr><td>Mix measured</td><td>71.5 ± 4.09</td><td rowspan="2">0.16</td></tr><tr><td>Mix calculated</td><td>77.2 ± 1.41</td></tr><tr><td>S. cuspidatum</td><td>83.1 ± 1.66</td><td rowspan="2">0.004</td></tr><tr><td>Betula spp.</td><td>63.7 ± 2.27</td></tr><tr><td>Mix measured</td><td>71.3 ± 1.46</td><td rowspan="2">0.64</td></tr><tr><td>Mix calculated</td><td>72.9 ± 1.96</td></tr></tbody></table>

706

707
Table 3. Mean (± 1 s.e., n = 4) remaining mass (%) after one year *in situ* incubation of *Molina caerulea* and *Betula spp.* in their native decomposition environment, in S. rubellum hummock and in S. cuspidatum lawn environments. *P* values correspond to the comparison of remaining mass of a litter type in *Sphagnum* environment to their native decomposition environment. Significant are in italics.

<table><thead><tr><th>Incubation site</th><th>Litter</th><th>Mass remaining ± s.e.</th><th>P</th></tr></thead><tbody><tr><td><em>M. caerulea</em></td><td></td><td>86.4 ± 2.84</td><td></td></tr><tr><td><em>S. rubellum</em> hummocks</td><td><em>M. caerulea</em></td><td>68.5 ± 2.56</td><td>0.0025</td></tr><tr><td><em>S. cuspidatum</em> lawns</td><td></td><td>71.6 ± 2.74</td><td>0.008</td></tr><tr><td><em>Betula spp.</em></td><td></td><td>67.4 ± 1.61</td><td></td></tr><tr><td><em>S. rubellum</em> hummocks</td><td><em>Betula spp.</em></td><td>59.6 ± 5.36</td><td>0.12</td></tr><tr><td><em>S. cuspidatum</em> lawns</td><td></td><td>63.7 ± 2.27</td><td>0.57</td></tr></tbody></table>

713

714
Table 4. Mean remaining mass after 64 days of incubation (± 1 s.e., n = 6, %), and mean water extractable organic carbon (WEOC) concentration (± 1 s.e., n = 42, mg g⁻¹ dry weight), CO₂ production (± 1 s.e., n = 42, µg C-CO₂ g⁻¹ dry weight h⁻¹), litter water content (± 1 s.e., n = 42, g H₂O g⁻¹ dry weight) and WEOC pH (± 1 s.e., n = 42) over 64 days of the laboratory experiment of *Sphagnum* spp. and vascular plant litters, in monoculture and in mixture (Mix). Significant differences are in italics.

<table><thead><tr><th></th><th><i>M. caerulea</i></th><th><i>S. rubellum</i></th><th>P</th><th>Mix measured</th><th>Mix calculated</th><th>P</th></tr></thead><tbody><tr><td>Remaining mass</td><td>96.1 ± 0.38</td><td>92.6 ± 0.52</td><td>&lt;0.0001</td><td>91.8 ± 0.53</td><td>93.8 ± 0.38</td><td>&lt;0.0001</td></tr><tr><td>WEOC</td><td>1.77 ± 0.12</td><td>3.40 ± 0.09</td><td>&lt;0.0001</td><td>3.51 ± 0.16</td><td>2.53 ± 0.09</td><td>&lt;0.0001</td></tr><tr><td>CO₂ production</td><td>19.7 ± 2.61</td><td>29.1 ± 4.58</td><td>0.06</td><td>35.9 ± 3.89</td><td>24.2 ± 2.78</td><td>0.01</td></tr><tr><td>Water content</td><td>1.28 ± 0.21</td><td>17.2 ± 0.66</td><td>&lt;0.0001</td><td>8.08 ± 0.66</td><td>8.96 ± 0.37</td><td>0.09</td></tr><tr><td>pH</td><td>5.09 ± 0.04</td><td>4.52 ± 0.08</td><td>&lt;0.0001</td><td>4.62 ± 0.06</td><td>4.80 ± 0.04</td><td>0.01</td></tr></tbody></table>

721

722
<table><caption>Table 5. Relative mean square error (RMSE) achieved when the Gogo et al. (2014) model was fitted to the data of this study.</caption><thead><tr><th></th><th>Remaining mass</th><th>WEOC</th><th>Cumulative CO<sub>2</sub></th><th>Sum</th></tr></thead><tbody><tr><td><i>S. rubellum</i></td><td>0.76</td><td>17.8</td><td>6.72</td><td>25.27</td></tr><tr><td><i>M. caerulea</i></td><td>0.16</td><td>11.5</td><td>5.89</td><td>17.58</td></tr><tr><td>Mixture measured</td><td>0.20</td><td>13.6</td><td>3.91</td><td>17.74</td></tr><tr><td>Mixture calculated</td><td>0.14</td><td>12.75</td><td>4.37</td><td>17.27</td></tr></tbody></table>
<table><caption>Table 6. Model parameters and percentage of change between measured mixture and expected mixture: (measured – expected) / expected x 100.</caption><thead><tr><th>Parameters</th><th></th><th>S. rubellum</th><th>M. caerulea</th><th>Mix. Measured</th><th>Mix. Calculated</th><th>% of difference</th></tr></thead><tbody><tr><td rowspan="4">Exo-enzyme catalysis</td><td>m<sub>c</sub></td><td>0.063</td><td>0.081</td><td>0.016</td><td>0.065</td><td>-75.5</td></tr><tr><td>a<sub>c</sub></td><td>0.0011</td><td>0.0004</td><td>8.27E-07</td><td>0.0007</td><td></td></tr><tr><td>b<sub>c</sub></td><td>0.0015</td><td>0.0019</td><td>0.0026</td><td>0.0018</td><td></td></tr><tr><td>initial rate (a<sub>c</sub> + b<sub>c</sub>)</td><td>0.0026</td><td>0.0023</td><td>0.0026</td><td>0.0024</td><td>8.1</td></tr><tr><td rowspan="4">Respiration</td><td>m<sub>r</sub></td><td>0.012</td><td>0.027</td><td>0.010</td><td>0.019</td><td>-46.1</td></tr><tr><td>a<sub>r</sub></td><td>5.52E-10</td><td>0</td><td>6.68E-10</td><td>1.38E-10</td><td></td></tr><tr><td>b<sub>r</sub></td><td>0.36</td><td>0.51</td><td>0.38</td><td>0.42</td><td></td></tr><tr><td>initial rate (a<sub>r</sub> + b<sub>r</sub>)</td><td>0.0122</td><td>0.0271</td><td>0.3793</td><td>0.4215</td><td>-10.0</td></tr></tbody></table>

729

730
**Figure captions**

**Fig. 1** Measured (mean, n = 6, dots) and modelled (lines, modelling based on Gogo et al., 2014) mass loss (a, b), WEOC (c, d) and cumulative CO₂ (e, f) in *Sphagnum rubellum* (black circles), *Molinia caerulea* (white triangles), and litters in mixture (measured – dark grey diamonds, and expected – light grey squares)

**Fig. 2** Kinetics of the exo-enzyme catalysis rate (a, b) and respiration rate (c, d) in *Sphagnum rubellum* (a) and *Molinia caerulea* (b) litters in monoculture and in measured (c) and expected mixture (d)

**Fig. 3** Relationship between SUVA₂₈₀ (L cm⁻¹ mg⁻¹, ± 1s.e., n = 6) and dissolved organic carbon (DOC, mg L⁻¹) of the WEOC extracted from *Sphagnum rubellum* (a) and *Molinia caerulea* (b) litters in monoculture and in measured (c) and expected mixture (d). The black line corresponds to the “conservation” line, where aromatic content experienced no variation (either input = output ≠ 0 or input = output = 0)

**Fig. 4** Kinetics of the C1/C2 (a, b), C3/C2 (c, d) and C4/C2 (e, f) ratios obtained after the PARAFAC analysis of the excitation-emission matrices of the WEOM extracted from *Sphagnum rubellum* (black circles) and *Molinia caerulea* (white triangles) litters in monoculture and in measured and expected mixture (measured – dark grey diamonds, and expected – light grey squares)

**Fig. 5** Relationship between modelled enzyme activity (a, b) and respiration (c, d) and the C4/C2 ratio in *Sphagnum rubellum* (black circles) and *Molinia caerulea* (white triangles) litters in monoculture and in measured and expected mixture (measured – dark grey diamonds, and expected – light grey squares). All correlations are significant (P < 0.05)

**Fig. 6** Relationship between modelled enzyme activity (a, b) and respiration (c, d) and the litter water content in *Sphagnum rubellum* (black circles) and *Molinia caerulea* (white triangles) litters in monoculture and in measured and expected mixture (measured – dark grey diamonds, and expected – light grey squares). All correlations are significant (P < 0.05).
<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

Days
<figure><img src="image_7.png" /><figcaption>Figure 2.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 3.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 4.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 5.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 6.</figcaption></figure>