Plant Ecol (2011) 212:219–228 DOI 10.1007/s11258-010-9816-8
Linking carbon balance to establishment patterns: comparison of whitebark pine and Engelmann spruce seedlings along an herb cover exposure gradient at treeline Sheel Bansal • Keith Reinhardt Matthew J. Germino
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Received: 22 April 2010 / Accepted: 10 July 2010 / Published online: 29 July 2010 Ó Springer Science+Business Media B.V. 2010
Abstract There is increasing evidence that landscape vegetation patterns near species’ range limits are associated with positive biotic interactions, such as in the alpine-treeline ecotone. In the northern Rocky Mountains, whitebark pine (Pinus albicaulis) is considered an early-successional species, able to establish in exposed microsites, while late-successional species such as Engelmann spruce (Picea engelmannii) are more dependent on neighboring vegetation to facilitate establishment. We compared ecophysiological traits associated with carbon balance of newly germinated seedlings of whitebark pine and Engelmann spruce along an herb cover gradient to (1) infer which ecophysiological properties explain the establishment success of seedlings, and (2) to assess differences in establishment patterns with respect to
All the authors contributed equally to this manuscript. S. Bansal K. Reinhardt (&) M. J. Germino Department of Biological Sciences, Idaho State University, 650 Memorial Drive, Pocatello, ID 83209, USA e-mail:
[email protected] K. Reinhardt Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA
distance from neighboring vegetation. We measured survival over 2 years, and concurrently measured gas exchange and water relations (photosynthesis, respiration, and transpiration), morphology [specific leaf area (SLA)], and biochemistry [chlorophyll fluorescence (Fv/Fm) and nonstructural carbohydrates]. Both species initially established in the most exposed microsites away from vegetation during their first growing season, but only pine persisted in exposed microsites to the end of the second growing season. Pine exhibited phenotypic traits to increase stress tolerance (e.g., higher soluble sugar concentrations, lower SLA) and improve carbon balance (e.g., greater water use efficiency, lower respiration, higher Fv/Fm) compared to spruce in exposed sites, but had lower carbon balance under herb cover. Superior establishment success of pine in exposed microsites at treeline could thus be attributed to a suite of intrinsic physiological advantages that are apparent at the earliest stage of development. Keywords Chlorophyll fluorescence Nonstructural carbohydrates Photosynthesis Picea engelmannii Pinus albicaulis Respiration Specific leaf area Transpiration
Introduction Present Address: S. Bansal Department of Forest Ecology and Management, Swedish University of Agricultural Science, 901 83 Umea˚, Sweden
Understanding how physiological mechanisms contribute to species’ range limits is important for
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assessing ecological responses to changes in the environment. For example, a mechanistic understanding of range limits of tree species is necessary for predicting biogeographic changes to forest cover, such as migration of forests to higher elevations or latitudes with climate change (Kupfer and Cairns 1996; Grace et al. 2002). Forest-alpine treelines are often used for studies of range limits, because they are conspicuous and ecologically important land cover boundaries that occur along sharp climate gradients (Tranquillini 1979). The upper elevation limit of trees results in part from inadequate adaptation of trees to physical conditions in the alpine, as for any climate-controlled plant-species boundary (Ko¨rner 2003). However, there is an increasing amount of evidence that biotic factors (plant–plant interactions) may play an important role in determining the altitude of treelines by affecting the establishment of trees above and beyond their current distribution (Callaway 1998; Arroyo et al. 2003; Germino et al. 2002; Smith et al. 2003; Maher et al. 2005; Bader et al. 2007). This may be especially so in the case of ‘‘diffuse’’ or ‘‘island’’ (vs. ‘‘abrupt’’) treeline ecotones, where the forest boundary consists of patchy, multi-age vegetation mosaics and is often co-dominated by two or more species (e.g., Harsch et al. 2009). While there are much correlative data linking the elevational limits of adult trees at treeline with abiotic conditions (e.g., 5–7°C growth limit global isotherm; Ko¨rner and Paulsen 2004), there is also much ecophysiological support linking treeline elevation with regeneration patterns of younger lifestages of trees (i.e., germinant and recently established seedlings; Smith et al. 2003, 2009). These seedling studies also provide evidence that ecological facilitation by adult trees, herbs, rocks, and even micro-topographies in stressful sub-alpine environments may be critical to successful establishment of young seedlings (Germino et al. 2002; Maher et al. 2005; Resler et al. 2005; Bader et al. 2007; Hughes et al. 2008). For example, Callaway (1998) found that shadeadapted subalpine fir (Abies lasiocarpa) saplings were highly aggregated and had greater growth around adult whitebark pine (Pinus albicaulis) individuals, forming clusters of multi-species tree islands. Maher et al. (2005) showed that tree and herb cover had additive, positive effects on conifer seedling survival and photosynthesis.
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Seedling recruitment patterns at the alpine-treeline ecotone may also be explained by species-specific physiological specializations to cope with environmental stress (Bansal and Germino 2010). Appreciable increases in tree abundance into otherwise unforested areas of diffuse alpine-treeline ecotones require colonization of new tree islands for advancement of treeline altitude. In turn, this depends on successful establishment of ‘‘pioneer’’ tree species into open areas that are able to endure high sunlight, radiative frost, and possibly dry, shallow soils. It is commonly asserted in the literature that whitebark pine is a pioneer species in the northern Rockies owing to its successful recruitment after fire, low leaf:sapwood area ratio, and greater water use efficiency compared to competing tree species (Callaway et al. 2000; Sala et al. 2001; Tomback et al. 2001). Once established, pioneer individuals may be able to provide ecological facilitation to later seral, shadetolerant species. One question that remains, though, is how early in terms of lifestages is ecological specialization between pioneer and later-successional species evident in seemingly temperature-limited, stressful environments? Additionally, does seedling ecophysiology correspond to the successional landscape patterns evident in diffuse treelines starting at the earliest lifestages? In the northern Rocky Mountains, USA, treelines are often diffuse, multi-species, and made up of clumps of multi-age tree islands and/or ribbon forest bands (Billings 1969; Germino et al. 2002). In this study, we compared how spatial patterns of neighboring herbs compared to establishment patterns of whitebark pine (pioneer species) and Engelmann spruce (Picea engelmannii, shade-adapted species) seedlings, and investigated what underlying physiological traits contributed to the observed establishment patterns. We asked which ecophysiological traits associated with carbon balance might allow whitebark pine greater initial success compared to cooccurring Engelmann spruce in exposed microsites at treeline. Physiological processes associated with carbon assimilation may be particularly important for understanding limitations to tree establishment, growth, and distributions at high elevations (Stevens and Fox 1991; Ko¨rner 1998; Bansal and Germino 2008). We predicted that whitebark pine seedlings would exhibit a suite of traits to improve carbon and water use efficiency (e.g., higher photosynthesis,
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lower respiration, lower transpiration) and to enhance tolerance to light and freezing stress (e.g., lower specific leaf area (SLA), higher soluble sugars) in exposed microsites, similar to previous studies on pioneer-species ecophysiology in stressful habitats (e.g., Chapin 1995; Braatne and Bliss 1999; Llambı´ et al. 2003). These expected traits of whitebark pine seedlings would allow establishment in exposed microsites away from herbs, while Engelmann spruce would be more prevalent immediately adjacent to neighboring herbs, where there would be a balance between competition for resources and protection from environmental stress by canopy cover.
Materials and methods Study area, experimental design, and seed propagation The study area was located on the west slope of Fred’s Mountain in the Teton Range in the Rocky Mountains, USA (43°47.26°N, 110°57.52°W). The continental climate of Fred’s Mountain is typical of the region, with mean annual temperature of 3°C, mean annual snowfall of 1300 cm, and only 3–4 months without permanent snow cover. During the study period (2007–2008), mean temperature during the growing season (July–September) was 13.4°C, with mean daily maximum and mean daily minimum temperatures of 22.9°C and 3.7°C, respectively. Differences in growing season mean, mean maximum, and mean minimum temperatures between the years were less than 1.5°C in all cases. Tree species in the study area were whitebark pine, subalpine fir, and Engelmann spruce. Herb cover was dominated by either tall forbs, cushion plants, or a blend of the two community types. Tall-forb species included Antennaria spp., Anemone tetonensis, Solidago multiradiata, Ligusticum filicinum, Geranium viscosissimum, Lomatium cous, Oxytropis spp., Hedysarum occidentale, and Delphinium nelsonii. The cushion community was dominated by Phlox pulvinata with forbs such as Hymenoxys grandiflora, and widerranging forbs such as Achillea millefolium, Phacelia sericea, Pedicularis groenlandica, and subalpine daisy (Erigeron spp.) that were common to all sites we examined. Graminoids were sub-dominant, and consisted of grasses such as Poa pattersonii and occasional sedges such as Carex hoodii.
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We established six sites along a 1.5 km transect at about 3000 m elevation, which coincides with the approximate altitude of treeline in this area. Each site was located away from tree islands and canopies, and was \0.5 ha in area. Within each site, 14 plots were randomly established that were approximately 0.5 m2 in size and had representative vegetation of the site. Seeds of whitebark pine and Engelmann spruce were sown in rows (1 species/row) within each plot among the natural herbaceous vegetation so that seedlings could potentially germinate under, adjacent, near, and away from herbs. Structures were installed over some of the plots to alter temperatures by 1–3°C as part of a different study (Germino unpubl.), but we found limited variation among the plots in properties reported in this study. Seeds of whitebark pine and Engelmann spruce were obtained from the U.S. Forest Service (Wind River Ranger District, Shoshone National Forest and Montpelier Ranger District, Caribou-Targhee National Forest, respectively). Seed sources were from localities between 2400 and 3000 m elevation that were \125 km from the study site, and are considered to be in the same transfer zone as the study site. Prior to outplanting, seeds were prepared and stratified according to Schopmeyer (1974). Briefly, seeds were imbibed under running water for 5 days, surface-sterilized with dilute hydrogen peroxide, and cold stratified at 3°C for 30 days. Seeds were then sown in late June 2007, immediately after snowmelt, so germination would coincide with natural germination in July. A comparable number of seeds within each plot were sown (N = 21 and 49 seeds/plot for whitebark pine and Engelmann spruce, respectively). Following outplanting, plots were watered twice daily with about 4 l (*4 mm) of water using a watering can to facilitate seed germination. Watering was tapered off in mid-July. Establishment patterns Seedling survival was assessed by presence–absence surveys that were conducted bi-weekly starting in early July 2007, however, only end-of-season data (September 2007 and September 2008) are reported in this study. We set up an ordinal classification system of microsites that was based on proximity of each seedling to the closest herb (herb cover rating, HCR). The four HCRs, from least to most canopy
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cover, were defined as: 1—seedling at least 10 cm away from nearest herb; 2—seedling between 5 and 10 cm away from nearest herb; 3—seedling adjacent to herb (\5 cm from nearest herb); 4—seedling covered by herb. Surviving seedlings within each microsite (HCR 1–4) were tallied and then divided by the total number of surviving seedlings across all microsites to give a frequency of survival for each microsite.
We calculated the projected leaf area for each seedling by removing all needles of the whole crown and laying them flat, digitally photographing the needles, and then quantifying the area using image processing software (Image J, Scion Co., Fredrick, MD). Following area measurements, needles were dried for 48 h at 70°C, and then weighed for drymass to ±0.1 mg. SLA was calculated as projected leaf area divided by leaf mass (cm2 mg-1).
Gas exchange and water relations
Chlorophyll fluorescence, nonstructural carbohydrates
Instantaneous gas exchange was measured using a portable photosynthesis system (model LI-6400, Li-Cor Biosciences, Lincoln, NE, USA) equipped with a CO2-controller and a red-blue LED chamber (model LI-6400-02b). Gas exchange variables were measured on entire seedlings and are reported on a silhouette leaf area basis according to Smith et al. (1991). Silhouette leaf area was determined by digitally photographing the seedling and objects of known size (for calibration) from the angle of the LI-6400 light source, and then quantified using image processing software (Image J, Scion Co., Fredrick, MD). Gas exchange was measured between 1000 and 1400 h local time on 7 days in August 2007, and on 4 days in August 2008. Both species were measured simultaneously and the combined number of gas exchange measurements between the 2 study years was N = 88 for Engelmann spruce and N = 118 for whitebark pine. During all gas exchange measurements chamber conditions were matched to ambient conditions for CO2 gas concentration (380 ppm), air humidity (26 ± 1.2%), and air temperature (20.6 ± 0.2°C). Light saturated photosynthesis (Asat) was measured with the chamber light source set at a light intensity of 1200 lmol m-2 s-1. Transpiration (E) was simultaneously measured during photosynthetic measurements, and water use efficiency (WUE) was calculated as Asat/E. Dark respiration (R) was measured by darkening the chamber (light intensity of 0 lmol m-2 s-1) and then waiting until gas exchange fluxes stabilized (approximately 4 min). Morphology At the end of the second growing season, all surviving seedlings were harvested to measure projected leaf area and needle biomass of whole crowns.
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Chlorophyll fluorescence was measured in the field during midday (1000–1400 h local time) in midAugust 2007 using a portable chlorophyll-a fluorometer (model MINI-PAM, Heinz Walz GmbH, Effeltrich, Germany). Needles were dark adapted for 25 min using leaf clips provided with the fluorometer. The ratio of variable to maximum fluorescence (Fv/Fm) was determined for the dark-adapted needles by measuring the fluorescence emission from PSII under a weak measurement light (Fo) and following a pulse of saturated light (Fm). From this, Fv was determined as Fm - Fo (Krause and Weis 1991; Maxwell and Johnson 2000). Fv/Fm values for healthy plants are typically around 0.83 (Bjo¨rkman and Demmig 1987), and lower values indicate photoinhibition. Nonstructural carbohydrate (NSC) is defined as starch plus soluble sugars (glucose, fructose, and sucrose); our protocol was modified from Hoch et al. (2002). Following measurements of drymass, we ground the needles to a fine powder and weighed to ±0.1 mg. We added 2 ml water to each sample and heated to 100°C with steam for 30 min to extract soluble sugars into aqueous solution, and then enzymatically treated a 200 ll aliquot of each sample with invertase, phosphoisomerase, and glucose hexokinase to convert sucrose, fructose, and glucose to 6-Phosphogluconate (Sigma Diagnostics, St. Louis, MO, USA). Oxidation of the soluble sugars to 6-Phosphogluconate resulted in an equimolar reduction of NAD to NADH, increasing absorbance of the solution at 340 nm, which was measured using a spectrophotometer (Synergy Microplate Reader, Biotek Instruments, Winooski, VT, USA), and was directly proportional to soluble sugar concentrations. The original sample (powder plus water) was then additionally treated with a high activity fungal
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We used chi-square analyses to test for microsite effects (HCR 1–4) on seedling survival at the end of each growing season for each species separately. We used ANOVAs to test for tree species and microsite effects on the response variables Asat, R, WUE, SLA, Fv/Fm, and NSC (total, starch, and soluble sugars) (SAS v9.2; SAS Institute, Cary, NC, USA). For the ANOVAs, tree species and HCR were fixed factors and site was a random factor. For NSCs from September 2008, HCR 1 was excluded for statistical analyses because of missing data for spruce seedlings. Tree species and herb effects on gas exchange variables did not significantly differ between years, and were pooled for analyses. NSCs and leaf areas were log transformed to meet the assumptions of normality and homoscedasticity of error variance.
Results Establishment patterns
Gas exchange and water relations Leaf-level photosynthesis (Asat) increased twofold in Engelmann spruce and sixfold in whitebark pine with increasing distance from herbs (Fig. 2a). There was no difference in photosynthesis between species, however, microsite effects on photosynthesis were significant (F3,56 = 5.35, P = 0.002). Leaf-level dark respiration (R) in whitebark pine was similar across all HCR, but R in spruce increased twofold in 50 40
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Data analysis
(P \ 0.001; Fig. 1). About 45% of all spruce seedlings at the end of the study were located in microsites directly adjacent to herbs (HCR = 3).
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alpha-amylase from Aspergillus oryzae (Clarase G-Plus, Genecore International, Rochester, NY, USA) to metabolize starch to glucose. The solution was re-analyzed for total NSC (starch and soluble sugars) with the procedure described previously. Starch was calculated as total NSC minus soluble sugars. All NSC data were normalized for drymass of needle tissue. The time-of-day that we sampled did not significantly affect NSC concentrations (Bansal and Germino 2009).
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At the end of the first growing season, both Engelmann spruce (P = 0.004) and whitebark pine (P \ 0.0001) were more abundant in exposed microsites (HCR 1–2) compared to protected microsites (HCR 3–4). Specifically, about 60 and 75% of all surviving Engelmann spruce (N = 56) and whitebark pine seedlings (N = 32), respectively, were present in microsites not overtopped or directly adjacent to herb cover in September 2007, when herb cover was undergoing senescence (Fig. 1). At the end of the second growing season, [80% of all surviving whitebark pine seedlings (N = 41) were in exposed microsites (P = 0.006), while \35% of Engelmann spruce seedlings (N = 36) were in open microsites
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under herb
Fig. 1 Frequency distribution of seedling abundances for whitebark pine and Engelmann spruce across an herb cover gradient (HCR 1–4) of microsites at the end of their first (September 2007; top panel) and second (September 2008; bottom panel) growing seasons. Percentages were calculated as the number of seedlings within each microsite divided by the total number of seedlings across all microsites. The levels of the herb cover gradient are defined as: 1 open-canopy microsite, seedlings [10 cm away from nearest herb; 2 seedlings 5–10 cm away from nearest herb; 3 seedlings adjacent (\5 cm) to herb; 4 seedlings covered by herb
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Chlorophyll fluorescence, nonstructural carbohydrates Midday Fv/Fm was about 20% greater in whitebark pine compared to Engelmann spruce across all herb cover microsites (F1,84 = 44.15, P \ 0.0001; Fig. 4a). Mean soluble sugar concentrations were approximately 7–10% drymass, and mean starch concentrations were relatively low (\7% of drymass) in both species. Soluble sugars were *20% greater in whitebark pine than in spruce (F1,30 = 5.00, P = 0.03; Fig 4b). Starch concentrations were 42% lower in whitebark pine (mean = 2.74 ± 0.5%) than in spruce (mean = 6.59 ± 1.4%; F1,30 = 6.12, P = 0.02). There were no significant effects of microsite or species 9 HCR interactions.
A sat (µmol m-2 s-1)
Whitebark pine had significantly lower SLA than Engelmann spruce (F1,50 = 22.09, P \ 0.0001). SLA of Engelmann spruce was between 0.15 and 0.12 cm2 mg-1 in the most and least herb cover, respectively, and SLA of whitebark pine was between 0.10 and 0.8 cm2 mg-1 across the herb cover gradient, although the trends were not significant (P = 0.13; Fig. 3).
a spruce pine
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In upper subalpine areas of the northern Rocky Mountains, whitebark pine is considered a pioneer species that is able to successfully establish in exposed sites, whereas co-occurring trees species such as Engelmann spruce and subalpine fir are considered to be shade-adapted species. Previous studies investigating early stages of tree recruitment in the upper subalpine have focused on older seedlings, saplings, and adult trees (Callaway 1998; Tomback et al. 2001; Bekker 2005). In this study, we show that the spatio-temporal patterns of adult tree species in the alpine-treeline ecotone (i.e., tree island
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Discussion
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Morphology
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open-canopy microsites (species 9 herb interaction, F3,56 = 6.49, P = 0.004; Fig. 2b). Water use efficiency was marginally greater in whitebark pine compared to Engelmann spruce across all HCR (F1,82 = 7.39, P = 0.079; Fig. 2c).
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under herb
Fig. 2 Instantaneous photosynthesis under saturating light (a), dark respiration (b), and water use efficiency (c) of whitebark pine and Engelmann spruce seedlings that were growing along the herb cover gradient. Measurements were conducted in August 2007 and 2008. See Fig. 1 caption for herb cover rating details
initiation) begins at the earliest life stages (establishment), and can be explained by differences in adaptive morphological and ecophysiological traits. Establishment patterns At our sites in the alpine-treeline ecotone, both whitebark pine and Engelmann spruce seedlings
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Fig. 3 Specific leaf area of whole crowns of whitebark pine and Engelmann spruce seedlings that were growing along the herb cover gradient. Data are from the end of the second growing season. See Fig. 1 caption for herb cover rating details
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exhibited similar patterns of initial survival, with the majority of seedlings surviving in exposed (HCR 1–2) microsites at the end of the first growing season (Fig. 1 top panel). However, only whitebark pine was able to persist in the most exposed microsites by the end of the second growing season (Fig. 1 bottom panel), while Engelmann spruce was relatively more abundant adjacent to herbs. The establishment patterns observed in our manipulative experiment are comparable to natural patterns reported by Maher and Germino (2006) across several mountain ranges in the northern Rockies. In that study, both whitebark pine and Engelmann spruce germinants occupied microsites with intermediate sky exposures, similar to the intermediate HCR observed here. After their first growing season, though, whitebark pine seedlings occupied microsites with greater sky exposure than Engelmann spruce seedlings. Gas exchange and water relations
Fv/Fm
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Soluble Sugar (% drymass)
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Fig. 4 Maximum photochemical efficiency (Fv/Fm, a) and soluble sugar concentrations in needles (b) in whitebark pine and Engelmann spruce seedlings that were growing along the herb cover gradient. Fv/Fm data are from August 2007, and soluble sugar concentrations data are from the end of the second growing season. No soluble sugar data were available for microsite 1 for spruce. See Fig. 1 caption for herb cover rating details
Overall, photosynthesis did not differ between the two species (Fig. 2a), suggesting that variation in net CO2 uptake at the leaf-level alone is not an overriding driver of variation in species distribution and regeneration patterns at treeline. Nevertheless, there were some parallels between survival along the herb cover gradient and carbon flux. For example, photosynthesis did match establishment patterns along the herb cover gradient during the first growing season, with greater abundance and carbon gain in the more exposed microsites (HCR 1–2). This suggests that leaf-level photosynthesis may be relatively important very early in development, when crowns are small and total leaf area is relatively low. In addition, whitebark pine was able to maintain constant and relatively low respiration rates across the exposure gradient compared to Engelmann spruce, even in the most exposed microsite (Fig. 2b). This allowed for greater carbon gain and carbon use efficiency in whitebark pine (A:R [ 3.8) compared to Engelmann spruce (A:R \ 2) in the more exposed microsites. Low respiration helps to ‘conserve’ carbon and improve carbon use efficiency (Maseyk et al. 2008), which may have contributed to greater abundance of whitebark pine seedlings in stressful microsites. However, under complete herb cover A:R was less than 1.0 for both species due to low photosynthesis,
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possibly contributing to the low abundances for these species in this microsite. Water use efficiency (WUE) was 2–3 times greater in whitebark pine than in Engelmann spruce in all microsites, except underneath herb cover (HCR = 4), where WUE was the same in both species (Fig. 2c). Although tree growth at high elevations is generally considered constrained by cool temperatures, drought stress may be an important local factor in the northern Rocky Mountains (Brodersen et al. 2006), particularly for seedlings that are still developing deep roots. Greater carbon and water use efficiency in whitebark pine compared to Engelmann spruce may be critical traits for establishment success at high elevations, especially in microsites without much protective herb cover (e.g., rocky terrain) that are prone to desiccation.
was able to maintain substantially higher Fv/Fm compared to Engelmann spruce, exhibiting resistance to low-temperature photoinhibition (Fig. 4a). Also, whitebark pine maintained greater soluble sugar concentrations compared to Engelmann spruce (Fig. 4b). Greater soluble sugar concentrations provide a level of cryoprotection, which may be an ¨ gren adaptive trait to cope with treeline climate (O et al. 1997; Janusz et al. 2001). Both of these biochemical traits (higher Fv/Fm and soluble sugars) likely aid whitebark pine’s superior establishment success at treeline, especially in open-canopy microsites, compared to Engelmann spruce.
Morphology
We have identified a suite of physiological distinctions associated with carbon balance between whitebark pine and Engelmann spruce that likely contributed to the spatio-temporal patterns of tree distributions and regeneration at treeline. Specifically, whitebark pine had greater soluble sugar concentrations in leaves and lower SLA, both of which can enhance tolerance to ¨ gren et al. 1997; light and freezing stress (e.g., O Dumais and Pre´vost 2008). Additionally, whitebark pine had superior carbon and water use efficiency compared to Engelmann spruce, which are traits of successful pioneer species in stressful upper-elevation locations (Chapin 1995; Braatne and Bliss 1999; Llambı´ et al. 2003). Establishment patterns in relation to herb cover also corresponded to differences in carbon balance. In particular, photosynthetic capacity matched initial establishment patterns during the first growing season for both species. Also, whitebark pine had relatively low respiration and low SLA compared to Engelmann spruce across all microsites, which likely allowed whitebark pine to survive in the most exposed microsites away from the protective canopy of surrounding herbs. The evident tradeoff for whitebark pine was an apparent inability to establish near or under neighboring vegetation. In these microsites, whitebark pine had negative carbon flux (A:R \ 1), and may explain how whitebark pine is eventually outcompeted by co-dominant conifers and why its distribution does not extend lower into the contiguous forest. Decreases in hydraulic efficiency with increasing size may also contribute to observed spatial patterns of older trees (Sala 2006).
Low SLA is a morphological trait that is fundamentally linked to increased needle longevity and longterm carbon gain (Reich et al. 2007; Wright et al. 2004; Mediavilla et al. 2008), and was lower in whitebark pine compared to Engelmann spruce (Fig. 3). Relatively low SLA in whitebark pine is contrary to patterns typical in early-successional species (e.g., Llambi et al. 2003), even in other pine-spruce dominated ecosystems (More´n et al. 2000), because it leads to lower photosynthesis per unit mass (data not shown) and suggests slower growth rates according to the economic spectrum of plant traits worldwide (Wright et al. 2004). However, this architectural strategy may enable whitebark pine to endure environmental stress as a young seedling in the alpine-treeline ecotone. At the same time, relatively high SLA in Engelmann spruce may have enhanced carbon gain under more shaded conditions, thus contributing to its relatively greater establishment success adjacent to herbs. Chlorophyll fluorescence, nonstructural carbohydrates In the treeline ecotone, the young seedlings likely experienced repeated cool nights followed by sunny mornings (i.e., conditions conducive to long-term photochemical damage; Ball et al. 1991; Germino and Smith 1999), particularly in the open-canopy microsites. Under these stressful conditions, whitebark pine
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Summary, relevance, and implications to treeline dynamics
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We emphasized traits associated with carbon balance because limitations on carbon uptake or use are currently the prevailing hypotheses for restrictions to tree growth at high elevations (Stevens and Fox 1991; Ko¨rner 1998). Our data demonstrate how multiple physiological mechanisms related to both carbon sources and sinks are simultaneously important to tree population dynamics at high elevations. Carbon assimilation (photosynthesis) was related to survival during the first year of growth, and carbon efflux and allocation (respiration and SLA) were important to the spatial distribution of each species. Furthermore, carbon flux balances (A:R) corresponded with patterns of establishment. Finally, we showed that water use efficiency can reveal potential limitations to establishment of conifer seedlings, and research pertaining to water relations at high altitudes is limited (Johnson et al. 2004; Brodersen et al. 2006; Reinhardt et al. 2009). Consequently, predictions of future treeline dynamics and forest migration, should account for ecophysiological traits associated with both carbon and water, particularly for establishing seedlings. Acknowledgments Funding for this study was provided by a grant from the Department of the Energy NICCR to MJG, seeds were provided by the USDA Forest Service, equipment was provided by W.K. Smith, and logistical support was provided by Grand Targhee Ski Resort management, in particular Grant Fleming and Andy Steele. Terry Peterson provided statistical consulting. Tristan Kelly, Dennis Demshar, Martha Inouye, Ben Morris, and Joe Timchak assisted in the field. We appreciate the comments of the anonymous reviewers that helped strengthen this manuscript.
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