We collected immature and adult female Pardosa milvina from Miami University's

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1 1 Supplementary Information: Fear of predation alters soil CO 2 flux and nitrogen content 2 3 Supplemental methods Organism collection and maintenance We collected immature and adult female Pardosa milvina from Miami University's Ecology Research Center ( N, W). Spiders were housed individually in plastic containers (7cm wide x 6cm tall) with a substrate of commercially available peat moss and potting soil (1:1 mixture), provided water ad libitum, and given two crickets (Acheta domesticus) once a week. All spiders were used in experiments one week after their most recent feeding. Sinella curviseta were derived from the Crossley Culture ( and reared communally under similar conditions with the exception that food was provided in the form of a sliced potato and baker's yeast. Only large (approx. 2mm) S. curviseta were used in experiments. All study organisms were maintained in an environmental chamber with a 13h:11h light:dark cycle, at 25ºC. No organisms were used in more than one trial Experimental setup The experiment was conducted within horizontally-oriented Mason jars (8.5cm wide x 15.5cm tall, 947mL), each containing 60g of 1:1 mixture of peat moss:potting soil. We added 1g of dried straw (3cm long pieces) on top of the soil to provide habitat structure and a substrate for microbe growth. Jars were cleaned with ethanol between trials. 23

2 CO 2 measurements We placed a glass vial with 10mL 0.25M NaOH centrally in each jar immediately after adding detritivores, and the vial was replaced with a new vial every 24h for four days. Total daily CO 2 flux was quantified by titration with 0.25M HCl to determine the quantity of CO 2 absorbed following established procedures [1] Soil content measurements We dried soil and straw at 60ºC for 48h and followed established protocols [2]. Samples were ground and analyzed for C and N content using a Flash 2000 Combustion NC soil analyzer (CE Elantech, Inc., Lakewood, NJ, USA). Sub-samples were taken to determine organic carbon content by ashing samples at 550ºC for 4h and subtracting post-ashed C content from pre-ashed C content and correcting for mass lost on ignition Statistical analyses We used simple linear regression to test the relationship between the consumption of detritivores by predators and the total daily CO 2 flux on the last day of the experiment (n = 21-32). We tested for correlation between consumption of detritivores by predators and soil content (i.e., %C, % organic C, %N, and C:N) using MANOVA. CO 2 flux dynamics were analyzed using repeated measures ANOVA, with treatment as a factor (n = 17-21). Differences between treatments in total C, organic C, and C:N were analyzed with one-way ANOVA (n = 18-20). All analyses were conducted on unmanipulated and corrected values (see main text), and Welch's tests were used instead of ANOVA when groups had unequal variances. All analyses were carried out using JMP (version 9.0; SAS Institute, Inc., Cary, NC, USA).

3 Supplemental results Detritivore survival Consumption of detritivores was negatively correlated with CO 2 flux on the last day of the experiment (r = 0.8, 95% CI = ; = R 2 = 0.65, p < 0.01; Figure 1). The proportion of detritivores surviving at the end of the experiment did not correlate with any measures of soil content (MANOVA: F 4,12 = 0.18, p = 0.72) CO 2 flux dynamics All treatments started at a similar state and fluctuated over time (Figure 2), with a significant time effect and an interaction between time and treatment despite there being no significant overall treatment effect (Table 1). Unmanipulated CO 2 flux values for the last day of the experiment illustrate the impact of detritivores, as they significantly increased CO 2 values (Figure 3, Table 2) Soil C, organic C, and C:N content All treatments resulted in a slight increase in soil carbon content compared to the control (Figure 4a, Table 4). This effect was driven by the addition of detritivores, as corrected values revealed no differences between the detritivore treatment and either the cue or predation treatment (Figure 4b, Table 4). The carbon in the soil was over 99% organic, so the treatment effects on organic carbon content are qualitatively the same as for total carbon (Figure 5, Table 5). Differences between treatments in soil C:N are a product of the impacts of detritivores, predator cues, and predation on carbon and nitrogen content, and their impacts on soil C:N were fairly uniform across treatments (Figure 6a). The impact of detritivores on soil nitrogen drove

4 70 statistical differences between treatments for corrected C:N values (Figure 6b, Table 6) References [1] Fisk MC, Schmidt SK, Seastedt TR Topographic patterns of above- and belowground production and nitrogen cycling in alpine tundra. Ecology 79, (doi: / (1998)079[2253:tpoaab]2.0.co;2) [2] Vanni MJ, Renwick WH, Bowling AM, Horgan MJ, Christian AD Nutrient stoichiometry of linked catchment-lake systems along a gradient of land use. Freshwater Biol. 56, (doi: /j x)

5 80 Tables of treatment effects on CO 2 flux and soil content Table 1. Treatment effects on unmanipulated CO 2 flux dynamics (repeated measures ANOVA). 83 F df p Time ,72 <0.01 Treatment 0.1 3, Time*Treatment 5.5 3,74 <0.01 Sample size: B (20), D (21), C (20), P (17)

6 Table 2. Treatment effects on unmanipulated CO 2 flux from the last day of the experiment. Statistics reported are Cohen's d, 95% confidence intervals, and F-values, degrees of freedom, and p-values. Treatments are: blank (B), cues (C), predation (P), detritivore (D). Symbols between treatment letters indicate relationships based on effect sizes. CO 2 (ml 24h -1 ) Cohen's d 95% CI F df p D > B 0.7 (0.0, 1.3) C = B 0.0 (-0.6, 0.6) 3.2 3, P = B -0.1 (-0.7, 0.6) Sample size: B (20), D (21), C (20), P (17)

7 Table 3. Treatment effects on unmanipulated soil N content. Statistics reported are Cohen's d, 95% confidence intervals, and F-values, degrees of freedom, and p-values. Treatments are: blank (B), cues (C), predation (P), detritivore (D). Symbols between treatment letters indicate relationships based on effect sizes. % nitrogen Cohen's d 95% CI F df p D > B 0.8 (0.2, 1.4) C = B 0.5 (-0.2, 1.1) 2.8 3, P = B 0.5 (-0.1, 1.2) Sample size: B (20), D (20), C (19), P (18)

8 Table 4. Treatment effects on soil C content. All tests were performed on unmanipulated and corrected values (see methods). Statistics reported are Cohen's d, 95% confidence intervals, and F-values, degrees of freedom, and p-values. Treatments are: blank (B), cues (C), predation (P), detritivore (D). Symbols between treatment letters indicate relationships based on effect sizes. % carbon % carbon (corrected) Cohen's d 95% CI F df p Cohen's d 95% CI F df p D > B 0.6 (0.0, 1.3) C = D 0.1 (-0.5, 0.8) C > B 0.8 (0.1, 1.4) 2.3 3, P = D 0.0 (-0.6, 0.6) 2.6 2, P = B 0.6 (-0.1, 1.2) C = P -0.1 (-0.8, 0.5) Sample size: B (20), D (20), C (19), P (18)

9 Table 5. Treatment effects on soil organic C content. All tests were performed on unmanipulated and corrected values (see methods). Statistics reported are Cohen's d, 95% confidence intervals, and F-values, degrees of freedom, and p-values. Treatments are: blank (B), cues (C), predation (P), detritivore (D). Symbols between treatment letters indicate relationships based on effect sizes. % organic carbon % organic carbon (corrected) Cohen's d 95% CI F df p Cohen's d 95% CI F df p D = B 0.6 (-0.1, 1.2) C = D 0.1 (-0.5, 0.8) C > B 0.7 (0.0, 1.3) 1.9 3, P = D 0.0 (-0.6, 0.6) 2.0 2, P = B 0.5 (-0.1, 1.2) C = P -0.1 (-0.8, 0.5) Sample size: B (19), D (20), C (19), P (18)

10 Table 6. Treatment effects on soil C:N. Statistics reported are Cohen's d, 95% confidence intervals, and F-values, degrees of freedom, and p-values. Treatments are: blank (B), cues (C), predation (P), detritivore (D). Symbols between treatment letters indicate relationships based on effect sizes. C:N C:N (corrected) Cohen's d 95% CI F df p Cohen's d 95% CI F df p D = B -0.3 (-0.9, 0.3) C = D 0.6 (0.0, 1.3) C = B 0.4 (-0.3, 1.0) 1.6 3, P = D 0.4 (-0.3, 1.0) 4.5 2, P = B 0.1 (-0.6, 0.7) C = P -0.4 (-1.1, 0.2) Sample size: B (20), D (20), C (19), P (18)

11 Figure 1. Relationship between detritivores consumed by predators and total CO 2 flux on the last day of the experiment Figure 2. Unmanipulated CO 2 flux dynamics (mean +SE) Figure 3. Unmanipulated CO 2 flux on the last day of the experiment. Box plots show median, first and third quartiles, greatest values within 1.5 interquartile range, and outliers Figure 4. Soil C content for both unmanipulated (A) and corrected values (B). Box plots show median, first and third quartiles, greatest values within 1.5 interquartile range, and outliers Figure 5. Soil organic C content for both unmanipulated (A) and corrected values (B). Box plots show median, first and third quartiles, greatest values within 1.5 interquartile range, and outliers Figure 6. Soil C:N for both unmanipulated (A) and corrected values (B). Box plots show median, first and third quartiles, greatest values within 1.5 interquartile range, and outliers.

12 CO 2 (ml 24h 1 ) Proportion of S i nel l a consumed by P ard osa

13 CO 2 (ml 24h 1 ) Blank Cues Predation Detritivore Day

14 CO 2 (ml 24h 1 ) Blank Cues Predation Detritivore

15 Blank Cues Predation Detritivore Cues Predation Detritivore % carbon A % carbon (corrected) B

16 Blank Cues Predation Detritivore Cues Predation Detritivore % organic carbon A % organic carbon (corrected) B

17 Blank Cues Predation Detritivore Cues Predation Detritivore C:N A C:N (corrected) B