Northeastern Naturalist Vol. 25, No. 2 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 265 2018 NORTHEASTERN NATURALIST 25(2):265–276 Sensitivity of Eleven Milkweed (Asclepias) Species to Ozone Abigail C. Myers1, Dennis R Decoteau2, Richard Marini2, and Donald D. Davis3,* Abstract - We exposed 11 milkweed species to ozone within continuous stirred-tank reactor (CSTR) chambers in a greenhouse to determine species sensitivity and potential use as bioindicators to detect phytotoxic levels of ambient ozone. Asclepias syriaca (Common Milkweed), A. ovalifolia (Oval-leaf Milkweed), A. sullivantii (Prairie Milkweed), A. speciosa (Showy Milkweed), A. asperula (Spider Milkweed), A. incarnata (Swamp Milkweed), A exaltata (Tall Milkweed), and A. curassavica (Tropical Milkweed) developed typical ozone-induced dark stipple on the adaxial surface of older leaves. Tropical Milkweed also exhibited significant premature defoliation (accelerated leaf senescence). Asclepias tuberosa (Butterfly Milkweed), A. hirtella (Green Milkweed), and A. verticillata (Whorled Milkweed) were tolerant to ozone. Foliar stipple on Common Milkweed increased with ozone concentration and time. In addition to Common Milkweed, a bioindicator commonly used to detect phytotoxic levels of ozone, the other 7 ozone-sensitive milkweed species should be evaluated further as potential ozone bioindicators. Introduction Tropospheric, ground-level, ambient ozone is the most widespread phytotoxic air pollutant in the US. (Krupa 1997, USEPA 2003). Ground-level ozone is a secondary air pollutant formed by chemical reactions between oxides of nitrogen (NOx) and volatile organic compounds (VOCs) in the presence of sunlight (Krupa et al. 2001). Significant sources of NOx and VOCs include automobile exhaust and combustion of fossil fuels. Ozone formed by the reaction of these precursors can travel long distances, depending on weather patterns, and is not restricted to urban or industrial areas. In the northeastern US, ground-level ozone is usually greatest during calm, stagnant, late summer days with temperature inversions. Minor amounts of localized ground-level ozone also can be produced by lightning strikes and advection from the upper atmosphere (Krupa 1997, Krupa et al. 2001). Plant injury is initiated when ozone enters leaf stomata during normal gas exchange. Once inside the leaf, ozone or its secondary products cause cellular injury, which may result in visible leaf symptoms (Skelly 2000). Richards et al. (1958) first described visible ozone-induced injury on broadleaved plants as an upper (adaxial) leaf surface “stipple” on Vitis spp. (grape). Leaf stipple is characteristic of ozoneinduced symptoms on most sensitive broadleaved plants, and it is used to evaluate ozone injury in the field (Manning and Federer 1980) and within controlled-environment exposure chambers (Kline et al. 2009, Seiler et al. 2014). 1Former Graduate Student in Environmental Pollution Control, The Pennsylvania State University, University Park, PA 16802. 2Department of Plant Science, The Pennsylvania State University, University Park, PA 16802. 3Department of Plant Pathology and Environmental Microbiology, and Penn State Institutes of Energy and the Environment, The Pennsylvania State University, University Park, PA 16802. *Corresponding author - ddd2@psu.edu. Manuscript Editor: Douglas DeBerry Northeastern Naturalist 266 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 Vol. 25, No. 2 Summary tables of ozone-sensitive plant species, usually based on ozoneinduced stipple, that list bioindicator plants useful to detect phytotoxic levels of ambient ozone have been published (USDOI 2003). Among the bioindicators listed are several species of ozone-sensitive Asclepias (milkweed). In addition to stipple, ozone-sensitive species may also develop premature defoliation, resulting in reduced photosynthesis and growth (Skelly 2000). Ozone-induced defoliation may cause changes in milkweed nectar production, which may be detrimental to butterflies and other pollinators, as well as reduced pollen germination and seed production (Hughes et al. 1990). Milkweeds are classified within the dogbane family (Apocynaceae, subfamily Asclepiadoideae). Woodson (1954) reported 108 indigenous North American species of Asclepias within 9 subgenera. It was recently estimated that ~75 Asclepias taxa are found in the US (Fishbein et al. 2011, USDANRCS 2017), most of which have not been evaluated for ozone sensitivity. Asclepias syriaca L. (Common Milkweed) is very sensitive to ozone (Bennett et al. 2006, Bergweiler et al. 2008, Davis 2007, Duchelle and Skelly 1981) and is often used as a bioindicator to detect phytotoxic levels of ozone (Manning and Federer 1980). Other milkweed species reported as very sensitive to ozone include A. curassavica L. (Tropical Milkweed) (Bolsinger et al. 1991, 1992; Hughes et al. 1990), A. exaltata L. (Tall Milkweed) (Chappelka et al. 2007, Souza et al. 2006), and A. incarnata L. (Swamp Milkweed) (Kline et al. 2008, Orendovici et al. 2003). Asclepias viridis Walter (Green Antelopehorn Milkweed) was rated as slightly sensitive (Davis 2002). Apocynum cannabinum L. (Indian Hemp Dogbane) and A. androsaemifolium L. (Spreading Dogbane), 2 other members of the dogbane family, were also reported to be sensitive to ozone (Davis 2007, Eckert et al. 1999, Kline et al. 2009). On 1 October 2015, the US Environmental Protection Agency (EPA) strengthened the US National Ambient Air Quality Standard (NAAQS) for ozone, reducing the standard from 75 ppb to 70 ppb ozone to help protect public health and welfare, and improve the health of plants and ecosystems (USEPA 2015). The new NAAQS for ambient ozone is based on the 4th-highest daily-maximum ozone concentration, averaged across 3 consecutive years for an average time of 8 h, not to exceed 70 ppb. However, it is unknown if the new NAAQS will protect plant species that are very sensitive to ozone. In this study, we exposed milkweed species to ozone concentrations below or near the current NAAQS of 70 ppb. Our objectives for this study were to (1) evaluate the ozone-sensitivity of various milkweed species and compare ozone-sensitivity of selected species with that of Common Milkweed (2012 and 2013 studies) and (2) characterize the ozone doseresponse of Common Milkweed (2014 study). Methods General We exposed milkweed plants to ozone within 16 continuous stirred-tank reactor (CSTR) chambers (Heck et al. 1975) in a greenhouse on The Pennsylvania State University campus, University Park, PA. Ambient air entering the Northeastern Naturalist Vol. 25, No. 2 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 267 greenhouse passed through activated-charcoal filters that reduced ozone concentrations within the greenhouse and exposure chambers to 5–8 ppb, which we considered the control concentration of ozone for all experiments. We connected the CSTR chambers to an ozone generator (except for control chambers) and to a computerized monitoring system that measured, controlled, and displayed ozone concentrations (Seiler et al. 2014). 2012 preliminary ozone-exposure study We conducted a preliminary study during fall 2012 to refine ozone exposure and monitoring techniques, and to select ozone-sensitive milkweed species to be utilized in the 2013 and 2014 studies. We selected the following Asclepias species for the 2012 study, based on seed availability: A. asperula (Decne.) Woodson (Spider Milkweed), A. hirtella (Pennell) Woodson (Green Milkweed), A. ovalifolia Decne. (Oval-leaf Milkweed), A. speciosa Torr. (Showy Milkweed), A. tuberosa L. (Butterfly Milkweed), and A. verticillata L. (Whorled Milkweed), as well as Common Milkweed, Swamp Milkweed, and Tall Milkweed. We germinated seeds of the 9 milkweed species and exposed the resultant seedlings to 5–8 ppb ozone (control), 75 ppb ozone, or 120 ppb ozone for 8 h/day (0900 hrs–1700 hrs), 5 d/week (Monday through Friday), for 4 weeks within CSTR chambers in a greenhouse. Preliminary exposure techniques are not reported in this section, but in the our subsequent 2013 and 2014 studies we utilized techniques that we developed or refined in 2012. Common Milkweed, Oval-leaf Milkweed, Showy Milkweed, Spider Milkweed, Swamp Milkweed, and Tall Milkweed exhibited typical ozone-induced stipple (Skelly 2000); thus, we selected them for the 2013 screening study. Butterfly Milkweed, Green Milkweed, and Whorled Milkweed were tolerant to ozone and we excluded them from further studies. After we completed the 2012 study, we obtained seeds of Tropical Milkweed and A. sullivantii Engelm. (Prairie Milkweed), and considered these 2 species for use in the 2013 study. All milkweed species selected are native to the US except Tropical Milkweed, which is native to the Neotropics as far north as Mexico (Woodson 1954). 2013 screening study: Exposure of 6 milkweed species to ozone Plant culture. We initially selected Common Milkweed, Oval-leaf Milkweed, Prairie Milkweed, Showy Milkweed, Spider Milkweed, Swamp Milkweed, Tall Milkweed, and Tropical Milkweed for the 2013 study. We placed seeds of each species into individual plastic bags containing ~20 g of soil moistened with water. Seeds were cold-stratified in a refrigerator at ~3 °C for 6 weeks, spread on the surface of 25 cm x 50 cm trays that were partially filled with topsoil, covered with potting mix, and watered. We placed the trays in plastic bags for 2 weeks to maintain high humidity and enhance seed germination. After germination, we placed individual seedlings in starter trays for 3 months, after which we repotted individual seedlings into 2-L pots containing Sunshine professional potting mix #1 (SUN GRO, Agawam, MA) and applied ~1 g granular Scotts Osmocote Plus fertilizer (15N-9P-12K; Scotts Miracle-Gro, Marysville, OH) to the soil surface. Seedlings of Oval-leaf Milkweed and Tall Milkweed grew poorly and were discarded. Northeastern Naturalist 268 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 Vol. 25, No. 2 Exposure to ozone. To allow acclimation to the exposure-chamber environment, we placed the plants of the remaining 6 species into the CSTR chambers 3 d prior to initiation of ozone exposures. At this time, plants were full-grown and at or near flowering. We placed 1 plant per species in each of the 16 CSTR chambers, where they were exposed to 5–8 ppb (control), 40 ppb, 75 ppb, or 120 ppb ozone, with 4 chambers maintained at each concentration. The 40-ppb concentration approximated background ozone in rural Pennsylvania (Orendovici-Best et al. 2010, Seiler et al. 2014). The 75-ppb ozone treatment represented the 2013 ozone NAAQS at time of study initiation. We chose the 120-ppb concentration because this level was high enough to ensure foliar-injury symptoms, allowing comparison of injury levels among more-tolerant species. We conducted ozone exposures from 15 July to 3 August 2013, 5 d/week (Monday to Friday), 7 h/day (0900 to 1600 hrs), in a squarewave design. We evaluated and recorded ozone-induced symptoms on Friday of each exposure week. During the weekend, plants remained within the greenhouse maintained at the background level of 5–8 ppb ozone. Leaf-injury evaluation. We visually assessed ozone-induced foliar injury on each plant using a modified Horsfall–Barratt scale (Horsfall and Barratt 1945). Symptom classes ranged from 0 to 5, where 0 = no injury, 1 = 1–6% injury, 2 = 7–25%, 3 = 26–50%, 4 = 51–75%, and 5 = >75% injury; we rated complete defoliation as 100%. To provide a single value for statistical analyses, we calculated the midpoint of each class as the mean of the minimum and maximum values. In addition, we randomly selected and tagged 6 leaves of varying age from each plant to follow visual-injury evaluations, including defoliation (100%) over time. We rated each of the 6 tagged leaves for leaf-level injury severity, using the 0–5 scale. We calculated mean ozone injury per plant as the percentage of the 6 leaves that had an injury rating ≥1, and overall injury rating (INJ) as the product of the mean of the 6 leaf-severity ratings (SEV) and the amount of injury at the plant level (AMT) as follows: INJ% = SEV% x AMT%. The experimental design was a 6 x 4 x 4 factorial in a randomized complete block, with 6 milkweed species, 4 time-periods (weeks) and 4 ozone concentrations. We initially analyzed the data as repeated measures analysis of variance (ANOVA) using SAS Proc Mixed (Littell et al. 2006). Species and time were considered as fixed effects (indicator variables), ozone concentrations as the regressor variable, and block as a random effect. However, the 3-way interaction— species x time x the quadratic effect of time and concentration—was significant, so we re-analyzed the data as 16 (4 weeks x 4 ozone concentrations), 1-way ANOVAs to test the null hypothesis that species’ responses were equal. We used Common Milkweed as the ozone-sensitive “standard” bioindicator (positive control) for all comparisons because of its reported high sensitivity to ozone. We employed Dunnett’s test (P = 0.05; Dunnett 1955) to compare mean injury values for each species within each combination of time and ozone concentration to those of Common Milkweed. Premature defoliation. Premature defoliation (accelerated senescence of older leaves) is a significant ozone response for plant species such as Tropical Milkweed Northeastern Naturalist Vol. 25, No. 2 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 269 (Hughes et al. 1990). As stated above, we rated complete defoliation as a 100% injury and recorded data weekly. We tabulated mean percent defoliation separately to illustrate the level of accelerated senescence for each milkweed species. We analyzed data using the same procedure as described for injury severity. 2014 dose-response exposure of Common Milkweed In 2014, we exposed only Common Milkweed to ozone in order to develop an ozone dose-response curve for this commonly used bioindicator. We treated seeds and resultant seedlings as described for the 2013 exposures. However, after we replanted individual seedlings into large containers, we performed weekly applications of Miracle-Gro® Liquid All-Purpose Plant Food (12N-4P-8K; Scotts Company, Marysville, OH) to the potting soil at a rate of 0.05 L fertilizer/7.5 L water. In early July 2014, we repotted individual Common Milkweed plants into 2-L pots, at which time the plants were full-grown, but not yet flowering. Plants were exposed to ozone from 17 July until 13 August, 0800 to 1400 hrs in a square-wave design, for 6 d/week (Monday through Saturday). Treatments consisted of 4 ozone concentrations (5–8 ppb [control], 30 ppb, 60 ppb, and 90 ppb), with 16 plants/chamber and 2 chambers replicated at each concentration. The 30-ppb concentration approximated background ozone levels in rural Pennsylvania (Orendovici-Best et al. 2010, Seiler et al. 2014). We chose the 60 ppb concentration because it was slightly less than the 70-ppb ozone concentration being considered at the time as a new ozone NAAQS. We selected the 90-ppb ozone level to maximize development of ozone-induced symptoms, allowing more robust comparisons among symptom responses of more tolerant species. We evaluated the plants for ozone injury each week, utilizing the 2013 protocol. We performed our analyses in SAS Proc Mixed with multiple regression in a block design (Littell et al. 2006). The response variable was ozone-injury level. We included as regressor variables linear and quadratic terms for ozone concentration and exposure time and set block as a random effect (Freund and Littell 2000). Results 2013 screening study: Exposure of 6 milkweed species to ozone Leaf-injury evaluation. We did not observe ozone injury on any milkweed species exposed to 5–8 ppb ozone (control, data not shown). Likewise, exposure to 40 ppb ozone for 1 or 2 weeks did not induce symptoms on any species (Table 1). However, following the 3-week exposure to 40 ppb ozone, Tropical Milkweed developed significantly greater injury than did Common Milkweed, the positive control. Following the 4-week exposure at this concentration, all species except Spider Milkweed developed symptoms. However, only Tropical Milkweed symptoms were significantly greater than those of Common Milkweed. Exposure to 75 ppb ozone for 1 week did not induce symptoms on any species. After exposure to 75 ppb ozone for 2 weeks, all species except Prairie Milkweed exhibited ozone injury, but injury ratings were not significantly different from those for Common Milkweed. Following exposure at this concentration for 3 and 4 weeks, Northeastern Naturalist 270 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 Vol. 25, No. 2 Table 2. Mean % leaf defoliation on 6 milkweed species exposed to 40 ppb, 75 ppb, or 120 ppb ozone for 1, 2, 3, or 4 weeks during 2013. We did not observe defoliation on controls exposed to 5–8 ppb ozone (data not shown). Mean defoliation percentages within columns followed by * are significantly different (P = 0.05) from mean percentages on Common Milkweed, the positive control, according to Dunnett's test. Exposure week for each ozone concentration 40 ppb ozone 75 ppb ozone 120 ppb ozone Species Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Common Milkweed 0.0% 0.0% 0.0% 4.2% 0.0% 0.0% 0.0% 12.5% 0.0% 0.0% 4.2% 12.5% Tropical Milkweed 0.0% 0.0% 45.8%* 70.8%* 0.0% 8.3% 37.5%* 45.8%* 0.0% 25.0%* 33.3%* 54.2%* Swamp Milkweed 0.0% 0.0% 0.0% 33.3%* 0.0% 0.0% 12.5%* 29.2%* 0.0% 0.0% 41.7%* 45.8%* Showy Milkweed 0.0% 0.0% 0.0% 25.0%* 0.0% 0.0% 12.5%* 29.2%* 0.0% 0.0% 29.2%* 29.2%* Prairie Milkweed 0.0% 0.0% 0.0% 12.5% 0.0% 0.0% 0.0% 4.2% 0.0% 0.0% 0.0% 8.3% Spider Milkweed 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 4.2% 20.8%* Table 1. Mean ozone injury (mean INJ%) on 6 milkweed species exposed to 40 ppb, 75 ppb, or 120 ppb ozone for 1, 2, 3, or 4 weeks during 2013. We did not observe injury on controls exposed to 5–8 ppb ozone (data not shown). Mean leaf-injury values within columns followed by * are significantly different (P = 0.05) from mean injury on Common Milkweed, the positive control, according to Dunnett’s test. Exposure week for each ozone concentration 40 ppb ozone 75 ppb ozone 120 ppb ozone Species Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Week 1 Week 2 Week 3 Week 4 Common Milkweed 0.000 0.000 0.000 0.038 0.000 0.002 0.008 0.208 0.006 0.049 0.115 0.553 Tropical Milkweed 0.000 0.000 0.403* 0.624* 0.000 0.148 0.403* 0.624* 0.000 0.333 0.660* 0.880 Swamp Milkweed 0.000 0.000 0.000 0.293 0.000 0.003 0.169 0.488 0.000 0.021 0.587* 0.587 Showy Milkweed 0.000 0.000 0.000 0.220 0.000 0.112 0.157 0.497 0.006 0.042 0.325 0.463 Prairie Milkweed 0.000 0.000 0.000 0.112 0.000 0.000 0.013 0.144 0.000 0.006 0.012 0.319 Spider Milkweed 0.000 0.000 0.000 0.000 0.000 0.002 0.015 0.117 0.002 0.095 0.122 0.463 Northeastern Naturalist Vol. 25, No. 2 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 271 all species exhibited injury symptoms, but only Tropical Milkweed developed symptoms significantly greater than those observe for Common Milkweed. Exposure to 120 ppb ozone for 1 week induced a trace of injury on Common Milkweed and Spider Milkweed. After 2 weeks’ exposure at this concentration, we noted light injury on Common Milkweed, Showy Milkweed, and Spider Milkweed, but injury levels on the latter 2 species were not significantly different from those of Common Milkweed. All species developed injury following the 3-week exposure to 120 ppb ozone, with injury levels on Tropical and Swamp Milkweed significantly greater than on Common Milkweed. The 4-week exposure to 120 ppb ozone resulted in severe injury on all species, but values for the 5 species were not significantly different from Common Milkweed. Premature defoliation. We did not observe premature defoliation on any species for any time in the control chambers (5–8 ppb, data not shown). Likewise, exposure to 40 ppb ozone for 1 or 2 weeks did not induce defoliation on any species (Table 2). However, following exposure to 40 ppb for 3 weeks, Tropical Milkweed exhibited 45.8% defoliation, which was significantly greater than the amount of defoliation on Common Milkweed (0.0%). Exposure to 40 ppb ozone for 4 weeks induced defoliation on all species except Spider Milkweed. Levels of defoliation of Tropical Milkweed (70.8%), Swamp Milkweed (33.3%), and Showy Milkweed (25.0%), were significantly greater than that of the standard Common Milkweed (4.2%), but defoliation of Prairie Milkweed (12.5%) was statistically similar to Common Milkweed. Following exposure to 75 ppb zone for 2 weeks, we observed slight defoliation (8.3%) on Tropical Milkweed, which was statistically similar to Common Milkweed (0.0%). After 3 weeks at this ozone level, defoliation of Tropical Milkweed (37.5%), Showy Milkweed (12.5%), and Swamp Milkweed (12.5%) were all significantly greater than defoliation of Common Milkweed (0.0%). Prairie Milkweed and Spider Milkweed did not exhibit defoliation. Exposure at 75 ppb ozone for 4 weeks induced significantly greater defoliation on Tropical Milkweed (45.8%), Showy Milkweed (29.2%), and Swamp Milkweed (29.2%) than Common Milkweed (12.5%). Prairie Milkweed (4.2%) was only slightly defoliated, and Spider Milkweed (0.0%) was not defoliated. Exposure to 120 ppb ozone for 1 week did not induce defoliation on any species. Following the 2-week exposure at this dose, only Tropical Milkweed exhibited defoliation (25.0%), which was significantly greater than Common Milkweed (0.0%). The remaining species were not defoliated. After the 3-week exposure to 120 ppb ozone, all species except Prairie Milkweed exhibited some level of defoliation. Defoliation levels for Tropical Milkweed (33.3%), Swamp Milkweed (41.7%), and Showy Milkweed (29.2%) were significantly greater than Common Milkweed (4.2%). Spider Milkweed exhibited a low level (4.2%) of defoliation. Following a 4-week exposure to 120 ppb ozone, all species exhibited defoliation. Defoliation levels for Tropical Milkweed (54.2%), Swamp Milkweed (45.8%), Showy Milkweed (29.2%), and Spider Milkweed (20.8%) were significantly greater than Common Milkweed (12.5%). Prairie Milkweed exhibited 8.3% defoliation at this level of ozone. Northeastern Naturalist 272 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 Vol. 25, No. 2 2014 dose-response exposure of Common Milkweed Common Milkweed plants did not exhibit ozone injury when exposed to 5–8 ppb ozone in the control chambers throughout the entire experiment, nor following exposure for 1 week at any ozone dosage (data not tabulated, but rather illustrated in Fig. 1). Only 1 of 32 plants exposed to 30 ppb ozone developed a trace of visible injury after week 3. Plants exposed to 60 ppb and 90 ppb ozone for 2 weeks developed injury values of 0.006 and 0.021, respectively. After 3 weeks of exposure, Common Milkweed plants in the 60 ppb and 90 ppb ozone chambers exhibited injury ratings of 0.020 and 0.078, respectively. Proc Mixed uses maximum-likelihood estimation for mixed models rather than ordinary least squares, so R2 values could not be estimated in these results. We employed Proc Mixed to obtain P-values for the independent variables and the regression coefficients. To obtain R2 values, we analyzed data using multiple regression in SAS Proc Reg (Freund and Littell, 2000), where injury was the response variable and weeks of exposure and ozone concentration were the regressor variables. The R2 values are likely low because variation explained by block was pooled into the error term. The (week) x (concentration) interaction (P = 0.0077), and the (week)2 x (concentration)2 interaction (P = 0.0001), were significant (R2 = 0.9701, adjusted R2 = 0.9596). The predictive model developed was: injury index = 0.0042 + 0.00459 (week) + 0.0003856 (concentration) – 0.000975 (week2) – 0.00000279 (concentration2) – 0.0003229 (week * concentration) + 0.00000208 (week2 * concentration2). This model indicates that ozone-induced injury on Common Milkweed is predicted to increase at an increasing rate over time and with concentrations of ≥ 30 ppb ozone (Fig. 1). Discussion In 2015, the EPA strengthened the ozone NAAQS, reducing the standard from 75 ppb to 70 ppb ozone (USEPA 2015). In our preliminary 2012 study and our 2013 Figure 1. Predicted values of ozone-injury index on Common Milkweed following exposure to 4 ozone concentrations during a 4-week period. Northeastern Naturalist Vol. 25, No. 2 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 273 screening study, we utilized 75 ppb ozone, approximating ozone concentrations in the NAAQS. Following exposure to 75 ppb ozone for 4 weeks in 2012 and 2013, mean ozone injury ranking (INJ%, Table 1) among species was Tropical Milkweed (0.624) > Showy Milkweed (0.497) > Swamp Milkweed (0.488) > Common Milkweed (0.208) > Prairie Milkweed (0.144) > Spider Milkweed (0.117) > Butterfly Milkweed (0.000) = Green Milkweed (0.000) = Whorled Milkweed (0.000). Based on ozone-induced stippling, we consider Tropical Milkweed, Showy Milkweed, Swamp Milkweed, and Common Milkweed to be sensitive to ozone; Prairie Milkweed and Spider Milkweed to be slightly tolerant to ozone; and Butterfly Milkweed, Green Milkweed, and Whorled Milkweed to be very tolerant. Although 75 ppb is slightly greater than the current NAAQS, and our exposure regime was different from the NAAQS, our findings suggest that Tropical Milkweed, Showy Milkweed, and Swamp Milkweed may be sensitive to ozone concentrations at or near the current NAAQS. Throughout the 2013 study, Tropical Milkweed usually had the highest levels of ozone-induced injury, often greater than Common Milkweed (Table 1). Hughes et al. (1990) also reported that Tropical Milkweed was very sensitive to ozone based on ozone-induced stipple. However, they also stated that stipple ratings of Tropical Milkweed often could not be conducted due to the high level of premature defoliation of stippled leaves. At the end of our 4-week exposure to 75 ppb ozone, mean rating of premature defoliation among the 6 species was as follows: Tropical Milkweed (45.8%) > Showy Milkweed (29.2%) = Swamp Milkweed (29.2%) > Common Milkweed (12.5%) > Prairie Milkweed (4.2%) > Spider Milkweed (0.0%). The high level of ozone-induced defoliation on Tropical Milkweed in our study and that of Hughes et al. (1990) is significant because the range of ozone concentrations used in both studies is near the current ozone NAAQS of 70 ppb. The order of rankings of leaf injury and premature defoliation are very similar because premature defoliation was rated as 100% injury (Symptom Class 5 in the modified Horsfall–Barratt scale for leaf injury). Although Tropical Milkweed is sensitive to ozone, defoliation of stippled leaves as reported by Hughes et al. (1990) and non-stippled leaves (this study) may be problematic when attempting to use this species as an ozone bioindicator. In both cases, after the leaves had dropped, the stippled or non-stippled leaves could not be rated, which confounded the data. Tropical Milkweed harbors a protozoan parasite (Ophryocystis elektroscirrha) that is infectious to Danaus plexippus (L.) (Monarch Butterfly) caterpillars, which may ingest this protozoan when feeding on Tropical Milkweed leaves (McLaughlin and Myers 1970, Satterfield et al. 2015). Thus, planting this bioindicator out of its natural range may not be wise. These factors may limit the usefulness of Tropical Milkweed as a bioindicator to detect phytotoxic levels of ambient ozone. Swamp Milkweed was the only milkweed species other than Tropical Milkweed to have stipple values significantly different than Common Milkweed, the positive control. However, ozone-injury values for Swamp Milkweed were only slightly greater, and usually not statistically different from those on Common Milkweed. Northeastern Naturalist 274 A.C. Myers, D.R Decoteau, R. Marini, and D.D. Davis 2018 Vol. 25, No. 2 Nevertheless, Swamp Milkweed also should be considered for use as an ozonesensitive bioindicator. Our model predicted that the level of ozone-induced injury on Common Milkweed would rise at an increasing rate with time and ozone concentration (Fig. 1). This finding suggests that injury to ozone-sensitive plants may occur below the current ozone NAAQS of 70 ppb. However, this study was conducted within controlled- environment chambers in a greenhouse, and our results must be interpreted with caution because chamber results may be different from those obtained in the field. Nevertheless, Common Milkweed remains a useful bioindicator (Manning and Federer 1980) to detect phytotoxic levels of ambient ozone. Also, Asclepias species not previously reported to be ozone-sensitive, or not evaluated, should be tested for field use as bioindicators of phytotoxic levels of ambient ozone. Whenever possible, utilization of new or untested milkweed bioindicators in field surveys should be conducted in conjunction with co-located ambient ozone monitors. 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