Unveiling below-ground species abundance in a

biodiversity experiment: a test of vertical niche

differentiation among grassland species

Liesje Mommer1,2*, Jasper van Ruijven2, Hannie de Caluwe1, Annemiek E. Smit-Tiekstra1,

Cornelis A.M. Wagemaker3, N. Joop Ouborg3, Gerard M. Bogemann1,

Gerard M. van der Weerden4, Frank Berendse2 and Hans de Kroon1

1Institute for Water and Wetland Research, Experimental Plant Ecology, Radboud University Nijmegen, PO Box 9010,

6500 GL Nijmegen, The Netherlands; 2Nature Conservation and Plant Ecology, Wageningen University, PO Box 47,

6700 AA Wageningen, The Netherlands; 3Institute for Water and Wetland Research, Molecular Ecology, Radboud

University Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands; and 4Institute for Water and Wetland

Research, Botanical garden, Radboud University Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands

Summary

1. Plant diversity has profound effects on primary production. Plant diversity has been shown to

correlate with increased primary production in nutrient-limited grassland ecosystems. This

overyielding has been attributed to vertical niche differentiation among species below-ground,

allowing for complementarity in resource capture. However, a rigorous test of this longstanding

hypothesis is lacking because roots of different species could not be distinguished in diverse

communities.

2. Here, we present the first application of a DNA-based technique that quantifies species

abundances in multispecies root samples. We were thus able to compare root distributions in

monocultures of two grasses and two forbs with root distributions in four-species mixtures. In order

to investigate if vertical niche differentiation is driven by soil nutrient depletion, the topsoil layer of

the communities were either nutrient-rich or -poor.

3. Immediately in the first year, 40% more root biomass was produced in mixtures than

expected from the monocultures, together with significant below-ground complementarity

effects, probably preceding above-ground overyielding. This below-ground overyielding

appeared not to be the result of vertical niche differentiation, as rooting depth of the community

tended to decrease, rather than increase in mixtures compared to monocultures. Roots thus

tended to clump in the very dense topsoil layer rather than segregate over the whole profile in

mixtures. The below-ground overyielding was mainly driven by enhanced root investments of

one species, Anthoxanthum odoratum, in the densely rooted topsoil layer without retarding the

growth of the other species.

4. Synthesis. Conventional ecological mechanisms, such as competition for nutrients, do not seem

to be able to explain the increased root investments of A. odoratum in mixtures compared to

monocultures, with apparently little effect on the root growth of the other species. Instead, the

observed root responses are consistent with species-specific root recognition responses. From a

community perspective, the observed early below-ground overyielding may initiate the recently

reported increased soil organic matter, mineralization and N availability and thus may ultimately

be responsible for the higher productivity at high plant species diversity.

Key-words: biodiversity, determinants of plant community diversity and structure, ecosystem

functioning, niche differentiation, nutrient depletion, root ecology, root interactions, species

interactions

*Correspondence author. E-mail: l.mommer@science.ru.nl

� 2010 The Authors. Journal compilation � 2010 British Ecological Society

Journal of Ecology 2010, 98, 1117–1127 doi: 10.1111/j.1365-2745.2010.01702.x

Introduction

Anthropogenic exploitation of the natural environment

causes dramatic changes in the composition of ecological

communities (Chapin et al. 2000; Hooper et al. 2005). The

decline of plant diversity is one of the major issues in this

context, as plants are the primary producers in terrestrial

ecosystems. After two decades of conducting biodiversity

experiments, it is well established that plants produce more

above-ground biomass when they grow in mixtures than

would be expected from their monocultures (Cardinale et al.

2007). Enhanced biomass production in diverse communities

has been explained by selection and ⁄or complementarity

effects (Loreau & Hector 2001). The selection effect assumes

that the most productive species dominates the biomass of

the species mixtures as a result of chance. Complementarity

or facilitation occur when species exhibit niche differentia-

tion allowing for resources capture that is complementary in

space or time. Cardinale et al. (2007) statistically showed

that above-ground overyielding is largely due to species com-

plementarity.

To date it is unclear whether above-ground overyielding and

complementarity are mirrored underground, as most experi-

ments have focused on shoot biomass only. Only a few biodi-

versity studies quantified total root biomass and report mixed

results, ranging from 50% below-ground overyielding (Tilman

et al. 2001; Dimitrakopoulos & Schmid 2004; Reich et al.

2004) to no overyielding (Spehn et al. 2000; Gastine, Scherer-

Lorenzen & Leadley 2003), or even reduced biomass partition-

ing to roots in mixtures compared to monocultures (Bessler

et al. 2009). Until now, below-ground complementarity has

never been studied in diverse communities, as relative abun-

dance at the species level was unknown for root mixtures. This

omission was largely due to a lack of technical possibilities to

explore root distribution in diverse communities. We recently

developed a molecular method to quantify the proportional

abundance of different species in mixed-species root samples

(Mommer et al. 2008), allowing for tests of below-ground

complementarity.

One commonly proposed mechanism to reach functional

complementarity is vertical niche differentiation below-

ground. This, for example, is assumed to occur between deep-

rooting dicots and shallower-rooting grasses (Parrish&Bazzaz

1976; Berendse 1981, 1983; Dimitrakopoulos & Schmid 2004;

von Felten & Schmid 2008; Levine & HilleRisLambers 2009).

Complementarity would increase if roots of a given species

grew away from zones of intense nutrient competition with

neighbours (Gersani, Abramsky & Falik 1998; Semchenko,

John&Hutchings 2007b), leading to a further vertical segrega-

tion of roots of the different species, reminiscent of the two

barnacle species in the classic example of realized niche differ-

entiation (Connell 1961). Nutrient depletion would then be a

driving force behind niche differentiation (Casper, Schenk &

Jackson 2003). Dicots are thus expected to root even deeper

and grasses even shallower in species-rich mixtures than in

monocultures. Such functional complementarity in vertical

root distribution of different species should in theory lead to a

deeper and more even distribution of roots for the community

as a whole, and may lead to below-ground overyielding. This

overproduction allows amore complete exploitation of the soil

column and its available nutrients and in the end a higher

above-ground biomass production.

Here, we report about a biodiversity experiment with four

grassland species – two grasses and two dicots – with a below-

ground focus. We use the molecular technique of Mommer

et al. (2008) to unravel species abundances in root mixtures to

test the following specific hypotheses: (i) vertical root distribu-

tion will be different among the four species, and these differ-

ences will be more pronounced in mixtures than in

monocultures, leading to vertical segregation of root systems

of the different species. As it takes time for root systems to

establish, the vertical segregation will increase over time. (ii)

Vertical segregation of root systems results in a deeper and

more even distribution of roots at community level, and leads

to below-ground overyielding.

As net nitrogen mineralization typically decreases with soil

depth, most of the roots are present in the topsoil layer. There-

fore, competition for nutrients, and thus nutrient depletion, is

expected to be most intense in the topsoil. To investigate

whether rooting patterns were driven by soil nutrient deple-

tion, we included a treatment with a nutrient-poor topsoil

layer, to simulate intense nutrient competition and experimen-

tally speed up vertical niche differentiation. The third hypothe-

sis is, therefore, as follows: (iii) If nutrient depletion is

intensifying root competition and driving vertical niche differ-

entiation, segregation of rooting patterns of the species should

be enhanced in the poor topsoil treatment compared to the

nutrient-rich control.

Materials and methods

EXPERIMENTAL SETUP AND MEASUREMENTS

Two grasses, Anthoxanthum odoratum L. and Festuca rubra L., and

two forbs, Leucanthemum vulgare Lamk. and Plantago lanceolata L.,

which were also used in another biodiversity experiment (van Ruijven

& Berendse 2005), were grown in replicatedmonocultures and 1:1:1:1

mixtures in a substitutive design (Fig. S1) in plots (50 · 50 cm; 70 cm

depth; grouped by three in polyethylene containers) in the Nijmegen

Phytotron (http://www.ru.nl/phytotron). Soil depth was 64 cm,

divided in a 52-cm thick layer of a rich soil mixture (2:1:1 (v:v:v)

sand : loamy sand : potting soil) and a 12-cm layer consisting of

nutrient-poor riverine sand. The order of the layers depended on the

treatment: in the nutrient-poor topsoil treatment the sand layer was

on top of the nutrient-rich layer, whereas in the nutrient-rich topsoil

treatment the order was reversed. To determine the difference in

nutrient status in the two topsoil types, extractable nutrient content

of the freshlymixed soil was determined at the start of the experiment.

Soil samples (20 g, 20 mm diameter, 0–10 cm deep) were diluted in

50-mL demineralized water, shaken for 1 h gently to dissolve the

nutrients in the water and analysed with an Auto Analyser 3 system

(Bran+Luebbe, Norderstedt, Germany) for nitrate and phosphate.

Values of total extractable nitrogen (N) and phosphorus (P) (mg kg)1

dry soil) were 12.5±1.3 and 0.6±0.1, respectively, for the nutrient-

poor topsoil, whereas these values were 910.4±63.1 and 5.8±0.3,

respectively, for the nutrient-rich topsoil (which was also the bulk

1118 L. Mommer et al.

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

soil; N = 5). At the bottom of all containers was a 4-cm layer with

pebbles, allowing drainage via the outlet at the bottom.

Assignment of the species (mono vs. mixture) · nutrient treat-

ments occurred randomly to a total of 36 plots (12 containers of three

plots each); resulting in monocultures N = 3–4; mixtures N = 4.

Different replicates of species · nutrients were always in different

containers. Five-week-old seedlings, originating from seeds of natural

populations occurring in the river forelands of the river Rhine, near

Nijmegen, the Netherlands, were planted in June 2006. In each plot

36 seedlings were planted (plant density 144 m2), but only the area of

the inner 4 · 4 plants (32 · 32 cm) was used for measurements to

avoid edge effects. In the growing season of 2006, plants were watered

2 L per plot three times a week with an automatic irrigation system

(PRIVA, de Lier, the Netherlands), giving constant moisture levels.

In 2007 this amount was increased to 4 L per plot, three times a week.

In winters, soil moisture levels weremaintained by wateringmanually

once a week.

In late September in 2006 and 2007, standing shoot biomass at the

end of the growing season (living and dead biomass) was harvested

by clipping 1–2 cm above soil surface, a standard practice for hay

meadows in which these species occur. In order to investigate stand-

ing root mass at the end of the growing season, soil cores (20 mm

diameter, two in monocultures, four in mixtures) were taken in seven

soil layers (0–6, 6–12, 12–18, 18–24, 24–40, 40–55, 55–66 cmdepth) at

the beginning of October 2006 and 2007. Distances to surrounding

individual plants were equal (see Fig. S1 in Supporting Information).

Roots (<2 mm) per soil increment were collected after carefully rins-

ing them with tap water. Fresh weight was determined with a micro-

balance (Sartorius, Nieuwegein, the Netherlands). Up to 100 mg of

fresh root material was stored at )80 �C for molecular analyses.

From a second subsample, root length was determined from root

scans (600 dpi, Epson expression 10000 XL; Regent Instruments,

Quebec, Canada) with WinrhizoTM software (‘very pale roots’-

option; Regent Instruments). Shoot and root dry weights were deter-

mined after drying at 70 �C for 48 h.

Quantitative real time polymerase chain reactions (RT-PCR) with

primers for non-coding species-specific markers were performed on

genomic DNA extracts from every mixed root sample separately

(Mommer et al. 2008). Using this technique, species abundance was

quantified in soil layers 1, 2, 4 and 6, comprising 60% (2006) and

80% (2007) of all root mass. Analyses were performed on the basis of

100 mg fresh rootmass. Datawere recalculated in terms of dry weight

as the ratio between dry weight and fresh weight of roots was constant

(mean±SE: 0.18±0.01,N = 50).

For the analysis ofN uptake, all shoot material of individual plants

(two per species per plot) was dried and pulverized. Of this shoot

material, 2–2.5 mg was used in a nitrogen analyser (EA 1110; Carlo

Erba – Thermo Electron, Milan, Italy) in combination with a mass

spectrometer (DEltaPlus; ThermoFinnigan, Bremen, Germany.

CALCULATIONS AND STATIST ICAL ANALYSES

All calculations and statistical analyses on roots were performed on

average plot values: i.e. root data of two cores were pooled in mono-

cultures, four cores in mixtures.

Mean rooting depth was calculated as the sum of the amount of

roots in soil layeri multiplied by the mean depth of layeri divided by

the total amount of roots in all layers. Analyses at the species level are

based on root mass density as determined from the four layers using

the RT-PCR technique. Coefficients of variation (CV) were deter-

mined as the standard deviation of root mass across the soil profile

divided by the mean. In order to compare the species results of mean

rooting depth and CV to the responses of the community as a whole,

analyses at the community scale were also based on these four soil

layers. However, analyses on the seven soil layers resulted in qualita-

tively similar results.

Net biodiversity effects, complementarity effects, selection effects

of shoot and root biomass were calculated according to Loreau &

Hector (2001). In this so-called additive partitioning method, the net

difference in yield for a mixture (DY, the net effect) is equal to the

observed yieldYOminus the expected yieldYE (i.e. the averagemono-

culture yield of the component species):

DY ¼ YO � YE

The net effect is assumed to be the sum of the complementarity

effect and the selection effect. The complementarity effect is calcu-

lated as:

N�mean ðDRYiÞ �mean ðMiÞ

in which N is the number of species, DRYi is the difference

between the observed and expected relative yield for species I and

Mi is the biomass in the monoculture of species i.

The selection effect is calculated as:

N� covarianceðDRYi;MiÞ

For roots, these biodiversity effects were calculated from the roots

in all seven soil layers. Species abundances of roots in soil layer 3 were

estimated from ratios in soil layer 4, roots in soil layers 5 and 7 from

ratios in soil layer 6. To compare observed and expected values,

deviation from expected values (D) were calculated as ln(observed))l-n(expected) for each species and layer. The expected value was always

based on the average of the monoculture(s); i.e. ¼ of the mono-

culture(s). Root : shoot ratios were calculated on the basis of above-

and below-ground biomass values perm2.

Statistical analyses were performed using full-factorial univariate

anovas (General Linear Model, SPSS 15.0), with diversity (monocul-

ture vs. mixture) and treatment (rich vs. poor topsoil layer) as fixed

factors. When analyses were performed at the species level, species

was also included as a fixed factor. Analyses were always run sepa-

rately for the two different years. In cases where D values were used,

significant levels of the intercepts of the predicted model were used to

determine whether these values deviated significantly from zero. This

was also done to evaluate net, complementarity and selection effects.

Further differences in performance (of species) between monoculture

and mixture were determined using t-tests (Compare Means, SPSS

15.0). If needed, variables were transformed in order to meet assump-

tions of anova.

Results

COMMUNITY EFFECTS: BELOW-GROUND

OVERYIELDING AND COMPLEMENTARITY

We observed a marked diversity-induced overyielding

below-ground in the rich topsoil treatment (Fig. 1; Table 1).

Immediately after the first growing season mixtures had

produced up to 35% more root biomass than expected on

the basis of the monocultures (mean relative yield total

RYT2006RichTop = 1.35). This below-ground overyielding,

also indicated by a significant net biodiversity effect (Table 1),

was mainly caused by a significant positive complementarity

effect. Selection effects were small and negative, indicating that

Below-ground species distributions in a biodiversity experiment 1119

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

the subordinate species contributed more to the root growth

stimulation than the highly productive, dominant ones. The

positive effect of diversity on root biomass in the rich topsoil

treatment increased over the second growing season (mean

RYT2007RichTop = 1.48; Fig. 1, Table 1).

Biomass accumulation both below- and above-ground was

only slightly reduced in the poor topsoil compared to the rich

topsoil treatment (Fig. 1). However, overyielding and comple-

mentarity effects were retarded in the poor topsoil compared

to the rich topsoil as they became only significant in the second

year (meanRYT2007PoorTop = 1.62; Fig. 1, Table 1).

Above-ground, no increase of biomass production was yet

apparent in the mixtures in the first 2 years (Fig. 1, Table 1),

which appears to be common in the first years of biodiversity

experiments, particularly in the absence of nitrogen-fixing

legumes (van Ruijven & Berendse 2005). For the rich topsoil

treatment, net biodiversity effects for the shootwere even nega-

tive in the second year (Table 1). The biodiversity effects for

the community as a whole were not apparent, as clear signifi-

cant effects in roots were counterbalanced by negative effects

in the shoot.

NO EVIDENCE FOR ENHANCED VERTICAL NICHE

DIFFERENTIAT ION IN MIXTURES

Six weeks after planting, all species had grown roots already to

the deepest soil layers (minirhizotron observations, data not

shown), but most roots were allocated to the upper layers.

Over both treatments, 30% and 40% of the dicot and grass

roots, respectively, were present in the first 12 cm of soil after

Roo

t bio

mas

s (g

m–2

)

–2000

–1500

–1000

–500

0

A. odoratum F. rubraL. vulgareP. lanceolataExpectedObserved

Sho

ot b

iom

ass

(g m

–2)

0

500

1000

1500

2000

*

Rich topsoil

*

*

20072006Poor topsoil

20072006

*

Fig. 1. Above- and below-ground biomass (g dry weight m)2) for the four monocultures (simple bars), expected and observed values for the

mixtures (hatched and open stacked bars, respectively) in the rich and poor topsoil treatment at the end of the growing seasons of 2006 and 2007.

Anthoxanthum odoratum and Festuca rubra are grasses, Leucanthemum vulgare and Plantago lanceolata are dicots. The expected values are based

on the average of the monocultures. Data are mean±SE, N = 3–4. Significant differences (P < 0.05) between expected and observed values

are indicated by an asterisk, indicating significant over- or underyielding.

Table 1. Biodiversity effects for root, shoot and total biomass (g m)2), as well as shoot nitrogen (N) accumulation (g m)2), in both treatments

(rich topsoil and poor topsoil) in 2006 and 2007. Significant deviations (P < 0.05) from the intercept of the predicted model on square-root

transformed values are given in bold. Data are mean±SE,N = 3–4 plots

Rich topsoil treatment Root Shoot Total N uptakeshoot

2006 Overall net effect 112.0±27.7 71.3±124.3 183.3±112.2 )3.61±2.59

Complementarity effect 112. 5±24.8 94.1±118.2 179.9±112.2 )3.45±2.55

Selection effect )0.5±0.02 )22.8±7.2 3.4±5.0 )0.15±0.05

2007 Overall net effect 232.3±94.7 )114.0±29.9 118.3±102.5 )1.28±0.31

Complementarity effect 315.0±104.4 )142.6±32.6 26.1±100.8 )1.53±0.29

Selection effect )82.7±9.7 28.6±12.6 92.2±24.2 0.25±0.06

Poor topsoil treatment

2006 Overall net effect 5.5±46.6 162.8±69.3 168.4±73.1 )0.21±1.24

Complementarity effect 7.6±46.9 138.0±64.6 171.5±77.3 )0.61±0.96

Selection effect )2.1±0.3 24.8±6.07 )3.2±10.2 0.41±0.31

2007 Overall net effect 342.8±158.6 )13.0±71.6 329.8±150.6 )0.35±0.55

Complementarity effect 390.8±166.2 )16.4±68.6 281.6±148.8 )0.48±0.59

Selection effect )48.0±7.6 3.4±4.5 48.2±25.8 0.13±0.03

1120 L. Mommer et al.

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

the first growing season. In this topsoil layer root densities were

rather high, ranging from 132 up to 623 m dm)3 for P. lanceo-

lata and F. rubra, respectively. These densities increased even

further in the second year as all species expanded their root sys-

tem mainly in this dense upper soil layer (Fig. 2): 70% of the

roots were allocated to the topsoil (top 12 cm).

Festuca rubra and P. lanceolata were the most selective

species with regard to root placement. In monocultures, these

species had 30% and 46% more roots in the rich than in

the poor top soil layers, respectively. In contrast, L. vulgare

had 20% less roots in the rich compared to the poor topsoil.

Root densities in rich and poor topsoil became more similar

after the second growing season (Fig. 2).

Fundamental niches in terms of mean rooting depth in

monocultures differed significantly among species (Table 2;

Fig. 2). The differences among the species were less pro-

nounced than anticipated, as they showed a wide distribu-

tion with considerable overlap. Moreover, there was only a

non-significant trend towards grasses rooting more superficial

than the dicots (mean rooting depth of grasses vs. dicots in

2006 12.1 and 13.9 cm, respectively; F = 2.81; d.f. = 1;

P = 0.12; Fig. 1). All species decreased their mean rooting

depth in 2007 compared to 2006. In contrast to our expecta-

tion, the poor topsoil treatment did not result in significant

deeper rooting of the species (Table 2; Fig. 2).

Applying the molecular tools to quantify species contribu-

tions showed that the niches shifted under interspecific compe-

tition, but the responses differed among species, treatment and

year (Fig. 2; Table 2). Leucanthemum vulgare rooted signifi-

cantly deeper in mixtures than in monocultures of the poor

topsoil treatment in the first year of the experiment (2006), but

not in the second year (2007). More pronounced was the

response of A. odoratum, rooting significantly shallower in

mixtures than in monocultures in the rich topsoil treatment

in both years. Festuca rubra and P. lanceolata did not show

significant differences in mean rooting depth in mixture vs.

0.00 0.25 0.50

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0

ExpectedObserved

Root mass density (g dm–3)0.00 0.25 0.50

**

0.0 0.5 1.0 1.5

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5

0.0 0.5 1.0 1.5

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5

0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5

*

A. o

dora

tum

Soi

l dep

th (

cm)

F. r

ubra

Soi

l dep

th (

cm)

L. v

ulga

reS

oil d

epth

(cm

)P

. lan

ceol

ata

Soi

l dep

th (

cm)

0.0 0.5 1.0 1.5

0–6

–18

–40

–65

0.0 0.5 1.0 1.5 2.0

0–6

–18

–40

–65

0.00 0.25 0.50

0–6

–18

–40

–65

*

0.00 0.25 0.50

0–6

–18

–40

–65

Rich topsoil

20072006

Poor topsoil

20072006

Fig. 2. Rooting patterns of different species in monocultures and mixtures. Observed (in actual four-species mixtures) and expected (based on

the respective monocultures) root distributions (circles and lines; g dm)3) and mean rooting depth (diamonds; cm) of the different species in both

treatments (rich or poor top soil; hatched bar indicates the position of the nutrient-poor sand layer within the container) and years. Anthoxant-

hum odoratum and Festuca rubra are grasses, Leucanthemum vulgare and Plantago lanceolata are dicots. Data are mean±SE, N = 3–4. Aster-

isks indicate significant differences inmean rooting depth betweenmonocultures andmixtures: *P < 0.05; **P < 0.01. Please note the different

scales of rootmass density among species and years.

Below-ground species distributions in a biodiversity experiment 1121

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

monoculture. While for two of the four species – A. odoratum

and (partly) L. vulgare – differences in mean rooting depth

were more pronounced in mixtures than in monocultures,

vertical segregation of root systems seemed to occur.

At the community level, the below-ground vertical segre-

gation of the two species did not result in a deeper and more

even distribution of roots. Mean rooting depth of the total

community was not increased in mixtures compared to

monocultures (Table 3). Different topsoil layers did not

affect the mean rooting depth of the community either

(Table 3). Moreover, there was a non-significant trend for

larger rather than smaller coefficients of variation of root

mass over soil layers in mixtures compared to monocultures

(Table 3), suggesting that root distributions tended to clump

rather than be more even in the diverse community.

ENHANCED ROOT GROWTH OF A. ODORATUM IN

MIXTURES

An appropriate null-hypothesis based on interspecific competi-

tive equivalence is that all species would produce¼ of the root

mass in the mixtures compared to the monocultures (dotted

line, Fig. 3). If species differ in competitive ability, some spe-

cies would do better (above the dotted line of Fig. 3), and some

worse (below the dotted line). Using the molecular tools to

unravel the species abundance in the mixtures, we found that

most root masses in mixtures were as high or higher than

expected from their respective monocultures, as most points

were above the dotted line, but not significantly different from

the 1 : 4 expectation (Fig. 3). Only a few root masses of L.

vulgare in mixtures were significantly lower than expected in

the null-hypothesis (indicated with ‘–’ in Fig. 3). In contrast,

most root masses of A. odoratum in mixtures, but also some

root masses of other species, were significantly larger than the

null hypothesis predicted (indicated with ‘*’ in Fig. 3). More-

over, the A. odoratum root masses of the topsoil were not sig-

nificantly different from the 1 : 1 line (indicated with an arrow

in Fig. 3), indicating that root mass in mixtures equals root

mass in monocultures in the top soil layer, while there were

only one-fourth of the number of individual plants in the mix-

tures than themonocultures.

In contrast to A. odoratum, the other grass species in the

experiment, F. rubra, was strikingly unresponsive to growth in

monoculture vs. mixture. Festuca rubra had by far the largest

root system (Fig. 1), but did not take advantage of the lower

root length density of the other species when growing in the

mixture (Fig. 3).

The described root patterns of A. odoratum and the other

species occurred in the rich top treatment immediately in the

first growing season, whereas in the poor topsoil treatment this

took more time. After two growing seasons, however, the pat-

terns were largely similar in the two treatments.

Table 3. Mean rooting depth (cm,MRD) and coefficient of variation of root mass over soil layers (CV) and anova results for the community as a

whole, calculated from root mass distribution over four soil layers (0–6; 6–12; 12–24; 40–55 cm depth). Treat. indicates the top soil treatment

(nutrient-rich or poor); Div. indicates the diversity level of the community (monoculture vs. four-species mixture). Mean rooting depths were

ln-transformed in order tomeet anova assumptions. *P < 0.05; †P < 0.10;N = 3–4 plots

Year Treat. Div.

MRD CV

Source D.f.

MRD CV

Mean±SE Mean±SE F-value F-value

2006 Rich top Mono 13.2±0.7 0.57±0.06 Div. 1 1.7 3.1

Mix 11.7±0.8 0.69±0.07 Treat. 1 0.3 3.9†

Poor top Mono 12.3±0.1 0.71±0.05 Div. · Tr. 1 0.6 0.1

Mix 11.9±0.7 0.80±0.04 Error (MS) 9 1.6 118.6

2007 Rich top Mono 6.4±0.02 1.36±0.02 Div. 1 0.3 0.9

Mix 6.1±0.2 1.47±0.01 Treat. 1 2.3 8.7*

Poor top Mono 6.5±0.7 1.32±0.07 Div. · Tr. 1 1.7 3.3

Mix 7.2±0.3 1.29±0.04 Error (MS) 9 0.4 46.7

Table 2. anova results, split per year, for mean rooting depth with

species, diversity (mono vs. mixtures) and treatment (i.e. rich vs. poor

topsoil) as fixed factors. Overall rooting depths were significantly

different among species. General diversity and treatment effects were

not apparent. To explore the significant interaction between species

and diversity, another anova, split per year and species, showed that

diversity effects were significant for Anthoxanthum odoratum in both

years (shallower rooting) and for Leucanthemum vulgare in 2006

(deeper rooting; analysis not shown). *P < 0.05; **P < 0.01;

***P < 0.001

Year Source d.f. F-value

2006 Species 3 17.5***

Diversity 1 0.77

Treatment 1 0.41

Species · Div 3 5.14**

Species · Treat 3 4.10*

Div · Treat 1 6.69*

Sp · Div · Treat 3 0.79

Error (d.f., MS) 38, 4.95

2007 Species 3 9.30***

Diversity 1 0.23

Treatment 1 0.14

Species · Div 3 3.95*

Species · Treat 3 1.40

Div · Treat 1 6.19*

Sp · Div · Treat 3 0.57

Error (d.f., MS) 38, 2.31

1122 L. Mommer et al.

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

Over all species, root : shoot ratios significantly increased

over time (analysis on transformed value (ln + 1): F = 112.7,

d.f. = 1; P < 0.001; Fig. 4), which was to be expected as

nutrients were depleted over time with the removal of above-

ground biomass from the plot. Root : shoot ratios were also

significantly higher in the poor topsoil than in the rich topsoil

(F = 9.2, d.f. = 1, P < 0.005; Fig. 4). However, the largest

allocation shift was observed in A. odoratum, where the

enhanced root mass production in mixtures resulted in a three-

fold root:shoot ratio increase compared to monocultures

(Fig. 4). The other species did not show significant changes in

root:shoot ratio in the rich topsoil treatment. In the poor top-

soil, F. rubra andP. lanceolata even had significantly decreased

root:shoot ratios in mixture compared to monoculture in the

first growing season (Fig. 4).

Discussion

While species differences in phenology of root growth (Fargi-

one & Tilman 2005), nitrogen preference (McKane et al. 2002;

von Felten et al. 2009) and specific interactions with soil

organisms (Kardol et al. 2007; van der Heijden, Bardgett &

van Straalen 2008)may all contribute to overyielding and com-

plementarity in species-rich communities, species differences in

rooting depth are commonly assumed to form a prime axis of

niche differentiation below-ground (Parrish & Bazzaz 1976;

Berendse 1981, 1983; Dimitrakopoulos & Schmid 2004;

Schenk & Jackson 2005; von Felten & Schmid 2008; Levine &

HilleRisLambers 2009). However, using a newmolecular tech-

nique that quantifies root distributions of individual species in

mixed communities, we have not been able to obtain support

for this longstanding hypothesis in ecology. Species indeed dif-

fered in their fundamental and realized niches regarding mean

rooting depth, but there was a wide overlap in their distribu-

tion. At the community level, overall rooting depth tended to

decrease, rather than increase, in mixtures compared to mono-

cultures, and roots tended to clump in the very dense topsoil

layer rather than segregate over the whole profile in mixtures.

Although the trend from the first to the second year was in the

opposite direction, it may take more than the two growing

0.02 0.5 1 5 10 15 20

0.1 0.2 0.5 1 2 3 4 5

Null-hypothesis

Soil depth

-

*

*

**

Mixture = Monoculture

Mixture = Monoculture

Null-hypothesis

*

*

0.1 0.2 0.5 1 2 3 4 5

Roo

t mas

s de

nsity

MIX

TU

RE

(g

dm–3

)

0.025

0.05

0.1

0.5

1

2

3

A. odoratumF. rubraL. vulgareP. lanceolata

Mixture = Monoculture

Null-hypothesis

Root mass density MONOCULTURE (g dm–3) Root mass density MONOCULTURE (g dm–3)

0.02 0.5 1 5 10 15 20

Roo

t mas

s de

nsity

MIX

TU

RE

(g

dm–3

)

0.002

0.01

0.1

1

10

Soil depth

Soi

l dep

thS

oil d

epth

*

*

-

-Null-h

ypothesisMixture = Monoculture

*

2007

Poor topsoil20

06Rich topsoil

Fig. 3. Root mass density in mixtures as a function of root mass density in monoculture of the different species in the different soil layers (0–6;

6–12; 12–24; 40–55 cm depth). Data are presented separately for the rich and poor topsoil treatment in both years. The dotted line represents the

null-hypothesis: in themixtures all species would produce¼of the rootmass of themonocultures, assuming that intraspecific competition equals

interspecific competition. Points that are significantly lower than expected from the null-model are indicated with – (only L. vulgare). Points that

are significantly larger than the null-model are indicated with *. The solid line indicates where the root biomass in mixture is equal to those of the

monocultures, despite the number of plant individuals being only one-fourth of the monocultures, and points not significantly different from this

line are indicated with an arrow (Anthoxanthum odoratum in topsoil layer only). Because root biomass declined with depth (Fig. 1), both axes can

also be read as the inverse of soil depth.Anthoxanthum odoratum and Festuca rubra are grasses, Leucanthemum vulgare and Plantoago lanceolata

are dicots. Data are mean±SE,N = 3–4.

Below-ground species distributions in a biodiversity experiment 1123

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

seasons of ourmeasurements before vertical niche complemen-

tarity becomes apparent below-ground.

As in other studies (Tilman et al. 2001; Dimitrakopoulos &

Schmid 2004; Reich et al. 2004), we observed below-ground

overyielding in our experiment, which was apparent immedi-

ately in the first year (Fig. 1, Table 1), preceding overyielding

above-ground. Below-ground overyielding mainly occurred in

the dense topsoil layers and increased over time (Fig. 2). This

was mainly the result of enhanced root growth ofA. odoratum,

with little reduction in root biomass of the other species. Here,

we discuss the possible underlying mechanisms and ecosystem

consequences of the enhanced standing rootmass with increas-

ing diversity.

A LIMITED ROLE OF NUTRIENT RESPONSES IN

UNDERSTANDING THE INTERSPECIF IC ROOT

RESPONSES

How can the increased root mass production of A. odoratum

in mixtures compared to monocultures be explained by

conventional mechanisms of resource foraging and biomass

allocation? Root mass increase could be the prelude to over-

all dominance if A. odoratum would be the competitively

superior species. However, root mass of the other species was

only slightly impeded (Fig. 3) and selection effects were

negative rather than positive (Table 1), underscoring that

A. odoratum was not the most productive species in our

experiment, neither above- nor below-ground (Fig. 1), and

never became dominant in mixtures. Indeed, this species

was always among the three least productive species out

of eight common grassland species in the 9-year time series

of the Wageningen biodiversity experiment (van Ruijven

& Berendse 2009). In the Wageningen monocultures of

A. odoratum, standing above-ground biomass was c.

200 g m)2 in the first year, and gradually decreased to c.

80 g m)2 after 9 years. Monocultures of F. rubra and other

species initially yielded 250–500 g m)2, but decreased to 180–

250 g m)2; which is still more than double the production of

A. odoratum. Moreover, A. odoratum did not have the high-

est relative abundance in 4- and 8-species mixtures over the

9 years (25–20%, respectively), since F. rubra (30–15% in

4- and 8-species mixtures, respectively) and other species (i.e.

P. lanceolata 40–30% in 4- and 8-species mixtures, respec-

tively; Centaurea jacea (70–40%, respectively)) have similar

or higher relative abundances. Finally, A. odoratum did not

outcompete the other species in 4- and 8-species mixtures

over the 9 years that the experiment was running.

The higher root mass of A. odoratum in mixture was associ-

ated with a remarkable threefold increase in root : shoot ratio

ofA. odoratum. A stimulation of root allocation of this magni-

tude seems at odds with known responses to nutrient depletion

of the soil (Poorter & Nagel 2000). We observed significant

effects related to nutrient depletion (significant effects of time

and topsoil treatment) on root : shoot ratio, but these were

not of such an extraordinary magnitude (Fig. 4). Moreover,

the root : shoot ratio shift of A. odoratum occurred indepen-

dent of the nutrient treatment of the topsoil, and in both years

of study.

Furthermore, the lack of essential differences between the

rich and poor topsoil treatment also indicates that it is not sim-

ply the availability of nutrients that determine the interspecific

root responses. Based on these arguments, we suggest that

SpeciesA. odoratum F. rubra L. vulgare P. lanceolata

nsns

ns

***

$

ns** ***

nsns ns

SpeciesA. odoratum F. rubra L. vulgare P. lanceolata

Roo

t : s

hoot

rat

io

0

1

2

3

4 ns

ns

ns

***

Roo

t : s

hoot

rat

io

0

1

2

3

4Monoculture Mixture

Rich topsoil Poor topsoil

2006

2007

Fig. 4. Allocation patterns to root and shoot given by root : shoot ratios of the different species in monoculture and mixture in both years (2006

and 2007) in the rich and poor topsoil treatment. Anthoxanthum odoratum and Festuca rubra are grasses, Leucanthemum vulgare and Plantago

lanceolata are dicots. Data are mean±SE, N = 3–4. Significant differences between monocultures and mixtures per species and year on

ln+1-transformed values are indicated with asterisks: **P < 0.01; ***P < 0.001; $P < 0.1.

1124 L. Mommer et al.

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

competitive and allocation responses to soil nutrients alone are

unlikely to drive the elevated root mass in mixtures vs. mono-

cultures.

ALTERNATIVE EXPLANATIONS FOR THE SPECIES-

SPECIF IC ROOT INTERACTIONS

Recent advances in root ecology suggest that root growth and

distribution can be tuned to the identity of neighbouring roots

(Schenk 2006; de Kroon 2007) by distinguishing roots of the

same physiological individual from roots of a different geno-

type of the same species (Maina, Brown & Gersani 2002;

Gruntman&Novoplansky 2004; Dudley &File 2007) or roots

of a different species (Huber-Sannwald et al. 1998; Bartelhei-

mer, Steinlein & Beyschlag 2006; Li et al. 2007; Semchenko,

Hutchings & John 2007a). Responses to neighbours range

from inhibition to stimulation of root growth (Mahall & Call-

away 1991; Li et al. 2006; Semchenko, John & Hutchings

2007b). The mechanisms behind root recognition responses

are yet obscure, but non-nutritious cues, such as root exudates

(Bais et al. 2006) and ⁄or root rhizosphere biota (Kardol et al.

2007; van der Heijden, Bardgett & van Straalen 2008) may be

involved since the root responses appear very species-specific

and density-dependent.

We would argue that the root biomass responses of

A. odoratum are consistent with the operation of such non-

nutritious cues: growth enhancement at low densities (i.e. in

mixtures) and ⁄or inhibition of root growth at high densities

(monocultures), without affecting the root growth of the

other species. Density-dependent regulation of root growth

in monocultures and mixtures may have occurred via accu-

mulation of autotoxic root exudates (Bais et al. 2006) and ⁄orhost-plant specific pathogens (Van der Putten & Peters 1997;

Westover & Bever 2001). Alternatively, root growth of

A. odoratum in mixtures may have been stimulated by the

actual presence of roots of other species, directly, due to

growth-stimulating root exudates from these species and ⁄orindirectly, due to root growth-promoting rhizobacteria (Bais

et al. 2006; Berg & Smalla 2009). For our study species,

A. odoratum, it has been shown that seedlings grow better

on natural root growth-promoting isolates of Bacillus of

other species than on its own isolates (Westover & Bever

2001), suggesting that root growth promoting bacteria may

play a role in grassland systems. A more indirect explanation

for the enhanced root growth of A. odoratum in diverse com-

munities would be that high plant diversities affect soil

microbial communities (Kowalchuk et al. 2002; Bartelt-

Ryser et al. 2005), leading to suppression of (root) pathogens

(i.e. soil suppressiveness (Weller et al. 2002); cf. studies in

leaves (Mitchell, Tilman & Groth 2002)), although it usually

takes several years for the microbial communities to build

up (Steinbeiss et al. 2008). Such pathogens should then be

more deleterious for A. odoratum than for the other species

of our system in order to explain our results.

We conclude that our results are consistent with non-

nutritional, density-dependent feedback mechanisms as

reported for the spread of invasive species (Bais et al. 2003;

Levine et al. 2006) and plant succession (Bever 1994; Kardol

et al. 2007). However, although the interactions below-

ground may be essentially non-nutritious and depending on

species identity, the effects may improve the nutritional sta-

tus in mixtures vs. monocultures (cf. Li et al. 2007) or

increase competitiveness for soil nutrients. Future research is

needed to demonstrate what interactions are really involved

and to pinpoint the exact mechanisms in this apparent spe-

cies-specific root recognition.

ECOSYSTEM IMPLICATIONS

The ecosystem implications of the below-ground overyielding

reported here and in other studies (Tilman et al. 2001;

Dimitrakopoulos & Schmid 2004; Reich et al. 2004) may be

considerable, as the higher root mass in these grassland

mixtures appears to persist with possibly important conse-

quences for the soil C andN budget. In theWageningen biodi-

versity experiment (van Ruijven & Berendse 2005, 2009), root

masses were 40% higher in four-species mixtures than in the

average monoculture after 9 years (L. Mommer & J. van

Ruijven, unpubl. data). Similarly, in the Cedar Creek biodiver-

sity experiment, total root mass was 50–200% higher in the

eight-species mixtures than in the monocultures, 12 years after

the start of the experiment (Fornara, Tilman & Hobbie 2009).

Given the high turnover rates of roots (Van der Krift &

Berendse 2002), a higher root mass production will lead to

increased organic matter input into the soil. Indeed, after

4 years in the Jena Biodiversity Experiment, carbon stocks

were significantly higher in plots with higher species diversity

(Steinbeiss et al. 2008). In the long-term, increased organic

matter input can increase mineralization and N-availability

(Fornara, Tilman & Hobbie 2009), which in turn is held

responsible for the higher productivity in more diverse plant

communities.

Conclusions

Our experimental grassland communities revealed below-

ground species complementarity as expected, but found no

evidence for the associated vertical niche differentiation that

is generally assumed. While the exact mechanisms for the

observed root responses remain obscure, the results are

consistent with species-specific non-nutritional root interac-

tions. The root growth stimulation in species mixtures may

be the trigger to enhanced carbon sequestration and N

availability in the soil, finally leading to enhanced above-

ground production in diverse plant communities. If these

novel interactions indeed play a role in below-ground over-

yielding, they may be the drivers of fundamental ecosystem

processes.

Acknowledgements

We thank Jelle Eygensteyn for N measurements; Jan van Walsem and Frans

Moller for help during harvests and Jean Knops, Wim van der Putten, Ronald

Pierik and Eric Visser for comments on an earlier version of the manuscript.

Below-ground species distributions in a biodiversity experiment 1125

� 2010 The Authors. Journal compilation � 2010 British Ecological Society, Journal of Ecology, 98, 1117–1127

L.M. is supported byNetherlands Organization for Scientific Research (NWO)

VENI grant 016091116.

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Received 08 February 2010; accepted 18 June 2010

Handling Editor:Marina Semchenko

Supporting Information

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Figure S1. Experimental setup of amixture plot.

Figure S2. Nutrient uptake at the community scale.

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