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Frontiers in Forests and Global Change ·

Volume 9 - 2026 | https://doi.org/10.3389/ffgc.2026.1814554

In situ warming causes thermal divergence in photosynthetic capacity between two boreal conifers classified as the same plant functional type

  • William R. Vaughn1,2*corresponding author
  • Anthony R. Taylor2
  • David A. MacLean2
  • Loïc D’Orangeville3
  • Chris B. Edge1
  • Robert W. Buchkowski2,4
  1. 1Natural Resources Canada, Canadian Forest Service - Atlantic Forestry Centre, Fredericton, NB, Canada
  2. 2Faculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, NB, Canada
  3. 3Department of Wood and Forest Sciences, Laval University, Québec City, QC, Canada
  4. 4Department of Biology, Western University, London, ON, Canada
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Abstract

The boreal forest in North America plays a major role in global vegetation–atmosphere interactions and is expected to experience significant warming by the end of this century; therefore, accurately predicting carbon dynamics in this region is increasingly important. Carbon dynamics are often predicted using terrestrial biosphere models, which rely on parameters such as the maximum rate of Rubisco carboxylation to simulate carbon uptake. Terrestrial biosphere models generally group species into broad plant functional types that may oversimplify species-specific responses to warming. To address this limitation, we conducted a field warming experiment using custom-built phytotrons to evaluate the photosynthetic responses of two-year-old balsam fir (Abies balsamea (L.)) and white spruce (Picea glauca (Moench)) seedings—two boreal conifers commonly grouped in the same plant functional type—under four treatments: control (no phytotron), ambient (phytotron with no warming), and +3 °C and +6 °C above ambient. Under the +6 °C treatment, stomatal conductance, transpiration, net assimilation, and maximum rate of Rubisco carboxylation in white spruce were significantly greater than those in balsam fir, while no major differences were observed between these species under the ambient and +3 °C treatments. Interspecific variation in gas exchange and thermal acclimation likely drove the observed divergence in photosynthetic capacity under the warmest treatment. These findings support a growing body of research indicating that grouping physiologically distinct species into broad plant functional types may misrepresent the effects of climate change on forest carbon dynamics in terrestrial biosphere models.

Keywords

Introduction

Under the high-emission climate change scenario, global warming of up to 5.7 °C is expected to occur by the end of this century (IPCC, 2023). Warming is anticipated to occur at a significantly faster rate in northern-latitude regions, like the boreal forest (Zhang X. et al., 2019). The boreal forest includes 2.23 x 106 km2 of forest land cover in North America (Brandt, 2009), has a large influence on global vegetation–atmosphere interactions, and is a major source of uncertainty in carbon uptake within terrestrial biosphere models (Fisher et al., 2018). Terrestrial biosphere models predict current and future carbon cycling using parameters that simulate net photosynthesis of vegetation, among other factors. However, there is a lack of empirical data regarding the temperature effects on key biochemical processes that regulate photosynthesis in boreal tree species (Smith and Dukes, 2013; Lombardozzi et al., 2015; Stinziano et al., 2019; Rogers et al., 2022)—specifically, the maximum rate of ribulose-1,5-bisphosphate carboxylation (V*cmax*) by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the maximum rate of electron transport (J*max*) in the light-dependent reactions, which supply the energy for carboxylation and for regeneration of ribulose-1,5-bisphosphate in the Calvin-Benson cycle. Moreover, these processes are generally grouped by plant functional type (Rogers, 2014; Luo et al., 2019), further reducing the precision of model outputs. By grouping species into plant functional types, species diversity in the model is reduced, which can lead to significant errors in the simulation of gross primary production and carbon balance of terrestrial ecosystems (Zaehle et al., 2005; Harper et al., 2016; Butler et al., 2022).

Process-based, terrestrial biosphere models typically estimate net photosynthesis with the Farquhar–von Caemmerer–Berry model (Farquhar et al., 1980), in which V*cmax* is a key parameter. Growth temperature significantly influences both V*cmax* and its thermal acclimation. The acclimation of V*cmax* to higher temperatures may involve lowering investment in Rubisco, thereby reducing basal V*cmax* (V*cmax* measured at a standard temperature of 25 °C (V*cmax25*)) and maintaining consistent carbon assimilation rates (Way and Yamori, 2014). However, if V*cmax25* remains unchanged in plants growing under higher temperatures (i.e., no acclimation), both V*cmax* and assimilation rates will be greater relative to plants growing under cooler temperatures (Way and Oren, 2010). In previous field warming studies, boreal tree species have shown significant increases in V*cmax* when measured at high growth temperatures and no change in V*cmax25*, suggesting V*cmax* acclimation did not occur (Stefanski et al., 2020; Bermudez et al., 2021; Dusenge et al., 2024). Recent literature highlights both the lack of consensus regarding the acclimation responses of photosynthetic capacity (biochemical limits of photosynthesis: V*cmax* and J*max*; Farquhar et al., 1980) to warming and the limited number of species that have been investigated (Kumarathunge et al., 2019; Crous et al., 2022; Dusenge et al., 2024).

Species-specific differences in stomatal responses to vapour pressure deficit (VPD) strongly influence carbon dioxide (CO2) uptake and, because VPD increases exponentially with warming, may ultimately exert the greatest limitation on photosynthetic capacity under future climate change (Urban et al., 2017; Kurepin et al., 2018; Dusenge et al., 2021; Reich et al., 2022; Stefanski et al., 2023). The onset of stomatal closure in response to moderate warming and VPD initially has little impact on gross primary production, as photosynthesis is less sensitive to small decreases in internal CO2 when internal CO2 concentration is already high (Zhang Q. et al., 2019). However, species with greater stomatal sensitivity to VPD show greater declines in stomatal conductance (g*s*) as temperature and VPD increase (Grossiord et al., 2020), leading to an instantaneous decrease in carbon uptake (Mirabel et al., 2023). The long-term reduction in g*s* under high VPD eventually leads to downregulation of the photosynthetic system due to sustained limitation of CO2 influx (Flexas and Medrano, 2002; Quebbeman and Ramirez, 2016; Dusenge et al., 2021), resulting in reduced photosynthetic capacity (i.e., reduced V*cmax25*) and gross primary production (Xu and Baldocchi, 2003; Way and Sage, 2008; Quebbeman and Ramirez, 2016). While photosynthetic capacity may shift with seasonal warming, the intrinsic water use efficiency (iWUE) and the ratio of intercellular to air CO2 concentration (C*i*/C*a*) often remain stable, reflecting coordinated stomatal regulation that maintains a relatively constant ratio of carbon gain to stomatal conductance (Xu and Baldocchi, 2003; Dusenge et al., 2021).

In terrestrial biosphere models, tree species are typically grouped into plant functional types based on similarities in resource capture, responses to disturbances, life history strategies, structural characteristics, and phenology (Smith et al., 1997; Wullschleger et al., 2014). These plant functional types are often assigned single, mean values for traits and key physiological processes, such as V*cmax* and the stomatal slope parameter (Rogers et al., 2022), constraining interspecies trait diversity (Saccone et al., 2017). In this study, we focus on balsam fir (Abies balsamea (L.)) and white spruce (Picea glauca (Moench))—two economically and ecologically important boreal tree species native to North America that are often grouped into the same plant functional type (e.g., Medvigy et al., 2009; Oleson et al., 2013; Melton and Arora, 2016; Magnani et al., 2023). While these species share some characteristics, they differ in their drought and shade tolerance (Niinemets and Valladares, 2006; Ravenscroft et al., 2010) and water-use strategies (Martínez-Vilalta et al., 2004; Stefanski et al., 2023), all of which may directly influence carbon uptake.

Balsam fir exhibits a more conservative water-use strategy relative to white spruce, reflected by its more positive water potential at 50% loss of stem hydraulic conductivity (Martínez-Vilalta et al., 2004; Hacke and Jansen, 2009; Choat et al., 2012) and lower embolism resistance in both roots and stems when compared with white spruce (Hacke and Jansen, 2009). Moreover, this species tightly regulates stomatal conductance in response to drought and high VPD (Oogathoo et al., 2020; Stefanski et al., 2023; Schulz et al., 2024), thereby maintaining leaf water potentials while restricting CO2 uptake (McDowell et al., 2008). In contrast, white spruce exhibits relatively low minimum seasonal water potentials and negative or narrow hydraulic safety margins (Urli et al., 2023), suggesting this species maintains stomatal conductance, and thus CO2 uptake, under high evaporative demand. Because these species-specific stomatal behaviors are not explicitly represented in the plant functional types used in terrestrial biosphere models (e.g., Ball-Berry model; Ball et al., 1987), stomatal conductance may be inaccurately modeled, potentially affecting estimates of gross primary production and evapotranspiration (Miner et al., 2017; Leng et al., 2024). The accurate modeling of gross primary production and evapotranspiration is essential for predicting changes in the global terrestrial carbon and water cycles under climate change (Jung et al., 2010; Peters et al., 2018).

In this paper, we investigated the effects of warming and VPD on the photosynthetic capacities of balsam fir and white spruce, two dominant boreal forest tree species in North America, which are both predicted to decline at their southern range limits (Rogers et al., 2017; Taylor et al., 2017; Cadieux et al., 2019; Lu et al., 2019). While white spruce ranges across the boreal forest from Newfoundland in the east to Alaska in the west (Abrahamson, 2015), balsam fir has a smaller distribution that overlaps the range of white spruce from Newfoundland to western Alberta (Uchytil, 1991). Direct comparisons of the effects of warming and high VPD on gas exchange in balsam fir and white spruce are rare; therefore, collecting these data is essential given their wide distributions. To achieve our objective, we implemented a novel experiment using custom-built phytotrons (small, temperature-controlled greenhouses) installed on a recently cleared forest site, allowing us to simulate future temperature regimes while preserving natural environmental conditions such as soil structure, precipitation, and light. We hypothesized that (1) relative to white spruce, balsam fir would exhibit lower stomatal conductance and transpiration rates under warming because of its more conservative water use strategy, characterized by a heightened sensitivity to increasing VPD; and (2) white spruce seedlings would exhibit greater carbon assimilation rates and V*cmax25* under warming than balsam fir because they will maintain sufficient CO2 supply and avoid downregulation of photosynthetic capacity.

Materials and methods

Site description and preparation

Our experiment site is located in the Acadia Research Forest (Figure 1a), one of the Canadian Forest Service’s two research forests, located approximately 20 km northeast of Fredericton, New Brunswick on Highway 10. This research forest is representative of the Acadian Forest Region of New Brunswick and Nova Scotia (Natural Resources Canada, 2026), which lies in the transitional zone between the boreal forest to the north and temperate forest to the south. This site was chosen because it is located near the southern range limits of many boreal tree species, where they are expected to experience the greatest impacts of climate warming (Fisichelli et al., 2014; Reich et al., 2022).

Composite image showing: **(a)** the site after standing trees were mulched; **(b)** the site within the surrounding landscape; **(c)** a plot template (used to establish plot size and set phytotron anchor points) on a recently cleared plot; **(d)** the interior of a phytotron prior to planting, showing the circulation fan (1), exhaust fan (2), heater (3), temperature sensor housed in a white solar shield (4), and control module (5); **(e)** white spruce seedlings planted on one half of a phytotron plot; and **(f)** the arrangement of randomly assigned control and treatment plots.
Figure 1. Composite image showing: (a) the site after standing trees were mulched; (b) the site within the surrounding landscape; (c) a plot template (used to establish plot size and set phytotron anchor points) on a recently cleared plot; (d) the interior of a phytotron prior to planting, showing the circulation fan (1), exhaust fan (2), heater (3), temperature sensor housed in a white solar shield (4), and control module (5); (e) white spruce seedlings planted on one half of a phytotron plot; and (f) the arrangement of randomly assigned control and treatment plots.

In July 2022, a 0.6-ha forested site (46°00′30″N 66°21′52″W)—composed predominantly of ~50-year-old red spruce (Picea rubens Sarg.), balsam fir and hardwood species—was cleared with a forestry mulcher (Figure 1b). The machine did not mulch into the organic layer; stumps were mulched until they were level with the existing soil surface. The site is characterized by imperfectly drained clay loam with a medium to coarse surface texture and a fine-textured parent material. As both balsam fir and white spruce are tolerant of fresh to moist soils (Uchytil, 1991; Abrahamson, 2015; Sutton, 2022), this site was considered suitable for testing the effects of warming on plant performance. In the northwest corner of this site, a smaller 0.1-ha area was fenced off for wildlife exclusion and surrounded by a one-meter-deep drainage ditch dug along the outside perimeter of the fence. Within the enclosure, eight 2.4 x 3.7 m plots were established in two rows of four and subsequently cleared of mulch down to the surface of the soil (Figure 1c). Based on 1991–2020 climate normals from the Fredericton International Airport weather station, located approximately 20 km from the site, the mean annual temperature is 5.8 °C, with mean January and July temperatures of –9.2 °C and 19.7 °C, respectively, and mean July VPD of 0.6 kPa (Environment and Climate Change Canada, 2025). Annual precipitation averages 1,022 mm and is distributed relatively evenly throughout the year.

Phytotron construction

Phytotrons were made with Bella® greenhouses (measuring 2.4 m wide, 3.7 m long, and 2.2 m high; Model 703730; Canopia by Palram, Allentown, PA, USA), which were assembled and fastened to pressure-treated lumber frames. Each phytotron received an exhaust fan (Model S14-E1; Canarm, Brockville, ON, Canada), a motorized backdraft damper (Model SX3216X16; Canarm, Brockville, ON, Canada), an air circulation fan (Model HAF14-ZO; Canarm, Brockville, ON, Canada), and a convection heater (Model 705777; Canopia by Palram, Allentown, PA, USA). A temperature control system (Figure 1d) was installed in each phytotron to maintain a programmed temperature differential above ambient temperature (air temperature measured at 1 m above the ground outside the phytotrons). The temperature control system consisted of a heater, an exhaust fan, two temperature/humidity sensors (one sensor inside the phytotron and one sensor outside the phytotron were each mounted at a height of 1 m above the ground; Model 4099; Adafruit Industries, New York, NY, USA), two solid-state relays (Model SSR-240D25; TE Connectivity, Galway, Ireland), and a HUZZAH32 microcontroller (Model 3591; Adafruit Industries, New York, NY, USA). The heater and exhaust fan in each phytotron were connected to heating and cooling relays controlled by the microcontroller, which continuously monitored internal and external (ambient) temperatures and activated either component to maintain the programmed temperature differential for each treatment. Additionally, during times when cooling was not necessary (e.g., overnight or during overcast days), the exhaust fans were programmed to vent for 1 min every 30 min to ensure ambient levels of relative humidity and CO2 were maintained inside each phytotron. Temperature and relative humidity data were recorded over the entire duration of the experiment.

The exteriors of the phytotrons used in our experiment were made of transparent, double-walled polycarbonate sheets, which marginally reduced light transmission by 20% (Palram-Canopia 8 ft. Wide Bella Greenhouse Series, 2025). This effect was anticipated, as it was also observed in the Spruce and Peatland Responses Under Changing Environments (SPRUCE) experiment (Hanson et al., 2017), which used double-walled enclosures to encapsulate treatment areas, as well. This is a limitation common to all warming experiments that use structures to trap incoming solar radiation, necessitating the inclusion of an ambient treatment to determine the effect of the phytotron structures.

Seed source and seedling preparation

In the spring of 2022, balsam fir and white spruce seed were sown in 45-cell trays at the Atlantic Forestry Centre Greenhouse from stored bulk seed collections that were harvested in New Brunswick. Prior to sowing, the trays were filled with a mixture of peatmoss and perlite. After germination, seedlings were fertilized with water-soluble 11:41:08 (N:P:K) starter fertilizer (Plant Products Inc., Leamington, ON, Canada) at a concentration of 50 ppm N for two weeks. After this period, seedlings were fertilized with water-soluble 20:08:20 (N:P:K) grower fertilizer (Plant Products Inc., Leamington, ON, Canada) at a concentration of 100 ppm N until late August. Finally, a water-soluble finisher fertilizer (08:20:30 (N:P:K); 35 ppm N; Plant Products Inc., Leamington, ON, Canada) was used until the end of the growing season. These seedlings were transplanted into 3.8 L square pots in the spring of 2023 and placed in an outdoor growing pad. The fertilizer regime during the 2023 growing season was identical to that of 2022.

In May of 2024, 18 trees of each species were planted in monospecific groups on opposite halves of each of the eight plots, selected at random (Figure 1e). Within each plot half, trees were planted in rows following an alternating pattern of four, three, four, three, and four trees per row, with 30 cm between rows and 60 cm between trees within rows. This planting configuration allowed for a 30 cm space between the outer trees and the phytotron structure and a 60 cm space between the monospecific groups of balsam fir and white spruce trees.

Experimental design

Phytotrons were anchored to the ground over six of the eight cleared plots (selected at random) and the other two were left as unmanipulated controls (referred to as controls from this point forward). Three treatments were randomly assigned to two replicate plots: ambient (tracking ambient air temperature), +3 °C above ambient (maintaining constant differential above ambient temperature), or +6 °C above ambient (Figure 1f). All treatments started on 10 June 2024. Competing vegetation within each plot was manually removed weekly.

Because phytotron structures completely covered the plots and prevented rainfall from reaching the ground, we irrigated each phytotron plot with an amount of water equal to the precipitation measured with a rain gauge installed on site after every precipitation event. Two soil water potential sensors (one in the centre of each plot half; Model 200SS; Irrometer Company, Inc., Riverside, CA, USA) and one soil temperature sensor (in the centre of each plot; Model 200TS; Irrometer Company, Inc., Riverside, CA, USA) were installed 15 cm below the surface of the soil to measure soil water potential and temperature in each plot over the course of the entire experiment.

Air temperature and relative humidity data collection continued until the end of the experiment (6 December 2024). Temperature and relative humidity data collected from each treatment were used to produce VPD data (see Noh and Lee, 2022). Connectivity issues between the temperature sensors and the cloud-based storage resulted in data gaps for the following dates: 29 June–1 July, 7 July, 10 August, 24 August–2 September, 6–8 September, and 12 October–4 November. To fill in the missing data, temperature and relative humidity data from a weather station located approximately 1 km from the site were used to estimate values for the control treatment for the periods where data were missing. The average temperature differences between the control and each treatment (calculated from periods with complete data at the experiment site) were then added to the weather station data to estimate values for the ambient, +3 °C, and +6 °C treatments to fill in the gaps. We compared on-site and weather station temperature data using root-mean-square-error (RMSE) and bias. Weather station temperature closely matched on-site measurements, with low error (RMSE = 0.49 °C) and minimal bias (−0.14 °C; Supplementary Table S1.1). On-site observations during these data gaps verified that the temperature control systems were functioning normally.

Sampling of assimilation and dark respiration

Using a LI-6800 portable photosynthesis system with a large leaf and needle chamber (LI-COR, Lincoln, Nebraska, USA), net photosynthesis (A*net*), dark respiration (R*dark*), stomatal conductance (g*s*), transpiration (E), and the ratio of intercellular to air CO2 concentration (C*i*/C*a*) were collected from fully expanded, current-year foliage. Three trees of each species were randomly selected from every phytotron in every treatment (n = 48). Each tree was measured four times in random order on different days between 9:00 a.m. and 2:00 p.m. from 17 July to 7 August using the same branch segments each time. For each sample, temperature in the LI-6800 cuvette was set to match the current temperature of the phytotron at the time of measurement under 1,000 μmol m−2 s−1 photosynthetic photon flux density (in the range of saturating light reported in past literature; see Man and Lieffers, 1997; Goodine et al., 2008; Benomar et al., 2016), 60% relative humidity, and 400 μmol mol−1 CO2. Net assimilation and g*s* were allowed to stabilize prior to measuring. To minimize measurement bias, gas exchange data were considered stable when the slope of CO2 assimilation was less than 0.1 µmol m−2 s−1 min−1 and the slope of stomatal conductance was less than 0.05 mol m−2 s−1 min−1. After A*net* was recorded, the cuvette lights were turned off, and R*dark* was measured once gas exchange rates stabilized again. Because the same branch segments were planned to be used in the A-C<sub>i</sub> measurements, the foliage was collected for projected area measurement after A-C<sub>i</sub> data collection. Samples that produced negative C*i* or E values were removed during data cleaning. This resulted in four less data points (all four measurements from one seedling) in the control treatment, three less in the ambient treatment, and one less each in the +3 and +6 °C treatments.

A-C*i* curves and calculations of V<sub>cmax</sub> and J<sub>max</sub>

Between 26 August and 18 September, photosynthetic CO2 response curves (A-C*i* curves) were produced for the same branch segments of the trees that were selected for gas exchange measurements. The measurements were taken sequentially at 400, 300, 200, 100, 30, 400, 400, 600, 800, 1,000, and 1,200 µmol mol−1 CO2 (Ca) using the LI-6800 portable photosynthesis system equipped with a large leaf and needle chamber. The cuvette conditions were set at 28 °C air temperature, 60% relative humidity, and 1,500 µmol m–2 s–1 photosynthetically active radiation for all trees. A light response curve was completed prior to the collection of the A-C*i* data to establish the saturating level of light for both species. We chose 28 °C, as it was approximately midway between daily maximum temperatures of the control and +6 °C treatments. Once A-C*i* data collection was complete, the projected leaf area of each sample was determined by collecting the branch segments, scanning the foliage, and processing the scanned images with ImageJ (Schneider et al., 2012).

The fitaci function from the ‘plantecophys’ package (Duursma, 2015) was used to evaluate the A-C*i* curves of every tree. This function uses the Farquhar–von Caemmerer–Berry model (Farquhar et al., 1980) to determine the maximum rate of Rubisco carboxylation (V*cmax*) and the maximum rate of electron transport (J*max*) values from A-C*i* curves. All A-C*i* curves were visually inspected and curves with possible erroneous data (e.g., curves with multiple negative A*net* or C*i* values, indicative of measurement errors) were removed, which resulted in one less curve in the ambient treatment. Because mesophyll conductance to CO2 was not measured, the reported V*cmax* and J*max* are considered apparent values, as they were derived from intercellular CO2 concentrations rather than from chloroplastic CO2 concentrations at the site of carboxylation. We determined values of V*cmax* and J*max* standardized to 25 °C (V*cmax25* and J*max25*), facilitating comparison with those reported in previous studies. The fitaci function standardizes photosynthetic parameters to 25 °C by fitting the measured A-C*i* curves using temperature-dependent parameters within the Farquhar model framework and expressing them at a common reference temperature.

Foliar nitrogen analysis and LMA

For each treatment and species combination, foliar nitrogen was measured using 10 branch samples and an elemental analyzer (Model ECS 4010; Costech Analytical Technologies Inc., Valencia, CA, USA). Leaf mass per area (LMA) was determined by scanning foliage from three branch samples per treatment/species combination, calculating leaf area using ImageJ, oven-drying samples at 70 °C for 48 h, and then dividing dry mass by leaf area. Nitrogen per unit leaf area (N*area*) was determined by multiplying LMA by foliar nitrogen concentration.

Statistical analyses

All results were analyzed with mixed effect linear regression models for each response variable using the ‘glmmTMB’ package (Brooks et al., 2025). Response variables included A*net*, R*dark*, g*s*, E, C*i*/C*a*, iWUE (A*net*/g*s*), V*cmax25*, J*max25*, J*max25*/V*cmax25*, LMA, and N*area*. Explanatory variables included treatment (control, ambient, +3 °C, and +6 °C), species and their interaction as fixed effects, with phytotron as a random effect. Because the LI-6800 cuvette temperatures were higher in the warmer treatments during point-sampling of gas exchange variables, a centered cuvette temperature variable—centered within each treatment—was included in the A*net*, R*dark*, g*s*, E, C*i*/C*a*, and iWUE models to reduce collinearity between treatment and cuvette temperature. These models also had seedling ID as an additional random effect, as repeated measurements of gas exchange were taken from each tree. For all response variables, two separate models were fitted: one including only the control and ambient treatments to evaluate the effect of phytotron presence (i.e., no phytotron vs. phytotron), and another including only the phytotron treatments (ambient, +3 °C, and +6 °C) to assess the effect of warming. Additionally, we fit models including all treatments (control, ambient, +3 °C, and +6 °C) to assess whether the overall response patterns observed within phytotron treatments were still present when analyzed together with the control treatments. Type III Wald chi-square tests were used to assess the significance of fixed effects and their interactions in all models. Type III tests were selected because interaction effects were of primary interest, and the gas exchange and A-C*i* measurements were slightly unbalanced following the removal of some observations during data quality checks, as previously described. Marginal and conditional R2 values were computed for each model to assess model fit. Residual normality and homoscedasticity of the models were evaluated using diagnostic plots, including Q-Q plots and plots of residuals versus fitted values. Post-hoc multiple comparisons of estimated marginal means were conducted for species within treatments and treatments within species for all models using the emmeans function from the ‘emmeans’ package (Lenth, 2025). Pairwise comparisons were adjusted using the Šidák method to control the family-wise Type I error rate associated with multiple comparisons.

Results

Effects of treatments on environmental conditions

Warming treatments were effective in producing distinct differences in both air temperature and VPD (Figure 2). Temperature and VPD in the ambient treatment (phytotron with no warming) closely tracked those of the control treatment (no phytotron). In contrast, the +3 °C, and + 6 °C treatments resulted in consistent increases in air temperature and VPD above those of ambient and control treatments (Table 1). Specifically, in the month of July (the hottest month), the average maximum temperature and VPD were 28.4 °C and 1.9 kPa, 29.0 °C and 1.9 kPa, 32.3 °C and 2.5 kPa and 34.7 °C and 3.0 kPa for the control, ambient, +3 °C, and + 6 °C, respectively. These effects extended belowground as well, with soil temperature patterns showing the same treatment differences (Figure 3), indicating that the warming treatments successfully increased both the air and soil temperatures. Although some variation in soil water potential was observed among treatments, no values below field capacity were observed (Figure 3), likely due to the site's imperfect drainage. Having not experienced soil water deficits, our results mainly reflect the impacts of increasing temperature and VPD.

Daily average air temperature (°C) and vapour pressure deficit (VPD; kPa) of all treatments from 12 June to 6 December 2024. Open circles represent treatment termination dates. Temperature and VPD data of the ambient and control treatments were nearly identical. Ambient is phytotron but no treatment and control is no phytotron.
Figure 2. Daily average air temperature (°C) and vapour pressure deficit (VPD; kPa) of all treatments from 12 June to 6 December 2024. Open circles represent treatment termination dates. Temperature and VPD data of the ambient and control treatments were nearly identical. Ambient is phytotron but no treatment and control is no phytotron.
Daily average soil temperature (°C) and water potential (MPa) of all treatments from 26 June to 30 October 2024. Red dashed line represents the water potential at field capacity, which typically occurs at approximately −0.033 MPa (Weil et al., 2017).
Figure 3. Daily average soil temperature (°C) and water potential (MPa) of all treatments from 26 June to 30 October 2024. Red dashed line represents the water potential at field capacity, which typically occurs at approximately −0.033 MPa (Weil et al., 2017).

Table 1. Average monthly maximum temperature (°C) and VPD (kPa) for all treatments (control, ambient, +3 °C, and +6 °C).

MonthAverage maximum temperature (°C)Average maximum VPD (kPa)
ControlAmbient+3 °C+6 °CControlAmbient+3 °C+6 °C
June26.2827.5229.8232.151.892.012.382.87
July28.3829.0032.2734.651.931.882.493.03
August26.0126.4629.5532.231.661.602.112.65
September23.1823.6326.8429.691.451.391.912.44
October14.5214.9117.6720.490.770.771.001.35
November7.429.5611.7213.480.330.490.560.65
December0.787.340.230.50

Bold text indicates data collection began after the beginning of the month or stopped before the end of the month. Months with no data collection are blank.

Point-sampling of gas exchange

Gas exchange models generally performed well, with conditional R2 values exceeding 0.5 for A*net*, g*s*, R*dark*, and E (Table 2). White spruce A*net*, R*dark*, and E all increased significantly from ambient to +6 °C (p < 0.05; Figure 4). In contrast, balsam fir exhibited a significant treatment effect only for A*net*, which decreased between +3 °C and +6 °C (p < 0.05; Figure 4). Neither species showed significant effects of treatment on g*s*; however, balsam fir exhibited a marginal reduction in g*s* from +3 °C to +6 °C (p = 0.071; Figure 4). Both species showed similar A*net*, g*s*, and E under the ambient and +3 °C treatments, although white spruce exhibited significantly higher R*dark* than balsam fir under ambient and +3 °C (p < 0.05; Figure 4). Notably, under +6 °C, white spruce A*net*, R*dark*, g*s*, and E were 68, 99, 72, and 67% higher, respectively, than those of balsam fir (p < 0.05; Figure 4; Supplementary Table S1.2). This pattern was also observed in the full model that included control and phytotron treatments (Supplementary Table S1.3). When comparing control and ambient treatments only, R*dark* was significantly higher in the control treatment for both balsam fir and white spruce (p < 0.05; Table 2; Supplementary Table S1.2), but no significant treatment effects were observed for A*net*, g*s*, and E models. Further, no significant effects were observed in any of the C*i*/C*a* and iWUE models (Table 2).

Boxplots showing point-sample data of gross assimilation (*A*<sub>*net*</sub>), dark respiration (*R*<sub>*dark*</sub>), stomatal conductance (*g*<sub>*s*</sub>), and transpiration (*E*) in 48 balsam fir and white spruce seedlings, each measured four times between 17 July and 7 August using an LI-6800 under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (*α* = 0.05).
Figure 4. Boxplots showing point-sample data of gross assimilation (A*net*), dark respiration (R*dark*), stomatal conductance (g*s*), and transpiration (E) in 48 balsam fir and white spruce seedlings, each measured four times between 17 July and 7 August using an LI-6800 under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (α = 0.05).

Table 2. Type III Wald chi-square (χ2) tests from an analysis of deviance assessing the modeled effects of treatment (control, ambient, +3 °C, and +6 °C), species (balsam fir and white spruce) and their interaction on gas exchange (net assimilation (A*net*), dark respiration (R*dark*), stomatal conductance (g*s*), transpiration (E), and intrinsic water use efficiency (iWUE)), A-C*i* (maximum rate of carboxylation (V*cmax*), maximum rate of electron transport (J*max*), and their ratio (J*max*/V*cmax*) standardized to 25 °C), and foliar attributes (leaf mass per unit area (LMA) and nitrogen per unit leaf area (N*area*)).

TreatmentSpeciesTreatment × SpeciesR2mR2c
χ2DFp-valueχ2DFp-valueχ2DFp-value
A. Phytotron treatments
Gas exchange
A<sub>net</sub>9.8820.0070.0410.83716.702<0.0010.370.73
R<sub>dark</sub>5.5720.0627.6910.0064.3120.1160.510.52
g<sub>s</sub>5.2220.0740.2110.6519.0720.0110.250.73
E4.2820.1170.3310.56412.6220.0020.470.80
C<sub>i</sub>/C<sub>a</sub>1.1120.5731.2410.2662.6220.2700.090.61
iWUE0.8220.6651.5210.2172.6220.2700.090.62
A-C*i*
V<sub>cmax25</sub>5.4820.0640.0310.86410.5820.0050.340.34
J<sub>max25</sub>7.8920.0190.2710.60311.4220.0030.350.35
J<sub>max25</sub>/V<sub>cmax25</sub>3.2320.1991.2510.2630.46620.7920.160.17
Foliar attributes
LMA7.3020.03013.411<0.0014.5420.1030.390.39
N<sub>area</sub>6.6720.03612.941<0.00110.4920.0050.340.77
B. Control versus ambient treatments
Gas exchange
A<sub>net</sub>0.0810.7733.1810.0751.9710.1600.140.78
R<sub>dark</sub>6.1910.0138.4710.0040.0210.8750.530.54
g<sub>s</sub>0.0710.7861.8210.1781.5710.2100.070.38
E0.0810.7822.8810.0902.5710.1090.200.42
C<sub>i</sub>/C<sub>a</sub>0.9310.3350.0510.8150.5510.4580.020.17
iWUE0.8810.3470.0510.8210.6210.4330.020.18
A-C*i*
V<sub>cmax25</sub>0.5010.4790.4810.4910.3510.5540.120.12
J<sub>max25</sub>0.5510.4600.5610.4560.7110.3990.150.15
J<sub>max25</sub>/V<sub>cmax25</sub>0.0110.9150.2210.6412.6810.1020.160.38
Foliar attributes
LMA0.9210.3373.0610.0800.5610.4550.330.33
N<sub>area</sub>1.9710.16111.3310.0010.1310.7180.430.72

A. Analyses comparing ambient (phytotron but no treatment), +3 °C, and +6 °C treatments, and B. analyses with control (no phytotron) and ambient treatments. The Marginal (R²m) and conditional (R²c) coefficients of determination are provided to indicate variance explained by fixed effects alone and by both fixed and random effects, respectively. Significant effects (α = 0.05) are shown in bold.

Photosynthetic capacity

Models for V*cmax25* and J*max25* had conditional R2 values of 0.34 and 0.35, respectively, in the phytotron treatments (Table 2). The main effects of treatment and species did not significantly influence V*cmax25* and J*max25*, however, we did observe a significant interaction between treatment and species (Table 2). Specifically, under +6 °C, V*cmax25* and J*max25* of white spruce were 50% and 47% greater, respectively, than those of balsam fir (p < 0.05; Figure 5; Supplementary Table S1.2), while no significant differences were observed under ambient and +3 °C (Figure 5). Additionally, white spruce V*cmax25* and J*max25* increased significantly from ambient to +6 °C, while balsam fir J*max25* decreased from +3 °C to +6 °C (p < 0.05; Figure 5). No significant effects on the J*max25*/V*cmax25* ratio were observed in the phytotron treatments (Table 2), and no significant effects were observed in the V*cmax25*, J*max25*, and J*max25*/V*cmax25* models that only included control and ambient treatments (Table 2). These responses in photosynthetic capacity were consistent with those from the full model that included control and phytotron treatments (Supplementary Table S1.3).

Boxplots of maximum Rubisco carboxylation rate (*V*<sub>*cmax*</sub>) and maximum electron transport rate (*J*<sub>*max*</sub>), standardized to 25 °C in balsam fir and white spruce seedlings under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (*α* = 0.05). Data reflect measurements from *A–C*<sub>*i*</sub> response curves collected between 26 August and 18 September.
Figure 5. Boxplots of maximum Rubisco carboxylation rate (V*cmax*) and maximum electron transport rate (J*max*), standardized to 25 °C in balsam fir and white spruce seedlings under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (α = 0.05). Data reflect measurements from A–C*i* response curves collected between 26 August and 18 September.

Foliar attributes

Both the LMA and N*area* treatment models performed well, with R2 values of 0.77 and 0.39, respectively. Treatment (p < 0.05) and species (p < 0.05) significantly influenced both LMA and N*area* as main effects; however, N*area* was also affected by an interaction between treatment and species (p < 0.05; Table 2; Figure 6). LMA and N*area* were greater in white spruce than balsam fir under the ambient treatment (p < 0.05; Figure 6), and balsam fir LMA and N*area* increased with warming from ambient to +6 °C (p < 0.05; Figure 6). When comparing control and ambient treatments, no significant treatment effects were observed for LMA or N*area*.

Boxplots of leaf mass per area (*LMA*) and leaf nitrogen per unit leaf area (*N*<sub>*area*</sub>) in balsam fir and white spruce seedlings under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (*α* = 0.05).
Figure 6. Boxplots of leaf mass per area (LMA) and leaf nitrogen per unit leaf area (N*area*) in balsam fir and white spruce seedlings under three phytotron treatments: ambient, +3 °C, and +6 °C. Treatment means are indicated with a black diamond inside each boxplot. Bold, uppercase letters indicate significant pairwise differences among treatments within species, and lowercase letters indicate significant differences among species within treatments based on estimated marginal means (α = 0.05).

Discussion

Under 6 °C of warming, white spruce trees exhibited 67% higher E and 72% higher g*s* relative to balsam fir, supporting our first hypothesis. A similar divergence in leaf-level gas exchange between two other boreal conifers was reported by Dusenge et al. (2021), who found that tamarack (Larix laricina (Du Roi) K. Koch) exhibited increased A*net* and g*s* with warming, while Anet and gs in black spruce (Picea mariana (Mill.) B.S.P.) were unaffected. Considering that soil moisture remained above field capacity across all treatments in our experiment, this observation likely reflects species-specific differences in responses to temperature and VPD, with white spruce maintaining higher rates of gas exchange than balsam fir under the +6 °C warming treatment. This, in turn, supports the greater carbon assimilation rates and V*cmax25* observed in white spruce under +6 °C warming, consistent with our second hypothesis. Because N*area* did not differ between balsam fir and white spruce under + 6 °C warming, the lower carbon assimilation rates and photosynthetic capacity observed in balsam fir in this treatment were unlikely the result of nitrogen limitation (Dang et al., 1997).

Interspecific variation in responses to warming

We observed a clear divergence in leaf-level gas exchange and photosynthetic capacity between balsam fir and white spruce under 6 °C of warming above ambient temperature. As we did not observe differences in C*i*/C*a* among treatments in either species, stomatal limitation was unlikely to have caused the observed decline in balsam fir A*net* in the +6 °C treatment at the time of measurement. While neither species exhibited significant effects of treatment on g*s*, the marginal decline in balsam fir g*s* from +3 °C to +6 °C and the significant difference in g*s* between balsam fir and white spruce under +6 °C suggest a temperature threshold in balsam fir was surpassed, which is consistent with this species’ non-linear response in sap flow to rising VPD (Oogathoo et al., 2020). A species with a non-linear stomatal response to rising VPD would not exhibit a steady decline in g*s* with increasing atmospheric evaporative demand (Oren et al., 1999; Grossiord et al., 2020). Consequently, the absence of a consistent decline in balsam fir g*s* across warming treatments is not unexpected. Despite this non-linear stomatal behavior, iWUE remained stable across treatments, supporting the optimization theory, which predicts coordinated regulation between carbon assimilation and g*s* (Cowan, 1978; Gardner et al., 2023).

Species with greater stomatal sensitivity to VPD tend to exhibit larger reductions in g*s* under higher temperatures (Grossiord et al., 2020), which reduces carbon uptake (Mirabel et al., 2023). Over time, sustained reductions in g*s* may limit CO2 diffusion sufficiently to drive downregulation of the photosynthetic apparatus (Wong et al., 1979; Flexas and Medrano, 2002; Ethier et al., 2006; Dusenge et al., 2021), ultimately decreasing photosynthetic capacity (Way and Sage, 2008; Quebbeman and Ramirez, 2016). The decline in balsam fir J*max25* from +3 °C to +6 °C and its lower R*dark* relative to white spruce under the +6 °C treatment are consistent with the lower maintenance respiration demands associated with reduced photosynthetic capacity (Hikosaka, 2005). However, because we observed only a marginal decrease in balsam fir g*s* from +3 °C to +6 °C and no significant change in white spruce g*s* with warming, we cannot confidently conclude that interspecific variation in stomatal behavior drove the divergence in photosynthetic capacity. Rather, the significant increases in white spruce V*cmax25* and J*max25* from ambient to +6 °C and the significant decline in balsam fir J*max25* from +3 °C to +6 °C suggest that these species vary in their temperature response of photosynthetic biochemistry (Crous et al., 2022). This differential response in thermal acclimation has been reported for tree species in boreal, temperate, and tropical biomes (Way and Oren, 2010; Crous et al., 2022). In some cases, like the in situ warming experiment conducted by Stefanski et al. (2020) in the southern boreal, no acclimation in V*cmax25* or J*max25* was observed. Due to this lack of consensus, further research is needed to determine the species-specific acclimation potential of photosynthetic capacity to future warming.

While both species were exposed to the same conditions in the +6 °C treatment, the 67% greater E in white spruce may have enhanced evaporative cooling and reduced its foliar temperature relative to balsam fir (Kolb and Robberecht, 1996; Birami et al., 2018; Sadok et al., 2021). Comparatively higher foliar temperature in balsam fir may explain the reduction in J*max25* under the +6 °C treatment, as warming can shift nitrogen investment from electron transport capacity toward carboxylation capacity to offset increased photorespiratory demand (Crous et al., 2022), which is consistent with the lack of change in balsam fir V*cmax25* across treatments. The increase in white spruce V*cmax25* and J*max25* under warming may reflect greater physiological plasticity in photosynthetic capacity, consistent with evidence that species differ in their ability to acclimate V*cmax25* and J*max25*through adjustments in gas exchange and nitrogen allocation under elevated temperature (Kattge and Knorr, 2007; Way and Yamori, 2014; Yamori et al., 2014; Bermudez et al., 2021; Dusenge et al., 2021; Crous et al., 2022).

Although both species maintained a consistent ratio of carbon uptake to water loss across treatments, balsam fir exhibited lower A*net* in the warmest treatment. In a glasshouse warming experiment conducted by Dusenge et al. (2020), black spruce biomass decreased with temperature due to a decline in A*net*, whereas tamarack biomass increased while A*ne*t was unaffected. While we have not yet reported growth data from our study, the greater photosynthetic capacity and A*net* observed in white spruce relative to balsam fir may confer a competitive advantage under future warming, as white spruce may eventually outpace balsam fir in growth. Overall, species-specific thermal acclimation potential may strongly influence productivity in the boreal forest under projected climate change (Yamori et al., 2014; Crous et al., 2022).

We did not observe negative effects of short-term warming projections (+3 °C) on photosynthetic capacities in either species, so significant divergences in their photosynthetic capacity may only occur under warming associated with long-term, high-emission scenario projections. However, because the trees did not experience soil water deficits during the experiment, we were unable to observe the interaction between drought and warming. Past manipulative experiments have shown that drought exacerbates the negative effects of high VPD on carbon assimilation (Will et al., 2013; Gao et al., 2017; Vaughn et al., 2021). Therefore, had soil water deficits occurred during the experiment, the divergence in photosynthetic capacity between balsam fir and white spruce may have occurred under cooler temperatures.

Higher rates of R*dark* were observed in control treatments relative to ambient in both balsam fir and white spruce seedlings. On an area basis, R*dark* has been observed to increase with increased light intensity in both balsam fir and white spruce (Lusk and Reich, 2000), as higher light levels are associated with greater photosynthetic capacity and leaf nitrogen content, which in turn increase maintenance respiration rates (Hikosaka, 2005). This trend is partially supported by the marginally higher V*cmax25* and N*area* in balsam fir and white spruce seedlings in the control treatment relative to the ambient treatment (Supplementary Table S1.2). Because temperature did not vary between the control and ambient treatments, the lower R*dark* in the ambient treatment was likely driven by reduced light transmission through the polycarbonate walls of the phytotrons. The reduction in light transmission would have also affected seedlings in the other phytotron treatments; however, its effects could not be isolated because temperature and VPD were incrementally higher in these treatments.

Implications for terrestrial biosphere models

Models that group species with contrasting physiological responses to warming (e.g., balsam fir and white spruce) into a common plant functional type may inaccurately project long-term forest carbon uptake under a high-emission climate change scenario, as species-specific productivity is likely to diverge. This may occur because many terrestrial biosphere models represent all species within a plant functional type using a common set of physiological parameters. For example, species within a common plant functional type are typically assigned the same stomatal slope parameter, which governs the response of g*s* to photosynthesis and environmental conditions. (Ball et al., 1987). Our findings and those of other field warming studies (Dusenge et al., 2021; Stefanski et al., 2023) do not support the use of this strategy because the effect of warming on gas exchange and carbon assimilation often varies among species that are commonly grouped together in plant functional types. Including species specific traits has been shown to substantially alter carbon uptake estimates by up to 30% in boreal regions (Butler et al., 2022) and improve the accuracy of biome-scale gross primary production projections, reducing the root mean square error of simulated boreal forest gross primary production by more than 40% relative to flux tower observations (Harper et al., 2016). Ultimately, more species-specific empirical data collected using a consistent measurement technique and covering a wide range of environmental conditions and seasonal variability is needed to further improve our knowledge of species-specific stomatal behavior (Miner et al., 2017; Leng et al., 2024).

Conclusion

The divergent responses of white spruce and balsam fir trees to elevated temperatures—particularly in stomatal conductance, carbon assimilation, and photosynthetic capacity—provide valuable insight into how these trees may respond under future climate warming. As our study examined seedlings that experienced only one year of warming, our observations may only reflect juvenile responses to warming and VPD. Therefore, more data will be collected over multiple years to confirm the observed patterns. The boreal forest constitutes more than 30% of North America’s total forest area, so even small changes in leaf-level physiology of dominant boreal species could have substantial impacts on global vegetation–atmosphere interactions. Our findings support the utilization of species-specific parameterization in terrestrial biosphere models and demonstrate the effectiveness of in situ warming experiments in capturing plant-environment interactions.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ffgc.2026.1814554/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Sanna Sevanto, Los Alamos National Laboratory (DOE), United States

Reviewed by: Ximeng Li, Minzu University of China, China

Jessica Schedlbauer, West Chester University, United States

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was received for this work and/or its publication. Funding for this project was provided by Natural Resources Canada—Canadian Forest Service.

Data availability

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://osf.io/weg97/overview?view_only=67c5918234474c91b403f238599c5b62.

Author contributions

WV: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. AT: Conceptualization, Supervision, Writing – review & editing. DM: Conceptualization, Supervision, Writing – review & editing. LD’O: Conceptualization, Supervision, Writing – review & editing. CE: Conceptualization, Supervision, Writing – review & editing. RB: Conceptualization, Supervision, Writing – review & editing.

Acknowledgements

We thank the staff at the Atlantic Forestry Centre greenhouse in Fredericton for assisting with the logistics of the experiment, and Declan McKillop and Mackenzie Good for assisting with data collection.

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Citation

Vaughn, W. R., et al. (2026). In situ warming causes thermal divergence in photosynthetic capacity between two boreal conifers classified as the same plant functional type. Frontiers in Forests and Global Change, Volume 9. https://doi.org/
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Copyright © 2026 His Majesty the King in Right of Canada.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: William R. Vaughn

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