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Methods for Experiment 298 -

Common garden design and establishment

Sampling locations From May 2013 to June 2015, we collected rhizome cuttings from aspen populations throughout the Midwestern U.S. range of Populus tremuloides and P. grandidentata. Collections of P. grandidentata represent the species western range edge in Minnesota and Iowa and interior populations in Minnesota and Wisconsin. Cuttings of P. tremuloides were collected from these sites as well as from small, isolated stands in Nebraska and from the South Dakota Black Hills. Finally, we collected cuttings from the hybrid P. x smithii aspens in the Niobrara River Valley (NRV) of north-central Nebraska. Though this hybrid can and does form across its parents overlapping ranges we were, in the work reported here, interested in the physiology of the NRV hybrid aspens. We therefore confined our sampling of hybrid trees to this stand. Due to the few remaining stands of the NRV hybrids, and the attempt to collect from the nearest parental species populations in all directions surround the NRV hybrids, many more collections were made of P. tremuloides, which occurs in closer proximity to the NRV than of P. grandidentata and P. x smithii. Common garden We established a common garden with all three aspen taxa grown out from collected rhizomes in the greenhouse. When collected, rhizomes were trimmed to 10 to 50 cm in length, transported to the University of Minnesota Plant Growth Facilities (St. Paul, MN), and propagated following root cutting protocols. Our common garden was located at Cedar Creek Ecosystem Science Reserve (CCESR, East Bethel, MN, a Long Term Ecological Research Site. The garden was located within the interior of the current ranges of both P. tremuloides and P. grandidentata and both species could be found occurring naturally within 0.5 km. The garden site has excessively drained, sandy soils and a humid continental climate (mean annual temperature: 6.2 degrees C, mean annual precipitation: 782 mm). Minimum winter temperatures average -15 degrees C and, though periodic summer droughts do occur, our common garden was located in a mesic site adjacent to a slough and wetlands. Trees produced in the greenhouse were randomly planted with respect to genotype at one meter square intervals in a cleared field that had been fenced to exclude deer. We watered all seedlings and mowed the space between them following establishment. Propagation success was variable among taxa and more of the P. grandidentata and P. x smithii stems failed than P. tremuloides. We transplanted 362 P. tremuloides, 42 P. grandidentata, and 31 P. x smithii stems for a total of 435 stems into the common garden.

agwe298 - Budbreak

Budbreak method.

Stems were collected from naturally occurring adults of the indicated Populus species at Cedar Creek in the winter of 2016, on the indicated dates. Stems were then stored in water in a warm, artificially lit greenhouse conditions and the number of days until budbreak was recorded.

agxe298 - Chlorophyll fluorescence

Chlorophyll fluorescence method

Stems were collected from common garden plants on 7 June, 2016. All branches were collected by clipping stems under water and the cut ends were kept in floral tubes filled with distilled water to maintain leaf hydration. Collected branches and leaves were kept in darkness at 20 degrees C for 24 hours prior to beginning treatments. The experimental group branches and leaves were placed in the freezer and cooled at the same rate as in the electrolyte leakage experiment to -7 degrees C. The temperature was selected based on earlier trials that revealed when a freezing response could be detected as well as consideration of the possible late-spring freezing temperatures plants might experience in the field. The consequences of freezing for photosynthetic function were assessed by measuring the maximum quantum efficiency (in the dark) using a pulse-modulated fluorometer (Hansatech Instruments, Norfolk, UK); per cent decline in Fv/Fm was calculated relative to the control value measured at four time intervals over 24 hours. Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.

agye298 - Common garden plant collection

Common garden plant collection method

Common garden plants were collected from natural populations across the Midwest. Genetic and morphological data are given in: Deacon, N. J., Grossman, J. J., Schweiger, A. K., Armour, I., and Cavender-Bares, J. (2017). Genetic, morphological, and spectral characterization of relictual Niobrara River hybrid aspens (Populus x smithii). American Journal of Botany, 104(12), 1878--1890. DOI 10.3732/ajb.1700268

agze298 - Electrolyte leakage and resistance to damage

Electrolyte Leakage Method

Stem segments without attached leaves or branches were collected from common garden plants while dormant (March 21, 2016), in early spring (May 17, 2016), and in late fall (October 10, 2016). A 10 cm segment from each of 12 P. grandidentata, 5 P. x smithii, and 38 P. tremuloides individuals was collected and placed in a floral tube filled with distilled water. Collected stems were kept cool and dark until processed in the Cavender-Bares lab at the University of Minnesota. Four pieces, each 1 cm in length, were cut from each stem and placed in tubes filled with 9 ml nanopure H2O and 1 steel shot ball and assigned randomly to control, -5 degrees C, -10 degrees C, and -15 degrees C freezing treatments. Control stem tubes were kept at 4 degrees C for the duration of the freezing process. We used a programmable temperature controller with a circulating pump in conjunction with a modified chest freezer to control the temperature and keep the plants in the dark. Temperatures were programmed to decline at 5 degrees C per hour and hold for 30 minutes at -5 degrees C, -10 degrees C, and 15 degrees C. Samples were removed for each temperature after the 30 minute hold. Pre and post autoclaved conductivity of the control and freezing treatment groups were measured using a conductivity probe. An index of injury (percent) was calculated to compare the electrolyte leakage caused by cell rupture at different freezing temperatures compared to the boiled electrolyte leakage (Flint, Boyce, & Beattie, 1967). Freezing tolerance was calculated as 100 less the index of injury such that positive values indicate higher tolerance. Publications cited in these methods: Flint, H.L., B.R. Boyce, and D.J. Beattie. 1967. Index of injury - A useful expression of freezing injury to plant tissues as determined by the electrolytic method. Can. J. Plant Sci. 47:229 - 230. Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.

ahae298 - Leaf osmotic potential and stomatal pore index

Instrumentation - Leaf osmotic potential

Leaf osmotic potential was calculated using a VAPRO Vapor Pressure Osmometer Wescor, Vapro 5520, Logan, Utah, USA).

Leaf osmotic potential and stomatal pore index methods

Leaf osmotic potential was determined from leaf discs from fully saturated plants following a modified protocol from (Bartlett et al., 2012). 6.35mm diameter leaf discs were cut using a paper hole punch from the same set of common garden plants as gas exchange and SPI measurements on the mornings of August 1 and 2, 2016. Tubes containing the leaf discs were immediately placed in liquid nitrogen. We removed the leaf discs from the liquid nitrogen and placed them in humidified plastic bags to thaw for approximately 5 minutes before disrupting them with 10 to 12 pin holes and placing them one at a time into the VAPRO Vapor Pressure Osmometer Wescor, Vapro 5520, Logan, Utah, USA). Osmometer readings in mmol kg-1 (x) were converted to MPa based on the van't Hoff relationship at 20 degrees C. SPI: Three to five leaves per plant were collected from the same plants as gas exchange measurements. Stomatal densities and guard cell lengths were determined from clear nail varnish impressions on the abaxial leaf surface between the mid-vein and the leaf margin and measured on images taken from a microscope camera using ImageJ software. Measurements were averaged over three regions of the impression at 20x and 40x magnification respectively. Total stomatal pore area index (SPI; a dimensionless index of stomatal pore area per lamina area) was calculated as stomatal density x guard cell length2 (Sack, Cowan, Jaikumar, & Holbrook, 2003). Publications cited in these methods: Bartlett, M. K., Scoffoni, C. , Ardy, R. , Zhang, Y. , Sun, S. , Cao, K. and Sack, L. (2012), Rapid determination of comparative drought tolerance traits: using an osmometer to predict turgor loss point. Methods in Ecology and Evolution, 3: 880-888. doi:10.1111/j.2041-210X.2012.00230.x SACK, L. , COWAN, P. D., JAIKUMAR, N. and HOLBROOK, N. M. (2003), The 'hydrology' of leaves: co - ordination of structure and function in temperate woody species. Plant, Cell & Environment, 26: 1343-1356. doi:10.1046/j.0016-8025.2003.01058.x Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.

ahbe298 - Cavitation

Cavitation methods

Stems were collected from common garden plants and returned to the lab. Vulnerability curves were constructed by measuring native hydraulic conductivity, then spinning stems to successively lower water potentials and remeasuring conductivity. Pre-dawn water potential was measured on freshly collected leaves. Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.

Instrumentation

Spinning carried out using a modified rotor (Sorvall Model RC-5B; Ivan Sorvall Inc., Norwalk CT, USA) and water potential measured using a Scholander pressure chamber.

ahce298 - Leaf gas exchange

Gas exchange methods

Gas exchange was measured on two to three leaves per unique genotype in the common garden plants. Plants were measured at midday (1200 - 1400) on 12-14 September, 2016 using a portable photosynthesis system (LICOR 6400-40, Licor, Inc., Lincoln, NE, USA) at ambient CO₂ (400 ppm), VPD, and light intensity of 1500 mmol m-2 s-1. Intrinsic water use efficiency (WUEi) was calculated as light saturated photosynthetic rate (Amax; mmol m-2 s-1) divided by leaf stomatal conductance (gs, mol m-2 s-1). Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.

ahde298 - Leaf phenology

Leaf phenology methods

We recorded the status of overwintering leaf buds to monitor expansion, bud break, and leaf maturation weekly from late March to June 2016. Developmental stage was scored on a 0-5 scale from dormant to fully expanded leaves. In this scale, 1 indicated bud swelling, 3 indicated leaf emergence, and 5 indicated full expanded, green leaves. Results from this data are published in: Deacon, N. J., J. J. Grossman, and J. Cavender-Bares. 2019 (in press) "Tradeoffs between freezing and drought tolerance mechanisms in two midwestern aspens, Populus tremuloides and P. grandidentata, and their hybrid, P. xsmithii." Ecology and Evolution.