Oecologia (2014) 175:457–470 DOI 10.1007/s00442-014-2923-9
Physiological ecology - Original research
Nitrogen‑addition effects on leaf traits and photosynthetic carbon gain of boreal forest understory shrubs Sari Palmroth · Lisbet Holm Bach · Annika Nordin · Kristin Palmqvist
Received: 14 August 2013 / Accepted: 5 March 2014 / Published online: 6 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Boreal coniferous forests are characterized by fairly open canopies where understory vegetation is an important component of ecosystem C and N cycling. We used an ecophysiological approach to study the effects of N additions on uptake and partitioning of C and N in two dominant understory shrubs: deciduous Vaccinium myrtillus in a Picea abies stand and evergreen Vaccinium vitisidaea in a Pinus sylvestris stand in northern Sweden. N was added to these stands for 16 and 8 years, respectively, at rates of 0, 12.5, and 50 kg N ha−1 year−1. N addition at the highest rate increased foliar N and chlorophyll concentrations in both understory species. Canopy cover of P. abies also increased, decreasing light availability and leaf mass per area of V. myrtillus. Among leaves of either shrub, foliar N content did not explain variation in light-saturated CO2 exchange rates. Instead photosynthetic capacity varied with stomatal conductance possibly reflecting plant hydraulic properties and within-site variation in water availability. Moreover, likely due to increased shading under P. abies and due to water limitations in the sandy soil under
Communicated by Hermann Heilmeier. S. Palmroth (*) Division of Environmental Science and Policy, Nicholas School of the Environment, Duke University, Box 90328, Durham, NC 27708‑0328, USA e-mail: [email protected] S. Palmroth · A. Nordin Department of Forest Ecology and Management, Swedish University of Agricultural Sciences (SLU), 901 83, Umeå, Sweden L. Holm Bach · K. Palmqvist Department of Ecology and Environmental Science, Umeå University, 90187 Umeå, Sweden
P. sylvestris, individuals of the two shrubs did not increase their biomass or shift their allocation between above- and belowground parts in response to N additions. Altogether, our results indicate that the understory shrubs in these systems show little response to N additions in terms of photosynthetic physiology or growth and that changes in their performance are mostly associated with responses of the tree canopy. Keywords Biomass allocation · Chlorophyll content · Photosynthetic capacity · Stomatal conductance · Vaccinium
Introduction Tree growth in the vast boreal forest [~10 % of land cover (Sabine et al. 2004)] is often limited by soil N (Tamm 1991; Vitousek and Howarth 1991). Soil fertility and other site-quality factors, including water holding capacity, drive the rate of stand development and, thus, the degree of canopy closure and allocation of site resources between trees and understory plants. The canopy in the European boreal coniferous forest is mostly composed of Pinus sylvestris (Scots pine) and Picea abies (Norway spruce). While both trees show high ecological plasticity, pure, fairly open Pinus sylvestris stands are more common on dry, low-fertility soils whereas Picea abies dominates on more mesic and fertile sites (Cajander 1949; Esseen et al. 1997). The associated continuum of understory communities mirrors the site-quality gradient as Vaccinium vitis-idaea and Calluna vulgaris dominate in Pinus sylvestris stands and V. myrtillus in Picea abies stands. The understory biomass is much lower than that of trees, but turns over faster and, for example in an Alaskan Picea mariana forest, the understory net
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primary production was estimated to account for 10–50 % of ecosystem production (O’Connell et al. 2003). Measured estimates of understory gross primary production (GPP) are, however, only available from a few stands throughout the boreal zone (Moren and Lindroth 2000; Widen 2002; Kolari et al. 2006; Misson et al. 2007; Kulmala et al. 2011). In a synthesis of studies on ecosystem fluxes of boreal and temperate forests, Misson et al. (2007) showed that the understory contribution to ecosystem GPP was 14 % on average, with the highest estimate reaching 39 %. These results clearly indicate that understory C uptake must be accounted for in ecosystem C-balance calculations. Regrettably, none of these ecosystem flux studies cover the wide fertility range found in boreal forests, limiting the scope of inference. To facilitate modelling efforts from stand to landscape, we need a better understanding of understory C uptake and partitioning of C and N among biomass pools along natural resource gradients and, also, under conditions where N availability is enhanced via atmospheric deposition or fertilization. Boreal understory species greatly differ in their ability to take up and utilize additional N (Nordin and Näsholm 1997; Manninen et al. 2009). When the two most common dwarf shrubs were grown in controlled conditions without an overstory, N-fertilized deciduous V. myrtillus doubled its biomass over 1 year, whereas evergreen V. vitis-idaea performed better than V. myrtillus at low N (Grelet et al. 2001). While deciduousness may mean increased plasticity, perhaps through a faster initial response, another fertilization study in a tree-less tundra community found no such effect (Karlsson 1985). In the latter study, the overall growth response of another deciduous shrub, Vaccinium uliginosum was neither faster nor stronger than that of V. vitis-idaea. In boreal forests, in turn, N fertilization typically increases tree biomass and leaf area (Axelsson and Axelsson 1986; Bergh et al. 1999; Nohrstedt 2001). The subsequent increase in shading, water limitations, or susceptibility of understory plants to biotic stressors may impose species-specific limitations to growth. Fertilization-induced changes in understory species composition are relatively well documented (Mäkipää 1995, 1999; Strengbom et al. 2002; Nordin et al. 2009; Gundale et al. 2011, 2013), yet the physiological responses underlying such shifts are not well understood. For individuals growing in a given light environment, N additions may increase the partitioning of C aboveground and leaf mass fraction (LMF) (Litton et al. 2007; Manninen et al. 2009; Poorter et al. 2012). Increased shading may also increase LMF but decrease total plant biomass. Yet, shaded plants often adjust leaf mass per area (LMA) even more than biomass partitioning between aboveground and belowground
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components (Poorter et al. 2012). Thus, where light availability decreases with increasing fertility, LMA likely decreases. Lower LMA (within a species) is associated with lower area-based photosynthetic capacity (Oren et al. 1986) but, for a given LMF, also with higher leaf area; thus C uptake per unit ground area may remain unchanged. Foliar N generally increases with soil N availability, yet how the ‘extra’ N is partitioned among and within leaves, and among the component processes of photosynthesis, varies with species and the availability of other resources (Evans 1989; Evans and Poorter 2001). In low light environments, if plant growth is not enhanced by N fertilization, the relative sink strength of various N storage pools can increase (Nordin and Näsholm 1997; Bubier et al. 2011). Excess N can be stored as photosynthetic enzymes (Lawlor et al. 1987; Warren et al. 2003), inorganic N (Gebauer et al. 1984) or, especially in boreal ecosystems, as free amino acids (Näsholm and Ericsson 1990; Nordin and Näsholm 1997). In sum, in forests where N addition results in increased canopy leaf area, causing increased shading of the understory, morphological and physiological responses of understory species may result in little effect on growth or photosynthesis with the extra N accumulating in storage. We assessed the effects of N additions on the C uptake and growth of understory plants. We focused on two shrubs in two different forest types; V. myrtillus in a mesic, fertile Picea abies stand (Svartberget) and V. vitis-idaea in a dry, nutrient-poor Pinus sylvestris stand (Åheden) in northern Sweden. These stands were located within ~1 km from each other and subjected to N addition at rates of 0, 12.5, and 50 kg N ha−1 year−1 over 16 and 8 years, respectively. At the time of our study, both forest stands were well adjusted to the N-addition regimes. Following an initial 3to 5-year adjustment phase to the new N regime, no further changes in understory species composition were recorded (Nordin et al. 2009; Gundale et al. 2011). Also, N additions had no effect on the mycorrhizal associates of the two shrubs in either stand (Ishida and Nordin 2010). We quantified a number of potential determinants of C uptake: relative light availability, leaf photosynthetic light response, LMA, and allocation of C and N among various biomass pools of the two shrubs. We hypothesized that N additions would lead to increased photosynthetic capacity of both shrubs (hypothesis 1). However, as the abiotic factors differed between the sites, we expected that the effects of N additions on growth would differ between the species. Specifically, we hypothesized that increased shading from the P. abies overstory would hamper the growth response of V. myrtillus to N addition (hypothesis 2), and that water limitation would hamper the expected growth response to N addition of V. vitis-idaea (hypothesis 3).
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Materials and methods Sites The experiment was performed at two sites, Svartberget and Åheden, ~1 km apart, within the Svartberget Experimental forest (64°14′N, 19°46′E), Vindeln, northern Sweden. Characteristics of the sites and details of the fertilization experiments can be found in Nordin et al. (2005) and Gundale et al. (2011) but this information is also briefly summarized here. The Svartberget site is a 100-year-old Picea abies (L.) Karst. forest growing on a gently sloping moraine. The field layer is dominated by Vaccinium myrtillus and Vaccinium vitis-idaea and the bottom layer by Hylocomium splendens (Hedw.) Br. Eur. and Pleurozium schreberi (Brid.) Mit. The Åheden site is a 60-year-old Pinus sylvestris L. forest growing on a fine sandy and silty glacial outwash sediments dominated by V. vitis-idaea and Calluna vulgaris (L.) Hull, and the bottom layer by Pleurozium schreberi. Annual mean temperature (1961–1990) in the study area is 1.5 °C and precipitation 591 mm, with the averages for July 14.1 °C and 69 mm, respectively (Vindeln-Sunnansjönäs, Swedish Meteorological and Hydrological Institute). The background atmospheric N deposition rate in this area is ~2 kg N ha−1 year−1 (Phil-Karlsson et al. 2009). The fertilization experiment started at Svartberget in 1996 and consists of six replicate square plots (1,000 m2) of each treatment [0 (control), 12.5 (N1) and 50 (N2) kg N ha−1 year−1] in a randomized block design. In 2003, an identical fertilization experiment (but in a completely randomized design) was set up at Åheden. The lower N-addition rate in these experiments was designed to mimic the highest deposition rates in boreal forests (Dentener et al. 2006), and the higher rate is in line with the typical long-term fertilization experiments in the area. Analyses of root‑to‑shoot ratio and C and N concentrations For biomass estimates, we sampled two 0.25-m × 0.25-m subplots from each treatment (control, N1, and N2) in all the six plots (n = 6) at both Svartberget and Åheden experiments between 23 and 26 August 2011. At Åheden, the subplots for V. vitis-idaea biomass sampling were laid out randomly in each plot. At Svartberget, the individuals of V. myrtillus tend to be more aggregated than randomly scattered in the fertilized stands (Strengbom et al. 2002) and our biomass sampling (targeting V. myrtillus) reflects the biomass where V. myrtillus occurs rather than the plot average. We harvested all V. vitis-idaea and V. myrtillus plants, above- and belowground parts except for fine roots, from the subplots and divided the biomass into various
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categories. For the deciduous V. myrtillus the four categories were leaves, current annual shoot, remaining shoot, and roots. For the evergreen V. vitis-idaea the five biomass categories included all the above and also leaves from previous years. These biomass fractions were summed and root-to-shoot ratios calculated. LMF was expressed as the fraction of leaf mass of total aboveground biomass, and leaf area ratio (LAR) as leaf area (=leaf mass divided by LMA; see details below) divided by aboveground biomass (m2 kg−1). Gas exchange measurements We measured gas exchange of healthy looking V. myrtillus leaves (one upper-crown leaf per plant, all plants of similar size) in three plots (n = 3) at Svartberget between 11 and 19 July 2011, yielding 12–16 individual photosynthetic light response curves per treatment. The gas exchange measurements at Åheden were carried out between 20 and 27 July 2011, also in three plots (n = 3). We sampled current-year leaves (CL) and previous year’s leaves (CL + 1) from V. vitisidaea (one of each per plant), yielding four to nine curves per treatment and age class. At both sites, we also collected ten additional leaves from each plant for N concentration, chlorophyll and LMA determinations (see below). We measured CO2 and H2O exchange rates of intact leaves in the field using a portable photosynthesis system LI-6400 (LI-COR, Lincoln, NE). Chamber conditions were kept as constant as possible: temperature at 20 °C, flow at 400 μmol s−1, CO2 concentration at 400 μmol mol−1 and relative humidity at 60–70 %. Photosynthetic photon flux density (PPFD) was first set to 200 μmol m−2 s−1 and then increased stepwise up to 500 μmol m−2 s−1, after which PPFD was decreased stepwise to zero. The width of the step was 20 μmol m−2 s−1 on the light-limited part of the curve and 50– 100 μmol m−2 s−1 otherwise, resulting in 16 (V. myrtillus) or 18 (V. vitis-idaea) points per curve. The leaves were collected for measurements of leaf area and leaf dry weight (see details below). The resulting light response curves were inspected visually and obvious outliers removed. We estimated dark respiration rate (Rd) as CO2 exchange at PPFD = 0 μmol m−2 s−1. To obtain estimates for the lightresponse-curve parameters we fitted a non-rectangular hyperbola to the data (Thornley 2002; Posada et al. 2009): 2 1 φI + Amax,g − 4θφIAmax,g ) − Rd , A= (φI + Amax,g − 2θ where A is CO2 exchange rate, I is instantaneous PPFD incident on leaf, ϕ is apparent quantum yield, Amax,g is light-saturated gross assimilation rate, and θ is curvature. We also recorded stomatal conductance (gs) and intercellular CO2 concentration. Because the weather conditions before and during the measurement campaigns were
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Table 1 Environmental data from a weather station at Svartberget showing mean values for the period of gas exchange measurements and the preceding week Week before
Experiment
Svartberget, Vaccinium myrtillus Air temp. (°C; min./max.) 17.7 (4.7/27.1) Soil temp. (°C; min./max.) 12.7 (11.6/13.9) RH (%) 66 Precipitation (mm) 0 23.2 Global radiation (MJ m−2)
14.5 (2.9/23.5) 13 (11.6/13.8) 76 30 17.1
Åheden, Vaccinium vitis-idaea Air temp. (°C; min./max.) Soil temp. (°C; min./max.) RH (%) Precipitation (mm)
14.2 (0.2/23.2) 13.1 (11.6/14.7) 71 11
17.9 (9.4/24.5) 15 (13.7/16.1) 76 26
18.4
16
Global radiation (MJ m−2)
temp. Temperature, min. minimum, max. maximum, RH relative humidity
similar at each site (Table 1), and the range of chamber conditions was narrow, we assume that the response curves represent growing conditions, i.e. treatments. Light availability Light availability under the main canopy in each stand was quantified through analysis of hemispherical images. Photographs were taken from all sampling locations (above biomass plots, and above individuals measured for gas exchange and sampled from transects as described below) under overcast conditions or at low sun angles. We used a Canon EOS 1000D equipped with an EX Sigma Circular Fisheye DC HSM 4.5 mm 1:2,8 and the pictures were analysed with Hemiview 2.1 (DeltaT devices, UK) updated with lens properties for the EX Sigma Circular Fisheye. From the output, we extracted indirect site factor (ISF), which is the percentage of indirect radiation reaching the observation point compared to that found in an open field and relates to canopy openness. Again, because the individuals of V. myrtillus are aggregated rather than randomly scattered across the P. abies stand, the corresponding ISF estimates reflect the environment where V. myrtillus occurs. We also note that the treatment plots were rather small relative to tree height (side of plot ≈1.6 × dominant height in the P. abies stand) allowing some tops of out-of-plot trees at low sun (elevation) angles to be captured by the fisheye images. This likely reduced the apparent differences in the mean ISF among the plots. However, ISF provides a realistic index of the variability in relative light availability among individuals.
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LMA and N concentration along transects Between 26 July and 3 August 2012, we carried out additional sampling to quantify the variability in LMA and foliar N concentration of both V. myrtillus and V. vitis-idaea in the P. abies stand at Svartberget. In three plots of each treatment, we established a transect from one corner of the plot to the opposite corner and sampled five leaves of each species at 2-m intervals along the transect (51 samples of five leaves per species and N treatment). A hemispherical picture was taken at each sampling point and the leaf samples were analysed for area, fresh and dry weight, and N concentration (see below). Laboratory measurements Areas of leaves sampled for gas exchange, and of the ten additional leaves collected from each gas exchange plant, were determined from digital images printed on paper, cut, weighed, and the weight converted to area based on the average area-to-weight ratio of the paper. Leaf area of the leaves from the 2012 transect was obtained from digital leaf images analysed with ImageJ image analysis software (http://rsbweb.nih.gov/ij/). All leaf samples were then dried (at 70 °C for 24 h) and LMA (g cm−2) was determined based on leaf area of fresh leaves and weight of dried leaves. Chlorophyll, C, and N concentrations were determined from ten leaves collected from the plants sampled for gas exchange. The leaves were ground and C and N concentrations of the milled material were analysed using an elemental analyser (EA Flash 1112; Thermo Fisher Scientific). For chlorophyll quantification, a sub-sample of the milled material was dissolved in MgCO3-saturated dimethyl sulphoxide at 60 °C for 40 min as in Palmqvist and Sundberg (2001). A similar procedure (excluding the chlorophyll content analysis) was applied to the harvested biomass fractions as well as to the leaf samples collected from the 2012 transect. Statistical analysis For each species and age class, we regressed LMA, chlorophyll content, Amax, and gs, with ISF and foliar N (using both area- and mass-based rates and concentrations) as well as Amax with gs. In these analyses individual sample points (or leaves) were used (see details above). Where a significant relation between any two variables was found (P 0.05).
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In addition, we used one-way ANOVA, for each species and age-class separately, to test for among-treatments differences in the gas exchange characteristics (n = 3) and the sizes of the biomass components (n = 6). The ANOVA performed reflected the experimental design at each site: the randomized block design at Svartberget and the fully randomized design (no blocks) at Åheden. When the assumptions for ANOVA were not met, transformations were attempted, and if these transformations were not successful, a t-test was used (equal variances not assumed). Simulations of potential daily C gain We evaluated the combined effect on potential daily C gain (per unit leaf area) of the variability in the parameters of the light response curve and the relative availability of light. Simulations were performed for one mid-summer day (day-of-year 183) close enough to the measurement campaigns but still including all possible sun angles, and assuming either clear skies or overcast conditions. The amount of incoming diffuse and direct PPFD was simulated half hourly based on the geographical location of the stands (Stenberg 1998). PPFD available for understory was estimated for each gas exchange sampling location using the estimates of ISF. We assumed that ISF was independent of the sun’s elevation and azimuth angles, which is not too unrealistic for low values of ISF and at low sun elevation angles, but likely results in underestimation of canopy openness directly above the observation point. We used half-hourly direct and diffuse PPFD estimated for each sampling point (i.e. each estimate of ISF) together with all the measured light response curves to calculate daily (24 h) CO2 exchange. We assumed, for simplicity, that daytime respiration is equal to Rd. Stand-level means of potential C gain for each observed ISF were obtained using all available response curves for each site. For V. vitis-idaea, we averaged the daily CO2 exchange rates of CL and CL + 1, reflecting equal proportions of each age class (Karlsson 1985).
Results Effects of N additions on understory light availability and leaf morphology Figure 1 combines all estimates of LMA as they relate to foliar N concentration (leaf N per unit mass; Nm), and light availability (as ISF). The 2011 gas exchange dataset consists of V. myrtillus leaves sampled from the Picea abies stand and V. vitis-idaea leaves sampled from the Pinus sylvestris stand. In the 2012 transect dataset, leaves of both species were sampled from the P. abies stand. Both
Fig. 1 Leaf mass per area (LMA) based on samples of ten leaves averaged for each individual of Vaccinium vitis-idaea from Åheden (a, b) and Vaccinium myrtillus from Svartberget (c, d) selected for gas exchange measurements as a function of indirect site factor (ISF) (a, c) and leaf N per unit mass (Nm) (b, d). Closed symbols 1-yearold leaves of V. vitis-idaea, dark grey symbols current-year leaves of V. vitis-idaea, open symbols V. myrtillus leaves. Also included are data on both V. vitis-idaea and V. myrtillus collected from transects at Svartberget Picea abies forest (light grey symbols). Solid line linear fit to 2011 data, broken lines linear fit to 2012 data. In a, LMA is related to ISF only in plots with N addition of 12.5 kg N ha−1 year−1 (N1) and plots with N addition of 50 kg ha−1 year−1 (N2) (P
Physiological ecology - Original research
Nitrogen‑addition effects on leaf traits and photosynthetic carbon gain of boreal forest understory shrubs Sari Palmroth · Lisbet Holm Bach · Annika Nordin · Kristin Palmqvist
Received: 14 August 2013 / Accepted: 5 March 2014 / Published online: 6 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Boreal coniferous forests are characterized by fairly open canopies where understory vegetation is an important component of ecosystem C and N cycling. We used an ecophysiological approach to study the effects of N additions on uptake and partitioning of C and N in two dominant understory shrubs: deciduous Vaccinium myrtillus in a Picea abies stand and evergreen Vaccinium vitisidaea in a Pinus sylvestris stand in northern Sweden. N was added to these stands for 16 and 8 years, respectively, at rates of 0, 12.5, and 50 kg N ha−1 year−1. N addition at the highest rate increased foliar N and chlorophyll concentrations in both understory species. Canopy cover of P. abies also increased, decreasing light availability and leaf mass per area of V. myrtillus. Among leaves of either shrub, foliar N content did not explain variation in light-saturated CO2 exchange rates. Instead photosynthetic capacity varied with stomatal conductance possibly reflecting plant hydraulic properties and within-site variation in water availability. Moreover, likely due to increased shading under P. abies and due to water limitations in the sandy soil under
Communicated by Hermann Heilmeier. S. Palmroth (*) Division of Environmental Science and Policy, Nicholas School of the Environment, Duke University, Box 90328, Durham, NC 27708‑0328, USA e-mail: [email protected] S. Palmroth · A. Nordin Department of Forest Ecology and Management, Swedish University of Agricultural Sciences (SLU), 901 83, Umeå, Sweden L. Holm Bach · K. Palmqvist Department of Ecology and Environmental Science, Umeå University, 90187 Umeå, Sweden
P. sylvestris, individuals of the two shrubs did not increase their biomass or shift their allocation between above- and belowground parts in response to N additions. Altogether, our results indicate that the understory shrubs in these systems show little response to N additions in terms of photosynthetic physiology or growth and that changes in their performance are mostly associated with responses of the tree canopy. Keywords Biomass allocation · Chlorophyll content · Photosynthetic capacity · Stomatal conductance · Vaccinium
Introduction Tree growth in the vast boreal forest [~10 % of land cover (Sabine et al. 2004)] is often limited by soil N (Tamm 1991; Vitousek and Howarth 1991). Soil fertility and other site-quality factors, including water holding capacity, drive the rate of stand development and, thus, the degree of canopy closure and allocation of site resources between trees and understory plants. The canopy in the European boreal coniferous forest is mostly composed of Pinus sylvestris (Scots pine) and Picea abies (Norway spruce). While both trees show high ecological plasticity, pure, fairly open Pinus sylvestris stands are more common on dry, low-fertility soils whereas Picea abies dominates on more mesic and fertile sites (Cajander 1949; Esseen et al. 1997). The associated continuum of understory communities mirrors the site-quality gradient as Vaccinium vitis-idaea and Calluna vulgaris dominate in Pinus sylvestris stands and V. myrtillus in Picea abies stands. The understory biomass is much lower than that of trees, but turns over faster and, for example in an Alaskan Picea mariana forest, the understory net
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primary production was estimated to account for 10–50 % of ecosystem production (O’Connell et al. 2003). Measured estimates of understory gross primary production (GPP) are, however, only available from a few stands throughout the boreal zone (Moren and Lindroth 2000; Widen 2002; Kolari et al. 2006; Misson et al. 2007; Kulmala et al. 2011). In a synthesis of studies on ecosystem fluxes of boreal and temperate forests, Misson et al. (2007) showed that the understory contribution to ecosystem GPP was 14 % on average, with the highest estimate reaching 39 %. These results clearly indicate that understory C uptake must be accounted for in ecosystem C-balance calculations. Regrettably, none of these ecosystem flux studies cover the wide fertility range found in boreal forests, limiting the scope of inference. To facilitate modelling efforts from stand to landscape, we need a better understanding of understory C uptake and partitioning of C and N among biomass pools along natural resource gradients and, also, under conditions where N availability is enhanced via atmospheric deposition or fertilization. Boreal understory species greatly differ in their ability to take up and utilize additional N (Nordin and Näsholm 1997; Manninen et al. 2009). When the two most common dwarf shrubs were grown in controlled conditions without an overstory, N-fertilized deciduous V. myrtillus doubled its biomass over 1 year, whereas evergreen V. vitis-idaea performed better than V. myrtillus at low N (Grelet et al. 2001). While deciduousness may mean increased plasticity, perhaps through a faster initial response, another fertilization study in a tree-less tundra community found no such effect (Karlsson 1985). In the latter study, the overall growth response of another deciduous shrub, Vaccinium uliginosum was neither faster nor stronger than that of V. vitis-idaea. In boreal forests, in turn, N fertilization typically increases tree biomass and leaf area (Axelsson and Axelsson 1986; Bergh et al. 1999; Nohrstedt 2001). The subsequent increase in shading, water limitations, or susceptibility of understory plants to biotic stressors may impose species-specific limitations to growth. Fertilization-induced changes in understory species composition are relatively well documented (Mäkipää 1995, 1999; Strengbom et al. 2002; Nordin et al. 2009; Gundale et al. 2011, 2013), yet the physiological responses underlying such shifts are not well understood. For individuals growing in a given light environment, N additions may increase the partitioning of C aboveground and leaf mass fraction (LMF) (Litton et al. 2007; Manninen et al. 2009; Poorter et al. 2012). Increased shading may also increase LMF but decrease total plant biomass. Yet, shaded plants often adjust leaf mass per area (LMA) even more than biomass partitioning between aboveground and belowground
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components (Poorter et al. 2012). Thus, where light availability decreases with increasing fertility, LMA likely decreases. Lower LMA (within a species) is associated with lower area-based photosynthetic capacity (Oren et al. 1986) but, for a given LMF, also with higher leaf area; thus C uptake per unit ground area may remain unchanged. Foliar N generally increases with soil N availability, yet how the ‘extra’ N is partitioned among and within leaves, and among the component processes of photosynthesis, varies with species and the availability of other resources (Evans 1989; Evans and Poorter 2001). In low light environments, if plant growth is not enhanced by N fertilization, the relative sink strength of various N storage pools can increase (Nordin and Näsholm 1997; Bubier et al. 2011). Excess N can be stored as photosynthetic enzymes (Lawlor et al. 1987; Warren et al. 2003), inorganic N (Gebauer et al. 1984) or, especially in boreal ecosystems, as free amino acids (Näsholm and Ericsson 1990; Nordin and Näsholm 1997). In sum, in forests where N addition results in increased canopy leaf area, causing increased shading of the understory, morphological and physiological responses of understory species may result in little effect on growth or photosynthesis with the extra N accumulating in storage. We assessed the effects of N additions on the C uptake and growth of understory plants. We focused on two shrubs in two different forest types; V. myrtillus in a mesic, fertile Picea abies stand (Svartberget) and V. vitis-idaea in a dry, nutrient-poor Pinus sylvestris stand (Åheden) in northern Sweden. These stands were located within ~1 km from each other and subjected to N addition at rates of 0, 12.5, and 50 kg N ha−1 year−1 over 16 and 8 years, respectively. At the time of our study, both forest stands were well adjusted to the N-addition regimes. Following an initial 3to 5-year adjustment phase to the new N regime, no further changes in understory species composition were recorded (Nordin et al. 2009; Gundale et al. 2011). Also, N additions had no effect on the mycorrhizal associates of the two shrubs in either stand (Ishida and Nordin 2010). We quantified a number of potential determinants of C uptake: relative light availability, leaf photosynthetic light response, LMA, and allocation of C and N among various biomass pools of the two shrubs. We hypothesized that N additions would lead to increased photosynthetic capacity of both shrubs (hypothesis 1). However, as the abiotic factors differed between the sites, we expected that the effects of N additions on growth would differ between the species. Specifically, we hypothesized that increased shading from the P. abies overstory would hamper the growth response of V. myrtillus to N addition (hypothesis 2), and that water limitation would hamper the expected growth response to N addition of V. vitis-idaea (hypothesis 3).
Oecologia (2014) 175:457–470
Materials and methods Sites The experiment was performed at two sites, Svartberget and Åheden, ~1 km apart, within the Svartberget Experimental forest (64°14′N, 19°46′E), Vindeln, northern Sweden. Characteristics of the sites and details of the fertilization experiments can be found in Nordin et al. (2005) and Gundale et al. (2011) but this information is also briefly summarized here. The Svartberget site is a 100-year-old Picea abies (L.) Karst. forest growing on a gently sloping moraine. The field layer is dominated by Vaccinium myrtillus and Vaccinium vitis-idaea and the bottom layer by Hylocomium splendens (Hedw.) Br. Eur. and Pleurozium schreberi (Brid.) Mit. The Åheden site is a 60-year-old Pinus sylvestris L. forest growing on a fine sandy and silty glacial outwash sediments dominated by V. vitis-idaea and Calluna vulgaris (L.) Hull, and the bottom layer by Pleurozium schreberi. Annual mean temperature (1961–1990) in the study area is 1.5 °C and precipitation 591 mm, with the averages for July 14.1 °C and 69 mm, respectively (Vindeln-Sunnansjönäs, Swedish Meteorological and Hydrological Institute). The background atmospheric N deposition rate in this area is ~2 kg N ha−1 year−1 (Phil-Karlsson et al. 2009). The fertilization experiment started at Svartberget in 1996 and consists of six replicate square plots (1,000 m2) of each treatment [0 (control), 12.5 (N1) and 50 (N2) kg N ha−1 year−1] in a randomized block design. In 2003, an identical fertilization experiment (but in a completely randomized design) was set up at Åheden. The lower N-addition rate in these experiments was designed to mimic the highest deposition rates in boreal forests (Dentener et al. 2006), and the higher rate is in line with the typical long-term fertilization experiments in the area. Analyses of root‑to‑shoot ratio and C and N concentrations For biomass estimates, we sampled two 0.25-m × 0.25-m subplots from each treatment (control, N1, and N2) in all the six plots (n = 6) at both Svartberget and Åheden experiments between 23 and 26 August 2011. At Åheden, the subplots for V. vitis-idaea biomass sampling were laid out randomly in each plot. At Svartberget, the individuals of V. myrtillus tend to be more aggregated than randomly scattered in the fertilized stands (Strengbom et al. 2002) and our biomass sampling (targeting V. myrtillus) reflects the biomass where V. myrtillus occurs rather than the plot average. We harvested all V. vitis-idaea and V. myrtillus plants, above- and belowground parts except for fine roots, from the subplots and divided the biomass into various
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categories. For the deciduous V. myrtillus the four categories were leaves, current annual shoot, remaining shoot, and roots. For the evergreen V. vitis-idaea the five biomass categories included all the above and also leaves from previous years. These biomass fractions were summed and root-to-shoot ratios calculated. LMF was expressed as the fraction of leaf mass of total aboveground biomass, and leaf area ratio (LAR) as leaf area (=leaf mass divided by LMA; see details below) divided by aboveground biomass (m2 kg−1). Gas exchange measurements We measured gas exchange of healthy looking V. myrtillus leaves (one upper-crown leaf per plant, all plants of similar size) in three plots (n = 3) at Svartberget between 11 and 19 July 2011, yielding 12–16 individual photosynthetic light response curves per treatment. The gas exchange measurements at Åheden were carried out between 20 and 27 July 2011, also in three plots (n = 3). We sampled current-year leaves (CL) and previous year’s leaves (CL + 1) from V. vitisidaea (one of each per plant), yielding four to nine curves per treatment and age class. At both sites, we also collected ten additional leaves from each plant for N concentration, chlorophyll and LMA determinations (see below). We measured CO2 and H2O exchange rates of intact leaves in the field using a portable photosynthesis system LI-6400 (LI-COR, Lincoln, NE). Chamber conditions were kept as constant as possible: temperature at 20 °C, flow at 400 μmol s−1, CO2 concentration at 400 μmol mol−1 and relative humidity at 60–70 %. Photosynthetic photon flux density (PPFD) was first set to 200 μmol m−2 s−1 and then increased stepwise up to 500 μmol m−2 s−1, after which PPFD was decreased stepwise to zero. The width of the step was 20 μmol m−2 s−1 on the light-limited part of the curve and 50– 100 μmol m−2 s−1 otherwise, resulting in 16 (V. myrtillus) or 18 (V. vitis-idaea) points per curve. The leaves were collected for measurements of leaf area and leaf dry weight (see details below). The resulting light response curves were inspected visually and obvious outliers removed. We estimated dark respiration rate (Rd) as CO2 exchange at PPFD = 0 μmol m−2 s−1. To obtain estimates for the lightresponse-curve parameters we fitted a non-rectangular hyperbola to the data (Thornley 2002; Posada et al. 2009): 2 1 φI + Amax,g − 4θφIAmax,g ) − Rd , A= (φI + Amax,g − 2θ where A is CO2 exchange rate, I is instantaneous PPFD incident on leaf, ϕ is apparent quantum yield, Amax,g is light-saturated gross assimilation rate, and θ is curvature. We also recorded stomatal conductance (gs) and intercellular CO2 concentration. Because the weather conditions before and during the measurement campaigns were
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Table 1 Environmental data from a weather station at Svartberget showing mean values for the period of gas exchange measurements and the preceding week Week before
Experiment
Svartberget, Vaccinium myrtillus Air temp. (°C; min./max.) 17.7 (4.7/27.1) Soil temp. (°C; min./max.) 12.7 (11.6/13.9) RH (%) 66 Precipitation (mm) 0 23.2 Global radiation (MJ m−2)
14.5 (2.9/23.5) 13 (11.6/13.8) 76 30 17.1
Åheden, Vaccinium vitis-idaea Air temp. (°C; min./max.) Soil temp. (°C; min./max.) RH (%) Precipitation (mm)
14.2 (0.2/23.2) 13.1 (11.6/14.7) 71 11
17.9 (9.4/24.5) 15 (13.7/16.1) 76 26
18.4
16
Global radiation (MJ m−2)
temp. Temperature, min. minimum, max. maximum, RH relative humidity
similar at each site (Table 1), and the range of chamber conditions was narrow, we assume that the response curves represent growing conditions, i.e. treatments. Light availability Light availability under the main canopy in each stand was quantified through analysis of hemispherical images. Photographs were taken from all sampling locations (above biomass plots, and above individuals measured for gas exchange and sampled from transects as described below) under overcast conditions or at low sun angles. We used a Canon EOS 1000D equipped with an EX Sigma Circular Fisheye DC HSM 4.5 mm 1:2,8 and the pictures were analysed with Hemiview 2.1 (DeltaT devices, UK) updated with lens properties for the EX Sigma Circular Fisheye. From the output, we extracted indirect site factor (ISF), which is the percentage of indirect radiation reaching the observation point compared to that found in an open field and relates to canopy openness. Again, because the individuals of V. myrtillus are aggregated rather than randomly scattered across the P. abies stand, the corresponding ISF estimates reflect the environment where V. myrtillus occurs. We also note that the treatment plots were rather small relative to tree height (side of plot ≈1.6 × dominant height in the P. abies stand) allowing some tops of out-of-plot trees at low sun (elevation) angles to be captured by the fisheye images. This likely reduced the apparent differences in the mean ISF among the plots. However, ISF provides a realistic index of the variability in relative light availability among individuals.
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LMA and N concentration along transects Between 26 July and 3 August 2012, we carried out additional sampling to quantify the variability in LMA and foliar N concentration of both V. myrtillus and V. vitis-idaea in the P. abies stand at Svartberget. In three plots of each treatment, we established a transect from one corner of the plot to the opposite corner and sampled five leaves of each species at 2-m intervals along the transect (51 samples of five leaves per species and N treatment). A hemispherical picture was taken at each sampling point and the leaf samples were analysed for area, fresh and dry weight, and N concentration (see below). Laboratory measurements Areas of leaves sampled for gas exchange, and of the ten additional leaves collected from each gas exchange plant, were determined from digital images printed on paper, cut, weighed, and the weight converted to area based on the average area-to-weight ratio of the paper. Leaf area of the leaves from the 2012 transect was obtained from digital leaf images analysed with ImageJ image analysis software (http://rsbweb.nih.gov/ij/). All leaf samples were then dried (at 70 °C for 24 h) and LMA (g cm−2) was determined based on leaf area of fresh leaves and weight of dried leaves. Chlorophyll, C, and N concentrations were determined from ten leaves collected from the plants sampled for gas exchange. The leaves were ground and C and N concentrations of the milled material were analysed using an elemental analyser (EA Flash 1112; Thermo Fisher Scientific). For chlorophyll quantification, a sub-sample of the milled material was dissolved in MgCO3-saturated dimethyl sulphoxide at 60 °C for 40 min as in Palmqvist and Sundberg (2001). A similar procedure (excluding the chlorophyll content analysis) was applied to the harvested biomass fractions as well as to the leaf samples collected from the 2012 transect. Statistical analysis For each species and age class, we regressed LMA, chlorophyll content, Amax, and gs, with ISF and foliar N (using both area- and mass-based rates and concentrations) as well as Amax with gs. In these analyses individual sample points (or leaves) were used (see details above). Where a significant relation between any two variables was found (P 0.05).
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In addition, we used one-way ANOVA, for each species and age-class separately, to test for among-treatments differences in the gas exchange characteristics (n = 3) and the sizes of the biomass components (n = 6). The ANOVA performed reflected the experimental design at each site: the randomized block design at Svartberget and the fully randomized design (no blocks) at Åheden. When the assumptions for ANOVA were not met, transformations were attempted, and if these transformations were not successful, a t-test was used (equal variances not assumed). Simulations of potential daily C gain We evaluated the combined effect on potential daily C gain (per unit leaf area) of the variability in the parameters of the light response curve and the relative availability of light. Simulations were performed for one mid-summer day (day-of-year 183) close enough to the measurement campaigns but still including all possible sun angles, and assuming either clear skies or overcast conditions. The amount of incoming diffuse and direct PPFD was simulated half hourly based on the geographical location of the stands (Stenberg 1998). PPFD available for understory was estimated for each gas exchange sampling location using the estimates of ISF. We assumed that ISF was independent of the sun’s elevation and azimuth angles, which is not too unrealistic for low values of ISF and at low sun elevation angles, but likely results in underestimation of canopy openness directly above the observation point. We used half-hourly direct and diffuse PPFD estimated for each sampling point (i.e. each estimate of ISF) together with all the measured light response curves to calculate daily (24 h) CO2 exchange. We assumed, for simplicity, that daytime respiration is equal to Rd. Stand-level means of potential C gain for each observed ISF were obtained using all available response curves for each site. For V. vitis-idaea, we averaged the daily CO2 exchange rates of CL and CL + 1, reflecting equal proportions of each age class (Karlsson 1985).
Results Effects of N additions on understory light availability and leaf morphology Figure 1 combines all estimates of LMA as they relate to foliar N concentration (leaf N per unit mass; Nm), and light availability (as ISF). The 2011 gas exchange dataset consists of V. myrtillus leaves sampled from the Picea abies stand and V. vitis-idaea leaves sampled from the Pinus sylvestris stand. In the 2012 transect dataset, leaves of both species were sampled from the P. abies stand. Both
Fig. 1 Leaf mass per area (LMA) based on samples of ten leaves averaged for each individual of Vaccinium vitis-idaea from Åheden (a, b) and Vaccinium myrtillus from Svartberget (c, d) selected for gas exchange measurements as a function of indirect site factor (ISF) (a, c) and leaf N per unit mass (Nm) (b, d). Closed symbols 1-yearold leaves of V. vitis-idaea, dark grey symbols current-year leaves of V. vitis-idaea, open symbols V. myrtillus leaves. Also included are data on both V. vitis-idaea and V. myrtillus collected from transects at Svartberget Picea abies forest (light grey symbols). Solid line linear fit to 2011 data, broken lines linear fit to 2012 data. In a, LMA is related to ISF only in plots with N addition of 12.5 kg N ha−1 year−1 (N1) and plots with N addition of 50 kg ha−1 year−1 (N2) (P
