Subordinate plant species enhance community resistance against drought in semi-natural grasslands

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1 Journal of Ecology 2013, 101, doi: / Subordinate plant species enhance community resistance against drought in semi-natural grasslands Pierre Mariotte 1,2 *, Charlotte Vandenberghe 2, Paul Kardol 3, Frank Hagedorn 4 and Alexandre Buttler 1,2,5 1 Ecole Polytechnique Federale de Lausanne (EPFL), School of Architecture, Civil and Environmental Engineering (ENAC), Laboratory of Ecological Systems (ECOS), Station 2, 1015 Lausanne, Switzerland; 2 Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Site Lausanne, Station 2, 1015 Lausanne, Switzerland; 3 Department of Forest Ecology and Management, Swedish University of Agricultural Science, Umea, Sweden; 4 Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Site Birmensdorf, Z urcherstrasse 111, 8903 Birmensdorf, Switzerland; and 5 Laboratoire de Chrono-Environnement, UMR CNRS 6249, UFR des Sciences et Techniques, 16 route de Gray, Universite de Franche-Comte, F Besancßon, France Summary 1. According to the insurance hypothesis, more diverse plant communities are more likely to be resistant to drought. Whilst many experiments have been carried out to determine the effects of plant diversity on plant community insurance, the results are still contradictory. 2. Here, we conducted a drought experiment where we tested whether the presence of subordinate species increases plant community insurance. In Swiss Jura grassland, we combined a removal experiment of subordinate species with a summer drought event using rainout shelters. 3. Plant community composition was determined after the drought and based on biomass measurements; we estimated resistance, recovery and resilience of the plant community for each combination of treatments. Moreover, to assess drought impacts on water-use efficiency (WUE), we analysed carbon isotope ratios (d 13 C values) in plant leaves of two dominants and two subordinates collected at the end of the drought period. 4. We showed that subordinate species are more resistant to drought and increased community resistance by enhancing their above-ground biomass production during the imposed drought. These patterns were associated with decreased competitiveness of dominant species whose biomass decreased during drought. Significant increase in d 13 C values in plant tissue under drought indicated a better WUE for the measured species. Interestingly, the WUE was significantly higher in plots where subordinates were removed. Recovery and resilience were not affected by the summer drought, but the absence of subordinates reduced overall above-ground biomass in both watered and drought plots. 5. Synthesis. We demonstrated that, independent of plant diversity, the presence of drought-resistant subordinate species increases plant community insurance against drought and, hence, is important for the functioning of grassland ecosystems. Key-words: biomass-dependent hypothesis, drought, ecosystem stability, global change, insurance hypothesis, isotopes, plant hierarchy, plant climate interactions, precipitation Introduction Climate is one of the major drivers of plant species distribution around the world, and climatic changes are expected to shift species distributions or modify their abundance and dominance patterns (Parmesan 2006; Lenoir et al. 2008). For certain regions in Europe, recent climate change models *Correspondence author. pierre.mariotte@epfl.ch predict a decrease in summer precipitation, resulting in severe droughts and increased water stress (Christensen et al. 2007). In Switzerland, the mean decrease in summer precipitation is expected to reach 20% in 2070 compared with 1990 (Frei et al. 2006). Water availability is a main driver of net primary production, and changes in precipitation play an important role in the potential global change impacts on terrestrial ecosystems (Ciais et al. 2005; Engler et al. 2011). High-elevation ecosystems are disproportionately vulnerable to climate change (Beniston et al. 1996; Nogues-Bravo et al. 2007), and 2013 The Authors. Journal of Ecology 2013 British Ecological Society

2 764 P. Mariotte et al. mountain grassland communities are expected to be greatly affected by drought events (Smith 2011a) with significant consequences for ecosystem functioning (Gilgen et al. 2010). Importantly, the response of plant communities to environmental stress depends on species diversity and composition, which both are main drivers of community stability (Loreau et al. 2001; Griffin et al. 2009). Understanding which component of the community (e.g. diversity, composition) promotes ecosystem maintenance during summer drought event remains essential (Smith 2011b). According to the diversity insurance hypothesis (Yachi & Loreau 1999), an increase in community diversity corresponds to an increase in the range of potential species responses to environmental perturbation. Therefore, more diverse communities would have a higher chance containing species that are well-adapted to summer drought and, thus, can compensate for the biomass loss of less-adapted species. Insurance of plant communities against perturbation includes three phases during which species diversity plays a role: resistance, recovery and resilience (Pimm 1984; Tilman & Downing 1994; Van Ruijven & Berendse 2010; Vogel, Scherer-Lorenzen & Weigel 2012). Resistance is the ability of the plant community to withstand the perturbation by maintaining biomass or reproductive outputs during the event. Recovery is the ability of the plant community to compensate for the loss of biomass or reproductive outputs due to the perturbation. Resilience is the ability of the plant community to return to its original state following the perturbation; resilience combines resistance and recovery to provide a measure of community stability (Van Ruijven & Berendse 2010; Vogel, Scherer-Lorenzen & Weigel 2012). Several drought experiments have been carried out to determine the effects of plant diversity on plant community insurance, but the results so far have been contradictory. Whilst decreasing resistance against drought with increasing species diversity has been shown for constructed communities (Pfisterer & Schmid 2002; Van Peer et al. 2004; De Boeck et al. 2008), increased resistance with increasing species diversity has been shown for natural grasslands (Tilman & Downing 1994; Kahmen, Perner & Buchmann 2005). In a recent mesocosm experiment, Van Ruijven & Berendse (2010) found that recovery after drought was enhanced with increasing species diversity, but effects were mainly due to the response of one single species; in their study, no effects were found on community resistance and resilience. Moreover, Wang, Yu & Wang (2007) suggested that susceptibility to drought is mainly biomass-dependent with high-biomass communities being less resistant to drought than low-biomass systems. In high-biomass communities, tall plants limit light availability for drought-resistant species. In contrast, in lowbiomass communities, light limitation is less, and droughtresistant species can compensate for the biomass loss of drought-sensitive species. In a multifactorial climate change experiment, Kardol et al. (2010a) showed that the proportion of subordinate species increased under dry compared with wet conditions; they suggested that dominant species responded most strongly to the direct impacts of drought, whereas subordinate species responded to the resulting decrease in the strength of competition interactions with the dominant species. Shifts in competitive interactions might thus act as a key factor in community responses to drought by favouring drought-resistant subordinate species, which then may compensate for the loss of drought-sensitive dominant species. Therefore, high species diversity associated with the presence of more resistant species may facilitate insurance to drought in low-productive grasslands where competitive interactions are likely to decrease between drought-resistant species (i.e. subordinates) and drought-sensitive species (i.e. dominants). One of the main factors determining plant drought resistance under conditions of water limitation is water-use efficiency (WUE). Indeed, improved WUE is recognized as a plant strategy to maintain biomass production during drought and corresponds to the ability of a species to maximize the utilization of water. The 13 C isotope discrimination of plants can be used as an integral measure for WUE, because it is sensitive to the ratio of the CO 2 in the chloroplast to the CO 2 in ambient air (ci/ca; Farquhar, Oleary & Berry 1982). An increase in d 13 C is coupled with a decrease in ci/ca, which is caused by changes in the ratio between photosynthesis and stomatal/mesophyll conductance (Farquhar, Ehleringer & Hubick 1989). Therefore, an increase in 13 C isotope discrimination corresponds to decreased stomatal conductance, that is, a plant adaptation for better WUE. The measurement of d 13 C values in plant leaves can thus provide insight in the drought resistance of plants and help identifying the most resistant species in the community (Condon et al. 2004). Semi-natural grasslands of the Swiss Jura Mountains are subjected to regular grazing and trampling disturbance and have in general low productivity but high diversity with up to 40 vascular plant species per square metre (Buttler, Kohler & Gillet 2009). Observations of plant species frequency and relative cover in these grasslands reveal distinct distributions, with some species found frequently with high relative cover (defined as dominant species) and others found frequently, but with low relative cover (defined as subordinate species; sensu Grime et al. 1987; Grime 1998; Olff & Bakker 1998). Species that generally do not persist over time and appear only briefly as seedlings that fail to survive are defined as transient species (sensu Grime 1998; i.e. low frequency and low relative cover). Dominant species are generally taller and account for a high proportion of the total community biomass, whilst subordinate species are smaller, grow under the canopy of dominants and account for a low proportion of the total community biomass. In these semi-natural grasslands, subordinate species seem to benefit from cattle activity because large grazers create gaps and areas of reduced root competition from dominants (Mariotte et al. 2012a) or because subordinates regrow better after grazing than dominants (Tahmasebi Kohyani et al. 2009). According to the mass ratio hypothesis (Grime 1998), ecosystem functioning is determined by the traits of the dominant species independent of changes in species richness, such as variation in the number of subordinate species. Although dominant species are clearly important for ecosystem functioning, there is growing

3 Subordinates promote resistance to drought 765 evidence that subordinate species are likewise important, especially for the maintenance of ecosystem functioning during periods of stress such as drought (Wang, Yu & Wang 2007; Kardol et al. 2010a). In this study, we examined how subordinate species influence community insurance to drought in semi-natural grasslands of the Swiss Jura. First, we manipulated plant communities using a removal experiment of subordinate species. Second, we manipulated water availability by imposing a severe summer drought using rainout shelters. Compared with constructed communities, plant removal experiments have the advantage of testing impacts of plant species presence in real ecosystems (Diaz et al. 2003). This approach was used, here, to test the diversity insurance hypothesis by focusing on one key component of diversity, that is, the presence of subordinate species. Specifically, we tested the role of subordinate species in drought effects on community resistance, recovery and resilience, and on ecosystem functioning (i.e. productivity) using community above-ground biomass measurements and stable carbon isotope natural abundance (d 13 C). We hypothesized that the absence of subordinate species decreases community biomass production and reduces plant community insurance to summer drought. Additionally, we also tested the biomass-dependent hypothesis (Wang, Yu & Wang 2007), which predicts that low-productive plant communities allow the persistence of drought-resistant subordinate species and, therefore, are more resistant to drought than high-productive communities. Materials and methods SITE DESCRIPTION The field experiment was carried out at an extensively grazed and species-rich (mean of 30 species per square metre) pasture situated on a calcareous slope (30 ; 1200 m a.s.l.) in the Swiss Jura Mountains (Agroscope Changins-W adenswil Research Station, La Fretaz, western Switzerland, E, N). The soil was classified as Cambisol eutric (World Reference Base for Soil Resources IUSS Working Group WRB 2006) and was not deeper than 40 cm. The climate is suboceanic with a mean annual precipitation of 1393 mm (data from MeteoSwiss station Bullet/La Fretaz, ). The pasture is grazed by cattle following a rotational system during the growing season from May to September. DEFINING DOMINANT, SUBORDINATE AND TRANSIENT SPECIES In June 2008, we selected a 25 by 25 m area where vegetation is very homogeneous, and we excluded cattle using electric fences. Within the cattle-excluded area, 49 plots of 1.2 by 1.2 m were established at a regular distance of 1.6 m from each other. In July 2008, absolute plant cover was determined within all plots using a modified Braun Blanquet index (1 = cover < 5%, 2a = 5 10%, 2b = 10 25%, 3 = 25 50%, 4 = 50 75%, 5 = %). A total of 37 species were identified and classified in competitive hierarchical groups following Grime (1998) and according to a frequency abundance curve based on species frequency (100% frequency means that the species is present in all plots) and cumulate relative cover (species ranked by relative cover in ascending order and cumulated, 100% cumulate relative cover correspond to the most abundant species). A species was classified as dominant if its frequency was > 70% and its cumulative relative cover was > 12%. A species was classified as subordinate if its frequency was > 70% and its cumulative relative cover was between 2% and 12%. The remaining species, which appear only briefly and fail to survive or show great fluctuations over time, were classified as transient species. This method yielded eight dominant species (Trifolium repens L., Cynosurus cristatus L., Festuca nigrescens Lam., Agrostis capillaris L., Carum carvi L., Ranunculus acris ssp. friesianus, Taraxacum officinale agg., Alchemilla monticola Opiz) and five subordinate species (Trifolium pratense L., Achillea millefolium L., Leontodon sp. [Leontodon autumnalis L. + Leontodon hispidus L. sl.], Cerastium fontanum ssp. vulgare, Veronica chamaedrys L.). Dominant and subordinate species selected in this experiment did not differ in terms of soil moisture preferences and were all indicators of average soil moisture conditions based on Ellenberg indicator values (F = 5orF = 6; Hill et al. 1999). REMOVAL EXPERIMENT From the 49 plots, we retained the 30 most homogenous ones according to above-ground biomass and species cover as determined in 2008 (Braun Blanquet index). We applied the following three treatments each with 10 replicates: control without removal (C), removal of all subordinate species (S) and partial biomass removal of dominant species (D). Equal amounts of biomass were removed in S and D treatments. Treatment D allowed us to distinguish the effects of biomass removal as such and effects of suppression of subordinate species. As removal of individuals of Cynosurus cristatus and Festuca nigrescens was not feasible, these species were not considered in the removal treatment D. To estimate the total biomass of the removed subordinate and dominant plants, we developed allometric models for each of the six dominant and five subordinate species used in the removal treatments. In July 2008, above- and below-ground parts of 30 individuals of each species were randomly sampled in the spare plots of the experiment. In the laboratory, plant material was cleaned, and all individuals were measured for the following traits: number of leaves, length of the largest leaf and length of the stem. Thereafter, plants were dried at 60 C for 72 h and then weighed. For each species, the above-ground plant trait with the highest predictability for total biomass was determined by testing correlations between the plant traits and total dry weight. Each model followed the same basic equation: b ¼ cx b eqn 1 where b is the total biomass of the individual plant, and x is the value of the plant trait (see Appendix S1 in Supporting Information). No correlations were found for Agrostis capillaris, and therefore, for this species, mean biomass of the 30 individuals was used to estimate total biomass in the field. Plant removals were carried out at the end of June in 2009, 2010 and First, each plot was divided in 144 cells of 10 by 10 cm, and all subordinate plants were pulled out by hand in each cell in a way that a maximum number of roots were removed whilst minimizing disturbance of the soil structure. Then, each S plot was randomly paired with a D plot, such that once the total biomass of removed subordinate species was determined (using the allometric models), an equal amount of biomass of dominant species was removed in the paired D plot. For each D plot, individuals of dominant species were

4 766 P. Mariotte et al. randomly selected, taken randomly amongst the 144 cells, pulled out and measured for traits (as described above) to determine the total removed biomass using the respective allometric model. We repeated the removal of individuals of dominant species until the same amount of biomass was removed as in the paired S plots. The estimated mean biomass removed in S and D plots (1 SE) was 17 3gm 2 in 2009, 3 1 in 2010 and 2 1 in 2011, which corresponded to 1.4%, 0.5% and 0.3% of the total community above-ground biomass in 2009, 2010 and 2011, respectively. Subordinate species present in the S plots during the second and third year of the experiment mainly resulted from germinating seeds. dominant, subordinate and transient species. We calculated recovery as the ratio of biomass that regrew after the drought (July 2011) to pre-drought biomass (July 2010). We calculated resilience as the ratio of total biomass after the drought (year 2011) to total biomass before the drought (year 2009), that is, the biomass produced the year after the drought relative to the biomass produced the year before the drought. As semi-natural grasslands are continuously perturbed by cattle activity, plant community resistance, recovery and resilience to permanent disturbance (i.e. grazing/biomass removal) were calculated in watered plots, serving as control comparisons to community insurance against drought. SUMMER DROUGHT To simulate a severe summer drought, we installed rainout shelters from 9 July to 6 September To control for shelter effects, we established shelters for both control (n = 15) and drought plots (n = 15). For control plots (hereafter referred to as watered plots ), water collected from the roof was added back to the plot. Combining removal (C, S, D) with drought (watered, drought) treatments resulted in five replicates of each treatment combination. Rainout shelters (adapted from Yahdjian & Sala 2002) were made of wooden frames (1.80 m m) covered with transparent foil that permitted 90% penetration of photosynthetically active radiation (Cello Flex 4SF; Puteaux SA, Clayes-sous-bois, France). The mean height of the shelters was 1.70 m to leave sufficient space for plant growth. Shelters had a 30 inclination following the soil surface topography, and on the lowest side, a gutter was installed to collect the intercepted precipitation in a storage tank (60 L). On the four sides of the frame, a foil strip was fixed to minimize border effects. To ensure sufficient air circulation, we left two open layers (15 cm), above the soil and under the roof. Control plots were watered after each rainfall event with an amount of water corresponding to the locally recorded precipitation of the event (Bullet/La Fretaz, MeteoSwiss station close to the study site). From the beginning of July until the end of September 2010, microclimatic variables were continuously monitored in the centre of three randomly chosen drought and three randomly chosen watered plots. Soil moisture (EC-5) and temperature (ECT) sensors were placed at 10 cm depth, and air temperature (EHT) sensors with radiation shield were placed at 50 cm above the soil surface. Data were recorded every 10 min using data loggers (Em50; Decagon Devices Inc., Pullman, WA, USA) fixed to the rainout shelter. RESISTANCE, RECOVERY AND RESILIENCE In each plot, above-ground biomass was harvested twice a year, in early July and early September 2009, 2010 and 2011 (see Fig. 1); these harvests simulated the rotational cattle grazing as described above. Cutting height of the vegetation was c. 3 cm above the soil level. At the 2010 harvests, above-ground plant biomass was collected in the central square of each plot (50 by 50 cm), separated by species, dried at 60 C for 72 h and weighted. Community insurance against drought may be biomass-dependent. Therefore, we took pre-drought biomass into account in our measurements of community insurance (Wang, Yu & Wang 2007; Van Ruijven & Berendse 2010; Vogel, Scherer-Lorenzen & Weigel 2012). We calculated resistance of the plant community to drought stress as the ratio of regrowth biomass during the drought (September 2010) to pre-drought biomass (July 2010), that is, the proportion of biomass that regrew during the drought. Resistance was calculated for the whole plant community as well as for groups of CARBON ISOTOPES Carbon isotope ratios (d 13 C values) were determined as an integral measure for WUE (Farquhar, Ehleringer & Hubick 1989; Seibt et al. 2008). Leaf material of two dominant species (Taraxacum officinale and Alchemilla monticola) and two subordinate species (Achillea millefolium and Veronica chamaedrys) was collected at the end of the drought treatment (September 2010) in control plots without removal (C). At the same time, leaf material of the two dominant species was also sampled in plots where subordinate species were removed (S). We chose these four species because they were present in all plots (i.e. drought and watered). Leaf material was ground to a fine powder, and we determined carbon isotope ratios (d 13 C) using an elemental analyzer (Euro EA 3000; HEKAtech, Wegberg, Germany) coupled to an isotope ratio mass spectrometer (Delta V Advantage; Thermo, Germany). STATISTICAL ANALYSIS Measures of community stability, that is, resistance, recovery and resilience, were compared between drought plots and watered plots for each removal treatment (C, D and S) to distinguish between the effects on community stability associated with the removal treatment and the effects associated with the drought treatment. Measures of community stability were analysed using two-way ANOVA with removal and drought (and their interactions) as fixed factors, followed by Tukey s post hoc tests. We ran a linear regression between post-drought biomass (September 2010) and pre-drought plant species diversity to test the diversity insurance hypothesis. We ran a linear regression between postdrought biomass (September 2010) and pre-drought biomass (July 2010) to test the biomass-dependent hypothesis. Finally, we ran a linear regression between post-drought biomass (September 2010) and pre-drought biomass of subordinates (July 2010) to test their contribution to community resistance to drought. To test the goodness-of-fit, statistical significance of linear regressions was obtained from ANOVAs. In plots without plant removal (C), carbon isotope measurements were analysed using two-way ANOVA with drought and plant species (and their interactions) as fixed factors. As the drought treatment had an overall significant effect on d 13 C values, we subsequently analysed carbon isotope values separately for watered plots and drought plots using one-way ANOVA with plant species as a fixed factor, followed by Tukey s post hoc tests. When we compared C and S plots, carbon isotopes measurements were analysed using three-way ANOVA with drought, removal and plant species (and their interactions) as fixed factors. Treatment effects on plant community composition were analysed using canonical correspondence analysis (CCA; after Hellinger

5 Subordinates promote resistance to drought 767 (a) (b) (c) (d) Fig. 1. Micrometeorological data from July to October 2010 for drought plots (grey lines) and watered plots (black lines). (a) Daily precipitation sum (data from MeteoSwiss station Bullet/La Fretaz), (b) mean daily air temperature at 50 cm above the soil level, (c) mean daily soil temperature at 10 cm depth, (d) mean daily soil moisture content at 10 cm depth. Black points correspond to the two harvests of community above-ground biomass. transformation), including drought and removal as explanatory variables. CCA was run using data of proportional species abundance in September 2010, that is, regrowth biomass during the drought. Subordinate species, which were absent in S plots, were left out of the CCA. Permutation tests (Monte Carlo) were used to assess the significance of these multivariate regression models. All statistical analyses were carried out using R version (R Development Core Team, 2011). Results SOIL MOISTURE The experimental drought reduced the amount of precipitation by 268 mm (Fig. 1), which represents about 20% of mean annual precipitation at our study site. The rainout shelters decreased soil moisture at 10 cm depth on average by 67% during the drought period, with a maximum decrease in soil moisture of 97% at the end of August. After the drought, from September to October, soil moisture remained lower in the drought plots by 35 40%. During the drought, the average air temperature increased by around 0.4 C and the average soil temperature by around 0.2 C in drought plots compared with watered plots. However, effects of drought on temperature were not significant (air temperature: F 1,116 = 0.137, P = 0.71; soil temperature: F 1,116 = 0.152, P = 0.70). COMMUNITY RESISTANCE, RECOVERY AND RESILIENCE Plant community resistance was significantly affected by the drought (F 1,24 = , P < 0.001) and removal treatments (F 2,24 = 3.583, P < 0.05), and importantly, there was a

6 768 P. Mariotte et al. significant drought 9 removal interaction effect (F 2,24 = 4.348, P < 0.05; Fig. 2a). In control plots, plant communities showed about 60% of regrowth, whilst there were no significant effects of the removal treatments. In drought plots, plant communities showed about 20% of regrowth in C and D plots, whereas community resistance was significantly lower in S plots with only 2.5% of the biomass regrowing after the drought treatment. Resistance of dominant and transient species against drought was similar to those of the whole plant community (Fig. 2b) but lower for dominant than for transient species; dominant species showed only 5% of regrowth in C plots, 3% in D plots and 0.09% in S plots. In contrast to dominant and transient species, subordinate species showed significantly stronger resistance (F 1,16 = 6.766, P < 0.05) in drought plots than in control plots (i.e. resistance to natural grazing disturbance); regrowth was about 500% in control plots and about 2000% in drought plots (Fig. 2b). The proportional biomass of species groups shifted in response to the drought treatment: dominant species decreased from 60% to 25%, whilst transient and subordinate species increased from 40% to 65% and from 2% to 12%, respectively. Pre-drought plant diversity had no effect on post-drought biomass (linear regression, F 1,8 = 0.579, P = 0.47), but postdrought biomass decreased with increasing pre-drought biomass (Fig. 3a). Further analysis showed a positive correlation between the pre-drought biomass of subordinate species and post-drought community biomass (F 1,8 = 5.464, P < 0.05; Fig. 3b). Together, these results demonstrate that the plant community was more resistant and produced more biomass after drought when containing high biomass of subordinate species. Plant community recovery was not affected by the drought and removal treatments (Fig. 2a), and community aboveground biomass recovered completely with about 100% of regrowth in all treatments. Plant community resilience was not affected by the drought treatment (Fig. 2a) but was significantly affected by the removal treatment (F 2,24 = 9.351, P < 0.001): regrowth was about 20% lower in plots where subordinate species were removed than in control plots without removal (Fig. 2a). Community above-ground biomass did not differ between removal treatments at the start of the experiment in 2009, and we did not detect significant differences in community aboveground biomass amongst years (2009, 2010, 2011) in control plots (watered and without plant removal, n = 5), indicating that natural annual variation was small during our 3-year experiment. Plant community composition (subordinate species excluded) was significantly affected by the drought (F 1,26 = 3.607, P < 0.001) and removal treatments (F 2,26 = 1.426, P < 0.05), as shown by results of CCA (Fig. 4). The first CCA axis, explaining 11.5% of the variation in community composition, discriminated between those (a) (b) Fig. 2. (a) Plant community resistance, recovery and resilience in watered plots (i.e. control for continuous grazing disturbance, white bars) and drought plots (grey bars) following the removal treatment. (b) Resistance of dominant, subordinate and transient species in drought plots (grey bars) and watered plots (i.e. control, white bars) following the removal treatment (C, D, S). Percentages correspond to the contribution of each species group to the community biomass. Significant treatment effects indicated in each graph (*P < 0.05, **P < 0.01, ***P < 0.001). Bars topped with different letters are significantly different (P < 0.05). Control = without removal, D = random removal of dominant biomass, S = removal of subordinate species.

7 Subordinates promote resistance to drought 769 (a) (b) Fig. 4. Species-treatment plot resulting from canonical correspondence analysis (CCA) showing composition of the plant community (subordinate species excluded) in September 2010 after the experimental summer drought. Treatments include removal (C, D and S) and drought (Watered, Drought). Axes 1 and 2 account for 11.5% (F 1,26 = 3.72, P < 0.001) and 5.3% (F 1,26 = 1.72, P = 0.04) of the explained variance, respectively. Control = without removal, removal D = random removal of dominant biomass, removal S = removal of subordinate species. Species names referring to species abbreviations are listed in Appendix S2 in Supporting information. Fig. 3. (a) Linear relationships between post-drought community biomass (September 2010) and (a) pre-drought community biomass (July 2010), and (b) pre-drought biomass of subordinate plant species (July 2010). Regressions include drought plots without plant removal and with partial removal of dominant biomass. plant communities that were watered and drought plots. Most dominant and transient species were associated with watered plots; only the dominant species Taraxacum officinale and the transient species Plantago media were associated with drought resistance. The second CCA axis, explaining 5.3% of the variation in community composition, discriminated plant communities between control plots (C and D) and plots where subordinate species were removed (S). The common grasses of the study site, Poa pratensis, Poa annua and Festuca pratensis, were associated with C and D plots, whilst some of the transient species (Stellaria graminea, Prunella vulgaris, Rumex acetosa and, in particular, Leucanthemum vulgare) were strongly associated with plots where subordinates species were removed. CARBON ISOTOPE COMPOSITION Carbon isotope ratios (d 13 C) of plant leaves collected in control plots (i.e. without removal) ranged between 31.5& and 27.9& (Fig. 5a). The d 13 C values were significantly higher in drought plots than in watered plots (F 1,31 = , P < 0.001). The interaction effect between plant species and drought was not significant (F 1,36 = 0.729, P = 0.40), indicating that the drought effect on d 13 C values was similar for dominant and subordinate species. When comparing d 13 C values of plant leaves collected in plots without removal (C) with plots that had subordinate species removed (S), there were significant main effects of the drought and removal treatments, and there was a significant drought 9 removal interaction effect (F 1,32 = , P < 0.01; Fig. 5b). In watered plots, d 13 C of plant leaves was higher in S plots than that in C plots, whereas no effects of the removal treatment were found in drought plots. Discussion Using a field experiment, we tested the effects of subordinate plant species removal, summer drought and their interactive effects on plant community insurance and plant community composition in semi-natural grasslands. The experimental summer drought significantly shifted plant community composition (Fig. 4), and the removal of subordinates decreased the resistance of the plant community to drought (Fig. 2). Following the removal treatments, plant community composition was different in communities without subordinates compared with intact communities. Some studies have shown that species removal generally affects the remaining plant community, but with a time-lag in the response of plant groups or plant species (Wardle et al. 1999; Symstad & Tilman 2001). Other

8 770 P. Mariotte et al. (a) (b) Fig. 5. Carbon isotope ratios (d 13 C) after the experimental summer drought in plant leaves of (a) two subordinate species (Veronica chamaedrys, Achillea millefolium) and two dominant species (Taraxacum officinale, Alchemilla monticola) in watered plots (white bars) and drought plots (grey bars) without plant removal (C), and (b) two dominant species (Taraxacum officinale, Alchemilla monticola) in watered and drought plots without plant removal (C) and with removal of subordinate species (S). Significant treatment effects indicated in each graph (*P < 0.05, **P < 0.01, ***P < 0.001). Bars topped with different letters are significantly different (P < 0.05), and differences between species in watered plots are represented by small letters and by capital letters in drought plots. studies have shown changes in plant composition in response to drought (Bloor et al. 2010; Craine et al. 2011; Weißhuhn, Auge & Prati 2011). Few studies so far have explicitly considered which species groups (e.g. functional groups or trait groups) are most sensitive to drought. In our study, however, we found that the proportion of dominant species decreased in response to drought, whereas the proportion of subordinate and transient species increased. Our results indicate that the changes in plant community composition through the removal of subordinates negatively affected community resistance. As expected, drought significantly reduced plant community resistance compared with the control (around 20% vs. 60%). However, the decline in resistance was about 10 times higher in plots where subordinates had been removed than in plots without removal. This finding can be explained by the lower resistance of dominant and transient species in communities without subordinates, indicating that, if present, the subordinates facilitated the regrowth of their dominant and transient neighbours during the drought. In constructed grassland communities, Jentsch et al. (2011) found that plant competitive and facilitative effects strongly increased in response to severe drought, and therefore, plant plant interactions might be a key factor in community resistance against drought. Further, subordinate species produced more biomass after the first cut, which simulated cattle grazing (Fig. 2b), and this confirms findings from a previous study which showed that subordinates regrow more after grazing (Tahmasebi Kohyani et al. 2009). Importantly, we showed that subordinate species insured the plant community to drought by a highly significant increase in their biomass in drought compared with watered plots. These findings may be explained by reduced competitiveness of dominant species, who were strongly negatively affected by the drought, and/or by a physiological adaptations of subordinates to drought such as better WUE or a deeper rooting system. Measurements of 13 C isotope discrimination have been used as an integral measure for plant WUE (Farquhar, Ehleringer & Hubick 1989; Seibt et al. 2008). Significant increase in d 13 C values in plant biomass in response to drought indicated a better WUE, which is supported by other drought experiments in temperate grasslands where leaf gas exchange has been measured (Gilgen et al. 2010; Otieno et al. 2012). Our results showed that drought effects on WUE were similar for the dominant and subordinate species, suggesting that dominant and subordinate species did not differ in their physiological adaptation to drought. Therefore, it seems most likely that the increase in resistance of subordinate species in drought plots resulted from reduced competition by dominant species (Kardol et al. 2010a). Mariotte et al. (2012a) showed that reduced root competition from dominant species increased the competitive abilities of subordinate species, which may then grow better below-ground and produce more above-ground biomass through root shoot competition interactions. In addition, Kahmen, Perner & Buchmann (2005) observed an increase in below-ground biomass after drought in a highly diverse grassland, which was strongly dependent on plant community composition. We suggest that the reduced competitiveness of dominant species benefits subordinate species, especially below-ground, and thereby enhances their above-ground biomass production during drought. Below-ground dynamics remain relatively understudied in biodiversity and climate change research, although it is increasingly recognized that plant soil interactions, such as microbial feedbacks may play an important role in the effects of subordinate species on drought insurance (e.g. Kardol et al. 2010b; Hawkes et al. 2011). We showed that the presence of subordinate species considerably increased plant community resistance against drought, but further analysis, at the plot level, also showed that the positive effects of subordinate species depended on the predrought community biomass. Whilst plant diversity had no impact on the resistance to drought, we demonstrated that plant communities were more resistant in low-productive plots than in high-productive plots and, importantly, that this was due to the increasing biomass of subordinate species when productivity decreased (Fig. 3). These findings provide further support for the biomass-dependent hypothesis as proposed by Wang, Yu & Wang (2007), showing that higher resistance to

9 Subordinates promote resistance to drought 771 drought observed in low-productive plots is linked to higher biomass of drought-resistant subordinate species. We, thereby, demonstrate the importance of low productivity for the persistence of subordinate species and, hence, for the plant community resistance to drought. These findings also emphasize the necessity to take into account the pre-drought biomass when testing the effects of drought on ecosystem functioning. The removal of subordinates had no significant effect on plant community recovery after drought. Nevertheless, we observed a trend of lower recovery where subordinates were removed. We hypothesize that subordinate species would have been more influential after repeated drought events, which are forecasted in the near future. Only few studies so far have explored effects of recurrent summer drought in grasslands. However, the few results so far indicate that there is no uniform grassland response to recurrent drought, but that sites with high annual precipitation are better buffered against summer drought (Gilgen & Buchmann 2009). Moreover, in a 5-year drought experiment, Jentsch et al. (2011) showed that climate extremes affected many ecosystem-regulating functions (e.g. gas exchange, water and nutrient cycling), whilst plant community biomass production was maintained. We suggest that future research should investigate recurrent drought event along productivity and/or precipitation gradients to better understand long-term plant community dynamics and changes in ecosystem functions under future climate scenarios. In the Swiss Jura Mountains, plant communities seem to be highly resilient to a single drought event, as our results showed that 1 year after the perturbation, plant communities produced the same amount of biomass in drought and control plots. However, the resilience of the plant community was significantly reduced in plots where subordinate species were removed (Fig. 2a), indicating that community above-ground biomass decreased and was negatively affected by the continuous, 3-year removal of subordinate species. Interestingly, in watered plots, the removal of subordinate species decreased the WUE of dominant species. We hypothesize that the absence of subordinates induces a stress for neighbouring species. The filtering hypothesis (Grime 1998) suggests that the important role of subordinate species on ecosystem functioning is related to facilitation of the recruitment of dominant species by creating favourable niches. Indeed, subordinates may increase water availability for neighbouring plants, for example, through interactions with mycorrhizal fungi, which have been found to benefit subordinate species more than dominant species (Grime 1987; Van der Heijden et al. 1998; Mariotte et al. 2012b). Therefore, our results also highlight the importance of subordinate species for ecosystem functioning as suggested by previous studies (Lyons et al. 2005; Polley et al. 2006; Mariotte et al. 2012c). Despite the difficulty of testing the insurance hypothesis, we have demonstrated that in species-rich grassland communities, subordinate species, a key component of plant diversity, are a main driver of community resistance to drought. Our findings show the importance of ecosystem-level impacts of these lowabundant plant species, which are generally not included in experiments with randomly assembled communities (Bardgett & Wardle 2010). In contrast, the plant removal approach, which we used here for subordinate species and which has been used in previous studies for plant functional groups (Wardle et al. 1999; Symstad & Tilman 2001) and early successional plant communities (Polley et al. 2006), has the advantage of testing for impacts of the loss of plant species in real ecosystems (Diaz et al. 2003). The negative effect of diversity on drought resistance, as observed in constructed grasslands (Pfisterer & Schmid 2002; Van Peer et al. 2004; De Boeck et al. 2008; Van Ruijven & Berendse 2010), might be due to the random assemblage of plant species, which generally neglects the competitive hierarchy amongst species in the community. However, more diverse communities have a higher chance of including drought-resistant subordinate species, which could provide an explanation for the positive effects of diversity on drought resistance, as observed in natural grasslands (Tilman & Downing 1994; Kahmen, Perner & Buchmann 2005). In addition to species richness per se, our subordinate species hypothesis provides additional explanation for biodiversity effects on community insurance against drought and highlights the importance of plant competitive hierarchies as key components of community stability. Future research should explicitly consider the mechanisms by which subordinate species maintain above-ground biomass production and increase community resistance during drought stress. We predict that below-ground plant traits, in particular interactions with microbial communities, play an important role. 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