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- MATERIALS AND METHODS
In outdoor common gardens, high latitude populations of deciduous tree species often display higher assimilation rates (A) than low latitude populations, but they accomplish less height. To test whether trends in A reflect adaptation to growing season length or, alternatively, are garden growth artefacts, we examined variation in height increment and ecophysiological traits in a range-wide collection of Populus balsamifera L. populations from 21 provenances, during unconstrained growth in a greenhouse. Rooted cuttings, maintained without resource limitation under 21 h photoperiod for 90 d, displayed increasing height growth, A, leaf mass per area and leaf N per area with latitude whereas stomatal conductance (gs) showed no pattern. Water-use efficiency as indicated by both gas exchange and δ13C increased with latitude, whereas photosynthetic nitrogen-use efficiency decreased. Differences in δ13C were less than expected based on A/gs, suggesting coextensive variation in internal conductance (gm). Analysis of A–Ci curves on a subset of populations showed that high latitude genotypes had greater gm than low-latitude genotypes. We conclude that higher peak rates of height growth in high latitude genotypes of balsam poplar are supported by higher A, achieved partly through higher gm, to help compensate for a shorter growing season.
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Variation in geographically-widespread species is produced either by phenotypic plasticity (acclimation to environmental conditions) or by adaptation of genotypes to specific environmental conditions (Conover & Schultz 1997). Understanding of these patterns has been advanced by numerous studies along resource gradients (Poorter 1999; Reich et al. 1999) and with latitude or altitude (see Farmer 1993; Howe et al. 1995; Jonas & Geber 1999). Most such studies are restricted to variation along a single transect or a limited number of populations. Species with extensive geographic ranges, however, have the potential to exhibit large intraspecific variation in physiology, morphology, phenology and growth rate, and thus constitute good models for the study of local and regional adaptation. In this regard there are numerous reports of differentiation in photosynthesis to contrasting environments in Populus species (e.g. Ceulemans et al. 1992). Such patterns have not, however, been explored in much detail in balsam poplar (Populus balsamifera L.), a transcontinental species with a wide range in the boreal zone across North America, from Colorado to Nunavut, and from Alaska to Newfoundland. Balsam poplar is very closely related to black cottonwood (P. trichocarpa Torr. & Gray); in fact, black cottonwood is considered to be a subspecies of P. balsamifera in many treatments. Black cottonwood is the first woody plant to have had its genome sequenced (Tuskan et al. 2006).
Latitude represents a complex environmental gradient, along which photoperiod, temperature, growing season length [frost-free days (FFDs)], moisture availability and soil nutrient status can all be expected to vary. Differences in growing season length have been reported to correlate with characters such as leaf nitrogen per area (Leaf N), specific leaf area and photosynthesis (see Reich, Walters & Ellsworth 1997; Diemer 1998). In a pot study using populations of Sitka alder [Alnus sinuata (Reg.) Rydb.] and paper birch (Betula papyrifera Marsh.) from British Columbia, Canada, Benowicz, Guy & El-Kassaby (2000b) found an intrinsic relationship between midsummer photosynthetic rate and subsequent levels of fall frost hardiness (as growing season length is reflected in the date of hardiness development). Similarly, Gornall & Guy (2007) reported that photosynthetic rates (A) increased with latitude of origin in five provenances of black cottonwood along with an increase in Leaf N, stomatal density (SD) and stomatal conductance (gs), with no trend in leaf mass per area (LMA). Intrinsic water use efficiency (WUEi) and carbon isotope discrimination did not vary with latitude, implying a common internal conductance (gm) to the diffusion of CO2 from the intercellular space to the site of carboxylation, but this was not assessed. Flexas et al. (2007) emphasized that comparisons of gm in different natural plant populations, and its possible influence over efficiencies of water-use and nitrogen-use, should be a research priority.
The trend towards increased photosynthesis with latitude may be a case of parallel evolution among deciduous trees. Genotypic differences found in common garden environments presumably reflect adaptive variation, but careful experimental work is necessary to distinguish traits under selection from other effects such as plasticity or artefacts of differential growth.
By and large, height increment is greater in low latitude populations than in high latitude populations when both are growing in a common garden. Such was the case in the study reported by Gornall & Guy (2007) – although the northern black cottonwood populations had the highest photosynthetic rates, they were also smaller in size, and it is possible that one result may have spawned the other. Photosynthetic rates may have declined in the larger low latitude trees because of increased self-shading, or because of higher rates of soil nitrogen depletion. Furthermore, if adaptation to shorter growing seasons has indeed resulted in higher rates of carbon assimilation, then there should be a similar effect on height increment when plants are kept free of other limitations, most particularly the short days (or long nights) that trigger the cessation of active shoot elongation.
By making use of the extensive Agriculture Canada Balsam Poplar (AgCanBaP) collection we set out to test the hypothesis that during free growth (i.e. without photoperiod, nutrient and water limitations) genotypes native to regions with short growing seasons would grow more rapidly than genotypes adapted to longer growing seasons, commensurate with higher A. The study aimed to answer the following questions:
Do climate-related patterns in A and related ecophysiological variables occur under greenhouse conditions when all genotypes are of similar age and size, and are grown without resource limitation?
If population-level variation in A exists in balsam poplar, is it reflected in variation in height increment during free growth under extended days?
Are the proximal causes for population-level variation in A in balsam poplar the same as those previously reported for the closely related black cottonwood?
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- MATERIALS AND METHODS
Several growth and physiological traits in P. balsamifera are strongly related to geographic and climatic variables (Table 2), which by nature co-vary over North America for physiographic reasons. Treeline and the species range for P. balsamifera are at generally higher latitudes in the west (Fig. 1), resulting in similar relationships with longitude as with latitude. Geography is also confounded by elevation that tends to be greater for provenances from the west and southwestern part of the range. In this context, the preferred measure for growing season length is FFD. Measures of temperature (MAT, AST) and CONT are generally consistent with FFD. Indices of annual and summer drought tend to be highest in the northwest of the species range because precipitation decreases towards the north and west (Table 1).
Table 2. Pearson correlations (r) between geographic, climatic and physiological variables for all 210 genotypes [those that are significant are set in bold (P < 0.05); bold * are significant after Bonferroni selection (P < 0.0003)]
During rapid growth in the greenhouse, plant height increment was positively correlated with both latitude and longitude of origin, along with CONT, ADI and SDI (Table 2). Similarly, height increment was negatively correlated with FFD and measures of temperature. The population KUU deviated from this pattern, for reasons unknown (Fig. 2a). Final height (not shown) had a similar pattern. Irrespective of their height increment, INU, KUU and DEN had the highest rates of photosynthesis (Fig. 2b). Variation in A was highly consistent with variation in LMA (Fig. 2c) and strongly correlated with Leaf N (r = 0.744, P = 0.0001). Indeed, there was no relationship between A and FFD if photosynthesis was expressed relative to leaf mass (not shown). Across all 210 genotypes, light-saturated photosynthetic assimilation (A) was significantly correlated with FFD and several other co-varying climate parameters (Table 2). PNUE showed opposite correlations with geographic and climatic variables relative to A, LMA, Leaf N, WUEi and δ13C. Among the physiological variables considered (Table 3), A was positively correlated with gs, Leaf N and CCI, but was inversely correlated with SD.
Figure 2. Mean values (±standard deviation) for (a) height increment over 18 d during peak growth (b) assimilation rate (A) and (c) leaf mass per unit area (LMA) during free growth across 21 populations of Populus balsamifera. Provenances are arranged from left to right according to increasing frost-free days. INU, Inuvik; KUU, Kuujjuaq; DEN, Denali National Park; FBK, Fairbanks; GIL, Gillam; NWL, Norman Wells; WHR, Whitehorse; HAY, Hay River; RNA, Rouyn-Noranda; MGR, Mount Groulx; STO, Stony Rapids; FTM, Fort McMurray; LOV, Love; STL, Stettler; GPR, Grande Prairie; SOU, Sioux Lookout; CGY, Calgary; CAR, Carnduff; MTN, Matane; ROS, Roseville; FRE, Fredericton.
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Table 3. Pearson correlations (r) among physiological variables for all 210 genotypes. [those that are significant are set in bold (P < 0.05); bold * are significant after Bonferroni selection (P < 0.001)]
| ||A||gs||WUEi||Leaf N||PNUE||δ13Cleaf||δ13Cwood||SD||CCI||LMA|
|WUEi|| || ||1||0.358*||−0.125||0.226*||0.300*||−0.208||0.267*||0.292*|
|Leaf N|| || || ||1||−0.831*||−0.087||−0.050||−0.096||0.475*||0.488*|
|PNUE|| || || || ||1||0.040||−0.063||−0.008||−0.354*||−0.464*|
|δ13Cleaf|| || || || || ||1||0.656*||−0.165||0.153||0.204|
|δ13Cwood|| || || || || || ||1||−0.089||0.085||0.242*|
|SD|| || || || || || || ||1||−0.063||−0.134|
|CCI|| || || || || || || || ||1||0.268*|
Even though SD was higher at lower latitudes, gs was not clearly related to any geographic or climatic variables (Table 2). In contrast, correlations between intrinsic water-use efficiency (WUEi) and the geographic and climatic variables closely paralleled A alone (Table 2), even though variation in both A and gs contributed strongly to WUEi (Table 3). Across all 210 genotypes, A and gs were also positively correlated with each other (r = 0.236, P < 0.001). Trends were different at the population level (not shown) in that gs was in this case not significantly correlated with WUEi, whereas A was (r = 0.831, P < 0.0007). Consequently, population differences in WUEi were largely determined by A. Population means for WUEi ranged from 28.6 to 39.0 µmol CO2 mmol−1 H2O (Fig. 3). Populations from the extreme northwest had higher WUEi than those from the southeast. For example, populations DEN and INU had greater WUEi (39.0 and 36.2 µmol CO2 mmol−1 H2O, respectively) than FRE (29.1 µmol CO2 mmol−1 H2O).
Figure 3. Mean values (±standard deviation) for intrinsic water use efficiency (WUEi) during free growth across 21 populations of Populus balsamifera. Provenances are arranged from left to right according to increasing frost-free days. INU, Inuvik; KUU, Kuujjuaq; DEN, Denali National Park; FBK, Fairbanks; GIL, Gillam; NWL, Norman Wells; WHR, Whitehorse; HAY, Hay River; RNA, Rouyn-Noranda; MGR, Mount Groulx; STO, Stony Rapids; FTM, Fort McMurray; LOV, Love; STL, Stettler; GPR, Grande Prairie; SOU, Sioux Lookout; CGY, Calgary; CAR, Carnduff; MTN, Matane; ROS, Roseville; FRE, Fredericton.
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There was variation in δ13C among population means (∼0.98‰ foliar, and ∼1.1‰ wood). Wood and foliar δ13C values were correlated with FFD, LAT and LON but not with summer temperature (Table 2). Wood and foliar δ13C were, as expected, strongly inter-correlated across all 210 genotypes (Table 3) and across populations (r = 0.799, P < 0.0007, not shown). In contrast, δ13C values were correlated with WUEi across all genotypes (Table 3), but not across populations (r = 0.185, not significant).
A canonical structure for the provenances under study was obtained using nine geoclimatic and seven ecophysiological traits, including height increment (Table 4). Two significant canonical variables were extracted (CLIM1 and CLIM2) that explained 17% and 26% of the variance in plant traits, respectively, during redundancy analysis. All climate predictor variables loaded highly on CLIM1 along with latitude and longitude, whereas measures of summer temperature (AST and CONT) were most strongly related to CLIM2. The strongest canonical loadings were seen for height increment and leaf N on CLIM1 and for δ13Cwood on CLIM2. PNUE had negative loading on CLIM1 whereas height increment, δ13Cleaf and δ13Cwood, were positively related to both CLIM1 and CLIM2. In contrast, although Leaf N, A, and WUEi were also positively related to CLIM1, they were negatively related to CLIM2. These tendencies indicate that trees from areas with short growing seasons and/or low summer temperatures had higher photosynthetic rates driven in part by elevated Leaf N. Figure 4 presents A as a function of FFD and Leaf N for all 210 genotypes. As shown by this figure, variation in Leaf N can account for considerable variation in A, both within and between populations, but FFD remains a significant predictor even when Leaf N is accounted for.
Table 4. Canonical structure between geoclimatic parameters and plant traits with their first two canonical variables, CLIM1 and CLIM2
|Geoclimatic variables||CLIM 1||CLIM 2||Response variables||CLIM 1||CLIM 2|
|ADI||0.39||0.29|| || || |
|SDI||0.82||0.12|| || || |
Figure 4. Assimilation rate (A) as a function of Leaf N and frost-free days (FFDs) across all 210 genotypes of Populus balsamifera. The dependent variable can be predicted from a linear combination of the independent variables using the equation A = 15.865 + (0.019 × Leaf N) − (0.019 × FFD).
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The relationship between WUE as determined by gas exchange and WUE as indicated by δ13C is related to the conductance of CO2 diffusion within the leaf (Evans & von Caemmerer 1996). Carbon isotope discrimination during photosynthesis calculated from both gas exchange data (Δi) and from δ13C values of leaf (Δleaf) and wood (Δwood) tissue are plotted together in Fig. 5. Wood is typically more enriched in 13C than leaf tissue, hence Δleaf and Δwood are not the same but parallel each other closely. They differ in elevation by roughly 0.5‰. In contrast, Δi is ∼2‰ greater than Δwood, but this difference depends largely on the value of b used in Eqn 3. Some offset is expected because of the diffusion gradient for CO2 from the intercellular space to the sites of carboxylation in the chloroplast, inversely proportional to the internal transfer conductance (gm). Regardless of the value chosen for b, there is a significant increase in Δi as a function of FFD, but not in Δwood and Δleaf, the latter determined on the exact same leaves as used in the gas exchange analysis. Put another way, the discrepancy between predicted and observed isotope discrimination decreases with latitude. The implication is that gm is increased in populations adapted to shorter growing seasons.
Figure 5. Carbon isotope discrimination calculated from δ13Cwood (□), δ13Cleaf (Δ) and intrinsic water use efficiency (○) across 21 populations of Populus balsamifera plotted against frost-free days (FFD).
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A–Ci curves were constructed for six of the populations shown in Fig. 5; three approaching the northern edge of the species range (≤122 FFD) and three from the south (≥161 FFD). Consistent with the previous gas exchange measurements, photosynthesis was higher in genotypes of northern provenance than in genotypes of southern provenance, a difference that was reflected in somewhat higher J and TPU, and possibly Vcmax and gs (Table 5). Day respiration did not differ. The most obvious contrast was a greater than twofold difference in gm, whereby increased gm was again associated with fewer FFD. Population differences in gm (nested within geography) were also nearly significant (P = 0.0521, not shown).
Table 5. Fitted A–Ci curve parameters (±standard error) estimated at 27°C on populations representative of North (INU, DEN, KUU) and South (STL, FRE, MTN) geography. Values reported for A and gs are at ambient CO2 (380 µL L−1). P is the probability of a difference between North and South
|North||14.89 ± 0.82||0.266 ± 0.01||87.95 ± 6.26||115.44 ± 6.97||8.12 ± 0.53||2.50 ± 0.19||0.447 ± 0.124|
|South||10.84 ± 0.32||0.218 ± 0.01||76.89 ± 4.30||93.20 ± 1.88||6.56 ± 0.11||2.83 ± 0.19||0.173 ± 0.030|