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Pedobiologia 52 (2009) 263—272

0031-4056/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

www.elsevier.de/pedobi

Carbon and nitrogen mobilisation by earthwormsof different functional groups as affected by soilsand content

O. Butenschoena,�, S. Marhanb, R. Langelc, S. Scheua

aDarmstadt University of Technology, Institute of Zoology, Schnittspahnstraße 3, 64287 Darmstadt, GermanybUniversity of Hohenheim, Institute of Soil Science, Department Soil Biology, Emil-Wolff-Strasse 27,70599 Stuttgart, GermanycGeorg-August-University Gottingen, Faculty of Forest Sciences and Forest Ecology,Centre for Stable Isotope Research and Analysis, Busgenweg 2, 37077 Gottingen, Germany

Received 18 April 2008; received in revised form 5 November 2008; accepted 6 November 2008

KEYWORDSStable isotopes;Soil organic matter;N-mobilisation;Lumbricidae

ee front matter & 2008pedobi.2008.11.001

ing author. Tel.: +49 615ess: o.butenschoen@gm

SummaryEarthworms play an important role in organic matter processing and nutrient cyclingin temperate ecosystems. It is known that earthworms preferentially ingest sandgrains mixed with organic material and it has been suggested that the mixture ofsand and organic material during the gut passage may play an important role in litterdegradation and nutrient release, which may accelerate assimilation of nutrients byearthworms and likely enhance plant growth. In a greenhouse experiment, weinvestigated the effect of the anecic earthworm species Lumbricus terrestris and theendogeic earthworm species Octolasion tyrtaeum separately and in combination oncarbon and nitrogen mobilisation from surface applied rye litter labelled with 13Cand 15N. By mixing arable soil with 25% sand, we investigated the effect of theavailability of sand. To quantify the mobilisation of 15N, three rye seedlings wereplanted in each microcosm and analysed for isotope signature after 3 months ofincubation. Mobilisation of 13C was quantified by analysing the incorporation of labelinto the soil and earthworm tissue.Irrespective of the addition of sand the biomass of L. terrestris decreased during theexperiment, whereas that of O. tyrtaeum increased in single species treatment andslightly decreased in the combined treatment with L. terrestris. The concentrationof 13C decreased while that of 15N increased in the tissue of both earthworm species,with the effect being more pronounced in L. terrestris for 13C and in O. tyrtaeum for15N. Both earthworm species increased shoot biomass, with the effect of L. terrestris(+80%) exceeding that of O. tyrtaeum (+28%) and maximum plant biomass in thecombined treatment (+92%). Earthworms did not affect the 15N concentration of rye

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1 16 3600; fax: +49 6151 16 6111.x.de (O. Butenschoen).

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plants, but sand significantly increased 15N concentration of plants, presumably dueto improving soil structure. Overall, the incorporation of 13C into the soil was lowand was significantly increased in presence of sand, with the highest enrichment intreatments without earthworms.The results indicate that the availability of sand does not increase effects ofearthworms on litter degradation, nutrient release and plant growth. Rather,independent of soil sand content earthworms increase plant growth, whereas thepresence of sand itself enhances the uptake of nitrogen from plant litter and theincorporation of litter carbon into the soil.& 2008 Elsevier GmbH. All rights reserved.

Introduction

Earthworms form the dominant macrofaunagroup in many terrestrial ecosystems and functionas ecosystem engineers, directly and indirectlymodifying the chemical, physical and biologicalproperties of the soil, and controlling ecosystemstructure and functioning (Jones et al., 1997;Lavelle, 1997). One of the most important mechan-isms is the control of decomposition of organicmatter, mostly resulting in increased nutrientavailability for plants and enhanced plant produc-tivity. Earthworm–plant interactions have beenstudied for a long time and in most studies,earthworms beneficially affected plant growth(see reviews, Brown et al., 1999; Scheu, 2003).However, the effects are variable and in somestudies earthworms did not affect or even reducedplant growth (Brown et al., 1999; Schmidt andCurry, 1999), indicating that the mechanismsresponsible for earthworm-mediated changes inplant growth need further attention.

A number of mechanisms are involved in earth-worm-mediated changes in plant growth includingroot feeding (Cortez and Bouche, 1992), seeddispersal (Milcu et al., 2006; Eisenhauer et al.,2008), increase of the soil pore volume (Kirkham,1982), alteration of the soil pH (Edwards and Lofty,1977), hormone-like effects (Dell’Agnola andNardi, 1987) and change of the soil structure(McInerney and Bolger, 2000). However, predomi-nantly earthworms affect plant growth by incorpor-ating organic matter into the soil and stimulatingmicrobial activity (Wurst et al., 2004). Sinceearthworms of different ecological groups preferdifferent habitats and feed on different foodresources they likely affect nutrient mineralizationand plant growth differently. Anecic earthwormspecies such as Lumbricus terrestris, for instance,incorporate litter material into the mineral soilthereby making it available for the soil food web(Bossuyt et al., 2005). Endogeic earthworm spe-cies, in contrast, primarily consume soil and

associated humified organic matter in the upperlayer of the mineral soil. However, due to selectivefeeding on organic materials and low assimilationefficiency little of the organic material ingested isincorporated into the biomass of earthworms(Whalen and Parmelee, 1999). The main part isvoided in casts containing particulate organicmaterial and nutrients excreted, such as urineand mucopolysaccharides. These casts serve ashabitat for microorganisms (Tiunov and Scheu,2000), which mineralize the organic matter thereinand release nutrients that contribute to plantnutrition.

Although a number of different soils, earthwormand plant species have been investigated, little isknown on the impact of soil texture on theseprocesses. It has been suggested that earthwormspreferentially ingest organic matter mixed withmineral soil over pure organic matter, with theingested minerals, in particular sand grains, facil-itating mechanical fragmentation of organic matterduring gut transition (Doube et al., 1997; Schul-mann and Tiunov, 1999). Since the reduced particlesize of organic matter not only accelerates assim-ilation of nutrients by earthworms but also nutrientaccessibility for microbial decay in casts, it mayincrease microbial mineralization of nutrients andstimulate plant growth.

Although earthworm casts are microbial hot-spots, earthworms are regarded to protect carbonfrom microbial mineralization due to reducedaccessibility and oxygen availability in casts (Tiunovand Scheu, 2000; McInerney et al., 2001; Marhanand Scheu, 2006). However, the texture of the soilsignificantly affects the incorporation and stabili-zation of carbon in soil aggregates. For example, ithas been shown that clay minerals protect organicmatter against microbial degradation (Feller andBeare, 1997), whereas the decomposition of or-ganic matter mixed with mineral soil is increased inparticular in coarse-textured soil (Hassink et al.,1997). Hence, both the stabilization of carbon inearthworm casts and the mobilisation of nitrogen

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Carbon and nitrogen mobilisation by earthworms of different functional groups 265

from litter enclosed into casts are likely to varywith soil texture.

The present study investigates the influence ofthe availability of sand on the mobilisation ofcarbon and nitrogen from leaf litter processed byanecic and endogeic earthworm species. We in-vestigated if the availability of sand increasesdegradation of leaf litter during the gut passageand result in (1) increased earthworm biomassdue to enhanced nutrient accessibility, (2) in-creased nitrogen mineralization and enhancedplant growth and (3) decreased carbon incorpora-tion into the mineral soil due to increased microbialaccessibility. The use of 15N and 13C labelledlitter allowed to quantify the uptake of nitrogeninto plants and the stabilization of carbon inthe mineral soil matrix. By using an anecic(L. terrestris) and an endogeic (Octolasion tyr-taeum) earthworm species, the effect of earth-worms of different ecological groups and theirinteraction was investigated.

Materials and methods

Experimental set-up

Soil samples were collected from an arable fieldclose to Bad Lauchstadt, Saxony-Anhalt, Germany.The mean long-term annual temperature at thestudy site is 8.7 1C and the mean annual precipita-tion is 484mm. The soil is a Mollisol, HaplicChernozem loam consisting of 6.1% sand, 79.0% siltand 14.9% clay. Total carbon and nitrogen concen-tration as measured in triplicate samples by anelemental analyser (NA 1500, Carlo Erba, Milan,Italy) were 1.7% (1.084 at% 13C) and 0.16%, respec-tively. Further details on the study site are given inKorschens (1994). Soil samples were taken from theupper 10 cm, transferred to the laboratory, sieved(4mm) to remove stones and larger plant residuesand defaunated for meso- and macrofauna byfreezing at �28 1C for 2 weeks. Then, the soilwas kept at 8 1C for 1 week to allow themicroorganisms to recover from the freezing andthawing procedure.

Adult O. tyrtaeum were sampled by hand in a130-year-old beech forest on limestone nearGottingen (southern Lower Saxony, Germany).Adult L. terrestris were extracted from an oak-beech forest 20 km south of Darmstadt (Hesse,Germany) by using 0.06% formalin solution. Earth-worms were washed twice in distilled water andkept in containers filled with experimental soil at5 1C for 2 weeks before they were used for the

experiment. Both earthworm species are amongthe most common and widespread species inneutral and base-rich pastures, gardens and arableland all over Europe.

15N and 13C labelled leaf litter were obtained bythe method described in Schmidt and Scrimgeour(2001). Fourteen boxes (180mm� 550mm�170mm) were filled with a mixture of sandy soilfrom an experimental field near Darmstadt (Hesse,Germany) and forest soil from the beech forestnear Gottingen (southern Lower Saxony, Germany).Eighty rye seeds (Secale cereal L., ‘‘Avanti’’,Saaten-Union GmbH, Isernhagen, Germany) weresown per box and watered with distilled water.When the plants were at the two-leaf stage, sixboxes were labelled with 13C and 15N by wateringthe plants once a week with a solution of 0.33 g15NH4

+ (99 at% 15N; Campro Scientific, Berlin, Ger-many) in 1500ml distilled water. Twice a week, theplants were labelled with 13C by spraying a solutionof 250ml distilled water containing urea (99 at%13C; Campro Scientific, Berlin, Germany) startingwith 0.032 and increasing to 0.323 g 13C urea at theend. The remaining eight boxes were watered oncea week with a solution of 0.44 g NH4

+-N (Merck,Darmstadt, Germany) in 2000ml distilled water.After 6 weeks, the plants were cut at ground leveland dried at 65 1C for 72 h. Aliquots of the labelledand unlabelled leaf litter were ground to powder,approximately 2.5mg were weighed into tincapsules and 15N and 13C concentration wereanalysed in triplicate samples. The leaf litter usedin the experiment was a mixture of three partslabelled and four parts unlabelled leaves resultingin a 13C and 15N content of 1.1170.01 at% (39.1070.64% C) and of 10.3670.68 at% (0.9570.04% N),respectively.

The experiment was carried out in 48 microcosmsconsisting of PVC tubes (height 310mm, diameter160mm) closed at the bottom by lids in a fullfactorial design with six replicates per treatment.The microcosms were equipped with ceramiclysimeters, which were connected by hoses with avacuum pump to allow drainage of the soil undersemi-natural conditions (�200 to �400 hPa) andsampling of leaching water from each microcosm inseparate bottles. Half of the microcosms were filledwith 6.82 kg fresh weight arable soil (equivalent to6.0 kg dry weight), the other half with 5.11 kgfresh weight arable soil homogenously mixed byhand with 1 kg silica sand with particle size63–250 mm and 500 g silica sand with particle size250–500 mm (Euroquarz, Dorsten, Germany). Priorto addition, the sand was washed twice withdistilled water. Soil was compacted by hand to abulk density of 1.06 g cm�3. Three rye seeds

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O. Butenschoen et al.266

(‘‘Avanti’’, Saaten-Union GmbH, Isernhagen, Ger-many) were sown in each microcosm and wateredwith distilled water. Two weeks later non-germi-nated seedlings were replaced. From each of themicrocosms, 100 g fresh weight soil were removed,thoroughly mixed with 7 g labelled leaf litter(chopped by scissors o20mm), and placed backinto the microcosms forming a shallow soillayer enriched in litter. Then, earthworms wereadded to the microcosms. In microcosms withO. tyrtaeum, four individuals with a total livebiomass of 0.9770.07 g, in those with L. terrestris,two individuals with a total live biomass of9.7470.64 g were added. Microcosms with bothearthworm species received four O. tyrtaeum(total live biomass of 0.9570.04 g) and twoL. terrestris individuals (total live biomass9.9570.13 g). The microcosms were placed in aclimate-controlled greenhouse at 18 1C for 12weeks and irrigated by hand with 90ml distilledwater per day.

Sampling and analytical procedure

During the experiment, leaching water of eachmicrocosm was collected at regular intervals andstored at 4 1C until further analysis. The leachedwater of each microcosm was weighed and theamount of ammonium (NH4

+-N) and nitrate (NO3�-N)

was analysed by distillation (Vapodest 20, Ger-hardt, Bonn, Germany) in triplicate pooled samplesof 2 weeks. Withered leaves were collected twotimes from the microcosms to avoid that they enterthe soil. Leaves were dried at 65 1C for 48 h andweighed. At the end of the experiment, plantswere cut at ground level, dried at 65 1C for 72 h andweighed. Soil cores were divided by hand in threelayers of 0–3 (H1), 3–10 (H2) and 10–30 cm (H3).Earthworms were collected by hand, counted,washed, dried for 1min on filter paper andweighed. Then, earthworms were killed by freezingat �20 1C, dried at 65 1C for 72 h and stored in adesiccator. Root-free soil samples were taken fromeach soil layer using a soil corer and dried at 65 1Cfor 72 h. The remaining soil of each layer wassubmerged in water and then roots were washed ona screen (1mm), dried at 65 1C for 72 h andweighed.

For isotope measurement, the anterior parts ofthe earthworms were used to avoid contaminationby gut material. Root-free soil samples of the twoupper soil layers (H1 and H2), roots of the twolower layers (H2 and H3) and shoots were ground toa powder by a ball mill (MM 200, Retsch, Haan,Germany). Due to contamination of roots of H1 with

labelled leaf litter, they were not included in theanalysis. Samples were dried at 65 1C for 72 h andapproximately 3mg of plant material, 15mg root-free soil and 1mg of gut-free earthworm tissuewere weighed into tin capsules. Stable isotoperatios were determined by a system of an ele-mental analyser (NA 1500, Carlo Erba, Milan, Italy)coupled with a trapping box (type CN, Finnigan,Bremen, Germany) and an isotope ratio massspectrometer (MAT 251, Finnigan, Bremen,Germany) (Reineking et al., 1993). V-PDB (PeeDee Belemnite) served as standard for d13C,atmospheric N2 for d15N. Acetanilide (Merck,Darmstadt, Germany) was used for internalcalibration. The amount of 13C and 15N of thesamples were calculated by subtracting naturalsignatures of retained control samples from themeasured 15N and 13C at% at the end of theexperiment and expressed as mg per g fresh (dry)weight.

Data analysis

Prior to statistical analysis data on the amount ofnitrogen leached, and shoot and root biomass ofthe sand treatments were multiplied by 1.25 toadjust for the 25% lower amount of soil (and 25%lower amount of carbon and nitrogen). Data onearthworm survival and live body mass and data oncarbon (12C, 13C) and nitrogen (14N, 15N) concen-tration of earthworm tissue were analysed by two-factor analysis of variance (ANOVA) with the factorssand (with and without addition) and earthworm(with and without L. terrestris and O. tyrtaeum).Data on shoot biomass were analysed by three-factor analysis of variance with the factors sand(with and without addition), L. terrestris (withand without) and O. tyrtaeum (with and without).Data on roots (biomass, 14N and 15N concentration)and soil (12C and 13C concentration) in differentsoil layers, and data on shoots (14N and 15Nconcentration) and nitrogen leached (nitrate andammonium) at different sampling dates wereanalysed by repeated measures analyses ofvariance (RM-ANOVA). Significant layer (time) �treatment interactions were investigated usingANOVA for each layer or sampling. Differencesbetween means were investigated using Tukey’sHSD test. Prior to analyses, data were inspected forhomogeneity of variance (Levene test). A statisticalprobability Po0.05 was considered significant.STATISTICA 7.0 (Statsoft, Tulsa, USA) and SAS 8.0(Statistical Analysis System, SAS Institute Inc.,Cary, USA) software packages were used forstatistical analyses.

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Carbon and nitrogen mobilisation by earthworms of different functional groups 267

Results

Earthworms

In microcosms with L. terrestris virtually all theleaf litter had been consumed by the end of theexperiment, while in control microcosms andmicrocosms with O. tyrtaeum only the leaf litterremained virtually unchanged on the soil surface.All L. terrestris individuals survived except onewhich died by accident. In contrast, L. terrestrisand the addition of sand significantly affected thesurvival of O. tyrtaeum; all individuals survived intreatments without L. terrestris whereas in pre-sence of L. terrestris survival of O. tyrtaeumdecreased by 25.0% and 54.2% in treatments withand without sand, respectively (F1,19 ¼ 5.4,P ¼ 0.031). The mean body mass of L. terrestrisdecreased during the experiment on average by28.7% irrespective of the treatments. In contrast,the mean body mass of O. tyrtaeum was signifi-cantly affected by the presence of L. terrestris(F1,18 ¼ 21.8, P ¼ 0.0001). In presence of L. terres-tris, it decreased by 4.2% and 6.0% in treatmentswith and without sand, respectively, whereas intreatments without L. terrestris, it increased by23.7% and 59.5% with and without sand, respec-tively (Figure 1).

Irrespective of the treatments, tissue carbonconcentration and concentration of 13C of bothearthworm species decreased during the experi-ment with the decrease being more pronounced inL. terrestris. In L. terrestris, tissue carbon con-

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Figure 1. Effect of the presence of Lumbricus terrestris[without L. terrestris (�Lt) and with L. terrestris (+Lt)]and availability of sand [without 25% sand (�S) and with25% sand (+S)] on the biomass of Octolasion tyrtaeum.Means of three replicates71 S.D.

centration decreased from an initial of 0.51 to0.46 g C g�1 body weight, whereas in O. tyrtaeum itdecreased from an initial of 0.49 to 0.45 g C g�1

body weight. In L. terrestris, concentration of 13Cdecreased from an initial of 5.54 to 5.02mg 13C g�1

body weight, whereas in O. tyrtaeum it decreasedfrom an initial of 5.35 to 5.21mg 13C g�1 bodyweight. Tissue nitrogen concentration of L. terres-tris decreased during the experiment from aninitial of 0.16 to 0.13 g N g�1 body weight and thatof O. tyrtaeum from an initial of 0.14 to 0.11 g N g�1

body weight; it was neither affected by thepresence of the other earthworm species nor bythe addition of sand. In contrast, irrespective ofthe treatments the tissue 15N concentration of bothearthworm species increased during the experi-ment with the increase being more pronounced inO. tyrtaeum. In L. terrestris, it increased from aninitial of 0.57 to 0.79mg 15N g�1 body weight,whereas in O. tyrtaeum it increased from an initialof 0.53–2.25mg 15N g�1 body weight.

Soil

The carbon concentration of the soil increasedduring the experiment, with the increase beingmore pronounced in H1 than in H2 (F1,8 ¼ 87.8,Po0.0001). It increased from an initial of 12.69 and16.93mgC g�1 soil dry weight in treatments withand without sand to 14.86 and 18.23mgC g�1 soildry weight in H1, and to 13.41 and 17.21mgC g�1

soil dry weight in H2 with and without sand,respectively. In H1, the increase in carbon concen-tration was most pronounced in the control treat-ments with 3.02mgC g�1 soil dry weight, and lesspronounced in presence of earthworms with anincrease of 0.77, 1.52 and 1.62mgC g�1 soil dryweight in presence of both earthworm species,L. terrestris and O. tyrtaeum, respectively(F3,8 ¼ 22.2, P ¼ 0.0003). Sand increased the con-centration of carbon in H1 by 2.16mgC g�1 soil dryweight compared with an increase of only1.30mgC g�1 soil dry weight in treatments withoutsand (F1,8 ¼ 18.7, P ¼ 0.002). In H2, both earth-worm species affected the concentration ofcarbon, but the effect depended on the availabilityof sand (sand� earthworm interaction; F3,8 ¼ 16.9,P ¼ 0.0007). Irrespective of the presence ofearthworms, the concentration of carbon increasedby 0.28mgC g�1 soil dry weight in treatmentswithout sand. In contrast, in treatments withsand the concentration of carbon increased by0.88, 1.37 and 0.72mgC g�1 soil dry weight in thecontrol treatment and in presence of L. terrestrisand O. tyrtaeum, respectively, but decreased by

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Figure 2. Effect of the presence of Lumbricus terrestris

O. Butenschoen et al.268

0.12mgC g�1 soil dry weight in presence of bothearthworm species.

The concentration of 13C increased during theexperiment with the increase being more pro-nounced in H1 than in H2 (F1,8 ¼ 88.8, Po0.0001).It increased from an initial of 0.138 and0.183mg 13C g�1 soil dry weight in treatments withand without sand to 0.161 and 0.198mg 13C g�1 soildry weight in H1, and to 0.145 and 0.187mg 13C g�1

soil dry weight in H2 with and without sand,respectively. In H1, the increase of 13C concentra-tion was most pronounced in the control treat-ments with 0.033mg 13C g�1 soil dry weight, andless pronounced in presence of earthworms with anincrease of 0.008, 0.016 and 0.017mgC g�1 soil dryweight in presence of both earthworm species, andin presence of L. terrestris and O. tyrtaeum,respectively (F3,8 ¼ 22.3, P ¼ 0.0003). Sand in-creased the concentration of 13C in H1 by0.024mg 13C g�1 soil dry weight compared with anincrease of only 0.014mg 13C g�1 soil dry weight intreatments without sand (F1,8 ¼ 18.4, P ¼ 0.003,Figure 2a). In H2, both earthworm species affectedthe concentration of 13C, but the effect dependedon the availability of sand (sand� earthworminteraction; F3,8 ¼ 16.7, P ¼ 0.0008). Irrespectiveof the presence of earthworms, the concentrationof 13C increased by 0.003mg 13C g�1 soil dry weightin treatments without sand. In contrast, in treat-ments with sand the concentration of 13C increasedby 0.009, 0.015 and 0.008mg 13C g�1 soil dry weightin the control treatment and in presence ofL. terrestris and O. tyrtaeum, respectively, butdecreased by 0.001mg 13C g�1 soil dry weight inpresence of both earthworm species (Figure 2b).

[without L. terrestris (�Lt) and with L. terrestris (+Lt)],Octolasion tyrtaeum [without O. tyrtaeum (�Ot) andwith O. tyrtaeum (+Ot)] and availability of sand [without25% sand (�S) and with 25% sand (+S)] on the amount of13C excess in (a) H1 (0–3 cm) and (b) H2 (3–10 cm). Meansof three replicates71 S.D.

Nitrogen leached

In total, 7725ml distilled water per microcosmwas added during the experiment. The availabilityof sand significantly increased total leaching ofwater with 2743 and 2026ml leached withand without sand, respectively (F1,35 ¼ 91.2,Po0.0001). Furthermore, L. terrestris reducedtotal leaching of water with 1962 and 2870mlleached with and without L. terrestris, respectively(F1,35 ¼ 142.7, Po0.0001). In the first 2 weeks ofthe experiment, before adding earthworms andleaf litter, mineral N predominantly leached asnitrate (88.6 mgNd�1); leaching of ammonium waslow (1.8 mg Nd�1). The availability of sand did notaffect ammonium leaching, but significantly in-creased leaching of nitrate with 95.7 and81.5 mgNd�1 leached with and without sand,respectively (F1,40 ¼ 17.6, P ¼ 0.0001). At the

second sampling, 2 weeks after the addition ofearthworms and leaf litter, no ammonium wasdetected and leaching of nitrate was signifi-cantly reduced to 21.0 mgNd�1 (F1,40 ¼ 1511.5,Po0.0001). The availability of sand significantlyreduced leaching of nitrate with 17.3 and24.8 mgNd�1 leached with and without sand,respectively (F1,40 ¼ 19.9, Po0.0001). Further-more, earthworms decreased leaching of nitratewith 25.3 mgNd�1 leached in the control treatment,22.7 mgNd�1 in presence of both species and19.5 mgNd�1 in presence of L. terrestris(F3,40 ¼ 5.0, Po0.005); leaching of nitrate was

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Carbon and nitrogen mobilisation by earthworms of different functional groups 269

lowest in presence of O. tyrtaeum with16.6 mgNd�1.

Plants

Both earthworm species significantly increasedtotal shoot biomass, with the effect beingmore pronounced in presence of L. terrestris(F1,40 ¼ 189.23, Po0.0001) than in presence ofO. tyrtaeum (F1,40 ¼ 14.64, P ¼ 0.0004) with anincrease by 80% and 28%, respectively; the pre-sence of both earthworm species increased totalshoot biomass by 92% (Figure 3).The averagenitrogen and 15N concentration of retained controlsamples of rye shoots were 9.2970.12mgN g�1 dryweight and 0.03670.0004mg 15N g�1 dry weight,respectively. At the end of the experiment, oldand young leaves contained 7.3271.39 and14.6572.75mgN g�1 dry weight, respectively(F1,16 ¼ 135.1, Po0.0001). Neither the availabilityof sand nor the presence of earthworms affectedleaf nitrogen concentration. Shoot 15N concentra-tion also differed between old and young leaveswith 0.1270.03 and 0.2470.05mg 15N g�1 dryweight, respectively (F1,16 ¼ 97.37, Po0.0001).Earthworms did not affect leaf 15N concentration,but sand significantly increased it from 0.1170.01

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Figure 3. Effect of the presence of Lumbricus terrestris[without L. terrestris (�Lt) and with L. terrestris (+Lt)]and Octolasion tyrtaeum [without O. tyrtaeum (-Ot) andwith O. tyrtaeum (+Ot)] on shoot biomass. Means of threereplicates71 S.D. Data are adjusted to the addition of25% sand.

to 0.1370.03mg 15N g�1 dry weight in old leaves(F1,16 ¼ 6.1, P ¼ 0.03) and from 0.2170.03 to0.2770.05mg 15N g�1 dry weight in young leaves(F1,16 ¼ 9.6, P ¼ 0.007).

Total root biomass significantly increased withsoil depth with 0.8670.63, 0.9970.36 and2.0770.92 g dry weight in H1, H2 and H3, respec-tively (F2,80 ¼ 48.43, Po0.0001). In H1, O. tyr-taeum significantly decreased total root biomassfrom 1.0570.73 to 0.6770.44 g dry weight(F1,40 ¼ 5.23, P ¼ 0.03), whereas in H3 L. terrestrissignificantly increased root biomass from1.4570.50 to 2.6970.82 g dry weight(F1,40 ¼ 36.83, Po0.0001). In H2, L. terrestrisincreased root biomass, but the effect dependedon the availability of sand (sand� L. terrestrisinteraction; F1,40 ¼ 4.49, P ¼ 0.04), with1.0570.39 and 0.7170.21 g dry weight withoutL. terrestris with and without sand, and 1.0870.27and 1.1570.43 g dry weight in presence ofL. terrestris with and without sand, respectively.L. terrestris increased the shoot-to-root ratio ofthe plants from 1.07 in the control treatmentto 1.36 in single species treatment, whereasO. tyrtaeum significantly increased it to 1.52(F1,40 ¼ 10.66, P ¼ 0.002); the presence of bothearthworms resulted in the highest shoot-to-rootratio of 1.60.

The average nitrogen and 15N concentrationof retained control samples of rye roots were8.7870.49 and 0.03370.018mg 15N g�1 dryweight, respectively. At the end of the experiment,the root nitrogen concentration was depleted butdid not differ between roots of H2 and H3 with5.4970.89mgN g�1 dry weight. Earthworms alsodid not affect root nitrogen concentration in H2 andH3, whereas the availability of sand decreased it inH2 with 5.2470.66 and 6.1470.67mgN g�1 dryweight in treatments with and without sand,respectively (F1,16 ¼ 10.15, P ¼ 0.006). In H3, theavailability of sand also decreased root nitrogenconcentration, but the effect was only marginallysignificant (F1,16 ¼ 4.48, P ¼ 0.05). Root 15N con-centration significantly differed between H2 and H3with 0.08470.015 and 0.06270.017mg 15N g�1 dryweight in H2 and H3, respectively (F1,16 ¼ 24.71,P ¼ 0.0001). Sand did not affect root 15N concen-tration in H2 and H3. In contrast, both earthwormspecies increased root 15N concentration in H2from 0.06870.013mg 15N g�1 dry weight in thecontrol to 0.09270.015, 0.09270.007 and0.08370.009mg 15N g�1 dry weight in treatmentswith L. terrestris, O. tyrtaeum and both earthwormspecies, respectively (F1,16 ¼ 11.44, P ¼ 0.004). InH3, L. terrestris increased root 15N concentration,but the effect depended on the availability of

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sand (sand � L. terrestris interaction; F1,16 ¼ 5.66,P ¼ 0.03), with 0.06270.020 and 0.04670.012mg 15N g�1 dry weight without L. terrestriswith and without sand, and 0.06470.017 and0.07570.015mg 15N g�1 dry weight in presence ofL. terrestris with and without sand, respectively.

Discussion

Earthworms

Contrary to our hypothesis, the availability ofsand did not affect earthworm biomass and tissue13C and 15N concentrations, suggesting that eithersand did not facilitate the mechanical fragmenta-tion of leaf litter during gut passage, or did notaccelerate assimilation of nutrients by earthworms.Irrespective of the treatment, the biomass ofL. terrestris decreased during the experiment,whereas that of O. tyrtaeum increased in singlespecies treatments and slightly decreased in thepresence of L. terrestris, indicating that in parti-cular food resources for L. terrestris were in shortsupply, but also that L. terrestris effectivelyexploited food resources of O. tyrtaeum. However,in the field earthworm communities presumably aredriven mainly by abiotic factors rather than bycompetition, (Briones et al., 1995; Ponge andDelhaye, 1995).

Besides suffering from the presence ofL. terrestris, 13C and 15N concentrations in earth-worm tissue indicate that O. tyrtaeum also bene-fited from the presence of L. terrestris. Theconcentration of 13C in the tissue of both earth-worm species slightly decreased with the depletionbeing more pronounced in L. terrestris than inO. tyrtaeum. In contrast, the concentration of 15Nin the tissue of both earthworm species increased,with the increase being more pronounced inO. tyrtaeum and being independent of L. terres-tris. This suggests that O. tyrtaeum has utilisedlitter components incorporated into the soil byL. terrestris.

Plant performance

Both earthworm species significantly increasedplant growth. This is consistent with previousstudies showing that earthworms promote plantgrowth (Brown et al., 1999; Scheu, 2003). However,our hypothesis that earthworms take up sandgrains, thereby facilitating decomposition of leaflitter and increasing nitrogen mineralization andthereby plant growth was not confirmed. Although

both earthworm species fed on the labelled leaflitter, they did not affect the concentration of 15Nin the rye plants, indicating that they did not affectmineralization of nitrogen from labelled leaf litter.Thus, we suggest that earthworms increased plantgrowth by other mechanisms, such as hormone-likeeffects, alteration of soil structure or the interac-tion with microorganisms. In addition, earthwormsincreased root biomass and shoot-to-root ratiowhich also might have beneficially affected shootbiomass.

Rather than in combination with earthworms theaddition of sand alone increased the concentrationof 15N in the rye plants, indicating that the sandincreased accessibility and microbial decay of theleaf litter material. Previous studies have shownthat organic matter associated with fine soilparticles is more protected from microbial miner-alization than material associated with coarse soilparticles (Rovira and Vallejo, 2002). The physicalprotection of organic matter in soil results frominaccessibility to microbial decay of organic matterattached to small soil pores (Six et al., 2002;Koegel-Knabner et al., 2008). Thus, the increasedmineralization of 15N from labelled leaf litter andincreased concentration of 15N in the rye plants inthe sand treatments at the end of the experiment islikely to be attributed to an increased microbialaccessibility of leaf litter material in more coarse-textured sandy soils.

Soil

By ingesting and fragmenting litter materials andmixing them with mineral soil during gut passage,earthworms significantly affect the transformationof organic matter and nutrient dynamics in soils.Although earthworms may accelerate the initialdecomposition of organic residues (Marhan andScheu, 2005), long-term studies have indicatedthat carbon in casts of earthworms is protectedfrom microbial mineralization (Bossuyt et al., 2005;Pulleman et al., 2005; Fonte et al., 2007). There isevidence that this process is mainly due to reducedmicrobial accessibility and oxygen availability incast aggregates when organic matter becomesenclosed in microaggregates within large castmacroaggregates formed by earthworms duringgut passage (McInerney et al., 2001). However,the process varies with soil texture and decom-position and mineralization of organic matter hasbeen shown to be faster in coarse-textured soil(Marhan and Scheu, 2005).

In the present study, the availability of sandpredominantly increased the incorporation of litter

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carbon into the soil by earthworms, indicating thatsand facilitates the stabilization of litter carbon inearthworm casts. This contrasts previous studiesinvestigating the mineralization of organic matterin coarse-textured soils. However, the incorpora-tion of litter carbon was generally low andprimarily occurred in the top layer, where thelabelled rye litter was added in the beginning of theexperiment indicating that effects of earthwormsand sand on carbon sequestration in soil weregenerally low.

Conclusions

In contrast to our expectations, the availabilityof sand did not accelerate assimilation of litternutrients by earthworms. Earthworm biomassand tissue 13C and 15N concentrations at the endof the experiment, however, indicate thatL. terrestris more effectively competed withO. tyrtaeum for resources. Furthermore, ourhypothesis that the availability of sand increaseslitter degradation during the earthworm gut pas-sage resulting in an enhanced release of nutrientsand plant growth was not confirmed. Although bothearthworm species significantly increased plantgrowth, presumably due to increased availabilityof nutrients in soil, this was independent of thepresence of sand. Moreover, both earthwormspecies did not affect the uptake of nitrogen outof the added litter. Rather, the addition of sandstimulated the decomposition of litter and therelease of nitrogen presumably by increasing soilmacropore space and thereby the accessibility oflitter to microorganisms. In addition, our data didnot support our hypothesis that the availability ofsand decreases the allocation of carbon into themineral soil due to increased microbial accessibil-ity. Overall, the results indicate that organicmatter dynamics during earthworms gut transitionare only little affected by soil texture, rather,independent of earthworm activity soil texturedetermines the mobilisation of carbon and nitrogenin soil.

Acknowledgments

Financial support was provided by the DeutscheForschungsgemeinschaft (DFG) within the scopeof the priority program SPP 1090 ‘‘Soils as sourceand sink for CO2’’. We thank Dr. Ines Merbach(Helmholtz-Zentrum fur Umweltforschung - UFZ,Versuchsstation Bad Lauchstadt) for providing thesoil for the experiment.

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