In previous work, we found no evidence of genetic variation for population growth rate among A. nerii populations collected from widely separated populations in eastern North America (Zehnder & Hunter, 2007). Here, to eliminate genetic variation from our study, we established an A. nerii colony from a single individual collected near Ann Arbor, Michigan, USA, and then maintained the colony on the low cardenolide milkweed, A. tuberosa, at the University of Michigan Biological Station (UMBS) in Pellston, Michigan.
A. syriaca seeds were locally collected in Pellston, MI, USA, and A. speciosa seeds were purchased from Prairie Moon Nurseries. After surface sterilization, seeds were cold-stratified for 6 weeks, sterilized again, and germinated. Seedlings were planted in deepotsTM containing Metromix 360 and Osmocote controlled release fertilizer in a glasshouse at the University of Michigan Biological Station (Pellston, MI, USA). In late June, when plants were 71 old, one individual of each species was placed in one of 40 outdoor, open-top controlled atmosphere chambers at UMBS, half of which were kept at ambient (average 400.7 ppm) or elevated (average 739.0 ppm, dawn until dusk) atmospheric CO2 concentrations. CO2 concentrations in chambers were monitored continuously using a LI-COR LI-6262 IRGA (LI-COR, Lincoln, NE, USA).
After 4 weeks under either elevated or ambient CO2, physical and chemical traits of each plant were measured. Plant height, leaf number, and stem diameter were recorded before and after growth in the chamber array to determine change in plant size.
Plants were dried for 7 days and then weighed.