Materials and Methods

Fifty 3-month-old C. occidentalis plants, indigenous to Kings Canyon National Park, CA, were propagated from seed and grown in a greenhouse. The plants were grown in 2.8-L pots (height = 18.4 cm, top diameter = 16.2 cm). Seventeen 4-year-old, seed-propagated F. anomala plants indigenous to Dinosaur National Monument, UT, and 30 9-month-old, vegetative-cutting-propagated P. pubens plants indigenous to Liberty County, FL, were grown in 14.6-L (height = 27.9 cm, top diameter = 30.2 cm) and 3.64-L (height = 15.9 cm, top diameter = 20.3 cm) pots, respectively. Containers of plants of each species were filled with soilless media (LC1 Sunshine Mix; Sun Gro Horticulture, Bellevue, WA) and were then inserted into larger pots [volume = 3 L, height = 15.2 cm, top diameter = 20.3 cm (C. occidentalis and P. pubens); volume = 22 L, height = 29.2 cm, top diameter = 35.6 cm (F. anomala)] covered with aluminum foil to minimize solar heat load of the rhizosphere of each plant.

There were five treatments: severe drought, moderate drought, moist (i.e., control), partial flood, and flood. These treatments represented the range of water stress levels that ornamental plants are commonly exposed to in managed landscapes. The moist treatment was considered the control as a result of conventional nursery practices both in the greenhouse and in the field to maintain a moist rhizosphere for optimal plant growth. Potted plants were irrigated to the water-holding capacity of drained root zones every 12, 8, and 4 d for plants in the severe drought, moderate drought, and moist treatments, respectively. The lower half of the aluminum foil-covered containers in the partial flood treatment was continuously saturated with a column of tap water. Entire root zones of the flooded plants were completely submerged by maintaining the water column in the pots at the surface of the medium. Within each species, assignment of treatment and greenhouse position of plants were completely random. The three species were separated spatially to facilitate intraspecific comparisons. Sample sizes per treatment were equal within C. occidentalis (n = 10) and P. pubens (n = 6). In F. anomala, severe drought and drought had four and the remaining treatments three. Treatments were imposed on 19 June and ended on 30 Aug. 2006.

When the plants in the severe drought, drought, and moist treatments were scheduled for irrigation on the same date (13 July and 6 Aug.), all plants were fertilized with 8.92 mm N (20N–20P–20K) (Plant Products, Brampton, ON, Canada) in tap water. Root zones of plants in the partial flood and flood treatments were fertilized after removing them from the foil-covered pots. Multiple 1000-W, high-pressure sodium lamps provided supplemental irradiance between 0600 and 2200 hr. Mean air temperature (27.7 °C) was determined with a datalogger (CR23X; Campbell Scientific, Logan, UT), which also logged irradiance data with a probe (QSO-ELEC; Apogee Instruments, Logan, UT) to determine mean photosynthetically active radiation [211 μmol·m⁻²·s⁻¹ (daily maximum = 495 μmol·m⁻²·s⁻¹)].

Net photosynthesis of the youngest, fully expanded leaf on the longest stem of each plant was measured during the middle of the day with a steady-state photosynthesis system (LI-6400; LI-COR, Lincoln, NE) with an LED light source (model 6400-02B; LI-COR) set at 1500 μmol·m⁻²·s⁻¹, which is above the light saturation point of several deciduous woody species (Dougherty et al., 1979; Landhäusser and Lieffers, 2001; McCarron and Knapp, 2001), before irrigating on 13 July, 6 Aug., and 30 Aug. Ambient CO₂ concentration in the cuvette was set at 400 μmol CO₂·mol⁻¹. Soil moisture content by volume of the upper 6 cm of the root zones was determined on each date before irrigation with a Thetaprobe (model ML2x; Delta-T Devices, Cambridge, UK), which was set at the factory-calibrated organic soil type option. Medium redox potential of each plant in the partial flood and flooded treatments was also measured at a soil depth of 10.7 cm with a platinum combination electrode (Ag+/AgCl, Accumet; Fisher Scientific, Hampton, NH) before measurement of net photosynthesis. At treatment termination, leaf area was measured with a leaf area meter (LI-3000; LI-COR) and roots, stems, and leaves of each plant were oven-dried at 65 °C for 3 d before determining dry weights of each.

Net photosynthesis of each species was analyzed with a two-factor, repeated-measures analysis of variance (ANOVA) to evaluate the effects of treatment, measurement date, and their interaction within a species. When evidence of an interaction was present, effects of the treatments at each measurement date (i.e., simple effects) were considered; otherwise, comparisons of plants in each treatment across time (i.e., main effects) were conducted. Significant interaction effects were partitioned into three tests: partial flood, control, and drought treatments by measurement date [4 degrees of freedom (df)]; flood and control treatments by measurement date (2 df); and severe drought and control treatments by measurement date (2 df). The goal of the study was to see how water stress treatments affected the plants compared with a presumably nonstressed, control treatment. Therefore, for each species, each stress treatment was compared individually with the control treatment rather than conducting multiple comparisons among all five treatments. When significant interactions necessitated comparisons among the treatments at each of the three measurement dates (i.e., comparisons of simple effects), the significance level was divided by three, which is a Bonferroni-type correction (Westfall et al., 1999), for each set of comparisons within a measurement date.

Mean dry weights of roots, stems, leaves, and whole plants were compared among the treatments within each species. Overall comparisons were made with a one-factor ANOVA and pairwise comparisons of the control plants to plants in each of the other treatments were made with t tests and corrected with Dunnett's method for multiple comparisons (Westfall et al., 1999). A significance level of P ≤ 0.05 was used for all statistical tests. For all estimates provided, we also provide the relevant 95% confidence intervals. Values of test statistics are presented for reported P values having no accompanying estimate; subscripts on F statistics are the numerator df (ndf) and denominator df (ddf), respectively (e.g., F_{ndf, ddf} ). Subscripts on t statistics are the df (e.g., t_df ). All analyses were conducted using the MIXED procedure in SAS/STAT version 9.1 (SAS, Cary, NC).

No prior estimates of within-species variability in photosynthesis were available when this experiment was conducted. To estimate within-species variability, one-factor ANOVAs were performed for each species when photosynthesis was measured after treatment initiation on 29 June 2006. Resulting mean square errors from the ANOVAs were considered measures of within-species variability. These measures were then used to calculate effect sizes detectable with 80% power on a 5% level test of no mean differences among treatments. The effect size was the ratio of the standard deviation of treatment means to the standard deviation of photosynthesis among plants within species. The detectable effect sizes were estimated to be 0.5149, 1.018, and 0.7076 of C. occidentalis, F. anomala, and P. pubens, respectively.

Results

Calycanthus occidentalis.

Averaged over time, mean net photosynthesis of C. occidentalis plants in the drought treatment was 3.40 μmol·m⁻²·s⁻¹ [95% confidence interval (CI): 2.07 to 4.72] less than that of plants in the control treatment (P < 0.0001) (Table 1). No differences in net photosynthetic rate were found, however, between the control and partially flooded treatments (1.03 μmol·m⁻²·s⁻¹, 95% CI: 2.36 to –0.29; P = 0.12) (Table 1).

Table 1.

Mean photosynthetic rate, root zone moisture content, leaf area, and plant dry weight of potted Calycanthus occidentalis Hook. & Arn. assigned to five soil–water treatments that were based on irrigation frequency.^z

View Table

Due to an interaction between treatment and time, comparisons between plants in the control and flood treatments (F_2,90 = 10.54; P < 0.0001) and control and severe drought treatments (F_2,90 = 4.57; P = 0.01) were analyzed at each time of measurement. Mean net photosynthesis of control plants was greater than that of flood plants on 13 July (t₁₃₅ = –8.65; P < 0.0001), 6 Aug. (t₁₃₅ = –3.84; P = 0.0006), and 30 Aug. (t₁₃₅ = –2.64; P = 0.0279) (Table 1). Since these three comparisons of simple effects were all significant and in the same direction, their main effects were compared. Across all three dates, the mean net photosynthetic rate of flooded plants was 5.53 μmol·m⁻²·s⁻¹ (95% CI: 4.20 to 6.86) lower than that from the control plants (t₄₅ = –8.40; P < 0.0001). Net photosynthetic rate of plants in the control treatment was greater than of those in the severe drought treatment on 13 July (t₁₃₅ = 7.89; P < 0.0001), 6 Aug. (t₁₃₅ = 3.86; P = 0.0006), and 30 Aug. (t₁₃₅ = 4.88; P < 0.0001) (Table 1). Averaging across time, the mean net photosynthetic rate of control plants was 6.08 μmol·m⁻²·s⁻¹ (95% CI: 4.76 to 7.74) higher than those in the severe drought treatment. Root zone water content of the medium ranged from 9.51% (severe drought treatment) to 76.5% (flood treatment) (Table 1). Medium redox potential over time of C. occidentalis plants in the partial flood and flood treatments was on average –307 (95% CI: –322 to –292) and –292 (95% CI: –302 to –282) mV, respectively. Anaerobic conditions generally occur at soil redox potentials 200 mV or less (Ponnamperuma, 1984).

No differences in leaf dry weight were found when comparing plants in the control treatment with those in the partial flood (–0.98 g, 95% CI: –6.48 to 4.52; P = 0.99) and severe drought (4.80 g, 95% CI: –0.70 to 10.30; P = 0.25) treatments, but leaf dry weight of the control plants was greater than that of plants in the drought (8.81 g, 95% CI: 3.16 to 14.5; P = 0.01] and flood (15.4 g, 95% CI: 9.87 to 20.9; P < 0.0001) treatments (Table 1). This overall pattern was consistent for leaf area and root, stem, and total dry weight data, except that control plants had greater leaf area (1363.4 cm², 95% CI: 988.12 to 1738.7; P < 0.0001) and root (63.8 g, 95% CI: 49.4 to 78.3; P < 0.0001), stem (8.99 g, 95% CI: 3.92 to 14.1; P = 0.003), and total (77.9 g, 95% CI: 57.5 to 97.8; P < 0.0001) dry weight than those in the severe drought treatment (Table 1).

Fraxinus anomala.

Mean net photosynthetic rates over time of F. anomala plants in the flooded and partially flooded treatments were, respectively, 10.0 μmol·m⁻²·s⁻¹ (95% CI: 7.41 to 12.6; P < 0.0001) and 7.49 μmol·m⁻²·s⁻¹ (95% CI: 4.89 to 10.1; P < 0.0001) less than that of plants in the control treatment (Table 2). No difference was found, however, between plants in the control and drought treatments (–0.87 μmol·m⁻²·s⁻¹, 95% CI: –3.31 to 1.57; P = 0.4518) (Table 2).

Table 2.

Mean photosynthetic rate, root zone moisture content, leaf area, and plant dry weight of potted Fraxinus anomala Torr. ex S. Watson assigned to five soil–water treatments that were based on irrigation frequency.^z

View Table

Simple effects of the severe drought versus control treatment were analyzed at each measurement period as a result of a significant interaction between these two treatments and time (F _2,24 = 9.54; P = 0.0009) (Table 2). Compared with the control treatment, the mean net photosynthetic rate of plants in the severe drought treatment was 9.10 μmol·m⁻²·s⁻¹ (95% CI: 3.13 to 15.1; P = 0.0015) lower on 13 July, but not different (–0.82 μmol·m⁻²·s⁻¹, 95% CI: –6.79 to 5.15; P = 0.7312) on 6 Aug. or 30 Aug. (–3.72 μmol·m⁻²·s⁻¹, 95% CI: –8.84 to 1.4; P = 0.0234) (Table 2). The water content of the root zones ranged from 29.4% (drought treatment) to 81.2% (partial flood treatment) (Table 2). Medium redox potentials over time of F. anomala plants in the partial flood and flood treatments were –280 (95% CI: –313 to –247) and –291 (95% CI: –335 to –248) mV, respectively.

Plants in the partial flood and flood treatments had less leaf, root, stem, and total dry weight than of those in the control treatment (Table 2). No differences in leaf, root, stem, and total dry weight were found between control plants and plants in the drought or severe drought treatments (Table 2). There was no evidence of differences in leaf area between control plants and those in the other treatments (Table 2).

Pinckneya pubens.

There were no observed differences in mean net photosynthesis over time between P. pubens plants in the control and drought treatments (1.41 μmol·m⁻²·s⁻¹, 95% CI: –1.18 to 4.00; P = 0.27) (Table 3). There were also no differences in mean net photosynthesis between plants in the control and partially flooded treatments (–0.31 μmol·m⁻²·s⁻¹, 95% CI: –2.90 to 2.28; P = 0.81) or between control plants and flood plants (2.38 μmol·m⁻²·s⁻¹, 95% CI: –0.20 to 4.98; P = 0.07) (Table 3).

Table 3.

Mean photosynthetic rate, root zone moisture content, leaf area, and plant dry weight of potted Pinckneya pubens Michx. assigned to five soil–water treatments that were based on irrigation frequency.^z

View Table

As a result of an interaction between the control and severe drought treatments and time (F _2,50 = 3.45, P = 0.04), comparisons of the severe drought treatment to control were made at each drought cycle. There was statistical evidence of the control having greater net photosynthetic rates than the severe drought treatment at each time point. Hence, averaging across time, plants in the control treatment had a mean net photosynthetic rate that was 6.33 μmol·m⁻²·s⁻¹ (95% CI: 3.74 to 8.92) higher than the severe drought plants (Table 3). Root zone water content of the medium spanned from 18.3% (severe drought treatment) to 78.6% (flood treatment) (Table 3). Medium redox potentials of P. pubens plants in the partial flood and flood treatments were –110 (95% CI: –211 to –9.64) and –200 (95% CI: –263 to –136) mV, respectively.

Although the stem dry weight of control plants was 6.60 g (95% CI: 0.65 to 12.55; P = 0.03) greater than that of plants in the severe drought treatment, there were no mean differences in leaf, root, or stem dry weight between plants in the control treatment and the other treatments (Table 3). There were also no differences in mean leaf area between plants in the control treatment and the other treatments (Table 3).

Discussion

Calycanthus occidentalis and F. anomala tolerate soil moisture extremes that are similar to edaphic conditions in their native habitats. Calycanthus occidentalis, which is often found near stream banks or in moist canyons (Hickman, 1993), appears to not be suitable for cultivation in areas with greater than ≈66% and lower than ≈30% soil moisture content (Table 1). Similarly, the tolerance of F. anomala to moderately and severely dry, but not overly moist, soil moisture conditions was expected based on the arid nature of its natural range (Welsh, 1987). In contrast, the capacity of P. pubens to tolerate water stress, particularly dry soil conditions, is greater than its ecology would imply (Delprete, 1996) (Table 3). We conclude that although P. pubens and F. anomala are adapted to a relatively wide range of soil–water conditions, as demonstrated by their capacity to fix and allocate carbon near optimal levels while exposed to sub- or supraoptimal soil moisture conditions (Tables 2 and 3), C. occidentalis appears to only grow well under sufficiently moist soil conditions with adequate drainage (Hickman, 1993). In addition, our data demonstrated that growth and photosynthesis are effective indicators of the performance of ornamental woody species, at least under greenhouse conditions (Tables 1, 2, and 3).

The F. anomala plants we evaluated were collected from the most northern area of its distributional range (Albee et al., 1988; Welsh, 1987). Based on the climate and ecology of northeastern Utah and observations of plants cultivated in Iowa and Illinois (J. R. Stewart, unpublished data), it is likely that conspecific plants from this source can tolerate harsher environmental conditions than assumed. However, other abiotic and biotic factors, including the emerald ash borer (Agrilus planipennis Fairmaire) that feeds on several Fraxinus species in the midwestern United States (McCullough and Roberts, 2002), need to be taken into consideration before widespread cultivation.

While a specific provenance of F. anomala was identified for potential horticultural use due to its putative tolerance of extreme soil-moisture conditions, no similar efforts were made to select P. pubens and C. occidentalis plants from specific provenances. Based on research on other potentially stress-resistant ornamental woody species (Balok and St. Hilaire, 2002; Bsoul et al., 2006), it is possible that some provenances of C. occidentalis exist that are located in relatively dry edaphic conditions. Further selection efforts could be made to identify drought-tolerant selections of this highly attractive species. Although P. pubens holds promise as a species that should be evaluated further in field trials, future work should also focus on identifying superior selections that are even more tolerant of limited soil–water conditions than our results demonstrate. Indeed, although severe drought plants photosynthesized at lower levels than control plants, there were no differences in carbon-allocation patterns between plants in the two treatments (Table 3). Although the high variation in allocation patterns may account for this finding, it is possible that P. pubens exhibits some form of physiological adjustment that increases its resource-use efficiency under dry conditions (Stewart and Graves, 2004).

Pinckneya pubens is relatively easy to propagate vegetatively (Dirr, 1998) and may do well under exposed or shaded conditions. Moreover, the native habitat of P. pubens appears to be shrinking and any effort to cultivate it could be considered a form of conservation (J. Sharma, personal communication). Pinckneya pubens is a promising horticultural species that should be evaluated further in the field to determine its ability to survive in a wider range of conditions.

Although the species were not compared statistically, it is interesting to note that the medium redox potentials of flooded and partially flooded P. pubens plants were substantially lower than the medium redox potentials of similarly treated plants of the other two species. Pinckneya pubens appears to have some physiochemical means of adjusting its rhizophere to anaerobic conditions. Other species have been found to adjust to low oxygen conditions by developing aerenchyma, adventitious roots, or expanded lenticels to improve gas exchange in roots (Crawford and Braendle, 1996; Ojeda et al., 2004; Schaffer et al., 1992). More research needs to be done to understand the means by which this intriguing species is able to tolerate flooded conditions as well as dry conditions. In addition, it leads to a more fundamental question as to why certain woody species from moist habitats are adaptable to soil moisture extremes [e.g., Fraxinus pennsylvanica Marshall (Whitlow et al., 1992) and Taxodium distichum (L.) Rich. (Nash and Graves, 1993)], but others are not [e.g., Cornus florida L. (Croker et al., 1998) and Acer pensylvanicum L. (Pijut, 2005)]. Pinckneya pubens and C. occidentalis both naturally occur in moist habitats, but P. pubens is substantially more adapted to drought and flooding events. The underlying mechanisms that enable some bottomland woody species to be drought-tolerant need to be explored further to help in making future selections of stress-tolerant species and cultivars.

Measuring the carbon fixation and allocation patterns of these species under a gradation of soil moisture levels provides a cursory estimate of their potential performance in the field. Widespread cultivation of each species may not be possible as a result of limitations related to cold hardiness, but further work is needed to determine if the cold hardiness of each taxa is greater than that required by the climate of their natural range as has been shown for other woody species (Flint, 1972; Parker, 1963; Schrader and Graves, 2003).

The involvement of horticulture in the introduction of invasive plant species has been recognized (Mack, 2005; Reichard and White, 2001) and provides incentive to evaluate the invasive potential of each of these species. Although the factors contributing to the invasiveness are numerous and complex (Lockwood et al., 2007; Lonsdale and Smith, 2001), there are a few traits that are hallmark characteristics of invaders that should be investigated. These include time to reproduction (Rejmánek and Richardson, 1996), fecundity (Stewart and Graves, 2006), flowering period (Mack, 2005), and seed germinability (Baker, 1974; Perrins et al., 1992).