1 Supporting Information 2 For: Do cities simulate climate change? A comparison of herbivore response to urban and 3 global warming 4 Elsa Youngsteadt, Adam G. Dale, Adam J. Terando, Robert R. Dunn, & Steven D. Frank 5 6 Text S1. Validation of historical abundance data 7 Our historical record of scale-insect abundance was an unintended byproduct of botanical 8 recordkeeping and is subject to some limitations. These data could incorporate collecting bias if 9 the original plant collectors favoured healthier or more perfect specimens. However, we suspect 10 that collecting bias had minimal impact on our results. M. tenebricosa are bark-colored, reach a 11 maximum of ~1 mm in diameter, and do not produce visible excrement; feeding damage is only 12 evident as a decline in tree condition when insects reach very high densities. At the densities we 13 observed on rural trees, scale insects were difficult to detect in the field. 14 Our ability to detect patterns in historical scale-insect abundance was nevertheless limited 15 by the fact that most sites were represented only by a single branch from a single tree visited in a 16 single year, and that we could only view one side of that branch. We used our samples from 20 17 rural forest sites collected in 2013 to suggest how population estimates based on a single branch 18 viewed on one side (herbarium-style sample) related to those based on a more exhaustive, 19 systematic sample. 20 As described in the main methods text, we collected 2 pressed (herbarium-style) 21 specimens with branch lengths equal to those of the corresponding historical specimens from 22 each of 2 – 3 trees per site. At the same time, we also made a systematic collection of 4 30-cm 23 samples from each of the same trees at the same sites. Herbarium-style specimens were affixed 1 24 to paperboard and examined under a dissecting microscope. The 30-cm samples were counted in 25 two ways: First, we taped samples to a sheet of paperboard and counted all visible scale covers 26 (1-sided counts). Then we re-examined the same 30-cm specimens on all sides and recorded the 27 total number of scale-insect covers (exhaustive counts). We used these data in the following 28 three analyses, performed in JMP 10.0.0. 29 First, we asked whether the herbarium record is likely to provide estimates of population 30 density that are correlated to those we would obtain with a more exhaustive sample. To address 31 this question, we drew one randomly selected herbarium-style specimen from each of our 20 32 sites, and compared the abundance estimates based on these single specimens to those based on 33 the exhaustive counts of the entire systematic sample (8 to 12 30-cm samples per site). 34 Abundance estimates (scale-insect covers per 2.5 cm) were log-transformed to meet assumptions 35 of linear correlation analysis, and the two estimates were highly correlated (r = 0.714, F1,18 = 36 18.675, P < 0.001). This result indicates that a census based on one herbarium-like sample 37 provides population estimates that are correlated to those derived from a more exhaustive count 38 using 8 – 12 samples per site. 39 Still, we acknowledge that our historical dataset likely contains many false negatives. 40 That is, some specimens on which we did not detect scale insects were collected at sites where 41 scale insects were actually present. However, we suggest that the chance of a false negative is 42 greater at a site with low population density than one with a high population density, so that false 43 negatives would have had a minimal effect on the overall shape of the historical abundance- 44 temperature relationship shown in the main text in Fig. 2 and Fig. 3. To test this hypothesis, we 45 used the 30-cm branch samples from the 20 rural forest sites. Since scale insects were detected at 46 all sites in the exhaustive counts (Table S2), any individual sample on which scale insects were 2 47 not detected in a 1-sided count was considered a false negative. The overall rate of false 48 negatives was 35% (that is, when affixed to paperboard and examined on one side, 35% of the 49 30-cm branches appeared to lack scale insects, even though M. tenebricosa was actually present 50 at all sites). To meet assumptions of linear regression, proportion of false negatives per site was 51 arcsine square root transformed, and exhaustive counts of insects on populated branches were log 52 transformed. The proportion of false negatives per site increased as insect density on populated 53 twigs decreased (r2 = 0.40, F1,18 = 12.08, P= 0.003). This result suggests that false negatives in 54 the historical dataset are most likely at sites where population density was very low, and 55 therefore do not qualitatively alter the results of our analyses of scale insect abundance. 56 Finally, we asked how many more scale insects are detected in exhaustive counts than in 57 one-sided counts. Here, we simply compared the exhaustive and 1-sided counts for the same 30- 58 cm samples. We pooled counts by site and found that that the exhaustive census detected about 59 3.3 times more scale-insect covers per site than did the 1-sided counts. The two counts were 60 related by the following linear regression equation: total count = -3.6 + (3.3 * 1-sided count); (r2 61 = 0.98, P < 0.001, SEintercept = 4.4, SEb = 0.1). Only 18% (range 0% to 57%) of scale covers 62 housed living scale insects at the time of collection. 63 64 65 66 67 68 69 3 0.0 0.2 0.4 0.6 0.8 1.0 Supplementary Figures and Tables Log scale covers per 2.5 cm 70 18 20 22 24 26 28 August average (°C) 71 72 Figure S1. Complete historical dataset showing log-transformed M. tenebricosa abundance with 73 Gaussian quantile regression model fits (τ = 0.75, 0.9, 0.95, 0.98). The data are the same as those 74 in Fig. 2 of the main text; this version differs in that log-transformed data are shown, without 75 urban data overlaid, and without truncation of the x-axis below 22 °C. 76 77 78 79 80 81 82 4 336 335 AIC 334 333 332 331 0 83 5 10 15 20 Number of years averaged 84 Figure S2. AICs for models relating probability of M. tenebricosa presence on 296 historical 85 samples to August average temperatures for 1 to 20 years up to and including the year of 86 collection. Although several time spans provide similar fits, the best fit (smallest AIC) was 87 obtained with the August average over 8 years (Table 1C). Specimen length was included as a 88 covariate in all models. 89 90 91 92 93 94 5 Log gloomy scale covers per 2.5 cm 0.4 0.3 0.2 0.1 0.0 24.0 95 24.5 25.0 25.5 26.0 26.5 27.0 Temperature (°C, average of 8 Augusts) 96 Figure S3. Scale insect density increased from past to present at 16 of 20 sites (black lines), 97 while prevailing temperatures also increased. Each line connects two samples collected at the 98 same location; in all cases, the historical sample is associated with the cooler temperature. 99 Coloured lines highlight samples representing sites where insect abundance remained constant 100 (orange) or declined (blue). 101 102 103 104 105 106 107 108 109 6 Table S1. The likelihood of detecting M. tenebricosa on an herbarium specimen increased with the average temperature of 8 Augusts at the specimen locality. Parameter whole model intercept temperature (°C) length (in) Estimate ± SE -14.17 ± 3.70 0.50 ± 0.14 0.02 ± 0.01 Wald Χ2 21.50 14.71 12.14 9.84 P < 0.001 < 0.001 < 0.001 0.002 AIC pseudo-r2 331.52 0.14 7 Table S2. Historical locality descriptions, 2013 coordinates, and 2013 M. tenebricosa abundance for historically sampled sites revisited in 2013. (Scale insect abundance is based on the 2013 exhaustive census using 4 30-cm twigs per tree and 2 - 3 trees per site.) Year of original M. tenebricosa Site description County sample Coordinates of 2013 samples covers per 2.5 cm Yates Pond Biological Area, mixed deciduous woods. Wake 1970 -78.6897, 35.7211 -78.6962, 35.7219 1.09 Yates Pond Biological Area, pond margin. Wake 1970 -78.6898, 35.7184 -78.6898, 35.7203 5.09 1 mi N of Bahama on Co Rd 1615. Durham 1971 -78.8894, 36.1849 -78.8893, 36.1849 0.09 Pine hardwoods forest on the old ferry road. Harnett 1972 -78.9231, 35.4756 -78.9189, 35.4722 0.30 NC Botanical Garden Oak-Hickory center section north slope, 385 ft. Orange 1972 -79.0309, 35.8972 -79.0297, 35.8964 0.70 Rich woodland along Cape Fear River in Raven Rock State Park ca 6 mi NW of Lillington off Co Rd 1257. Harnett 1973 -78.8948, 35.4452 -78.8951, 35.4455 0.58 Mixed woodland in Cliffs of Neuse State Park ca 3 mi WNW of Seven Springs off Co Rd 1742. Wayne 1973 -77.8907, 35.2350 -77.8925, 35.2330 0.29 Mixed woodland on north slope with white pine facing Deep and Rocky Rivers at confluence ca. 5 mi W of Moncure off CoRd 1959. Chatham 1973 -79.1531, 35.6172 -79.1530, 35.6166 0.61 Pocosin, Bladen Lakes State Forest 3 miles N of White Lake on NC 701. Bladen 1978 -78.4820, 34.6952 -78.4827, 34.6970 0.60 Camp Durant, bank of upper lake near granitic flat rocks. Wake 1981 -78.5867, 35.8899 -78.5866, 35.88967 0.98 Goose Creek State Park, park roadside shoulder before road to campsites. Beaufort 1981 -76.912, 35.4738 -76.9115, 35.4736 0.54 Duke Forest near the end of Route 751 along the forest road between gates 7 and 3; ca. 3 mi from Duke West Campus. Tree 6 m tall. Durham 1988 -78.9768, 36.0115 -78.9766, 36.0121 0.26 Gate 34 of Duke Forest off Eubanks Rd (SR 1727) about 10 mi SW of Duke West Campus and 2 mi N of Calvender. Tree 5 m tall on border of low woods. Orange 1988 -79.0958, 35.9678 -79.0949, 35.96811 0.17 Hillsborough division of Duke Forest in western outskirts of Hillsborough just off US 70 on West Hill St at Gate 30, the stone quarry of Duke University. Tree 6 m tall. Orange 1988 -79.1287, 36.0818 -79.1317, 36.0804 0.27 Weedy abandoned field along Old Oxford Highway (1004) about 0.7 mi SW of Fairntosh or 3.5 mi from Granville County Line, tree 5 m tall. Durham 1988 -78.8420, 36.0936 -78.8402, 36.0961 0.83 1.6 km S on NC 902, from the intersection of US 64 and NC 902; Pittsboro Lake park, E side of NC 902, collected on W side of Lake. Tree 8 m tall x 15 cm diameter. Mixed hardwood/pine forest on edge of lake. Chatham 1992 -79.1864, 35.7138 -79.1867, 35.7142 0.15 Schenck Forest. Wake 1997 -78.7219, 35.8164 -78.7235, 35.8178 0.81 South side of Falls Lake along lake shore adjacent to the Cheek Rd bridge traversing falls lake 2 mi SE of Redwood; lake shore along western end of bridge. Disturbed area, trash prevalent. Durham 1997 -78.7591, 36.0433 -78.7594, 36.0434 0.94 Northern edge of Falls Lake on the eastern side of County Rd 1900, east of NC50, lake edge, in full sun. Wake 1997 -78.6785, 36.05803 -78.6808, 36.0584 0.05 From I-95, exit 49, highway 53, highway 242N, first unpaved road on left following Jones Lake State Park entrance, follow fork to right to Salters Lake gate, continue on dirt road to lake shore, lacustrine fringe forest, closed woods. Bladen 1997 -78.6260, 34.6986 -78.6265, 34.6985 0.25 110 8