DOI: 10.5061/dryad.v6wwpzgs3
title: Interspecific variation and elevated CO2 influence the relationship between plant chemical resistance and regrowth tolerance

description: 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.
description: We grew four species of milkweed (<i>A.
 syriaca, A. speciosa, A. incarnata,</i> and <i>A.
 curassavica</i>) under ambient (400 ppm) and elevated (760 ppm)
 concentrations of atmospheric CO<sub>2 </sub>at the University
 of Michigan Biological Station (UMBS). To manipulate atmospheric
 CO<sub>2 </sub>concentrations, we used an outdoor array
 consisting of 40 open-top chambers, with 20 chambers maintained at ambient
 CO<sub>2</sub> and 20 chambers maintained at elevated
 CO<sub>2 </sub>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 <i>A. speciosa</i> and
 <i>A. curassavica</i> were obtained from commercial sources
 (Prairie Moon Nurseries, Winona, USA) and seeds of <i>A. incarnata
 </i>and <i>A. syriaca</i> were collected locally
 (Cheboygan county, MI). We surface sterilized all seeds following a
 six-week cold stratification period (for all but tropical <i>A.
 curassavica</i>), and germinated seeds on moist filter paper for 1
 week. We planted seedlings in 983 cm<sup>3</sup> deepots
 <sup>TM</sup> (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 CO<sub>2</sub>
 array. Once in the array, potted plants were maintained under their
 CO<sub>2</sub> 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 <i>A. speciosa</i> and<i> A. syriaca</i>
 used in this study, and not all milkweed species were represented in every
 chamber. Overall, our eight treatments (2 CO<sub>2</sub>
 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
 CO<sub>2</sub>) conditions for three weeks due to external
 limitations on use of the chambers. However, by re-growing clipped plants
 under ambient CO<sub>2</sub> 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 <i>Asclepias</i> 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. https://doi.org/10.1073/pnas.0802368105 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. https://doi.org/10.1146/annurev-arplant-042916-040856 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. https://doi.org/10.1111/j.1365-2311.2009.01101.x Vannette RL, Hunter MD (2011) Genetic variation in expression of defense phenotype may mediate evolutionary adaptation of <i>Asclepias syriaca </i>to elevated CO<sub>2</sub>. Glob Chang Biol 17:1277–1288. https://doi.org/10.1111/j.1365-2486.2010.02316.x Tao L, Hunter MD (2012) Does anthropogenic nitrogen deposition induce phosphorus limitation in herbivorous insects? Glob Chang Biol 18:1843–1853. https://doi.org/10.1111/j.1365-2486.2012.02645.x
description: <b>defense_trait_data</b> Data on
 chemical resistance, plant regrowth tolerance, and other variables
 measured for each milkweed individual
 <b>cardenolide_community_data</b>
 Data on detected cardenolide peaks from each milkweed sample in
 community matrix form