Wood properties and ring width responses to long-term atmospheric CO2 enrichment in field-grown loblolly pine (Pinus taeda L.)
Frank W. Telewski Fax: 1517 432 1090; e-mail: firstname.lastname@example.org
Loblolly pine (Pinus taeda L.) were grown in the field, under non-limiting nutrient conditions, in open-top chambers for 4 years at ambient CO2 partial pressures (pCO2) and with a CO2-enriched atmosphere (+ 30 Pa pCO2 compared to ambient concentration). A third replicate of trees were grown without chambers at ambient pCO2. Wood anatomy, wood density and tree ring width were analysed using stem wood samples. No significant differences were observed in the cell wall to cell lumen ratio within the latewood of the third growth ring formed in 1994. No significant differences were observed in the density of resin canals or in the ratio of resin canal cross-sectional area to xylem area within the same growth ring. Ring widths were significantly wider in the CO2-enrichment treatment for 3 of 4 years compared to the ambient chamber control treatment. Latewood in the 1995 growth ring was significantly wider than that in the ambient control and represented a larger percentage of the total growth-ring width. Carbon dioxide enrichment also significantly increased the total wood specific gravity (determined by displacement). However, when determined as total sample wood density by X-ray densitometry, the density of enriched samples was not significantly higher than that of the ambient chamber controls. Only the 1993 growth ring of enriched trees had a significantly higher maximum latewood density than that of trees grown on non-chambered plots or ambient chambered controls. No significant differences were observed in the minimum earlywood density of individual growth rings between chambered treatments. These results show that the most significant effect of CO2 enrichment on wood production in loblolly pine is its influence on radial growth, measured as annual tree ring widths. This influence is most pronounced in the first year of growth and decreases with age.
Many studies have explored the influence of enriched partial pressure of atmospheric CO2 (pCO2) on woody plant growth. Reviews of these studies document an enhanced rate of photosynthesis, with increased above- and below- ground biomass more consistently observed in controlled-environment studies (Eamus & Jarvis 1989; Dewar & Cannell 1992; Idso & Kimball 1993; Ceulemans & Mousseau 1994; Telewski & Strain 1994; Wullschleger, Post & King 1995; Eamus 1996; Tissue, Thomas & Strain 1997). A portion of the increased biomass is represented by increased xylem production in stems, branches and roots. However, few studies have characterized changes in anatomy and density that occur within the xylem of trees exposed to CO2 enrichment, and most of those that have were conducted on plants grown in growth chambers or greenhouses. Definition of the changes that may occur in the structure of xylem produced in response to CO2 enrichment under field conditions will be significant to wood production for forestry, forest ecology and interpretation of the relevance of past controlled-environment CO2 enrichment studies on woody plants. A projected continued increase in atmospheric CO2 above the preindustrial concentration may influence wood quality. In addition, knowledge of how the increased photosynthate is used in the xylem tissue will be useful to modellers of carbon partitioning and sequestering. Finally, the knowledge of what types of changes can be expected for a variety of species will be useful to dendrochronologists using tree rings in attempting to reconstruct the influence of increasing concentration of atmospheric pCO2 on forest communities over time.
Xylem anatomy and wood properties have been studied in only a handful of woody species (Table 1). Most of these experiments were conducted using potted seedlings grown in chambers or greenhouses (Atkinson & Taylor 1996; Conroy et al. 1990; Donaldson et al. 1987; Doyle 1987; Telewski & Strain 1987, 1994). The results of these past studies have been called into question because of the restriction of root growth within a container for long-term exposure studies (Graybill & Idso 1993; Körner 1995).
Table 1. .
Tree species analysed for changes in wood structure and anatomy in past studies. 0 = no change in the observed parameter; + = an increase in the observed parameter; n.a. = data not measured or unavailable
Hättenschwiler, Schweingruber & Körner (1996) reported on a complex interaction between nitrogen availability, competition and pCO2 enrichment in Norway spruce (Picea abies) grown in large containers (100 × 70 × 36 cm) within growth chambers simulating montane climatic conditions. Although pCO2 enrichment at low nitrogen concentration increased wood density, density decreased with increasing nitrogen availability. Enrichment in pCO2 did not enhance radial growth regardless of nitrogen availability (Table 1).
Only one study has observed changes in xylem anatomy and wood properties in response to pCO2 enrichment under non-restrictive root growth conditions in the field. Rogers et al. (1983) reported no significant differences in biomass or wood density of Pinus taeda in response to enrichment; significant increases in wood density were reported for Liquidambar styraciflua (Table 1).
A 4 year field study on Pinus taeda conducted in open-top chambers under non-restrictive root growth conditions reported a 90% increase in tree biomass and an increase in tree height in response to pCO2 enrichment (Tissue et al. 1997). Using stem tissues derived from this experiment, we investigated the effect of pCO2 enrichment on radial growth, wood anatomy and wood density in loblolly pine (Pinus taeda). On the basis of earlier studies on this species grown under controlled environmental conditions (Rogers et al. 1983; Telewski & Strain 1987, 1994), we hypothesized that annual growth increments, measured as tree rings, will exhibit increases without significant changes in wood specific gravity (measured by displacement) or wood density (measured by X-ray densitometry) in response to pCO2 enrichment, and these results will support earlier studies on this species grown under controlled-environment conditions (Telewski & Strain 1987, 1994).
MATERIALS AND METHODS
The selection, propagation, treatment and pCO2 enrichment of plants and the experimental design follows that of Tissue, Thomas & Strain (1996, 1997). Loblolly pine (Pinus taeda) seedlings were germinated in April 1992 under greenhouse conditions at the Duke University Phytotron with CO2 partial pressures (pCO2) that were automatically monitored and controlled at ambient pCO2 or + 30 Pa CO2 above ambient pCO2 (Hellmers & Giles 1979). After germination, the seedlings were inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Mycorr Tech Inc., Pittsburgh, PA). In May 1992, 24 seedlings were transplanted to each of three chambers for both pCO2 treatments. The cylindrical open-top chambers were each 3 m in diameter and 3 m tall (Rogers et al. 1983) and were situated in the Duke Forest. The native soil in each plot was excavated to 1 m and replaced with a 1:1:1 (v/v) mix of a native clay soil, topsoil and sand mixture representative of soil in a recently abandoned agricultural field. Soil mineral N concentrations at the beginning of the experiment were 8·44 ± 3·55 mg N kg–1 soil (mean ± SE, n = 12), with 80% of N as NO3 and 20% as NH4 (Tissue et al. 1996). Treatment groups included: non-chamber plot at ambient pCO2; chamber at ambient pCO2; and chamber + 30 Pa pCO2 enrichment. In the third year of the experiment the 3 m diameter chambers were replaced with 5 m diameter chambers. Chamber height was increased to 4 m in the third year and to 5 m in the fourth year. A subsample of three trees was harvested from each chamber and from the three non-chamber treatments at the end of the third and fourth years. Growth conditions, including seasonal variations in pCO2 in the three treatments, precipitation, photosynthetic photon flux density and air temperature, have been described (Tissue et al. 1996, 1997).
Basal stem sections were removed from two harvested trees per chamber or the non-chamber replicate treatments (six plants per CO2 treatment) during the 3 year and 4 year samplings. The samples derived from the third year were preserved in FAA (formalin:acetic acid [80%]:ethanol; 1:1:8). Disks were cut from the stem sections and each disk was divided into quarters. Each quarter section was prepared for standard paraffin embedding and sectioning. Sections 20 μm thick were cut from each quarter section, mounted on glass slides and stained with saffrin and fast green (Beryln & Miksche 1976). The density of resin canals was determined within the latewood region of the third year's growth ring by manual counting within a circular ocular target with an area of 4·91 mm2 as determined under 40× magnification (4× objective, 10× ocular), one count per quarter section. The number of resin canals per mm2 was calculated by dividing the number of resin canals within the circular target by the area of the target. The ratio of cell wall to cell lumen areas was determined within a rectangular window of 12 658 μm2 and the ratio of wood area to resin canal area was determined within a 3158 632 μm2 rectangular window, one window per quarter section, using a Macintosh computer equipped with image analysis software (NIH Image 1·51, National Institutes of Health, Bethesda, MD).
The second set of wood samples representing the 4 year harvest were air dried before processing. Cross sections were sanded and polished to expose clearly the growth ring structure. A section 5 mm thick was cut from the polished end using a band saw and the growth rings measured using a tree ring measuring machine (Henson Measuring Machine, Kurt Zahn, Mission Viejo, CA).
Wood density was determined by displacement specific gravity and by X-ray densitometry. For specific gravity, wood blocks 1 cm × 1 cm × stem diameter were cut from the cross sections. All tissues to the outside of the cambium were removed. The samples were extracted in a water, ethanol, ethanol:toluene (1:1), toluene sequence using Soxhlet columns. The samples were then dried at 38 °C to constant weight. The final constant weight was recorded and the blocks lightly coated in paraffin. The coated blocks were submerged in water and the displaced volume recorded. Specific gravity was calculated by dividing the block dry weight by the displacement volume. Sample preparation for the X-ray densitometry required milling 1 mm thick samples through the diameter of each cross section of extractive-free wood using a twin-bladed saw. These samples were then radiographed and scanned using an optical microdensitometer (Model 3 CS, Joyce-Loebl, Gateshead, UK) at the Laboratory of Tree-Ring Research, University of Arizona, Tucson. Average sample density, average ring density, maximum latewood density and minimum earlywood density were determined from the radiographs using a cellulose nitrate step wedge calibration curve.
Data were tested for normality and transformed using natural logs, arcsin square roots or square roots where necessary to normalize variances among CO2 treatments. Means and standard errors were calculated for each parameter and the main effects of CO2 on each measured parameter were tested for statistical differences with the analysis of variance (ANOVA) Type I general linear models procedure (GLM) using a computer statistical software package (SAS/STAT 6·12, SAS Institute Inc., Cary, NC). The statistical design consisted of two classes: class = treatment, which includes ambient pCO2 and ambient + 30 Pa pCO2, and class = chamber, with open-top chamber or non-chamber plot. This is an unbalanced design as there is no non-chamber ambient + 30 Pa pCO2 treatment — hence the application of the GLM procedure. The random chamber block term was nested within pCO2 concentrations in the GLM procedure. Scheffe tests were used for a comparison of dependent variable means among CO2 treatments. Treatment effects were considered significant if P < 0·05 for tree ring measurements and P < 0·10 for all other measured parameters.
No significant differences were observed between treatments for the anatomical features of cell wall:lumen area, resin canal area:tracheid area and resin canal density measured within the 1994 growth ring (Table 2).
Table 2. .
Anatomical characteristics within the latewood of the 1994 growth ring. Values are group mean ± SE (n
= 3). No significant differences were observed. n.s. = not significant at P
The growth rings produced during the first 2 years (1992 and 1993) were significantly wider (P=0·0001, P=0·0003, respectively) in trees enriched with +30Pa pCO2 above ambient concentration than both ambient controls. By the fourth year, there was no significant difference between the non-chambered ambient control and the enrichment treatment. The 1995 growth ring of the chambered ambient treatment was significantly narrower (P = 0·0137) than the non-chambered and enrichment treatments (Table 3). The earlywood portion of the growth rings was significantly wider in the enrichment treatment than it was in the chambered ambient control within the 1993 (P = 0·0002) and 1995 (P = 0·0015) growth rings and the 1993 (P = 0·0002) growth ring of the non-chambered control. The non-chambered control treatment had a significantly wider earlywood band (P = 0·0037) in the 1994 growth ring compared to the enrichment treatment and chambered ambient control (Table 3). The latewood bands of the enrichment treatment tend to be wider than either non-chambered or ambient chamber controls. The 1995 growth ring of the enrichment treatment had a significantly wider latewood band (P = 0·0001) compared to non-chambered or chambered ambient controls (Table 3).
Table 3. .
Annual growth ring widths of 4-year-old loblolly pine trees. A mean ± SE (n
= 3) followed by a different letter within a column is significantly different from other means at P
The wood produced under conditions of enrichment had the greatest overall sample specific gravity. This was a significantly greater specific gravity that the chambered ambient controls (P = 0·0882), but not significantly different from the non-chambered controls (Table 4).
Table 4. .
Wood specific gravity determined by displacement and tree ring density determined by X-ray densitometry of 4-year-old loblolly pine trees stems. A mean ± SE (n
= 3) followed by a different letter within a column is significantly different from other means at P
No significant differences were observed in the overall sample wood density as determined by X-ray densitometry, although there are consistently higher densities in the enrichment treatments compared to the ambient chambered controls (Tables 4 & 5). With the exception of the 1993 growth ring, no significant differences were observed in the minimum earlywood density (MEWD) between treatment groups. The 1993 growth ring MEWD is significantly greater in the non-chambered control compared to the enrichment treatment (P = 0·0669, Table 5). Similar results are reported for the maximum latewood density (MLWD) with the only significant increase in MLWD observed in the 1993 growth ring of the enrichment treatment compared to the non-chambered or ambient chamber controls (P = 0·031, Table 5).
Table 5. .
Minimum earlywood and maximum latewood tree ring density determined by X-ray densitometry of 4-year-old loblolly pine tree stems. A mean ± SE (n
= 3) followed by a different letter within a column is significantly different from other means at P
The results of the present study — specifically increased radial growth and essentially no change in wood density as determined by X-ray densitometry in response to CO2 enrichment — support earlier findings reported for potted, greenhouse-grown loblolly pine exposed to enriched pCO2 concentrations (Telewski & Strain 1987). The greatest significant differences between ambient chambered controls and trees grown under CO2 enrichment were observed in annual growth-ring width. Enrichment with CO2 resulted in greater biomass production (Tissue et al. 1997) and greater radial wood production in the loblolly pine seedlings, measured as total stem diameter (Tissue et al. 1997) and as individual ring widths (Table 3). A 93% increase in ring width, or a 280% increase in total ring transverse area, calculated from ring width, in response to pCO2 enrichment was observed in the 1992 growth ring. This large increase in the 1992 ring width in response to enrichment is consistent with the 217% increase in leaf area produced by trees during the first growing season in response to pCO2 enrichment (Tissue et al. 1997). This initially large rate of growth, expressed as ring width, leaf area and total biomass, was not sustained in subsequent years. However, absolute production of biomass continued to increase each year in trees grown in enriched pCO2 (Tissue et al. 1997). This continued increase in biomass is reflected in subsequent increases in response to pCO2 enrichment in annual ring width (15–37%) and total annual ring transverse area (49–86%). The greater increase in annual ring transverse area is a result of the initial larger circumference of the vascular cambium caused by accelerated cambial growth in the enrichment treatment.
Of 13 measured density values determined by X-ray densitometry (Tables 4 & 5), only one exhibited a significant increase in response to pCO2 enrichment compared to ambient chambered controls. This significantly greater value was reported for maximum latewood density in the 1993 growth ring. This does not appear to be a response to prevailing weather conditions, as all chamber treatments were exposed to similar seasonal temperature ranges, and all treatments received the same amount of rainfall (Tissue et al. 1997). This may be a reflection of available sugar produced during the 1992 growing season. Sugar content was higher during the winter/spring of 1992/93 than subsequent winter/spring season periods during the experiment (Tissue et al. 1997). Although not significant, enrichment does appear to produce slightly higher MEWDs and MLWDs compared to the ambient chambered control, an observation consistent with the earlier study (Telewski & Strain 1987).
The small increase in density, significant in only one of four maximum latewood density values, in combination with a wider latewood band — significantly wider in the last formed growth ring (Table 3) — could account for the observed increase in total wood specific gravity in response to enrichment (Table 4). Not only was the last-formed latewood band of the enriched trees significantly wider than the ambient control, it accounted for a higher percentage of total ring width (+ 30 Pa pCO2 = 24·9% total ring width, ambient control = 20·6% total ring width). The observed higher percentage of latewood within a growth ring of enriched trees is similar to those calculated from the data presented for the last-formed growth ring of Norway spruce grown under high nitrogen availability in Hättenschwiler et al. (1996): 560 cm3 m–3pCO2 = 21·1% total ring width, 420 cm3 m–3pCO2 = 12·9% total ring width.
In contrast to earlier studies, no significant changes were observed in wood anatomy in response to any of the treatments within the latewood of the 1993 growth ring (Table 2, Telewski & Strain 1987, 1994). These data suggest that the significant increases observed in the specific gravity are, in part, a result of increased latewood volume within a growth ring rather than changes in wood density determined by the volume of cell wall material versus lumen space.
Wood volume and strength are important considerations for the timber industry and the influence of any projected increase in pCO2 on tree growth will be important to silviculture (Hättenschwiler et al. 1996). Ring width is an indicator of volume growth, and specific gravity and density are indicators of wood strength. These results suggest that projected increases in the atmospheric content of CO2 may result in increased wood production without a loss in structural strength in loblolly pine, when this economically important species is grown in managed plantations with non-limiting nitrogen availability. Wood strength may increase if the increase observed in the specific gravity and latewood width continues beyond the juvenile growth phase in this species. Unfortunately, the low values for average density and specific gravity indicate that wood produced in this experiment is still in the juvenile state. In mature loblolly pine, average wood density is 520–660 kg m–3 (Panshin & deZeeuw 1980), whereas the average density of wood in this study was below 400 kg m–3. It is not known how mature wood development will be influenced by CO2 enrichment under controlled conditions or in the natural environment.
An increase in wood production beyond the scope of this 4 year study would only be possible if the observed increase in radial growth, expressed as tree rings, and biomass production (Tissue et al. 1997) were maintained after cambial maturation and canopy closure. In part, this is dependent upon the ability of an individual tree to continue increasing leaf area as it matures within a forest canopy. In this study, biomass production by loblolly pine appears not to be limited by leaf area index after four growing seasons (Tissue et al. 1997).
It is difficult to interpret the differences observed between chamber-grown trees and non-chamber-grown tree, because of possible chamber effects. Although soil nutrients and precipitation were constant between the chambers and non-chamber plots, small variations in temperature were recorded. The maximum air temperature was always higher in chambered plots, with a difference usually less than 1·5 °C. The greatest difference in temperature was recorded in June 1993 when the chamber plots averaged 2·9 °C higher than non-chamber plots (Tissue et al. 1996, 1997). No comparison was made of daily photosynthetic photon flux density between chamber and non-chamber plots. Although wind velocity was not monitored in this study, another chamber effect may be the reduction in, or absence of, wind within the chamber plots. Chambered trees are more protected from the direct influence of wind. Wind-induced stem movement enhances radial growth, decreases height growth and increases wood density in loblolly pine (Telewski 1990; Telewski & Jaffe 1981, 1986). Stems of non-chamber trees were shorter and thicker than those of chamber-grown trees (Tissue et al. 1996), suggesting a thigmomorphogenetic effect. A long-term (15 years) free-air CO2 experiment (FACE) started at the Duke Forest in August 1996 will be monitored for differences in wood properties and anatomical characteristics in the absence of any chamber effects.
Nitrogen availability appears to be a critical limiting factor in the response of individual tree species to CO2 enrichment (Kirschbaum et al. 1994; Hättenschwiler et al. 1996; Prior et al. 1997). Nitrogen was not limiting in the present study. The increased radial growth of loblolly pine, expressed as increased annual ring width (Table 3), is in agreement with the increased radial growth reported for longleaf pine (Pinus palustris), a closely related, sympatric species (Prior et al. 1997). Both species are native to the south-eastern USA and produce greater radial growth in response to CO2 enrichment when nitrogen is not limiting. The increase in radial growth was greater than that observed in non-limiting nitrogen soils at ambient CO2 concentrations (Table 3, Prior et al. 1997). This is in contrast to the results reported for the montane species Norway spruce, in which the radial stem increment was not significantly increased in response pCO2 enrichment when nitrogen availability was high (Hättenschwiler, Schweingruber & Körner 1996). Hättenschwiler, Schweingruber & Körner (1996) also reported an increase in wood density by CO2 enrichment, but decreased by increased nitrogen availability. CO2 enrichment significantly increased wood specific gravity (Table 4) and latewood maximum density in one annual growth ring (Table 5) in loblolly pine. It may be possible, based on the results of Hättenschwiler et al. (1996), that the small increase in wood specific gravity in loblolly pine could be greater if grown under limited nitrogen concentrations.
It may be difficult to interpret how these results may be useful to dendrochronological studies. To date, most dendrochronological studies investigating a possible pCO2 enrichment effect in the environment have focused on timberline conifer species, including Pinus longaeva (Graybill 1987; LaMarche et al. 1984), Pinus aristata, Pinus flexilis (Graybill 1987), Pinus sylvestris (Hari et al. 1984; Hari & Arovaara 1988), Pinus contorta (Jacoby 1986; Peterson et al. 1990), Pinus albicaulis (Peterson et al. 1990), Pinus balforania (Graumlich 1991) and Pinus cembra (Nicolussi, Bortenschlager, & Körner 1995). Many of these species are usually not limited with regard to canopy closure as they grow in fairly open-canopy timberline forests at maturity, but may be limited by lower nitrogen availability on these sites. Controlled enrichment studies reporting changes in radial growth have been conducted on several conifer species; however, only three — loblolly pine, radiata pine and Norway spruce — have been studied anatomically and for wood density. These species were studied as seedlings or saplings. In their natural habitat, they will form closed-canopy forests as they mature, possibly resulting in a reduced rate of radial growth.
On the basis of the results of studies on these species, it can be concluded that the influence of pCO2 enrichment on wood production and annual tree ring characteristics can vary between and within species, resulting in increased radial growth or density or both or no increase in either parameter (Tables 2 and 3; Conroy et al. 1990; Hättenschwiler et al. 1996; Prior et al. 1997). Such mixed results support an assumption of a complex genetic–environmental interaction (Becker 1991; Conroy et al. 1990; Graybill & Idso 1993; Kienast & Luxmoore 1988; Nicolussi, Bortenschlager, & Körner 1995), which may help to explain why a ‘CO2 fertilization effect’ has not been consistently observed in past dendrochronological studies (Graumlich 1991; Kienast & Luxmoore 1988).
We thank Will Cook and Jeff Pippen of Duke University for their many services through the 4 years of this study. We also thank Sandra Hudson and Anne Plovanich-Jones of Michigan State University and three anonymous reviewers for editorial comments on the manuscript. The research was supported by the Department of Energy, CO2 Research Division (contract DE-FG05–87ER60575), the Electric Power Research Institute (contract RP3041–02) and NSF grant BSR87–06429 for support of the Duke University Phytotron.