All Methods

Methods for Experiment 238 -

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. 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.

aaoe238 - Growth, gas exchange and hydraulic conductivity of greenhouse grown willows under pre-drought conditions

Pre-drought hydraulics and growth

We selected a pair of terminal, fully expanded leaves and covered one with parafilm and aluminum foil so that it could equilibrate with the stem. In the afternoon, we measured gas exchange on the transpiring leaf using a LICOR 6400 and immediately measured the leaf water potential of both leaves with a pressure chamber. We used this data to calculate leaf and plant hydraulic conductivity. Before the drought, we also harvested a total of eight plants per species, which we dried at 70 ?C for 5 days to measure stem, root and foliage biomass. 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.

aaqe238 - Total basal area of all willow and poplar species present in field plots

Field plot establishment and survey

Fifty 10 m ? 30 m plots were randomly established in three preserves: Cedar Creek Ecosystem Science Reserve LTER (Cedar Creek), Helen Allison Savannah Scientific and Natural Area (SNA), and Boot Lake SNA. Prospective plots were chosen by selecting grid cells on a map using a random number generator. Permanent plots were only established when there were willow and/or poplar species present. Since willows and poplars primarily occur in and near wetlands, we also randomly selected ten wetlands at Cedar Creek at which to establish plots. The plots were oriented to minimize environmental heterogeneity: for example, plots near bodies of water were positioned to run parallel to the water. When there were no obvious environmental gradients, the plots were oriented north to south. In each of the plots, we characterized species abundances by measuring their total basal area. For plants that had less than 12 stems, we measured the basal diameter of every stem. For plants with over 12 stems, we divided the stems into 4 mm size classes, measured six individuals in each size class, and counted the remaining stems. For the three tree species our study, we measured dbh instead of basal diameter. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

aare238 - Functional traits measured in a greenhouse common garden

Adult functional traits

Wood density was measured on 4-cm segments of one to two year old distal branches from six plants per species. We removed their bark and pith and estimated stem volume using water displacement. Turgor loss point was measured on turgid leaves removed from three well-watered plants per species. Sampling was completed predawn and leaf mass was adjusted when the initial water potential was less than -2 MPa. Repeated measurements were taken with a pressure chamber and a balance to create pressure volume curves using standard procedures. To determine SPI, we measured stomatal density in three sections of the leaf and stomatal aperture on nine stomata per leaf using impressions made of clear nail varnish. We calculated a cumulative stomatal density of the abaxial and adaxial sides of the leaves and calculated SPI based on the equation: SPI = stomatal density ? (stomatal aperture)2. We calculated SPI for four individuals per species. Lastly, we measured SLA on three fully-expanded, sun leaves, from eight per species. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

Adult functional traits

Seedling functional traits

Seeds were collected in the field, and viability tests were conducted weekly for thirteen weeks. At each time interval, we monitored the germination of six sets of 50 seeds per species. The seeds were germinated on moist filter paper in petri dishes and kept in a well-lit window. After the germination experiment, we transplanted seedlings into 2.5 cm diameter pots containing potting soil to monitor their growth. After seven weeks, we harvested forty plants per species, calculated their RER (cm day-1) and determined their dry mass. We did a second harvest of the same size seven weeks later to calculate RGR. Two of the species, S. candida and P. deltoides demonstrated mortality before the second harvest, resulting in only 15 and 25 individuals, being harvested at that point. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

aaue238 - Sequences of the nuclear gene ADH for thirteen species of willows and two poplars

DNA sequencing

We collected leaf material from central and northern Minnesota including multiples locations across Cedar Creek LTER. We deposited voucher specimens in the University Minnesota Herbarium and sequenced the nuclear gene, ADH (alcohol dehydrogenase). TheADH sequencing was completed at the University of Minnesota Using primers obtained from Belyaeva et al. (pers. com.). We samples one to two individuals per species and cloned 7-12 copies of the known polyploidy species (Salix lucida, S. serissima, S. discolor and S. humilis). We extracted DNA using a Dneasy Plant Mini Kit (Qiagen, Valencia, CA). The PCR reaction mixture consisted of 10-20 μg genomic DNA, 0.12 μM each primer, 0.1 mMdNTP, 0.9 ? Taq buffer, 1.8 mM MgCl2, 50 U/mL Taq polymerase (Sigma, St. Louis, MO, USA). The thermal cycling was 94?C (1 min), 50-54?C (1 min), 72?C (2 min) for 38 cycles and a final extension of 72?C (7 min). We cloned the polyploid species using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) and cleaned the plasmid DNA using a QIAprep Miniprep kit (Qiagen). The remaining diploids were cleaned using a GenElute PCR Clean-up kit (Sigma) and directly sequenced using an ABI Prism 3730xl Prism Analyzer (Applied Biosystems Inc., Foster City, CA, USA). See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

aave238 - Depth to the water table measurements and soil analysis for the field plots

Environmental conditions monitoring

We measured depth to the water table monthly in a 1.5 m long PVC pipe (2.5 cm diameter) buried in the center of each plot for two years (Aug. 2007- July 2009). We did not take measurements during the winter when the soil was frozen and did not measure depth to the water table in plots that were perennially saturated. The plots were divided into three 10 m ? 10 m subplots, and soil samples were collected in two subplots per plot in June 2008. We took ~15 cores per subplot (180.5 cm deep, 2.54 cm in diameter) and kept the samples in a cooler until we returned from the field. The samples were dried on trays in the sun, put through a 1.8 mm sieve and submitted to the University of Minnesota Analytical Laboratory within 48 hours of being collected. Each sample was analyzed for extractable phosphorus (Bray-1 method), exchangeable potassium and nitrate concentration along with pH and percent organic matter using standard protocols. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

Field plot establishment

aawe238 - Functional traits measured under field conditions in native populations

Field sampling

We sampled plants from a subset of plots that exhibited different levels of water availability. We divided the plots into four categories based on their depth to the water table in Aug. 2008. These categories were: (1) saturated, (2) water within 0 - 0.5 m of the surface, (3) water within 0.5 ? 1 m of the surface and (4) dry without water within 1m of the surface. We sampled each species in two plots per water availability category. The only exceptions were the tree species (S. amygdaloides, S. nigra, and P. deltoides) and the clonal species (S. interior and P. tremuloides), which we sampled in more than two plots because the tree species occurred at low densities, and we wanted to ensure that we sampled unique genotypes of all the clonal species. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.

Functional traits

Wood density was measured on 4-cm segments of one to two year old distal branches from six plants per species. We removed their bark and pith and estimated stem volume using water displacement. Turgor loss point was measured on six plants from two plots per species in the lowest and highest water availability categories where they occurred. These stem samples were collected in the morning and allowed to rehydrate in water for 3 hrs before we took measurements. Repeated measurements were taken with a pressure chamber and a balance to create pressure volume curves using standard procedures. To determine SPI, we measured stomatal density in three sections of the leaf and stomatal aperture on nine stomata per leaf using impressions made of clear nail varnish. We calculated a cumulative stomatal density of the abaxial and adaxial sides of the leaves and calculated SPI based on the equation: SPI = stomatal density ? (stomatal aperture)2 . We calculated SPI for six individuals per species. Lastly, we measured SLA on three fully-expanded, sun leaves, from ten individuals per species in the field in each water availability category. See Savage and Cavender-Bares. Habitat specialization and the role of trait lability in structuring diverse willow (genus: Salix) communities. Ecology (accepted 2011) for more details.