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Methods for Experiment 234 -

Greenhouse conditions

We propagated six willow species (Salix bebbiana, S candida, S. discolor, S. pedicellaris, S. petiolaris and S. pyrifolia) from seeds collected at Cedar Creek in a greenhouse at the University of Minnesota. Temperatures averaged 20?C and plants were illuminated for 12 hours per day. Plants were well-watered and fertilized for 2 years. Three weeks before the start of the experiment, we transplanted the plants into 6.25 liter treepots. Plants were watered to field capacity and allowed to dry out over 4 weeks in June. We conducted a follow-up experiment on nine individuals of S. bebbiana and S. petiolaris that were propagated the following year. See Savage and Cavender-Bares. Contrasting drought survival strategies of sympatric willows (genus Salix): consequences for coexistence and habitat specialization. Tree physiology (2011) vol. 31 pp. 604 for more details.

aape234 - Phospholipid fatty acids (PFLA) on decomposed litter

Stem hydraulics

We calculated stem hydraulic conductivity and percent loss in conductivity (PLC) on 8 plants per species under pre-drought conditions and 8?10 plants per species under drought conditions. We measured water potential of each plant using a leaf pressure chamber before measuring hydraulic conductance. We measured hydraulic conductance gravimetrically by running 10 mmol KCl solution through stem segments. We used segments that were 22cm long to determine hydraulic conductance and trimmed stems to 5mm to measure percent loss in conductivity. We were unable to determine PLC in S. candida because of difficulties with measuring hydraulic conductance after flushing. See Savage and Cavender-Bares. Contrasting drought survival strategies of sympatric willows (genus Salix): consequences for coexistence and habitat specialization. Tree physiology (2011) vol. 31 pp. 604 for more details.

aase234 - Photoprotective leaf pigments and chlorophyll fluorescence measurements during a greenhouse drought experiment

Physiological measurements

We took measurements on six individuals per species before the drought, day 15 of the drought and day 30 of the drought. We collected the following data: (1) predawn leaf water potential using a Scholander pressure chamber, (2) chlorophyll fluorescence using a LICOR 6400 and (3) gas exchange using a LICOR 6400. Chlorophyll fluorescence was measured on both dark- and light- acclimated leaves. Gas exchange was measured at a light intensity of 1200 umol m-2. After taking chlorophyll fluorescence measurements, we collected leaf discs and put them in liquid nitrogen. Samples were analyzed by HPLC to determine leaf pigment concentrations. See Savage et al. Willow species (genus Salix) with contrasting habitat affinities differ in their photoprotective responses to water stress. Functional Plant Biology (2009) vol. 36 pp. 300 for more details.

aate234 - Gas exchange, dieback, leaf water potential and chlorophyll content during a greenhouse drought experiment

Leaf senescence and dieback

We monitored leaf dieback on 20 leaves on 10 individuals per species. On 10 days during the drought, we estimated necrosis on each leaf and used this data to estimate when species experienced 50% dieback. We also measured relative leaf chlorophyll content using a SPAD-502 on two fully expanded leaves per plant eight times. We calibrated the SPAD meter by measuring leaf chlorophyll content in two leaves per species using HPLC. See Savage and Cavender-Bares. Contrasting drought survival strategies of sympatric willows (genus Salix): consequences for coexistence and habitat specialization. Tree physiology (2011) vol. 31 pp. 604 for more details.

The progression of the drought

We measured predawn water potential on one leaf per plant on seven individuals per species throughout the drought. We estimated soil moisture by weighing the pots and calculating the percent of water in the soil after factoring out dry plant weight, pot weight and soil dry weight. On the same plants, we measured leaf gas exchange on each plant?s first, fully expanded, living leaf using ambient CO₂ a light intensity of 1200?mol m-2s-1. See Savage and Cavender-Bares. Contrasting drought survival strategies of sympatric willows (genus Salix): consequences for coexistence and habitat specialization. Tree physiology (2011) vol. 31 pp. 604 for more details.