Symbiotic N
2
®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 diered signi®cantly in their15N atom%-excess. However, when several reference species were combined, the enriched15N 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 the15N 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.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Alpine ecosystem;15N isotope dilution;15N natural abundance;Trifolium;Lotus
1. Introduction
Symbiotic N2 ®xation of leguminous plants is
im-portant for the worldwide nitrogen budget (Evans and Barber, 1977). Plants that symbiotically ®x N2occur 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 environ-ments is unclear (Sprent, 1985).
Alpine areas are characterized by a relatively stress-ful climate with low temperatures, short growing sea-sons, and often also by low soil pH. These conditions restrict organic decomposition and microbial
trans-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
®x-ation (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 aected by
cli-matic conditions at high altitudes (Cralle and Heichel, 1982). Kessler et al. (1990) reported that low tempera-ture has a more negative eect on symbiotic N2
®x-ation than on plant growth. However, Svenning et al. (1991) showed that in a cold climate in Norway, adap-tation of both legumes and rhizobia occurs. Therefore,
0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 1 2 - 2
www.elsevier.com/locate/soilbio
it is still not clear whether symbiotic N2 ®xation at
high altitudes in the Alps is eective or not.
Very few investigators have studied symbiotic N2
®x-ation 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 contri-bution of symbiotic N2®xation to the legume's N
bud-get in natural species-rich permanent grassland along an altitudinal gradient. An enriched 15N isotope di-lution method using various reference species was established towards a complex permanent grassland and evaluated with the15N natural abundance method. Another aim was to determine the adaptability of rhi-zobia 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 inocu-lation 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 m2each, 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 hetero-geneity. At 2600 m a.s.l., one site of 2.25 m2was used as an experimental area. The 100 m2 (2.25 m2) exper-imental 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 (Sternpyr-anometer, 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 measure-ments 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 enriched15N isotope dilution method (Danso et al., 1993). The enriched15N isotope dilution method depends upon dierences in isotopic composition of the various sources of N available for plant growth, i.e. soil N, fertilizer N and atmospheric N2. The
pro-portion of N derived from symbiosis (%Nsym) was calculated for individual legume species at each alti-tude for each regrowth period as (McAulie 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 Arrhenatheretum Trifolium pratenseL.,T. repensL.,Lotus corniculatusL.,Vicia sativaL. 1380 Trisetetum T. pratenseL.,T. repensL.,L. corniculatusL.,V. sativaL.
1900 Nardetum T. pratenseL.,T. repensL.,L. corniculatusL.,T. thaliiVill.
2100 Nardetum T. nivaleSieber,T. alpinumL.,L. alpinus(DC.) Schleicher,T. badiumSchreber 2300 Nardetum/Curvuletum T. alpinumL.,L. alpinus(DC.) Schleicher
2600 Curvuletum T. alpinumL.,L. alpinus(DC.) Schleicher
%Nsym
1ÿ
15N atom%-excess in the legume
15N atom%-excess in the reference plant
!
100:
Two to three plants that do not ®x N2but 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 el-evation, symbiotic N2®xation was calculated using an
average of three±nine dierent reference species (Table 4). The N applied for15N labeling at each site
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 mÿ2of water following the
15
N application. The 15N atom%-excess of the enriched (NH4)2SO4solution 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 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 Dystric cambisol 4.9 9.1 20.7 65.3 5.6
1380 Dystric cambisol 8.5 12.3 27.9 52.3 4.6
1900 Spodi-dystric cambisol 15.2 13.6 23.4 50.8 4.1
2100 Humic podsol 19.7 14.8 18.4 47.1 4.1
2770 Spodi-dystric cambisol 29.3 20.8 18.5 31.4 3.1
aSource: H. Conradin (Swiss Federal Research Station for Agroecology and Agriculture).
Table 3
Microclimatic conditions during the growing season at the experimental sites (precipitationP, means, maximum, minimum of airTa and soil temperatureTsand daily radiationRdsnow not measured)
Altitude (m a.s.l. ) 900 1380 1900 2100 2770
Growing season May±October May±October June±October June±October July±October
1996 P(mm) 677 800 573 508 315
Ta(mean)(8C) 12.8 10.9 6.9 5.3 1.3
Ta(max)(8C) 31.7 30.2 20.8 18.9 13.1
Ta(min)(8C) ÿ2.3 ÿ2.0 ÿ5.0 ÿ6.0 ÿ8.7
Ts(mean)(8C) 16.7 13.9 11.4 8.3 5.4
Ts(max)(8C) 35.2 26.7 30.7 18.0 19.3
Ts(min)(8C) 5.3 4.9 1.8 0.4 0.12
Rd(MJ m
ÿ2
dÿ2) 14.2 14.2 14.3 13.6 13.0
1997 P(mm) 455 540 585 567 215
Ta(mean)(8C) 13.4 11.8 9.1 7.5 3.9
Ta(max)(8C) 31.7 30.5 22.0 20.2 15.2
Ta(min)(8C) ÿ5.7 ÿ5.2 ÿ8.0 ÿ9.4 ÿ13.0
Ts(mean)(8C) 17.0 14.2 12.9 10.7 7.6
Ts(max)(8C) 35.7 25.2 27.9 23.0 20.8
Ts(min)(8C) 1.6 2.2 ÿ0.2 0.3 ÿ0.6
Rd(MJ mÿ2dÿ2) 14.7 14.9 15.7 17.1 16.6
1998 P(mm) 525 555 619 580 357
Ta(mean)(8C) 13.6 10.2 8.6 7.0 5.0
Ta(max)(8C) 35.2 30.6 25.0 23.1 16.7
Ta(min)(8C) ÿ1.0 ÿ1.6 ÿ4.5 ÿ6.3 ÿ7.5
Ts(mean)(8C) 17.8 13.0 12.9 9.0 9.0
Ts(max)(8C) 39.4 27.6 30.7 24.1 22.8
Ts(min)(8C) 5.6 4.3 1.5 0.2 0.4
Rd(MJ m
ÿ2
®rst harvest each year. The assessment of symbioti-cally-®xed N commenced in 1997.
In an area about 1 km2from 2000 to 2700 m a.s.l., representing the upper limit of legumes in this region, about 100 plants of Trifolium alpinum and Lotus alpi-nus each, were examined for a red pigment inside the nodules (which is indicative of eective N2®xation).
2.3.15N 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 dierences in 15N concentration, the term d15N (-) is commonly used (Shearer and Kohl, 1986):
d15N -
atom%15N sample ÿatom%15N standard
atom%15N standard
!
1000,
where atmospheric N2is the standard.
The following formula is used to calculate the per-centage of symbiotically ®xed N (Ledgard and Peoples, 1988):
%Nsym
d15N in the reference plantÿd15N in the legume
d15N in the reference plantÿB
!
100,
whereB is15N enrichment, relative to atmospheric N2,
of the legume grown solely with atmospheric N2.
2.4. Sample preparation and15N analyses
All dried (658C for 48 h) plant material was ground in sequence using a Cyclotec 1093 sample mill (Teca-tor, 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 for15N concentration by a continuous-¯ow mass spectrometer (Europa Scienti®c,
Table 4
15N atom%-excess of individual reference species and mean of all legume species after application of15N at ®ve altitudes. Values are means2
SEM (n= 2±109, averaged over 1997 and 1998; at 2600 m a.s.l., data are for 1997, cv: coecient of variation; n.p.: not present or not measured; n.d.: no determination)
15N atom%-excess
Altitude (m a.s.l.) 900 1380 1900 2100 2300 2600
Reference species
Arrhenaterum elatius 0.043620.0019 n.p. n.p. n.p. n.p. n.p.
Anthoxantum odoratum/alpinum 0.080920.0036 n.p. n.p. 0.271520.0157 0.220320.0219 n.p. Agrostis tenuis/rupestris 0.048320.0023 0.044620.0027 0.074220.0085 0.141220.0311 0.067120.0135 n.p.
Cynosurus christatus 0.095020.0047 n.p. n.p. n.p. n.p. n.p.
Dactylis glomerata 0.074220.0033 0.067920.0026 n.p. n.p. n.p. n.p.
Leontodon hispidus/helveticus 0.042620.0019 n.p. n.p. 0.077720.0077 0.071920.0038 0.052820.0052
Lolium perenne 0.053520.0023 0.080620.0083 n.p. n.p. n.p. n.p.
Salvia pratensis 0.031220.0013 n.p. n.p. n.p. n.p. n.p.
Trisetum ¯avescens 0.101920.0050 0.119620.0069 n.p. n.p. n.p. n.p.
Achillea millefolium n.p. 0.070520.0050 n.p. n.p. n.p. n.p.
Festuca rubra n.p. n.p. 0.079220.0065 0.165620.0170 n.p. 0.075120.0196
Nardus stricta n.p. n.p. 0.053820.0050 0.053620.0024 0.051620.0033 n.p.
Phleum alpinum n.p. n.p. 0.138920.0151 n.p. n.p. n.p.
Potentilla aurea n.p. n.p. 0.062820.0074 0.090620.0048 0.069320.0056 n.p.
Hieracium pilosella n.p. n.p. 0.083520.0151 n.p. n.p. n.p.
Campanula barbata n.p. n.p. n.p. 0.085320.0215 n.p. n.p.
Carex curvula n.p. n.p. n.p. n.p. n.p. 0.041820.0097
p>Freference species 0.0001 0.0001 0.0001 0.0001 0.0001 0.3363
noverall 487 256 178 343 199 13
cv (%) 42 41 62 62 72 49
Legume species
mean 0.013420.0009 0.012120.0010 0.015520.0013 0.009520.0010 0.009120.0012 0.006720.0014
noverall 215 111 75 123 90 6
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 and20.3d per mil (per thousand) for the natural abundance mass spectrometer.
2.5. Speci®city of rhizobia
To determine the adaptability of rhizobia to high altitude conditions, the speci®city of rhizobia was stu-died. For this, Lotus corniculatus (cv. Leo, FENACO, Winterthur, Switzerland),Trifolium pratense(cv. RuÈtti-nova, FENOCO, Winterthur, Switzerland) and T. alpi-num (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 dierent 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 N-free 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
®x-ation (%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
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 ex-perimental sites between 2000 and 2700 m a.s.l. (above which they do not grow) had apparently eective root nodules.
3.2. Validation of %Nsym assessment
15
N atom%-excess values of the various reference species diered signi®cantly at each altitude (Table 4). Values ranged from 0.0312 atom%-excess in Salvia
pratensis to 0.1019 atom%-excess in Trisetum
¯aves-cens at 900 m a.s.l., and from 0.0536 atom%-excess in
Nardus strictato 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 speciesxsite,P> 0.0244).
At all sites, d15Nvalues of the reference species dif-fered signi®cantly (Table 6). The means d15N of all reference species were positive at the lower sites and Table 6
Natural abundance of15N 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 potentialBvalues (Bvalue is 15
N enrichment, rela-tive to atmospheric N2, of the legume grown solely with atmospheric N2). Means2SEM of 12±48 replicates in 1998; cv: coecient of variation;
n.p.: not present or not measured
d15N
Altitude (m a.s.l.) 900 1380 1900 2100 2300
Reference species
Arrhenaterum elatius 2.7720.30 n.p. n.p. n.p. n.p.
Anthoxantum odoratum/alpinum 3.0020.27 n.p. n.p. ÿ0.2621.11 0.0120.85
Agrostis tenuis 2.1020.10 10.9220.76 4.4221.32 ÿ0.9221.50 n.p.
Dactylis glomerata 2.2820.24 4.6220.48 n.p. n.p. n.p.
Leontodon hispidus/helveticus 3.3220.17 4.5920.24 n.p. ÿ1.8220.14 ÿ1.6820.30
Trisetum ¯avescens 1.9720.18 3.3920.50 n.p. n.p. n.p.
Festuca rubra n.p. 4.4820.32 ÿ0.6520.19 0.9820.53 n.p.
Nardus stricta n.p. n.p. 0.4220.20 ÿ0.8420.23 0.3620.47
Phleum alpinum n.p. n.p. 1.9020.45 n.p. n.p.
Potentilla aurea n.p. n.p. ÿ0.2620.17 ÿ2.7820.24 ÿ2.6420.52
Mean 2.6020.10 5.2820.30 0.6720.25 ÿ1.0320.27 ÿ0.9920.47
p>Freference species 0.0001 0.0001 0.0001 0.0001 0.0016
noverall 212 140 96 108 48
cv (%) 50 46 281 249 203
Legumespecies
Mean ÿ0.2620.11 1.0920.55 ÿ0.5920.11 ÿ0.2020.21 ÿ0.8420.17
noverall 36 24 12 12 12
cv (%) 179 182 44 269 49
Bvalue ÿ10
ÿ2 ÿ10ÿ2 ÿ10ÿ2 0ÿ11 0ÿ11
%Nsym 7963
114 564473 8537147 8750108 711384
SEM 24 212 224 29 219
cv (%) 26 94 126 168 127
Table 7
Percentage of leguminous plants nodulating after inoculation with a soil extract from two dierent sources: 2500 m a.s.l. where onlyTrifolium alpinumandLotus alpinusgrow and 1380 m a.s.l. where, among other legume species,T. pratenseandL. corniculatusgrow. 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 Legume species tested for nodulation
Lotus corniculatus Trifolium pratense Trifolium alpinum
Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2
2500 m a.s.l. (Trifolium alpinum, Lotus alpinus) 100% 100% 12.5% 4.2% 100% 100% 1380 m a.s.l. (T. pratense, L. corniculatus) 100% 100% 100% 100% 87.5% 66.7%
negative at the higher sites. With the exception at 1380 m a.s.l., d15N values for legume species were negative. Particularly at 900 and 1380 m a.s.l., d15N of the legume species were clearly dierent from the d15N
values of the reference species. Percentage of plant N derived from symbiotic N2 ®xation, (%Nsym)
aver-aged over all legumes was between 56 and 87% when applying an average Bvalue ofÿ1 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. whereTrifolium alpinumand
Lotus alpinus grow, all of the L. corniculatus and T.
alpinum seedlings formed apparently eective nodules
(Table 7); in contrast, in two separate experiments, only 12.5 and 4.2% of theTrifolium pratense seedlings formed apparently eective 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. pratenseandL. corniculatusseedlings formed appar-ently eective nodules. In this case, only 87.5 and 66.7% of the T. alpinum seedlings formed apparently eective 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
alti-tudinal gradient (Table 5). Furthermore, all legumes showed apparently eective nodules. This demon-strates 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
®x-ation has been quanti®ed over such an altitudinal gra-dient 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
®x-ation under arctic and alpine conditions (Wojcie-chowski 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 re-duction method, and thus, only produced data about
N2®xation over short sample periods (min-h).
Never-theless, 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 bud-gets.
Experiments in growth chambers often show that symbiotic N2 ®xation is inhibited by unfavorable
con-ditions 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 expla-nation may be that in the short-term laboratory exper-iments 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 geno-types at each altitude. This suggests that such investi-gations 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 coevolution-ary adaptation of rhizobia and plants at high altitudes with relatively stressful climate, and in fact, may explain discrepancies between the results from the pre-sent study and results reported in the literature. Ek-Jander and Fahraeus (1971) also found thatRhizobium trifolii isolates from a subarctic environment in Scandi-navia, 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 Rhizobiumfrom 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 consist-ently high N2 ®xation along the altitudinal gradient
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
®x-ation at higher amounts of soil N due to dierent physiological regulation of the nitrogenase activity. Furthermore, at the altitudinal limit of legumes, only plants with high symbiotic N2 ®xation may be
com-petitive.
The small changes in %Nsym from the ®rst to the second year of our study (Table 5) may have been due to dierent weather conditions. Annual and seasonal changes in precipitation and temperature could have in¯uenced the N availability, which is known to aect %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 enriched15N isotope dilution technique and the15N natural abundance method for studying symbiotic N2®xation in permanent grassland using various reference species
In our study, the enriched15N isotope dilution tech-nique has been applied for the ®rst time in species-rich, low N input permanent grassland, using a wide range of reference species for the calculation of %Nsym.
The signi®cant dierences in 15N atom%-excess among the reference species (Table 4) illustrate that the choice of reference species has major eects upon the calculated %Nsym. In our study, the dierent values of 15N atom%-excess among the reference species led to values of %Nsym from 57% (Salvia pra-tensis) 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 dierences among refer-ence species have been interpreted by several authors in terms of dierent rates of N uptake at dierent soil depths and at dierent times (reviewed in Chalk, 1985; Ledgard et al., 1985a, 1985b; Danso et al., 1993).
There are various reasons to suppose that the in-clusion of several reference species makes enriched15N 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 ¯a-vescens). 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 dierent soil and climatic condition at the sites may result in dierent behavior in terms of spatial and temporal N uptake by the reference species (KoÈr-ner 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 dierently within the group of reference species at the dierent sites (Table 4).
Even though we report considerable dierences in
15
N atom%-excess between the reference species, there is a clear dierentiation in 15N atom%-excess between reference and legume species (Table 4). There is, there-fore, 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 d15N in both legume and reference species decreased with increasing altitude. This, together with the variability of d15N (coecient of variation in Table 6) and the signi®cant dierences in d15Namong the reference species (Table 6), results in a less accurate assessment of the proportion of symbioti-cally-®xed N (Domenach and Corman, 1984; Ledgard and Peoples 1988). Low d15N values have also been found in other studies, and have attributed to rela-tively high use of N derived from atmospheric depo-sition by plants (Vitousek et al., 1989; Gebauer and Schulze, 1991; Garten, 1993; Bowman et al., 1996). Nadelhoer et al., 1996 suggested reasons for the low d15Nin arctic tundra ecosystems, including distinct iso-topic fractionation during soil N transformation. Fur-thermore, as a result of very dierent soil and climatic conditions along the altitudinal gradient and due to the dierent legume species, site- and species-speci®c isotope fractionation during N2 ®xation must be
expected. This would lead to a wide range of Bvalues (Bvalue is15N enrichment, relative to atmospheric N2,
of the legume grown solely with atmospheric N2
applying these B values, means of %Nsym were com-parable to the measures derived from the15N 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 dierences in15N values between reference plants and legumes are con-vincing evidence for high rates of symbiotic N2
®x-ation.
4.3. Conclusion
The present study provides basics to measure sym-biotic N2 ®xation in low N input, permanent
grass-lands. 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 DuÈr-steler and Werner Wild and the students Lukas RuÈtti-mann, 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 read-ing a draft of the manuscript, and M. Schoenberg for editing the language.
References
Aktin, O.K., Botman, B., Lambers, H., 1996. The relationship between the relative growth rate and nitrogen economy of alpine and lowland Poa species. Plant, Cell and Environment 19, 1324± 1330.
Arnone, J.A., 1999. Symbiotic N2 ®xation in a high Alpine
grass-land: eects of four growing seasons of elevated CO2. Functional
Ecology 13, 383±387.
Boller, B.C., NoÈsberger, J., 1987. Symbiotically ®xed nitrogen from ®eld-grown white and red clover mixed with ryegrasses at low levels of15N-fertilization. Plant and Soil 104, 219±226.
Bowman, W.D., Schardt, J.C., Schmidt, S.K., 1996. Symbiotic N2
-®xation in alpine tundra: ecosystem input and variation in ®x-ation rates among communities. Oecologia 108, 345±350. Chalk, P.M., 1985. Estimation of N2®xation by isotope dilution: an
appraisal of techniques involving15N enrichment and their
appli-cation. Soil Biology & Biochemistry 17, 389±410.
Cralle, H.T., Heichel, G.H., 1982. Temperature and chilling sensi-tivity of nodule nitrogenase acsensi-tivity of unhardened alfalfa. Crop Science 22, 300±304.
Danso, S.K. A., Hardarson, G., Zapata, F., 1993. Misconceptions and practical problems in the use of 15N soil enrichment tech-niques for estimating N2®xation. Plant and Soil 152, 25±52.
Domenach, A.M., Corman, A., 1984. Dinitrogen ®xation by ®eld grown soybeans: statistical analysis of variations ind15N and pro-posed sampling procedure. Plant and Soil 78, 301±313.
Ek-Jander, J., Fahraeus, G., 1971. Adaptation ofRhizobiumto sub-arctic environment in Scandinavia. In: Lie, T.A., Mulder, E.G. (Eds.), Biological Nitrogen Fixation in Natural and Agricultural Habitats. Martinus Nijho, The Hague, pp. 129±137.
Ellenberg, H., 1982. Vegetation Mitteleuropas mit den Alpen. Ulmer, Stuttgart.
Evans, H.J., Barber, L.E., 1997. Biological nitrogen ®xation for food and ®bre production. Science 179, 332±339.
Expert, J.M., Jacquard, P., Obaton, M., LuÈscher, A., 1997. Neighborhood eect of genotypes of Rhizobium leguminosarum biovar trifolii, Trifolium repens and Lolium perenne. Theoretical and Applied Genetics 94, 486±492.
Garten, C.T., 1993. Variation in foliar15N abundance and the avail-ability of soil nitrogen on Walker Branch watershed. Ecology 74, 2098±2113.
Gebauer, G., Schulze, D.E., 1991. Carbon and nitrogen isotope ratios in dierent compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge, NE Bavaria. Oecologia 87, 198±207.
Graham, P.H., 1991. Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Canadian Journal of Microbiology 38, 475±484.
Hùgh-Jensen, H., Schjoerring, J.K., 1994. Measurement of biological dinitrogen ®xation in grassland: comparison of the enriched15N dilution and the natural abundance methods at dierent nitrogen application rates and defoliation frequencies. Plant and Soil 166, 153±163.
Haselwandter, K., Hofmann, A., Holzmann, H.P., Read, D.J., 1983. Availability of nitrogen and phosphorus in the nival zone of the Alps. Oecologia 57, 266±269.
Holzmann, H.P., Haselwandter, K., 1988. Contribution of nitrogen ®xation to nitrogen nutrition in an alpine sedge community (Caricetum curvulae). Oecologia 76, 298±302.
Jacot, K.A., 1999. Signi®cance of legumes and symbiotic N2®xation
for grassland ecosystems along an altitudinal gradient in the Alps, ETH, PhD-thesis 13378, 91 pp.
Johnson, D.A., Rumbaugh, M.D., 1986. Field nodulation and acety-lene reduction activity of high-altitude legumes in the western United States. Arctic and Alpine Research 18, 171±179.
Karagatzides, J.D., Lewis, M.C., Schulman, H.M., 1985. Nitrogen ®xation in the high arctic tundra at Sarcpa Lake, Northwest Territories. Canadian Journal of Botany 63, 974±979.
Kessler, W., Boller, B.C., NoÈsberger, J., 1990. Distinct in¯uence of root and shoot temperature on nitrogen ®xation by white clover. Annals of Botany 65, 341±346.
KoÈrner, Ch., Renhardt, U., 1987. Dry matter partitioning and root length/leaf area ratios in herbaceous perennial plants with diverse altitudinal distribution. Oecologia 74, 411±418.
Ledgard, S.F., 1989. Nutrition, moisture and rhizobial strain in¯u-ence isotopic fractionation during N2®xation in pasture legumes.
Soil Biology & Biochemistry 21, 65±68.
Cycling in Agricultural Ecosystems. CAB, Wallingford, pp. 351± 367.
Ledgard, S.F., Simpson, J.R., Freney, J.R., Bergersen, F.J., 1985a. Field evaluation of 15N techniques for estimating nitrogen ®x-ation in legume-grass associations. Australian Journal of Agricultural Research 36, 247±258.
Ledgard, S.F., Simpson, J.R., Freney, J.R., Bergersen, F.J., 1985b. Eects of reference plant on estimation of nitrogen ®xation by subterraneaum clover using 15N methods. Australian Journal of Agricultural Research 36, 663±676.
LuÈscher, A., Connolly, J., Jacquard, P., 1992. Neighbor speci®city betweenLolium perenneandTrifolium repensfrom a natural pas-ture. Oecologia 91, 404±409.
LuÈscher, A., Jacquard, P., 1991. Coevolution between interspeci®c plant competitors? Trends in Ecology and Evolution 6, 355±358. McAulie, C., Chamblee, D.S., Uribe-Arango, H., Woodhouse,
W.W., 1958. In¯uence of inorganic nitrogen on nitrogen ®xation by legumes as revealed by15N. Agronomy Journal 50, 334±337.
Nadelhoer, K., Shaver, G., Fry, B., Giblin, A., Johnson, L., McKane, R., 1996.15N natural abundance and N use by tundra plants. Oecologia 107, 386±394.
Nesheim, L., Boller, B., 1991. Nitrogen ®xation by white clover when competing with grasses at moderately low temperatures. Plant and Soil 122, 47±56.
Nesheim, L., Oyen, J., 1994. Nitrogen-®xation by red clover (Trifolium pratenseL.) grown in mixtures with timothy (Phleum pratense L.) at dierent levels of nitrogen. Acta Agriculturae Scandinavica, Section B-soil and Plant Science 44, 28±34. Peoples, M.B., Gault, R.R., Scammell, G.J., Dear, B.S., Virgona, J.,
Sandral, G.A., Paul, J., Wolfe, E.C., Angus, J.F., 1998. the eect of pasture management on the contribution of ®xed N to the N-economy of ley-framing system. Australian Journal of Agricultural Research 49, 459±474.
Rheder, H., Schafer, A., 1978. Nutrient turnover studies in alpine ecosystems. Part IV: Communities of the central Alps and com-parative survey. Oecologia 34, 309±327.
Sanford, P., Pate, J.S., Unkovich, M.J., 1994. A survey of pro-portional dependence of subterraneum clover and other pasture legumes on N2®xation in south-west Australia utilizing
15
N natu-ral abundance. Austnatu-ralian Journal of Agricultunatu-ral Research 45, 165±181.
Schinner, F., 1982. Soil microbial activities and litter decomposition related to altitude. Plant and Soil 65, 87±94.
Schulman, H.M., Lewis, M.C., Tipping, E.M., Bordeleau, L.M.,
1988. Nitrogen ®xation by three species of Leguminosae in the Canadian high arctic tundra. Plant, Cell and Environment 11, 721±728.
Seresinhe, T., Hartwig, U.A., Kessler, W., NoÈsberger, J., 1994. Symbiotic nitrogen ®xation of white clover in a mixed sward is not limited by height of repeated cutting. Journal of Agronomy and Crop Science 172, 279±288.
Shearer, G., Kohl, D.H., 1986. N2 ®xation in ®eld settings:
esti-mations based on natural abundance. Australian Journal of Plant Physiology 13, 699±744.
Sparrow, S.D., Cochran, V.L., Sparrow, E.B., 1995. Dinitrogen ®x-ation by seven legume crops in Alaska. Agronomy Journal 87, 34±41.
Sprent, J.I., 1985. Nitrogen ®xation in arid environments. In: Wickens, G.E., Goodin, J.R., Field, D.V. (Eds.), Plants for Arid Lands. George Allen and Unwin, London, pp. 215±229.
Svenning, M.M., Junttila, O., Solheim, B., 1991. Symbiotic growth of indigenous white clover (Trifolium repens) with local Rhizobium-leguminosarum biovar trifolii. Physiologia Plantarum 83, 381±389.
Thompson, J.D., Turkington, R., 1990. The in¯uence of Rhizobium leguminosarum biovar trifolii on the growth and neighbor re-lationships of Trifolium repens and three grasses. Canadian Journal of Botany 68, 296±303.
Turkington, R., Harper, J.L., 1979. The growth, distribution and neighbor relationships ofTrifolium repensin a permanent pasture. Part IV: Fine scale biotic dierentiation. Journal of Ecology 67, 245±254.
Unkovichz, M.J., Pate, J.S., Sanford, P., Armstrong, E.L., 1994. Potential precision of thed15N natural abundance method in ®eld
estimates of nitrogen ®xation by crop and pasture legumes in south-west Australia. Australian Journal of Agriculture Research 45, 119±132.
Vitousek, P.M., Shearer, G., Kohl, D.H., 1989. Foliar 15N natural
abundance in Hawaiian rainforest: patterns and possible mechan-isms. Oecologia 78, 383±388.
Wojciehowski, M.F., Heimbrook, M.E., 1984. Dinitrogen ®xation in alpine tundra, Niwot Ridge, Front Range, Colorado, U.S.A. Arctic and Alpine Research 16, 1±10.
Zanetti, S., Hartwig, U.A., LuÈscher, A., Hebeisen, T., Frehner, M., Fischer, B.U., Hendrey, G.R., Blum, H., NoÈsberger, J., 1996. Stimulation of symbiotic N2®xation inTrifolium repensL. under
elevated atmospheric pCO2 in a grassland ecosystem. Plant
