RESEARCH New Phytol.

(2000), 147, 73–85

Dynamics of root systems in native

grasslands : effects of elevated atmospheric
CO
#
J. A. A R N O N E I I I",#*, J. G. Z A L L E R",$, E. M. S P E H N", P. A. N I K L A U S",
C. E. W E L L S%    C. K O$ R N E R"
"Department of Integrative Biology, University of Basel – Botanical Institute,
SchoW nbeinstrasse 6, CH-4056 Basel, Switzerland
#Desert Research Institute, Division of Earth and Ecosystem Sciences,
2215 Raggio Parkway, Reno, NV 89512, USA
$The Ecology Center, Utah State University, Logan, UT 84322–5230, USA
%Department of Environmental and Resource Sciences, University of Nevada-Reno, Reno,
NV 89557, USA
Received 4 November 1999 ; accepted 8 March 2000


The objectives of this paper were to review the literature on the responses of root systems to elevated CO in intact,
#
native grassland ecosystems, and to present the results from a 2-yr study of root production and mortality in an
intact calcareous grassland in Switzerland. Previous work in intact native grassland systems has revealed that
significant stimulation of the size of root systems (biomass, length density or root number) is not a universal
response to elevated CO . Of the 12 studies reviewed, seven showed little or no change in root-system size under
#
elevated CO , while five showed marked increases (average increase 38%). Insufficient data are available on the
#
effects of elevated CO on root production, mortality and life span to allow generalization about effects. The
#
diversity of experimental techniques employed in these native grassland studies also makes generalization difficult.
In the present study, root production and mortality were monitored in situ in a species-rich calcareous grassland
community using minirhizotrons in order to test the hypothesis that an increase in these two measures would help
explain the increase in net ecosystem CO uptake (net ecosystem exchange) previously observed under elevated
#
CO at this site (600 vs 350 µl CO l−" ; eight 1.2-m# experimental plots per CO level using the screen-aided CO
# # # #
control method). However, results from the first 2 yr showed no difference in overall root production or mortality
in the top 18 cm of soil, where 80–90% of the roots occur. Elevated CO was associated with an upward shift in
#
root length density : under elevated CO a greater proportion of roots were found in the upper 0–6-cm soil layer,
#
and a lower proportion of roots in the lower 12–18 cm, than under ambient CO . Elevated CO was also associated
# #
with an increase in root survival probability (RSP ; e.g. for roots still alive 280 d after they were produced under
ambient CO , RSP l 0.30 ; elevated CO , RSP l 0.56) and an increase (48%) in median root life span in the
# #
deepest (12–18 cm) soil layer. The factors driving changes in root distribution and longevity with depth under
elevated CO were not clear, but might have been related to increases in soil moisture under elevated CO
# #
interacting with vertical patterns in soil temperatures. Thus extra CO taken up in this grassland ecosystem during
#
the growing season under elevated CO could not be explained by changes in root production and mortality.
#
However, C and nutrient cycling might be shifted closer to the soil surface, which could potentially have a
substantial effect on the activities of soil heterotrophic organisms as CO levels rise.
#
Key words : root turnover, root production, root mortality, CO enrichment, global change, species-rich grassland,
#
missing carbon, root life span.

pheric CO , with special emphasis on root dynamics ;


The central aims of this paper are to synthesize the
current literature on the responses of root systems of
intact, native grassland ecosystems to elevated atmos-
*Author for correspondence (fax j1 775 673 7485 ; e-mail
jarnone!dri.edu).
#
and to present empirical results from the first 2 yr of
a study of root dynamics (especially of production
and mortality), conducted in a species-rich lowland
calcareous grassland in Switzerland, exploring the
possibility that increased root mortality might help
to explain the fate of the ‘ extra ’ carbon apparently
 74
RESEARCH J. A. Arnone III et al.
Table 1. Effects of elevated CO (mean difference as percentage change from ambient CO ) on fine-root biomass, length density production and mortality in native (in situ)
# #
or intact (monoliths) grasslands

CO treatments
#
(µl CO l−") and Observed Standing root
#
Experimental environmental Stage of soil depth biomass, number Root Root
Grassland system conditions* experiment (cm) Method or length production mortality Reference

Tallgrass prairie In situ 345, 685 Second yr 0–40 Coring No difference Mo et al. (1992)
C Kansas, ventilated
%
USA closed
chambers
Tallgrass prairie In situ 350, 700 OTC First 2 yr 0–15 Ingrowth cores j125% (Year 1) Owensby et al.
C \C mix j17% (Year 2) (1993)
$ %
Kansas, USA
Shortgrass Large cores 350, 700 growth Second growing 0–45 Entire core j23% (C ) Hunt et al.
$
steppe C or C chambers season harvested No difference (C ) (1996)
$ % %
Colorado, (PFD l 550
USA µmol m−# s−")
Mediterranean Monoliths 350, 700 First growing 0–20 Entire monolith No difference Navas et al.
annual glasshouse (full season harvested (1995)
Montpellier, sun)
France
Pasture turf Monoliths 350, 700 growth 217 d 0–25 Coring j50% Newton et al.
New Zealand chambers (1994)
(PFD l 480
µmol m−# s−")
Pasture turf Monoliths 350, 525, 700 324 d 0–30 Sequential cores j72% 350–525 Newton et al.
(sandy) growth j0% 525–700 (1995)
New Zealand chambers
(PFD l 500 Additional 105 d Coring j39% (wet soil) Newton et al.
µmol m−# s−") (to 429 d) j21% (dry soil) (1996)
350–700 ppm CO
#
 RESEARCH CO and native grassland root systems
Natural turf Monolith 350, 600 Two growing 0–25 Entire monolith j41% j20% j21% Fitter et al.
Nardus Juncus glasshouse (full seasons harvested\ (cumulative (cumulative (1996, 1997)

#
on peaty gley sun) minirhizotrons births) deaths)
UK
Calcareous Monoliths 350, 600 Two growing 0–25 Coring\ j48% j30% j29% Fitter et al.
UK glasshouse (full seasons minirhizotrons (1996, 1997)
sun)
Calcareous In situ 360, 700 OTC 3 yr 0–20 Minirhizotrons j25% (dry years) Sindhøj et al.
Sweden No difference (wet (2000)
years)
Calcareous In situ 350, 600 SACC First 4 yr 0–8 Coring No difference Leadley et al.
Switzerland Years 1 and 2 0–18 Minirhizotron No difference No difference No difference (1999) ; Arnone
(550 m) Year 1 Year 1 et al. (this study)
Calcareous Monoliths 350, 600 Second growing 0–20 Coring\entire No difference Sto$ cklin et al.
Switzerland glasshouse (full season monolith (1998)
(550 m) sun)
Salt marsh In situ 343, 681 OTC Second yr 0–15 Ingrowth cores j83% (C ) Curtis et al.
$
Maryland, C (Scirpus) No difference (1990)
$
USA C (Spartina) (C )
% %
Mixed C \C No difference
$ %
(mixed)
Alpine In situ 350, 600 OTC Year 2 (cool) Ingrowth cores No difference Scha$ ppi &
Switzerland Year 3 (warmer) 0–10 Small and No difference (Year 2) Ko$ rner (1996)
(2480 m) ingrowth cores (Year 3) j27% (Year 3) Ko$ rner et al.
Year 5 0–20 Large cores j12% (Year 5) (1997)

*PFD, photon flux density ; OTC, open-top chamber ; SACC, screen-aided CO control.
#

75
76 RESEARCH J. A. Arnone III et al.
taken up by ecosystems under elevated CO during
# $
the growing season (Stocker et al., 1997). The
literature on the effects of CO enrichment on root
#
systems of individual grass species growing in
nonnative systems (e.g. agricultural fields) or under
relatively artificial conditions (e.g. single plants in
pots on potting mix, or planted communities on
homogenized soils) has been reviewed extensively by
Rogers et al. (1994, 1996) and by Pritchard & Rogers
(2000).
Nonmanaged and extensively managed native
grasslands of the world account for more than half of
the grassland biome, and store tremendous amounts
of C in their soils (Schimel, 1995). On a global scale,
the grassland biome accounts for 25% of all
terrestrial land surface and 10% of global C stores
(mostly in soils ; Schimel, 1995 ; Schlesinger, 1997).
Because most (60–90%) of the net primary pro-
ductivity in grassland systems occurs below ground,
and root biomass pools often exceed above-ground
standing biomass pools by as much as two to five
times (Speidel, 1976 ; Stanton, 1988), the contri-
bution of root litter to the pool of soil organic matter
could greatly exceed the contribution of shoot litter.
Thus the way in which root systems of native
grasslands respond to elevated atmospheric CO will
#
probably play a large role in defining ecosystem C
cycling in the future, and in determining the
potential for grassland ecosystems to sequester C.
The literature on responses of intact native
grassland root systems to elevated CO is limited. Of
#
the 12 studies we identified (Table 1 ; with the study
by Hunt et al. counted twice because of separate
evaluation of C and C systems, but not including
$ %
the study presented in the current paper), six were
conducted in the field (in situ), and six in intact
monoliths that were removed from the field and
exposed to CO treatments in glasshouses or growth
#
chambers. With so few studies, and with so many
different experimental methodologies (CO levels ;
#
study duration ; soil depth sampled ; sampling
method ; parameters measured ; Table 1), it is
extremely difficult to discern general patterns of CO
#
response, even with respect to the two most common
measures of root response – standing biomass, length
or numbers (size) of roots (all 12 studies) ; and root
production (five studies). Seven of the 12 studies
reported little ( 20%) or no increase in the size of
root systems under elevated CO (C grasslands : Mo
# %
et al., 1992, 0% ; Hunt et al., 1996, 0% ; C
$
grassland : Navas et al., 1995, 0% ; Sto$ cklin et al.,
1998, 0% ; Leadley et al., 1999, 0%) ; Scha$ ppi &
Ko$ rner, 1996 and Ko$ rner et al., 1997, j6%, 2-yr
mean ; Sindhøj et al., 2000, j12%, 2-yr mean). The
average stimulatory response to elevated CO in
#
these low-responding systems (excluding the C
%
grassland of Mo et al., 1992 and Hunt et al., 1996) is
approx. j4%. The average stimulatory effect of the
other five studies reporting stronger effects was 38%
(C grassland : Hunt et al., 1996, j23% ; Newton
et al., 1994, j50% ; Newton et al., 1996, j30%,
2-yr mean ; Fitter et al., 1996, 1997, j41 and j48%
in Nardus\Juncus turf and calcareous grassland,
respectively). The explanation for low- versus high-
responding systems is unclear, but might be related
to length of growing season or overall soil resource
availability. For example, four of the seven low-
responding systems were conducted in the field
(possibly a shorter growing season), whereas all the
high responding systems were conducted in growth
chambers or glasshouses (possibly a longer growing
season). This last piece of speculation makes the
need for more field studies abundantly clear. Finally,
only two studies (Fitter et al., 1996, 1997) have
directly measured the effects of elevated CO on root
#
mortality in intact native grasslands (j21 and
j29%). No complete data set exists for native
grasslands that addresses the dynamic responses of
roots (production, mortality and fluctuations in root-
length density) to elevated atmospheric CO in situ.
#
Yet the available data clearly indicate that under-
standing the dynamic responses of grassland root
systems to elevated CO is key to understanding
#
ecosystem and global C cycling (Curtis et al., 1994 ;
Jackson et al., 1996, 1997).
Two almost universal sets of responses of plants
and plant communities to elevated atmospheric CO
#
are (1) stimulation of growing-season leaf and canopy
photosynthesis and net ecosystem CO exchange
#
(NEE ; Jackson et al., 1994 ; Wolfenden & Diggle,
1995 ; Drake et al., 1996 ; Owensby et al., 1996 ;
Stocker et al., 1997) ; and (2) a reduction in leaf
diffusive conductance (Farquhar & Wong, 1984 ;
Morison & Gifford, 1984 ; Morison, 1985 ; Tyree
& Alexander, 1993 ; Knapp et al., 1996 ; Lauber &
Ko$ rner, 1997) and some reduction in plant canopy
evapotranspiration (Jackson et al., 1994 ; Field et al.,
1995 ; Samarakoon & Gifford, 1995 ; Owensby et al.,
1997 ; Stocker et al., 1997 ; Arnone & Bohlen, 1998).
CO -induced increases in net ecosystem CO ex-
# #
change have also been associated with increases in net
primary productivity (NPP, net biomass production
plus all biomass losses, e.g. litter, exudation, herbi-
vory). Numerous studies have reported that much
of the increase in NPP might occur below ground, as
evidenced by increases in allocation to root systems
(Rogers et al., 1994) and soils via increases in live-
root rhizodeposition (e.g. cell sloughing, exudation
(O’Neill, 1994 ; Cardon, 1996 ; Darrah, 1996) and
fine root mortality (Pregitzer et al., 1995 ; Canadell et
al., 1996 ; Fitter et al., 1997). The positive effect of
high CO on overall soil water availability (Knapp et
#
al., 1996 ; Owensby et al., 1997 ; Arnone & Bohlen,
1998 ; Niklaus et al., 1998b) might also stimulate
above-ground (ANPP) and below-ground (BNPP)
productivity by enhancing soil nutrient availability,
particularly in seasonally dry grassland systems
where high CO might extend water availability into
#
 RESEARCH CO and native grassland root systems
#
dry periods (Hungate et al., 1997a). In fact, increases
in soil moisture under elevated CO might contribute
#
most to the stimulation of biomass production under
elevated CO in natural ecosystems (Hungate et al.,
#
1997b ; Owensby et al., 1997 ; Leadley et al., 1999).
We began our root dynamics studies in 1994 with
hopes of explaining the fate of the ‘ extra ’ C
apparently fixed during the growing season by
ecosystems under elevated CO (Stocker et al., 1997)
#
by finding increased root mortality under elevated
CO . Since then only two other studies have assessed
#
this directly, using minirhizotrons. Pregitzer et al.
(1995) reported a 250% increase in fine root
mortality in young stands of Populus under elevated
CO in plots with no additional N, while Fitter et al.
#
(1996, 1997) found a 29% increase in root mortality
in intact monoliths of calcareous grassland and a
21% increase in native turf on peaty soil.
The objectives of the present study were to
determine : (1) whether root mortality increases in
native calcareous grassland communities (Leadley &
Korner, 1996 ; Huovinen-Hufschmid & Ko$ rner,
1998) exposed to elevated atmospheric CO in the
#
field, and whether this increase could help to explain
the apparent sizable increases in net ecosystem CO
#
exchange observed during the growing season at
elevated CO (Stocker et al., 1997 ; but also see
#
Niklaus et al., 2000) ; (2) whether elevated CO alters
#
the size and vertical distribution of plant-community
root-length density (RLD) and how this varies
across seasons ; and (3) whether root longevity is
affected by high CO and by depth. By addressing
#
these objectives we aimed to elucidate and quantify
root-system responses that might affect ecosystem
function (e.g. C and nutrient cycling).
  
Field site and experimental design
A full description of the lowland grassland site
(500–530 m above sea level) in northwestern
Switzerland and details of the experimental design,
are given by Leadley & Ko$ rner (1996) and Leadley
et al. (1999). We used 16 1.2-m# research plots
containing intact, native, species-rich grassland
communities. Each plot was equipped with low-
stature screen-aided CO control windscreens
#
(Leadley et al., 1997) for controlling atmospheric
concentrations. Beginning 19 March 1994, CO
#
concentrations in eight of these plots were main-
tained at 360 µl CO l−" and in the remaining eight at
#
600 µl CO l−". The plots were arranged in a
#
randomized complete block design over a 0.2-ha area
on a south-facing 20m slope. The aspect of the slope
combined with warm summer temperatures (Fig. 1)
resulted in high evapotranspiration and, combined
77
with the relatively shallow, calcareous soil (transition
Rendzina with a 15 cm topsoil underlain with an
extremely rocky subsoil : Ogermann et al., 1994),
contributed to strong topsoil drying in June–August
(Niklaus et al., 1998b). Plant communities in each
plot consisted of an average of 31 plant species
(graminoids, forbs and leguminous forbs). The grass
species Bromus erectus dominates the site (Houvinen-
Hufschmid & Ko$ rner, 1998), which for many
decades before 1993 was grazed by cattle (Schla$ pfer
et al., 1998). From early 1993, however, the parcel of
land used in our study was fenced off from grazing
and mown (clipped) twice a year, in June and
October. The switch to clipping had very little effect
on plant-species composition or abundance
(Schla$ pfer et al., 1998).
In late February 1994 we installed one transparent
butyrate minirhizotron tube 5 cm in diameteri100
cm long in each experimental plot at an angle of
c. 35m to the plane of the soil surface. The tubes
were inserted through the 15-cm deep A horizon and
into the upper 3 cm of the rocky subsoil. This allowed
us to observe roots in the top 18 cm of soil, the layer
in which 80–90% of all roots occur (J. Arnone,
unpublished). Before installing the tubes we etched
an observation track (18 mm widei54 cm long) on
the outside upper surface of each tube. We then
divided each track into 45 frames 12 mm high and 18
mm wide. The tube bottoms were capped before
insertion into pre-cored cylindrical holes, and the
top 10 cm of each tube wrapped in opaque tape and
stoppered to prevent light penetration and entry of
debris and insects.
We were unable to distinguish among roots of the
more than 30 species growing in each experimental
plot and thus only considered responses of the root
system of the entire plant community. On 28 April
1994, we recorded video images of roots in all 45
frames in each minirhizotron using a Bartz BCT-2
Minirhizotron Camera (Bartz Technology Co.,
Santa Barbara, CA, USA) attached to a Hi-8 Sony
Camcorder (all mounted on a backpack). We re-
peated this on 15 more dates up to 6 April 1996. All
45 frames were used to quantify RLD through the
soil profile. This was accomplished by viewing
undigitized video tapes and counting intersects with
gridlines drawn on an overhead transparency and
placed over the video monitor (Tennant, 1975). We
also digitized three of the 45 video images per tube
for each of the first 10 sampling dates (first year of
CO treatment) to quantify root production and
#
mortality (based on both number of roots and root
length). On each sampling date we recorded each
new root that appeared in (production) and each root
that disappeared from (mortality) the three frames,
which were located at depths of 3, 9 and 15 cm, and
were chosen to represent the 0–6, 6–12 and 12–18 cm
soil layers, respectively. We also measured the length
and diameter of new and disappearing roots using
78 RESEARCH J. A. Arnone III et al.

30
(a)
Calculations and statistical analyses

Mean daily air temperature

Total annual root length production was calculated
20 for each depth (frame) in each experimental plot by
summing length increments produced 19 March
at 1 m (°C)

1994 to 6 April 1995. Total annual root length mor-

10 tality was calculated in the same way over the same
period by summing root length that disappeared
from view. Both measures were expressed as cm root
0
length per cm# of minirhizotron observation area.
The RLD at installation of the minirhizotron tubes
–10
was considered to be zero. The roots that became
visible on the minirhizotron tubes from late
30
(b) February 1994 to late April 1994 (first sampling
Mean daily soil temperature

date) were considered root production. Statistics for

20 the 0–18 cm category were calculated as the mean of
the three depths. Cumulative root production and
at 5 cm (°C)

mortality during the first year (19 March 1994 to 6

10 April 1995) were calculated using numbers of roots
and expressed as number of roots per cm# of
minirhizotron observation area. For this period we
0 also calculated the percentage of whole-tube root
length that was present in each soil layer for each plot
and date as an expression of the vertical distribution
–10
of roots through the soil profile. We did this on each
250 date for each plot by calculating the difference
Total monthly precipitation (mm)

between the percentage of roots found in the 0–6 cm

200
150
100
50
0 in life span analyses.
1994
Month/year
Fig. 1. Mean daily (a) air temperature (1 m above ground
level) and (b) soil temperature (5 cm depth), and (c)
monthly total precipitation during the study period. No
temperature data are available for the first 6 months of
1994. Arrows in (c) indicate times of mowing with clippers
to 5 cm.
racker2 software (Duke University, Durham,
NC, USA). A root or root segment was considered
‘ dead ’ when it had disappeared from the frame. All
16 sampling dates and a total of 15 000 individual
root observations during the 2-yr study period were
used to analyse effects of CO on root system size
# hi(t) l h (t)exp(β xi j…jβkxik)
measured as RLD. We analysed root production and
mortality for only the first year of CO treatment,
#
using the three frames per tube (5000 individual root
observations).
(c)
layer and the percentage of roots found in the 12–18
cm layer.
The date of a root birth was defined as midway
between the date it was first observed and the date of
the preceding observation. The date of root death
was defined as midway between the date it was last
observed and the date of the following observation.
Roots that were still alive on the last minirhizotron
observation date were recorded as ‘ censored ’ for use
The effects of CO level and depth (0–6, 6–12 and
1995 1996 #
12–18 cm) on the risk of root mortality were
estimated using a stratified Cox proportional hazards
regression (Cox, 1972 ; Allison, 1995 ; Wells &
Eissenstat, 2000) performed with SAS PROC
PHREG (SAS Institute, Cary, NC, USA). All
cohorts of roots produced during the experiment
were combined for this analysis, and life span data
were stratified by block in order to control for the
potential effects of site location on root longevity.
The hazard (hi) is defined as the instantaneous risk
of mortality at time t, conditioned on survival to t
(Eqn 1). In a proportional hazards model, the hazard
of an individual at time t is written as the product of
two components, a baseline hazard function (h ) and
!
a linear function of k covariates which is exponen-
tiated (Allison, 1995) :
Eqn 1
! " "
PROC PHREG employs the method of partial
likelihood (Cox, 1972) to estimate the β coefficient
(parameter estimate) associated with each covariate
 RESEARCH CO and native grassland root systems
#
(xik) in the model and calculates a chi-square statistic
used to test the null hypothesis that each β is equal to
zero. A negative parameter estimate indicates that
increasing values of the covariate are associated with
a decreasing risk of mortality and longer life spans.
Likewise, a positive parameter estimate indicates
that increasing values of a covariate are associated
with an increasing risk of mortality and shorter life
spans.
An estimate of the survivor function for each
CO idepth combination was generated using the
# 0
BASELINE statement of PHREG. The survivor
function (S(t) ; henceforth referred to as root survival
probability ; Eqn 2) is defined as the probability (P)
that a root’s life span (T or failure time) is greater
than time t :
S(t) l PoT
tq Eqn 2
Median life spans for each treatment combination
were calculated as the number of days until the
survival probability 0.50. Carbon dioxide, depth
and block were included as variables when evaluating
their effects on median life span using ANOVA.
Effects of CO on total annual root length
# disappeared from the minirhizotron surface during that
production and root length mortality averaged across
all three depths were analysed using an unpaired
Student’s t-test (n l 8). To test effects of CO and
#
soil depth on total annual root length production,
root length mortality and root longevity, we used a
two-way ANOVA in which CO effects were tested
# #
against the plot-within-CO term and depth effects
#
against the overall model error term. Time-course
minirhizotron data (cumulative root production and
mortality across all depths and at each of the three
depths, and RLD over time across all depths and at
each of the three separate depths) were analysed by
repeated-measures ANOVA to assess potential CO
#
effects over time, again with the experimental plot as
the statistical unit (n l 8). We also used repeated
measures to evaluate effects of elevated CO on the
#
vertical distribution of RLD. For this analysis we
used the difference between percentage allocation to
roots in the 0–6-cm layer and percentage allocation
to roots in the 12–18-cm layer calculated for each
plot and date. All model terms were fitted using a
Type III ANOVA. Post hoc linear contrasts were
used to test for differences between treatment means
(Fisher’s LSD test). Data were transformed before
ANOVA as necessary to ensure homogeneity of
variance and normal distributions. Data shown in
the figures are means and standard errors of the
mean.

Root length production and mortality
The total amount of root length produced per unit
area of minirhizotron observation surface in the top
79
Mortality
(%)
(a) (b)
59 ±4
0–18 56 ±3
0 2 4 6 0 2 4 6
Depth (cm)
(c) (d)
73 ±4
0–6
73 ±3
6–12 50 ±9
45 ± 7
Amb. CO2 46 ±6
12–18
Elev. CO2 48 ±11
3 6 9 0 3 6 9
Root length Root length
production mortality
(cm cm– 2) (cm cm–2)
Fig. 2. (a, c) Mean total root length production and (b, d)
mortality measured early March 1994–April 1995, ex-
pressed as cm root length per cm# of minirhizotron
observation surface, broken out by soil layer in (c, d).
Hatched bars, ambient CO ; closed bars, elevated CO .
# #
Root length appearing on the minirhizotron tubes from
early March 1994 (time of tube installation) to 28 April
1994 (first video recording) was considered new root
production. No mortality could be observed during this
period. Percentage mortality is expressed as the percentage
of root length produced at any time up to 6 April 1995 that
period. MeanpSE, n l 8 plots.
18 cm of soil did not differ significantly between
plant communities maintained at ambient and el-
evated atmospheric CO (PCO l 0.5671 ; Fig. 2a) in
#
the first year of the study. The same held for the total
amount of root length mortality (i.e. disappeared,
Fig. 2b ; PCO l 0.6680). Although differences were
#
not statistically significant, mean values of root
length production and mortality were lower under
elevated CO than under ambient CO in all three
# #
soil layers (Fig. 2c,d). Root length production and
mortality decreased with increasing soil depth
(Pdepth l 0.0074), with 40–50% of the production
and mortality occurring in the topmost layer (0–6 cm)
under both ambient and elevated CO .
#
At all soil depths and both CO levels, root length
#
mortality was proportional to root length production
in the first year (Fig. 2). The percentage of root
length produced that died in the 1 yr observation
period also decreased with depth (Pdepth l 0.0213),
with the highest percentage mortality occurring in
the topmost soil layer (73%). Averaged across all
three soil layers, approx. 57% of the root length
produced in the first year died in that same year.
Root diameter averaged over the year and all soil
layers was remarkably similar under ambient CO
#
(0.17 l 0.01 mm ; median diameter l 0.15 mm ;
PCO l 0.8245) and elevated CO (0.16 l 0.01 mm ;
# #
median diameter l 0.15 mm). Thus changes in root
length appear to correspond well to changes in root
biomass (‘ true ’ production and mortality, from a C
mass balance perspective, not testable experimen-
tally). Tissue density of roots sampled from cores
80 RESEARCH J. A. Arnone III et al.

All depths: 0–18 cm 0–6 cm 6–12 cm 12–18 cm

Cum. fine root mortality Cum. fine root production 20
(number cm–2) (a) (c) (e) (g)

15
Amb. CO2

10

5 Elev. CO2

0
(b) (d) (f) (h)
(number cm–2)

10

J F M AMJ J A S ON D J F M AM J J J F M AMJ J A S ON D J F M AM J J J F M AMJ J A S O N D J F M AM J J J F MAMJ J A SON D J F M AM J J

1994 1995 1994 1995 1994 1995 1994 1995

Month and year
Fig. 3. Mean cumulative root production (a, c, e, g) and mortality (b, d, f, h) for all depths (a, b) and for
individual depths (c, d ; e, f ; g, h) expressed as number of roots per cm# of minirhizotron observation surface
(meanpSE, n l 8 plots), mid-March 1994 to 6 April 1995. Down arrows in each of the upper panels indicate
mowing dates. Open symbols, ambient CO ; closed symbols, elevated CO .
# #

All depths: 0–18 cm 0–6 cm 6–12 cm 12–18 cm

Root length density (cm cm–2)

8 (a) (b) (c) (d)

0
1994 1995 1996 1994 1995 1996 1994 1995 1996 1994 1995 1996
Year
Fig. 4. Seasonal fluctuations in mean root-length densities, in cm root length per cm# of minirhizotron
observation surface, for (a) all depths, (b) 0–6 cm, (c) 6–12 cm and (d) 12–18 cm (meanpSE, n l 8 plots)
recorded early April 1994–April 1996. Arrows, mowing dates. Open symbols, ambient CO ; closed symbols,
#
elevated CO .
#

(Leadley et al., 1999) remained unaffected by corresponded to summer soil drying (Niklaus et al.,
elevated CO (data not shown). 1998b) and heating, and to cold winter temperatures,
#
Rates of root production and mortality varied respectively (Fig. 1).
throughout the year (Fig. 3 ; Ptime 0.05 for data in
all panels), with generally lower mean rates observed
Root length density
under ambient CO . However, no statistically signifi-
#
cant differences between CO treatments were During the first year of CO treatment, RLD
# #
detected. Here again, mortality was proportional to increased dramatically in all soil layers from the start
production, with periods of rapid production ac- of CO treatment (19 Mar 1994), through the first
#
companied by high mortality at all depths (e.g. 28 ‘ growing season ’ (9 Nov 1994) and winter ‘ dormant
Apr–5 July 1994 ; 6 Sept–6 Nov 1994 ; 28 Feb–6 Apr period ’ (9 Nov 1994 to 28 Feb 1995), and on
1995), and periods of slow production accompanied into the first part of the second growing season
by low mortality. The two slow periods of root (28 Feb–6 Apr 1995 ; Fig. 4). During this period,
production and mortality (5 July–6 Sept 1994, and however, RLDs in plant communities exposed to
6 Nov 1994 to 28 Feb 1995), compared to all other elevated CO did not differ from those in plant
#
periods during the year, occurred immediately communities exposed to ambient CO (PCO range :
# #
following summer and fall mowing (slow period vs 0.7866–0.9001 ; Fig. 4). As with root production and
remaining periods, P l 0.0397). These two periods mortality (Fig. 3), the rate of increase in RLD also
 RESEARCH CO and native grassland root systems 81
#

Percentage of whole-tube root length

50 (a) (b)

40

Line of equal
30 distribution of roots
in all three layers

20

10 Ambient CO2 0– 6 cm Elevated CO2 0 – 6 cm

6 –12 cm 6 –12 cm
12–18 cm 12–18 cm
0
1994 1995 1996 1994 1995 1996
Year
Fig. 5. Vertical distribution of root-length densities, in cm root length per cm# of minirhizotron observation
surface, in each of the three soil layers in grassland plots maintained under (a) ambient (open symbols) or (b)
elevated (closed symbols) atmospheric CO quantified between 28 April 1994 and 12 April 1996 (meanpSE,
#
n l 8 plots). Squares, 0–6 cm soil layer ; triangles, 6–12 cm layer ; diamonds, 12–18 cm layer. Arrows, mowing
dates. Shaded area, difference between the mean percentage of root length observed in the topmost and bottom-
most soil layers.

slowed following mowing and increased with canopy

regrowth (P 0.05 for each depth). However, Ambient CO2
0–6
Depth of soil layer (cm)

changes in soil water availability (Zaller & Arnone, Elevated CO2

1997 ; Niklaus et al., 1998b) and temperature (Fig. 1)
during the growing season also might have played a
role in controlling the rate of change in RLD. 6–12
During the second year of CO treatment, we did
#
not observe any effects of CO on RLD (PCO all
# #

0.50). Fluctuations abounded, however. Between 12–18 +48 % (P = 0.068)
the start of the second year of CO treatment (6 Apr
#
1995) and 2 June 1995, RLD in all soil layers and
both CO treatments plummeted from 9 to 3 cm
# 0 100 200 300
cm−# (Fig. 4a). This drop returned RLDs to levels Median root life span (d)
equal to those observed during the 1994 summer
dry-soil period ; and they remained low for another 3 Fig. 6. Median root life span (meanpSE, n l 8 plots) for
the first year (19 March 1994 to 6 April 1995 ; including
months (until 3 Aug 1995), spanning the 1995 censored roots), calculated as the number of days until the
summer dry-soil period, until they rose again to survivor function dropped below 0.5 (see the Materials
reach a second-year maximum of 5 cm cm−# in the and Methods section ; PCO l 0.451 ; ANOVA : Pdepth l
fall (24 Oct 1995). The start of another decline 0.008 ; PCO ×depth l 0.145).# Hatched bars, ambient CO ;
# #
in RLD (to 4 cm cm−# by 12 Apr 1996) coincided closed bars, elevated CO .
#
with the 1995 October mowing. We observed the
same patterns in all three soil layers. CO (16.8p1.8%) than that measured in plots
#
Elevated CO affected the vertical distribution maintained at ambient CO (10.2p1.2%) assessed
# #
(deployment) of RLD during several extended over these periods. Under elevated CO , a mean of
#
periods in the 2-yr study (Fig. 5). Throughout these 41p1% of all root length during these periods was
periods (28 Apr–6 Nov 1994, first seven sampling present in the 0–6 cm soil layer and 24p1% in the
dates ; 3 Aug 1995 to 12 Apr 1996, last three 12–18 cm layer. Under ambient CO , a mean of
#
sampling dates), RLDs under both CO treatments 38p1% of all root length during these periods was
#
were highest in the 0–6-cm layer and lowest in the present in the 0–6 cm soil layer and 28p1% in the
12–18-cm layer (e.g. PCO l 0.0471 for topmost vs 12–18 cm layer. Between these periods of greater
#
bottom-most layer). In communities maintained vertical root length stratification was a period of
under elevated CO , however, the vertical strati- lower stratification (winter and spring 1995) in which
#
fication of root length was more pronounced than it the distribution of root length among the soil layers
was in those maintained at ambient CO . This was was more equal under both ambient and elevated
#
indicated by a significantly greater difference (PCO l CO (Fig. 5). This interim period appeared to be
# #
0.0512) between the percentage of root length longer (not tested statistically) in plots under
present in the topmost soil layer (0–6 cm) and the ambient CO (7.5 months) than in plots under ele-
#
bottom-most layer (12–18 cm) in plots kept at high vated CO (5 months).
#
82 RESEARCH J. A. Arnone III et al.

Table 2. Results of proportional hazards regression performed on root life span data collected 28 April
1994 to 6 April 1996 pooled across all cohorts (dates of root production)

Parameter Wald
Variable df estimate SE chi-square P
chi-square

Cohort 1 k0n000461 0n0005354 0n74032 0n3896

CO 1 k0n339474 0n16819 4n07374 0n0436
#
Depth 1 k0n020022 0n03131 0n40894 0n5225
DepthiCO 1 0n057413 0n02041 7n91077 0n0049
#
Data were stratified by block. Cohort was included as a covariate to determine whether the risk of mortality differed
across production dates.

1.0
(a) (b) (c)
Root survival probability

0 – 6 cm 6 –12 cm 12 –18 cm
0.8
Elevated CO2
0.6

0.4

0.2
Ambient CO2
0.0
0 100 200 300 400 0 100 200 300 400 0 100 200 300 400
Time after root produced (d)
Fig. 7. Root survival probability curves for roots produced under ambient (open symbols) or elevated (closed
symbols) CO within each of three soil depths (a, b, c) in the first year of the experiment in a native calcareous
#
grassland in Switzerland. Arrows, mowing dates. Higher probabilities are associated with longer root life spans.
See Table 2 for associated PHREG P values.

seasonally high temperatures (25mC) occurring in

Root longevity
July and lowest temperatures in late December–
In the first year of CO treatment, elevated CO had February. Soil temperature at a depth of 5 cm
# #
no effect on median root life spans when viewed never dropped below 0mC, although air temperature
across all depths (Fig. 6). However, the longevity of measured 1 m above ground did drop as low as
roots in both CO treatments increased with depth k10mC on a number of occasions.
#
(Pdepth l 0.0081). In the 12–18-cm soil layer, Despite the continuation of rainfall throughout
longevity of roots in communities maintained at the summer months (Fig. 1c), the surface soil in all
elevated CO (median life span 274 d) was almost plots dried substantially (Niklaus et al., 1998b) by
#
50% greater (P l 0.0680) than that observed under the end of June in both 1994 and 1995. This
ambient CO (174 d). condition persisted to the end of August in both
#
Also during the first year, elevated CO signifi- years. Soil water content in the top 0–10 cm in all
#
cantly decreased the risk of root mortality (PCO l plots during these months dropped from field
#
0.0046), and this effect became more pronounced capacity ($35 % vol) to below 20%, and even as low
with increasing depth (PCO ×depth l 0.0049 ; Table 2). as 10% (Niklaus et al., 1998b). Under elevated CO ,
# #
Consequently, root survival probability was en- however, the water content in the A horizon was
hanced under elevated CO in the two deeper soil consistently greater than that measured under ambi-
#
layers (Fig. 7). However root survival probability ent CO . For example, mean soil water content for
#
did not differ between ambient and elevated CO in the period extending from April 1995–April 1996
#
the deeper soil layers until at least 150 d after root was 10% greater under elevated CO (33 vs 30%
#
production, suggesting that the effect of elevated CO vol). During a 6-wk dry period in summer 1995 soils
#
was to prolong the life of older roots, rather than to were 15% moister under high CO (26.8 vs 23.3%
#
prevent the mortality of younger roots. vol ; Zaller & Arnone, 1997).


Environmental and soil conditions
The data on root production, mortality and longevity
Air and soil temperatures during the 2-yr study from the first year of the study (Figs 2, 3, 6, 7) and
period were typical for this site (Fig. 1a, b) with the RLD data from the first 2 yr (Figs 4, 5) indicate
 RESEARCH CO and native grassland root systems
#
that elevated CO had relatively few of the hypo-
# Root length density and vertical shifts in RLD under
thesized effects on root systems in these grasslands
but did cause a number of unexpected changes in
root systems that might affect ecosystem function.
Root production, mortality and missing below-ground
C
The absence of a detectable change in root mortality,
production (Figs 1, 3) or RLD (Fig. 4), even after an
entire year of exposure to elevated CO , leads us to
#
reject our original hypothesis that a CO -induced
#
stimulation in root mortality would help to account
for any of the extra CO taken up during the growing
#
season in grasslands under elevated CO . As root
#
diameter was not affected by high CO and no
# %
changes in root tissue density were observed, we can
infer that CO had no detectable effect on the
# $
production and mortality of root biomass in the top
18 cm of soil, where most roots occur. Further, the
absence of a CO effect in the first 2 yr of the study,
#
the period when the most pronounced CO effects
#
are often seen (Norby et al., 1992 ; Oechel et al.,
1994), suggests that the patterns observed in the first
2 yr might have persisted into the following 3 yr of
the larger study. In fact, Leadley et al. (1999) found
no CO effect on root biomass at our site during the
#
first 4 yr, suggesting that RLD probably also
remained unaffected by elevated CO in years 3 and
#
4. In the only other study to examine the effects of
high CO on root dynamics in intact calcareous
# #
grassland (monoliths), both root mortality and
production were stimulated by elevated CO (Fitter
#
et al., 1996, 1997 ; Table 1). Fitter et al. (1996, 1997)
also reported the same for a Nardus\Juncus grassland
monolith on more acidic soil. In a 4-yr study of
alpine grassland, root production in ingrowth cores
was slightly stimulated by high CO (Scha$ ppi &
#
Ko$ rner, 1996 ; Ko$ rner et al., 1997 ; Table 1). Thus it
appears from the very limited data on intact systems
that the effects of elevated CO in grasslands might
#
vary from zero to a stimulation of as much as 30%,
depending on grassland type and experimental
conditions.
The absence of change in annual root mortality in
our study indicates that, in order to explain the
increase in net ecosystem CO exchange observed
#
under elevated CO during the growing season (270
# #
d ; Stocker et al., 1997), C must be getting into the
soil by other means. One possibility might be via
increased production and mortality of mycorrhizal
hyphae (analogous to that reported by Rillig et al.,
1998). Another might be via a large stimulation of
root exudation or other live-root rhizodeposition
(suggested by data from short-term studies in
artificial grass systems (Cardon, 1996 ; Gorrison,
1996 ; Ineson et al., 1996 ; Hungate et al., 1997b ;
Cheng & Johnson, 1998).
83
high CO
#
The consistent absence of statistically detectable
CO effects on RLD at any depth and at any time in
#
the 2-yr observation period (Fig. 4) suggests that the
same size of root system can sustain the larger
(j20%, Leadley et al., 1999) and physiologically
more active (Stocker et al., 1997) plant canopies
present under elevated CO . A variety of possible
#
mechanisms might underlie this observation. Nu-
trient uptake efficiency of root systems, expressed in
units of nutrients taken up per unit root length,
might increase under elevated CO (BassiriRad et
#
al., 1996). Another possible explanation is that a
shift takes place in use of nutrient species from those
for which root foraging is more advantageous (NH -
N) to those in which root foraging is less ad-
vantageous (e.g. NO -N ; G. Berntson, pers. comm.).
The marked decrease in soil NO -N under elevated
$
CO in the calcareous grassland examined in this
#
study (Niklaus et al., 1998a) supports this hypo-
thesis. It also supports the notion that increased
colonization of roots by mycorrhizal fungi (not
measured in our study but reported elsewhere)
(Godbold et al., 1997 ; Rygiewicz et al., 1997 ; Rillig
et al., 1998), as well as the maintenance of a larger
network of extramatrical mycorrhizal hyphae, which
the data of Rillig et al. (1999) suggest, could
compensate for the lack of an increase in root length
at high CO . However other studies indicate that this
#
response to elevated CO may be the result of not
accounting for the size or stage of development of
root systems (Staddon, 1998 ; Staddon et al., 1999 ;
Fitter et al., 2000), and that morphological and
functional responses of mycorrhizas to elevated CO
#
may be most closely linked to the responses of root
systems themselves. Yet another possibility is that
plant demand for nutrients might not be altered
significantly by elevated CO (Niklaus et al., 1998a),
#
perhaps because of higher ‘ nutrient use efficiency ’ of
biomass production. In this case, investing more C
in root systems would be energetically disadvant-
ageous for plants. Evidence supporting the possi-
bility of higher nutrient use efficiency under ele-
vated CO in the grassland we studied is the con-
#
sistently greater (j20%) amounts of above-ground
biomass and lower C : N ratios observed under
elevated CO relative to the lack of change in root
system size (Fig. 5 ; Niklaus et al., 1998a ; Leadley et
al., 1999). As with most in situ studies or in relatively
undisturbed systems (Table 1), the actual causes of
the patterns observed in our study are not clear.
The tendency for greater root growth at shallower
depths under elevated CO (Fig. 5) might have been
#
due to higher soil moisture levels in the 0–6 cm layer.
Higher moisture availability (from mid-1995 ;
Niklaus et al., 1998b) might also have increased
nutrient availability simply by enabling microbial
84 RESEARCH J. A. Arnone III et al.

mineralization to continue longer into dry periods community productivity). Thus it is impossible to
in high-CO soils. Although routine assays con- generalize about the effects of elevated CO on root
# #
ducted at our site suggest that nutrient availability in longevity, or about the causes that might underlie
these grasslands did not increase under elevated CO these effects, in native grasslands.
#
(Niklaus et al., 1998a), the existence of such
favourable soil zones could have stimulated root 
proliferation into this surface zone (Eissenstat &
Caldwell, 1988 ; Jackson & Caldwell, 1989). Greater The results of our field study indicate that continuing
increases in atmospheric CO might have little
surface rooting under elevated CO has also been
# #
observed in cotton (Prior et al., 1994), oak scrubland impact on root mortality and production in some
(Day et al., 1996), wheat (van Vuuren et al., 1996), native grassland systems, rather than increasing
and in intact calcareous grassland monoliths (Fitter them as originally hypothesized. Changes in root
mortality could not account for any extra CO taken
et al., 1997). The end result of these shifts might be #
up under elevated CO during the growing season by
a movement of C and nutrient cycling, and possibly #
C sequestration, to shallower soil layers. Effects of the calcareous grassland ecosystem studied. Our
disturbance resulting from installation of mini- findings suggest that overall ecosystem C and
rhizotron tubes (Hendrick & Pregitzer, 1996 ; Majdi, nutrient cycling via root production and mortality in
1996 ; Joslin & Wolfe, 1999) might have contributed the A horizon are likely to remain unchanged as
atmospheric CO levels rise. Within this soil layer,
to the continual increase in RLD in both ambient #
and elevated CO treatments into the winter of the however, a shift in RLD away from the bottom of
# the A horizon to the top suggests that C cycling, C
first year (Fig. 4) followed by a drop in RLDs in the
second year, but these potential effects do not deposition and nutrient cycling might shift upward
to shallower soil layers in the future under high CO .
preclude reliable and robust treatment comparisons. #
The size of root systems in these grassland com-
munities cannot be expected to change under future
Root longevity and demography under high CO CO levels, but roots in deeper soil layers might live
# #
for longer than under current CO levels. These
The causes of the increase in root longevity observed #
seemingly subtle effects of CO on root-system
at depth in both CO treatments (Figs 6, 7), and the
# #
further enhancement under elevated CO , are behaviour might still profoundly affect the activity of
# heterotrophic organisms living in the soil (possibly
unclear. Lower soil temperatures at depth, and lower
root tissue C : N ratios under elevated CO (C. earthworm activity ; Zaller & Arnone, 1997). It
# might be these effects that ultimately have the
Ko$ rner, pers. obs.), could have contributed to lower
root respiration rates, and root C economies that greatest impact on ecosystem C cycling and plant
favour longer life spans (Marshall & Waring, 1985 ; community structure (Zaller & Arnone, 1999) as
CO levels rise.
Eissenstat & Yanai, 1997 ; Eissenstat et al., 2000). #
Greater root life span with depth has also been
reported in alfalfa (Medicago sativa ; Goins &               
Russelle, 1996) and lemon (Citrus volkamericana ; We are grateful to Gunnar Hirschel, Edith Totschnig,
Kosola et al., 1995), but Hendrick & Pregitzer (1992) Christine Cardella and Petra Bockmu$ hl for their great
noted greater survivorship among shallower roots of assistance with minirhizotron installation, videotaping in
sugar maple (Acer saccharum). Although the relative the field, digitizing and cataloguing root images, and image
stimulatory effect of elevated CO on soil water analysis. Paul Leadley and Reto Stocker helped ensure
#
availability in our study was more pronounced in the that the field facility functioned properly and without
uppermost soil layer (Niklaus et al., 1998b), moister interruption. Valuable discussions of the results with
soils at depth under elevated CO might also have Daniel Obrist enhanced the quality of this manuscript, as
# did three anonymous reviews, and the editorial contri-
contributed to greater root longevity. Pregitzer et al.
butions from Roger Kreidberg. The Swiss National
(1993) reported the occurrence of longer-lived roots
Science Foundation provided financial support for this
in water patches. The effects of CO on root longevity
# project with a grant from the Swiss Priority Program on
that we observed contrast with those of Pregitzer et the Environment to C. Ko$ rner, J. Arnone and B. Schmid
al. (1995), who found decreased root longevity in (NF-SPPU 5001–035214). Completion of data analysis
Populus trees growing at low soil N under elevated and this manuscript was made possible through a generous
CO , as well as with those of Fitter et al. (1997), who grant from the Treubel-Fonds of Basel, Switzerland to
#
also showed a tendency toward decreased root J. A. This research was conducted as part of the Global
longevity in monoliths of two native grassland Change and Terrestrial Ecosystems (GCTE) network of
communities under elevated CO . However, the core research projects.
#
effects of elevated CO on root physiology and life
#
history are likely to be complex and modulated 
directly and indirectly by multiple factors (e.g. CO
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