Plants from cuttings of the two species and natural hybrids Trait differences between taxa in the field can reflect genetic differences, spatial differences in the environment, or effects of the maternal environment during an earlier growth period. To eliminate current environmental effects, we measured gas exchange traits for potted plants under common conditions. In summer 2003, we collected 300 stem cuttings from plants along the Piute transect. These cuttings were placed in water until they rooted, and then planted in 4-inch pots with a 1 : 1 mixture of pumice and potting soil (Sunshine Soil Mix, Sun Gro Horticulture, Vancouver, Canada). Plants were transplanted into 1-gallon pots after 1 month and placed outside at the University of California, Irvine (UCI). The potted plants were watered regularly and fertilized with Miracle Grow (The Scotts Company, Marysville, OH, USA; 15-30-15 NPK with trace elements) every 3 wk. Gas exchange measurements were taken on potted plants while they were blooming during the spring of 2005 using a portable gas exchange system with full-spectrum lamps (Li-Cor 6400; Li-Cor, Lincoln, NE, USA).
We measured instantaneous photosynthetic rate (Amax), transpiration (E), stomatal conductance (gc), and internal CO2 concentration (ci). Intrinsic WUE was calculated as Amax/gc, which was highly correlated with Amax/E (r = 0.845, n = 30) and with ci (−0.865). Unless otherwise noted, light was set at a photosynthetically active radiation (PAR) intensity of 1800 µmol m−2 s−1, leaf temperature at 26°C (close to ambient in the common garden), and CO2 at 37 Pa (ambient). Measurements were taken on plants in a randomized order between 10:00 and 14:00 h. Leaf to air VPD during measurement was similar for the three plant types (F2,30 = 2.69, P = 0.085) and ranged from 1.37 to 2.38 kPa.
The leaves of these species are small (average of 1.73 cm2 for P. newberryi and 0.66 cm2 for P. davidsonii), so, in order to achieve measurable carbon flux, we placed a small branchlet consisting of several overlapping leaves in the leaf chamber at one time, a common method for plants with small leaves (Ding et al., 1991; Royer et al., 2005; Maherali et al., 2006). Leaf area in the cuvette was determined in two ways. First, the branchlets were scanned in the overlapping configuration they formed while in the chamber, and silhouetted leaf area was calculated. However, it is possible that overlapping leaves still photosynthesized, so we also separated each leaf and calculated total leaf area. SLA did not differ significantly between the species, so this method was similar to determining carbon gain per unit of leaf mass. The two methods of calculating leaf area yielded the same conclusions, so we only report results for silhouetted leaf area.
Variables were analyzed by ANOVA, with plant type as a fixed factor, supplemented with two planned comparisons, using the CONTRAST statement in Proc GLM of sas (SAS Institute, Cary, NC, USA). The first comparison was between the two parent species. The second comparison was between the mean of the hybrids and the mid-parent value (the average of the mean values of P. newberryi and P. davidsonii) to test for hybrid vigor. In the absence of environmental effects, first-generation hybrids would not differ from the mid-parent value if physiological characters combined additively. Including VPD as a covariate in the analyses did not change the results, so we present only the ANOVA.
To determine the light intensity at which maximum photosynthetic rate was reached, in a separate experiment we measured photosynthetic rate at PAR intensities of 0, 50, 100, 500, 1000, 1500, 1700, 1900, 2100, 2300, and 2500 µmol m−2 s−1. Light compensation point and light saturation were calculated using Photosyn Assist (Dundee Scientific, Scotland, UK), and plant types were compared by ANOVA. The two species did not differ in light saturation point (F2,20 = 0.164, P = 0.850) or light compensation point (F2,20 = 2.159, P = 0.144). We used a PAR intensity of 1800 µmol m−2 s−1 in all other measurements, as it is a typical saturating light intensity for alpine plants (Korner, 1999; Campbell et al., 2005).
We also measured the photosynthetic rate of P. newberryi, P. davidsonii, and natural hybrids every 2°C from 4 to 35°C to determine whether they differed in the temperature at which they reached maximum photosynthetic rate. Potted plants were taken from the common garden outside at UCI and placed in a walk-in growth chamber with grow lamps, and were allowed 1 h to equilibrate to 4°C. Light in the leaf chamber was set at a PAR intensity of 1800 µmol m−2 s−1. The temperature in the chamber was slowly raised by 2°C increments, and plants were given 15–30 min to equilibrate to each new temperature before a measurement was taken. For each individual, we noted the temperature at which maximum photosynthetic rate was reached and analyzed those temperatures by ANOVA with plant type as a fixed factor. As temperatures in the field decrease with elevation (see Results), we expected that, if there was a difference, the alpine P. davidsonii would reach Amax at a lower temperature than P. newberryi.
Plants grown from seeds generated by hand-pollination To compare gas exchange among natural hybrids, F1 plants, and various backcrosses, we grew different types of plants from seed. During spring 2004, we hand-pollinated three types of plants (P. davidsonii, P. newberryi, and natural hybrids) that themselves were grown from cuttings in pots at UCI. For each study transect, we made nine crosses representing all factorial combinations of the three types as the mother crossed with the three types as the father. These crosses represent pure parents, reciprocal F1 hybrids, reciprocal backcrosses, and crosses between natural hybrids. For each cross, 10–15 mother plants were crossed with 1–6 different father plants. Seeds were germinated in spring 2005 and plants were grown outside in pots at UCI. Growing plants from seed allowed us to eliminate any effects of early maternal environment that may have influenced the performance of cuttings. During fall 2006, we measured instantaneous gas exchange rates of the nine cross types (when none of the plants was flowering). As in the previous potted plant measurements, environmental levels were controlled, and all plant types were measured over the same range of VPD (from 1.11 to 2.42 kPa), which did not differ significantly depending on the cross type (F8,116 = 0.504, P = 0.851). Amax, E, and WUE were analyzed by two-way ANOVA with type of mother and type of father as fixed factors. WUE was again calculated as Amax/gc, which was highly correlated with Amax/E (r = 0.880, n = 125) and ci (−0.977). We performed four planned comparisons for all gas exchange variables. First, we compared the mean values of the two pure parental crosses. Next, we compared the reciprocal F1 hybrids to see whether the direction of the cross influenced gas exchange. The third comparison tested for effects of hybrid generation by comparing the average value of both F1s and the value of later generation hybrid (natural hybrid-by-natural hybrid) crosses. The final comparison, to test for hybrid vigor, was between the average of the F1s and the mid-parent value.
Field populations To determine whether gas exchange differences among the two species and hybrids seen in the common gardens could also be detected under field conditions, we took gas exchange measurements on three populations along the Lee Vining transect during the summer of 2007. The three populations represented P. newberryi at 2382 m, morphological hybrids at 2888 m, and P. davidsonii at 3505 m elevation. Each population was measured between 10:00 and 13:00 h on a sunny day when plants were in full bloom (16 May for P. newberryi, 12 June for hybrids, and 27 June for P. davidsonii). The distances between populations and differences in phenology prevented us from making same-day measurements in all populations. These field results must be viewed with caution, as any observed differences in gas exchange could reflect seasonal changes as well as differences in spatial environment and genetic background.
As before, we measured instantaneous Amax, E, gc, and ci. Intrinsic WUE was again calculated as Amax/gc, which was highly correlated with Amax/E (r = 0.954, n = 42) and ci (r = −0.997, n = 42). Light was set at a PAR intensity of 1800 µmol m−2 s−1, leaf temperature at 26°C (close to ambient; range 23–29°C), and CO2 at 37 Pa. Amax, E, gc, and WUE were analyzed by ANCOVA with plant type (P. newberryi, natural hybrid, or P. davidsonii) as a fixed categorical factor and leaf to air VPD as a covariate. We included this covariate to eliminate one source of variation across the season, as the three populations differed slightly in VPD values during measurement (P. newberryi mean = 2.537 kPa, hybrid mean = 2.384 kPa, P. davidsonii mean = 3.052 kPa; F2,39 = 67.80, P < 0.0001).