Functional Ecology 2013, 27, 1064–1074
doi: 10.1111/1365-2435.12094
Herbivory differentially alters litter dynamics of two functionally contrasted grasses bastien Ibanez*,1, Lionel Bernard2, Sylvain Coq3,4, Marco Moretti1, Sandra Lavorel3 and Se Christiane Gallet2 1
Swiss Federal Research Institute WSL, Community Ecology Research Unit, Bellinzona, Switzerland; 2Laboratoire de Savoie, 73376 Le Bourget-du-lac, France; 3Laboratoire d’Ecologie d’Ecologie Alpine, UMR CNRS 5553, Universite Joseph Fourier, B.P. 53, 38041 Grenoble Cedex 9, France; and 4Centre Alpine, UMR CNRS 5553, Universite d’Ecologie Fonctionnelle et Evolutive (CEFE), CNRS, 1919 Route de Mende, 34293 Montpellier Cedex 5, France
Summary 1. Herbivores can have contrasted impacts on litter quality and litter decomposition, through an alteration of leaf chemistry and leaf senescence. Depending on the context, herbivores can induce defensive secondary compounds and thus slow down litter decomposition or accelerate decomposition by short-cutting nutrient resorption. 2. Almost nothing is known for grasses, which contain smaller amounts of secondary compounds than forbs and trees. Because grasses span a gradient from exploitative species having a low C : N ratio and induced defences, to conservative species having a high C : N ratio and constitutive defences, we hypothesize that the litter dynamics of functionally contrasted grasses may be differentially altered by herbivores. 3. In a mesocosm experiment, we assessed the litter decomposition rate of two subalpine grasses, the more exploitative Dactylis glomerata and the conservative Festuca paniculata, in the presence of two grasshopper species, Chorthippus scalaris and Euthystira brachyptera. We hypothesized that decomposition patterns depending on grass species and herbivory could be explained by the C : N ratio and the total phenolic content of fresh, senescent and decomposed leaves. 4. Herbivory by grasshoppers induced the accumulation of phenolics in the fresh leaves of D. glomerata, but most of these compounds were lost during senescence. The decomposition rate of D. glomerata senescent leaves did not depend on herbivory, phenolics and N content or C : N ratio. In contrast, herbivory did not induce any phenolic accumulation in the grazed leaves of F. paniculata, but during senescence, phenolics disappeared in greater proportions in grazed leaves than in ungrazed leaves, probably due to the physical alteration of grazed leaves. Herbivory slowed down the decomposition rate of F. paniculata, which was correlated to the phenolic concentration of senescent leaves, but not to the C : N ratio or N content. 5. Herbivory by grasshoppers differentially altered the litter decomposition rate of the two functionally contrasted grasses, having no effect on D. glomerata and slowing down F. paniculata. Thus, the combination of chemical and physical modifications of leaves by grazing and their interaction with grass traits may have either accelerating or decelerating effects on litter decomposition, with potentially complex outcomes at the ecosystem level. Key-words: flavonoids, grasshoppers, leaf senescence, litter decomposition, phenolic acids, phenolics, plant secondary metabolites
Introduction Below- and above-ground linkages mediated by the effects of herbivores on plants and soil processes are recognized as a major driver of terrestrial ecosystems (Frank & *Correspondence author. E-mail:
[email protected]
McNaughton 1993; Bardgett & Wardle 2010). Herbivores can affect soil processes either directly, through their frass, their cadavers and the alteration of soil parameters like temperature and moisture, or indirectly, through their effects on plant community composition and on the quantity and quality of plant litter and root exudates (Hunter 2001; Bardgett & Wardle 2003).
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society
Herbivory alters grass litter The alteration of litter quality deserves specific attention because it is one of the most complex below- and aboveground linkages. Indeed, herbivory can induce different types of secondary compounds in plants such as polyphenols, flavonoids and phenolic acids (Oksanen et al. 1987; Cipollini et al. 2008; Ibanez, Gallet & Despres 2012), with potentially different consequences for litter decomposition (Findlay et al. 1996; Schimel, Cates & Ruess 1998; Schweitzer et al. 2005). For example, tannins could slow down decomposition, while phenolic acids have been reported to exert an accelerating effect (Schimel, Cates & Ruess 1998; H€attenschwiler et al. 2011). Also, herbivory by insects can alter litter quality and decomposition through a limitation of nutrient resorption in leaves, which accelerates decomposition (Chapman et al. 2003; Sariyildiz et al. 2008). Moreover, Chapman, Schweitzer & Whitham (2006) proposed that herbivory induces recalcitrant compounds in deciduous trees, while it limits nutrient resorption in conifers, so that the herbivory–litter quality linkage depends on the plant taxa considered. Hence, the complexity of herbivory–litter quality linkage may be responsible for the observation that responses of litter decomposition to herbivory are idiosyncratic (Bardgett & Wardle 2003; Schweitzer et al. 2005; Chapman, Schweitzer & Whitham 2006; Frost et al. 2012). To date, almost all the studies that track the influence of herbivores on litter quality and litter decomposition have focused on woody plants, but little specific work has been conducted on grasses. Studies that investigated the effects of grazing-induced grass species replacement suggest that herbivory tends to reduce litter quality and slows down its decomposition (Semmartin et al. 2004; Garibaldi, Semmartin & Chaneton 2007). However, the effects of herbivory on litter quality and decomposition of grazed vs. ungrazed grasses are almost unexplored. In a study with large grazing herbivores, Semmartin, Garibaldi & Chaneton (2008) found that grazing either enhanced or retarded litter decomposition of two functionally contrasted grass species (Lolium multiflorum and Paspalum dilatatum) and hypothesized that the contrasting N economy and photosynthetic metabolism of the plants involved might explain this idiosyncrasy. Indeed, grasses are functionally heterogeneous, as are trees. There is a contrast between exploitative species, which are fast growing with a low C : N ratio and conservative species, with opposite traits (Grime 1977). Additionally, secondary compounds might be at play in grasses, in a similar fashion to deciduous trees. Although grasses contain no tannins (Barbehenn & Peter Constabel 2011), they contain relatively small amounts of phenolics and relatives such as phenolic acids and flavonoids (Sanchez-Moreiras, Weiss & Reigosa-Roger 2003). Moreover, grasses cover a broad functional range from conservative species that tend to rely on constitutive defences like toughness and silica to exploitative species able to accumulate secondary compounds (Cebrian & Duarte 1994; Rosenthal & Kotanen 1994).
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We therefore hypothesize that (i) herbivory of grasses by insects affects litter decomposition through the alteration of both the C : N ratio and the amount of secondary compounds in leaves and that (ii) functionally contrasted grasses are impacted by herbivory in different ways. To test these hypotheses, we manipulated herbivory by grasshoppers in experimental communities (mesocosms) of two grass species with contrasting traits and compared the litter decomposition of consumed and unconsumed grasses. We then explored the chemistry of the fresh, senescent and decomposed leaves, in order to explain observed decomposition patterns.
Materials and methods STUDY AREA, PLANT AND INSECT SPECIES
The experiment was conducted at the Station Alpine Joseph Fourier, at the Col du Lautaret (2100 m) in the central French Alps. All plants and grasshoppers were collected in the neighbouring subalpine grasslands, on the south-facing aspect of the commune of Villard d’Arene, ranging from 1700 to 2100 m (see Quetier et al. 2007 for a detailed description of the site and its vegetation). As model plants, we used two dominant perennial tussock species, that is, the conservative Festuca paniculata (L.) Schinz & Thell., which has tough leaves and high C/N, and the exploitative Dactylis glomerata (L.), which has tender leaves and low C/N (Gross, Suding & Lavorel 2007). As model herbivores, we used two Gomphocerinae grasshoppers, the habitat generalist Chorthippus (Stauroderus) scalaris Fischer–Waldheim, present in most plant communities in the study area, and the habitat specialist Euthystira brachyptera Ocskay, restricted to tall-grass meadows and shaded environments.
HERBIVORY EXPERIMENT
In early May 2010, single tillers of both species were separated from large tussocks and planted in individual godets filled with potting soil. One month later, in early June 2010, they were planted in large pots (Ø 45 cm, h 50 cm) filled with a standard mixture of 2/3 of sand, 1/6 of vermiculite and 1/6 of potting soil and fertilized with 3 g of low leaching rate fertilizer (Fertiltop, 16 8 10 + 4 MgO + oligoelements) in order to reduce nutrient limitation. In each plot, we planted 24 individual plants, according to the following design: ‘D’ treatment: 21 D. glomerata and three F. paniculata individuals (i.e. 7 : 1) ‘F’ treatment: three D. glomerata and 21 F. paniculata individuals (i.e. 1 : 7) ‘50’ treatment: 12 D. glomerata and 12 F. paniculata individuals (i.e. 1 : 1) The plants mixtures were designed for the purpose of a companion study focusing on the effects of herbivory on plant–plant competition (Ibanez et al. in press). One month later, the pots were covered by an insect nylon mesh at 50 cm above the ground and four adult grasshoppers were introduced as follows in each of the plant composition treatments: ‘Cs’ treatment: two males and two females of C. scalaris ‘Eb’ treatment: two males and two females of E. brachyptera ‘CsEb’ treatment: one male and one female of each species ‘0’ treatment (control): no grasshoppers. Grasshopper density corresponded thus to 25 individuals m 2, which is about 5–10 times the observed natural densities in the
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1064–1074
1066 S. Ibanez et al. surrounding communities (S. Ibanez, pers. obs.). In total, 48 mesocosms were set, with four replicates for each combination of plant composition and herbivory treatments (three plant treatments, four grasshopper treatments, 48 = 3 9 4 9 4). Mesocosms were checked weekly, and the dead grasshopper bodies removed and replaced by living individuals of the same species and sex, in order to keep the density and proportion of the species, and thus herbivory pressure constant. The grasshoppers were present in the mesocosms from the 29 June 2010 until the 27 September 2010 and from the 27 June 2011 until the 10 September 2011 when the experiment was stopped. The grasshoppers were removed earlier in 2011 to allow the harvest of fresh leaves before the beginning of senescence. The same mesocosms were studied during these two consecutive years. All the individual plants had several herbivory marks at the end of each season, so that the herbivory pressure was substantial enough to affect leaf chemistry. Grasshoppers were not selective regarding plant species, they consumed D. glomerata and F. paniculata according to their relative availabilities in the mesocosms, but due to the greater tolerance of D. glomerata, herbivory increased the competitive ability of D. glomerata with respect to F. paniculata (Ibanez et al. in press).
DECOMPOSITION EXPERIMENT
On 2 November 2010 (about 4 months after the introduction of the grasshoppers), senescent leaves on both plant species were harvested from the plants in each mesocosm and dried for 48 h at 40 °C before the decomposition experiment. One hundred milligram of senescent leaves of each species (D. glomerata and F. paniculata) was used for the D treatment and F treatment, respectively. For the 50 treatment, we used 50 mg of both plant species. The bulked senescent leaves were then put in 5 cm 9 4 cm litter bags with distinct mesh size, large mesh size (1500 lm) and small mesh size (68 lm, 68PES4/135; DIATEX, St-Genis-Laval, France), for the purpose of another experiment focusing on the relative contribution of microbial and animal decomposers. In order to disentangle the potential effects of herbivory on the quality of senescent leaves and on the mesocosms’ soil environmental conditions, the litter bags were placed on the ground at two distinct places, namely in the mesocosm in which the senescent leaves were harvested (experimental conditions) and in a neighbouring natural grassland dominated by F. paniculata (field conditions). Therefore, for each mesocosm treatment, four litter bags were designed, while both mesh size (small vs. large) and location (mesocosm vs. field) factors were combined for a total number of 192 litter bags (48 9 2 9 2). The litter bag experiment started mid-November 2010 and ended mid-May 2011. At this subalpine site, there is substantial winter time decomposition, especially under a thick snowpack (Saccone et al. 2012). The decomposed leaves were extracted from the litter bags and dried for 24 h at 40 °C. The percentage of mass loss during the November–May interval was calculated as: % mass loss = 1009 (mass before decomposition mass after decomposition)/mass before decomposition.
LEAF CHEMISTRY
For each mesocosm, an additional sample (10 mg) randomly sampled from the senescent leaves harvested for the decomposition experiment was dried for 48 h at 40 °C only, because higher temperatures could alter phenolic compounds and kill the microbes present at the leaf surface that might be involved in decomposition (Gracßa, B€arlocher & Gessner 2005). Senescent leaves were then ground to powder and analysed for carbon and nitrogen concentration using a CHN analyser (CHS NA1500; Carbo Erba Instrument, Milan, Italy). At the end of the decomposition experiment,
10 mg of the decomposed leaves of each litter bag was analysed in the same way. Ten milligram of fresh leaves harvested in August 2011 was also analysed for carbon and nitrogen concentration. Fresh leaves were collected at the very end of the experiment to avoid artificial herbivory during the experiment. Leaf phenolics were extracted twice in a 70 : 30 ethanol–water solution under reflux at 100 °C. After filtration, the two extracts were bulked and the final volume measured. The total phenolic compound concentration was determined with Folin–Ciocalteu reagent after evaporation under vacuum. The total phenolic concentration was calculated by comparison with a calibration curve made with gallic acid and expressed in milligram of gallic acid equivalent per gram of dry mass. Separation and UV characterization of the different monomers of phenolic compounds were performed using high-performance liquid chromatography (HPLC) coupled with diode array detector. Injection volume was 20 lL on RP C18 column at 15 mL min 1 flow. Phenolic compounds were separated depending on their hydrophilic character and molecular weight. Two different gradient methods were used depending on the phenolic class. For low-molecular-weight phenolic monomers (including phenolic acids and aldehydes characterized by one aromatic ring), the percentage of acetonitrile in acetic acid (at 05% in distilled water) ranged from 0% to 20% during 45 min. For flavonoids, the percentage of methanol in acetic acid (at 05% in distilled water) ranged from 10% to 60% during 30 min. The HPLC technique provides for each sample a chromatogram of several peaks that are recorded at the maximum wavelength of absorbance (see Appendix S1 in Supporting Information for examples of chromatograms). Each peak corresponds to a single compound or a group of related compounds. Preliminary analysis revealed that the flavonoid and phenolic acid contents in senescent and decomposed leaf extracts were too low to be measured, and we thus restricted our analysis to fresh leaves.
STATISTICAL ANALYSIS
We used the free R software (R Development Core Team 2011) for all analyses. The significance threshold was fixed to 005. The C : N ratios of the fresh, senescent and decomposed leaves were square-root transformed prior to analysis. Total phenolics were log-transformed to achieve normality. Preliminary analyses showed that the different combinations of grasshopper species (Cs, Eb and CsEb treatments) gave similar results (data not shown). Thus, to gain analysis power, we defined the herbivore factor as the presence (either Cs, Eb or CsEb treatments) or absence (O treatment) of grasshoppers.
Herbivory experiment The effect of the presence of grasshoppers on leaf parameters was tested with type III ANOVA tests with the presence/absence of grasshoppers and leaf state (fresh, senescent and decomposed) as factors. We used the ‘ANOVA’ function from the ‘car’ R package (Fox & Weisberg 2011), which is suitable for unbalanced designs. When the ANOVAs were significant, for each leaf response variable, the pairwise statistical differences between factor levels (i.e. the presence/absence of grasshoppers and the leaf state, fresh, senescent, decomposed) were tested with Tukey’s tests using the glht function of the multcomp R package (Hothorn, Bretz & Westfall 2008). The heterogeneity of variances was taken into account with the ‘sandwich’ R package (Zeileis 2006). We calculated the percentage of variation of total phenolics during senescence as follows, with Px standing for the concentration of phenolics in leaf state Pfresh)/Pfresh. We calculated ‘x’: % variation = 1009 (Psenescent the percentage of variation during decomposition similarly, using Pdecomposed and Psenescent. No statistical tests were made on these
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 1064–1074
Herbivory alters grass litter
Results HERBIVORY EXPERIMENT
Consistent with previous measurements at the site (Gross, Suding & Lavorel 2007), ungrazed F. paniculata fresh leaves had a larger C : N ratio (mean SD: 202 41) than ungrazed D. glomerata fresh leaves (175 19, Fig. 1). The C : N ratio of D. glomerata leaves was not affected by the presence of grasshoppers, but it was affected by leaf state (Table 1); in particular, decomposed
50
ab ab
a
bc
40
ab
c
30
Dry mass loss of senescent leaves (%)
We evaluated the influence of plant species identity and of the presence/absence of herbivores on the percentage of mass loss in the litter bags. We also checked whether the location and mesh size of the litter bags interacted with the plant and herbivore treatments. We included the four factors (plant species, herbivores, location, mesh size) as well as the interactions in 25 mixed models (Bates, Maechler & Bolker 2011) with mesocosms as a random factor. Mixed models were required here because there were several litter bags for each mesocosm, whereas in the leaf chemistry analysis, there is a single sample per mesocosm. The 25 models ranged from an intercept model (one parameter) to the model including all factors and all interactions (24 parameters, Table 1). For each model, we calculated the Bayesian Information Criterion (BIC), the Akaike Information Criterion (AIC) and the corrected AIC (AICc). The model having the lowest BIC and AICc values was considered as the best model, in the sense that it included the factors and their interactions that best explained litter mass loss. Nonadditive effects in the mixed litter of F. paniculata and D. glomerata were not significant and therefore not considered any further. We further evaluated the influence of leaf chemistry on litter decomposition, by calculating three mixed models, each including either the C : N ratio, the N content or the phenolic content. All three models included mesocosms as a random factor and location and mesh size as covariates. We evaluated these models for both plant species separately because leaf chemistry was not independent from plant species identity. The significance of each leaf chemistry factor was then evaluated by a Wald chisquare test, using the ‘ANOVA’ function from the ‘car’ R package (Fox & Weisberg 2011).
No herbivores Herbivores
20
Decomposition experiment
60
percentages because they would have been redundant with the tests described above.
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Festuca paniculata
Dactylis glomerata
Mixed litter
Fig. 1. Percentage of dry mass loss of the senescent leaves in function of the plant species identity (Dactylis glomerata or Festuca paniculata) and the herbivory treatment (grazed in grey or ungrazed leaves in white). Each box represents the lower quartile, the median (bold line) and the upper quartile. The notches correspond roughly to a 95% confidence interval. The letters above each box correspond to the result of the Tukey’s test, and boxes that do not share any letter are significantly different.
leaves had a lower C : N ratio than fresh and senescent leaves (Fig. 1a). The interaction term between leaf state and herbivory was significant (Table 1), where the C : N ratio of grazed leaves was generally slightly lower than the C : N ratio of ungrazed leaves, except for senescent leaves that had the opposite pattern (Fig. 1a). The pairwise comparisons (Tukey’s test) revealed that the C : N ratio of the decomposed leaves in the presence of herbivores was lower than the C : N ratio of both herbivory treatments of the fresh leaves and the senescent leaves with herbivores (Fig. 1a). In contrast, the presence of grasshoppers increased the C : N ratio of F. paniculata leaves overall (Table 1), although pairwise comparisons between grazed and ungrazed leaves of individual leaf states were not significant (Tukey’s test, Fig. 1b). Leaf state also affected
Table 1. ANOVA table (type III tests) of the C : N ratio (square-root transformed) and the phenolic concentration (log-transformed) in function of leaf state (fresh, senescent, decomposed) and herbivory (presence/absence of grasshoppers) C : N ratio
Dactylis glomerata Leaf state Herbivory Leaf state : Herbivory Residuals Festuca paniculata Leaf state Herbivory Leaf state : Herbivory Residuals
Phenolic concentration
Sum Sq
d.f.
F-value
P(>F)
0836 0029 0343 5195
2 1 2 106
8525 0595 3499
