Global analysis of nitrogen and phosphorus limitation: methods
Relevant studies were identified by searching titles and abstracts of publications returned from searches on ISI Web of Science using combinations of key words such as nitrogen, phosphorus, nutrient, enrichment, fertilization and bioassay. We also included studies summarized in previously published syntheses (DiTommaso & Aarssen 1989; Elser et al. 1990; Tanner et al. 1998; Downing et al. 1999b) and searched all subsequent papers citing those syntheses. For studies that included additional manipulations (such as grazer exclusion), we included only treatment combinations using the unmanipulated controls (grazers at natural densities). Studies including such secondary manipulations were a small subset of our data. Studies were included if they involved (minimally) independent manipulations of both N and P availability or (ideally) full factorial manipulations of N and P. (Some studies involved both N and P enrichment but did not apply, or report data from, both treatments in all individual experiments. Thus, the numbers of observations for N and P responses are not necessarily identical.) By including only studies that manipulated both N and P, we minimized potential biases induced by investigator focus on particular limiting nutrients thought to be most important in particular kinds of ecosystems. Furthermore, we analysed the data in two ways, one in which all data were included and another in which only data from fully factorial experiments were included. The overall patterns were the same for the two approaches. Thus, we present the results for the more inclusive data set in order to increase the scope of habitats and experimental approaches encompassed.
We included only studies that reported mean community-level biomass or production responses of autotrophs to nutrient enrichment. Single-species responses were eliminated unless drawn from a mono-dominant community in the judgment of the original authors or, if several species from a community were individually assayed for N and P response, an average across all species was taken for a given study. The preferred metric was biomass per unit area (terrestrial, wetland, benthic) or volume (pelagic). We also accepted proxy variables that are known to be correlated with standing biomass, such as chlorophyll concentration (most common in phytoplankton studies), ash-free dry mass, carbon mass, biovolume, percent cover and primary production. Many studies in forests and other systems dominated by woody plants and a small percent of marine benthos studies reported incremental rates (change in height or radius) rather than standing biomass. Inclusion of these studies did not qualitatively change the results of our analyses, and so we present results from the larger inclusive data set. Studies involving organism counts were excluded because of the orders-of-magnitude discrepancies in organism size among systems, and the expected inverse relation between organism size and abundance (Cohen et al. 1993; Cyr et al. 1997).
We defined a study as a temporally and spatially distinct experiment with internally consistent controls. Multiple studies could be reported from within one publication, for instance, if the same experimental treatments were performed in multiple streams with differing water quality or for water samples obtained from different stations along an oceanographic transect. When multiple measures were reported over time in a single experiment, we generally used the last temporal sample to avoid phases of transient dynamics in order to capture measures closer to when the system approached a potential equilibrium with the added nutrients. Exceptions were made to standardize duration within systems or to avoid excessively long incubations (mainly for bioassays with freshwater or marine phytoplankton). Data for multiple sampling dates in extended studies were averaged if phenological changes necessitated the use of mean values over all samples instead of the final value in order to be more ecologically relevant. In these cases, we used the most robust values by deferring to the working knowledge and intuition of the original authors.
We used the ln-transformed response ratio as our primary effect size metric RRX = ln (Eâ C), where E is the measured value of the response variable in enrichment treatment X (N or P or N P) and C is its value in the unenriched control treatment. RR is one of the most frequently used effect metric in ecological meta-analysis (Hedges et al. 1999; Lajeunesse & Forbes 2003). Unlike Hedge s d, the ln-response ratio does not require a measure of sample variability. Moreover, in comparisons across systems where response variables and experimental designs can differ considerably, the analysis of change relative to the control is more meaningful than standardized absolute differences between means.
For each study, we used a unique study identifier linked to the citation of the publication and obtained the following information wherever possible. We categorized the system as marine, terrestrial, or freshwater and the stratum within each system by assigning aquatic studies to either pelagic or benthic subcategories and the terrestrial to either aboveground or belowground. Some studies in wetlands and salt marshes were difficult to categorize. For these, we used the operational approach that studies addressing submersed or floating macrophytes, or microalgae growing on them, were classified as aquatic (marine or freshwater), whereas studies on above-water rooted plants were termed terrestrial. For studies involving submersed macrophytes, when nutrients were added to the sediments, only responses of the macrophytes were included. When nutrients were added to the overlying water, only responses of the epiphytes were included. Finally, we also created a standardized set for habitat subcategories consisting of: grassland â meadow; tundra; forest â shrubland; wetland; stream; lake pelagic; lake benthos; marine benthos (hard bottom), marine benthos (soft bottom); or marine pelagic. We also entered supporting data about incubation conditions and the local environment, including concentrations of available nutrients (nitrate, ammonium, soluble reactive phosphorus).