Introduction 1995 1999 1992 1999 1996 2002 1998 1998 2001 2002 1996 1999 2000 1998 2001 2002 2002 1992; 1994 1996 2001 2003 1996 2002 1985 1990 1996 1997 2004 2006 Trifolium repens 2006 1999 T. repens In this study we tested the following specific hypotheses: In the absence of herbivores, systemically induced ramets of clonal plants perform worse than uninduced ramets of the same genotype. This is due to costs of defense induction when defense is not needed. In the presence of herbivores, induced ramets of clonal plants perform better than uninduced plants, due to an enhanced protection through induced defense. T. repens Materials and methods Study organisms T. repens Spodoptera exigua 2004 Pre-growth of plant material We started the experiment with 64 cuttings of each of the five genotypes. The cuttings were planted in pairs in plastic trays (16 cm × 12 cm × 5 cm) using sterilized clay grains as a substrate (Seramis; Masterfoods, Germany). Each tray was fertilized weekly with 50 ml full-strength Hoagland solution before the start of the experiment. At the beginning of the experiment, all cuttings consisted of a main stolon with at least eight fully developed ramets. If present, side branches were removed immediately before starting the experiment. Experimental design 1 2 T. repens Fig. 1 white gray circles upper drawing wavy black lines lower drawing  Systemic induction of resistance S. exigua 2006 T. repens In order to enhance plant interactions, induced and control plants were grown together in the competition tray. Since all plants growing together belonged to the same genotype, induction effects cannot be confounded with genetic differences in plant traits, including competitive ability, between induced and control plants. All measurements described below were performed on ramets growing in the competition trays. Costs of SIR Costs of defense induction were measured as a reduction in plant performance. Costs can be measured after initial herbivore damage (and consequent defense induction) in the absence of subsequent herbivore attacks. To quantify costs of defense induction we measured the following traits which are known to be closely related to plant performance and fitness: total biomass production, relative biomass allocation to leaves, petioles, stolons, and roots, number and length of the main and side stolons and number of ramets on the main and side stolons. We also measured the petiole length, petiole dry mass, leaf area, leaf dry mass of the fourth and fifth youngest ramets of each cutting. Benefits of SIR To quantify benefits of SIR we exposed the plants in the competition tray to a second, controlled herbivory attack (referred to as “herbivory treatment”). We released five fourth instar caterpillars on day 16 in the competition tray and then added two and three more on day 17 and 18, respectively, to achieve substantial levels of herbivore damage. The plants were harvested 19 days after the start of the experiment. We quantified benefits of induced resistance by scoring herbivory damage in the induced and in the control plants. At the time of harvesting each ramet on the main stolon was classified according to the leaf area consumed. We visually estimated the damage and assigned each ramet a damage category ranging from 0 to 3. The values corresponded to the following amounts of damage: 0 = no damage, 1 = 1–33%, 2 = 33–66% and 3 = 66–100% of leaf area consumed. We also recorded the position of the damaged ramet on the stolon to investigate possible intra-clonal variation in the damage pattern according to ramet age. In addition to the degree of damage, we measured the dry mass of leaves, petioles, stolons and roots in induced and control plants. Herbivore preference test S. exigua M P n Statistical analysis  df 1984 1990 Costs of SIR Repeated measures ANOVA was performed to test for costs of defense induction in number and length of the main and side stolons and number of ramets on the main and side stolons, relative biomass allocation to roots, stolons, petioles and leaves and petiole length, petiole dry mass, leaf area, leaf dry mass of the fourth and fifth youngest ramets. Defense induction (induced vs. control) was considered a within-subjects effect, and genotype was treated as a between-subjects effect. Absolute dry masses of roots, stolons, petioles and leaves were analyzed using two-way repeated measures ANOVA (within-subjects effect—defense induction; between-subjects effects—plant genotype and herbivory). Benefits of SIR The amount of damage in the herbivory treatment was assessed with doubly repeated measures ANOVA using ramet age and defense induction as repeated factors and genotype as main effect. The analysis included a profile analysis (SAS procedure GLM; profile statement) to test for differences in the degree of damage between adjacent ramets on the stolons. To correct for differences in the developmental stage of different cuttings we used only the six youngest ramets of each cutting in the damage analysis. All analyses were conducted with SAS 9.1 (SAS Institute, Cary, N.C.). Results Costs of SIR 1 2 P 3 Table 1 Repeated measures ANOVA for effects of genotype, herbivory and defense induction on roots, stolons, petioles, leaves and total dry mass Source df Root Stolon Petioles Leaves Total MS F MS F MS F MS F MS F Between-subject effects Genotype (Gen) 4 981 7.20*** 3,710 4.12*** 3,741 10.97*** 11,002 10.85*** 60,377 8.99*** Herbivory (Herb) 1 28.1 0.21 78.6 0.09 1,646 4.83* 4,234 4.18* 11,926 1.78 Gen × Herb 4 49.3 0.36 124 0.14 105 0.31 292 0.29 1,171 0.17 Error 30 136  899  342  1,014  6,714  Within-subject effects Induction (Ind) 1 79.8 0.81 574 1.97 416 5.11* 98.6 0.37 1,879 0.99 Ind × Gen 4 53.9 0.54 83.1 0.28 50.7 0.62 367 1.37 1,054 0.55 Ind × Herb 1 106 1.07 57.1 0.20 11.1 0.14 160 0.60 1,143 0.60 Ind × Gen × Herb 4 139 1.40 542 1.86 159 1.95 265 0.99 3,525 1.85 Error 30 99.0  292  81.3  268  1,901  P P Table 2 Costs of systemic induced resistance (SIR). Repeated measures ANOVA for effects of genotype and defense induction on relative dry mass allocation to roots, stolons, petioles and leaves on plants without an herbivory treatment in the competition tray Source df Roots Stolons Petioles Leaves MS F MS F MS F MS F Between-subjects effects Genotype (Gen) 4 29.8 6.61**  496 14.57*** 22.7 3.54* 35.7 7.3** Error 15 4.5  127  6.4  4.9  Within-subject effects Induction (Ind) 1 12.0 2.06 0.04 0.01 5.3 1.92 35.8 7.32* Ind × Gen 4 2.2 0.38 8.2 1.16 1.8 0.64 10.9 2.24 Error 15 5.8  7.1  2.8  4.9  P P P Table 3 Costs Benefits  Root (mg) Stolons (mg) Petioles (mg) Leaves (mg) Total (mg) Costs Uninduced 15.6 ± 3.0 (4.5 ± 0.7%) 103.2 ± 6.7 (34.9 ± 1.0%) 72.6 ± 4.8 (24.9 ± 0.6%) 107.5 ± 8.3 (35.7 ± 0.7%) 298.9 ± 21.1 Induced 11.3 ± 2.1 (3.4 ± 0.6%) 96.1 ± 6.0 (34.8 ± 1.0%) 67.3 ± 4.5 (24.2 ± 0.5%) 106.8 ± 8.3 (37.6 ± 0.6%) 281.6 ± 19.1 Benefits Uninduced 14.5 ± 2.2 (5.1 ± 0.8%) 99.5 ± 4.8 (37.6 ± 0.8%) 62.8 ± 3.7 (23.8 ± 0.8%) 90.1 ± 5.6 (33.5 ± 0.8%) 266.9 ± 13.2 Induced 14.8 ± 3.7 (4.8 ± 0.9%) 95.8 ± 6.1 (36.8 ± 1.1%) 59.0 ± 4.5 (22.6 ± 0.6%) 95.1 ± 7.6 (35.8 ± 0.6%) 264.7 ± 19.7 4 P Table 4  Costs of SIR. Repeated measures ANOVA for effects of genotype and defense induction on plant fitness and performance-related traits in the absence of herbivores Source df Ramet no. main stolon Length main stolon Ramet no. side stolons Length side stolons Side stolons number Fourth ramet petiole length Fourth ramet area MS F MS F MS F MS F MS F MS F MS F Between-subjects effects Genotype (Gen) 4 2.38 3.41* 23.5 5.4*** 71.9 6.72** 30.8 1.63 16.0 12.97*** 18.0 14.93*** 8.57 15.36*** Error 15 0.69  4.31  10.7  18.9  1.23  1.20  0.55  Within-subject effects Induction (Ind) 1 1.80 12.13** 7.57 2.45 1.25 0.25 0.78 0.23 0.15 0.31 4.38 5.22* 0.10 0.23 Ind × Gen 4 0.17 1.19 3.92 1.27 1.83 0.37 4.10 1.22 0.12 0.24 0.35 0.42 0.09 0.21 Error 15 0.14  3.08  4.95  3.37  0.51  0.84  0.46  P P P 4 P P Benefits of SIR S. exigua 5 P 2 2 Table 5 Benefits of SIR. Doubly repeated measures ANOVA for effects of genotype, defense induction and ramet age on leaf area loss due to herbivory Source df MS F Between-subjects effects Genotype (Gen) 4 1,102 2.39† Error 15 461  Within-subjects effects Induction (Ind) 1 6,847 63.92*** Ind × Gen 4 244 2.28 Error (induction) 15 107  Age 5 18,988 133.0*** Age × Gen 20 630 4.41*** Error (age) 75 142  Ind × Age 5 125 1.22 Ind × Age × Gen 20 172 1.67† Error (Ind × Age) 75 103  P P Fig. 2  asterisks P ns  1 P P 3 3 F P 5 P 2 2 2 5 5 P Discussion 1996 1999 Costs of SIR Biomass production and allocation Lepidium virginicum 2005 1997 Reduction in developmental growth rate 1992a 1992b 1997 1998 Reduction in petiole length T. repens 1997 1997 2006 1990 2003 2004 2005 Chenopodium album Benefits of SIR Reduced damage 1995 Nicotiana tabaccum 2000 1963 2004 1999 T. repens In conclusion, the present study shows that in the short term, the activation of early-warning responses in clonal plant networks has both costs and benefits. In the absence of herbivores, the performance of the induced phenotype was compromised as compared to the uninduced phenotype in terms of potential competitive ability. In the presence of herbivores, the induced phenotype was favored by suffering considerably less herbivore damage suggesting potential advantages for the phenotype correctly matching its environment. Whether this represents an adaptive value of the induced responses remains to be demonstrated in longer-term studies where the initial small changes observed in our study can be measured directly in terms of fitness. The long-term balance of costs and benefits of induced resistance in clonal plant networks is likely to be strongly context dependent and a function of the match between spatio-temporal aspects of systemic defense expression and the feeding behavior of herbivores.