Abstract Wetlands are among the most important

ecosystems on Earth both in terms of productivity and

biodiversity, but also as a source of the greenhouse gas

CH4. Microbial processes catalyzing nutrient recycling

and CH4 production are controlled by sediment phys-

ico-chemistry, which is in turn affected by plant activity

and the foraging behaviour of herbivores. We per-

formed field and laboratory experiments to evaluate

the direct effect of herbivores on soil microbial activity

and their indirect effects as the consequence of

reduced macrophyte density, using migratory Bewick’s

swans (Cygnus columbianus bewickii Yarrell) feeding

on fennel pondweed (Potamogeton pectinatus L.) tu-

bers as a model system. A controlled foraging experi-

ment using field enclosures indicated that swan

bioturbation decreases CH4 production, through a de-

crease in the activity of methanogenic Archaea and an

increased rate of CH4 oxidation in the bioturbated

sediment. We also found a positive correlation be-

tween tuber density (a surrogate of plant density dur-

ing the previous growth season) and CH4 production

activity. A laboratory experiment showed that sedi-

ment sterilization enhances pondweed growth, proba-

bly due to elimination of the negative effects of

microbial activity on plant growth. In summary, the

bioturbation caused by swan grazing modulates CH4

cycling by means of both direct and indirect (i.e. plant-

mediated) effects with potential consequences for CH4

emission from wetland systems.

Keywords Multitrophic interactions Æ Methane

cycling Æ Shallow lakes Æ Bewick’s swans Æ Fennel

pondweed

1 Introduction

Wetlands are among the most productive ecosystems

on Earth, mainly due to the rapid recycling of nutri-

ents mediated by the hydrological regime and the

coupled activity of water and soil bacteria (Mitsch

and Gosselink 2000). Wetlands are also among the

most prominent sources of atmospheric CH4, con-

tributing 55% to the annual global CH4 emission to

the atmosphere (Houghton et al. 2001; LeMer and

Communicated by Christian Korner

Electronic supplementary material Supplementary material isavailable in the online version of this article athttp://dx.doi.org/10.1007/s00442-006-0445-9 and is accessible for authorized users.

P. L. E. Bodelier (&) Æ M. Stomp Æ H. J. LaanbroekDepartment of Microbial Wetland Ecology,Netherlands Institute of Ecology (NIOO-KNAW),Centre for Limnology, Rijksstraatweg 6,3631AC, Nieuwersluis, The Netherlandse-mail: p.bodelier@nioo.knaw.nl

M. StompAquatic Microbiology, Institute for Biodiversityand Ecosystem Dynamics, University of Amsterdam,Nieuwe Achtergracht 127, 1018 WS Amsterdam,The Netherlands

L. Santamaria Æ M. KlaassenDepartment of Plant–Animal Interactions,Netherlands Institute of Ecology (NIOO-KNAW),Centre for Limnology, Rijksstraatweg 6,3631AC, Nieuwersluis, The Netherlands

L. SantamariaMediterranean Institute for Advanced Studies(IMEDEA, CSIC-UIB), Miquel Marques 21,07190, Esporles, Mallorca, Illes Balears, Spain

Oecologia (2006) 149:233–244

DOI 10.1007/s00442-006-0445-9

123

PLANT ANIMAL INTERACTIONS

Animal–plant–microbe interactions: direct and indirect effectsof swan foraging behaviour modulate methane cyclingin temperate shallow wetlands

Paul L. E. Bodelier Æ Maayke Stomp ÆLuis Santamaria Æ Marcel Klaassen ÆHendrikus J. Laanbroek

Received: 14 September 2005 / Accepted: 26 April 2006 / Published online: 31 May 2006� Springer-Verlag 2006

Roger 2001; Segers 1998). Both productivity and CH4

emission are facilitated by the simultaneous action of

aerobic as well as anaerobic microbes in the many

oxic–anoxic interfaces in wetlands. The latter are

created by wetland plants which introduce oxygen

into the sediments by radial oxygen loss (cf. Bodelier

2003) thereby influencing rhizosphere microbes di-

rectly. The direct facilitation of microbial activity in

the rhizosphere, mobilizes nutrients and renders them

available to the plants thereby enhancing plant pro-

ductivity (Bodelier et al. 1996; Caffrey and Kemp

1991; Christensen et al. 1994; Karjalainen et al. 2001;

Pedersen et al. 1995; Risgaard-Petersen and Jensen

1997). The enhanced oxidation of NH3 to NO3– in the

rhizosphere of submerged macrophytes (e.g. Ottosen

et al. 1999; Risgaard-Petersen and Jensen 1997),

which is of potential benefit for the plants since it

enhances the supply of N in a form preferred by the

plants, is a good example of the latter.

Since the emission of CH4 is the outcome of the

anoxic production by methanogenic microbes (in part

driven by organic C availability) and the aerobic con-

sumption by methanotrophic bacteria, aquatic macro-

phytes play a significant role in the cycling and

emission of CH4 from wetland ecosystems (Boon and

Mitchell 1995; Heilman and Carlton 2001; Kankaala

and Bergstrom 2004; Sorrell et al. 2002). The previ-

ously mentioned NH3-oxidizing bacteria also have the

ability to consume CH4 (Bodelier and Frenzel 1999)

and have to be taken into account when assessing CH4

cycling in rhizospheres of macrophytes.

Considering the above, herbivores such as ducks and

swans can influence CH4-cycling microbes in a direct

and an indirect way. Grubbing activity of waterfowl

results in spatial and temporal oxygenation of the

sediment thereby directly affecting CH4-consuming

bacteria by providing these obligatory aerobic

microbes with oxygen they need and exposing the

obligatory anaerobic CH4-producing microbes to

harmful habitat conditions. Swans foraging on macro-

phyte tubers may cause bioturbation down to 30 cm

according to the burial depth of the tubers (Santamaria

and Rodriguez-Girones 2002). As already discussed

above waterfowl can thus create oxic/anoxic interfaces

analogous to the roots of macrophytes with similar

impact on sediment microbes. Besides bioturbation

waterfowl can also influence microbial activity directly

by nutrient input via faeces, with possible feedbacks to

vegetation (Bazely and Jefferies 1989; Walker et al.

2003). Grazing by herbivores such as ducks and swans,

is likely to modulate the effects of macrophytes on

CH4-cycling microbes and hence on CH4 emission

from wetland ecosystems, in an indirect way. Grazing

by waterfowl may have severe effects on macrophyte

density (Idestam-Almquist 1998; Santamaria and

Rodriguez-Girones 2002; Sondergaard et al. 1997),

thereby potentially reducing the rhizosphere effect on

sediment microbes as discussed above.

Despite their potential importance, the direct

effects of herbivores and the indirect effects of plant–

herbivore interactions on sediment microorganisms

have received very limited attention, particularly in

aquatic ecosystems. In this study we perform field and

laboratory experiments to evaluate the direct effect of

grubbing activities of herbivorous swans on microbial

activity, and their indirect effects as the result of

herbivore-mediated differences in root density. We

hypothesize that direct effects by bioturbation will

stimulate aerobic while hampering anaerobic

microbes. Indirect effects through reduction of the

rhizosphere effect will reduce all microbial activity.

To test these hypotheses, we manipulated swan

grazing pressure in the field and studied its effects on

sediment physico-chemistry and microbiology. In

addition, a laboratory experiment was used to address

plant-mediated, indirect effects of herbivory on soil

microbiology, mediated by a reduction in plant den-

sity. We pay specific attention to CH4 cycling, both

because this is a process of particular importance in

wetlands and shallow lakes, and because it is likely to

be among the most affected by the herbivores and

plants under study.

2 Materials and methods

2.1 Study system

The study was performed in the Lauwersmeer

(53�22¢N, 06�13¢E), a shallow freshwater lake

(2,000 ha, of which 750 ha is < 70 cm deep) that

represents one of the most important stopovers of

Bewick’s swans during their autumn migration. The

submerged vegetation that covers its extensive shal-

low areas is dominated by fennel pondweed , a

pseudo-annual angiosperm that survives the winter

by means of asexual propagules (below-ground tu-

bers). Bewick’s swans (Cygnus columbianus bewickii

Yarrell) migrate annually between their breeding

grounds in the Pechora Delta (northern Russia) and

their wintering sites in the Netherlands and the UK

(Beekman et al. 2002). During migration, the swans

make several stopovers in which they feed exten-

sively on the starch-rich, subsurface tubers of Pota-

mogeton pectinatus L. (fennel pondweed). Swans

start feeding on the tubers immediately after their

234 Oecologia (2006) 149:233–244

123

arrival in early October and abandon the lake to

continue their migration or to feed in nearby crops

and grasslands by mid-to-late November.

2.2 Field experiment: direct effects of foraging

swans on microbial processes

Since flocks of foraging Bewick’s swans remain in their

pondweed foraging areas around the clock, progres-

sively exploiting the tuber banks, it is difficult to

measure grubbing effects directly in the field without

disturbing the whole flock or leaving considerable time

between the observations made before and after swan

foraging. For this reason, we decided to cage six cap-

tive animals in an enclosure built in a pondweed field

exploited regularly by swans, which allowed for sam-

pling both immediately before and after the swans’

foraging bout.

The experiment took place during November 2000

in the Nieuwe Robbegat, a protected area within the

Lauwersmeer that is closed to the public and where

hunting is prohibited. A cage (10·10 m, 2 m high,

made of fishing net) was installed in September, before

arrival of the Bewick’s swans to the area. The cage was

made of 13 heavy wooden stakes connected with steel

cables to which the fishing nets were fastened. On

November 15, immediately after the Bewick’s swans’

departure from the foraging area, the plot was sampled

using two different sampling grids covering the com-

plete 10·10 m area equally spaced as displayed in

Fig. 1. To determine the density of fennel pondweed

tubers 25 cores (30 cm length, 10 cm diameter) were

taken. Nine equally spaced (see Fig. 1) cores (20 cm

length, 3.8 cm diameter) were used to for microbio-

logical parameters and sediment physico-chemistry

(see Supplement S1). Subsequently, six Bewick’s swans

were placed inside the enclosure and were allowed to

forage while being observed from a boat at approxi-

mately 150 m distance. The six swans that were re-

leased in this cage originated from the wild but had

been kept in captivity prior to the experiment for

several years. Their behaviour appeared very natural

(Nolet et al. 2002). The treatment resulted in a net

feeding time (i.e. time that the swans had their heads

under water) over the enclosed area of 12.0 foraging

swan hours. The choice of the number of swans used

was based on observations on free-living swans in the

Lauwersmeer area (Nolet et al. 2001). The grazing

pressure observed in the cage is in the same range as

observed in the field (Nolet 2004; Nolet et al. 2001).

Subsequently, swans were removed from the enclosure

on 17 November and cores for tuber and soil samples

were taken again in the plot (as above).

2.3 Grazing pressure

All cores were stored outdoors in a cooling box until 20

November, then transported to the laboratory for

processing. To estimate tuber bank biomass, the sedi-

ment sampled in the cores was washed through a 3-mm

sieve. Tubers were then separated, counted and

weighed individually [fresh weight (f.w.), accuracy

0.001 g], as described by (Nolet et al. 2001). Although

both (i.e. tuber and sediment) sampling grids differed

in grid size and number of nodes (see Fig. 1), they

coincided in the specific locations where sediment

cores were sampled. Since there were more cores

measuring tuber density than those sampled to mea-

sure microbial activity and soil chemistry, grazing

pressure at the different sediment-sampling points was

estimated as the difference in the average number of

tubers (before minus after grazing) using the from six

to nine ‘‘grazing pressure’’ cores in the immediate

vicinity of each ‘‘microbial’’ core. This resulted in a

running window of grazing pressure estimation, single

grazing pressure cores being used in from one to three

grazing pressure estimates at the ‘‘microbial’’ core

location.

2.4 Microbiological activity

Sediment sampled in the cores was sieved ( < 2 mm),

thoroughly mixed, separated into sub-samples (for

10m

10m

Tuber sampling point “Microbial” sampling point

Fig. 1 Schematic presentation of the experimental plot withsampling grids for tuber density analyses (grey circles) andmicrobial and sediment physico-chemical analyses (black squares )

Oecologia (2006) 149:233–244 235

123

microbial incubations and physico-chemical measure-

ments) and incubated immediately for the microbial

activity measurements.

2.4.1 Potential CH4 production

Potential CH4 production (PMP) was determined as

described by (Bodelier et al. 2000a). Ten grams of

sediment was transferred to 150-ml flasks and 10 ml

sterile demineralised water was added. The deminer-

alised water had been deoxygenated with N2 prior to

addition. The flasks were closed with rubber stoppers

and the residual CH4 and O2 was removed by flushing

with N2 for 30 min. The flasks were incubated statically

in the dark. CH4 accumulation in the headspace was

monitored by GC analysis. The CH4 production po-

tential was calculated using the linear regression

coefficient of the linear part of the CH4 increase curve.

2.4.2 Potential CH4 oxidation

The potential CH4 oxidation (PMO) activity of the

sediment was determined as described by Bodelier and

Frenzel (1999). In short, 10 g sediment was transferred

to 150-ml flasks and diluted 1:1 (w/v) with sterile

demineralised water. The flasks were closed with rub-

ber stoppers and 1.3 ml pure CH4 (10,000 p.p.m.v.) was

added. The flasks were incubated on a shaker

(150 r.p.m.) at 20�C. The decrease in CH4 in the

headspace was monitored by GC analysis of regularly

taken sub-samples. Since CH4 depletion curves typi-

cally display bi-phasic kinetics, an initial PMO

(PMOini) and an induced PMO (PMOind) depletion

rate can be distinguished. PMOini indicates active

methanotrophic biomass in the field, whereas the

PMOind is a measure for total methanotrophic abun-

dance in the sample (i.e. including bacteria that were

not active in situ) (Gilbert and Frenzel 1998). The

PMO rates (initial and induced) were calculated using

the slope of the regression analyses on the linear parts

of the CH4 depletion curves.

2.4.3 Potential NH4+ oxidation activity

Potential NH4+ oxidation (PAO) was determined as

described by Bodelier and Frenzel (1999). In short,

10 g sediment was transferred to 150-ml flasks covered

with aluminium foil. Twenty microlitres of PNA med-

ium [0.33 g (NH4)2SO4, 0.14 g K2HPO4, 0.027 g

KH2PO4 per l] was added. The flasks were incubated at

20�C on a shaker at 150 r.p.m. The oxidation of NH4+

was monitored by taking three samples (1 ml) of the

mixture in the first 6 h followed by 14 samples spread

evenly over the following 7 days. The samples were

centrifuged at 13,000 r.p.m. for 10 min and the super-

natant stored at –20�C for analyses of NO3–/NO2

– con-

centrations. The PAO rates were calculated using the

slope of the linear regression analyses on the NO3– and

NO2– increase curves.

2.5 Sediment physico-chemistry

Sediments subsamples used for physico-chemical

measurements were dried at 40�C and analysed to

measure pH (extracted in water and in CaCl2),

%CaCO3, %organic matter, total N, total and avail-

able P, Na+, K+, Mg2+, Fe3+, Cl-, SO42–, NH4

+, NO3– and

conductivity (all according to Troelstra et al. 1995).

Separate subsamples were used to determine water

content (weight loss of 5-g sediment samples dried at

105�C for 24 h).

2.6 Laboratory experiment: indirect effects

of foraging swans on microbiological processes

2.6.1 Experimental design and plant cultivation

The experiment aimed at determining the effect of

plant density on microbial activity rates and the po-

tential effects of the latter (mediated by changes in

sediment chemistry) on plant growth. Plants were

grown in beakers from tubers originally collected in the

Lauwersmeer. Sixteen treatments were used, resulting

from a factorial combination of sediment type (sandy

vs. clay rich), sediment microbiota (control vs. sterile

sediment) and planting densities (0, 1, 3 and 6 plants/

beaker, i.e. including a control without plants). For

every treatment six replicate beakers were used. Plant

densities correspond to the variation in tuber densities

observed in the field as a result of varying grazing

intensities.

Pondweed tubers used in the experiment were ob-

tained from the institute’s tuber stock. They belonged

to six different clonal lines originally collected in the

Lauwersmeer (Hangelbroek et al. 2002), one of each

being used for each replicate aquarium (i.e. integrating

random factors aquarium and clone in a single factor).

Individual tuber mass was standardised to a relatively

narrow range (0.040–0.125 g f.w.). Tubers were sur-

face-sterilised [5 min in NaHClO3 (5%) followed by

thorough rinsing in sterilised water (Bodelier et al.

1997)] and pre-sprouted for 6 days in microtiter trays

filled with clean sand and water. Sprouted tubers were

randomly assigned to beakers and planted in plastic

236 Oecologia (2006) 149:233–244

123

beakers (700 ml volume, 14.4 cm height, 8.9 cm upper

diameter, 6.5 cm lower diameter) filled with approxi-

mately 535 ml sediment. Sandy and clay-rich sediment

were obtained from swan foraging fields at the Lauw-

ersmeer (near Nieuwe Robbengat and Zoutkamp,

respectively) after the swans had left the foraging

areas. Tubers, stones and other large particles were

removed using a 2-mm sieve [for a description of sed-

iment granulometry at those sites, see Nolet et al.

(2001)]. Sterile sediment was obtained by autoclaving

wet sediment samples twice (1 h at 121�C, on 2 sub-

sequent days). In all cases, sediment was placed in the

pots and covered with a layer of sterile aquarium sand

to minimize siltation and nutrient release to the water

column.

Per treatment two replicate beakers were placed in

each of six separate aquaria, this made a total of six

repeated-measures replicates (i.e. treatments and their

interactions were fully nested within each random

factor aquarium). The experiment was labour inten-

sive; hence, replicates were not grown simultaneously

but in a ‘‘ladder’’ design (i.e. we initiated two repli-

cates every fortnight). Each aquaria had a separated

water pump (Eheim Aqua Boss) for mixing and aer-

ation. The aquaria were filled with tap water and

placed in a growth chamber (water temperature 22�C,

day length 16 h, irradiance 150 lmol photons m–2 s–1

at 1 cm under the water surface). Water loss due to

evaporation was compensated for by adding demin-

eralized water. Plants were grown for 6 weeks before

harvesting for plant biomass determination, sediment

microbiological assays and sediment physico-chemistry

measurements.

2.7 Pre-harvest analyses

2.7.1 Plant growth rates

Plant height (length of the longest shoot including the

terminal leaf, to the nearest tip in mm) was measured

weekly and used to obtain non-destructive estimates of

the relative growth rate (RGR; obtained from logistic

regression fits).

2.7.2 Pore water physico-chemistry

Pore water was sampled at weekly intervals in four out

of six aquaria, by means of Rhizon soil solution sam-

plers (Eijkelkamp, the Netherlands) connected to

Venoject blood collecting tubes as described by Bo-

delier et al. (1997). Pore water samples taken during

the experiment were analysed for CO2, CH4, HCO3–,

NH4+, NO–

3 and NO–2 concentrations and pH. HCO3

–

content was determined by titration with HCl. CO2 and

CH4 concentrations in the headspace of the tubes were

determined according to Bodelier and Frenzel (1999),

after which tubes were frozen (–20�C) for further

analyses. Following careful thawing, NH4+, NO3

– and

NO2– were measured using a Technicon Traacs 800

auto-analyser (Technicon Instruments, Tarrytown,

N.Y.).

2.8 Post-harvest analyses

2.8.1 Harvesting procedure

The harvest of six aquaria was carried out in a period

of 6 weeks. Every 2 weeks, two aquaria, containing

two replicate beakers of every treatment, were har-

vested. We removed the aboveground plant parts and

the cover of aquarium sand from every beaker and

transferred the sediment (including plant roots and

rhizomes) to a glass beaker filled with 600 ml auto-

claved and N2-deoxygenated demineralised water.

Agitation for 10 min with a magnetic stirrer was used

to produce a soft slurry, from which the roots were

carefully picked out. Roots and sediment slurries were

used immediately for separate analyses of most

microbiological and physico-chemical variables, while

sub-samples, used to estimate the numbers of CH4- and

NH3-oxidizing bacteria, were stored at 8�C until fur-

ther analyses (usually the next day).

2.8.2 Plant biomass

Aboveground parts (shoots+leaves), roots+rhizomes

and tubers were dried (for 48 h at 70�C) to determine

their respective dry weights.

2.9 Microbiological analyses

2.9.1 PMO of sediment and roots

The PMO activity of the sediment (PMOsediment) was

determined as described above for the field experiment

using 20 g slurry (as described in the harvest procedure

section). The PMO associated with roots (PMOroots)

was determined as follows: 0.3 g root material was

transferred to 12-ml tubes with butyl rubber stoppers

and 120 ll pure CH4 (10,000 p.p.m.v.) was added. The

tubes were incubated in the dark. The concentration of

CH4 in the headspace was monitored twice a day for 8–

9 days by GC analysis. Before removing 100 ll air

from the headspace, 100 ll air was injected to keep the

Oecologia (2006) 149:233–244 237

123

pressure stable. After 6 days 1 ml O2 was added. After

incubation the dry weight of the roots was determined

by drying them at 70�C for 48 h.

2.9.2 PAOsediment and PAOroots

PAOsediment was determined as described for the

field experiment using 20 g slurry. The PAOroots was

determined by transferring 0.3 g root material to 150-

ml flasks covered with aluminium foil. The roots were

incubated with 25 ml PNA medium after which the

same procedure was followed as with the sediment

slurries.

2.9.3 Potential CH4 production of sediment and roots

Potential CH4 production of the sediment (PMPsedi-

ment) was determined as described for the field

experiment using 60 g slurry. The PMP associated with

roots (PMProots)was determined by transferring 0.3 g

root material to 12-ml tubes and flushing them with N2.

The concentration of CH4 in the headspace was mon-

itored twice a day for 8–9 days by GC analysis. Before

removing 100 ll air from the headspace, 100 ll N2 was

injected to keep the pressure stable.

2.9.4 Numbers of CH4 oxidizers in sediment and

associated with roots

The number of CH4 oxidizers (NMO) was determined

by means of the most probable number (MPN) method

according to (Bodelier and Frenzel 1999). In order to

extract the cells from the sediment particles, 10 g slurry

was diluted 1:5 with phosphate buffer saline (PBS). For

the roots, 50 ml PBS was added to 1 g roots. Glass

beads (3 mm, 10 g) were added to both extracts of

sediment and roots. The extracts were shaken for 4 h at

150 r.p.m. and subsequently diluted 1:10 with PBS. A

sub sample of 100 ll of the suspension was serially

diluted 1:1 in sterile microtiter plates (Nunc, Denmark)

containing growth medium for methanotrophs (NMS).

The plates were incubated for 4 weeks at 25�C in gas-

tight jars containing 20% CH4 in air. Inoculated plates

without CH4 served as controls. Wells that were turbid

were considered positive. MPNs were obtained from

statistical tables (Rowe et al. 1977).

2.9.5 Numbers of NH4+ oxidizers in sediment and

associated with roots

The numbers of NH3 oxidizers (NAO) was determined

by the MPN method as described by Bodelier and

Frenzel (1999). The same sediment and root extracts

were used as described for the determination of the

numbers of CH4 oxidizers. A sub sample of 1 ml of the

suspension was serially diluted 1:3.25 in sterile micro-

titer plates (Nunc) containing growth medium for

NH3-oxidizing bacteria (Bodelier and Frenzel 1999).

After an incubation period of 2 months in the dark at

25�C, NH4+ oxidation was scored in each well by

checking for the presence of NO2– and NO3

– by adding

Griess Ilosvay reagents (Spektroquant test, Merck,

Darmstadt) and Zinc powder. MPNs were obtained

from statistical tables (Rowe et al. 1977).

2.10 Sediment physico-chemistry

Sediment-slurry subsamples used for physico-chemical

measurements were dried at 40�C and analysed as

described in the field experiment section on sediment

physico-chemistry.

2.11 Gas analyses

CH4 in headspace samples was analysed using a

HP5340 GC equipped with a flame ionization detector.

He was used as the carrier gas, synthetic air and H2 as

burning gases. The oven temperature was 80�C. Cali-

bration was performed using standard gas mixtures.

CO2 was measured also with a HP5340 GC, equipped

with a thermal conductivity detector. He was used

as the carrier gas, oven temperature was 50�C and

the calibration was performed using a standard gas

mixture.

2.12 Statistical analyses

2.12.1 Field observations

To simplify the analysis and avoid problems of

redundancy and multi-colinearity (arising from the use

of multiple, non-independent and sometimes highly

correlated variables), we reduced all sediment and

pore water variables to the first two to three axes ex-

tracted by a principal components analyses (PCA). The

number of PCA axes used as independent variables in

the multiple regressions discussed below was chosen

based of the proportion of variance explained by each

axis (up to a threshold of 12%, at which the cumulative

percentage of explained variance started to level off).

We performed two different analyses.

First, we estimated the relative change in microbial

activity as (before–after)/before (n=9) and fitted it to

a multiple regression model with grazing pressure

238 Oecologia (2006) 149:233–244

123

[=relative change in tuberbank biomass, in g f.w. m–2,

estimated as (before–after)/before; n=9] and relative

change in sediment physico-chemistry [=first and sec-

ond axes of a PCA based on the relative change in the

physico-chemical variables from before to after graz-

ing, estimated as (before–after)/before; n=9 for each

variable] as independent variables. With this approach,

changes in microbial activity are directly linked to

grazing pressure and changes in sediment chemistry,

and the differences are independent of the spatial

structure on which they are over imposed (under the

assumption that over the 2-day period, the only factor

potentially causing differences in soil microbiology and

physico-chemistry was the experimentally imposed

swan grazing).

Second, we used the initial values (n=9) of microbial

activity to make a multiple regression on the initial

values of tuber density and sediment physico-chemistry

(using the first three axis of a PCA of all sedi-

ment+pore water variables). The purpose was to assess

whether a surrogate of plant density during the growth

season (i.e. tuber density) was correlated with micro-

bial activity in the sediment, after accounting for the

effect of soil heterogeneity in physico-chemical com-

position.

2.13 Laboratory experiment

All variables were compared by means of ANOVAs,

with all fixed factors (sediment type, sterilization, tuber

density and their second- and third-order interactions)

nested within the random factor ‘‘aquarium’’ (=clone,

since each clone was in a different aquarium). In these

ANOVAs, all sediment and pore water variables were

reduced to the first two axes of a PCA (for the same

reason as mentioned above). Data were log-trans-

formed to assure homoscedasticity and normality of

residuals whenever necessary. All statistical tests were

performed using STATISTICA 6.0 for Windows

(Tulsa, Okla.).

3 Results

3.1 Field experiment

Swan grazing resulted in a 9% decrease in average

tuber-bank biomass, from 71.0 to 64.6 g f.w. m–2

which is in the range of foraging yields observed for

free-living swans in the same area (Nolet 2004).

However, due to the strong small-scale spatial vari-

ation in grazing pressure, swan consumption varied

within the plot from zero to 62% of the tuber-bank

biomass removed. Grazing pressure had direct effects

on PMP and PMOini: results of the multiple

regression indicate that grazing pressure affected

PMP significantly and negatively, and PMOini posi-

tively and marginally significantly (Table 1; Fig. 2),

and had no effect on PMOind and PAO. In addition,

there were significant effects of sediment physico-

chemistry on PMP (i.e. a positive correlation with

PCA axis 1, indicating negative correlations with

conductivity, Cl, Na, K and organic matter). The

effect of grazing pressure on PMP is, however, not

attributable to an indirect effect, mediated by chan-

ges in sediment physico-chemistry: separate regres-

sions indicated that axis 1 of sediment physico-

chemistry was not significantly correlated with graz-

ing pressure [axis 1: F(1,7)=0.47, P=0.51], although

the effect of axis 2 was marginally significant

[F(1,7)=4.33, P=0.076; intercept=0.129±0.560, slope=

1.859±0.893].

Tuber density had a marginally significant, positive

effect on PMOind, and no effect on PMOini, PMP, or

Table 1 Results of the multiple regression analyses withbackwards removal of variables, for the effect of burrowingswans and sediment physico-chemistry on the microbial activity

in the sediment of the Lauwersmeer. PMOini Initial potentialCH4-oxidizing activity, PMP potential CH4-producing activity,NS not significant

Intercept Grazing pressure (%) Sediment physico-chemistry Adjusted R2

PCA axis 1a PCA axis 2b

PMOini Estimate±SE 0.047±0.094 0.302±0.150 0.28t (7) 2.01c

PMP Estimate±SE 0.066±0.144 –0.849±0.306 0.172±0.066 0.180±0.098 0.50t (5) –2.77* 2.60* 1.83 NS

*P < 0.05aVariables with loadings >0.7 were organic matter, Na, K, conductivity, Cl, all (–)bVariables with loadings >0.7 were total P, Mg, both (+)cP < 0.10

Oecologia (2006) 149:233–244 239

123

PAO (Table 2). PMP showed a significant correlation

with sediment physico-chemistry, through a positive

correlation with PCA axis 2 (i.e. with conductivity, K

and Mg). Indirect effects of swan grazing, mediated by

a reduction in tuber production, are thus most likely to

affect PMOind.

3.2 Laboratory experiment

PMProots, PAOroots and numbers of NH4+ oxidizers in

the sediment and on roots were undetectable, thus they

were not included in the statistical analyses. Plant

density had significant effects on PMOsediment and

PMOroots (Supplement S2), but did not influence

NMO or PAO. The effect on PMO was, however,

opposite to that found in the field. PMOroots de-

creased with plant density and, while PMOsediment

increased with plant density in sandy sediment, it de-

creased with plant density in clay-rich sediment

(Fig. 3). In the absence of plants, PMOsediment did

not differ between sediment types.

PMP showed a strong heteroscedasticity, largely

caused by the predominance of non-detectable activi-

ties in sandy sediment. In clay-rich sediment, higher

activities were measured; non-parametric analyses

using Friedman ANOVA indicated a marginally sig-

nificant increase in PMP activity when plants were

present, as compared to the treatment without plants

(ANOVA v2=7.20, df=3, P=0.066; n=6).

Plant growth was significantly affected by sediment

microbial activity. Sediment sterilization resulted in

increased biomass yield (total dry weight) and shoot

length, while plant RGR and shoot-to-root ratio de-

creased (Supplement S2; Fig. 4). Concerning sediment

chemistry, tuber density had a significant effect on axis

1 (i.e. a negative correlation with conductivity, inor-

ganic anions and cations, organic matter, total N and P

in the sediment), which showed much larger scores for

the two highest tuber densities. These results are con-

sistent with the depletion of nutrients and ions related

to plant growth. Sediment type and sterilization had

significant and marginally significant effects on axis 2,

respectively (mainly representing pore water variables;

-1.2 -0.8 -0.4 0.0 0.4 0.8-0.6

-0.3

0.0

0.3

0.6

-0.8

-0.4

0.0

0.4

0.8

1.2

Grazing pressure

Rel

ativ

e C

hang

e in

PM

OR

elat

ive

Cha

nge

in P

PMO

MP

PMP

Fig. 2 Effect of swan grazing pressure on the potential activityof CH4-producing Archaea (potential CH4 production; PMP)(upper graph) and CH4-oxidizing bacteria (potential CH4-oxidizing activity; PMO) (lower graph). Upper graph Partialresidual plot derived from a multiple regression model, lowergraph standard regression fit

Table 2 Results of the multiple regression analyses withbackwards removal of variables, to determine the effect oftuber density and soil physico-chemistry on the rates of bacterial

activity measured before swan grazing. Only significant effectsare shown. For abbreviations, see Table 1

Intercept Tuber density Sediment physico-chemistry Adjusted R2

PCA axis 1a PCA axis 2b PCA axis 3c

PMOind Estimate±SE 83.59±8.36 0.23±0.11 0.31t (7) 2.14d

PMP Estimate±SE 1.97±0.14 –0.26±0.07 0.63t (7) –3.84**

**P < 0.01aVariables with loadings >0.7 were SO4 (–0.92), total N (0.91), total P (0.89), Cl (–0.72) and pHH2O (–0.82)bVariables with loadings >0.7 were K (0.91), Mg (0.84) and conductivity (0.82)cVariables with loadings >0.7 were pHCaCl2

(–0.68), CaCO3 (–0.61)dP < 0.10

240 Oecologia (2006) 149:233–244

123

Supplement S2). N and NH4+ concentrations in the pore

water were higher, and CO2 lower in sterile sediment.

CH4 was undetectable in the porewater during the

whole experiment.

4 Discussion

The bioturbation caused by swan grubbing as tested in

the field experiment (i.e. direct effects) seemed to have

little bearing on NH3 oxidation, yet it modulated CH4

cycling microbiological processes through a combina-

tion of effects, with possible consequences for CH4

emission from the studied shallow lake ecosystem. The

direct effect of grubbing was a decrease in the activity

of methanogenic Archaea and an increase in the rate of

CH4 oxidation in the bioturbated sediment, which

confirms our hypotheses with respect to the expected

direct effects. Obviously, this would result in a net

reduction of CH4 emission from the lake under study.

However, theoretically swan grazing may release CH4

that is trapped in the sediment, which may compensate

for the emission reduction as the consequence of the

microbiological processes which are affected.

Although the field experiment was designed to test

the direct effects of bioturbation, tuber density at time

of sampling is regarded as a surrogate for plant density

in the previous growing season and hence also with

respect to the indirect (plant density) effects conclu-

sions can be drawn from the field trial. Interestingly,

swan grazing resulted in a (marginally significant) in-

crease in the initial rate of CH4 oxidation (PMOini,

indicating the active methanotrophic biomass when

taken from the field), while tuber density had marginal,

positive effects on the induced activity of CH4-oxidiz-

ing bacteria (PMOind, representing the total methan-

otrophic biomass present). Hence, in the field situation

direct and indirect effects are thus likely to act at dif-

ferent temporal scales. While the immediate effect of

swan burrowing affects the instantaneous activity of

active methanotrophic biomass in the short-term (i.e.

PMOini), pondweed abundance during the previous

growing season leads to enhanced total methano-

trophic biomass, detectable as higher PMOind over

longer periods of time (several months later).

At first sight, the indirect effects tested in the labo-

ratory experiment seem to be at odds with the positive

effect of tuber density on CH4 oxidation as observed in

the field. In clay-rich sediment, the presence of plants

resulted in increased CH4 production and decreased

CH4 oxidation (in both roots and sediment); in sandy

sediment, PMOroots and PMOsediment showed

opposite trends and PMP was negligible. It is, however,

important to note that such an experiment focuses on

the short-term response of the microbial community to

plant growth, while field data probably indicate its

overall, long-term result. Short-term decreases in

PMO, and increases in PMP in clay-rich sediment are

probably the result of root C exudation and subsequent

oxygen limitation for CH4-oxidizing bacteria due to

heterotrophic microbial respiration. This effect will be

enhanced by the low gas diffusivity of clay-rich sedi-

ment, and the reduced redox potential will lead to even

higher rates of root C exudation by the plants. Since

the increase in PMP did not result in higher CH4

availability in the short-term (CH4 concentration in the

Numbers of tubers per pot

Met

hane

pro

duct

ion

(nm

olC

H4.

g.-1

.h-1

)M

etha

ne o

xida

tion

(µm

ol C

H4.

g.-1

.h-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2B

0 1 3 60.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6C

0.00

0.02

0.04

0.06

0.08

0.10

AClay-rich

Sandy

Fig. 3a–c Microbial activity in beakers filled with sandy andclay-rich sediment in which different densities of pondweedplants were cultivated. a CH4 oxidation in sediment. b CH4

oxidation in roots. c CH4 production in sediment. Bars indicatemeans±SEs

Oecologia (2006) 149:233–244 241

123

pore water was undetectable throughout the experi-

ment), we must conclude that the increased PMOind

observed in the field probably reflects a long-term re-

sponse to increased substrate availability, which re-

quires the accumulation of CH4 over much longer

periods of time than the duration of our laboratory

experiment (months instead of weeks). Finally, the

decrease in PMOroots (in both sand and clay) can be

explained by nutrient uptake effects: increased root

density results in decreased nutrient concentrations (N

and P) around the roots, thus decreasing PMO activity.

CH4 oxidation around roots of wetland plants seems to

depend strongly on the availability of N, for which

CH4-oxidizing bacteria can not compete for with plants

(Bodelier and Laanbroek 2004; Bodelier et al. 2000b).

The reduced activity of CH4-oxidizing bacteria with

increasing root biomass would theoretically lead to

higher CH4 emission. The latter will be less strong

when plant density is reduced by swan grazing.

An additional feed-back loop in the network of

relationships relating pondweeds, swans and sediment

microorganisms involves the negative effect that

microbial activity had on plant growth. The reasons for

this effect are unclear. It could be attributed to an

enhanced nutrient supply as a by-product of sediment

sterilization. However, plant shoot-to-root ratio de-

creased in sterilized sediment, contrary to what would

be expected if plants were released from nutrient lim-

itation (given that the sediment is the main source of

nutrients). Alternatively, it could be the result of de-

creased N supply to the pore water caused by bacterial

activity. In particular, N and NH4+ concentrations in the

pore water were higher (and CO2 lower) in sterile

sediment, particularly in clay-rich sediment. Moreover,

while the initial NH4+ concentration was comparable

for the sterile and non-sterile treatments, it decreased

over time in the non-sterile treatment from

0.39±0.12 mM in week 1 to 0.13±0.02 mM in week 4

but did not vary over time in the sterile treatment.

Although several studies have shown that NH3 oxi-

dizers are weak competitors against plants for NH4+

(Bodelier et al. 1998, 2000a), microbial activity could

have reduced the nutrient availability in the pore water

when plants were starting to grow (and thus not able to

compete for nutrients).

Numbers of tubers per pot

Sho

ot-r

oot r

atio

Tot

al p

lant

bio

mas

s (d

ry w

eigh

t)

0.0

0.5

1.0

1.5

2.0

A

1 3 60.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5B

Non-sterile, clay-richNon-sterile, sandySterile, clay-richSterile, sandy

Fig. 4 Total biomass yield (a) and shoot-to-root ratio (b) ofpondweed plants grown in beakers filled with sterile and non-sterile, sandy and clay-rich sediment. Bars indicate means±SEs

Swans

Tubers

Pondweed

CH4 productionCH4 consumption

CH4 emission

-

- -++/-

-

--

+/-

+/- = Sand/clay

Fig. 5 Schematic presentation of the modulating mechanism ofdirect and indirect effects of swan grazing on CH4 emission fromthe investigated shallow lake ecosystem. The solid arrowsindicate relationships analysed in this study. The dotted arrowsindicate proposed effects on CH4 emission as the consequence ofthe analysed relationships.+ indicates a positive and – a negativeeffect of the different modules in this multitrophic system as theconsequence of swan grazing. ± indicates that a positive effect isobserved for sandy sediment and a negative effect for clayeysediment

242 Oecologia (2006) 149:233–244

123

In summary, we have demonstrated that the forag-

ing activity of swans modulates CH4 cycling in shallow

wetlands, while it seems to have little bearing on NH3

oxidation. Swan bioturbation decreased CH4 emission

both directly (through a decrease in the activity of

methanogenic bacteria and an increase in the rate of

CH4 oxidation in the bioturbated sediment) and indi-

rectly (through a decrease in the population density of

fennel pondweed, which reduced its positive effect on

the rates of CH4 production). The matrix of direct and

indirect effects of swan grazing on CH4 cycling

microbiological processes and the resultant CH4

emission are presented in Fig. 5. Collectively the

grazing activities of swans can potentially result in re-

duced emission of CH4 from shallow lake ecosystems

(see Fig. 5). The scheme also highlights that the extent

of the swan grazing effect on CH4 emission will partly

depend on the sediment clay content.

Besides its direct importance for CH4 cycling in

wetlands, the interactions between herbivores and

sediment microbes described here is likely to result in

increased stability of the swan–pondweed relationship.

Given that microbial activity seemed to have negative

effects on plant growth, particularly in clay-rich sedi-

ment (which promotes anaerobic conditions), the

combined effects of sediment oxygenation and reduced

activity of anaerobic bacteria are likely to benefit those

tubers surviving swan predation.

Effects of herbivory on belowground processes and

their feedback mechanisms has been intensively

investigated in terrestrial systems (cf. Ayres et al. 2004;

Bardgett and Wardle 2003; Walker et al. 2003). In

these studies microbiological processes are mainly af-

fected by altered input of C by plants as a direct effect

of the action of the herbivores on the plants or by

nutrient input (e.g. N) via faeces of waterfowl. These

kinds of multi-trophic interactions have only rarely

been investigated in aquatic systems (Connors et al.

2000; Walker et al. 2003). Moreover, direct effects of

larger vertebrates on microbiological processes have,

to our knowledge, only been reported in two studies. In

one case muskrats stimulated nitrification through

bioturbation (Connors et al. 2000) and in another N

fixation was stimulated in a tidal saltmarsh as the

consequence of grazing activities by geese (Bazely and

Jefferies 1989). The observed bi-trophic and multi-

trophic relationships in our study may have direct ef-

fects on an important ecosystem function, namely the

emission of the greenhouse gas CH4 to the atmosphere.

In that light the presented swan–pondweed–microbe

system represents an important model system for

studying the importance of multitrophic interactions in

relation to ecosystem functioning.

Acknowledgements Thijs de Boer, Oscar Langevoord andKlaas Siewertsen provided technical support in the field andlaboratory. Special thanks are due to Wiecher Smant (NIOO-KNAW, Centre for Terrestrial Ecology) for carrying out part ofthe chemical analyses. This publication is publication number3839 of the Netherlands Institute of Ecology (NIOO-KNAW),Centre for Limnology, Nieuwersluis, the Netherlands. The au-thors declare hereby that all experiments described in the pres-ent study comply with the current laws in the Netherlands.

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