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.