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1 2 3 1 SUPPLEMENTAL SEED INCREASES NATIVE SEEDLING ESTABLISHMENT IN 4 5 6 2 ROADSIDE PRAIRIE RESTORATION 7 8 3 9 10 11 4 Submission Category: Research Article 12 1 1,3 1,2 13 5 Authors: Jessica L. Riebkes , Mark E. Sherrard , Laura L. Jackson 14 15 6 Affiliations: 1Department of Biology, University of Northern Iowa, Cedar Falls, Iowa, 50614- 16 17 18 7 0421 For Peer Review 19 20 8 2 Tallgrass Prairie Center, 2412 West 27th Street, Cedar Falls, Iowa, 50614-0294 21 22 9 3 Corresponding Author: Mark Sherrard, [email protected] 23 24 25 10 Running head: Supplemental seed in roadside prairie restoration 26 27 11 28 29 12 Author contributions: JLR, LLJ conceived and designed the research; JLR performed the 30 31 32 13 experiment; JLR, MES analyzed the data; JLR, MES, LLJ wrote and edited the manuscript; MES 33 34 14 made the figures. 35 36 37 15 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Restoration Ecology Page 2 of 38

1 2 3 1 ABSTRACT 4 5 6 2 Tallgrass prairie restorations are plagued by high seed costs and low rates of seedling 7 8 3 establishment. Many restorations suffer high rates of seed loss to granivores, yet to date, there 9 10 11 4 are no established protocols to minimize their impact. In this study, we tested whether the 12 13 5 application of supplemental (sacrificial) seed reduces native seed consumption and increases 14 15 6 native seedling establishment in roadside prairie restoration. We applied supplemental birdseed 16 17 18 7 to a random subset of researchFor plots Peer at three roads Reviewide prairie restoration sites and compared rates 19 20 8 of seed consumption and early native seedling establishment between supplemental seed plots 21 22 9 and control plots. All three roadside restorations were seeded in fall 2014, immediately following 23 24 25 10 the first frost. To assess native seed consumption, we monitored rates of seed removal from 26 27 11 “seed cards” during the first 14 days of the restorations. To assess early seedling establishment, 28 29 12 we identified and counted all native seedlings in mid-July of the first restoration year. The 30 31 32 13 application of supplemental seed did not reduce rates of seed consumption, which were very low 33 34 14 during the early stages of these restorations, but did increase early native seedling establishment. 35 36 37 15 Native seedling establishment was approximately 37% higher in supplemental seed plots than in 38 39 16 control plots across restoration sites. The application of supplemental seed may have increased 40 41 17 seedling establishment by reducing consumption of native seed during winter and spring. Our 42 43 44 18 results suggest that supplemental seed is a practical, inexpensive technique for increasing 45 46 19 seedling establishment in roadside prairie restoration. 47 48 20 49 50 51 21 52 53 22 Keywords: buffet experiment, roadside restoration, seed predation, seedling establishment, 54 55 23 supplemental seed, tallgrass prairie restoration 56 57 58 59 60 Page 3 of 38 Restoration Ecology

3 1 2 3 24 IMPLICATIONS FOR PRACTICE 4 5 6 25 • This study suggests that it is possible to mitigate seed loss due to granivores and increase 7 8 26 native seedling establishment in roadside prairie restoration by using supplemental seed. 9 10 11 27 • One mechanism through which increased seedling establishment could improve the 12 13 28 success of roadside prairie restoration is by reducing weed biomass. High weed biomass 14 15 29 can delay or prevent native establishment, reduce native richness and diversity, and 16 17 18 30 increase managementFor costs Peer in prairie restoration. Review 19 20 31 • In this study, supplemental seed increased the cost of roadside prairie restoration by 21 22 23 32 approximately 16% while boosting seedling establishment by approximately 37%. This 24 25 33 suggests that practitioners may be able to reduce native seeding rates, and therefore net 26 27 34 costs, by using supplemental seed in roadside prairie restoration. 28 29 30 35 31 32 36 33 34 37 35 36 37 38 38 39 39 40 41 40 42 43 44 41 45 46 42 47 48 49 43 50 51 44 52 53 45 54 55 56 46 57 58 59 60 Restoration Ecology Page 4 of 38

4 1 2 3 47 INTRODUCTION 4 5 6 48 The tallgrass prairie ecosystem, which once covered approximately 100 million ha of North 7 8 49 America, now occupies less than 3 percent of its original expanse (Sampson & Knopf 1994; 9 10 11 50 Smith 1998; Smith et al. 2010). Restoration practitioners are attempting to reestablish this 12 13 51 endangered ecosystem and the services it once provided, but these projects are notoriously costly 14 15 52 (Gerla et al. 2012). A low-diversity seed mixture (20-30 species) can cost between $500 and 16 17 -1 -1 18 53 $1500 ha , while a high-diversityFor Peermixture (50-70 Review species) can cost as much as $5000 ha 19 20 54 (Prairie Moon Nursery 2013). Low seedling establishment is a major contributor to this cost. 21 22 55 Restoration practitioners typically sow between 400 and 950 pure live seeds (PLS) m-2 to achieve 23 24 -2 25 56 a final stand density of 30 adult plants m (Smith et al. 2010; Williams 2010): an establishment 26 27 57 rate of 3.1 – 7.5%. Identifying the causes of seed loss in native prairie restoration, and 28 29 58 developing protocols to minimize this loss, could improve restoration success. 30 31 32 59 33 34 60 Small vertebrate granivores, such as meadow voles, field mice, and birds, can be a significant 35 36 37 61 cause of seed loss in tallgrass prairie restorations. Previous studies have shown that these 38 39 62 granivores can reduce seed number in prairie restorations (Howe & Brown 1999; Clark & 40 41 63 Wilson 2003; Hemsath 2007) and alter the composition of the emerging community (Howe & 42 43 44 64 Brown 1999, 2000). Further, a recent study found that small vertebrate granivores reduce native 45 46 65 seedling emergence by approximately 30% in newly restored prairies (Pellish et al. in press). 47 48 66 This reduction in seedling emergence could lead to higher weed establishment, which could 49 50 51 67 delay or prevent native seedling establishment, increase management costs, and reduce the 52 53 68 overall quality of the restoration (Schramm 1990; Blumenthal et al. 2003, 2005; Grman & 54 55 69 Suding 2010; Dickson et al. 2010; Martin & Wilsey 2012; Nemec et al. 2013). To date, there are 56 57 58 59 60 Page 5 of 38 Restoration Ecology

5 1 2 3 70 no established protocols for reducing the impact of small vertebrate granivores in native tallgrass 4 5 6 71 prairie restoration. 7 8 72 9 10 11 73 One technique that could reduce vertebrate granivory during prairie restoration is the use of 12 13 74 chemical feeding deterrents. Capsaicin, for example, is an effective feeding deterrent against 14 15 75 mammalian granivores (Levey et al. 2006) and increases seedling recruitment for some prairie 16 17 18 76 species during restorationFor (Hemsath Peer 2007). Similarl Reviewy, the fungicide Thiram emits a sulfurous 19 20 77 odor that repels birds and deer mice from agricultural seed (Nolte & Barnett 2000; Ngowo et al. 21 22 78 2005). However, this technique may not be feasible for prairie restoration because of the variable 23 24 25 79 morphology of prairie seeds (size, shape, and texture) and the environmental exposure that seeds 26 27 80 experience before germination. Another technique that could reduce vertebrate granivory during 28 29 81 prairie restoration is the application of supplemental (sacrificial) seed. The use of supplemental 30 31 32 82 seed is based on the evolutionary principles of mast seeding (Janzen 1971; Kelly 1994; Kelly et 33 34 83 al. 2000; Kelly & Sork 2002) and optimal diet theory (Sih & Christensen 2001). Mast seeding is 35 36 37 84 the intermittent production of large synchronized seed crops. In high seed years, the seed crop 38 39 85 satiates granivores, reducing overall seed loss. Optimal diet theory suggests that granivores 40 41 86 should preferentially consume seeds that provide higher net energy intake per unit handling time 42 43 44 87 (Janzen 1971; Pulliam & Brand 1975; Kerley & Erasmus 1991; Sih & Christensen 2001). Based 45 46 88 on these principles, providing granivores with an abundant, higher-calorie seed source should 47 48 89 reduce consumption of native seeds during restoration. If so, the application of supplemental 49 50 51 90 seed could be a practical, inexpensive technique for improving restoration success. 52 53 91 54 55 56 57 58 59 60 Restoration Ecology Page 6 of 38

6 1 2 3 92 Roadsides are an integral component of restoration efforts in Iowa, U.S.A. (Smith 1998; Houseal 4 5 6 93 & Smith 2000). In 1988, Iowa legislation established the integrated roadside vegetation 7 8 94 management (IRVM) program and the Living Roadway Trust Fund to support the restoration 9 10 11 95 and protection of native vegetation in the state’s roadsides (Brandt et al. 2015). Since that time, 12 13 96 more than 20,000 ha of roadside have been restored to native prairie (Iowa Department of 14 15 97 Transportation 2017). In addition to improving the aesthetics of Iowa’s roadsides, these 16 17 18 98 restoration projects reduceFor soil erosion, Peer improve wReviewater quality, reduce herbicide use, and provide 19 20 99 valuable habitat for wildlife (Christiansen & Lyons 1975; Flynn 1994; Ries et al. 2001; Brandt et 21 22 100 al. 2015). Improving the success of these roadside restoration projects will contribute to the 23 24 25 101 overall recovery of the tallgrass prairie ecosystem. 26 27 102 28 29 103 In this study, we examine the effect of supplemental seed addition on roadside prairie restoration. 30 31 32 104 We applied supplemental birdseed to research plots at three roadside restoration sites and 33 34 105 compared rates of seed consumption and native seedling establishment between supplemental 35 36 37 106 seed plots and control plots. We predicted that the application of supplemental seed would 38 39 107 reduce consumption of native seeds during the initial stages of the roadside restoration and 40 41 108 increase early native seedling establishment. 42 43 44 109 45 46 110 47 48 111 49 50 51 112 52 53 113 54 55 56 57 58 59 60 Page 7 of 38 Restoration Ecology

7 1 2 3 114 METHODS 4 5 6 115 Study Sites 7 8 116 This research was conducted in 2014 and 2015 at three county roadside restoration sites in Iowa, 9 10 11 117 U.S.A. One site was located in Linn County, Iowa and two sites were located in Benton County, 12 13 118 Iowa (Fig. S1). For convenience, we refer to these as the Linn, Benton North (Benton N) and 14 15 119 Benton South (Benton S) sites throughout the article. The roadsides were regraded in summer 16 17 18 120 2014 and seeded with Fornative prairie Peer vegetation in Reviewfall 2014 by the Linn and Benton County 19 20 121 Secondary Roads Departments. 21 22 122 23 24 25 123 Linn - The Linn County restoration site was 0.11 ha in size and located in Marion, Iowa (42° 2’ 26 27 124 6.6” N; 91° 31’ 13” W, Fig. S1). The site was on the south side of Marion Airport Road, which 28 29 125 runs east to west. Prior to restoration, the site was dominated by nonnative grasses, including: 30 31 32 126 Agropyron repens L. (Quack grass), Bromus inermis Leyss. (Smooth brome), Medicago sativa L. 33 34 127 (Alfalfa), Poa pratensis L. (Kentucky bluegrass), and Trifolium pratense L. (Red clover). 35 36 37 128 Management consisted of biannual roadside mowing and spot-spraying or mowing of noxious 38 39 129 weeds. The soil at Linn is classified as Klinger-Maxfield silty clay loams (Natural Resources 40 41 130 Conservation Service 2013). In summer 2014, the vegetation and a layer of topsoil were stripped 42 43 44 131 from the site. On August 6 2014, the site was hydroseeded with a cover crop of Avena sativa L. 45 46 132 (Common oats; 5.6 g m-2), Elymus canadensis L. (Canada wild rye; 0.11 g m-2), and Secale 47 48 133 cereale L. (Cereal rye; 5.6 g m-2) using a Finn T-90 hydroseeder (Finn Corporation, Fairfield, 49 50 51 134 OH, U.S.A.) and then cultipacked with a Reinco 6-foot mulch disc cultipacker (Reinco Inc., 52 53 135 Fairfield, NJ, U.S.A.). 54 55 136 56 57 58 59 60 Restoration Ecology Page 8 of 38

8 1 2 3 137 On September 26 2014, we established six research plots at Linn. The plot sizes were 37 m (east 4 5 6 138 to west) × 5 m (north to south). The first frost at Linn occurred on October 30 2014. On 7 8 139 November 12 2014, the Linn County Secondary Roads Department drill-seeded the site with a 9 10 -2 -2 11 140 30-species mixture (Table S1) at the rate of 1.174 g m (576.5 seeds m ) using a Truax grass 12 13 141 drill (Truax Company, Inc., New Hope, MN, U.S.A.). The site was mowed on June 23 2015, to 14 15 142 manage weeds and prevent the cover crop from becoming the dominant vegetation. 16 17 18 143 For Peer Review 19 20 144 Benton N and Benton S - The Benton County restoration sites were located near Atkins, Iowa 21 22 145 (Benton N: 41° 59’ 7” N; 91° 51’ 13” W, Benton S: 41° 57’ 29” N; 91° 51’ 13” W, Fig. S1). 23 24 25 146 Benton N was 0.3 ha in size and Benton S was 0.55 ha in size and both were located on the east 26 27 147 side of 33rd Avenue / West 28 th Street in a portion of the roadside that runs north to south. Prior 28 29 148 to restoration, both sites were dominated by the nonnative grass Bromus inermis Leyss. (Smooth 30 31 32 149 brome) and management consisted of occasional spraying with 2-4D or Milestone for noxious 33 34 150 weeds. The soils at Benton N and Benton S are classified as Kenyon loam (Natural Resources 35 36 37 151 Conservation Service 2013). In late summer 2014, the vegetation and a layer of topsoil were 38 39 152 stripped from the site to make the ditch wider, less steep, and to create a shoulder for the road. 40 41 153 On September 8 2014, the sites were planted with a cover crop of Triticum aestivum (Winter 42 43 - 44 154 wheat; 5.6 g m ). The cover crop was drill-seeded on the bottoms and foreslopes with a Truax 45 46 155 Flex II drill (Truax Company, Inc., New Hope, MN, U.S.A.) and hydroseeded on the backslopes 47 48 156 using a Finn hydroseeder (Finn Corporation, Fairfield, OH, U.S.A.). On September 26 2014, 49 50 51 157 eight plots were established at Benton N and 15 plots were established at Benton S. Each plot 52 53 158 was 10 m (east to west) × 37m (north to south). The first frost at the Benton sites occurred on 54 55 159 October 23 2014. On October 29 2014, the Benton County Secondary Roads Department drill- 56 57 58 59 60 Page 9 of 38 Restoration Ecology

9 1 2 3 160 seeded the foreslopes and bottoms of both sites with a 35-species mixture (Table S1) at a rate of 4 5 -2 -2 6 161 5.6 g m (3014.9 seeds m ) using a Truax Flex II drill (Truax Company, Inc., New Hope, MN, 7 8 162 U.S.A.). The backslopes were hydroseeded with the same seed mixture on the same day at the 9 10 -2 11 163 same seeding rate (5.6 g m ) using a Finn hydroseeder (Finn Corporation, Fairfield, OH, 12 13 164 U.S.A.). 14 15 165 16 17 18 166 In spite of their close proximityFor and Peer identical see dingReview protocol, we treated Benton N and Benton 19 20 167 S as separate sites because of differences in ditch profile and hydrology. In particular, Benton N 21 22 168 was a steeper, dryer, upland site, while Benton S was a flatter, wetter, lowland site. 23 24 25 169 26 27 170 Supplemental Seed 28 29 171 The supplemental seed mixture consisted of an equal proportion (by mass) of four types of 30 31 32 172 birdseed: black oil sunflower ( Helianthus annuus L.), Nyjer thistle ( Guizotia abyssinica L. f. 33 34 173 Cass.), white millet ( Panicum miliaceum L.), and cracked corn ( Zea mays L.). All seed was 35 36 37 174 obtained from Cedar River Milling Company (Waterloo, IA, U.S.A.). We added mineral salt to 38 -2 39 175 the supplemental seed mix at the rate of 4.1 g m to increase palatability (Weeks & Kirkpatrick 40 41 176 1976; Robbins 1983). To ensure that the supplemental seed would not germinate, we roasted the 42 43 44 177 sunflower and millet seeds at 180°C for 30 minutes (Corbineau et al. 2002). To confirm that the 45 46 178 seed was not viable, we attempted to germinate approximately 1 kg of roasted seed and detected 47 48 179 no germination. The thistle seed was pre-sterilized and the corn was cracked, which renders the 49 50 51 180 embryos non-viable. 52 53 181 54 55 56 57 58 59 60 Restoration Ecology Page 10 of 38

10 1 2 3 182 We applied the supplemental seed treatment immediately after the native species were seeded 4 5 6 183 (October 29 2014 at Benton N and Benton S; November 12 2014 at Linn). Supplemental seed 7 8 184 was applied to approximately half of the research plots: three (of six) plots at Linn, four (of 9 10 11 185 eight) plots at Benton N, and eight (of 15) plots at Benton S (Fig. S1). The plots receiving the 12 13 186 supplemental seed were chosen randomly. Supplemental seed was applied at ten times the 14 15 187 seeding rate of the native seed (56 g m -2 at Benton N and Benton S; 11.74 g m -2 at Linn) using a 16 17 18 188 hand broadcast seed spreaderFor (PlantMates Peer model Review76300, PlantMates LLC, Pasadena, TX, 19 20 189 U.S.A.). 21 22 190 23 24 25 191 Seed Removal Experiment 26 27 192 To assess granivory during the initial stages of these restorations, we monitored the rate of seed 28 29 193 removal from “seed cards” using a buffet-style experiment (e.g., Westerman et al. 2003; 30 31 32 194 Heggenstaller et al. 2006). The seed cards consisted of 30 well-filled Echinacea pallida seeds 33 34 195 glued to an 11 cm x 14 cm piece of coarse sand paper (3M Paper Sheet 346U, 36 Grit, aluminum 35 36 37 196 oxide commercial D-weight, 3M Company, Maplewood, MN, U.S.A.). To attach the seeds, we 38 39 197 applied a base layer of aerosol spray adhesive (3M Super 77 Multipurpose Adhesive Aerosol, 40 41 198 3M Company, Maplewood, MN, U.S.A.) to the sand paper, placed the 30 seeds on the adhesive, 42 43 44 199 waited 24 hours, and then covered the seeds with another layer of aerosol spray adhesive. 45 46 200 Previous research suggests that the adhesive and sandpaper do not attract or deter predators from 47 48 201 the seeds (Westerman et al. 2003). We allowed the adhesive to dry for 48 hours before placing 49 50 51 202 the cards in the field. Seed cards were affixed to the soil using 5.1 cm roofing nails. We chose 52 53 203 Echinacea pallida because of seed morphology (relatively small, yet manageable to work with 54 55 204 and identify for counting) and because it was in the seed mixture at each restoration site. 56 57 58 59 60 Page 11 of 38 Restoration Ecology

11 1 2 3 205 4 5 6 206 We conducted two seed card trials in this experiment. First, we conducted a seven-day trial 7 8 207 before the sites were seeded (“pre-planting trial”: September 26 – October 3 2014) to assess 9 10 11 208 background levels of granivory at each site. All remaining seeds were counted on the seventh 12 13 209 day of this trial (October 3 2014) to quantify seed loss. Any seed that was missing, chewed, or 14 15 210 broken was considered consumed. Second, we conducted a 14-day trial after the sites were 16 17 18 211 seeded (“post-plantingFor trial”: October Peer 29 – Novembe Reviewr 12 2014 at Benton N and Benton S; 19 20 212 November 12 – 26 2014 at Linn). Remaining seeds were counted on the seventh and 14 th day of 21 22 213 this trial to quantify seed loss. We used the same protocol in the pre- and post-planting trials. 23 24 25 214 Specifically, we placed seven seed cards, equidistant to one another, along a transect at the center 26 27 215 of each plot (Fig. S2). To assess passive seed loss to factors such as wind, rain, adhesive failure 28 29 216 or flaws in card design, we placed a control seed card next to one, randomly-selected seed card 30 31 32 217 in each plot. The control seed card was placed inside a metal cage (32 cm x 14 cm x 8 cm) and 33 34 218 surrounded by insect barrier cloth (Agribon + AG 15, 118’’ X 50’, lightweight grade) to exclude 35 36 37 219 small vertebrate granivores and invertebrate granivores respectively. We compared rates of seed 38 39 220 loss between the 7-day pre-planting trial and the seventh day of the post-planting trial. In the 40 41 221 post-planting trial, we compared rates of seed loss between supplemental seed plots and control 42 43 44 222 plots. 45 46 223 47 48 224 Seedling Establishment Experiment 49 50 51 225 We identified and counted all established seedlings at each roadside restoration site on July 20 - 52 53 226 24 2015. Because two seeding techniques were used at the Benton sites (drill seeding on 54 55 227 foreslopes and bottoms, hydroseeding on backslopes), we divided the ditch into three sections 56 57 58 59 60 Restoration Ecology Page 12 of 38

12 1 2 3 228 (foreslope, bottom, backslope; Fig. S2) at each site and quantified seedling establishment within 4 5 6 229 each section. The bottoms were defined as having a 0-5 degree slope, while the foreslopes (the 7 8 230 section next to the road) and backslopes were defined as having a slope equal to or greater than 9 10 11 231 five degrees as determined by a clinometer. In each plot, we identified and counted all seedlings 12 2 13 232 in five, 0.1-m quadrats in each section of ditch profile (i.e., 15 quadrats per plot). To minimize 14 15 233 variation and provide a buffer between adjacent plots, all sampling was localized in the central 16 17 18 234 five meters (of the longFor axis) of eachPeer plot (Fig. S 2).Review Within that five-meter sampling area, 19 20 235 quadrats were placed at five random positions along a transect at the center (by width) of each 21 22 236 ditch section (Fig. S2). All native seedlings from the planted seed mixtures were counted and 23 24 25 237 identified; however, we pooled the two Liatris species and the two Carex species to avoid 26 27 238 misidentification of similar species (Natural Resources Conservation Service 2006; Williams 28 29 239 2010). Any identified seedlings that were not in the seed mixture were counted as weeds. 30 31 32 240 33 34 241 Data Analysis 35 36 37 242 In the seed removal experiment, we counted the remaining seeds on each seed card and 38 39 243 quantified seed loss for each plot as the seven-card average. Less than one percent of seeds were 40 41 244 lost from control cards, and consequently, we did not correct the data for passive seed loss. We 42 43 44 245 analyzed the seed removal data using two different general linear models. First, we compared 45 46 246 seed loss between the seven-day pre-planting trial and the first seven days of the post-planting 47 48 247 trial in a model that had trial (pre-planting vs. post-planting) and site as fixed factors. For this 49 50 51 248 analysis, we only used data from control plots in the post-planting trial because of the potential 52 53 249 confounding effect of the supplemental seed addition. Second, we compared seed loss between 54 55 250 supplemental seed plots and control plots at the end of the 14-day post-planting trial in a model 56 57 58 59 60 Page 13 of 38 Restoration Ecology

13 1 2 3 251 that had treatment (supplemental seed plots vs. control plots) and site as fixed factors. The 4 5 6 252 treatment × site term was removed from both models because of non-significance. Removal of 7 8 253 this term did not alter the significance of any factor in either model. 9 10 11 254 12 13 255 In the seedling establishment experiment, we counted the total number of established native 14 15 256 seedlings in the five 0.1-m2 quadrats and divided by 0.5 to compute the number of established 16 17 2 18 257 native seedlings per m For in each section Peer of the ditch Review profile. To test whether the application of 19 20 258 supplemental seed affected seedling establishment, we used a general linear model with 21 22 259 treatment, site, and section of ditch profile as fixed factors. 23 24 25 260 26 27 261 In both the seed removal experiment and seedling establishment experiment, we inspected the 28 29 262 data for normality using boxplots and scatter plots (qqnorm plots) of model residuals-versus- 30 31 32 263 predicted values. Data from the seed removal experiment did not require transformation. Data 33 34 264 from the seedling establishment experiment was cube-root transformed to improve normality. All 35 36 37 265 data were analyzed in R (v. 3.1-109, R Core Team 2013). 38 39 266 40 41 267 42 43 44 268 45 46 269 47 48 270 49 50 51 271 52 53 272 54 55 273 56 57 58 59 60 Restoration Ecology Page 14 of 38

14 1 2 3 274 RESULTS 4 5 6 275 Seed Removal Experiment 7 8 276 Granivores consumed significantly more seed in the seven-day pre-planting trial (24.8%) than in 9 10 11 277 the first seven days of the post-planting trial (4.3%, F=26.15, p<0.001, Fig. 1, Table S2). In the 12 13 278 14-day post-planting trial, granivores only consumed 7.4% of seeds and seed consumption did 14 15 279 not differ significantly between supplemental seed plots and control plots ( F=1.35, p=0.256, Fig. 16 17 18 280 1, Table S3). For Peer Review 19 20 281 21 22 282 Seedling Establishment Experiment 23 24 -2 25 283 On average, 128.4 ± 12.9 native seedlings m (mean ± SE) established at the three roadside 26 27 284 restoration sites (Fig. 2). Seedling establishment differed between sites ( F=3.52, p=0.035, Table 28 29 285 S4) and was significantly higher at Benton N (184.2 ± 32.3) than at Linn (103.6 ± 29.4). 30 31 32 286 Seedling establishment at Benton S (107.2 ± 11.0) did not differ significantly from either of the 33 34 287 other two sites. Because the seeding rate was lower at Linn than at the Benton sites, the seedling 35 36 37 288 establishment rate was higher at Linn (18%) than at Benton N (6.1%) and Benton S (3.6%). The 38 39 289 seedling establishment rates of each species are summarized in Table S5. 40 41 290 42 43 44 291 Seedling establishment was significantly higher in supplemental seed plots (157.3 ± 19.3) than in 45 46 292 control plots (99.6 ± 16.0) across roadside restoration sites ( F=13.11, p<0.001, Fig. 2, Table S4). 47 48 293 Seedling establishment was higher in supplemental seed plots than in control plots at each 49 50 51 294 individual site; however, this difference was only significant at Linn (Fig. 2). 52 53 295 54 55 296 Seedling establishment differed significantly between sections of the ditch profile across 56 57 58 59 60 Page 15 of 38 Restoration Ecology

15 1 2 3 297 restoration sites (F=10.32, p<0.001, Table S4, Fig. 3). Seedling establishment was significantly 4 5 -2 6 298 higher on the foreslopes (196.1 ± 25.5 seedlings m ) than on the bottoms (87.9 ± 15.3 seedlings 7 8 299 m-2) and backslopes (101.3 ± 19.6 seedlings m-2). On the backslopes, seedling establishment was 9 10 11 300 marginally higher in supplemental seed plots than in control plots (p=0.07; Fig. 3). Conversely, 12 13 301 supplemental seed addition did not affect seedling establishment on the foreslopes or bottoms 14 15 302 (Fig. 3). There was a marginally significant site × section of ditch profile term (F=2.41, p=0.057, 16 17 18 303 Table S4), which may Forhave occurred Peer because seedling Review establishment on the foreslopes and 19 20 304 bottoms was highest at Benton N while seedling establishment on the backslopes was highest at 21 22 305 Linn (Fig. 3). Seedling establishment was higher in supplemental seed plots than in control plots 23 24 25 306 in every site × section of ditch profile treatment combination; however, this difference was never 26 27 307 statistically significant (Fig. 3). 28 29 308 30 31 32 309 33 34 310 35 36 37 311 38 39 312 40 41 313 42 43 44 314 45 46 315 47 48 316 49 50 51 317 52 53 318 54 55 319 56 57 58 59 60 Restoration Ecology Page 16 of 38

16 1 2 3 320 DISCUSSION 4 5 6 321 In this study, we tested whether the application of supplemental seed influences the success of 7 8 322 roadside prairie restoration. To do this, we compared rates of native seed consumption (seed 9 10 11 323 removal experiment) and native seedling establishment (seedling establishment experiment) 12 13 324 between supplemental seed plots and control plots at three newly restored roadside sites. In the 14 15 325 seed removal experiment, we found that the rate of seed loss was very low during the initial 16 17 18 326 stages of these restorationsFor and was Peer not influenced Review by the supplemental seed addition. In the 19 20 327 seedling establishment experiment however, we found that seedling establishment was 21 22 328 approximately 37% higher in supplemental seed plots than in control plots across restoration 23 24 25 329 sites. Collectively, our results suggest that the application of supplemental seed increases native 26 27 330 seedling establishment in roadside prairie restoration and that this may be due to lower 28 29 331 consumption of native seed during winter and spring. 30 31 32 332 33 34 333 Supplemental seed may have increased seedling establishment in roadside restorations by 35 36 37 334 reducing consumption of native seed. Two mechanisms through which supplemental seed could 38 39 335 reduce native seed consumption are predator satiation and predator manipulation. Predator 40 41 336 satiation is related to seed density. At higher seed densities, granivores consume a lower 42 43 44 337 proportion of available seed (e.g., Cardina et al. 1996; Edwards & Crawley 1999), thus leaving 45 46 338 more native seeds to germinate and become established. This interpretation is consistent with the 47 48 339 evolutionary principle of mast seeding as a defense against granivores (Janzen 1971; Kelly 49 50 51 340 1994). Predator manipulation is related to optimal diet theory. Preferential consumption of the 52 53 341 more abundant, higher-calorie birdseed may have resulted in lower consumption of the less 54 55 56 57 58 59 60 Page 17 of 38 Restoration Ecology

17 1 2 3 342 abundant, lower-calorie native seed (Janzen 1971; Kerley & Erasmus 1991; Sih & Christensen 4 5 6 343 2001). These two mechanisms are not mutually exclusive and could be operating in combination. 7 8 344 9 10 11 345 The effect of supplemental seed on seedling establishment was remarkably consistent in our 12 13 346 study, suggesting that this technique may improve the success of a wide variety of roadside 14 15 347 restoration projects. The three restoration sites used in our study differed in profile and 16 17 18 348 hydrology, and were restoredFor using Peer different seed Review mixtures, seeding rates, and seeding methods. 19 20 349 In spite of this variability, native seedling establishment was always higher in supplemental seed 21 22 350 plots than control plots (at every site, in every section of the ditch profile, and in every site × 23 24 25 351 section of ditch profile treatment combination) although the effect was not always significant. 26 27 352 Previous studies have shown that granivores are an important cause of seed loss in prairie 28 29 353 restorations and significantly reduce native seedling emergence (e.g., Howe & Brown 1999; 30 31 32 354 2000; Clark & Wilson 2003; Pellish et al. 2017). Our study supports these conclusions and 33 34 355 suggests that the application of supplemental seed is an effective technique for reducing this 35 36 37 356 impact and improving the success of roadside restoration projects. 38 39 357 40 41 358 Differences in seedling establishment between sections of the ditch profile could be related to 42 43 44 359 soil moisture. Seedlings on the roadside bottoms had to endure periods of standing water, which 45 46 360 may have lead to higher mortality and lower establishment in this section. On the foreslopes and 47 48 361 backslopes, soil moisture may have been influenced by aspect (the direction a slope faces). In the 49 50 51 362 Northern hemisphere, north- and east-facing slopes are relatively wetter than south- and west- 52 53 363 facing slopes at the same elevation. This can lead to higher establishment on north- and east- 54 55 364 facing slopes in roadside restorations (Bochet & García-Fayos 2004; Bochet et al. 2007). We 56 57 58 59 60 Restoration Ecology Page 18 of 38

18 1 2 3 365 found mixed support for this interpretation. Seedling establishment was indeed higher on the 4 5 6 366 east-facing foreslope than the west-facing backslope at Benton N (ANOVA: F = 12.503, p = 7 8 367 0.004) and marginally higher on the east-facing foreslope than the west-facing backslope at 9 10 11 368 Benton S (ANOVA: F = 3.251, p = 0.083); but seedling establishment was not higher on the 12 13 369 north-facing backslope than the south-facing foreslope at Linn (ANOVA: F = 0.195, p = 0.703). 14 15 370 In spite of these overall differences in seedling establishment, the influence of supplemental seed 16 17 18 371 on seedling establishmentFor did not Peerdiffer between seReviewctions (non-significant treatment × section 19 20 372 term: F = 0.58, p = 0.58, Table S4). This suggests that practitioners should apply supplemental 21 22 373 seed evenly to all sections of the ditch profile, regardless of differences in soil moisture. 23 24 25 374 26 27 375 In contrast to our prediction, the application of supplemental seed did not reduce native seed 28 29 376 consumption during the initial stages of roadside restoration. Our seed removal experimental did 30 31 32 377 however highlight temporal variation in rates of seed predation. Seed predation varies temporally 33 34 378 based on consumer presence and activity (Howe & Brown 2000; Heggenstaller et al. 2006). In 35 36 37 379 our study, we detected significantly higher seed removal in the seven-day pre-planting trial, 38 39 380 which occurred before the first frost, than the first seven days of the post-planting trial, which 40 41 381 occurred after the first frost. These results suggest that invertebrates, which would have been 42 43 44 382 present in the pre-planting trial and absent from the post-planting trial, are an important cause of 45 46 383 seed loss in roadside restoration. The impact of invertebrate granivores on prairie restoration is 47 48 384 poorly understood, but previous studies have shown that invertebrates are an important cause of 49 50 51 385 seed loss for some prairie and weed species in the Midwestern United States (Clark & Wilson 52 53 386 2003; Gaines & Gratton 2010). In contrast to invertebrates, mammalian granivores may have 54 55 387 avoided these roadside restoration sites because there was low vegetative cover in the roadsides 56 57 58 59 60 Page 19 of 38 Restoration Ecology

19 1 2 3 388 and surrounding agricultural fields (Heggenstaller et al. 2006; Baraibar et al. 2009). Rodents can 4 5 6 389 be important consumers of prairie seed during winter (Westerman et al. 2008), when seed loss 7 8 390 was not being monitored in our study. Future studies should attempt to quantify seed loss 9 10 11 391 throughout the restoration process to better understand peak predation times for different 12 13 392 granivores. This data would help practitioners determine the optimum time to apply 14 15 393 supplemental seed. Although seed loss was low during the initial stages of these restoration, 16 17 18 394 consumption of nativeFor seed was likelyPeer higher in conReviewtrol plots than supplemental seed plots at 19 20 395 some point during the restoration process because of the ultimate difference in seedling 21 22 396 establishment between treatments. 23 24 25 397 26 27 398 Management Implications 28 29 399 One way that supplemental seed could improve the outcome of roadside restoration projects is by 30 31 32 400 reducing weed establishment. Restorations with high native establishment typically have fewer 33 34 401 weeds (Blumenthal et al. 2003, 2005; Middleton et al. 2010; Carter & Blair 2012; Nemec et al. 35 36 37 402 2013). The establishment of weeds can delay or even prevent native seedling establishment, and 38 39 403 ultimately reduce native richness and diversity in prairie restorations (Blumenthal et al. 2003; 40 41 404 Martin and Wilsey 2012). High weed biomass can also increase the management costs ofprairie 42 43 44 405 restoration. Another way that supplemental seed could improve roadside restoration success is by 45 46 406 reducing granivore-induced effects on community composition. Granivores can alter community 47 48 407 composition by preferentially consuming certain species (Howe & Brown 1999). The presence 49 50 51 408 of high-calorie birdseed may reduce the likelihood that native species with relatively high-calorie 52 53 409 seeds will be preferentially consumed and thereby increase the overall richness and diversity of a 54 55 410 restoration. 56 57 58 59 60 Restoration Ecology Page 20 of 38

20 1 2 3 411 4 5 6 412 Because seed costs are already a prohibitive aspect of prairie restoration, it is important to 7 8 413 consider the cost and benefits of supplemental seed. Using the native seed mixture (IRVM 9 10 -2 -2 11 414 Diversity Mix) and seeding rates (prairie seed = 1.174 g m , supplemental seed = 11.74 g m ) at 12 -1 13 415 Linn as an example, the cost of native seed was $811 ha (personal communication, Kristine 14 15 416 Nemec, University of Northern Iowa) and the cost of supplemental seed was $129 ha -1. 16 17 18 417 Therefore, supplementalFor seed increased Peer the total seReviewed cost of the restoration project by 19 20 418 approximately 16% ($940 ha -1). Practitioners may be able to offset this additional cost by 21 22 419 reducing native seeding rates. The application of supplemental seed increased native seedling 23 24 25 420 establishment by 37%. If restoration practitioners could achieve the same native seedling 26 27 421 establishment with 37% fewer native seeds, it would reduce the total seed cost to $640 ha -1 28 29 422 (prairie seed: $511 ha -1; supplemental seed: $129 ha -1): a 21% reduction in seed cost. It should be 30 31 32 423 noted however, that an increase or decrease in native seeding rates does not always correspond to 33 34 424 a proportional increase or decrease in native seedling establishment (Williams and Smith 2007). 35 36 37 425 Future research should examine different combinations of native and supplemental seeding rates 38 39 426 to determine the optimum combination from an economic perspective. 40 41 427 42 43 44 428 Roadside restoration projects are an important component of restoration efforts in Iowa, U.S.A. 45 46 429 (Smith 1998; Houseal & Smith 2000) and contribute to the overall recovery of the tallgrass 47 48 430 prairie ecosystem. There are several unique challenges associated with roadside restoration 49 50 51 431 projects, including: slope, aspect, disturbance (e.g., gravel deposition via snowplow), and 52 53 432 environmental heterogeneity (e.g., soil moisture, light and nutrient availability, soil type). Our 54 55 433 study suggests that supplemental seed significantly increases native seedling establishment 56 57 58 59 60 Page 21 of 38 Restoration Ecology

21 1 2 3 434 during the critical early stages of roadside restoration projects. This technique could help 4 5 6 435 practitioners overcome the challenges of roadside restoration. 7 8 436 9 10 11 437 12 13 438 ACKNOWLEDGEMENTS 14 15 439 We thank J. Abernathy, K. Elgersma, G. Houseal, K. Nemec, D. Williams, and J. Young for 16 17 18 440 helpful comments and Fordiscussions Peer on the manuscript. Review We thank R. Roman and B. Bonar for 19 20 441 seeding and managing the roadside sites. We thank A. Andrews, J. Cubit, W. Pingel, O. 21 22 442 Schouten, C. Swanson, and D. Williams for assistance in the field. We thank D. Williams for 23 24 25 443 assistance with seedling identification. This work was supported by the Living Roadway Trust 26 27 444 Fund and the University of Northern Iowa. 28 29 445 30 31 32 446 33 34 447 35 36 37 448 38 39 449 40 41 450 42 43 44 451 45 46 452 47 48 453 49 50 51 454 52 53 455 54 55 456 56 57 58 59 60 Restoration Ecology Page 22 of 38

22 1 2 3 457 LITERATURE CITED 4 5 6 458 Baraibar B, Westerman PR, Carrión E, Recasens J (2009) Effects of tillage and irrigation in 7 8 459 cereal fields on weed seed removal by seed predators. Journal of Applied Ecology 9 10 11 460 46:380-387 12 13 461 Blumenthal DM, Jordan NR, Russelle MP (2003) Soil carbon addition controls weeds and 14 15 462 facilitates prairie restoration. Ecological Applications 13:605-615 16 17 18 463 Blumenthal DM, JordanFor NR, Svenson Peer EL (2005) ReviewEffects of prairie restoration on weed 19 20 464 invasions. Agriculture, Ecosystems & Environment 107:221-230 21 22 465 Bochet E, García-Fayos P (2004) Factors controlling vegetation establishment and water erosion 23 24 25 466 on motorway slopes in Valencia, Spain. Restoration Ecology 12:166-174 26 27 467 Bochet E, García-Fayos P, Alborch B, Tormo J (2007) Soil water availability effects on seed 28 29 468 germination account for species segregation in semiarid roadslopes. Plant and Soil 30 31 32 469 295:179-191 33 34 470 Brandt J, Henderson K, Uthe J (2015) Integrated Roadside Vegetation Management Technical 35 36 37 471 Manual. Tallgrass Prairie Center, University of Northern Iowa, Cedar Falls, Iowa, U.S.A. 38 39 472 Cardina J, Norquay HM, Stinner BR, McCartney DA (2011) Postdispersal predation of 40 41 473 velvetleaf ( Abutilon theophrasti ) seeds. Weed Science 44:534-539 42 43 44 474 Carter DL and Blair JM (2012) High richness and dense seeding enhance grassland restoration 45 46 475 establishment but have little effect on drought response. Ecological Applications 47 48 476 22:1308-1319 49 50 51 477 Christiansen PA, Lyons DL (1975) Roadside vegetation management. Report to the Iowa 52 53 478 Department of Transportation, Ames, Iowa, U.S.A. 54 55 479 Clark DL, Wilson MV (2003) Post-dispersal seed fates of four prairie species. American Journal 56 57 58 59 60 Page 23 of 38 Restoration Ecology

23 1 2 3 480 of Botany 90:730-735 4 5 6 481 Corbineau F, Gay-Mathieu C, Vinel D, Come D (2002) Decrease in sunflower ( Helianthus 7 8 482 annuus ) seed viability caused by high temperature as related to energy metabolism, 9 10 11 483 membrane damage and lipid composition. Physiologia Plantarum 116:489-496 12 13 484 Dickson TL, Wilsey BJ, Busby RR, Gebhart DL (2010) Melilotus officinalis (yellow sweet 14 15 485 clover) causes large changes in community and ecosystem processes in both the presence 16 17 18 486 and absence ofFor a cover crop. Peer Biological InvasionsReview 12:65-76 19 20 487 Edwards GR, Crawley MJ (1999) Rodent seed predation and seedling recruitment in mesic 21 22 488 grassland. Oecologia 118:288-296 23 24 25 489 Flynn L (1994) Iowa roadsides return to native prairies. Roads and Bridges 32:61 26 27 490 Gaines HR, Gratton C (2010) Seed predation increases with ground beetle diversity in a 28 29 491 Wisconsin (USA) potato agroecosystem. Agriculture, Ecosystems & Environment 30 31 32 492 137:329-336 33 34 493 Gerla PJ, Cornett MW, Ekstein JD, Ahlering MA (2012) Talking big: lessons learned from a 35 36 37 494 9000 hectare restoration in the northern tallgrass prairie. Sustainability 4:3066-3087 38 39 495 Grman E, Suding KN (2010) Within-year soil legacies contribute to priority effects during 40 41 496 establishment of California grassland plants. Restoration Ecology 18:664-670 42 43 44 497 Heggenstaller AH, Menalled FD, Liebman M, Westerman PR (2006) Seasonal patterns in post- 45 46 498 dispersal seed predation of Abutilon theophrasti and Setaria faberi in three cropping 47 48 499 systems. Journal of Applied Ecology 43:999-1010 49 50 51 500 Hemsath CM (2007) Quantifying granivory in a reconstructed prairie: affects of season, species, 52 53 501 seed predators, sacrificial food, and the chemical deterrent capsaicin. MS Thesis, 54 55 502 University of Northern Iowa 56 57 58 59 60 Restoration Ecology Page 24 of 38

24 1 2 3 503 Houseal G, Smith D (2000) Source-identified seed: The Iowa roadside experience. Ecological 4 5 6 504 Restoration 18:173-183 7 8 505 Howe HF, Brown JS (1999) Effects of birds and rodents on synthetic tallgrass communities. 9 10 11 506 Ecology 80:1776-1781 12 13 507  (2000) Early effects of rodent granivory on experimental forb communities. Ecological 14 15 508 Applications 10:917-924 16 17 18 509 Iowa Department of TransportationFor Peer (2017) Integrated Review Roadside Vegetation Management, 19 20 510 https://iowadot.gov/lrtf/integrated-roadside-vegetation-management/irvm (accessed on 21 22 23 511 August 30 2017) 24 25 512 Janzen DH (1971) Seed predation by animals. Annual Review of Ecology and Systematics 26 27 513 2:465-492 28 29 30 514 Kelly D (1994) The evolutionary ecology of mast seeding. Trends in Ecology & Evolution 31 32 515 9:465-470 33 34 516 Kelly D, Harrison AL, Lee WG, Payton IJ, Wilson PR, Schauber EM (2000) Predator satiation 35 36 37 517 and extreme mast seeding in 11 species of Chionochloa (Poaceae). OIKOS 90:477-488 38 39 518 Kelly D, Sork VL (2002) Mast seeding in perennial plants: Why, how, where? Annual Review of 40 41 519 Ecology and Systematics 33:427-447 42 43 44 520 Kerley GIH, Erasmus T (1991) What do mice select for in seeds? Oecologia 86:261–267 45 46 521 Levey DJ, Tewksbury JJ, Cipollini ML, Carlo TA (2006) A field test of the directed deterrence 47 48 49 522 hypothesis in two species of wild chili. Oecologia 150:61-68 50 51 523 Martin LM, Wilsey BJ (2012) Assembly history alters alpha and beta diversity, exotic-native 52 53 524 proportions and functioning of restored prairie plant communities. Journal of Applied 54 55 56 525 Ecology 49:1436-1445 57 58 59 60 Page 25 of 38 Restoration Ecology

25 1 2 3 526 Middleton EL, Bever JD Schultz PA (2010) The effect of restoration methods on the quality of 4 5 6 527 the restoration and resistance to invasion by exotics. Restoration Ecology 18:181-187 7 8 528 Natural Resources Conservation Service (2006) Central region seedling ID guide for native 9 10 11 529 prairie plants. United States Department of Agriculture, Elsberry, Missouri, U.S.A. 12 13 530 Natural Resources Conservation Service (2013) Web soil survey: custom soil resource report. 14 15 531 http://websoilsurvey.nrcs.usda.gov/ (accessed September 2013) 16 17 18 532 Nemec KT, Allen CR,For Helzer CJ, PeerWedin DA (2013) Review Influence of richness and seeding density on 19 20 533 invasion resistance in experimental tallgrass prairie restorations. Ecological Restoration 21 22 534 31:168-185 23 24 25 535 Ngowo V, Mulungu LS, Lodal J, Makundi RH, Massawe AW, Leirs H (2005) Evaluation of 26 27 536 thiram and cinnamamide for protection of maize seeds against multimammate mice, 28 29 537 Mastomys natalensis , in Tanzania. Belgian Journal of Zoology 135:179-181 30 31 32 538 Nolte DL, Barnett JP (2000) A repellent to reduce mouse damage to longleaf pine seed. 33 34 539 International Biodeterioration & Biodegradation 45:169-174 35 36 37 540 Pellish CA, Sherrard ME, Leytem, Jackson LL (2017) Small vertebrate granivores reduce 38 39 541 seedling emergence in native tallgrass prairie restoration. Restoration Ecology (DOI: 40 41 542 10.1111/rec.12557) 42 43 44 543 Prairie Moon Nursery (2013) Cultural guide & pricing, https://www.prairiemoon.com/home.php 45 46 544 (accessed 30 Sep 2013) 47 48 545 Pulliam HR, Brand MR (1975) The production and utilization of seeds in plains grassland of 49 50 51 546 southeastern Arizona. Ecology 56:1158-1166 52 53 547 R Core Team (2013) R: A language and environment for statistical computing. R Foundation for 54 55 548 Statistical Computing, Vienna, Austria. https://www.R-project.org/ 56 57 58 59 60 Restoration Ecology Page 26 of 38

26 1 2 3 549 Ries L, Debinski DM, Wieland ML (2001) Conservation value of roadside prairie restoration to 4 5 6 550 butterfly communities. Conservation Biology 15:401-411 7 8 551 Robbins C (1983) Wildlife feeding and nutrition. Academic Press, New York, New York, U.S.A. 9 10 11 552 Sampson F, Knopf F (1994) Prairie conservation in North America. Bioscience 44:418–421 12 13 553 Schramm P (1990) Prairie restoration: A twenty-five year perspective on establishment and 14 15 554 management. Pages: 169-177 In: Smith DD, Jacobs CA (eds) Proceedings of the Twelfth 16 17 18 555 North AmericanFor Prairie Conference, Peer Cedar Review Falls, Iowa, U.S.A. 19 20 556 Sih A, Christensen B (2001) Optimal diet theory: when does it work, and when and why does it 21 22 557 fail? Animal Behaviour 61:379-390 23 24 25 558 Smith D (1998) Iowa prairie: original extent and loss, preservation and recovery attempts. 26 27 559 Journal of Iowa Academy of Sciences 105:94-108 28 29 560 Smith D, Williams D, Houseal G, Henderson K (2010) The Tallgrass Prairie Center guide to 30 31 32 561 prairie restoration in the upper Midwest. University of Iowa Press, Iowa City, Iowa, 33 34 562 U.S.A. 35 36 37 563 Weeks HP, Kirkpatrick CM (1976) Adaptations of white-tailed deer to naturally occurring 38 39 564 sodium deficiencies. Journal of Wildlife Management 40:610–625. 40 41 565 Westerman PR, Wes JS, Kropff MJ, Van Der Werf W (2003) Annual losses of weed seeds due to 42 43 44 566 predation in organic cereal fields. Journal of Applied Ecology 40:824-836 45 46 567 Williams D (2010) The Tallgrass Prairie Center guide to seed and seedling identification in the 47 48 568 upper Midwest. University of Iowa Press, Iowa City, Iowa, U.S.A. 49 50 51 569 Williams D, Smith DD (2007) The effects varying seeding rates of prairie grasses and forbs on 52 53 570 native plant establishment in a prairie reconstruction. Iowa Department of Transportation 54 55 571 Internal Report, Cedar Falls, Iowa, U.S.A. 56 57 58 59 60 Page 27 of 38 Restoration Ecology

27 1 2 3 572 SUPPORTING INFORMATION 4 5 6 573 Figure S1. Locations and plot layouts of the three roadside restoration sites. 7 8 574 Figure S2. Photo of roadside (highlighting sections) and schematic diagram of plots. 9 10 11 575 Table S1. Seed mixtures and seeding rates for the Linn and Benton County sites. 12 13 576 Table S2. General linear model: seed removal experiment (trial effect). 14 15 577 Table S3. General linear model: seed removal experiment (treatment effect). 16 17 18 578 Table S4. General linearFor model: seedlingPeer establishm Reviewent experiment. 19 20 579 Table S5. Average number of established seedlings m-2 of each species at each site. 21 22 580 23 24 25 581 26 27 582 28 29 583 30 31 32 584 33 34 585 35 36 37 586 38 39 587 40 41 588 42 43 44 589 45 46 590 47 48 591 49 50 51 592 52 53 593 54 55 594 56 57 58 59 60 Restoration Ecology Page 28 of 38

28 1 2 3 595 FIGURE CAPTIONS: 4 5 6 596 7 8 597 Figure 1. Seed consumption at three roadside prairie restoration sites. Seed consumption was 9 10 11 598 quantified for each plot as the average number of seeds removed from seven seeds card after 12 13 599 seven or 14 days. We present the data as the average of all plots (±1SE) at all three sites. Seed 14 15 600 consumption was significantly higher in the seven-day pre-planting trial (grey triangles) than in 16 17 18 601 the first seven days of Forthe post-planting Peer trial. Fo r Reviewthis analysis, we only used data from control 19 20 602 plots in the post-planting trial (grey circles) because of the potential confounding effect of 21 22 603 supplemental seed addition. Seed consumption did not differ between supplemental seed plots 23 24 25 604 (white circles) and control plots (grey circles) in the 14-day post-planting trial. 26 27 605 28 29 606 Figure 2. The number of established seedlings per m-2 in supplemental seed plots (white bars) 30 31 32 607 and control ploys (grey bars) at three roadside restoration sites, as well as the three-site average. 33 34 608 Data presented are means ±1SE. Significant differences between treatments, based on Tukey 35 36 37 609 post hoc tests, are indicated with asterisks. Dashed lines represent site averages (across 38 39 610 treatments) and significant differences between sites are indicated with different letters. 40 41 611 42 43 -2 44 612 Figure 3. The number of established seedlings per m in supplemental seed plots (white bars) 45 46 613 and control ploys (grey bars) in each section of the ditch profile at three roadside restoration 47 48 614 sites, as well as the three-site average. Data presented are means ±1SE. Significant differences 49 50 51 615 between treatments, based on Tukey post hoc tests, are indicated with asterisks (° indicates a 52 53 616 marginally significant [p < 0.10] difference). Dashed lines represent section averages (across 54 55 617 restoration sites). 56 57 58 59 60 Page 29 of 38 Restoration Ecology

29 1 2 3 618 Figure 1. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 619 31 32 620 33 34 35 621 36 37 622 38 39 623 40 41 42 624 43 44 625 45 46 47 626 48 49 627 50 51 628 52 53 54 629 55 56 630 57 58 59 60 Restoration Ecology Page 30 of 38

30 1 2 3 631 Figure 2. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 632 31 32 633 33 34 634 35 36 635 37 38 39 636 40 41 637 42 43 638 44 45 46 639 47 48 640 49 50 51 641 52 53 642 54 55 643 56 57 58 644 59 60 Page 31 of 38 Restoration Ecology

31 1 2 3 645 Figure 3. 4 5 6 7 8 9 10 11 12 13 14 15 16 646 17 18 For Peer Review 19 647 20 21 648 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Restoration Ecology Page 32 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 Benton N Benton S Linn 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Figure S1 . Above: County map of Iowa highlighting the location of the three restoration sites in Benton 56 and Linn Counties. Below: Plot layout at the three restoration sites: Benton N, Benton S, and Linn. 57 58 59 60 Page 33 of 38 Restoration Ecology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Figure S2. Above: Photograph of a roadside restoration site, highlighting the three sections of the ditch 50 51 profile. Center: Schematic diagram of one research plot in the seed card experiment. Seed cards were 52 placed equidistant to one another (spanning all three sections of the ditch profile) along a transect located 53 the center of the longest plot axis. A control card was placed next to one randomly selected seed card in 54 each plot. Below: Schematic diagram of one research plot in the seedling establishment experiment. 55 Sampling occurred in a 5 m strip located at the center of the longest plot axis, creating a 16 m buffer 56 between the sampling area and adjacent plot on both sides. Five 0.1-m quadrats (represented by Xs) were 57 58 randomly placed along a transect located at the center (by width) of each ditch section. 59 60 Restoration Ecology Page 34 of 38

1 2 3 Table S1. Seed mixtures for the Linn and Benton County sites. Species are listed alphabetically 4 5 and nomenclature is based on USDA plants database (USDA-NRCS 2015). 6 7 Linn Benton 8 g m -2 seeds m -2 g m -2 seeds m -2 9 Amorpha canascens 0.02 9.88 0.07 42.07 10 11 Andropogan gerardii Vitman 0.13 47.44 0.57 201.72 12 Asclepias incarnata L. . . 0.09 15.13 13 Asclepias tuberosa L. 0.02 2.66 0.07 11.29 14 Astragalus canadensis L. 0.02 10.08 0.07 42.87 15 16 Baptisia alba (L.) Vent. 0.01 0.84 0.06 3.57 17 Bouteloua curtipendula (Michx.) Torr. 0.13 28.47 0.57 121.03 18 Carex bicknellii Britton For Peer 0.00Review 11.68 0.01 49.64 19 20 Carex vulpinoidea Michx. . . 0.01 31.52 21 Chamaecrista fasciculata (Michx.) Greene 0.12 7.41 0.52 31.52 22 Dalea purpurea Vent. 0.02 12.97 0.10 55.16 23 Desmodium canadense (L.) DC. 0.01 1.63 0.04 6.93 24 25 Echinacea pallida Nutt. 0.02 4.50 0.10 19.12 26 Elymus canadensis L. 0.11 20.56 0.48 87.41 27 Eryngium yuccifolium Michx. 0.02 5.56 0.09 23.64 28 29 Helenium autumnale L. . . 0.03 136.58 30 Heliopsis helianthoides (L.) Sweet 0.03 7.01 0.13 29.79 31 Lespedeza capitata Michx. 0.01 3.95 0.06 16.81 32 33 Liatris aspera Michx. 0.02 13.84 0.10 58.83 34 Liatris pycnostachya Michx. 0.03 11.42 0.13 48.54 35 Monarda fistulosa L. 0.02 43.24 0.07 183.86 36 Oligoneuron rigidum (L.) Small 0.01 12.16 0.04 51.69 37 38 Panicum virgatum L. . . 0.48 235.34 39 Penstemon grandiflorus Nutt. 0.01 5.19 0.04 22.06 40 Ratibida pinnata (Vent.) Barnhart 0.02 25.95 0.10 110.31 41 42 Rudbeckia hirta L. 0.02 56.83 0.07 241.64 43 Ruellia humilis Nutt. 0.01 1.54 0.04 6.56 44 Scirpus atrovirens Willd. . . 0.01 144.99 45 Silphium lacinatum L. 0.01 0.20 0.04 0.83 46 47 Sorghastrum nutans (L.) Nash 0.13 56.93 0.57 242.06 48 Sporobolus compositus (Poir.) Merr. 0.13 132.84 0.57 564.81 49 Symphyotrichum novae-angliae (L.) G.L. Nesom 0.01 19.57 0.04 83.21 50 51 Tradescantia ohiensis Raf. 0.03 7.91 0.12 33.62 52 Verbena stricta Vent. 0.01 8.30 0.04 35.30 53 Zizia aurea (L.) W.D.J. Koch 0.02 5.98 0.07 25.42 54 55 1.17 576.54 5.61 3014.88 56 57 58 59 60 Page 35 of 38 Restoration Ecology

1 2 3 Table S2 . General linear model comparing seed loss between the seven-day pre-planting trial 4 5 6 and the first seven days of the post-planting trial. We only used data from the control plots of the 7 8 post-planting trial because of the potential confounding effect of supplemental seed addition. 9 10 11 Trial (pre-planting vs. post-planting) and site were fixed factors in the model. The treatment × 12 13 site interaction term was removed from the model because it was not significant. Removal of this 14 15 term did not alter the significance of any factor. Data presented are degrees of freedom ( df ), sum 16 17 18 of squares (SS), mean Forsquares (MS), Peer F-statistics ( ReviewF), and P-values ( p). Significant terms are 19 20 indicated in bold. 21 22 23 24 25 Factor df SS MS F p 26 27 trial 1 378.20 378.20 26.15 <0.001 28 site 2 6.81 3.41 0.24 0.792 29 30 residuals 23 332.64 14.46 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Restoration Ecology Page 36 of 38

1 2 3 Table S3. General linear model comparing seed loss between treatments (control plots vs. 4 5 6 supplemental seed plots) and sites in the 14-day post-planting trial. Treatment and site were fixed 7 8 factors in the model. The treatment × site interaction term was removed from the model because 9 10 11 it was not significant. Removal of this term did not alter the significance of any factor. Data 12 13 presented are degrees of freedom ( df ), sum of squares (SS), mean squares (MS), F-statistics ( F), 14 15 and P-values ( p). Significant terms are indicated in bold. 16 17 18 For Peer Review 19 20 Factor df SS MS F p 21 22 treatment 1 3.28 3.28 1.35 0.256 23 24 site 2 18.55 9.28 3.82 0.036 25 26 residuals 25 60.65 2.43 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

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1 2 3 Table S4. General linear model comparing the number of established seedlings between roadside 4 5 6 restoration sites (site), treatments (control plots vs. supplemental seed plots), sections of the ditch 7 8 profile (section) and their interactions. Site, treatment, and section were all fixed factors in the 9 10 11 model. Data were cube-root transformed to improve normality. Data presented are degrees of 12 13 freedom ( df ), sum of squares (SS), mean squares (MS), F-statistics ( F), and P-values ( p). 14 15 Significant terms are indicated in bold. 16 17 18 For Peer Review 19 20 Factor df SS MS F p 21 22 site 2 6.809 3.405 3.52 0.035 23 24 treatment 1 12.676 12.676 13.11 <0.001 25 26 section 2 19.953 9.977 10.32 <0.001 27 site × treatment 2 2.842 1.421 1.47 0.24 28 29 treatment × section 2 1.112 0.556 0.58 0.58 30 31 site × section 4 9.323 2.331 2.41 0.057 32 33 site × treatment × section 4 1.202 0.301 0.31 0.87 34 35 residuals 66 63.821 0.967 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Restoration Ecology Page 38 of 38

1 2 3 Table S5: Average number of established seedlings per m2 of each species at each roadside 4 5 restoration site and at all three sites combined (overall). Data presented are means and standard 6 errors (in parentheses). Species are ordered from most to least common across sites. 7 8 Overall Linn Benton N Benton S 9 Rudbeckia hirta 18.18 (2.70) 27.67 (4.45) 24.13 (4.48) 10.71 (1.96) 10 Echinacea pallida 11.79 (1.30) 4.67 (0.87) 14.50 (2.83) 13.29 (1.71) 11 12 Sorghastrum nutans 9.79 (2.04) 4.44 (0.70) 21.17 (6.11) 5.57 (1.40) 13 Verbena stricta 9.52 (1.31) 4.33 (0.59) 13.25 (3.02) 9.62 (1.80) 14 Heliopsis helianthoides 7.79 (0.79) 4.22 (0.69) 10.50 (1.89) 7.76 (0.87) 15 16 Penstemon grandiflorus 7.71 (1.16) 1.33 (0.41) 10.75 (2.54) 8.71 (1.62) 17 Ratibida pinnata 7.00 (1.29) 13.22 (1.53) 16.25 (2.97) 6.67 (1.07) 18 Andropogan gerardii For 6.79Peer (1.22) 3.89 Review (0.78) 13.42 (3.07) 4.24 (1.27) 19 Eryngium yuccifolium 6.36 (0.83) 1.22 (0.20) 9.00 (2.27) 7.05 (0.85) 20 21 Bouteloua curtipendula 6.24 (1.01) 4.67 (0.79) 7.58 (1.94) 6.14 (1.54) 22 Zizia aurea 5.05 (0.61) 5.67 (0.59) 4.58 (1.14) 5.05 (0.88) 23 Ruellia humilis 3.29 (0.46) 0.44 (0.12) 4.75 (1.15) 3.67 (0.56) 24 25 Sporobolus compositus 3.02 (0.87) 8.44 (1.69) 1.83 (0.60) 1.38 (0.46) 26 Carex sp. 2.86 (1.18) 3.00 (0.49) 7.50 (3.95) 0.14 (0.08) 27 Monarda fistulosa 2.57 (0.47) 1.78 (0.40) 3.58 (1.17) 2.33 (0.55) 28 Tradescantia ohiensis 2.38 (0.41) 0.00 (0.00) 1.58 (0.40) 3.86 (0.71) 29 30 Desmodium canadense 2.24 (0.32) 1.22 (0.19) 3.17 (0.81) 2.14 (0.38) 31 Elymus canadensis 2.24 (0.47) 4.89 (0.84) 1.50 (0.35) 1.52 (0.42) 32 Dalea purpurea 2.14 (0.43) 0.89 (0.20) 4.92 (1.19) 1.10 (0.34) 33 34 Liatris sp. 1.60 (0.30) 1.00 (0.29) 1.58 (0.71) 1.86 (0.36) 35 Lespedeza capitata 1.50 (0.27) 1.33 (0.21) 2.33 (0.72) 1.10 (0.28) 36 Asclepias incarnata 1.10 (0.22) . 1.92 (0.58) 1.10 (0.24) 37 Oligoneuron rigidum 0.76 (0.15) 1.00 (0.13) 0.92 (0.38) 0.57 (0.18) 38 39 Asclepias tuberosa 0.69 (0.18) 0.89 (0.15) 0.75 (0.43) 0.57 (0.22) 40 Chamaecrista fasciculata 0.67 (0.16) 2.00 (0.21) 0.67 (0.31) 0.10 (0.07) 41 Astragalus canadensis 0.52 (0.14) 0.67 (0.15) 0.92 (0.38) 0.24 (0.10) 42 43 Baptisia alba 0.31 (0.10) 0.00 (0.00) 0.58 (0.28) 0.29 (0.13) 44 Amorpha canascens 0.21 (0.11) 0.44 (0.21) 0.17 (0.12) 0.14 (0.08) 45 Panicum virgatum 0.14 (0.08) . 0.25 (0.14) 0.14 (0.14) 46 Helenium autumnale 0.10 (0.05) . 0.17 (0.12) 0.10 (0.07) 47 48 Symphyotrichum novae-angliae 0.05 (0.03) 0.11 (0.05) 0.00 (0.00) 0.05 (0.05) 49 Scirpus atrovirens 0.02 (0.02) . 0.00 (0.00) 0.05 (0.05) 50 Silphium lacinatum 0.02 (0.02) 0.11 (0.05) 0.00 (0.00) 0.00 (0.00) 51 52 53 54 55 56 57

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