We addressed issues of the response of leaf area longevity to changes in CO2 and N supplies in a subset of monoculture plots for 10 species of a larger study of elevated CO2, N fertilization, and decreases in biodiversity (the BioCON experiment, Reich et al., 2001). Each plot is 2 m × 2 m, situated on low-N, sandy soils, and distributed among six 20-m diameter experimental areas (rings). Each monoculture is replicated twice at two factorial levels of N and CO2 supplies. Monocultures were planted in 1997 with 12 g m−2 of seed. Throughout the 1998 growing season, three rings were exposed to ambient atmospheric CO2 concentrations and three to elevated CO2 using the free-air CO2 enrichment system (Lewin et al., 1994). In the three elevated CO2 rings, sufficient CO2 was added during daylight hours to maintain the atmosphere at 550 µmol CO2 mol−1; the three ambient rings were treated identically but without the additional CO2. Half of the plots were amended with 4 g N m−2 yr−1 in the form of NH4NO3 applied over three dates. CO2 treatment began in April, 1998 and the first N treatment began in May 1998.
Leaf longevity was measured in monocultures of 10 species: 4 C3 grasses (Agropyron repens, Bromus inermis, Koeleria cristata, and Poa pratensis), 4 C4 grasses (Andropogon gerardii, Bouteloua gracilis, Schizachyrium scoparium, and Sorghastrum nutans) and 2 C3 forbs (Achilleamillefolium and Solidago rigida) with similar census techniques as Craine et al. (1999). Leaf longevity was determined on three randomly chosen individual plants in each plot. A grass individual was defined as a set of leaves that appeared to share the same basal meristem; a forb individual was defined as those leaves that shared the same stem emerging from the ground. Plants were first censused on day 139 of 1998 (May 19) and recensused every 2 wk until day 258 of 1998 (September 15). This period represents the majority of the growing season for the 10 species we sampled. Leaves that had been damaged by herbivory or other factors were removed from the data set. Individual plants that had died were replaced by new plants. Less than 5% of the individuals were replaced and < 2% of all leaves were excluded from the analysis due to damage.
For forbs, for each census date, if a leaf was judged to be approx. fully expanded, the date was recorded as the ‘birth’ date, a numbered 1 × 2 cm paper tag was attached at the base of the leaf with floss, and the maximum width and length of the leaf recorded. We estimate that, on average, expansion of forb leaves required c. 2 wk. During the census, the maximum width and length of previously tagged leaves were recorded, previously marked leaves were classified as being green or senesced, and new leaves tagged and measured. All leaves that were present at the beginning of the experiment or subsequently produced on the selected individual during the census period were tagged and quantified in this manner.
New grass leaves were marked at the base of the leaf with a permanent marker to uniquely identify each leaf on an individual plant that had been demarcated with its own tag. There was no observed difference between the longevity of marked leaves in comparison to unmarked leaves on adjacent plants. With each census date, the average width of the leaf, the total length of the leaf and the length of the senesced portion of the leaf were recorded. The senesced length was determined as the leaf length from the leaf tip to the interface of the green nonsenescent tissue and the brown, senescent tissue. If the senescence was noncontinuous, the total senescence length was measured in an according manner.
After leaves were measured, the corresponding areas of the leaves or sections of leaves were determined. For each grass leaf, the green and senesced areas were determined as the product of the width of the grass leaf and the length of the green or senesced portion of the leaf. For each forb species, we developed an allometric equation that predicts leaf area from length and maximum width. 12–19 leaves of various sizes from each of the four ambient CO2 plots of each forb species were collected in September, 1999. Subsequently, the length, maximum width, and area were determined for each leaf. Leaf area was determined with a Li-Cor LI-3000 Leaf Area Meter (Li-Cor Inc., Lincoln NE). For each species, a stepwise regression model was run for leaf area with length, width, and the squares of both measures. For Solidago rigida, the equation for predicting area was: −0.22 + 0.075 Length2 + 1.3 Width2 (r2 = 0.99). For A.millefolium, the equation for predicting area was: 0.043 + 0.011 Length2 + 0.60 Width2 (r2 = 0.91). N treatment was not found to affect the relationships between area and length or width.
The calculations that we used to determine the longevity of an average unit of leaf area were derived from Craine et al. (1999). The new calculations reflect using leaf area as a standard unit to compare species instead of individual leaves for forbs or leaf length for grasses and eliminate any bias that might be associated with an individual’s leaves of different sizes having different life-spans. In brief, the total leaf area that was both born and senesced in a plot over the census period and data on the dates of the censuses are used to calculate the total number of days lived by all the leaf area (leaf area-days) both born and senesced during the period. Division of the leaf area-days of all the leaves in a plot by the area of the leaves that had been both born and senesced during the census period gives the average longevity of the leaf area that had been both born and senesced during the census period. This calculation of the longevity of leaf area represents an average longevity over the census period, but does not compare longevity at different times of the year that may be associated with seasonal or ontogenetic differences among species.
Using data on the total leaf area present at each census, determining leaf area longevity first required determining the total number of leaf area-days that had been both born and senesced during the census using the following equation:
- ( Eqn 1a)
- ( Eqn 1b)
- ( Eqn 1c)
- ( Eqn 1d)
(i, first census date; j, last census date; An, cumulative leaf area observed or produced by census date n;An +1, cumulative leaf area observed or produced by the census following census n;Sn, cumulative leaf area senesced by census date n;Sn +1, cumulative leaf area senesced by the census following census n;kn, time interval between census n and n + 1; nS, the date at which the amount of leaf area senesced is equivalent to the amount of leaf area at the first census (S = Ai); and nl, the date at which the cumulative leaf area is equal to the amount of leaf area senesced by the last census date (A = Sj).)
The first two parts of the equation represent the total number of leaf area-days for all leaves tracked during the census, which is calculated as the difference between the total number of leaf area-days for total Eqn 1a, Fig. 1a) and senesced leaves (Eqn 1b, Fig. 1b) and is equivalent to the area between the curves for the census period used. The second part of the equation (Eqn 1c, Fig. 1c) represents the number of leaf-days for leaves that may have been present at the beginning of the census period, but should not be included in the determination of leaf area longevity as the date of birth of the leaf area was unknown and hence leaf area longevity could not be determined. The third part of the equation (Eqn 1d, Fig. 1d) represents the number of leaf area-days for leaves that had not senesced by the end of the census period. Similarly, this leaf area should not be included in the calculations of leaf area longevity as it is unknown when this leaf area would have senesced. These two quantities are subtracted from the total number of leaf area-days to provide the total number of leaf area-days for only those leaves that were both produced and senesced within the census period (Fig. 1e). Leaf area longevity is equivalent to the average horizontal distance between the two curves and was calculated by dividing the censused leaf area-days for which birth and death dates are known (Eqn 1a–d) by the total amount of leaf area that was born and senesced during the census (Ai− Aj). The dates nl and ns were calculated by linear interpolation based on the relevant census measures and dates. All analyses were computed with JMP vs 3.2.2 (SAS Institute, Cary, NC, USA).
Figure 1. Calculation of leaf area longevity of a species. Curve A (squares) represents the cumulative amount of leaf area (green or senesced) that was observed over the entire census period (Eqn 1a). Curve B (triangles) represents the cumulative amount of senesced leaf area over the entire census period (Eqn 1b). The areas under the curves are the total number of days that leaf area was green or senesced (Area under A) or the total number of days that leaf area was senesced (Area under B). The difference between these two curves represents the total number of leaf area-days for unsenesced leaves. Area C represents the total number of leaf area-days for leaves that were present at the beginning of the census (date of birth unknown) (Eqn 1c). Area D represents the total number of leaf area days for leaves that did not senesce during the census period (date of death unknown) (Eqn 1d). Subtracting Areas C and D from the area between Curves A and B provides the total number of leaf area days for leaves that were both born and senesced within the census period (Area E). Dividing Area E by the total area of leaves in Area E (the height of E) provides the estimate of leaf area longevity, equivalent to the average horizontal distance of Area E. Also represented are the first (ni) and last (nj) census dates and the interpolated ‘census dates’nl and ns (see text for details).
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For each species, average leaf area longevity and the standard errors of the means were computed with plots serving as replicates for a species. As an index of the total amount of leaf area that we sampled for a species, we also have provided the mean and standard error of leaf area sampled per individual that had both been produced and senesced during the census period. The average leaf area longevity and leaf area sampled were also calculated for all species at each level of CO2 and each N level. Differences in leaf area longevity and leaf area among species and treatments were determined with a linear regression model. Leaf area longevity was modelled with an additive general regression model that included the functional group of the species, the identity of the species (nested within functional group), the CO2 treatment category, the N treatment category, the interaction between CO2 and N treatment categories, and the interactions between functional group identity and both CO2 and N treatments. Due to the few number of replicates for each species at each factorial combination, we did not test for species-specific responses to elevated CO2 or N (i.e. no CO2 * species or N * species interactions).
In 5-yr-old monocultures (Cedar Creek LTER experiment E111), we had measured leaf longevity for 7 species that were also measured in the BioCON study (A.repens, A.gerardii, K.cristata, P.pratensis, S.scoparium, S.rigida, and S.nutans) using equivalent techniques (Craine et al., 1999). Soils in the 5-yr-old monocultures had similar amounts of soil N 0–20 cm (0.063%), but less soil C 0–20 cm (0.47 vs, 0.57% for BioCON) (D. A. Wedin, unpublished; P. B. Reich, unpublished). Even after 5 yr, biomass was much less in E111 than BioCON (Table 2), indicating much lower productivity in E111 than BioCON. We also compare average leaf percentage N (P. B. Reich, unpublished), root biomass, mid-June extractable NO3−, and N mineralization rate mid-June to mid-July (Reich et al., 2001) for the 7 species from BioCON and the 5-yr-old monocultures (J. M. Craine, unpublished) with t-tests and correlations. To examine the relationship of leaf longevity and these 4 measures among the 7 species in BioCON we ran a principal components analysis with the BioCON data and examined the loadings of these measures on the first axis.
Table 2. Results of the model that predicts leaf longevity
| ||F ratio||Prob > F||F ratio||Prob > F|
|Fxnl||14.5||< 0.001|| 4.6|| 0.01|
|Species[Fxnl]||12.1||< 0.001||14.3||< 0.001|
|CO2|| 5.1|| 0.03|| 1.4|| 0.23|
|Nitrogen|| 4.8|| 0.03|| 5.0|| 0.03|
|CO2 * Nitrogen|| 0.0|| 0.99|| 0.1|| 0.79|
|CO2 * Fxnl|| 3.7|| 0.03|| 0.2|| 0.79|
|N * Fxnl|| 0.9|| 0.40|| 2.3|| 0.11|