Functional Ecology 2007 21, 844–853
Leaf traits affect the above-ground productivity and quality of pasture grasses Blackwell Publishing Ltd
L. DA S. PONTES*, J.-F. SOUSSANA*†, F. LOUAULT*, D. ANDUEZA‡ and P. CARRÈRE* *INRA, UR874 Grassland Ecosystem Research, F-63100 Clermont-Ferrand, France, ‡INRA, UR1213 Unité de Recherche sur les Herbivores, F-63122 St-Genès-Champanelle, France
Summary 1. By comparing plant species under the same experimental field conditions, the direct effects of plant traits on ecosystem processes can be studied. We have analyzed the role of leaf traits (leaf lamina dry matter content, LDMC; leaf lamina N content, LNC and specific leaf lamina area, SLA) for the annual above-ground primary productivity (ANPP) and quality (pepsin-cellulase digestibility, crude protein content) for herbivores of 13 perennial C3 pasture grass species. 2. These relationships were investigated over 2 years with monocultures grown in a fully factorial block design crossing the plant species, the cutting frequency and the N supply factors. 3. The within species variation in leaf traits, ANPP, digestibility and protein content was less than between species variation. Species ranks for leaf traits were conserved among N supply and cutting frequency levels. Highly significant (P < 0·001) between species allometric relationships were found for LNC × SLA and SLA × LDMC, with common slopes but differences in intercept and shifts among factor levels. 4. The between species variation in ANPP was strongly (P < 0·001) and negatively correlated with the fresh-matter based leaf N content (i.e. LDMC × LNC) and was not affected by SLA, apparently because of a trade-off between SLA and leaf lamina fraction. Digestibility increased with SLA and declined with LDMC. Protein content increased with both fresh and dry-matter based LNC. 5. N supply increased LNC and SLA but reduced LDMC. Cutting frequency increased LDMC and reduced LNC. In response to cutting frequency, changes in digestibility and in fresh-matter based LNC were positively correlated. 6. We conclude that the between species variation in the annual production of digestible energy and of proteins by pasture grasses is controlled in an additive way by two leaf traits: LNC and LDMC. Key-words: grassland, leaf dry-matter content, leaf nitrogen content, primary productivity, specific leaf area Functional Ecology (2007) 21, 844–853 doi: 10.1111/j.1365-2435.2007.01316.x
Introduction Understanding the role of species for ecosystem functioning (e.g. net primary productivity, litter decomposition rate, herbivory) is one of the major objectives of studies in ecology (Chapin et al. 2000). Various attempts to generalize plant species effects on ecosystem processes have focused on single traits, called ‘functional effect traits’ (Díaz & Cabido 2001; Lavorel & Garnier 2002). Individual traits should, however, not be considered in isolation, because some pairs of traits are sufficiently © 2007 INRA. Journal compilation © 2007 British Ecological Society
†Author to whom correspondence should be addressed. E-mail:
[email protected]
closely coordinated (Wright et al. 2004) to be thought of as forming a single dimension of strategy variation compounded from several traits (Westoby & Wright 2006). By comparing plant species under the same experimental field conditions, the direct effects of plant traits on productivity can be studied (Craine et al. 2002). This experimental design allows studying the within species variation in trait values in response to stress and disturbance factors without interferences between species. There are, however, few published studies (e.g. Craine et al. 2002; Fargione & Tilman 2006) analyzing relationships between productivity and plant traits under experimental field conditions. Grazing, which is one of the most globally widespread land uses (Díaz et al. 2006), has a major importance 844
845 Leaf traits affect grass productivity and quality
for primary productivity and chemical composition of pasture species (e.g. Briske, Fuhlendorf & Smeins 2005). Several studies have shown that leaf traits of dominant species vary along gradients of disturbance by grazing and cutting (e.g. Díaz, Noy-Meir & Cabido 2001; Cingolani, Posse & Collantes 2005; Louault et al. 2005; Díaz et al. 2006). Grazing avoidance traits (such as high leaf dry matter content, LDMC) are usually associated with low palatability (Wardle et al. 1998; Cornelissen et al. 1999; Díaz et al. 2001). Grazing tolerance would be favoured by a high specific leaf area (SLA) which increases shoot regrowth ability (Westoby 1999) and by a high leaf N content (LNC) which increases leaf quality and selectivity by herbivores (Pérez-Harguindeguy et al. 2003; Cingolani et al. 2005). With 13 co-occurring perennial C3 pasture grass species grown in monocultures, we have investigated the role of three leaf traits (LDMC, LNC and SLA) for the above-ground net primary productivity (ANPP) and for the nutritive value (pepsin-cellulase digestibility and crude protein content) for herbivores. To assess species plasticity in response to nitrogen and disturbance, the grass species were compared in a fully factorial block design crossing the N supply and the cutting frequency factors.
Materials and methods Thirteen perennial C3 grasses that co-occur in seminatural mesic grasslands were studied: Alopecurus pratensis, Anthoxanthum odoratum, Arrhenatherum elatius, Dactylis glomerata, Elytrigia repens, Festuca arundinacea, F. rubra, Holcus lanatus, Lolium perenne, Phleum pratense, Poa pratensis, Poa trivialis and Trisetum flavescens. These species are all among the 20 most widely distributed Poaceae species in the French Massif Central (Antonetti et al. 2006). Seeds of these grass species were collected in their native habitat, within 20 km of the study site, in moderate to high fertility semi-natural mountain grasslands managed by grazing and cutting in the French Massif Central.
© 2007 INRA. Journal compilation © 2007 British Ecological Society, Functional Ecology, 21, 844–853
The experiment was established in an upland area of central France (Theix, 45°43′N, 03°01′E, 870 m a.s.l.) on a granitic brown soil (Cambic soil, FAO) (43% sand, 36% silt, 21% clay, pH (H2O) 6·2, 5·2% OM). The local climate is semi-continental, with a mean annual temperature of 9 °C ranging from 1 °C in January to 20 °C in August and an average annual rainfall of 760 mm. A factorial complete block design crossing three factors (plant species, cutting frequency and N supply) with three replicates per treatment combination was used. In May 2001, the grass monocultures were sown in rows (eight rows 18 cm apart) in each plot (2·8 × 1·5 m). The cutting frequency and N fertilizer treatments
were started in spring 2002. Two cutting frequencies (bimonthly, three cuts per year, and monthly, six cuts per year, C– and C+, respectively) and two levels of mineral N (NH4NO3) supply (12 and 36 g N m–2 year–1 at N– and N+, respectively, supplied in split applications after each cut) were compared. The N supply levels were established to obtain limiting and non-limiting N nutrition at N– and N+, respectively (Pontes 2006). Cutting frequencies were selected to simulate defoliation frequencies found in hay meadows (C–) and in grazed pastures (C+). In a given cutting treatment, all plots were cut simultaneously at 6 cm height with a mower (Haldrup, Logstor, Denmark). One cut out of two (cuts number 2, 4 and 6) was in common to the two cutting treatments. Phosphorus (8 g P2O5 m–2 year–1) and potassium (24 g K2O m–2 year–1) were supplied at non-limiting rates for growth. Soil volumetric water content was followed fortnightly in the twelve D. glomerata plots using time domain reflectometry (TDR, Trime-FM, Medfield, USA). When soil water content was below 10%, all plots were irrigated. A total of 100 mm of water was supplied in five applications during summers of 2003 (between July 7 and August 15) and 2004 (between June 28 and August 5).
- ( ) At each cutting date, the fresh harvested biomass of each individual plot was automatically collected by the mower and weighed. A subsample was immediately taken, weighed and dried at 60 °C for 48 h to determine the dry matter (DM) content of the harvested biomass and calculate the ANPP of each plot (g DM m–2). The annual ANPP (g DM m–2 year–1) was calculated as the sum of the six (C+) and three (C–) cuts performed each year.
The subsamples used to determine DM content were ground with a 1 mm screen through a Cyclotec sample mill (Model 1093 FOSS TECATOR Inc., Höganäs, Sweden). Each forage sample was analyzed via nearinfrared reflectance spectroscopy (NIRS) for crude protein (CP) content and enzymatic pepsin-cellulase dry matter digestibility (DMD). NIR spectra were collected with a Foss-NIRSystems 6500 monochromator (FOSS-NIRSystems, Silver Spring, MD, USA) which scans the spectral range of 400–2500 nm. Modified Partial Least Square (MPLS) calibration equations were developed using 144 samples which were selected from among all the spectra collected (n = 1512). The calibration set was analyzed for CP (using Kjeldahl N × 6·25) and DMD (Aufrère & Demarquilly 1989). All spectra and reference data were recorded and managed with the software Version 1·5 (Infrasoft International, Port Matilda, PA, USA). For CP and
846 L. Da S. Pontes et al.
DMD, respectively, the results of calibration statistics obtained were: minimum and maximum range (101–288, 492–870 g kg–1), standard error of cross-validation (6·6, 28 g kg–1) and r2 of cross-validation (0·92 for both).
Leaf traits were measured in June and in September 2003 and 2004, 3 weeks after a cut, which was common to the C– and C+ cutting treatments. Ten tillers were collected at random in each plot, avoiding 20 cm edges, cut with scalpels at ground level and kept in a cold box. In the laboratory, at plant level, the sheath length was first measured (SL). The tiller base was cut in de-ionized water and was then placed at 4 °C in the dark for at least 6 h to allow for full rehydration (Garnier et al. 2001a). After rehydration, the lamina of the youngest fully expanded leaf of each of the ten individuals was measured (LL), weighed and their area was measured with an electronic planimeter (LI 3100, Li-cor, Lincoln, NE, USA). The leaves were then oven dried at 60 °C for 48 h and weighed. Leaf dry matter content (LDMC, leaf lamina dry mass/leaf lamina fresh mass) and specific leaf area (SLA, leaf lamina area/leaf lamina dry mass) were calculated. The leaf lamina N content (LNC) was determined with an elemental analyser (Carlo Erba Instruments, CNS NA 1500, ThermoFinnigan, Milan, Italy) for each sample. The fresh matter based leaf N content (LNCF, g N g–1 FM) was calculated as LNC × LDMC.
© 2007 INRA. Journal compilation © 2007 British Ecological Society, Functional Ecology, 21, 844–853
Means of traits are means of two measurement dates (June and September) and of 2 years (2003 and 2004). Pontes (2006) has shown that species ranks are conserved between seasons and years. For each plot, annual means of DMD and CP were calculated as a weighted average, based on the harvested dry matter (ANPP) value at each cut. This procedure allows the calculation of a mean annual quality for the total herbage harvested during the 2 years. Analyses of variance () were performed using the statistical analysis package SAS ( Institute 2000, ver. 8, Cary, NC, USA) with the species, cutting regime, N supply and block factors. Plant tissue composition data (LDMC, LNC, CP and DMD) were transformed prior to using the Arcsin (square root) function and ANPP data were transformed by square root to normalize the data. Relationships among leaf traits were studied after log-transformation of values using standardized major axis (SMA) regression (Wright et al. 2004). SMA slopes were calculated as the linear regression slope divided by the correlation coefficient (r) (Sokal & Rohlf 1995). Statistical comparisons among treatments for SMA slopes and intercepts were performed according to Warton et al. (2006) in R (Ihaka & Gentleman 1996). Relationships between productivity, nutritive value and leaf traits, were analyzed by (Statgraphics
Plus, Manugistics, Rockville, MA, USA) using species means within each block (fixed factor) and within each N supply and cutting frequency level (n = 39). between responses to treatment factors were performed for ANPP and nutritive value and leaf traits. Spearman’s rank coefficients were used to compare species ranks for traits among treatments.
Results The species and N supply factors were significant for all variables analyzed (Table 1). The species factor was the single largest source of variation (44%–91% of the total variance explained) for all variables but CP. The cutting frequency factor was significant for all variables but SLA. Despite a significant effect for leaf traits, the block factor accounted only for a small share of the total variance (Table 1). For LNC, the species × N supply and the N supply × cutting frequency interactions were significant. For ANPP, CP and DMD the first order interactions were significant (except the species × N interaction for CP and DMD, and the cut × N interaction for DMD). Relative responses to cutting frequency and N supply were plotted as box plots indicating the variability observed among species (Fig. 1). Data (means per species within each N supply and cutting frequency levels) used to calculate these relative responses are shown in Supplementary Material (Supplementary Tables S1 and S2). For all species, LNC was increased by N supply and reduced by cutting frequency (Fig. 1a,c). In the same way, for most species, SLA (Fig. 1a,c) was increased by N supply (except for D. glomerata) and was reduced by cutting frequency (except for D. glomerata, Ph. pratense and P. trivialis). Only A. pratensis displayed a positive response of LNCF (i.e. the leaf N content per unit fresh matter, mg N g–1 FM) to cutting frequency (Fig. 1c). Except for F. arundinacea and P. trivialis, the relative changes of LDMC (Fig. 1a,c) were negative in response to N supply and positive in response to cutting frequency. The ANPP was usually increased by the N supply and reduced by the cutting frequency factors and displayed large but highly variable relative responses among species (Fig. 1b,d). Only two species (L. perenne and P. trivialis) had their production increased at a high compared to a low cutting frequency. There was a considerable variability of the grass species in the relative response to N supply (+4·8% to +52% increase between N– and N+). In contrast, nutritive value variables (DMD and CP) were stimulated by cutting frequency and N supply with relatively small variation among species (Fig. 1b,d). For all traits, as well as for productivity and nutritive value variables, the species ranking was highly correlated (P < 0·001) between the cutting frequency and N supply (Table 2) treatments.
847 Leaf traits affect grass productivity and quality
Table 1. Statistical significance of F ratios in ’s for leaf traits, productivity and nutritive value of the grass populations LDMC
Species Cut N Block Species × Cut Species × N Cut × N
LNC
SLA
ANPP
CP
DMD
df
VE
F
P
VE
F
P
VE
F
P
VE
F
P
VE F
P
VE F
P
12 1 1 2 12 12 1
74 2·3 5·4 2·8 – – –
54 20 47 12 – – –
*** *** *** *** ns ns ns
44 6·3 36 6·4 – 1·5
