Plant diversity a�ects culturable soil bacteria in

experimental grassland communities

ANDREÂ STEPHAN, ANDREA H. MEYER and BERNHARD SCHMID

Institut fuÈr Umweltwissenschaften, UniversitaÈt ZuÈrich, Winterthurerstr. 190, CH-8057 ZuÈrich, Switzerland

Summary

1 Utilization of carbon sources by culturable soil bacteria can be assessed with

BIOLOG microtiter plates (contain 31 C sources). We used this technique to inves-

tigate bacterial community structure at various levels of plant diversity. Plant diver-

sity levels were replicated and we investigated the in¯uence of three plant

functional groups, grasses, legumes and non-leguminous herbs, as well as the in¯u-

ence of individual plant species.

2 Catabolic activity and catabolic diversity of culturable soil bacteria were used to

estimate their density (abundance) and functional diversity, respectively. Both

increased linearly with the logarithm of plant species number and with the number

of plant functional groups in experimental grassland ecosystems. These e�ects may

have been caused by an increased diversity and quantity of material and energy

¯ows to the soil. They may also have been mediated by increased diversity of soil

microhabitats via a stimulation of the soil fauna.

3 The presence of particular plant species or functional groups in the di�erent

experimental communities stimulated the activity and functional diversity of the

culturable soil bacteria in addition to their contribution via plant diversity. The

legume Trifolium repens had the strongest e�ect and may be regarded as a keystone

species with regard to plant±microbial interactions in the systems studied.

Key-words: bacterial activity, bacterial diversity, BIODEPTH, keystone species,

log-linear diversity e�ect, plant diversity

Journal of Ecology (2000) 88, 988±998

Introduction

The loss of biodiversity is one of the major threats

to the world's ecosystems in the 21st century (Peters

& Lovejoy 1992). Ecosystem processes are strongly

a�ected by biodiversity, but the functional relation-

ship between the two depends on the system consid-

ered (SchlaÈ pfer et al. 1999; SchlaÈ pfer & Schmid

1999). In all cases, however, processes must be

maintained so that the ecosystem can continue to

exist in either a constant or a changing environment.

Functional redundancy of similar species may stabi-

lize ecosystem processes during occasional species

extinctions (Baskin 1994), but this ability appears to

be limited (Tilman & Downing 1994). However,

changes in ecosystem processes may themselves lead

to a decline in biodiversity and thus to further

reductions in ecosystem function, thereby starting a

self-reinforcing feedback cycle.

Many experiments have been established to exam-

ine the role of biodiversity, particularly plant diver-

sity, in maintaining ecosystem function (for a recent

review see Schmid et al. 2001). One example is the

European BIODEPTH project (Biodiversity and

Ecological Processes in Terrestrial Herbaceous

Ecosystems) in which an experiment speci®cally

designed to assess the e�ects of plant species number

on ecosystems, independently of the e�ects of spe-

cies identity, has been carried out at eight sites

(Diemer et al. 1997; Hector et al. 1999). Each parti-

cular level of species number was represented by a

variety of species compositions.

Interactions between plant communities and soil

bacteria have been reported (Grayston & Germida

1991; Grayston & Campbell 1996; Sharma et al.

1998; Staddon et al. 1998). Experiments at the Swiss

BIODEPTH site therefore included an assessment

of culturable soil bacteria to allow an analysis of the

e�ects of plant species number on soil biota. We

used commercial microtiter plates with multiple car-

bon sources (BIOLOG Ecoplates) as a sensitive butCorrespondence: Bernhard Schmid (fax�41 1635 5711;

e-mail: bschmid@uwinst.unizh.ch).

Journal of

Ecology 2000,

88, 988±998

# 2000 British

Ecological Society

rapid screening method to measure the activity and

functional diversity of a particular group of soil bac-

teria (Garland 1996; Smalla et al. 1998). Such bac-

teria can easily be cultivated and the functional

diversity indicated by this technique re¯ects the

diversity of their carbon-oxidation pathways. We

asked whether bacterial activity and/or bacterial

diversity increase with increasing plant diversity and

investigated the in¯uence of individual plant species

and plant functional groups on both aspects of the

biota.

Materials and methods

FIELD SITE AND EXPERIMENTAL DESIGN

The Swiss site of the European BIODEPTH project

is a former arable ®eld overlaying calcareous nutri-

ent-rich soil, situated at Lupsingen (47�270N, 7�410

E, 439m a.s.l.) in the Jura Mountains near Basel

(Diemer et al. 1997). Soil analyses were carried out

before establishment of the experiment (as described

in Diemer et al. 1997) and yielded the following

values (mean � standard error): texture, loam; pH,

7.20�0.04 (0.01-M CaCl2; pH meter 761, Knick,

Berlin, Germany); total carbon, 3.74�0.04mg gÿ1

(CHNS-analyser; LECO-932, St. Joseph, Michigan,

USA); extractable nitrate, 81.05�0.77mg gÿ1

(CaCl-solution 1 : 4); extractable phosphate, 1.61�

0.05 mg gÿ1 (saturated CO2-solution); extractable

potassium, 5.83�0.24 mg gÿ1 (saturated CO2-solu-

tion); extractable magnesium, 14.02�0.51mg gÿ1

(CO2-solution 1 : 10).

Two replicate blocks, each consisting of 32 plots,

8� 2m, separated by at least 1m, were established

in April 1995. A pool of 48 local grassland species

belonging to 13 plant families was selected and used

to assemble 32 mixtures of plant species at ®ve

diversity levels (Table 1).

Plant species number increased in geometric series

from monocultures to mixtures of 32 species (Table

1). The log2-transformed plant species number is

hereafter referred to as plant species richness or

PSR. A number of di�erent communities was

assembled at each PSR level according to a

restricted random sampling procedure from the spe-

cies pool (Diemer et al. 1997; Joshi et al. 2000). All

mixtures contained at least one grass, and within

each level there were roughly equal numbers of mix-

tures containing only grasses, mixtures with one

other functional group legumes or non-leguminous

herbs, hereafter referred to as forbs, and where

appropriate mixtures of all three functional groups.

The number of functional groups (hereafter referred

to as plant functional diversity, PFD) therefore ran-

ged from 1 (in 1 to 8 species mixtures) to 3 (in 4±32

species mixtures) and was used as an additional

treatment variable.

Each mixture was sown into one of the plots in

each block in May 1995. Pilot germination studies

Table 1 Species pool (a) and the 32 communities assembled from them (b). PSR� log2-transformed species number, each

community is identi®ed by a number and a lower-case letter. Nomenclature follows Binz & Heitz (1990)

(a)

Grasses Abbrev. Legumes Abbrev. Forbs Abbrev.

Agropyron repens AgR Anthyllis vulneraria AV Achillea millefolium AM

Agrostis stolonifera AgS Lathyrus pratensis LaP Ajuga reptans AjR

Agrostis tenuis AT Lotus corniculatus LC Anthriscus sylvestris AnS

Alopecurus pratensis AP Medicago sativa MS Bellis perennis BP

Anthoxanthum odoratum AO Onobrychis viciifolia OV Campanula patula CP

Arrhenatherum elatius AE Trifolium pratense TP Centaurea jacea CJ

Cynosurus cristatus CC Trifolium repens TR Centaurea scabiosa CS

Dactylis glomerata DG Vicia cracca VC Crepis biennis CB

Festuca ovina FO Daucus carota DC

Festuca pratensis FP Galium verum GV

Festuca rubra FR Geranium pratense GP

Holcus lanatus HL Heracleum sphondylium HS

Lolium perenne LoP Knautia arvensis KA

Phleum pratense PhP Leucanthemum vulgare LV

Poa pratensis PoP Pimpinella major PM

Trisetum ¯avescens TF Plantago lanceolata

Potentilla erecta

Prunella vulgaris

Ranunculus acris

Salvia pratensis

Sanguisorba o�cinalis

Scabiosa columbaria

Silene vulgaris

Taraxacum o�cinale

PL

PE

PV

RA

SP

SO

SC

SV

TO

989A. Stephan,

A.H. Meyer &

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# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

were carried out and sowing densities adjusted so

that for each community total seedling density was

500mÿ2 and seedlings of all component species were

initially present at equal frequencies.

Each plot was further subdivided into four sub-

plots of 2� 2m of which two were subsequently

sampled. Although one of these was subjected to

trampling, disturbance had little e�ect and the two

types of subplots were not therefore separated in the

®nal analyses. All communities were mown twice a

year as is typical for this type of extensively mana-

ged permanent grassland.

We wanted to simulate the consequences of biodi-

versity loss due to plant species extinctions, and

therefore removed seedlings of any non-sown species

as they appeared (Spehn, Joshi, Alphei, Schmid &

KoÈ rner 2000; Spehn, Joshi, Schmid, Diemer &

KoÈ rner 2000). Except at the highest diversity all

initially sown species survived in all plots at the

Swiss site, and PSR therefore remained constant.

Although a few species did die out in the analysed

portions of some 32-species plots, the numbers

always remained closer to the initial value than to

any other PSR level within the experiment (between

24�1.8 in 1996 and 27�1.7 species in 1997 per 2

� 2-m plot). An analysis of the e�ective species

number (the exponential of the Shannon diversity

index, see below, calculated from above-ground

Table 1 continued

(b)

Species no

PSR

1

0

2

1

4

2

8

3

32

5

1a DG 2a DG 3a FP 4a AO 5a AgR 5b AT

LoP HL AE AgS AO

1b LoP PoP CC AP AE

2b AE TF DG AE DG

1c PoP FP FP CC FO

3b CC HL DG FP

1d AE 2c PoP DG PoP FR HL

TF FP TF LoP LoP

1e TF LoP PoP PhP

2d LoP 4b AP TF PoP

1f FP TR 3c AE AO AV LaP

FR CC LaP LC

1g TP 2e AE TF FR LC MS

TP TR LoP OV TP

1h TR PoP TP TR

2f FP 3d HL LaP TR VC

1i PL PL LoP TP AM AM

PoP AjR AjR

1j TO 2g PoP TP 4c AP AnS CP

TO AE BP CJ

3e DG DG CS DC

FR FP CB GP

TF HL DC HS

RA TF GV KA

PL KA LV

3f AE RA PL PM

CC RA PL

PoP 4d AE SP PE

AM DG SO PV

FR SC RA

3g AE LoP SV SO

LoP LC TO TO

LaP TR

TO AM

DC

3h DG

FP 4e FP

LC LoP

PL PoP

TF

TP

TR

KA

TO

990Plant diversity

a�ects soil

bacteria

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

plant biomass proportions) within small 0.2� 0.5m

subplots in the second year of the experiment

showed a very strong congruence between designed

and realized values (linear regression: R2� 0.89, n�64).

Plant diversity treatments a�ected both biomass

allocation patterns (Spehn, Joshi, Schmid, Diemer &

KoÈ rner 2000) and soil characteristics (Spehn, Joshi,

Alphei, Schmid & KoÈ rner 2000). Above-ground bio-

mass increased by 143% from the lowest to the

highest diversity in 1997 and, although total below-

ground plant biomass was not a�ected, that of ®ne

roots also increased signi®cantly. Soil moisture dur-

ing the growing season was not in¯uenced by diver-

sity treatments, but soil temperature decreased

slightly with increasing diversity. The slight increase

in substrate-induced respiration suggested that soil

microbial biomass may increase with increasing

plant diversity, and although in-situ decomposition

of cellulose and birch-wood was not a�ected, both

numbers and biomass of earthworms were strongly

positively correlated with diversity. The presence of

legumes in the experimental plant communities

often had signi®cant e�ects on the activity of soil

fauna.

SAMPLING

Over a 4-day period in the third week of August in

each of 1997 and 1998, we took two soil samples of

100�20mg bulk soil (no rhizosphere, no living or

dead root parts) at a depth of 3 cm in each of the 64

plots (except that in 1997, accidentally, four plots

were not sampled and four other plots were sampled

twice). Preliminary sampling in three of the 64 plots

had shown that the highest activity of culturable soil

bacteria occurred at a depth of 2 cm and the second

highest at a depth of 4 cm. In 1997, the two samples

were taken in the same (undisturbed) subplot at

points separated by 2m, whereas in 1998, one sam-

ple was taken in each subplot. The soil samples were

placed directly into 2.5-mL Eppendorf tubes in the

®eld and were thereafter treated blindly.

EXTRACTION AND INCUBATION OF

BACTERIA

Soil bacteria were extracted within 6 h after sam-

pling. After shaking (Vortex, full speed) for 20min

in 1mL 0.2% Tetrasodium-pyrophosphate, the sam-

ples were allowed to settle for 3min. An aliquot

(150mL) of the supernatant was then diluted 100-

fold with 0.9% NaCl before 100mL were transferred

to each well of the BIOLOG Ecoplate (BIOLOG

Inc., Hayward, CA, USA).

Each of the 96 wells of an Ecoplate contained

dried nutrient solutions, containing a single carbon

compound, and a redox-colourant (tetrazolium vio-

let) (three replicate wells for each of 31 C sources

and no-carbon control). When culturable bacteria

grow, the oxidation of the C source forms NADH

which can be quanti®ed by its reduction of the col-

ourant. The microtiter plates were incubated at 22�C and read with a spectrophotometer (Microplate

Reader 3550, BIO-RAD, Hercules, CA, USA) at

590 nm. Absorbance values were recorded after 72

and 120 h in 1997, but as some of the latter exceeded

the maximum recordable, we decided to shorten the

incubation times in 1998 to 48, 72 and 96 h. We pre-

sent only data after 72 h: analyses using other incu-

bation times did not give di�erent or further

information.

RESPONSE VARIABLES

We took the mean of the three replicate wells per

plate containing a particular substrate and sub-

tracted the absorbance value for the control on the

same individual plate to calculate the catabolic

activity in the use of that C source (Ai). Negative Ai

values were set to zero. Overall catabolic activity

was calculated as the sum of activities for all 31 C

sources and, if divided by the number of C sources,

is equivalent to the AWCD (average well colour

development) calculated by Garland (1996). For

every sample, Ai values were used to calculate rich-

ness S, the Simpson index D0 (the reciprocal of the

dominance index), Shannon index of diversity H0,equitability of D0 and equitability of H0. We used

the formulae given in Begon et al. (1990):

S � number of C sources with Ai > 0;

D0 � 1=XSi�1

P2i ;

H0 � ÿXSi�1

Pi � lnPi

!;

equitability of D0 � D0=S;

equitability of H0 � H0=ln S;

where

Pi � Ai=XSi�1

Ai:

Richness simply represents a numerical measure

of the diversity of C sources that are utilized by a

sample. The Simpson and Shannon indices are com-

bined measures of the `number' and `abundance'

aspects of this diversity, but `number' can be

removed by dividing by richness or the natural loga-

rithm of richness, respectively. Equitabilities there-

fore re¯ect only the `abundance' aspect of the

991A. Stephan,

A.H. Meyer &

B. Schmid

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

diversity in C source utilization: equitability reaches

a maximum if C sources that can be used have the

same Ai values, whereas low values indicate that one

or a few C sources have much higher Ai values than

any others.

STATISTICAL ANALYSIS

The general linear model (GLM) approach to analy-

sis of variance (ANOVA) was used to analyse the

data by means of the Genstat 5 software (release 3;

Payne et al. 1993). The split-plot design involved a

block and plot factor and treatment factors as in

Table 2 (a similar approach to that used by Meyer &

Schmid 1999), to allow calculation of F-values for

signi®cance tests. Block and plot e�ects were used to

eliminate variation caused by spatial di�erences

within the experimental site. Sample mass was used

as a covariate in both years; ®ne root length per

plot and ®ne root biomass per plot (data from

Spehn, Joshi, Alphei, Schmid & KoÈ rner 2000) were

included as further covariates in a separate ANOVA

of 1997 data (results not presented).

Each level of PSR was represented by several

communities, and the e�ect of PSR itself therefore

could be tested against the variation among commu-

nities within PSR levels, to determine the e�ect of

species number per se, unconfounded by any parti-

cular species composition occurring at a particular

PSR level (see Hector et al. 1999). PSR levels could

be ordered along a continuous axis, with a logarith-

mic scale giving better ®ts than the untransformed

plant species number, allowing us to test for signi®-

cant linear contrasts of PSR and the deviation from

linearity. Parsimony, the small deviations from line-

arity and previous reports that biodiversity e�ects

are generally linear at the logarithmic scale (Hector

et al. 1999; Schmid et al. 2001) led us to prefer this

method to polynomial contrasts using untrans-

formed species number. Deviation from linearity

was non-signi®cant and small enough for it to be

omitted in all ®nal analyses of the e�ects of both

PSR and functional diversity (PFD) within PSR (i.e.

`eliminating PSR', see Payne et al. 1993).

Despite allocating large amounts of time and

labour to setting up and running the experiment, it

was only possible to include 32 plant communities.

P<0.1 was therefore regarded as a (`marginal') sig-

ni®cance level in testing the e�ects of PSR and PFD

to reduce the risk of making type-II errors (i.e. not

rejecting the null hypothesis if in fact it is false). In

addition to the P-values for PSR and PFD, we

report partial coe�cients of determination (R2) as a

measure of e�ect size (Cohen 1977). These coe�-

cients measure the proportion of the variation in a

response that is explained by an independent vari-

Table 2 Dummy or skeleton analysis of variance for all characters measured (see text for further details)

Source of variation d.f. Mean square Variance-ratio

Covariate(s) (cov) 1 (n)1 MScov MScov/MSr

Sample mass (w) 1 MSw MSw/MSr

Plot total (pt) 63 MSpt MSpt/MSr

Block (b) 1 MSb MSb/MSp

Mixtures (m) 31 MSm MSm/MSp

Diversity treatments (d) 2 MSd MSd/MSmi

Species richness loglinear (s) 1 MSs MSs/MSmi

No. functional groups linear (f) 1 MSf MSf/MSmi

Mixture identities (mi) 29 MSmi MSmi/MSp

Taxon 1 (t1) 1 MSt1 MSt1/MSp

Taxon 2 (t2) 12 MSt2 MSt2/MSp

Deviation (mid) 272 MSmid MSmid/MSp

Plot (p) 31 MSp MSp/MSr

Year (y) 1 MSy MSy/MSy.pt

Year�plot total 573 MSy.pt MSy.pt/MSr

Residual (r) 1263 MSr

Total (t) 2483 MSt

Values for sources in italics were obtained by totalling the sum of squares and degrees of freedom of their subordinate

contrasts. The deviation of the plant species and functional group diversities from the loglinear and linear contrasts,

respectively, and the interaction of species and functional diversity are not shown since they had small mean squares and

were omitted in all ®nal analyses. Interactions of year with treatment factors were similarly omitted.1For the 1997 data, we also tested ®ne root length and ®ne root biomass as covariates after sample mass (data from Spehn,

Joshi, Alphei, Schmid & KoÈ rner 2000), the d.f. for the covariates was then equal to 3, the number of covariates tested.2We usually tested only one taxon at a time: the second taxon then falls away and the deviation of mixtures identities has

d.f. 28.3Four plots were not sampled in 1997 and seven samples were lost due to defective BIOLOG plates, leading to smaller

degrees of freedom than for a full design without missing values.

992Plant diversity

a�ects soil

bacteria

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

able in a GLM. They were calculated according to

Rosenthal & Rosnow (1985) as

R2 � F-valueeffect � d:f:effect=�F-valueeffect� d:f:effect � d:f:denominator�:

The variation among communities within a parti-

cular PSR and PFD level could be tested for signi®-

cance at the plot level, because each particular

community occurred exactly once in each of the two

blocks. The factor year was used to test for tem-

poral variation in the response variables.

The full ANOVA was done for all response vari-

ables. Analyses of individual Ai values are not pre-

sented as these values were well re¯ected by the

overall catabolic activity. The e�ect of PSR, the fac-

tor in which we are particularly interested, is pre-

sented as a linear regression of the Ai adjusted for

the covariate sample mass. The analyses of indivi-

dual and overall activity and of diversity values used

untransformed data because residuals were normally

distributed and homoscedasticity was not improved

by transformation.

Results

INFLUENCE OF COVARIATES

Sample mass was positively correlated with all

response variables tested (P<0.001) and all ana-

lyses were therefore adjusted for sample mass.

Neither ®ne-root length nor ®ne-root biomass per

plot, which were measured in 1997 and included in a

separate analysis for that year, were signi®cantly

correlated with overall catabolic activity or any of

our measures of catabolic diversity or equitability,

although they have been shown to be positively cor-

related with PSR (Spehn, Joshi, Alphei, Schmid &

KoÈ rner 2000). These additional covariates were not

therefore included in any of the analyses reported

here.

EFFECTS OF DIVERSITY TREATMENTS

Overall catabolic activity increased linearly with

PSR (i.e. log2-transformed species number) (partial

R2� 0.28, P� 0.002, Fig. 1a) and therefore increased

faster at low plant species number than at higher

levels. For a given level of PSR, overall catabolic

activity increased with PFD but with only marginal

signi®cance (partial R2� 0.11, P� 0.062, Fig. 2a).

Of the 31 C sources tested, 15 showed a signi®cant

increase in activity with PSR at P<0.05, and a

further 13 had positive slopes: only three had nega-

tive slopes, none of which were signi®cant (Table 3).

Increased PSR also resulted in greater catabolic

diversity as measured by richness (partial R2� 0.14,

P� 0.037), and by both Shannon (partial R2� 0.21,

P� 0.010) and Simpson indices (partial R2� 0.24, P

� 0.005, Fig. 1b). The positive e�ect of PFD on

catabolic diversity was again rarely more than mar-

ginally signi®cant (richness, R2� 0.20, P� 0.013;

Shannon, R2� 0.10, P� 0.088; Simpson, R2� 0.11,

P� 0.065, Fig. 2b).

The equitabilities of the Simpson and Shannon

indices also showed signi®cant positive correlations

Fig. 1 Relationship between PSR (log2 plant species number) and (a) overall catabolic activity of culturable soil bacteria on

31 C sources (dimensionless absorbance values) and (b) the Simpson index of catabolic diversity in the use of these sources.

Least-square means, adjusted for the covariate sample mass, are shown �1 SE for each of the 32 plant communities, and

may be slightly displaced horizontally for clarity. Communities represented by squares contained legumes: hatched,

Trifolium repens only; open, other legume species; ®lled, both T. repens and other legume species. There were eight samples

for most communities (two years� two blocks� two samples per plot). The regression lines shown for PSR have partial R2

of 0.28 (a) and 0.24 (b).

993A. Stephan,

A.H. Meyer &

B. Schmid

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

Fig. 2 Relationship between PFD (number of plant functional groups) for particular levels of PSR and (a) overall catabolic

activity (dimensionless absorbance values) and (b) the Simpson index of catabolic diversity. Predicted means (from a GLM

including the covariate sample mass, PSR, and PFD)�1 SE are shown for the 10 possible combinations (of PSR and

PFD). Each combination is represented by between one and four communities (see Table 1) except for monocultures (PSR

� 0, n� 10). PSR� 0 (*), PSR� 1 (W), PSR� 2 (&), PSR� 3 (&) or PSR� 5 (~). Parallel regression lines for PFD

within a PSR level give partial R2 of 0.11 in both cases. See `Materials and methods' for further explanations.

Table 3 Catabolic activity of bulk soil bacteria on 31 C sources. Slope parameter of a linear regression model against PSR

(log2-transformed plant species number) for each of 31 C sources used, plus their corresponding standard errors (SE) and

the error probability (P) of the slope being di�erent from zero. NS� not signi®cant

C source Mean Slope SE P

a-D-lactose 0.9978 0.1010 0.0355 <0.01

D,L-a-glycerol phosphate 0.8140 0.0979 0.0275 <0.01

D-glucosaminic acid 0.7301 0.0790 0.0263 <0.01

Pyruvic acid methyl ester 0.6326 0.0755 0.0207 <0.01

D-galacturonic acid 1.0390 0.0736 0.0258 <0.01

b-methyl-D-glucoside 0.7745 0.0616 0.0237 <0.05

a-cyclodextrin 0.3728 0.0615 0.0169 <0.01

D-cellobiose 0.9566 0.0615 0.0400 NS

L-arginine 0.9942 0.0610 0.0260 <0.05

2-hydroxy benzoic acid 0.4616 0.0561 0.0181 <0.01

Phenylethylamine 0.4965 0.0530 0.0219 <0.05

Glucose-1-phosphate 0.4929 0.0493 0.0210 <0.05

Tween 80 0.9271 0.0483 0.0225 <0.05

N-acetyl-D-glucosamine 0.8674 0.0404 0.0226 (<0.1)

Glycogen 0.1797 0.0360 0.0111 <0.01

L-threonine 0.0803 0.0320 0.0071 <0.001

Putrescine 0.7349 0.0315 0.0150 <0.05

Tween 40 1.0736 0.0299 0.0186 NS

Itaconic acid 0.2465 0.0227 0.0191 NS

g-hydroxybutyric acid 0.8039 0.0212 0.0260 NS

L-serine 0.8058 0.0204 0.0197 NS

D-malic acid 0.3332 0.0173 0.0101 NS

D-xylose 0.3080 0.0163 0.0182 NS

L-asparagine 0.9808 0.0160 0.0233 NS

a-keto butyric acid 0.1265 0.0120 0.0116 NS

Glycyl-L-glutamic acid 0.1348 0.0105 0.0038 <0.05

i-erythritol 0.0285 0.0064 0.0038 NS

4-hydroxy benzoic acid 0.3515 0.0040 0.0157 NS

L-phenylalanine 0.0829 ÿ 0.0008 0.0097 NS

D-mannitol 0.7085 ÿ 0.0158 0.0170 NS

D-galactonic acid g-lactone 0.4672 ÿ 0.0184 0.0120 NS

994Plant diversity

a�ects soil

bacteria

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

with PSR (P� 0.006 and P� 0.013, respectively),

but were not further in¯uenced by PFD.

EFFECTS OF PARTICULAR FUNCTIONAL

GROUPS OR SPECIES

Functional group identity

The e�ect of the presence or absence of grasses

could not be tested because all communities, except

some of the monocultures, contained grasses. Forbs

did not have any signi®cant e�ect on any response

variable (P>0.1 for all signi®cance tests). Legumes

had positive e�ects on overall catabolic activity (P

<0.001, Fig. 1a) as well as on all indices of cata-

bolic diversity (Simpson: P� 0.001, Fig. 1b;

Shannon: P� 0.002; richness: P� 0.021; Simpson

equitability: P� 0.009; Shannon equitability: P�0.014).

Species identity

Eighteen plant species occurred in communities at

more than one PSR level and these were tested for

their individual in¯uences on overall catabolic activ-

ity and on all diversity indices (Table 4) but only

one species, the legume Trifolium repens had highly

signi®cant e�ects. The signi®cant or marginally sig-

ni®cant e�ects found for four grass and one forb

species, are close to the number expected by chance

given the high number of tests (six response vari-

ables� 18 plant species tested). The separate ana-

lyses for each of the 31 C sources showed that, for

more than half of them, Ai increased signi®cantly in

samples from communities with T. repens (P<

0.001 in six cases, 0.001EP<0.01 in six cases, 0.01

EP<0.05 in three cases, 0.05EP<0.1 in two

cases). Fewer signi®cant e�ects were found for

other species and could again have occurred by

chance.

The e�ect of inclusion of Trifolium repens on

catabolic activities and diversities was similar to

that of adding legumes as a functional group (see

Fig. 2). In fact, except for the equitability indices, T.

repens explained more of the variation than did

legumes as a functional group, whereas no other

legume species had a signi®cant in¯uence. When we

tested legumes as a functional group before adding

T. repens within legumes as a contrast to the analy-

sis, the e�ect of T. repens on catabolic activity, rich-

ness and Shannon and Simpson indices was still

signi®cant at P<0.05, although equitabilities were

not signi®cantly in¯uenced. When T. repens was

tested ®rst, the remaining legume species only

remained signi®cant at P<0.05 for overall cata-

bolic activity. The e�ects of the functional group

legumes were therefore mainly due to T. repens.

Table4Signi®cancesanddirectionofe�

ects

particularplantspecieshaveoncatabolicactivityanddiversity

(tests

wereonly

madeforspeciesthatoccurred

atmore

thanonePSR-level).NS�notsigni®cant

Overallcatabolicactivity

Richness

Sim

pson-index

Shannon-index

EquitabilityofSim

pson-index

EquitabilityofShannon-index

Trifolium

repens

�<

0.001

�<

0.001

�<

0.001

�<

0.001

�<

0.001

�<

0.001

Poapratensis

±<

0.05

NS

±<

0.1

±<

0.1

±<

0.1

±<

0.1

Dactylisglomerata

NS

NS

�<

0.1

�<

0.1

�<

0.1

�<

0.1

Taraxacum

o�cinale

NS

NS

NS

NS

�<

0.1

�<

0.05

Trisetum

¯avescens

NS

NS

NS

NS

±<

0.1

±<

0.05

Lolium

perenne

�<

0.1

NS

NS

NS

NS

NS

12other

plantspecies

NS

NS

NS

NS

NS

NS

995A. Stephan,

A.H. Meyer &

B. Schmid

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

EFFECTS OF YEAR

In 1998 overall catabolic activity (P� 0.004) and all

diversity indices were signi®cantly higher than in

1997.

Discussion

EFFECTS OF PLANT DIVERSITY ON

ACTIVITY AND FUNCTIONAL DIVERSITY OF

CULTURABLE SOIL BACTERIA

Our results show that plant species richness (PSR)

and plant functional diversity (PFD) have a positive

in¯uence on overall catabolic activity and catabolic

diversity of the culturable bacterial community in

the bulk soil in an experimental grassland ecosys-

tem. Although these bacteria represent only a small

fraction of the taxa present in the soil, we consider

them to be a useful indicator group for measuring

the e�ects of the autotrophic plant components on

the bacterial decomposers in such a system. The

increased oxidation of the C sources supplied

re¯ects an increased bacterial density (Haack et al.

1995) while the increase in catabolic diversity re¯ects

the use of di�erent carbon-oxidation pathways and

therefore functional diversity (Insam, Amor, Renner

& Crepaz 1996; évreaÊ s & Torsvik 1998; Sharma

et al. 1998; Staddon et al. 1998). It is unlikely that a

single genotype or low-level taxonomic unit could

express so much plasticity in C-source utilization,

and this functional diversity is therefore probably

related to taxonomic diversity (Haack et al. 1995;

Buyer & Drinkwater 1997; Baath et al. 1998; Ibekwe

& Kennedy 1998; évreaÊ s & Torsvik 1998).

However, plants show redundancy among taxa

within functional groups (see review in Schmid et al.

2001), and a similar situation may exist within bac-

terial communities, so that functional diversity

would provide a minimum estimate of taxonomic

diversity. We therefore conclude that both activity

and diversity at least of culturable soil bacteria

increase with increasing plant diversity.

We were unable to detect a di�erential response

to plant diversity in the use of a large range of car-

bon sources. Half of those tested, including carbo-

hydrates, amino acids, carboxylic acids,

phosphorylated compounds, polymers, esters and

amines, showed the same pattern as overall cata-

bolic activity. It has previously been suggested that

carbohydrates, carboxylic acids and amino acids can

be used to discriminate between the bacteria of dif-

ferent soil types (Grayston & Campbell 1996).

The diversities of di�erent trophic levels may be

expected to be linked if one level is limiting the

other in a bottom-up (e.g. energy ¯ow; Cody 1975;

Tonn & Magnusson 1982; Brown & Southwood

1983) or top-down process. In our study, higher

plant diversity may have in¯uenced the soil bacteria

by increasing the diversity of litter, the heterogeneity

of soil microhabitats, or energy and material ¯ows

from the vegetation to the soil. Insam, Rangger,

Henrich & Hitzl (1996) and Sharma et al. (1998)

have described positive e�ects of litter quality on

functional diversity of soil bacteria, although

Wardle et al. (1997) found that manipulating litter

diversity directly, rather than (as here) via the diver-

sity of the living plant community, had no e�ect.

The density of earthworms increased by 63% across

our range of diversities (Spehn, Joshi, Alphei,

Schmid & KoÈ rner 2000), suggesting that e�ects of

plant diversity on bacterial activity and diversity

were probably mediated to some extent by increased

heterogeneity of soil microhabitats. Above-ground

biomass increased with increasing PSR (Spehn,

Joshi, Schmid, Diemer & KoÈ rner 2000) and

enhanced ¯ow to the soil may well also have con-

tributed to the positive e�ect on bacteria.

EFFECTS OF INDIVIDUAL PLANT SPECIES

AND PLANT FUNCTIONAL GROUPS ON

ACTIVITY AND FUNCTIONAL DIVERSITY OF

CULTURABLE SOIL BACTERIA

The presence of particular plant species and plant

functional groups (as well as their number) in the

experimental communities has important e�ects on

culturable soil bacteria. The e�ect of legumes as a

functional group was mainly due to just one of the

four species tested (Trifolium repens). Catabolic

activity and diversity of soil bacteria taken from

under a monoculture of this plant species was as

high as in soil from the most diverse plant commu-

nities (see Fig. 1). Legumes form speci®c symbioses

with rhizobial bacteria and it is not therefore sur-

prising that one of them is crucial in the e�ect on

bacterial communities. Although in plants the term

`keystone' species is usually used to describe the

e�ects of a strong competitor on other plant species

(Bond 1993; Troumbis et al. 2000), it can be equally

well applied here, provided that expanding the use

of this term is accompanied by speci®cation of the

a�ected process. The e�ects of some grass and forb

species could not easily be explained and might have

represented statistical type-I errors.

Individual plant species or functional groups

clearly can a�ect the activity and diversity of cultur-

able soil bacteria. The strong e�ects of these species,

however, contrast with many which make a generic

contribution via the overall level of plant diversity.

EFFECTS OF TEMPORAL VARIATION

The di�erences between the two study years might

have been caused by external factors or a succes-

sional e�ect. The sampling period in 1998 was,

unlike that in 1997, preceded by intense rainfall,

although the increased catabolic activity and diver-

sity in 1998, the fourth year after sowing, compared

996Plant diversity

a�ects soil

bacteria

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998

with 1997, suggests successional development of the

underlying soil.

FURTHER CONCLUSIONS ON ECOSYSTEM

FUNCTIONING AND CONSERVATION OF

BIODIVERSITY

Our results show a positive in¯uence of plant diver-

sity on C-source utilization patterns in soil samples

and thus on the activity and functional diversity of

culturable bacteria in the bulk soil (Haack et al.

1995; Insam Amor, Renner & Crepaz 1996). The

relationship may be a mutual one in that plants may

also pro®t from diverse soil bacterial communities,

e.g. mediated by better nutrient mineralization,

growth stimulation, and enhanced antibiosis to

pathogens (Grayston & Germida 1991; Kim et al.

1998; Shah et al. 1998). Mutual relationships

between plant and soil organismic diversity have

also been suggested for the plant±arbuscular mycor-

rhizal system (Van der Heijden et al. 1998). Our

results underline the importance of biodiversity and

species conservation at the di�erent trophic and

taxonomic levels in grassland ecosystems.

Acknowledgements

This project was supported by a grant from the

Swiss Federal O�ce for Education and Science

(Project EU-1311 to B.S.) to join the EU-funded

BIODEPTH project and by grant no. 5001±44628 of

the Swiss National Science Foundation. We would

like to thank P. Breitinger for help in the ®eld and

H. Brandl, J. Joshi, M. Ke ry, E. Spehn and two

anonymous referees for discussion and critical com-

ments on earlier versions of this manuscript. Once

more, L. Haddon did a great job improving the ®nal

version of a manuscript written by authors for

whom English is a second language.

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Received 30 September 1999

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998Plant diversity

a�ects soil

bacteria

# 2000 British

Ecological Society

Journal of Ecology,

88, 988±998