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Soil Biology & Biochemistry 32 (2000) 1043±1052
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Symbiotic N2 ®xation of various legume species along an
altitudinal gradient in the Swiss Alps
Katja A. Jacot, Andreas LuÈscher, Josef NoÈsberger, Ueli A. Hartwig*
Institute of Plant Sciences, ETH-Zurich, 8092 Zurich, Switzerland
Accepted 15 December 1999

Abstract
Symbiotic N2 ®xation may be an important source of N for legumes in alpine ecosystems, though, this has hardly been
investigated. Symbiotic N2 ®xation in nine legume species in permanent grassland over an altitudinal gradient (from 900 up to
2600 m a.s.l.) was investigated in the Swiss Alps on strictly siliceous soils. To assess symbiotic N2 ®xation, an enriched 15N
isotope dilution method was established for low N input, permanent grasslands and was evaluated with the 15N natural
abundance method. The non-N2-®xing reference species used in both methods di€ered signi®cantly in their 15N atom%-excess.
However, when several reference species were combined, the enriched 15N isotope dilution method was reliable and led to the
conclusion that up to their altitudinal limit, legumes may acquire from 59% to more than 90% of their N through symbiotic N2
®xation depending on the species. These ®ndings were con®rmed by the 15N natural abundance method. Even at the legumes'
altitudinal limit all plants investigated showed apparently active nodules. Moreover, a clear host-microsymbiont speci®city
between plant and rhizobia was evident at high altitudes. This suggests that symbiotic N2 ®xation is well adapted to the climatic
and acidic soil conditions in the Alps and contributes, up to the altitudinal limit, a signi®cant amount of N to the N nutrition of

legumes. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Alpine ecosystem;

15

N isotope dilution;

15

N natural abundance; Trifolium; Lotus

1. Introduction
Symbiotic N2 ®xation of leguminous plants is important for the worldwide nitrogen budget (Evans and
Barber, 1977). Plants that symbiotically ®x N2 occur in
several nutritionally and climatically stressed arctic,
subarctic and alpine environments, though, the degree
to which they use symbiotically-®xed N as opposed to
inorganic soil N pools for N nutrition in such environments is unclear (Sprent, 1985).
Alpine areas are characterized by a relatively stressful climate with low temperatures, short growing seasons, and often also by low soil pH. These conditions
restrict organic decomposition and microbial trans-

* Corresponding author. Tel.:+41-1-632-49-30; fax:+41-1-632-11-53.
E-mail address: ueli.hartwig@ipw.agrl.ethz.ch (U.A. Hartwig).

formation of N and, thus, N availability in the soil
(Jacot et al., 1999). Under these conditions, symbiotic
N2 ®xation may represent an important source of N
for plants. On the other hand, non-symbiotic N2 ®xation (Holzmann and Haselwandter, 1988), snow melt
water, and precipitation (Haselwandter et al., 1983)
also contribute to nitrogen nutrition. Even where rates
of mineralization are low, N input through rain and
run-o€ water could still enable a small increase in
plant biomass at high altitudes if it were not for other
limiting factors.
Symbiotic N2 ®xation is likely to be a€ected by climatic conditions at high altitudes (Cralle and Heichel,
1982). Kessler et al. (1990) reported that low temperature has a more negative e€ect on symbiotic N2 ®xation than on plant growth. However, Svenning et al.
(1991) showed that in a cold climate in Norway, adaptation of both legumes and rhizobia occurs. Therefore,

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 1 2 - 2

1044

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

it is still not clear whether symbiotic N2 ®xation at
high altitudes in the Alps is e€ective or not.
Very few investigators have studied symbiotic N2 ®xation under alpine conditions (Wojciechowski and
Heimbrook, 1984; Johnson and Rumbaugh, 1986;
Holzmann and Haselwandter, 1988; Bowman et al.,
1996). Only one study quanti®ed N2 ®xation at high
altitudes under extreme conditions; Bowman et al.
(1996) found substantial N2 ®xation in Trifolium
species at Niwot Ridge in Colorado.
The aim of our study was to examine the contribution of symbiotic N2 ®xation to the legume's N budget in natural species-rich permanent grassland along
an altitudinal gradient. An enriched 15N isotope dilution method using various reference species was
established towards a complex permanent grassland
and evaluated with the 15N natural abundance method.
Another aim was to determine the adaptability of rhizobia to high altitude conditions. To investigate this,
the speci®city of rhizobia towards the host plants at

their upper altitudinal limit was studied in an inoculation experiment.

2. Material and methods
2.1. Experimental area and sites description
The study was conducted on the south slope of the
upper Rhine valley between Sumvitg and Trun
(approx. 45 km southwest of Chur in the eastern Alps
in Switzerland; 46845'N, 8857 'E). The geology of this
area is uniform and dominated by gneiss of granitic
composition (siliceous soil substrate). All studies were
made on species-rich permanent grassland (historically
used as pasture), on areas of 100 m2 on each of the
four sites from 900 up to 2100 m a.s.l. Four additional
subsites of 2.25 m2 each, were used at 2100 m a.s.l. An
experimental area at 2300 m a.s.l. was split into 16

subsites of 2.25 m2 each due to topographical heterogeneity. At 2600 m a.s.l., one site of 2.25 m2 was used
as an experimental area. The 100 m2 (2.25 m2) experimental areas of each major site were separated into 20
(4) plots, 12 of which were chosen randomly and used
as replicates. All four plots at the smaller sites were

used as replicates. The sites are described in Tables 1
and 2. Microclimatic data were collected during the
growing season (Table 3). Air temperature at 2 m
above ground and soil temperature 2.5 cm below, were
measured every 30 min, and total radiation (Sternpyranometer, Phillipp Schenk, A) every 10 min. Data
were collected with a data logger (Sky DataHog 2,
Wales, UK). Precipitation was collected 50 cm above
the ground with a funnel (f18 cm) attached to 5 litre
PE-bottles. The bottles were in PVC tubes (f18 cm)
covered with aluminium foil. Precipitation measurements were summed every 2 weeks.
2.2. Symbiotic N2 ®xation as determined by the enriched
15
N isotope dilution method
N2 ®xation was assessed for all legume species at
each site using the enriched 15N isotope dilution
method with various reference species (Tables 4 and
5). Individual plants were marked and harvested each
year. Symbiotic N2 ®xation was assessed for three
regrowth periods at 900 m a.s.l., for two regrowth
periods at 1380 m a.s.l., and for one regrowth period

at higher altitudes. Symbiotic N2 ®xation was assessed
using the enriched 15N isotope dilution method (Danso
et al., 1993). The enriched 15N isotope dilution method
depends upon di€erences in isotopic composition of
the various sources of N available for plant growth,
i.e. soil N, fertilizer N and atmospheric N2. The proportion of N derived from symbiosis (%Nsym) was
calculated for individual legume species at each altitude for each regrowth period as (McAuli€e et al.,
1958):

Table 1
Characterisation of vegetation (dominant vegetation, Ellenberg, 1982, and legume species) in the experimental area in the Vorderrhein valley (for
more information about plant species see also Table 5)
Altitude (m a.s.l.)

Dominant vegetation

Legume species

900
1380

1900
2100
2300
2600
2770

Arrhenatheretum
Trisetetum
Nardetum
Nardetum
Nardetum/Curvuletum
Curvuletum
Curvuletum

Trifolium pratense L., T. repens L., Lotus corniculatus L., Vicia sativa L.
T. pratense L., T. repens L., L. corniculatus L., V. sativa L.
T. pratense L., T. repens L., L. corniculatus L., T. thalii Vill.
T. nivale Sieber, T. alpinum L., L. alpinus (DC.) Schleicher, T. badium Schreber
T. alpinum L., L. alpinus (DC.) Schleicher
T. alpinum L., L. alpinus (DC.) Schleicher

None

1045

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

Table 2
Characterisation of soil (soil type, FAO-UNESCO, 1974), content of organic matter, clay, silt, and sand, and pH of the experimental sites in the
Vorderrhein valley
Altitude (m a.s.l.)

Soil typea

Organic matter

Clay (%)

Silt (%)

Sand (%)

pH(CaCl2)

900
1380
1900
2100
2770

Dystric cambisol
Dystric cambisol
Spodi-dystric cambisol
Humic podsol
Spodi-dystric cambisol

4.9
8.5
15.2
19.7
29.3

9.1
12.3
13.6
14.8
20.8

20.7
27.9
23.4
18.4
18.5

65.3
52.3
50.8
47.1
31.4

5.6

4.6
4.1
4.1
3.1

a

Source: H. Conradin (Swiss Federal Research Station for Agroecology and Agriculture).

%Nsym ˆ
15

1ÿ

15

N atom%-excess in the legume
N atom%-excess in the reference plant

also varied according to elevation from 1.6 kg
haÿ1yearÿ1 to 2.4 kg haÿ1yearÿ1. These amounts of N
are low (3±15%) compared to the total N yield of the
plant community (Jacot et al., 1999). N was supplied
as a solution of 15N-enriched (NH4)2SO4(1 l mÿ2). All
sites were watered with 1 l mÿ2 of water following the
15
N application. The 15N atom%-excess of the
enriched (NH4)2SO4 solution was 10% in 1996. Due to
low amounts of 15N atom%-excess in the plants, the
excess was increased to 15% in 1997 and 1998. A
homogeneously-labeled soil pro®le is a prerequisite for
reliable results using the 15N-isotope dilution method.
Therefore, the site was labeled two or three times
(intervals of 2 to 3 weeks) during each regrowth
period. The ®rst labeling was done 7 weeks before the

!

 100:
Two to three plants that do not ®x N2 but which grew
close (max. 5 cm) to each legume, served as reference
plants (Table 4). An appropriate reference species must
absorb N from the same nutrient pool as the legume
does, and use similar proportions of tracer N and soil
N during each phase of growth. Depending on elevation, symbiotic N2 ®xation was calculated using an
average of three±nine di€erent reference species
(Table 4). The N applied for 15N labeling at each site

Table 3
Microclimatic conditions during the growing season at the experimental sites (precipitation P, means, maximum, minimum of air Ta and soil
temperature Ts and daily radiation Rd snow not measured)
Altitude (m a.s.l. )
Growing season
1996

1997

1998

P (mm)
Ta(mean)(8C)
Ta(max)(8C)
Ta(min)(8C)
Ts(mean)(8C)
Ts(max)(8C)
Ts(min)(8C)
Rd (MJ mÿ2dÿ2)
P (mm)
Ta(mean)(8C)
Ta(max)(8C)
Ta(min)(8C)
Ts(mean)(8C)
Ts(max)(8C)
Ts(min)(8C)
Rd (MJ mÿ2dÿ2)
P (mm)
Ta(mean)(8C)
Ta(max)(8C)
Ta(min)(8C)
Ts(mean)(8C)
Ts(max)(8C)
Ts(min)(8C)
Rd (MJ mÿ2dÿ2)

900

1380

1900

2100

2770

May±October
677
12.8
31.7
ÿ2.3
16.7
35.2
5.3
14.2
455
13.4
31.7
ÿ5.7
17.0
35.7
1.6
14.7
525
13.6
35.2
ÿ1.0
17.8
39.4
5.6
15.1

May±October
800
10.9
30.2
ÿ2.0
13.9
26.7
4.9
14.2
540
11.8
30.5
ÿ5.2
14.2
25.2
2.2
14.9
555
10.2
30.6
ÿ1.6
13.0
27.6
4.3
15.0

June±October
573
6.9
20.8
ÿ5.0
11.4
30.7
1.8
14.3
585
9.1
22.0
ÿ8.0
12.9
27.9
ÿ0.2
15.7
619
8.6
25.0
ÿ4.5
12.9
30.7
1.5
16.2

June±October
508
5.3
18.9
ÿ6.0
8.3
18.0
0.4
13.6
567
7.5
20.2
ÿ9.4
10.7
23.0
0.3
17.1
580
7.0
23.1
ÿ6.3
9.0
24.1
0.2
15.4

July±October
315
1.3
13.1
ÿ8.7
5.4
19.3
0.12
13.0
215
3.9
15.2
ÿ13.0
7.6
20.8
ÿ0.6
16.6
357
5.0
16.7
ÿ7.5
9.0
22.8
0.4
16.5

1046

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

®rst harvest each year. The assessment of symbiotically-®xed N commenced in 1997.
In an area about 1 km2 from 2000 to 2700 m a.s.l.,
representing the upper limit of legumes in this region,
about 100 plants of Trifolium alpinum and Lotus alpinus each, were examined for a red pigment inside the
nodules (which is indicative of e€ective N2 ®xation).
2.3.

15

The following formula is used to calculate the percentage of symbiotically ®xed N (Ledgard and Peoples,
1988):
%Nsym ˆ
d15 N in the reference plant ÿ d15 N in the legume
d15 N in the reference plant ÿ B

!

 100,

N natural abundance

Legume species (Trifolium pratense, T. alpinum,
Lotus corniculatus, and L. alpinus ) and reference
species in a legume free area (0.25 m2) (Table 6) were
collected at least 100 m from the enriched sites. When
determining small di€erences in 15N concentration, the
term d15 N (-) is commonly used (Shearer and Kohl,
1986):
d15 N…- † ˆ
atom%15 N…sample† ÿ atom% 15 N…standard †
atom% 15 N…standard †

!

 1000,

where atmospheric N2 is the standard.

where B is 15N enrichment, relative to atmospheric N2,
of the legume grown solely with atmospheric N2.
2.4. Sample preparation and

15

N analyses

All dried (658C for 48 h) plant material was ground
in sequence using a Cyclotec 1093 sample mill (Tecator, HoÈganaÈs, Sweden) and a ball mill of type MM2
(Retsch, Arlesheim, Switzerland) to a very ®ne powder.
After redrying (358C for 24 h), the samples (1 mg of
leguminous plants and 2 mg of reference plants) were
weighed in tin caps (40 ml, LuÈdi, Flawil, Switzerland).
The samples were analyzed for 15N concentration by a
continuous-¯ow mass spectrometer (Europa Scienti®c,

Table 4
15
N atom%-excess of individual reference species and mean of all legume species after application of 15N at ®ve altitudes. Values are means 2
SEM (n = 2±109, averaged over 1997 and 1998; at 2600 m a.s.l., data are for 1997, cv: coecient of variation; n.p.: not present or not measured;
n.d.: no determination)
15

Altitude (m a.s.l.)
Reference species
Arrhenaterum elatius
Anthoxantum odoratum/alpinum
Agrostis tenuis/rupestris
Cynosurus christatus
Dactylis glomerata
Leontodon hispidus/helveticus
Lolium perenne
Salvia pratensis
Trisetum ¯avescens
Achillea millefolium
Festuca rubra
Nardus stricta
Phleum alpinum
Potentilla aurea
Hieracium pilosella
Campanula barbata
Carex curvula
p > Freference species
noverall
cv (%)
Legume species
mean
noverall
cv (%)

N atom%-excess

900

1380

1900

2100

2300

2600

0.043620.0019
0.080920.0036
0.048320.0023
0.095020.0047
0.074220.0033
0.042620.0019
0.053520.0023
0.031220.0013
0.101920.0050
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
0.0001
487
42

n.p.
n.p.
0.044620.0027
n.p.
0.067920.0026
n.p.
0.080620.0083
n.p.
0.119620.0069
0.070520.0050
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
0.0001
256
41

n.p.
n.p.
0.074220.0085
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
0.079220.0065
0.053820.0050
0.138920.0151
0.062820.0074
0.083520.0151
n.p.
n.p.
0.0001
178
62

n.p.
0.271520.0157
0.141220.0311
n.p.
n.p.
0.077720.0077
n.p.
n.p.
n.p.
n.p.
0.165620.0170
0.053620.0024
n.p.
0.090620.0048
n.p.
0.085320.0215
n.p.
0.0001
343
62

n.p.
0.220320.0219
0.067120.0135
n.p.
n.p.
0.071920.0038
n.p.
n.p.
n.p.
n.p.
n.p.
0.051620.0033
n.p.
0.069320.0056
n.p.
n.p.
n.p.
0.0001
199
72

n.p.
n.p.
n.p.
n.p.
n.p.
0.052820.0052
n.p.
n.p.
n.p.
n.p.
0.075120.0196
n.p.
n.p.
n.p.
n.p.
n.p.
0.041820.0097
0.3363
13
49

0.013420.0009
215
51

0.012120.0010
111
45

0.015520.0013
75
40

0.009520.0010
123
67

0.009120.0012
90
57

0.006720.0014
6
n.d.

1997

n.p.
n.p.
n.p.
n.p.
n.p.
n.p.
8822
8822
n.d.
0.0547
13
6
n.d.

1998

8322
n.p.
n.p.
n.p.
n.p.
n.p.
8921
8621
0.0045
0.0014
134
15

Cambridge, UK) in the Stable Laboratory Facility of
the University of Saskatchewan, Saskatoon, Canada.
The precision of these instruments were 2 0.0002
atom% for the enriched mass spectrometer and 2 0.3d
per mil (per thousand) for the natural abundance mass
spectrometer.

2300

2600

1047

8223
n.p.
n.p.
n.p.
n.p.
n.p.
9121
8622
0.0001
0.0001
65
11
0.8673
8622
8822
n.p.
n.p.
n.p.
8822
8923
8721
0.4918
0.0003
177
16
8621
8422
n.p.
n.p.
n.p.
8123
9321
8724
0.0001
0.0001
165
7
0.2565
7823
7723
5925
n.p.
8022
n.p.
n.p.
7322
0.0002
0.0213
87
24

a

7123
7822
6723
8222
n.p.
n.p.
n.p.
7421
0.0002
0.0001
137
18
0.0001
7722
7721
7122
7824
n.p.
n.p.
n.p.
7621
0.0203
0.0001
234
27
6822
6923
6422
7723
n.p.
n.p.
n.p.
6821
0.0133
0.0001
251
19
0.0001
Legume species:
Lotus corniculatus/alpinus
Trifolium pratense/nivale
T. repens
Vicia sativa
T. thalii
T. badium
T. alpinum
Mean
p > Flegume species
p > Fareference species
noverall
cv (%)
p > Fyear

1997

E€ect of the reference species on %Ndfs; for details on reference species see Table 5.

7822
7623
6524
n.p.
7723
n.p.
n.p.
7422
0.0051
0.0001
90
15
0.6361
8023
8222
7522
9121
n.p.
n.p.
n.p.
8221
0.0001
0.0001
119
11

1997
1998

1380

1998

1997

1900

1998

1997

2100

1998

1997

2.5. Speci®city of rhizobia

900
Altitude (m a.s.l. )

Table 5
Percentage of plant N derived from symbiotic N2 ®xation (%Nsym) for legume species at ®ve altitudes measured by the 15N isotope dilution method. Values are means2SEM (n = 12±75, derived from 2 to 21 replicates of each of 3±9 reference species per legume species, cv: coecient of variation; n.p.: not present; n.d.: no determination)

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

To determine the adaptability of rhizobia to high
altitude conditions, the speci®city of rhizobia was studied. For this, Lotus corniculatus (cv. Leo, FENACO,
Winterthur, Switzerland), Trifolium pratense (cv. RuÈttinova, FENOCO, Winterthur, Switzerland) and T. alpinum (ecotype, Grosse Scheidegg, 2300 m a.s.l., 50 km
southeast of Bern, E. Schweizer Samen AG, Thun,
Switzerland) were grown in a growth chamber (18/13
8C day/night, 80% relative humidity, 16 h photoperiod
at a photosynthetically active photon ¯ux density of
500 mmol mÿ2 sÿ1) and inoculated with di€erent soil
extracts (see Table 7). The experiment was conducted
twice (experiment 1: soil samples taken in June 1998;
and experiment 2: soil samples taken in July 1998). In
each experiment, ®ve soil subsamples from an area of
4 m2 were combined to one soil sample. Soil extracts
were made from 100 g fresh soil in 950 ml of sterile Nfree nutrient solution and ®ltered through ®lter paper
(No. 5893). The seeds were sterilized with 70% ethanol
for 5 min, put into sodium hypochlorite for 5 min, and
then washed with double distilled water (Milli-Q Plus,
Millipore Corporation, Kloten, ZuÈrich). The three-day
old seedlings were put into glass beakers (100 cm3),
®lled with autoclaved vermiculite and 50 ml of sterile
N-free nutrient solution and inoculated with soil
extracts (5 and 20 ml, respectively). The plants were
watered twice during the experiment with 50 ml sterile
N-free nutrient solution, and were examined for
nodules after 26 to 38 days.
2.6. Statistical analyses
Analyses of variance were carried out using the
GLM procedure of the statistical analysis package
SAS (SAS Institute, Cary, NC).

3. Results
3.1. Symbiotic N2 ®xation
The proportion of N derived from symbiotic N2 ®xation (%Nsym) was high in all legume species along
the altitudinal gradient, with values ranging from 59 to
93% (Table 5). At 900 and 1380 m a.s.l., %Nsym was
about 10% higher in 1997 than in 1998. At the other
sites, %Nsym remained the same over time. With one

1048

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

Table 6
Natural abundance of 15N …d15N) of individual reference species and mean of all legume species at ®ve altitudes. Percentage of plant N derived
from symbiotic N2 ®xation (%Nsym) of all legume species calculated with a range of various potential B values (B value is 15N enrichment, relative to atmospheric N2, of the legume grown solely with atmospheric N2). Means2SEM of 12±48 replicates in 1998; cv: coecient of variation;
n.p.: not present or not measured
d15N
Altitude (m a.s.l.)
Reference species
Arrhenaterum elatius
Anthoxantum odoratum/alpinum
Agrostis tenuis
Dactylis glomerata
Leontodon hispidus/helveticus
Trisetum ¯avescens
Festuca rubra
Nardus stricta
Phleum alpinum
Potentilla aurea
Mean
p > Freference species
noverall
cv (%)
Legume species
Mean
noverall
cv (%)
B value
%Nsym
SEM
cv (%)

900

1380

1900

2100

2300

2.7720.30
3.0020.27
2.1020.10
2.2820.24
3.3220.17
1.9720.18
n.p.
n.p.
n.p.
n.p.
2.6020.10
0.0001
212
50

n.p.
n.p.
10.9220.76
4.6220.48
4.5920.24
3.3920.50
4.4820.32
n.p.
n.p.
n.p.
5.2820.30
0.0001
140
46

n.p.
n.p.
4.4221.32
n.p.
n.p.
n.p.
ÿ0.6520.19
0.4220.20
1.9020.45
ÿ0.2620.17
0.6720.25
0.0001
96
281

n.p.
ÿ0.2621.11
ÿ0.9221.50
n.p.
ÿ1.8220.14
n.p.
0.9820.53
ÿ0.8420.23
n.p.
ÿ2.7820.24
ÿ1.0320.27
0.0001
108
249

n.p.
0.0120.85
n.p.
n.p.
ÿ1.6820.30
n.p.
n.p.
0.3620.47
n.p.
ÿ2.6420.52
ÿ0.9920.47
0.0016
48
203

ÿ0.2620.11
36
179
ÿ10ÿ2
7963
114
24
26

1.0920.55
24
182
ÿ10ÿ2
5644
73
212
94

ÿ0.5920.11
12
44
ÿ10ÿ2
8537
147
224
126

ÿ0.2020.21
12
269
0‡1
ÿ1
8750
108
29
168

ÿ0.8420.17
12
49
0‡1
ÿ1
7113
84
219
127

pratensis to 0.1019 atom%-excess in Trisetum ¯avescens at 900 m a.s.l., and from 0.0536 atom%-excess in
Nardus stricta to 0.2715 atom%-excess in Anthoxantum
alpinum at 2100 m a.s.l. Nevertheless, the values of
15
N atom%-excess of the reference plants were always
much higher than those of the leguminous plants
(Table 4). In 1997, the ranking of the reference species
according to 15N atom%-excess changed with site
(ANOVA, reference species x site, P > 0.0244).
At all sites, d15 N values of the reference species differed signi®cantly (Table 6). The means d15 N of all
reference species were positive at the lower sites and

exception (2100 m a.s.l. in 1998) %Nsym varied signi®cantly among the legume species at all sites. All of the
200 legume plants examined in the vicinity of the experimental sites between 2000 and 2700 m a.s.l. (above
which they do not grow) had apparently e€ective root
nodules.
3.2. Validation of %Nsym assessment
15

N atom%-excess values of the various reference
species di€ered signi®cantly at each altitude (Table 4).
Values ranged from 0.0312 atom%-excess in Salvia

Table 7
Percentage of leguminous plants nodulating after inoculation with a soil extract from two di€erent sources: 2500 m a.s.l. where only Trifolium
alpinum and Lotus alpinus grow and 1380 m a.s.l. where, among other legume species, T. pratense and L. corniculatus grow. The experiment was
conducted twice (Exp. 1: samples taken in June 1998 and Exp. 2: samples taken in July 1998). Percentages were calculated for each experiment
separately. Extracts were tested with 24 seedlings of each legume species for each experiment
Inoculum source and respective legumes

2500 m a.s.l. (Trifolium alpinum, Lotus alpinus )
1380 m a.s.l. (T. pratense, L. corniculatus )
no inoculum (sterile control)

Legume species tested for nodulation
Lotus corniculatus

Trifolium pratense

Trifolium alpinum

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Exp. 1

Exp. 2

100%
100%
0%

100%
100%
0%

12.5%
100%
0%

4.2%
100%
0%

100%
87.5%
0%

100%
66.7%
0%

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

negative at the higher sites. With the exception at 1380
m a.s.l., d15 N values for legume species were negative.
Particularly at 900 and 1380 m a.s.l., d15 N of the
legume species were clearly di€erent from the d15 N
values of the reference species. Percentage of plant N
derived from symbiotic N2 ®xation, (%Nsym) averaged over all legumes was between 56 and 87% when
applying an average B value of ÿ1 for the three lower
sites (Sanford et al., 1994; Unkovich et al., 1994;
Peoples et al., 1998) and a B value of 0 for the two
higher sites (Bowman et al., 1996) (Table 6).
3.3. Speci®city of rhizobia
When the legume species were inoculated with soil
extract from 2500 m a.s.l. where Trifolium alpinum and
Lotus alpinus grow, all of the L. corniculatus and T.
alpinum seedlings formed apparently e€ective nodules
(Table 7); in contrast, in two separate experiments,
only 12.5 and 4.2% of the Trifolium pratense seedlings
formed apparently e€ective nodules. When the legume
species were inoculated with soil extract from 1380 m
a.s.l., where T. pratense and L. corniculatus grow, all
T. pratense and L. corniculatus seedlings formed apparently e€ective nodules. In this case, only 87.5 and
66.7% of the T. alpinum seedlings formed apparently
e€ective nodules.

4. Discussion
4.1. Signi®cance of symbiotic N2 ®xation for legumes in
the Swiss Alps
Each legume species obtained a high proportion of
N through symbiotic N2 ®xation along the whole altitudinal gradient (Table 5). Furthermore, all legumes
showed apparently e€ective nodules. This demonstrates that, even at the upper altitudinal limit of the
individual species, symbiotic N2 ®xation was important
for the N budget of these legumes. To the best of our
knowledge, this is the ®rst time that symbiotic N2 ®xation has been quanti®ed over such an altitudinal gradient and including the limiting climatic conditions for
a legume. Only two studies quanti®ed symbiotic N2
®xation with respect to the N budget of legume plants
at high altitudes; Bowman et al., 1996 reported high
symbiotic N2 ®xation for Trifolium species on Niwot
Ridge, Colorado and Arnone (1999) in the Swiss Alps.
Other investigators have reported symbiotic N2 ®xation under arctic and alpine conditions (Wojciechowski and Heimbrook, 1984; Karagatzides et al.,
1985; Johnson and Rumbaugh, 1986; Holzmann and
Haselwandter, 1988; Schulman et al., 1988; Sparrow et
al., 1995). These investigators used the acetylene reduction method, and thus, only produced data about

1049

N2 ®xation over short sample periods (min-h). Nevertheless, their studies also indicate that arctic and alpine
legume plants are capable of symbiotic N2 ®xation
under extreme climatic conditions, though they do not
quantify the contribution of ®xation to plant N budgets.
Experiments in growth chambers often show that
symbiotic N2 ®xation is inhibited by unfavorable conditions such as, low temperature and low soil pH, to a
greater extent than plant growth (Kessler et al., 1990;
Nesheim and Boller, 1991; reviewed in Graham, 1991).
In our study, both the air and soil temperatures
decreased gradually with increasing altitude (Table 3);
the soil pH values were also very low at high altitudes
(Table 2). It is therefore surprising that with increasing
altitude in our study, symbiotic N2 ®xation was not
reduced more strongly than plant growth. The explanation may be that in the short-term laboratory experiments such as those by Kessler et al., 1990, the same
genotypes of legume and rhizobium were used in all
treatments, while we investigated the indigenous genotypes at each altitude. This suggests that such investigations have to be done under ®eld conditions where
legume and rhizobia are adapted to the appropriate
conditions (Turkington and Harper, 1979; Thompson
and Turkington, 1990; LuÈscher and Jacquard, 1991;
Svenning et al., 1991; LuÈscher et al., 1992; Expert et
al., 1997). It is indeed evident from the present study
that rhizobia from the upper altitudinal limit of
legumes are more speci®c towards alpine legumes than
towards legumes commonly growing at lower altitudes
(Table 7). This suggests evolutionary and coevolutionary adaptation of rhizobia and plants at high altitudes
with relatively stressful climate, and in fact, may
explain discrepancies between the results from the present study and results reported in the literature. EkJander and Fahraeus (1971) also found that Rhizobium
trifolii isolates from a subarctic environment in Scandinavia, grew faster, nodulated their hosts earlier, and
exhibited higher rates of acetylene reduction at low
temperatures than isolates from more southern areas.
Similarly, Svenning et al. (1991) showed that plants
from the north in Norway gave higher yield when
nodulated by Rhizobium from the north than from the
south. It has also been shown that rhizobia vary in
their tolerance to low pH (reviewed in Graham, 1991).
The results of lowland experiments and the consistently high N2 ®xation along the altitudinal gradient
found in the present study suggest that the ratio of soil
N availability to the N demand of legumes is relatively
constant. This agrees with studies conducted at high
altitudes, where net soil N mineralization was low
(Rheder and Schafer, 1978; Schinner, 1982; Haselwandter et al., 1983; Jacot et al., 1999). The accumulation of organic matter in the soil (Table 2) at higher
altitudes re¯ects a low rate of mineralization, which

1050

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

may lead to higher values of symbiotically ®xed N. An
alternative explanation of the present results could be
that alpine legumes do not reduce symbiotic N2 ®xation at higher amounts of soil N due to di€erent
physiological regulation of the nitrogenase activity.
Furthermore, at the altitudinal limit of legumes, only
plants with high symbiotic N2 ®xation may be competitive.
The small changes in %Nsym from the ®rst to the
second year of our study (Table 5) may have been due
to di€erent weather conditions. Annual and seasonal
changes in precipitation and temperature could have
in¯uenced the N availability, which is known to a€ect
%Nsym (Boller and NoÈsberger, 1987; Nesheim and
Oyen, 1994; Sereshine et al., 1994; Zanetti et al., 1996).
However, since the amounts of symbiotically ®xed N
are high, this increase in %Nsym from one year to the
next is not considered to be important for the legume
plant's N budget.
4.2. Validation of the enriched 15N isotope dilution
technique and the 15N natural abundance method for
studying symbiotic N2 ®xation in permanent grassland
using various reference species
In our study, the enriched 15N isotope dilution technique has been applied for the ®rst time in speciesrich, low N input permanent grassland, using a wide
range of reference species for the calculation of
%Nsym.
The signi®cant di€erences in 15N atom%-excess
among the reference species (Table 4) illustrate that
the choice of reference species has major e€ects upon
the calculated %Nsym. In our study, the di€erent
values of 15N atom%-excess among the reference
species led to values of %Nsym from 57% (Salvia pratensis ) to 87% (Trisetum ¯avescens ) at 900 m a.s.l.,
and from 82% (Nardus stricta ) to 97% (Anthoxantum
alpinum ) at 2100 m a.s.l. Such di€erences among reference species have been interpreted by several authors
in terms of di€erent rates of N uptake at di€erent soil
depths and at di€erent times (reviewed in Chalk, 1985;
Ledgard et al., 1985a, 1985b; Danso et al., 1993).
There are various reasons to suppose that the inclusion of several reference species makes enriched 15N
isotope dilution a more reliable method for assessing
N2 ®xation. First, the values of %Nsym are dependent
on more than one, possibly extreme, 15N atom%excess of a reference species (for example, Trisetum ¯avescens ). Second, a large number of reference species is
likely to be more representative of the root horizon
and N uptake over the experimental period; either
excluding or adding a single reference species is not
likely to cause much change in the %Nsym value.
Finally, we have chosen more than one site-adapted
reference instead of one particular reference species,

because the di€erent soil and climatic condition at the
sites may result in di€erent behavior in terms of spatial
and temporal N uptake by the reference species (KoÈrner and Renhardt, 1987; Aktin et al., 1996). Moreover,
it is very likely that particular reference species exist as
distinct ecotypes at various altitudes. It is evident that
the species (for example Agrostis tenuis/rupestris ) were
ranked di€erently within the group of reference species
at the di€erent sites (Table 4).
Even though we report considerable di€erences in
15
N atom%-excess between the reference species, there
is a clear di€erentiation in 15N atom%-excess between
reference and legume species (Table 4). There is, therefore, strong evidence for high symbiotic N2 ®xation by
legumes. Even if the reference species with the lowest
15
N atom%-excess at each site were used, the resulting
%Nsym (between 55% at 900 m a.s.l. and 78% at
2600 m a.s.l.) values would support the conclusion
that symbiotic N2 ®xation contributes signi®cantly to
the N budget of the legume.
Symbiotic N2 ®xation was also measured using the
15
N natural abundance method (Table 6). We found
that the values of d15 N in both legume and reference
species decreased with increasing altitude. This,
together with the variability of d15 N (coecient of
variation in Table 6) and the signi®cant di€erences in
d15 N among the reference species (Table 6), results in a
less accurate assessment of the proportion of symbiotically-®xed N (Domenach and Corman, 1984; Ledgard
and Peoples 1988). Low d15 N values have also been
found in other studies, and have attributed to relatively high use of N derived from atmospheric deposition by plants (Vitousek et al., 1989; Gebauer and
Schulze, 1991; Garten, 1993; Bowman et al., 1996).
Nadelho€er et al., 1996 suggested reasons for the low
d15 N in arctic tundra ecosystems, including distinct isotopic fractionation during soil N transformation. Furthermore, as a result of very di€erent soil and climatic
conditions along the altitudinal gradient and due to
the di€erent legume species, site- and species-speci®c
isotope fractionation during N2 ®xation must be
expected. This would lead to a wide range of B values
(B value is 15N enrichment, relative to atmospheric N2,
of the legume grown solely with atmospheric N2
(Shearer and Kohl, 1986; Ledgard, 1989; Hùgh-Jensen
and Schjoerring, 1994; Sanford et al., 1994; Unkovichz
et al., 1994; Peoples et al. (1998)). An appropriate estimate of the B value for each site and each legume
species is a prerequisite for the natural abundance
method. However, in multi-site studies with many
legume species, an appropriate assessment of the Bvalue is hardly feasible. According to Sanford et al.
(1994), Unkovich et al. (1994) and Peoples et al.
(1998), an averaged B value of ÿ1 would be appropriate for the lower sites and according to Bowman et al.,
1996, one of 0 for the higher sites can be used. When

K.A. Jacot et al. / Soil Biology & Biochemistry 32 (2000) 1043±1052

applying these B values, means of %Nsym were comparable to the measures derived from the 15N enriched
sites (Table 6). In view of the high sensitivity of the
calculation of %Nsym to the B value (Table 6), the
assessment of %Nsym by the natural abundance
method is very delicate. However, as with the enriched
15
N isotope dilution method, distinct di€erences in 15N
values between reference plants and legumes are convincing evidence for high rates of symbiotic N2 ®xation.
4.3. Conclusion
The present study provides basics to measure symbiotic N2 ®xation in low N input, permanent grasslands. From the results it can be concluded that up to
the altitudinal limit of legumes, the N2 ®xing symbiosis
is well adapted to the particular conditions and that
symbiotic N2 ®xation contributes signi®cantly to the N
nutrition of legume species at all altitudes.

Acknowledgements
This study was supported by a grant from the Swiss
National Science Foundation (31-45626.95 to U.A.H.).
We are very greatly indebted to G. Parry (University
of Saskatchewan (Saskatoon) Canada), for conducting
the 15N analysis. We thank the technicians Anni DuÈrsteler and Werner Wild and the students Lukas RuÈttimann, Christian Bernasconi, and Martina Battini for
their invaluable assistance during the experiment. Dr
H. Conradin carried out soil taxonomy and Dr. M.
Baltisberger veri®ed plant taxonomy. We also thank
A. NaÈgeli from the extension service, the 'BuÈndner
OberlaÈnder Bauernverband', the local community and
farmers in Sumvitg and Trun for their cooperation.
We thank Professor Dr. Ch. van Kessel for valuable
discussion concerning 15N techniques, Professor Dr.
P.J. Edwards and Dr. M.B. Peoples for critically reading a draft of the manuscript, and M. Schoenberg for
editing the language.

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