Technical Comments

Comment on “Unexpected reversal of C3 versus C4 grass response to elevated CO2 during a 20-year field experiment”

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Science  10 Aug 2018:
Vol. 361, Issue 6402, eaau1073
DOI: 10.1126/science.aau1073


Reich et al. (Reports, 20 April 2018, p. 317) assert that the responses of C3 and C4 grass biomass to elevated CO2 “challenge the current C3-C4 [elevated CO2] paradigm,” but these responses can be explained by the natural history of the experimental plants and soils without challenging this paradigm.

Reich et al. (1) explain that positive responses of plant biomass to elevated CO2 have disappeared in C3 grasses and appeared in C4 grasses over the 20 years of the BioCON experiment. They assert, as do the authors of the associated Perspective (2), that these results challenge current expectations of C3 and C4 plant responses to elevated CO2. Additional context should be made available to qualify this assertion. The pattern documented by Reich et al. can be explained by considering the natural history of the experimental plants and soils, without challenging general expectations of C3 and C4 grass responses to elevated CO2 in the absence of other limitations.

The soil at the BioCON experimental field, which was not described in the paper or its supplement, was an excessively drained outwash sand, originally described as a Typic Udipsamment (3). When the experiments at the Cedar Creek Ecosystem Science Reserve (including BioCON) were initiated, topsoil was bulldozed away from the experimental field to remove existing savannah vegetation and seedbank. The field was then fumigated with methyl bromide (4). Remaining subsoil would have been composed of >90% sand, with little organic matter aside from coatings on sand mineral surfaces. Therefore, despite its 20-year duration, the BioCON experiment documents responses in a disturbed, developing soil. Although results from this experiment might be relevant to agricultural or urbanized soils (5), extrapolating to plant communities in mature, undisturbed soils worldwide is problematic (6, 7).

Several publications from BioCON have demonstrated the importance of plant species identity, species richness, and functional group diversity in moderating responses to CO2 and N enrichment [e.g., (810)], yet Reich et al. have used results from monocultures and four-species assemblages of only C3 or C4 grasses to make a broad statement about the general responses of C3 and C4 grasses to elevated CO2. Despite variation among species, the C4 grasses as a group tend to have higher nitrogen use efficiencies than C3 grasses, reflecting their relatively smaller investment of N in photosynthetic carboxylation enzymes (11). Given the individual characteristics of the eight experimental grass species (Table 1) (12) and the initial seeding rate of 12 g seed/m2 for all plots (8), the C3 grasses would be expected to fill their plots faster than the C4 grasses, which they did; the C3 grasses grew greater overall biomass per plot than the C4 grasses in the first few years (1). Short-lived positive responses of the C3 plant biomass to elevated CO2 might also be expected, because their higher overall leaf N contents allow for some dilution of N; increased aboveground biomass with diluted N under elevated CO2 was indeed observed in early years (13).

Table 1 Traits of the grass species grown in the BioCON experiment (12).
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The low fertility and water-holding capacity of the experimental soils, however, would favor the experimental C4 grass species over time, because their fertility requirements—and, in some cases, their water requirements—are lower than in the C3 species grown (Table 1). This advantage would not be obvious in the earlier years of the experiment, because of the slower growth rates and longer lifespans of the C4 species relative to the C3 plants grown (Table 1), but C4 biomass would be expected to increase relative to C3 plants over time in these conditions, with associated increases in organic matter additions to the soil from roots and litter. Eventually, the C4 plots would accumulate more organic matter, providing carbon substrate for N-mineralizing microbes, as well as increased soil nutrient and water-holding capacity. These changes would alleviate N and H2O limitations in the C4 plots relative to the C3 plots, leading to further enhancements in annual biomass accumulation and nitrogen mineralization rates. Therefore, the observed shifts in relative response to elevated CO2 over time relate to the differential nutritional requirements also inherent in C3 and C4 photosynthetic metabolism, as well as to experimental conditions. Consequently, the observations do not disagree with general expectations of C3-C4 dynamics under elevated CO2 when no other limitations are present.

In addition to methods used to prepare the site before treatment application, the statistical design of the free-air CO2 enrichment (FACE) arrangement is also important. The authors state that the 88 one- and four-species, C3-only and C4-only plots analyzed constitute a fully factorial experiment. In fact, these plots are a subset of a broader experiment where three ambient and three elevated FACE rings provide blocked CO2 treatments, and N, functional group, and species richness treatments are applied as fully factorial split-plot treatments within the blocks (10). The monoculture and four-species plots analyzed in this paper are unevenly distributed among three ambient and three elevated FACE rings. This unbalanced design usually means that the model sum of squares for overall treatment effects is not equal to the sum of individual treatment sums of squares, which precludes straightforward repeated-measures analysis (14). The authors do not describe how their statistical analysis addresses these limitations, nor do they mention any multiple-test correction to the P values obtained in this and earlier reports of nonindependent response variables over the years of the experiment.

We recognize that long-term (20-year) experiments such as BioCON are invaluable and provide unique information; however, before extrapolating to a broader, ubiquitous inference, attention should be given to both the statistical details and the broader context of the environmental limitations associated with the location. In low-n FACE experiments, as described here, underlying variability in soils, particularly nutrient availability, could have an outsized impact on result interpretation [e.g., (15)].

The general theory of C3-C4 dynamics under elevated CO2, and its use in the Earth System Models that encode it, is a fundamental aspect of plant biological responses to rising carbon dioxide. Questioning this aspect should be encouraged. However, we would caution that additional research is necessary before the C3-C4 dynamic in response to CO2 is invalidated.


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