leaf natural 15n abundance and total n concentration as...

O Springer-Verlag 1999 Oecologia (1999) 120:171-182

J.C. Roggy M.F. Prévost F. Gourbiere H. Casabianca - J. Garbaye - A.M. Domenach

leaf natural 15N abundance and total N concentration as potential indicators of plant N nutrition in legumes and pioneer species in a rain forest of French Guiana

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Received 20 December 1997 /Accepted 19 November 1998

Abstract The suitability of the natural I5N abundance and of total N concentration of leaves as indicators of the type of plant N nutrition in a rain forest of French Guiana were tested. Leaf samples from primary legume species, non-legumes (pioneer species) and from the non-N2-fixing species Dicorynia guianensis were analyzed. Both 6I5N and total leaf N varied widely (-1 < 6I5N ao) < 7 and 1 < leaf N(%) < 3.2) suggesting possible distinctions between diazotrophic and non-fixing plants. The 6I5N also revealed two statistically distinct oups of non-N2- fixing species (6I5N = 5.14 f 0.3 vs 6 N - 1.65 f 0.17) related to the different ecological behaviors of these species in the successional processes. We conclude that the 6I5N signature of plant leaves combined with their total N concentration may be relevant indica" for identifying functional groups within the community of non-N2-fixing species, as well as for detecting diazotrophy. Despite the variability in the 615N of the non-N2-fixing species, N2-fìxing groups can still be identified, provided that plants are simultaneously classified taxonomically, by

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J.C. Roggy (@) Silvolab Guyane, Station de recherches forestières INRA BP 709, F-97387 Kourou Cedex. Guvane Francaise

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e-mail: [email protected] M.F. Prévost Sivolab Guyane, Laboratoire d'écologie végétale, ORSTOM, BP165, F-97323 Cayenne Cedex, Guyane Française F. Gourbiere A.M. Domenach Laboratoire d'kologie microbienne des sols, CNRS UMR, 5557 Université Claude Bernard Lyonl, 43 boulevard du 11 Novembre, F-69622 Villeurbanne Cedex, France H. Casabianca Laboratoire Central d'Analyses CNRS, Echangeur de Solaize, BP 22, F-69390 Vemaison, France J. Garbaye Laboratoire de Microbiologie forestihe INRA, Centre de Nancy, F-54280 Champenow, France \\ - - -

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their leaf 6I5N and total N concentration and by the presence or absence of nodules. The variability in the 6I5N of the non-fixing species is discussed.

Key words Tropical rain forest * 615N * Symbiotic N2 fixation - Tree legumes - Pioneer species

Introduction

Wet tropics are c peratures and abundant moisture. These factors have important effects on the rates of ecosystem processes such as primary production, nutrient cycling, decom- position, and other microbial processes (Jordän 1983; Vitousek 1984). High temperature and high humidity also result in a high potential for weathering parent rocks and leaching of nutrients. Hence, the availability of nutrients in tropical soils is very limited other ecosystems (Jordan 1985).

The relationships between nutrients forests have recently been considered &th particular

efficiency (Vitousek and Sandford 1986; Medina?"&d Cuevas 1989, 1994), especially as rain forests are now known to have more efficient nutrient cycling'systems than temperate forests. This concept supports the possibility of low nutrient losses from the system as well.as nutrient limitation to primary production (Vitousek 1984). Even though there is evidence that P is the lim-

reports also indicate substantial losses of N in highly leached Ultisols and Oxisols (Pleysier and Juo 1981; Arora and Juo 1982). Thus we still do not know enough about the nutrient relations and the nutritional constraints in tropical forest ecosystems, and more information is needed, particularly on the possible contribution of N2-fixing trees to the nitrogen cycle.

It has long been acknowledged that the natural isotopic composition of soil N is often different from that of atmospheric N2 (Hoëring and Ford 1960), the natural

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attention being paid to nutrient cycling and nutrieGuse 1

iting element in most rain forests (Vitousek 1984), many I

Fonds Documentaire ORSTOM Cote: &sr19660 . + fx:,?d

172

I5N abundance of which is stable (Mariotti 1983) and commonly used as a standard for 6I5N measurements (6I5N = O%,). Soil N generally has,positive values of 6I5N, but there can be exceptions, depending on the ecosystem type (Broadbent et al. 1980; Shearer and Kohl 1986; Vitousek et al. 1989). Soil 15N enrichment is generally attributed to a very slight discrimination against I5N during biological N2 fixation combined with a generally larger discrimination against I5N in N2 production during denitrification; the enzymatic fractionation factor ß ranging from l to 1.002 for N2 fixation (Minagawa and Wada 1986; Högberg 1997) and from 1 to 1.033 for denitrification (Mariotti et al. 1982; Yoshida et al. 1989; Högberg 1997): This difference makes it possible to distinguish the foliar natural I5N abundance of non-Nz-fixing plants subsisting on the soil N from that of potentially N2-fixing plants in the same location, which take N from the atmosphere and from the soil (Amarger et al. 1977; Bardin et al, 1977; Delwiche et al. 1979; Shearer and Kohl 1986; Domenach et al. 1989).

But plants lacking diazotrophic symbionts may still have low 6I5N values Domenach et al. 1989; Binkley et al. 1992). The foliar 6 5N in non-fixing plants has been shown to vary in many natural ecosystems (Hansen and Pate 1987; Pate et al. 1993), and the explanations for these variations have been synthesized by Handley and Raven (1 992). Ectomycorrhizal plants have been suggested to use both organic and inorganic soil N forms with different 6I5N signatures (Högberg 1990; Yone- yama et al. 1991; Högberg and Alexander 1995). Han- dley et al. (1993) suggested that the endomycorrhizal fungi could fractionate soil N. This variability of foliar 6I5N among non-fixing plants ofteqmakes it difficult to distinguish them from Nz-fixing ones (e.g., see Hansen and Pate 1987; Pate et al. 1993). However, Delwiche et al. (1979) and Virginia and Delwiche (1982) have shown that the isotopic approach can be combined with measurement of the total N concentration in leaves to give a good indicator for diazotrophy and a means of screening for N2-fixing plants in natural ecosystems.

The functional aspects of biodiversity were recently investigated by measuring ”N and I3C isotopes in 21 representative, ecologically important rain forest tree species in French Guiana (Guehl et al. 1998). The results demonstrated the possible occurrence of different trophic categories of patterns of Nnutrition among species, giving an interesting starting point for investigations into functional groups in the tree species community.

This study therefore. investigated whether the isotopic approach, combined with N concentration analysis, could be used to screen for actively performing N2-fixing species in a French Guiana rainforest. We also wanted to see if the 6I5N signature of tree foliage, which is related to the N source used by the plant, could be a potential indicator for functional groups in the species community. On the basis of the results of Guehl et al. (1998); we investigated legumes, a potentially nodulated family (Sprent 1995) and pioneer species (Prévost 1983). The latter are at the beginning of the regeneration be-

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havior continuum of silvigenesis (“gap species” of Bro- kaw 1985), while legumes are mostly at the end of it. (“primary species” of Brokaw 1985). The specific eco- physiological behavior of the pioneer species sets them apart from the legumes and all other primary species (Whitmore 1975).

Materials and methods

Sampling site

The studied area was the Piste de St. Elie in the ECEREX research zone (Sarrailh 1984) in French Guiana (5”18’N, 53’30’ W). The climate is equatorial with an annual rainfall of 3250 mm (Sabatier 1983) and a monthly temperature that varies slightly around 26°C (Boyé et al. 1979). The soils are Oxisols (Van Wambeke 1975) with a ferralitic cover developed over the weathered mantle of a Precambrian metamorphic rock (Bonidoro shale: Lévêque 1967; Boulet 1990). We used the botanical data of Sabatier et al. (1997), who made an inventory and measured all woody plants with a diameter at breast height (dbh) of 10 cm or more in a plot of 10 ha. A total of 6156 trees (493 taxa) were positioned and identiiied according to the terminology of Boggan et al. (1997) which refers to the taxonomy of Cronquist (1981). There are 200 species per hectare in the stand and “rare” species [species with few individuals) are predominant. The spatial variability of the floristic composition is great from one hectare to another. Thus, rainforest plant communities are described as “mosaics of treefall-created patches” (Richards 1952; Huston 1994). More than 50% of the trees in the stand are members of three families: the Lecythidaceae (26.2%), the Leguminoseae (sensu lato) with the Caesalpiniaceae being the most represented (18.5%), and the Chrysobalanaceae (7.2%). However, families with the largest number of individuals do not necessarily have the largest species richness (Sabatier and Prévost 1990). For the legume trees, only a few species with a high density of individuals constitute the “floristic background” of the forest.

Sampling strategy

Sampling was focused in the stand on two groups of tree species: 57 legume tree species c‘primary species”, 169 individuals) and 20 pioneer species (“gap species”, 55 individuals) were sampled. The primary species other than legumes were not sampled. This sampling was non-random with exclusive classes and the sampling strategy followed that of Domenach and Corman (1984) and Shearer and Kohl (1986). Leaves were collected from trees neigh- boring the non-N2-fixing reference species Dicoryniu guimensis (Caesalpiniaceae, 45 individuals). This species was taken as the non-fixing reference that is essentially ubiquitous and abundant in the forest cover (Guehl et al. 1998). Leaf samples were shot down during the rainy season from February to June 1995. Since 6l5N may depend on leaf age (Domenach et al. 1989), only the young fully expanded leaves from the upper canopy were collected.

Sample analysis

The leaf laminae (i.e., without petioles or rachis) were oven-dried at 60°C for 48 h, milled to a fine powder and packed in air-tight containers. Nitrogen isotope ratios (15N/14N) and total nitrogen concentrations were measured as described by Casabianca (1993), using an elementar analyser (SCA, CNRS, Solaize, France) cou- pled with a mass spectrometer (Finnigan Mat, DS, Bremen, Ger- many). The precision of measurement was 0.2 s%, (standard error, n = 1000). Results are reported as per cent N [leaf N (%)I and aI5N a,) where:

173

a15N(X,) = 1000(atom %I5N sample-atom % ”N standard)

where the standard is atmospheric N2 (atom%”N = 0.3663) (Mariotti 1983).

/atom%”N standard

Statistical analysis

The densities of most of the species studied in the stand were low. At least three replicates were taken for each species whenever possible; then the non-parametric Mann-Whitney U-test was used to test for significant differences from the reference species for both variables (P < 0.05). Otherwise, trends were determined according to whether individuals were within or outside the confidence interval of individuals in the reference species (P<0.02 for 6”N and P<O.O5 for leaf N; Statview 4.02 sofware).

Root nodule and N2 fixation

The N2-fixing status of the species was confìmed by looking for root nodules. When nodules were found, roots which carried them were traced up to the base of the parent tree and its identity recorded. Acetylene reduction assays were then used as an indicator of N:! fixation: excised roots with attached nodules were immediately placed in 100-ml flasks with rubber membrane stoppers. Part of the air of the flask was withdrawn and replaced by the same volume of C2H2 to give 11% CzHz (VIV) in the gas phase of the incubation vessels. Control flasks with C2H2 but without samples were included, as were control flasks with samples but without C2H2. The flasks were then incubated at ambient temperature for 30 min and 5-ml samples of the tube atmosphere were then taken with a gas-tight syringe, placed in a 15-ml Vacutainer and transported to the laboratory. There, 0.5-ml gas samples were analyzed for C2H4 on a Girdel 3000 gas chromatograph with a flame ionization detector calibrated with an ethylene standard (detection threshold: lo-” mol CzH4). The chromatograph characteristics are a 2-m stainless steel column containing activated alumina, at a column temperature of 160°C. Nitrogen was the carrier gas with a flow rate gf 35 ml s-’.

Results

Sample composition

The stand was rich in both legume and pioneer species but with few individuals of each species (Fig. la). The distribution of individuals within the sampled species reflects this feature (Fig. lb). Triplicate sampling was rare, except for the Caesalpiniaceae: Dicorynia guianensis (reference species, n = 45), Eperua falcata (n = 18), Bocoa prouacensìs (n = 15), Crudia bracteata (n = 13) and Vouacapoua americana (n = 11). These species made up most of the legume floristic background of the stand. The pioneer species all had the same features, with “long-lived” pioneers (Carapa procera, n = 9; Xylopìa nitida, n = 8; and Goupìa glabra, n = 7 ) being better represented than “short-lived” ones.

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General distribution of leaf 6I5N and N concentration

The species sampled gave a bimodal distribution for mean 6I5N (Fig. 2a). Ei hty-two percent of the species ranged from -1 to 3 N and 18% from 4 to 7 6I5N, suggesting great variability in 615N values between

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A 36 34 32

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I 2 3 4 5 6 7 8 IO II 14 I5 17 ?I 25 47 69 71 78 80 172383 Number of trees

34 J- L 32 - 30 - 28 - 26 -

M 24-

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I- ” . . . . . . I 2 3 4 6 7 8 9 II 13 15 , I 8 35

., 1 Number of trees

Fig. 1 Frequency distribution of individuals in legume and pioneer tree species in the stand (a) and in the sample (b) in a rain forest of French Guiana (Piste de St. fie). Asterisked is the non-N2-fïxing reference species Dicorynia guimensis

plants. The individuals in the reference species’ gave an unimodal 615N distribution, with values of 4-6 615N (Fig. 2c). This range of 615N values was superimposed on that of one group of sampled species, suggesting identical 615N signatures of plant foliage.

The distributionS.of the mean leaf N concentration in sampled species and the leaf N concentration in the reference individuals are given in Fig. 2b,d. The observed variability between species was close to that observed for individuals in the reference species. How- ever, 22 species were below the threshold of 2%, which appeared to be the mean for the reference species.

Statistical analysis

Coefficients of linear correlation between 6I5N and leaf N within species were calculated for species with at least three replicates (Table 1). No significant correlations were found between these variables for either legumes or pioneer species, except for two species (Crudia bracteata and Laetia procera). These results indicated that the variables were independent and could be analyzed separately.

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a Fig. 2 Frequency distribution 9 of leaf 6I5N and total leaf N concentration in the tree species (a, b mean values) and in the individuals of the non-N2-fixing reference tree species D. guiunensis (c, d) sampled in a rain forest of French Guiana (Piste de St. Elie) I 1 ru

- 1 0 1 2 9 1 5 a15N I953

2 2,s 3 5,5 Leaf N I%)

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Leaf N I%)

I The non-parametric Mann-Whitney U-tests were

conducted to test for significant differences (P < 0.05) from the reference species. The 6I5N mean rank given by the test formostlegumes (86%) andpioneer species (80%) was lower than the reference (Table 2) and these differences were significant (P < 0.01). Two species showed no significant differences (Inga sp4 and Loreya mespiloides). One showed a higher 615N mean rank (Zygia racemosa). The statistical results confirmed that species were dis- tributed bimodally as observed in Fig. 2a.

Fourteen percent of the legume species had statistically lower values for leaf N than the reference (Eperua spp., P<O.OI) and 58% had statistically higher values (P < 0.05).

Sixty percent of the pioneer species had statistically lower values for leaf N than the reference (P< 0.01) and no species had a statistically higher value. These results showed that the legume species (including the reference species) had higher leaf N concentrations than the pioneer species.

Classification of species into the 6I5N x leaf N plan

There were two statistically separate groups of plant species on the basis of 6V.N. The group with low 615N

values contained species with 6I5N of -lX0 to 3%, (Fig. 2a). These values are close to those of fixed NZ, the 6I5N of which was -2%, to O%, (Yoneyama 1987). These species are thus likely to be dependent on fìxation for their N sup ly. Non-N2-fìxing plants may nevertheless have low 6 N values, but surveys indicated that the occurrence of high leaf N concentration and low 6I5N in plants was a good indicator of N2 fixation (Virginia and Delwiche 1982). Guehl et al. (1998) found a mean leaf N concentration of 2% for the non-Nz-fìxing reference species D. guìanensis.

The mean leaf N concentration of the reference species was quite high (2.17zk0.05) and this value was used as threshold to divide species with low 6I5N values into statistically separate clusters of plants (Fig. 3a). The “supposed non-Nz fixers” group of species (mean 6I5N = 5.14&0.30 and mean leaf N = 2.61 ztO.09) had 6I5N values statistically similar to the reference species (6I5N = 4.93zt0.10). The “supposed NZ fixers” group (mean 6I5N = 2.O9=kO0.18 and mean leaf N = 2.48f 0.05) included species with 6I5N values significantly lower than the reference species and no distinct or significantly higher leaf N concentrations. The “uncertain other plants” group (mean 6I5N = 1.653~0.17 and mean leaf N = 1.69 f 0.04) contained species with 615N and leaf N concentrations significantly lower than the

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175

reference. There was no statistical difference between this group and the “supposed Nz fixers” for 6I5N but there was a significant difference in leaf M concentration. This group is mainly made up of pioneer species, except for one leguminous genus (Eperua).

Table 1 Coefficients of linear correlation (r) for regression of leaf S1’N with total leaf N concentration for legumes and pioneer species collected in a rain forest, Piste de St. Elie, French Guiana. Correlation coefficients and probabilities were calculated for species with at least three replicates (n: number of samples)

Plant type n r value P value

Legumes Alexa wachenheimii Bocoa prouacensis Crudia bracteata Dicorynia guianensis Eperua falcata Eperua grandifora Inga fmckoniana Inga spp. Inga sp4 Inga stipularis Paramachaerium

ormosioides Sclerolobium melinonii Swartzia polyphyIIa Vouacapoua americana Zygia racemosa

6 15 13 45 18 8 4 3 8 4 4

4 6 11 7

0.125 0.230

-0.612 0.107 0.209

0.480 0.714

0.868

-0.142

-0.043

-0.768

-0.848 0.387 0.246

-0.256

0.814 0.408 0.026 0.4846 0.404 0.738 0.520 0.494 0.920 0.132 0.23 1

0.152 0.448 0.466 0.579

The standard error and the standard deviation of means are reported for each group, and for both variables (Fig. 3a). The first indicates a significant increase in the leaf N concentration from the “uncertain other plants” species to the other groups; the latter that there is a continuum of leaf N concentration between these groups. This agrees with the species distribution (Fi!. 2b). We also note that the reference 615N is high (6l N = 4.93&0.1) compared to that of fixed Nz (2 < 615N < O) and that the total variability [measurement error (0.2%,) + variability (0.1%,)] is low.

The distribution of some legumes and pioneer species in the different groups is shown in Figs. 3b,c

Species with no replicates

The confidence intervals were calculated for the reference species (6I5N P<O.01 and leaf N: P<0.05). Le- gume and pioneer species with fewer than three replicates were clustered according to whether they were below or beneath the lower value of the confidence interval for each variable of the reference species. The sampled species divided into three clusters (Figs. 4a,b) and legumes had no species below the lower value for the leaf N concentration.

Pioneer species Carapa procera 9 0.236 0.540 Goupia glabra 7 0.538 0.212 Taxonomic composition of the clusters Laetia procera 7 0.835 0.020 Loreya mespiloides 4 -0.387 0.612 Table 3 lists the 77 species sampled together with ‘their

6I5N and leaf N concentration. Both variables varied Xylopia nitida 8 0.057 0.894

Table 2 Summary of Mann-Whitney U-test kesults for the legume and pioneer tree species collected in a rain forest, Piste de St. Elie, French Guiana. For species with at least three replicates, the non-

parametric Mann-Whitney U-test was used to test significant differences with the non-N2-iìxing reference species D. guiunensis for both S1’N and percent leaf N variables

6l’N variable

Mean rank Reference P Mean rank Reference P

Percent leaf N variable

mean rank mean rank

Legumes Alexa wachenheimii Bocoa prouacensk Crudia bracteata Eperua falcata Eperua grandifora Inga fanchoniana Inga spp. Inga sp4 Inga stipularis Paramachaerium ormosioides Sclerolobium melinonii Swartzia polyphylla Vouacapoua americana Zygia racemosa

Carapa procera Goupia glabra Laetia procera Loreya, mespiloides Xylopia nitida

Pioneer species

4.3 8.4

14.5 9.6 4.6 2.9 2.3

30.4 12.1 2.5 2.7 3.5 6.5

41.43

5 4.5

10.9 12 4.5

28.9 37.9 33.8 41 31 27 26 28.4 28.1 27 27 29 33.9 24.2

32 30 30 26.1 31

*** *** *** *** *** *** *** NS * *** *** *** *** ***

*** *** *** NS ***

.42.3 33.4 48.7 17.9 4.5

46.4 30.2 39 24.5 44.4 46.5 37.8 37 46.8

7.4 17.7 12.7 34.6 11

23.8 29.5 23.9 37.6 31 23.1 24.1 24.9 25 23.3 23.1 24.4 26.4 23.3

31.5 27.8 28.7 24.1 29.9

*** NS *** *** *** *** NS

NS ** *** *** * NS ***

*** NS

NS *** ***

* P < 0.05; ** P < 0.02; ***P, s 0.01; NSnon-sig~ikant

176

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0 ...............................................”...*? ........*..........I................

Fig. 3 Relationship between leaf ”N abundance (SI5N %J and total leaf N concentration (% leaf N) in rain forest tree species (a, vertical and horizontal lines indicate SE (short lines) and SD (long lines) of means), and in the individuals of some of these species @, legumes; c, pioneers; each data point represents a separate individual tree), compared to the non-N2-fixing reference species Dicorynia guianensis (crosses, n = 45) at the Piste de St Elie, French Guiana. Open circle supposed non-NZ-fixers: a (n= 19), b (Zygia racemosa), c (Loreya mespiloides); closed circle supposed N2-fixers: a (n = 70), b (Swartzia spp.), c (Goupia glabra); square uncertain other plants: a (n=57), b ( E p e m grandijlora), c (Carapa procera). .

between species. One group, the “supposed N2 fixers” (n = ,124 individuals) could fix N. Legumes made up 85% of this cluster with 67% of the sampled Ca- esalpiniaceae, 67% of the Mimosaceae and 89% of the

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1,s 2 2.5 3 33 Leaf N (95)

Fig. 4 Relationship between leaf 15N abundance (S’% yd and total leaf N concentration (leafN %) in rain forest tree species compared to the non-N2-fixing reference species Dicorynia guianensis. (cross, n=45 4 at the Piste de St Elie, French Guiana Vertical lines indicate the 6’ N confidence interval for individuals at 98% level, horizontal lines indicate the leaf N confidence interval for individuals at 95% level, broken lines indicate lower threshold values for both variables. Closed circles legumes (a) and pioneers @), each data point represents a separate species with 1 or 2 individuals

Papilionaceae, as well as 35% of the pioneer species (Bignoniaceae, Cecropiaceae, Flacourtiaceae, Me- lastomataceae, Rubiaceae, and Celastraceae). But the pioneer species remained statistically different from the legumes in terms of leaf N concentration (Table 4).

The second group was the non-N2-Wng group (“supposed non-N2 hers”, n = 41 individuals). The legumes made up 78% of this group with 11% of the sampled Papilionaceae, 22% of the Caesalpiniaceae, and 33% of the Mimosaceae plus 20% of the pioneer species. All the pioneer species were Melastomataceae and there was no statistical difference between the species in terms of leaf N concentration (Table 4).

The third group of species (“uncertain other plants”, n = 58 individuals) comprised mostly pioneer species (82% of the species). This group contained 45% of the pioneer species sampled (Annonaceae, Clusiaceae, Fla- courtiaceae, Meliaceae, Rubiaceae, Rutaceae, Tiliaceae, and Ulmaceae) and two Caesalpiniaceae: E. falcata and E. grandijlora (Detarieae subfamily). There were no

177

Table 3 N2-fixing ability of some legumes and pioneer tree species collected in a rain forest, Piste de St. Elie, French Guiana: evidence from leaf S15N fio) and total leaf N concentration (leaf N %). Values are means f SE (n number of samples). For species with at least three replicates, the non-parametric Mann-Whitney U-test was used to test significant differences with the reference species, for both variables (P < 0.05); otherwise trends were determined ac-

cording to whether individuals were within or outside the confidence interval of individuals in the reference species at 98% for the 6”N and 95% for the leaf N. 2 Under nodulation: ne not examined, - plants without nodules, + plants with nodules (for positive reports, roots were always traced up to the base of the tree). 3: ARA acetylene reduction assay used an indicator of efficient N2 fixation: + positive reports, neo not carried out

Plant typea n 615N (%o1 Leaf N (%) Nodulation ARA3 occurs2

Reference species Caesalpiniaceae

Supposed Nz fixers Legumes Caesalpiniaceae

Dicorynia guianensis

Bocoa prouacensis Crudia bracteata Peltogyne venosa Sclerolobium spp. .

S. albiflorum S. melinonii

Senna quiquangulata (sarmentos shrub)

Swartzia spp. S. arborescens S. guianensis S. panacoco S. polyphylla

Tachigalia paniculata Vouacapoua americana

Abarema spp. A. jupunba A . mataybifolia

Balizia pedicellaris Inga spp.

I. alba I. cayennensis I. cinnamomea I. fanchoniana I. gracilifolia I. leiocalycina I. paraensis I. pezizifera I. rubiginosa I. sarmentosa I. semialata I. splendens

I. strpularis I. thibaudiana I. tubaeformis

Mimosaceae

I. sp:

Stryphnodendron polystachyum

Alexa wachenheimii Andira coriacea Hymenolobium cf. flavum Ormosia spp.

Papilionaceae

O. melanocarpa Ormosia sp.

Paramachaerium ormosioides Poecilanthe hostmanii Vatairea erythrocarpa

Pioneer species Bignoniaceae

Cecropiaceae Jacaranda copaia

Cecropia. spp. C. obtusa C. sciadophylla

45

15 13 1 5 1 4 1

11 1 2 2 6 1

11

4 2 2 1

26 1 1 1 ) 4 1 1 1 2 1 1 1 2 3 4 1 1 1

6 2 1 4 2 2 4 2 1

2

2 1 1

4.93 2c 0.10

1.92 f 0.34 3.83 f 0.27 1 .O8 1.32 f 0.73 3.00 0.90 f 0.77 0.55

0.64 f 0.25 1.56 0.58 f 0.85 1.19 f 0.93 0.32 f 0.23 3.20 2.64 f 0.20

2.07 f 0.24 1.78 f 0.41 2.35 f 0.15 0.87 1.60 f 0.26 3.29 0.82 2.82 1.19 f 0.88 2.14 0.24 1.71 2.36 f 0.49 0.76 1.6 1 .O7 1.36 f 0.74 1.84 f 0.7 2.11 f 1.2 1 .O3 0.04 1.02

2.66 f 0.35 0.62 f 0.40

1.08 f 0.33 1.65 f 0.15 0.51 f 0.08 0.73 2k 0.11 2.50 f 0.22 2.88

-0.56

1.47 f 0.16

1.74 f 0.13 1.87 1.61

2.17 & 0.05

2.21 f 0.06 2.82 f 0.08 1.52 3.01 f 0.10 2.80 3.06 f 0.11 1.55

2.50 f 0.08 2.71 2.55 f 0.19 2.37 f 0.06 2.49 f 0.13 1.90 2.36 f 0.08

2.21 f 0.17 2.11 i 0.08 2.30 f 0.40 1.72 2.47 f 0.08 2.58 2.12 2.65 3.04 f 0.12 2.36 1.72 2.44 3.01 f 0.03 2.68 2.59 1.85 2.36 f 0.07 2.31 f 0.19 2.09 f 0.12 3.17 1.78 2.2

2.72 f 0.16 1.97 f 0.03 2.02 2.00 f 0.07 1.90 f 0.10 2.10 f 0.00 2.74 rt 0.05 2.15 f 0.08 1.80

2.10 f 0.14

2.49 f 0.21 2.71 2.28

-

- + ne

ne + +

+ + + + + -

+ + + ne + + + + + ne + + ne + + + + + ne + + + + + + + + ne

-

- -

neo

+ neo

nco nco + + +

+ + +

neo nco nco + + + + 4

+ + 4-

+ + + + + + + +

178

Table 3 (contd.)

Plant typea n . 6’” Um) Leaf N (%) Nodulation ARA3 occurs’

Celastraceae Goupia glabra

Flacourtiaceae Banara guianensis

Melastomataceae Loreya arborescens

Rubiaceae Palicourea guianensis

Supposed non-N2 h e r s Legumes Caesalpiniaceae

Cassia spruceana Crudia aromatica Dialium guianensis Sclerolobium guianensis

Mimosaceae Inga spp.

I. acrocephala I. edulis I. huberi I. lomatophylla I. sp4 I. jenmanii

Parkia spp. Pseudopiptadenia suaveolens Zygia racemosa

Dipteryx odorata Papilionaceae

Pioneer species Melastomataceae

Loreya mespiloides Miconia spp.

M. fragilis M. tschudyoides

Be[lucia grosssularioides Uncertain other plants Legumes Caesalpiniaceae

Eperua spp. E. falcata E. grandjlora

Pioneer species Annonaceae

Clusiaceae Xylopia nitida

Vismia spp. V. latifolia V. sessilifolia

Flacourtiaceae

Meliaceae

Rubiaceae

Rutaceae

Tiliaceae

U Im a c e a e

Laetia procera

Carapa procera

Isertia spiciformis

Zanthoxylum spl

Apeiba glabra

Trema guianensis

7

1

1

2

1 2 1 1

16 2 1 1 2 8 2 2 2 7

1

4 3 1 2 1 t

26 18 8

8

4 2 2

7

9

1

1

1

1.

1.40 f 0.7

0.81

2.44

2.12 f 0.28

4.90 5.21 f 0.49 4.36 4.74

4.93 f 0.35 3.84 f 1.15 4.91 4.43 5.40 f 1.20 5.33 f 0.54 4.19 f 1.19 6.63 f 2.41 4.62 i= 0.32 5.67 f 0.15

5.04

3.87 f 0.56 4.41 =k 0.56 3.82 4.91 f 0.45 6.62

1.48 4 0.17 1.52 f 0.19 1.39 f 0.40

0.96 =t 0.25

0.96 f 0.56 0.01 f 0.07 1.90 k 0.34

2.50 f 0.47

1.34 f 0.29

0.18

2.90

2.95

2.99

1.95 3: 0.13

2.24

2

1.99 f 0.32

1.70 2.76 f 0.16 2.51 2.56

2.46 f 0.08 2.59 -I 0.30 3.23 2.27 2.10 f 0.10 2.43 f 0.05 2.5 f 0.4 2.55 f 0.26 1.81 f 0.01 2.97 f 0.14

1.9

2.35 4 0.11 1.73 rt 0.24 2.21 1.49 i 0.06 2.41

1.69 f 0.05 1.83 i 0.04 1.37 f 0.06

1.75 & 0.07

1.41 f 0.11 1.26 f 0.17 1.46 f 0.01

i.77 f 0.11

1.56 f 0.07

1 .O3

1.07

1.48

1.43

i- + +

a Classification according to the taxonomy of Cronquist

Y.

179

Table 4 Summary of isotopic patterns of 615N ao) and total leaf N concentration (leaf N %) in tree species collected in a rain forest, Piste de St. Elie, French Guiana. Values are meansf SE (n number

of individuals). Values with the same superscript are not statistically different (I‘< 0.01). (For the reference species: n = 45, 6”N = 4.93 f O.lO%,”, leaf N = 2.17f0.05’)

Leaf N (%) Plant type n 6% a3 Legumes Pioneers Legumes Pioneers Legumes Pioneers

Supposed Nz fixers 110 15 1.90 f 0.13b 1.58 f 0.35b 2.43 f 0.04’ 2.07 f O.Ogd Supposed non-N2 fixers 33 8 5.17 f 0.22’ 4.42 f 0.46‘ 2.51 f 0.07’ 2.12 f 0.14C3d Uncertain other species 26 32 1.48 f 0.17b 1.78 f 0.25b 1.69 f 0.05e 1.59 f 0.05e

significant differences between the legumes and the pioneer species for 6”N or leaf N concentration (Table 4).

Observed nodulation

The N2-fìxing status of the species indicated by isotope analysis was confirmed by looking for root nodules. Nodules occurred on most of the “supposed N2 fixers” species, except for the pioneer species @able 3). All Mi- mosaceae and Papilionaceae had nodules, while the Ca- esalpiniaceae, B. prouacensis and V. americana had none. A recent survey however found typical bacterial nodules on B. prouacensis and “unidentified black glossy nodules” on V. americana (Béreau and Garbaye 1994). V. pallidior was also found to be nodulated in the Amazon region of Brazil (Moreira et al. 1992). The acetylene reduction assays revealed efficient N2 fixation for all the species tested (Table 3). No nodules were found in the “supposed non- N2 fixers” group of species except for the Inga genus, despite the high 615N values. Finally, no nodules were found in the “uncertain other plants” group, confirming the putative non Na-fixing status of these species.

Discussion

Relevance of the 615N method for screening N2-fixing plants

As suggested by Virginia and Delwiche (1982), our results show that measurements of natural 15N abundance are relevant indicators of symbiotic diazotrophy in plants when combined with measurements of total leaf N. A significant correlation between the two variables is not necessary, as indicated by our results.

The presence or absence of nodules on the species has codinned the indications given by the 615N results in about 80% of cases for the N2-fixing species and in 90% of cases for the non-N2-fixing species. A few Inga species were found to be nodulated despite their high 615N values. However, the 6I5N signature in young leaves reflects the’form of the N supply during growth. Thus, we as- sume that assimilation rather than fixation contributed to the N suppl of these species at the time of sampling.

The high 6 N value of the reference species, compared to that for fixed N2, allowed the detection of trees with a small contribution oK4ixed N2 to their N supply.

75

The low variability in this value also indicated a low spatial heterogeneity of the 6”N for this soil N source. The supposed non-N2-fixing species had 615N values close to that of the reference species. This suggests that these species could used the same soil N source and, thus, that the 615N of D. guianensis is representative of that of the primary non-N2-fixing species. These results confirm that D. guianensis is a good reference species, as suggested by Guehl et al. (1998) in other stands.

The 615N varied greatly among non-N2-fixing plants (Table 4) suggesting that plant-available soil N cannot be a single source. The 615N data also indicate that non- -fixing plants are clearly organized into two statistically distinct groups. Such organization undoubtedly pro- vides much information about the functioning of the non-N2-fixing species. This also allows the 615N method to be successfully used to screen for N2-fixing species, in contrast to Handley et al. (1994) who found that statistically different gröups of non-fixing plants made it impossible to distinguish them from the fixing ones. They concluded that Shearer and Kohl’s (1986) 1’ inear two-source model could not be used for estimating N2 fixation in situ, in such cases. Our results are also consistent with those of Handley et al. (1994) on the irrel- evance of the linear two-source model when there are more than two soil N sources. However, it is possible to estimate N2 fixation in our system. The system is sli htly more complex and, despite the variability in the N of the non-N2-fixing species, N2-fixing groups could still be identified, provided that plants are simultaneously classified taxonomically; by the presence or absence of nodules, their leaf 615N, and total N concentration.

B

Possible causes of 6I5N variability in non-N2-fixing species

Several authors (Salati et al. 1982; Hansen and Pate 1987; Vitousek et al. 1989; Högberg 1990; Pate et al. 1993) have reported that 615N analysis failed to discriminate putative N2-fixing plants from non-Nz-fixing ones in some ecosystems. In most cases, the 615N varied widely among non-N2-fixing plants. Many surveys suggested that this could be due to the mycorrhizal status of species in non-fixing plant communities (Högberg 1990; Handley et al. 1993; Högberg and Alexander 1995). Ectomycorrhizal symbioses are rare in the rain forest of French Guiana and have been found on Polygonaceae

180

and Nyctaginaceae species (Béreau et al. 1997). Endo- mycorrhizal (VA) symbioses prevail in this type of forest, and were found on all tree species examined by Béreau and Garbaye (1994). Thus, it seems unlikely that the ecto- or endomycorrhizal status of the trees can explain the differences in S15N between non-fixing plants. A second explanation could be that N isotope fractionation occurs during uptake by plants (Yoneyama et al. 1991). Our results do not allow us to speculate on this, but this could be an interesting aspect of the functional diversity in the rain forest. A third possibility is that the N available to plants may come from different pools (NH;, NO,) or depths in the soil, with different S1 5N signatures resulting in wide variability among non- k i n g plants (Handley and Raven 1992; Nadelhoffer and Fry 1994). The differences in S15N among N compounds are due to the fractioning values of metabolic reactions. Processes such as nitrification’ discriminate against ”N, leading to ”N-depleted NO, compared to NH: (Domenach and Chalamet 1977; Mariotti et al. 1981; Shearer and Kohl 1986).

Rain forests on Oxisols have high rates of N mineralization and nitrification (Vitousek 1984; Vitousek and Denslow 1986; Vitousek and Sanford 1986; Vitousek and Matson 1988; Robertson 1989; Neill et al. 1997). Maggs (1991) and Medina and Cuevas (1994) also showed that soil N was mineralized in NHt and NO, at similar rates and to similar degrees. On the other hand, the nitrate in tropical soils can be immobilized in the lower horizons at amounts comparable to those found in cultivated soils (Wetselaar 1962; Kinjo and Pratt 1971; Cahn et al. 1992). This limits losses by leaching from the topsoil and provide a storage of rb available to deeply rooted nitrate-using plants. Several studies have shown that the use of NO, or NH: by tree species in rain forests depends on their ecological behavior (Stewart et al. 1988, 1992; Freeden et al. 1991). Bazzaz (1984) and Tumbull et al. (1996) showed the preferential use of NO; by the pioneer species and of NHZ by the primary species. The pioneer species are also known to be deeply- rooted (Whitmore 1975; Hartshorn 1978).

Most of the non-N2-fixing species with a low SI5N were found to be pioneers and examination of their root systems (including the genus Eperuu) confirmed their capacity to explore deep soil horizons (personal obser- vation). The Eperua species have already been men- tioned for their low leaf SI5N (Guehl et al. 1998) and are known as “little treefall gap-adapted species” (For et

are primary forest species. This is consistent with the NO,/NH: hypothesis discussed above and indicates how measurement of natural I5N abundance can iden-

. tify “functional plant groups.”

1989). Most of the non-N2-fixing species with high S* 5 N

.

Variations in leaf N concentrations

The highest total leaf concentrations were found in legumes, with no significant differences between fixing

and non-fixing plants within this family. This indicates that forest legumes can extract large amounts of soil N, and that N is not limiting, in contrast to other findings in similar forests (Vitousek 1984). This also suggests that N2 fixation by rain forest legumes should not be considered as a competitive advantage for N nutrition, but rather as one of several ways of ob- taining N.

The low total leaf N concentrations of most pioneer species may have several explanations. Damage by her- bivores is common in tropical rain forests (Hartshorn 1978). The young leaves of pioneer species are heavily grazed while the leaves of primary species are less so (Coley 1983; Newbery and De Foresta 1985). Plants often adapt to grazing by producing carbon-based compounds, and pioneers have a high leaf phenol concentration (Coley 1983; Newbery and De Foresta 1985). We reported the total leaf N concentration as percent of dry matter and variations in the leaf C/N ratio can explain the low N concentrations in the pioneers rather than a limiting N nutrition. The leaf N concentration increases from the pioneer species to the primary legumes. This continuum of leaf N concentrations seems to be superimposed on that of the regeneration behaviors.

In conclusion, the SI5N signature of plant foliage is a relevant indicator of functional groups in the community of non-N2-fixing plants, as well as a good tool for determining diazotrophy when combined with analysis of total leaf N. We also conclude that measuring the natural abundance of 15N can be successfully extended to larger tree screenings in tropical rain forests. However, we need a better understanding of the factors contributing to the observed variability before this method can be used to quantify biological N2 fixation and for developing general models of plant N nutrition.

Acknowledgements The present work was initiated during a PhD thesis with cofinancial support from INRA and ORSTOM within the SILVOLAB group (CIRAD, ENGREF, INRA, ONF, ORS- TOM) in French Guiana. The authors wish to thank the CIRAD- Forêt in Kourou for. their financial support, the staff of INRA in Kourou and of ORSTOM in Cayenne for initiating J.C.R. to forest research. Other financial support was provided by the SOFT research program of the French Ministry of the Environment.

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leaf natural 15n abundance and total n concentration as...

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