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Interspecific variation and elevated CO2 influence the relationship between plant chemical resistance and regrowth tolerance

Cite this dataset

Decker, Leslie; Hunter, Mark D. (2020). Interspecific variation and elevated CO2 influence the relationship between plant chemical resistance and regrowth tolerance [Dataset]. Dryad.


To understand how comprehensive plant defense phenotypes will respond to global change, we investigated the legacy effects of elevated CO2 on the relationships between chemical resistance (constitutive and induced via mechanical damage) and regrowth tolerance in four milkweed species (Asclepias). We quantified potential resistance and tolerance tradeoffs at the physiological level following simulated clipping/mowing, which are relevant to milkweed ecology and conservation. We examined the legacy effects of elevated CO2 on four hypothesized tradeoffs between: 1) plant growth rate and constitutive chemical resistance (foliar cardenolide concentrations), 2) plant growth rate and mechanically induced chemical resistance, 3) constitutive resistance and regrowth tolerance, and 4) regrowth tolerance and mechanically induced resistance. We observed support for one tradeoff between plant regrowth tolerance and mechanically induced resistance traits that was, surprisingly, independent of CO2 exposure. Across milkweed species, mechanically induced resistance increased by 28% in those plants previously exposed to elevated CO2.  In contrast, constitutive resistance, and the diversity of mechanically induced chemical resistance traits declined in response to elevated CO2 in two out of four milkweed species. Finally, previous exposure to elevated CO2 uncoupled the positive relationship between plant growth rate and regrowth tolerance following damage. Our data highlight the complex and dynamic nature of plant defense phenotypes under environmental change and question the generality of physiologically-based defense tradeoffs.


We grew four species of milkweed (A. syriaca, A. speciosa, A. incarnata, and A. curassavica) under ambient (400 ppm) and elevated (760 ppm) concentrations of atmospheric CO2 at the University of Michigan Biological Station (UMBS). To manipulate atmospheric CO2 concentrations, we used an outdoor array consisting of 40 open-top chambers, with 20 chambers maintained at ambient CO2 and 20 chambers maintained at elevated CO2 from May through August of 2015. Chambers were 1 m high cubes with an octagonal top of diameter of 0.8 m composed of a PVC frame and clear plastic walls.

Seeds of A. speciosa and A. curassavica were obtained from commercial sources (Prairie Moon Nurseries, Winona, USA) and seeds of A. incarnata and A. syriaca were collected locally (Cheboygan county, MI). We surface sterilized all seeds following a six-week cold stratification period (for all but tropical A. curassavica), and germinated seeds on moist filter paper for 1 week. We planted seedlings in 983 cm3 deepots TM (6.9 cm diameter by 35.6 cm height) containing Metromix 360 (SunGro Horticulture, Vancouver, BC) and Osmocote controlled release fertilizer [N:P:K:16:16:16 ppm N (g/g)] (ICL Specialty Fertilizers, Dublin, USA) on 5-May-2015. Germinated seedlings were watered daily and grown in the UMBS greenhouse for two weeks before they were moved to randomly assigned chambers in the CO2 array. Once in the array, potted plants were maintained under their CO2 treatments for three months. To minimize the entrance of herbivores into the chambers, we placed fine mesh coverings over the openings of each chamber and physically removed any herbivores that we observed during daily visual inspections. Within each chamber, we grew as many as seven plants of each milkweed species. Low germination success limited the number of A. speciosa and A. syriaca used in this study, and not all milkweed species were represented in every chamber. Overall, our eight treatments (2 CO2 treatments x 4 milkweed species) combined for a total of 442 plants.

Three months following the initial transfer of plants into the array, we simulated clipping/mowing by cutting all plants at the soil line. Many milkweed habitats important to the specialist herbivores associated with milkweed are located near roadways and agricultural fields that are regularly mowed. Properly timed mowing can improve reproduction and decrease predator abundance of certain milkweed specialists, including the monarch butterfly. Thus, our simulated mowing represents an ecologically relevant stress regularly experienced by many milkweed plants.

The aboveground biomass that we removed was dried at 60ºC, weighed, and used to calculate growth rate prior to damage (below). Cut plants were watered, moved to the UMBS greenhouse, and maintained under identical (ambient CO2) conditions for three weeks due to external limitations on use of the chambers. However, by re-growing clipped plants under ambient CO2 we are able to isolate the legacy effects of altered carbon availability prior to damage on regrowth tolerance, and potential tradeoffs between growth and resistance. Thus, we can examine the repercussions of previous energetic allocation decisions made by plants under carbon enriched conditions in comparison to those under ambient conditions. After a three-week period, the aboveground regrowth plant material was harvested, dried at 60ºC, and weighed as a measure of regrowth tolerance. 

For a measure of growth rate prior to damage, we divided the aboveground dry biomass of the plant by 64 days (the number of days since the seedling had been transferred to soil) following Agrawal & Fishbein (2008). Similarly, to calculate plant regrowth rate following mechanical damage, we divided the mass of the regrowth material by 21 days (the length of time plants were allowed to regrow following damage). Differences in regrowth rate following damage are important for the competitive success and ultimate fitness of plants (Züst and Agrawal 2017).

We collected samples of the original aboveground foliage, and the regrowth foliage of each plant for cardenolide analysis using established methods (Zehnder and Hunter 2009; Vannette and Hunter 2011; Tao and Hunter 2012). Roughly 20 mg of dried plant material was ground in a ball mill, deposited in 1 mL methanol, and stored at -10°C prior to analysis. Cardenolides were extracted, separated and quantified with a 0.15mg/mL digitoxin internal standard (Sigma Chemical Company, St. Louis, Missouri, USA), by reverse-phase high-performance liquid chromatography (HPLC) on a Waters Acquity UPLC with PDA detector (Waters Corporation, Milford, MA, USA). Peaks with symmetrical absorbance between 217-222 nm were identified as cardenolides. Cardenolide concentrations were calculated as the sums of all separated peak areas, corrected by the concentration of the internal digitoxin standard and sample dry mass. We used digitoxin as an internal standard because it is absent from Asclepias and because purified standards remain unavailable for a majority of milkweed cardenolides. We recognize that cardenolides may differ in their concentration-area relationships, and our estimates of cardenolide concentration should be considered as measured in digitoxin-equivalents. Because milkweed plants were grown in field mesocosms which excluded herbivores all season, the foliar cardenolides measured from plants prior to simulated damage represent natural levels of constitutive resistance. Conversely, the foliar cardenolide concentrations of regrown tissue following clipping, represent mechanically induced resistance.

Agrawal AA, Fishbein M (2008) Phylogenetic escalation and decline of plant defense strategies. Proc Natl Acad Sci USA 105:10057–60.

Züst T, Agrawal AA (2017) Trade-Offs Between Plant Growth and Defense Against Insect Herbivory: An Emerging Mechanistic Synthesis. Annu Rev Plant Biol 68:513–534.

Zehnder CB, Hunter MD (2009) More is not necessarily better: the impact of limiting and excessive nutrients on herbivore population growth rates. Ecol Entomol 34:535–543.

Vannette RL, Hunter MD (2011) Genetic variation in expression of defense phenotype may mediate evolutionary adaptation of Asclepias syriaca to elevated CO2. Glob Chang Biol 17:1277–1288.

Tao L, Hunter MD (2012) Does anthropogenic nitrogen deposition induce phosphorus limitation in herbivorous insects? Glob Chang Biol 18:1843–1853.

Usage notes


Data on chemical resistance, plant regrowth tolerance, and other variables measured for each milkweed individual


Data on detected cardenolide peaks from each milkweed sample in community matrix form


National Science Foundation, Award: DEB-1256115

National Science Foundation, Award: DEB-1257160

Division of Environmental Biology, Award: 1257160

Division of Environmental Biology, Award: 1256115