Geoderma 237–238 (2014) 1–8

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Geoderma

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Both altitude and vegetation affect temperature sensitivity of soil organicmatter decomposition in Mediterranean high mountain soils

Alba Gutiérrez-Girón a,⁎, Eugenio Díaz-Pinés b, Agustín Rubio c, Rosario G. Gavilán a

a Departamento de Biología Vegetal, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spainb Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology, Garmisch-Partenkirchen, Germanyc Departamento de Silvopascicultura, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politécnica de Madrid, Madrid E-28040, Spain

⁎ Corresponding author.E-mail address: algutier@ucm.es (A. Gutiérrez-Girón).

http://dx.doi.org/10.1016/j.geoderma.2014.08.0050016-7061/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 April 2014Received in revised form 6 August 2014Accepted 9 August 2014Available online xxxx

Keywords:Respiration sensitivity to temperatureMicrobial respirationShrub encroachment

The aim of this work was to study the sensibility to warming of soil organic matter (SOM) decomposition in Med-iterranean high mountain areas. Thus, we investigated the effects of temperature, C availability and vegetation in aMediterranean high-mountain area in relation to SOM decomposition patterns. Along an altitudinal gradient (from2100 to 2380 m a.s.l.) in Central Spain mountains, we assessed the altitudinal shifts in soil organic C (SOC), soil ni-trogen (N), dissolved organic carbon (DOC),microbial biomass C (MBC),microbial respiration,microbial respirationsensitivity to temperature (Q10) and C availability index (CAI). Furthermore, we tested the differences in SOM de-composition rates between grasslands and shrub vegetation. SOC, DOC, N content, MBC, microbial respiration andCAI decreased, while Q10 increased with increasing altitude. In the grassland, MBC and microbial respiration werepositively correlated to SOM. Q10was positively correlated to pH and negatively correlated to substrate-inducedmi-crobial respiration. Soils below shrubs showed lowermicrobial respiration rates, lower CAI, and higherQ10 than soilsbelow grassland. HoweverMBC, DOC and soil N content were higher below shrubs. The results suggest that a rise intemperature would enhance SOM decomposition rates in grasslands more dramatically at higher altitudes, sincethey are more sensitive to temperature increases. The SOC accretion observed below shrubs may be due to thelower respiration rate of soil microorganisms, possibly determined by lower C substrate availability below shrubs.This result suggests a higher recalcitrance of shrub litter compared to grassland litter. Nevertheless, SOC in shrublandmay be released at a higher rate due to its higher temperature sensitivity.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Soil organic matter represents an important carbon reservoir in theterrestrial biosphere. It is estimated that SOC is roughly double that ofthe atmosphere or aboveground vegetation (Schimel, 1995). In soils in al-pine areas – as in other cold ecosystems – C stocks are particularly impor-tant, since low temperatures limit decomposer activity (Schlesinger andAndrews, 2000) and keep C immobilized in soils during long periods oftime (Körner, 2003). The forecasted global climate change would haveimportant effects on the SOM decomposition, and these effects are ex-pected to be greater in soils in the coldest regions (Melillo et al., 2002).Changes in SOMdecomposition rates could result in changes in biochem-ical cycles thatwould affect the structure and functioning of these ecosys-tems. Indeed changes in the structure and function of the ecosystemswould alter carbondioxide (CO2) efflux from the soil into the atmosphere,provoking positive feedbacks, enhancing the greenhouse effect. Never-theless, it is very uncertain whether ecosystems turn from a sink to asource – or vice versa – under climate change conditions, since several

factors affect the net balance between C loss through SOMdecompositionand C gain from primary productivity (Bardgett, 2005; Davidson andJanssens, 2006; Powlson et al., 2011).

SOM decomposition can be understood as a function of microbialcommunity (in terms of microbial biomass and specific composition)and activity (decomposition), and their dependence on environmentalfactors. Such environmental factors are mainly soil moisture (Chenet al., 2000; Schimel et al., 1999), soil temperature (Bekku et al., 2004;Kirschbaum, 1995), and substrate quantity (Vance and Chapin, 2001)and quality (Nadelhoffer et al., 1991; Vance and Chapin, 2001). Oneissue of long debate is the effect of increasing temperatures on SOMde-composition, since microbial respiration is sensitive to temperaturechanges. Specifically, soils in cold ecosystems often show higher tem-perature sensitivity of SOM decomposition when compared withother ecosystems (Bekku et al., 2004), making them more vulnerableto the impacts of increasing temperature. However, the factors deter-mining the temperature sensitivity of SOM decomposition are stillunder debate (Fierer et al., 2006; Vanhala et al., 2008). Existing researchalong altitudinal gradients showed controversial results with regard tothe temperature sensitivity of SOMdecomposition. Increases in temper-ature sensitivity have been observed with increasing elevation in soiland microbial respiration by Sjögersten and Wookey (2002), Kätterer

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et al. (1998) and Niklińska et al. (1999); while other authors have re-ported higher temperature sensitivity at lower altitudes (Lipson,2007), or found no differences between altitudes (Niklińska andKlimek, 2007; Schindlbacher et al., 2010). Although the knowledge ofthe environmental drivers of SOM decomposition in cold climateregions has improved in the last decade (e.g. Bekku et al., 2004;García-Pausas et al., 2008; Lipson, 2007; Lipson et al., 2000) little isknown about SOM decomposition responses to environmental factorsin Mediterranean high-mountain areas. Mediterranean ecosystemsshow particular environmental conditions, mainly driven by summerdrought, that make their functioning different to temperate and coldecosystems, and they may be particularly affected by climate warmingdue to temperature and rain pattern changes (Engler et al., 2011;Schröter et al., 2005).

High-mountain environments harbor ecosystems that are very sen-sitive and vulnerable to global changes (Schröter et al., 2005). Shrub en-croachment has occurred in recent decades in several Iberianmountainareas (Molinillo et al., 1997; Roura-Pascual et al., 2005), even in the areasurrounding the study site (Muñoz-Jiménez and García-Romero, 2004;Sánz-Elorza et al., 2003) due to land use and climate changes. Shrub en-croachment into mesic grasslands in the Pyreneanmountains showed aslight increase in SOC stocks (Montané et al., 2007) related to a higherrecalcitrance of shrub litter relative to grass litter (Montané et al.,2010). Nevertheless, the particular climate conditions of Mediterraneanareas that cause the dominance of dry grasslands in the study area maygive rise to different effects of vegetation changes on SOM decomposi-tion to those reported in northern mountains (Jackson et al., 2002).We are not aware of any study assessing the effects of vegetation chang-es on SOM dynamics in Mediterranean high-mountain soils.

Altitudinal gradients inmountains are correlatedwith climate gradi-ents and changes on vegetation communities. Therefore altitudinalshifts in SOM decomposition and its dependence on environmentalfactors make these comparisons useful for the assessment of climate-and vegetation-related impacts (Diaz et al., 2003; Niklińska andKlimek, 2007; Schindlbacher et al., 2010). The aim of this study is toevaluate the mechanisms responsible for differences in SOM decompo-sition patterns along an altitudinal gradient in Mediterranean high-mountains, involving both temperature shifts and changes in the vege-tation community, as a proxy for detecting feedbacks due to climatechange. Specifically, we evaluated the altitudinal shifts in SOC andtotal soil N, DOC and MBC. We also assessed the altitudinal shifts ofmicrobial respiration, temperature sensitivity and C substrate limi-tation. Finally, we tested for differences in soil and microbial pa-rameters between shrub and grassland vegetation to evaluate thepotential effects on SOM decomposition patterns due to shrub en-croachment in Mediterranean high-mountain grasslands. We hy-pothesized a decrease in SOM and therefore in SOC and soil Ncontent with increasing altitude, as plant activity is expected to bemore strongly limited by low temperatures than microbial activity(García-Pausas et al., 2007). Moreover, we expected a decrease inboth microbial respiration and C availability with elevation (Fiereret al. (2009)). Additionally, we surmised that the soil microbial res-piration in colder sites were more sensitive to temperature in-creases than those of warmer sites (Schindlbacher et al., 2010;Vanhala et al., 2008), and therefore Q10 of microbial respirationwould increase with elevation. With regard to differences in shruband grassland vegetation we hypothesized that – as proposed byJackson et al. (2002) for dry plant communities – in shrubland veg-etation, organic C pools were larger than in grasslands.

2. Materials and Methods

2.1. Study Site

The study was conducted in the Sierra de Guadarrama (40° 47′N, 3°57′ W), a mountain range located in Central Spain. Parent material of

the area consists of siliceous plutonic andmetamorphic rocks (i.e. gran-ite and gneiss). The vegetation in the area is constituted by shrub com-munities of the broom Cytisus oromediterraneus Rivas Mart. et al. anddwarf juniper Juniperus communis subsp alpina (Suter) Čelak., andshort and dry perennial grasses dominated by Festuca curvifolia Lag. exLange. Above the tree-line limit (1900–2000 m a.s.l.), both shrub andgrass communities form amosaic. Over 2200ma.s.l, the vegetation is al-most dominated by grasses of F. curvifolia accompanied by cushionplants. The soils at the sites are Umbrepts and Orthents under shrubcommunities, and Cryumbrets under high-mountain grassland commu-nities (Hoyos et al., 1980).

At the nearest weather station (2.3 km away), located in theNavacerrada pass at 1890 m a.s.l., the mean annual temperature is6 °C and annual precipitation is 1350 mm, with a slight dry periodfrom May to October with less than 10% of the total annual rainfall.These Mediterranean high-mountain ecosystems can be under snowcover from late October to mid April; however, the snow cover lengthgreatly varies between years as a consequence of varying Mediterra-nean climate conditions, and it often has a discontinuous duration dur-ing the winter due to the heterogeneous snow fall and wind effects.These ecosystems are also subjected to short growing seasons, lowwin-ter temperatures and strong winds.

2.2. Soil Temperature Measurements

Soil temperatures under shrubland and grassland vegetation wereassessed by means of temperature records from the GLORIA target re-gion in the Sistema Central mountains (www.gloria.ac.at). We usedthe records from 16 temperature dataloggers (Geoprecision M-log5W) buried at 10 cmdepth and distributed in the fourmain compassdirections (Pauli et al., 2004) on four summits in the Sierra deGuadarrama mountains ranging from 2100 to 2280 m a.s.l. In order toassess the soil temperature conditions, we calculatedmean annual tem-perature, mean temperature of the coldest month and mean tempera-ture of the warmest month using soil temperature records of 2008and 2009. The temperature records of two extra dataloggersinstalled at a depth of 10 cm in the highest site (i.e. 2380 m a.s.l.)were used to calculate the soil temperature variation linked to thealtitudinal gradient.

2.3. Sampling Collection

To assess altitudinal shifts on SOMdecompositionwe collected sam-ples in early July 2009 at four different altitudes along an altitudinal gra-dient (i.e., 2100, 2200, 2300 and 2380 m a.s.l.). At each altitudinal level,six 1 × 1 m2 plots with a well-developed vegetation cover wereselected. Inside each plot we randomly collected one soil block of10 × 10 × 10 cm3 for measurements of soil parameters and microbi-al activity.

To compare the vegetation effect (grasses vs shrubs) on soil andmicrobial parameters we collected mineral soil samples underC. oromediterraneus shrubs in sites at 2100 m a.s.l. and 2200 m a.s.l.where this type of vegetation was present. We randomly selectedsix shrub individuals of C. oromediterraneus close to each of the 1× 1 m2 plots of grassland previously sampled (6 shrubs for each al-titude and 12 in total). One soil block sample of 10× 10× 10 cm3was col-lected closer to the center of each individual shrub, under each of the 12shrubs.

After collection, all the soil samples were kept cold during trans-portation to the laboratory. Soil samples were passed through a2 mm-sieved, roots were manually removed and a sufficient quantityof soil samples was preserved frozen at −20 °C until further analyses.Prior to microbial measurements started, soil samples were thawedduring 24 h at 5 °C.

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2.4. Soil Parameter Measurements

Soil organic carbon (SOC mg C g−1 soil dw) was measured using aTotal Organic Carbon Analyzer TOC 5000A (Shimadzu, Kyoto, Japan).Total soil N (Nsoil mg N g−1 soil dw) was determined by the Kjeldahlmethod, followed by determination of NH4

+–N in distillate by titration(SM 702 Trinitro, Metrhohm AG, Switzerland) (Kandeler, 1995). SoilpH measurements were made in 1:2.5 soil suspension of soil:water (g:g) (pH-meter micro pH2000, CRISON, Barcelona, Spain). Soil water con-tent (SWC % g g−1 soil dw) was determined gravimetrically by drying2–3 g of fresh soil at 105 °C during 24 h.

2.5. Microbial Biomass Measurements

Microbial biomass was measured by the fumigation–extractionmethod (Brookes et al., 1985; Vance et al., 1987). Soil samples (10 gfresh weight) were extracted in 50 ml 0.5 M K2SO4 for 30 min on anautomatic shaker. Simultaneously with the extraction, another sub-sample (10 g fresh weight) was fumigated with chloroform for 24 h at25 °C to provoke the lysis of themicrobial cells and release of microbialintracellular C. Because in dry soil microorganisms cells are less affectedby chloroform (Sparling andWest, 1989), we rewetted the soil samplesto 25% of gravimetric SWC immediately before the fumigation, as rec-ommended by Sparling and West (1989). Subsequently, fumigatedsamples were extracted similarly to unfumigated samples. Extracted Cwas determined in extracts using a Total Organic Carbon Analyzer TOC5000A (Shimadzu, Kyoto, Japan). Microbial C flush (difference in ex-tractable C between unfumigated and fumigated samples) was convert-ed to microbial biomass C (MBC μg C g−1 soil dw) using an extractionfactor of 0.35 (Sparling et al., 1990). Dissolved organic C of soil(DOC μg C g−1 soil dw) was determined as the extracted C of theunfumigated samples (Dannenmann et al., 2009).

2.6. Microbial Respiration, Temperature Sensitivity and C-Limitation ofMicrobial Activity

For soil respirationmeasures, each sample was adjusted to 60% of itssoil–water field capacity. In 500 ml sealed jars, 15 g of soil was incubat-ed in dark conditions at 22 °C, 17 °C, 15 °C and 7 °C during 24h, 48 h, 96h and 144 h respectively. CO2 emission was measured by the titrationmethod (Öhlinger, 1995). An alkali trap of 15ml NaOH 0.1 Nwas placedinside each jar to absorb the CO2 produced. After incubation, 5 ml ofBaCl2 0.5 Nwas added to each trap, and consumption of NaOHwasmea-sured by titration (SM 702 Trinitro, Metrohm AG, Switzerland) with aHCl 0.1 N solution.

Substrate-induced respiration (RSIR) was measured following a modi-fied method from Anderson and Domsch (1978). 15 g soil samples wereset to 60% of their soil–water field capacity and 10 mg glucose g−1 freshsoil was added to each sample using a mixture of glucose and talcumpowder (1:3 w:w). Samples were incubated in dark conditions for 4 hat 22 °C, and CO2 emission was measured by the titration method(Öhlinger, 1995) as previously described. To calculate the respirationrate of soil samples at each temperature we used the formula:

Rr ¼ Vblank−Vsampleð Þ � N � 22 � 1000dw � t

where Rr is the respiration rate of soil, i.e. CO2 flux (mg CO2 kg−1 h−1);Vblank (ml) is the volume of HCl used in the blank, Vsample (ml) is thevolume of HCl used in the sample, N is the normality of the HCl solution,22 is a conversion factor (1ml of HCl 1.0 N corresponds to 22mg of CO2),dw is the dry weight (g) of the soil sample and t (hours) the incubationtime.

C limitation of soil microbial activity was evaluated calculating the Cavailability index (CAI) (Cheng et al., 1996). CAI was calculated as:

CAI ¼ R22 ¨C

RSIR

where R22 °C is the respiration rate at 22 °Cwithout added substrate andRSIR is the respiration rate with added glucose at 22 °C.

Temperature sensitivity of microbial respiration was assessed calcu-lating the Q10 values of each sample, considering respiration values atthe four experimental temperatures (i.e. 7 °C, 12 °C, 17 °C and 22 °C).To calculate the Q10 values of each sample, we first fitted the measuredrespiration rates to the van't Hoff equation (1898):

Rr ¼ αeβT

where α and ß are fitted parameters and T is the temperature. Then Q10

was calculated for each sample using the formula:

Q10 ¼ eβ10:

The function for the Q10 estimation was calculated using Sigmaplot10.0 (Systat Inc., Point Richmond, California, USA).

2.7. Statistical Analysis

Normality and homogeneity of variances were checked for all data.We carried out polynomial regression analysis based on first and secondorder models to test for variation along the altitudinal gradient of SOC,Nsoil, soil C:N ratio, DOC, MBC, R22 °C and RSIR, Q10 and CAI values. Thevalues of shrub samples were not included in the regression models toavoid confusing effects with vegetation. We selected best-fit equationsto assess the altitudinal trends of the variables studied. Differences insoil andmicrobial parameters between grassland and shrubland vegeta-tion were tested by means of two-way ANOVA analysis, with vegetationand altitude as themain factors and the interaction termvegetation× al-titude.When the interaction termwas significant, the differences at eachaltitude were tested by means of a Student's t-test. Differences in soiltemperatures between grassland and shrubland were tested by meansof ANCOVA analysis, considering altitude as a covariate. All statisticalanalyses were done using SPSS v15 (SPSS Inc., Chicago, Illinois, U.S.A.).

3. Results

3.1. Elevation Shifts of Soil Factors and Microbial Activity in Grasslands

Within the altitudinal gradient, the mean annual soil tempera-ture in the study area varied by 1.9 °C. At the sampling date, theSWC did not show any significant variation along the altitudinal gra-dient (Fig. 1a, P = 0.127), and showed relatively low values that did notexceed on average 9% (±2.25 SE), indicating that water shortage in sum-mer occurs even at the highest altitudes.

The results showed significant shifts in most of the soil parametersstudied due to the altitude (Fig. 1). The altitudinal shifts in Nsoil andSOC (P b 0.001 and P = 0.001 respectively, Fig. 1b, c) indicated a de-crease in SOMwith altitude. DOC shifts (P= 0.016) showed that the la-bile C fraction decreasedwith altitude (Fig. 1d). The increase in C:N ratiowith increasing altitude (P= 0.032, Fig. 1e) indicated an altitudinal var-iation in the quality of SOM.

The results revealed significant altitudinal shifts in microbialbiomass, microbial activity and their dependence on C availability andtemperature (Fig. 2). MBC showed a decrease with increasing altitude(P = 0.04, Fig. 2a). Similarly, microbial basal respiration and substrate-induced microbial respiration rates (i.e. R22 °C and RSIR respectively)decreased with altitude (P b 0.001 and P b 0.001 respectively, Fig. 2b,c). A shift in Q10 showed that the temperature sensitivity of microbial

Fig. 1.Altitudinal shifts in soil properties in grassland and vegetation effects. Changes in soil water content (SWC) (a), Nsoil (b), SOC, (c), dissolved organic carbon (DOC) (d), soil C:N ratio (C:N)(e) and pH (f) along the altitudinal gradient. Error bars represent standard errors (n=6). Results of polynomial regression (second order model) and linear regression are given. Regressionmodels do not include soil samples below shrubs to avoid confusing the effects of vegetation types. Soil samples below grassland (solid symbols), soil samples below shrubs (open symbols).

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respiration significantly increased with altitude (P= 0.004, Fig. 2d). Fur-thermore, CAI values indicated that substrate addition strongly affectedmicrobial respiration rates, often by an order of magnitude. Moreover,the altitudinal shift in CAI shows that soils at higher altitudes weremore C-substrate limited (P = 0.037, Fig. 2e).

3.2. Correlation of Soil Properties andMicrobial Parameters in Grassland Soils

MBC was positively related to microbial respiration rates (i.e. RSIR

and R22 °C) and to SOC, DOC and Nsoil (Table 1). Additionally, RSIR

and R22 °C were also related to soil pH. Soil C:N ratio and SWC didnot show any correlation with microbial parameters, but C:N was posi-tively correlated to pH (Table 1). Q10 was only negatively correlated toRSIR and positively correlated to pH (Table 1). Finally, CAI was positivelycorrelated to R22 °C but not to RSIR (Table 1).

3.3. Differences in Soil Properties andMicrobial Activity Between Shrub andGrassland

The results indicated significant differences in soil microclimaticconditions (Tables 2 and 3), SOM properties and microbial parametersbetween shrubs and grasses (Figs. 1 and 2, Table 3). Soils below shrubsremained cooler in the warmest month and warmer in the coldest

month (Table 2). Soil moisture below shrubs was significantly higherat the sampling date (Table 3). Comparisons of soil and microbial pa-rameters between shrubland and grassland showed that while SOC,Nsoil, DOC and MBC were significantly higher below shrubs, R22 °C rateand CAI were significantly lower (Table 3). In addition soil C:N ratiodid not show significant differences when comparing both vegetationtypes (Table 3). Furthermore, pH and RSIR showed a significant interac-tion between vegetation type and altitude (Table 3). At 2100m a.s.l. RSIR

was significantly higher below shrubs (P = 0.008), but at 2200 m a.s.l.there was no significant difference between the plant canopies (P =0.385). Altitudinal differences in pH were marginally significant, and acomparison of vegetation cover showed significantly lower values at2200 m a.s.l., while at 2100 m a.s.l. there was no significant differencebetween grassland and shrublands. Soil respiration below shrubs exhib-ited significantly higher sensitivity to temperature than soil respirationbelow grasslands.

4. Discussion

4.1. Altitudinal Trends in SOM Decomposition

As we hypothesized, we observed a decline in SOM contents andmicrobial respiration rates with increasing altitude within grasslands.

Fig. 2. Altitudinal shifts in microbial parameters in grassland and vegetation effects. Changes in MBC (a), R22° C (b), RSIR (c), Q10 (d) and CAI (e) along the altitudinal gradient. Error barsrepresent standard errors (n=6). Results of polynomial regression (second ordermodel) and linear regression are given. Regressionmodels do not include soil samples below shrubs toavoid confusing the effects of vegetation types. Soil samples below grassland (solid symbols), soil samples below shrubs (open symbols).

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The decrease in organic C pools (i.e. SOC, DOC, andMBC) with elevationwas in accordance with the SOM altitudinal shifts observed by García-Pausas et al. (2007), Cioci et al. (2008) and Djukic et al. (2010a). Altitu-dinal shifts in SOM decomposition rates (i.e. R22 °C and RSIR) were con-sistent with the decrease observed in other cold ecosystems in the

Table 1Pearson's correlations between soil and microbial parameters (n = 24). Correlation tests doSignificance levels are (*) P b 0.05; (**) P b 0.01; (*** )P b 0.001.

Nsoil SOC C:N pH SWC

Nsoil •

SOC 0.984*** •

C:N ns ns •

pH −0.543** −0.486* 0.450* •

SWC ns ns ns ns •

DOC 0.745*** 0.757*** ns ns nsMBC 0.705*** 0.740*** ns ns nsR22 0.746*** 0.709** ns −0.455* nsRSIR 0.741*** 0.716*** ns −0.533* nsQ10 ns ns ns 0.515* nsCAI ns ns ns ns ns

Alaskanmountains (Sveinbjörnsson et al., 1995). Our results also showeda positive relationship between SOMandmicrobial biomass, andmicrobi-al activity in accordance with Fierer et al. (2009). Although soil N avail-ability may affect soil microbial activity (Allen and Schlesinger, 2004;Joergensen and Scheu, 1999; Yoshitake et al., 2007), the slight increase

not include soil samples below shrubs to avoid confusing the effects of vegetation types.

DOC MBC R22 °C RSIR Q10

•

0.673*** •

0.578** 0.526** •

0.461* 0.561** 0.682*** •

ns ns ns −0.623** •

ns ns 0.714*** ns ns

Table 2Soil temperature (10 cmdepth)differences betweengrass and shrub vegetation after con-sidering altitude as covariate. Significance levels are (*) P b 0.05; (**) P b 0.01; (***)P b 0.001. Period of time considered: from 2008 to 2009. For grassland vegetationn = 22, for shrubland vegetation, n = 10.

Temperatures Vegetation Altitude Mean (SE)

F P F P Grassland Shrubland

Mean annual temperature°C

4.379 * 1.90 ns 6.51 (0.19) 5.55 (0.26)

Mean temperature of thecoldest month °C

13.63 *** 2.71 ns −0.58 (0.12) 0.38 (0.16)

Mean temperature of thewarmest month °C

33.12 *** 5.41 * 18.39 (0.27) 17.26 (0.54)

6 A. Gutiérrez-Girón et al. / Geoderma 237–238 (2014) 1–8

in the soil C:N ratio with elevation (which may indicate a SOM qualityshift), was not relevant for microbial activity and therefore for SOM de-composition along the altitudinal gradient.

Alongwith the decrease in SOC decomposition rates, the variation inCAI indicated an increase in C substrate limitation for soil microbial ac-tivitywith elevation. Therefore, our results suggest that,microbial respi-ration is strongly constrained by C availability, since the range of CAIvalues indicated differences of an order of magnitude between RSIR

and R22 °C. This result agrees with García-Pausas and Paterson (2011),who observed that SOM decomposition was strongly regulated by Cavailability in the surface horizons of soils in the Pyrenean mountains.Furthermore, DOC has often been considered a good indicator of C sub-strate for microbial activity (Cook and Allan, 1992; Jandl and Sollings,1997; Smolander and Kitunen, 2002) and the significant correlation be-tween DOC and MBC and microbial respiration rates supports their tightrelationship. Nevertheless, one of the key issues which remains unclearis the factors controlling the bioavailability of DOC as a substrate for mi-crobial respiration.

Our temperature sensitivity of microbial respiration showed valueswithin the range of those reported in arctic and alpine ecosystems(e.g. Bekku et al., 2004; Lipson et al., 2002). Q10 values in our studyarea were higher than values calculated in temperate ecosystems (seethe review of Q10 values by Bekku et al., 2004), and also higher thanthose reported by Díaz-Pinés et al. (2014) in lowlands (about 1200m a.s.l.) for pine and oak forests of Central Spain. The increase in Q10

values alongwith elevation is consistentwith a decrease in soil temper-ature with elevation, and therefore agrees with the higher temperaturesensitivity found at sites with lower soil temperatures reported byVanhala et al. (2008) and Schindlbacher et al. (2010).

Temperature sensitivity of SOM decomposition is affected by otherfactors, such as substrate availability (Fierer et al., 2006; Gershensonet al., 2009) and microbial community composition (Lipson, 2007;Lipson et al., 2002). The results of the correlations showed that withingrasslands, Q10 was not affected by C substrate availability, asmeasuredby CAI. Nevertheless Q10 was positively correlated to pH and negativelycorrelated to RSIR. Soil pH variations have often been related tomicrobial

Table 3Results of vegetation effects on soil properties and microbial activity tested by two-way ANOVP-values (0.05 b P b 0.1) are indicated as (ns). For vegetation n = 12, for altitude n = 12.

Vegetation Altitude

F P F P

Nsoil 16.1 *** 14.5 ***SOC 11.5 *** 14.7 ***C:N 2.9 ns 0.64 nspH 34.2 *** 3.6 (ns)DOC 24.3 *** 2.3 nsSWC 49.9 *** 0.05 nsMBC 16.9 *** 10.8 ***R22 11.8 *** 14.6 ***RSIR 4.4 * 11.2 ***CAI 19.5 *** 6.9 **Q10 9.2 *** 4.7 *

community structure, and are particularly related to the bacteria:fungiratio in soils (Baath and Anderson, 2003; Blagodatskaya and Anderson,1998; Djukic et al., 2010b); while RSIR is an index of potential microbialrespiration under optimum conditions, and showed relationships withmicrobial community structure (Lipson, 2007; Lipson et al., 2002).These significant correlations therefore suggest that Q10 variationare related to changes in the soil microbial community structurealong the altitudinal gradient, as observed by Djukic et al. (2010b),which might be driven by the soil temperature variation along thealtitudinal gradient.

Although altitudinal gradient correlates to variations in temperatureas well as to precipitation, in this study we did not assess the effects ofsoil moisture on RSIR and R22 °C, as we measured samples whosemoisture was set at the optimum in the laboratory. Moreover, theSWC of fresh samples showed low and very similar values among alti-tudes, since the summer drought had already started by the samplingdate. Therefore we were unable to detect any relationship between theSWC of fresh samples and the altitudinal shifts of soil and microbialparameters.

4.2. Vegetation Effects on SOM Decomposition

Upon our results, plant community composition clearly affects SOMdecomposition patterns in the uppermost 10 cm of the soil profile. Asexpected, soils below shrubs showed the highest values of every Cpool measured. The differences in SOC observed when comparingshrub and grasslands were larger than those reported by Montanéet al. (2007). Jackson et al. (2002) proposed that differences in SOC con-tent between grassland and shrub vegetationwould depend on climaticconditions and predicted a SOC enhancement after encroachmentunder dry climates. Therefore the greater differences in SOC that we ob-served when comparing grassland and shrub vegetationmay be relatedto climatic differences, since most soil samples included in the study ofMontané et al. (2007) were frommesic grasslands, whereas we studieddry grasslands.

Montané et al. (2010) proposed that the higher SOC contents ob-served under shrub communities may be related to a higher recalci-trance of shrub litter, since they observed lower SOM decompositionrates in shrub litter. In accordance with their findings, we observedthat SOM decomposition was lower below shrubs (Fig. 2). Higher recal-citrance of shrubland SOC is further supported by the greater C substratelimitation observed below shrubs in comparison to grasslands. More-over, the higher Q10 values observed in soils are consistent with the“carbon quality-temperature” hypothesis (Fierer et al., 2005, 2006)that predicts a higher temperature sensitivity of microbial activitywhen increasing recalcitrance of soil C substrates. On the other hand,the significantly higher values of DOC observed below shrubsmay indi-cate that a part of DOC may be resistant to microbial attack, and mayconstitute a non-labile fraction of DOC, as shown by Kalbitz et al.

A. Significance levels are (*) P b 0.05; (**) P b 0.01; (***) P b 0.001. Marginally significant

Vegetation × altitude Mean (SE)

F P Grassland Shrubland

0.52 ns 5.40 (0.48) 7.41 (0.41)0.31 ns 67.4 (5.9) 89.5 (5.7)0.59 ns 12.5 (0.19) 12.0 (0.20)

28.7 *** 4.9 (0.07) 4.6 (0.04)0.21 ns 407 (27) 681 (54)0.18 ns 5.7 (0.5) 16.0 (1.3)0.05 ns 1849 (173) 2741 (189)0.13 ns 16.1 (1.3) 11.3 (1.1)

11.2 *** 125 (4.3) 137 (7.0)0.68 ns 0.131 (0.010) 0.082 (0.005)3.9 (ns) 3.33 (0.14) 3.97 (0.19)

7A. Gutiérrez-Girón et al. / Geoderma 237–238 (2014) 1–8

(2003) after testing the biodegradability of DOC in different types ofsoils. The non-labile fraction of DOC has been reported to be often con-stituted by phenolic compounds which frequently originate from mi-crobial lignin degradation (Kalbitz and Kaiser, 2008). In addition, theunexpected absence of any difference in the soil C:N ratio observed be-tween leguminosae shrubs and grasslands may be related to the afore-mentioned higher chemical recalcitrance of C substrate under shrubs,whichmay further enhance SOC contents more strongly than N contentis enhanced by the quality of the leguminosae litter (McCulley et al.,2004). Finally, an alternative factor related to the lower SOM decompo-sition observed below shrub may be a potential negative effect of N indecomposition of recalcitrant compounds like lignin, as recently ob-served by Duboc et al. (2014) in histosols along an alpine gradient.

In viewof the increasing interest in C sequestration andmitigation ofclimate change processes we want to emphasize that our results showthat the potential mitigation of CO2 emissions to the atmosphere dueto shrub encroachment in the study area is still uncertain (Powlsonet al., 2011). Our results show that shrub encroachment of F. curvifoliagrasslands may contribute to enhanced SOC accumulation. However,in the frame of climate change and increasing temperatures we ob-served that soils under shrub vegetation show higher temperaturesensitivity.

5. Conclusions

Our findings suggest that an increase in temperature will accelerateSOM decomposition in the study area particularly in soils at a higheraltitude which are more sensitive to soil temperature changes interms of soil respiration. Moreover the results showed that C cyclingmay be strongly altered if temperature increases were also to cause en-croachment of grasslands, as the shrub vegetation determines changesin microclimatic soil conditions, C substrate availability, temperaturesensitivity of the microbial community and SOM decomposition rates.The higher SOC content in shrubland may be determined by the slowerSOM decomposition in shrublands related to a lower C-substrate avail-ability for microbial activity. However, SOC in shrubland may be releasedat a higher rate due to its higher temperature sensitivity.We propose thatdifferences in SOMdecomposition responses to the environmental factorsmight be related to different C substrate qualities and differences incomposition of the microbial community.

Acknowledgments

We thank the staff of the Cuenca Alta del Manzanares NatureReserve for the permission to work in this area. We thank BeatrizOrtiz and Dr. Paz Andrés for the help with the methodology of soilanalysis. We also thank Prudence Brooke-Turner for the English re-vision. This study was supported financially by the projects CGL2008-901/BOS and AGL2010-16862/FOR from the Ministry of Sci-ence and Technology, and by the pre-doctoral grant FPU-2005-0999 to AGG, funded by the Spanish Ministry of Education.

References

Allen, A.S., Schlesinger, W.H., 2004. Nutrient limitations to soil microbial biomass andactivity in loblolly pine forests. Soil Biol. Biochem. 36, 581–589.

Anderson, J.P.E., Domsch, K.H., 1978. A physiological method for the quantitativemeasurement of microbial biomass in soils. Soil Biol. Biochem. 10, 215–221.

Baath, E.,Anderson, T.H., 2003. Comparison of soil fungal/bacterial ratios in a pH gradientusing physiological and PLFA-based techniques. Soil Biol. Biochem. 35, 955–963.

Bardgett, R.D., 2005. The Biology of Soil. A Community and Ecosystem Approach. OxfordUniversity Press, Oxford.

Bekku, Y.S.,Nakatsubo, T.,Kume, A.,Koizumi, H., 2004. Soil microbial biomass, respirationrate, and temperature dependence on a successional glacier foreland in Ny-Ålesund,Svalbard. Arct. Antarct. Alp. Res. 36, 395–399.

Blagodatskaya, E.V.,Anderson, T.H., 1998. Interactive effects of pH and substrate quality onthe fungal-to-bacterial ratio and qCO2 of microbial communities in forest soils. SoilBiol. Biochem. 30, 1269–1274.

Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation andthe release of soil nitrogen: a rapid direct extraction method to measure microbialbiomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842.

Chen, H.,Harmon, M.E.,Griffiths, R.P.,Hicks, W., 2000. Effects of temperature and moistureon carbon respired from decomposing woody roots. For. Ecol. Manag. 138, 51–64.

Cheng, W., Zhang, Q., Coleman, D.C., Ronald Carroll, C.,Hoffman, C.A., 1996. Is availablecarbon limiting microbial respiration in the rhizosphere? Soil Biol. Biochem. 28,1283–1288.

Cioci, C., Corti, G.,Agnellia, A., Cocco, S., 2008. Role of the altitude on the organic matterpreservation in soils under a secondary prairie on the Majella massif (Italy).Agrochimica 42, 20–32.

Cook, B.D.,Allan, D.L., 1992. Dissolved organic carbon in old field soils: total amounts as ameasure of available resources for soil mineralization. Soil Biol. Biochem. 24,585–594.

Dannenmann, M.,Simon, J.,Gasche, R.,Holst, J.,Naumann, P.,Kögel-Knabner, I.,Knicker, H.,Mayer, H.,Schloter, M.,Pena, R.,Polle, A.,Rennenberg, H.,Papen, H., 2009. Tree girdlingprovides insight on the role of labile carbon in nitrogen partitioning between soilmicroorganisms and adult European beech. Soil Biol. Biochem. 41, 1622–1631.

Davidson, E.A., Janssens, I.A., 2006. Temperature sensitivity of soil carbon decompositionand feedbacks to climate change. Nature 440, 165–172.

Diaz, H.F.,Grosjean, M.,Graumlich, L., 2003. Climate variability and change in high elevationregions: past, present and future. Clim. Chang. 59, 1–4.

Díaz-Pinés, E.,Schindlbacher, A.,Godino, M.,Kitzler, B.,Jandl, R.,Zechmeister-Boltenstern, S.,Rubio, A., 2014. Effects of tree species composition on the CO2 and N2O efflux of aMediterranean mountain forest soil. Plant Soil. http://dx.doi.org/10.1007/s11104-014-2200-z.

Djukic, I., Zehetner, F., Tatzber, M.,Gerzabek, M.H., 2010a. Soil organic-matter stocks andcharacteristics along an Alpine elevation gradient. J. Plant Nutr. Soil Sci. 173, 30–38.

Djukic, I.,Zehetner, F.,Mentler, A.,Gerzabek, M.H., 2010b. Microbial community compositionand activity in different Alpine vegetation zones. Soil Biol. Biochem. 42, 155–161.

Duboc, O.,Dignac, M.-F.,Djukic, I.,Zehetner, F.,Gerzabek, M.H.,Rumpel, C., 2014. Lignin de-composition along an Alpine elevation gradient in relation to physicochemical andsoil microbial parameters. Glob. Chang. Biol. 20, 2272–2285.

Engler, R.,Randin, C.F.,Thuiller, W.,Dullinger, S.,Zimmermann, N.E.,Araújo, M.B.,Pearman,P.B., Le Lay, G.,Piedallu, C.,Albert, C.H.,Choler, P., Coldea, G.,De Lamo, X.,Dirnböck, T.,Gégout, J.-C.,Gómez-García, D.,Grytnes, J.-A.,Heegaard, E.,HØIstad, F.,Nogués-Bravo,D.,Normand, S., Puscas, M., Sebastiá, M.-T., Stanisci, A., Theurillat, J.-P., Trivedi, M.R.,Vittoz, P.,Guisan, A., 2011. 21st century climate change threatens mountain flora un-equally across Europe. Glob. Chang. Biol. 17, 2330–2341.

Fierer, N., Craine, J.M.,McLauchlan, K., Schimel, J.P., 2005. Litter quality and the tempera-ture sensitivity of decomposition. Ecology 86, 320–326.

Fierer, N.,Colman, B.P.,Schimel, J.P.,Jackson, R.B., 2006. Predicting the temperature depen-dence of microbial respiration in soil: a continental-scale analysis. Glob. Biogeochem.Cycles 20, GB3026 (1–10).

Fierer, N.,Strickland, M.S.,Liptzin, D.,Bradford, M.A.,Cleveland, C.C., 2009. Global patternsin belowground communities. Ecol. Lett. 12, 1238–1249.

García-Pausas, J., Paterson, E., 2011. Microbial community abundance and structure aredeterminants of soil organic matter mineralisation in the presence of labile carbon.Soil Biol. Biochem. 43, 1705–1713.

García-Pausas, J.,Casals, P.,Camarero, L.,Huguet, C.,Sebastià, M.-T.,Thompso, R.,Romanyà, J.,2007. Soil organic carbon storage in mountain grasslands of the Pyrenees: effects of cli-mate and topography. Biogeochemistry 82, 279–289.

García-Pausas, J.,Casals, P.,Camarero, L.,Huguet, C.,Thompson, R.,Sebastià, M.-T.,Romanyà,J., 2008. Factors regulating carbon mineralization in the surface and subsurface soilsof Pyrenean mountain grasslands. Soil Biol. Biochem. 40, 2803–2810.

Gershenson, A., Bader, N.E., Cheng, W., 2009. Effects of substrate availability on thetemperature sensitivity of soil organic matter decomposition. Glob. Chang. Biol. 15,176–183.

Hoyos, A.,Hernando, J.,Cuchi, M.J.,Egido, J.A., 1980. Caracterización de suelos del SistemaCentral. II. Umbrepts. Anales de edafología y agrobiología. 39, pp. 721–741.

Jackson, R.B.,Banner, J.L., Jobbagy, E.G.,Pockman, W.T.,Wall, D.H., 2002. Ecosystem carbonloss with woody plant invasion of grasslands. Nature 418, 623–626.

Jandl, R., Sollins, P., 1997. Water-extractable soil carbon in relation to the belowgroundcarbon cycle. Biol. Fert. Soils 25, 196–201.

Joergensen, R.G., Scheu, S., 1999. Response of soil microorganisms to the addition ofcarbon, nitrogen and phosphorus in a forest Rendzina. Soil Biol. Biochem. 31,859–866.

Kalbitz, K.,Kaiser, K., 2008. Contribution of dissolved organic matter to carbon storage inforest mineral soils. J. Plant Nutr. Soil Sci. 171, 52–60.

Kalbitz, K., Schmerwitz, J., Schwesig, D.,Matzner, E., 2003. Biodegradation of soil-deriveddissolved organic matter as related to its properties. Geoderma 113, 273–291.

Kandeler, E., 1995. Total nitrogen. In: Schinner, R., Öhlinger, R., Kandeler, E., Margesin, R.(Eds.), Methods in Soil Biology, pp. 403–406 (Berlin Heidelberg New York).

Kätterer, T., Reichstein, M.,Andrén, O., Lomander, A., 1998. Temperature dependence oforganic matter decomposition: a critical review using literature data analyzed withdifferent models. Biol. Fertil. Soils 27, 258–262.

Kirschbaum, M.U.F., 1995. The temperature dependence of soil organic matter decompo-sition, and the effect of global warming on soil organic C storage. Soil Biol. Biochem.27, 753–760.

Körner, C., 2003. Alpine Plant Life: Functional Plant Ecology of HighMountain Ecosystems.Springer, Berlin.

Lipson, D.A., 2007. Relationships between temperature responses and bacterial communitystructure along seasonal and altitudinal gradients. Microb. Ecol. 59, 418–427.

Lipson, D.A.,Schmidt, S.K.,Monson, R.K., 2000. Carbon availability and temperature controlthe post-snowmelt decline in alpine soil microbial biomass. Soil Biol. Biochem. 32,441–448.

8 A. Gutiérrez-Girón et al. / Geoderma 237–238 (2014) 1–8

Lipson, D.A.,Schadt, C.W.,Schmidt, S.K., 2002. Changes in soil microbial community struc-ture and function in an alpine dry meadow following spring snowmelt. Microb. Ecol.43, 307–314.

McCulley, R.L.,Archer, S.R., Boutton, T.W.,Hons, F.M., Zuberer, D.A., 2004. Soil respirationand nutrient cycling in wooded communities developing in grassland. Ecology 85,2804–2817.

Melillo, J.M.,Steudler, P.A.,Aber, J.D.,Newkirk, K.,Lux, H.,Bowles, F.P.,Catricala, C.,Magill, A.,Ahrens, T.,Morrisseau, S., 2002. Soil warming and carbon-cycle feedbacks to theclimate system. Science 298, 2173–2176.

Molinillo, M., Lasanta, T.,García-Ruiz, J.M., 1997. Managing mountainous degraded land-scapes after farmland abandonment in the central Spanish Pyrenees. Environ.Manag. 21, 587–598.

Montané, F., Rovira, P., Casals, P., 2007. Shrub encroachment into mesic mountain grass-lands in the Iberian Peninsula: effects of plant quality and temperature on soil Cand N stocks. Glob. Biogeochem. Cycles 21, GB4016.

Montané, F., Romanyà, J., Rovira, P., Casals, P., 2010. Aboveground litter quality changesmay drive soil organic carbon increase after shrub encroachment into mountaingrasslands. Plant Soil 337, 151–165.

Muñoz-Jiménez, J.,García-Romero, A., 2004. Modificaciones climáticas y evolución de lacubierta vegetal en las áreas culminantes de la Sierra de Guadarrama (SistemaCentral Español) durante la segunda mitad del siglo XX: las altas cuencas delVentisquero de la Condesa y de Valdemartín. Cuadernos de Investigación Geográfica.30, pp. 117–149.

Nadelhoffer, K.J.,Giblin, A.E., Shaver, G.R., Laundre, J.A., 1991. Effects of temperature andsubstrate quality on element mineralization in six arctic soils. Ecology 72, 242–253.

Niklińska, M.,Klimek, B., 2007. Effect of temperature on the respiration rate of forest soilorganic layer along an elevation gradient in the Polish Carpathians. Biol. Fertil. Soils43, 511–518.

Niklińska, M.,Maryański, M.,Laskowski, R., 1999. Effect of temperature on humus respirationrate and nitrogenmineralization: implications for global climate change. Biogeochemis-try 44, 239–257.

Öhlinger, R., 1995. Soil respiration by titration. In: Schiner, E., Öhlinger, R., Kandeler, E.,Margesin, R. (Eds.), Methods in Soil Biology. Springer, New York, pp. 95–98.

Pauli, H.,Gotffried, M.,Hohenwallner, D.,Reiter, K.,Casale, R.,Grabherr, G., 2004. The GLORIAField Manual — Multi-Summit Approach. European Commission, Luxembourg.

Powlson, D.S.,Whitmore, A.P.,Goulding, K.W.T., 2011. Soil carbon sequestration to miti-gate climate change: a critical re-examination to identify the true and the false. Eur.J. Soil Sci. 62, 42–55.

Roura-Pascual, N.,Pons, P.,Etienne, M.,Lambert, B., 2005. Transformation of a rural landscapein the eastern Pyrenees between 1953 and 2000. Mt. Res. Dev. 25, 252–261.

Sánz-Elorza, M.,Dana, E.D.,González, A., Sobrino, E., 2003. Changes in the high-mountainvegetation of the central Iberian Peninsula as a probable sign of global warming.Ann. Bot. 92, 273–280 (London).

Schimel, D.S., 1995. Terrestrial ecosystems and the carbon cycle. Glob. Chang. Biol. 1,77–91.

Schimel, J.P.,Gulledge, J.M.,Clein-Curley, J.S., Lindstrom, J.E.,Braddock, J.F., 1999. Moistureeffects on microbial activity and community structure in decomposing birch litter inthe Alaskan taiga. Soil Biol. Biochem. 31, 831–838.

Schindlbacher, A., de Gonzalo, C., Díaz-Pinés, E., Gorría, P., Matthews, B., Inclán, R.,Zechmeister-Boltenstern, S.,Rubio, A.,Jandl, R., 2010. Temperature sensitivity of forestsoil organic matter decomposition along two elevation gradients. J. Geophys. Res. 115(G03018), 1–10.

Schlesinger,W.H.,Andrews, J.A., 2000. Soil respiration and the global carbon cycle. Biogeo-chemistry 48, 7–20.

Schröter, D., Cramer, W., Leemans, R.,Prentice, I.C.,Araújo, M.B.,Arnell, N.W.,Bondeau, A.,Bugmann, H., Carter, T.R., Gracia, C.A., de la Vega-Leinert, A.C., Erhard, M., Ewert, F.,Glendining, M.,House, J.I.,Kankaanpää, S.,Klein, R.J.T.,Lavorel, S.,Lindner, M.,Metzger,M.J.,Meyer, J.,Mitchell, T.D., Reginster, I.,Rounsevell, M., Sabaté, S., Sitch, S., Smith, B.,Smith, J., Smith, P., Sykes, M.T., Thonicke, K., Thuiller, W.,Tuck, G., Zaehle, S., Zierl, B.,2005. Ecosystem service supply and vulnerability to global change in Europe. Science310, 1333–1337.

Sjögersten, S.,Wookey, P.A., 2002. Climatic and resource quality controls on soil respira-tion across a forest–tundra ecotone in Swedish Lapland. Soil Biol. Biochem. 34,1633–1646.

Smolander, A., Kitunen, V., 2002. Soil microbial activities and characteristics of dis-solved organic C and N in relation to tree species. Soil Biol. Biochem. 34,651–660.

Sparling, G.P.,West, A.W., 1989. Importance of soil water content when estimating soilmicrobial C, N and P by the fumigation–extraction methods. Soil Biol. Biochem. 21,245–253.

Sparling, G.P., Feltham, C.W., Reynolds, J.,West, A.W., Singleton, P., 1990. Estimationof soil microbial c by a fumigation–extraction method: use on soils of high or-ganic matter content, and a reassessment of the kec-factor. Soil Biol. Biochem.22, 301–307.

Sveinbjörnsson, B.,Davis, J.,Abadie, W.,Butler, A., 1995. Soil carbon and nitrogen mineraliza-tion at different elevations in the Chugach Mountains of South-Central Alaska, U.S.A.Arct. Alp. Res. 27, 29–37.

van't Hoff, J., 1898. Lectures of Theoretical and Physical Chemistry. Part 1. ChemicalDynamics. Edward Arnold, London.

Vance, E.D.,Chapin III, F.S., 2001. Substrate limitations to microbial activity in taiga forestfloors. Soil Biol. Biochem. 33, 173–188.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soilmicrobial biomass C. Soil Biol. Biochem. 19, 703–707.

Vanhala, P.,Karhu, K.,Tuomi,M.,Björklöf, K.,Fritze, H.,Liski, J., 2008. Temperature sensitivity ofsoil organic matter decomposition in southern and northern areas of the boreal forestzone. Soil Biol. Biochem. 40, 1758–1764.

Yoshitake, S.,Uchida, M.,Koizumi, H.,Nakatsubo, T., 2007. Carbon and nitrogen limitationof soil microbial respiration in a High Arctic successional glacier foreland nearNy-Alesund, Svalbard. Polar Res. 26, 22–30.