Tree propagation and drought treatment application
Root material from all experimental aspen genotypes was collected in June 2015 from various locations in Wisconsin. The cytotype of each genotype was determined via flow cytometry as described in Mock et al. (2012) and triploid cytotypes were confirmed using mitotic chromosome counts . In October 2017, root cuttings from all genotypes were planted in 4-l plastic flats filled with equal parts of torpedo sand and field topsoil (Keleney Topsoil, Madison, Wisconsin, USA). Once root cuttings produced new shoots, we cut them into sections of ca. 10 cm length. Shoots were individually grown in 0.5-l pots (60% torpedo sand, 40% field topsoil) and received 2 g of 6-month-release fertilizer (Nutricote total, Type 100 Blend, NPK 13-13-13, Arystra Lifescience, Broadway, NY, USA). By May 2018, shoots had developed into ~ 40 cm tall trees. These were planted into outdoor mesocosms (1.5 × 1.5 m) in a 20 cm deep layer of 60% torpedo sand and 40% field topsoil. One tree of each genotype was planted in each mesocosm (i.e. 8 different diploids and 8 different triploids per mesocosm). A randomly selected triploid tree was always planted next to a randomly selected diploid tree. To minimize potential edge effects, a border of single “buffer trees” was planted around the experimental trees of each mesocosm. All trees were planted at a 20 x 20 cm spacing. One week after planting, each tree received 3.5 g of 6-month-release fertilizer. Trees were watered frequently and allowed to grow for an additional eight weeks before applying the experimental treatments.
Several measures were taken to control the soil water content of the mesocosms. To intercept rainfall, we installed transparent, plastic covers over all mesocosms. To prevent infiltration of water from surrounding soil, the soil layer in each mesocosm was elevated on top of a 7.5-cm high layer of perforated plastic flats. A layer of root barrier fabric was placed between the soil layer and the supporting plastic flats. Finally, the soil surface in each mesocosm was covered with a 6-cm layer of leaf mulch to reduce evaporative drying.