Interactions Between Elevated Atmospheric CO2 and Defoliation on North American Rangeland Plant Species at Low and High N Availability

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Grass and Forage Science The Journal of the British Grassland Society The Ofﬁcial Journal of the European Grassland Federation

Interactions between elevated atmospheric CO2 and defoliation on North American rangeland plant species at low and high N availability

D. R. LeCain*, J. A. Morgan*, G. L. Hutchinson*, J. D. Reeder* and F. A. Dijkstra† *U.S. Department of Agriculture, Agricultural Research Service, Rangeland Resources Research Unit, Crops Research Laboratory, Fort Collins, CO, USA, and †Faculty of Agriculture, Food and Natural Resources, The University of Sydney, Level 4, Biomedical Building, 1 Central Avenue, Eveleigh, NSW 2015, Australia

Abstract Keywords: semi-arid rangeland, CO2, defoliation, nitro- 15 Although common disturbances of grazing lands like gen, C3 grass, C4 grass, forb, root, biomass, N recov- plant defoliation are expected to affect their sensitivity ery, forage quality. to increasing atmospheric CO2 concentration, almost no research has been conducted to evaluate how important such effects might be on the direct responses of Introduction rangelands to CO2. This growth chamber experiment About 40% of terrestrial ecosystems are classified as subjected intact plant–soil cylinders from a Wyoming, rangelands (Suttie et al., 2005). Rangeland ecosystems USA, prairie to a 3-way factorial of CO2 (370 vs. are not characteristically productive lands, but they )1 720 lLL ), defoliation (non-clipped vs. clipped) and support most of the world’s managed livestock in )2 soil nitrogen (control vs. 10 g m added N) under addition to large herds of native ungulates (Campbell simulated natural climatic conditions. Above- and et al., 1997). The productivity of rangelands may be below-ground biomass and N dynamics of the func- slowly increasing owing to the fertilization effects of tional groups C3 grasses, C4 grasses and forbs were rising levels of atmospheric CO2 (Polley, 1997; Morgan investigated. CO2 and defoliation had independent et al., 2001a, 2004b). However, the consequences of influences on biomass and N parameters of these rising atmospheric CO2 concentrations on plant–herbi- rangeland plants. Growth under CO2-enriched condi- vore dynamics like defoliation, that influence the tions enhanced above-ground biomass 50% in C3 ecology of rangelands, are relatively unexplored. grasses alone, while shoot N concentration declined Elevated CO2 is well known to increase above- 15 16% in both C3 and C4 grasses. Plant-soil N uptake ground plant productivity (Korner, 2000; Morgan et al., was unaffected by CO2 treatment. In contrast, defolia- 2004a; De Graaff et al., 2006). Owing to greater photo- tion had no effect on biomass, but increased tissue N synthetic enhancement, C3 plant growth is stimulated concentration 29% across all functional groups. With- more by elevated CO2 than C4 species (Korner, 2000; out additional N, forage quality, which is in direct Reich et al., 2001). However, there are several reports of relation to N concentration, will decline under increas- improved productivity in C4 rangeland species (Read ing atmospheric CO2. Increased dominance of C3 1996; LeCain and Morgan, 1998; Wand et al., 1999; grasses plus reduced forage quality may necessitate Owensby et al., 1999; LeCain et al., 2003). This has been changes in grazing management practices in mixed- attributed to improved water-use efficiency and soil- species rangelands. water status resulting from reduced stomatal conduc-

tance and plant transpiration under elevated CO2 (Volk et al., 2000; Nelson et al., 2003). This stomatal response

Correspondence to: D. R. LeCain, U.S. Department of Agri- occurs similarly in both C3 and C4 grasses (Wand et al., culture, Agricultural Research Service, Rangeland Re- 1999) and is a major factor boosting productivity in sources Research Unit, Crops Research Laboratory, 1701 CO2-enriched semi-arid rangelands (Morgan et al., Centre Avenue, Fort Collins, CO 80526, USA. 2011). In many temperate rangelands (such as much E-mail: [email protected] of the USA Great Plains), C3 grasses are the major forage during spring while C4 grasses are the primary mid-late- Received 29 April 2011; revised 19 October 2011 season forage; forbs are typically a less important food

350 2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 doi: 10.1111/j.1365-2494.2011.00847.x CO2, defoliation and N interactions on rangeland plant species 351

source (Milchunas et al., 1995). Rangeland forb pro- expected to be a major factor in rangelands as many duction may be increased by higher atmospheric CO2, rangeland soils are N limiting for plant growth (Hungate presumably because most rangeland forbs are C3 pho- et al., 1997). Many studies report reduced plant-tissue N tosynthesis types (Reich et al., 2001; Teyssonneyre concentration under elevated CO2 (Wilsey et al., 1997; et al., 2002; Polley et al., 2003). In mixed-species Morgan et al., 2004a; Milchunas et al., 2005a; Reich rangelands, CO2-induced shifts in the balance of C3 et al., 2006) because of improved nitrogen-use effi- and C4 grasses and forbs have the potential to signif- ciency or reduced soil-N availability (King et al., 2004; icantly alter community structure and ecosystem pro- Wand and Midgley, 2004). Although productivity may cesses, including animal grazing processes (Campbell increase under elevated CO2, reduced tissue N and and Stafford Smith, 2000; Morgan et al., 2007). protein concentration will have negative consequences In most rangelands, there is more plant matter below for domestic and indigenous fauna (Campbell and than above ground (Milchunas and Lauenroth, 1993, Stafford Smith, 2000; Milchunas et al., 2005a). Con- 2001). Although there are many reports on the influ- versely, defoliation can increase N concentration in ence of elevated CO2 on root productivity, the results regrowth tissue (Milchunas et al., 1995, 2005a; Mikola vary widely between ecosystems and sampling methods et al., 2005), but in the long term, reduced soil-N and a conceptual understanding of root responses has availability under elevated CO2 may lessen this re- not emerged (Hebeisen et al., 1997; Wilsey et al., 1997; sponse. Morgan et al., 2001b; Milchunas et al., 2005b; De Graaff Our objective was to investigate how defoliation and et al., 2006). In a field study on the shortgrass steppe of soil-N availability interact to affect the above- and Colorado, we reported little change in root biomass below-ground productivity (assessed as plant biomass) after 5 years of elevated CO2 (LeCain et al., 2006). and tissue N responses of a semi-arid grassland to However, studies investigating the interaction of ele- growth under present-day [CO2] and CO2-enriched vated CO2 and grazing on root growth are rare conditions. In this experiment, we utilized perennial (Augustine et al., 2010). plant-intact soil microcosms extracted from a northern As it is difficult to evaluate how grazing might USA mixed-grass prairie and subjected them to manip- interact with CO2 levels in sparsely stocked rangelands, ulations of defoliation, CO2 and N. Responses were defoliation has been used to simulate grazing. Grazing evaluated for the three major plant functional groups, or defoliation alone can increase or decrease plant C3 grasses, C4 grasses and forbs. Our hypotheses were as productivity depending on grazing intensity and the follows: grazing tolerance of species (Hendon and Briske, 2002). H1: Elevated CO2 will increase shoot biomass in forbs Forbs are generally thought to be less grazing tolerant and C3 grasses, with little response observed in C4 than grasses because of herbivory-exposed meristems grasses and roots. Defoliation will reduce shoot

(Heady and Child, 1994). While CO2 enrichment might biomass in forbs, but elevated CO2 will improve be expected to enhance plant recovery from defoliation, recovery from defoliation. because of more available CO2 and soil water (Wand H2: Enhanced shoot biomass under elevated CO2 will and Midgley, 2004), this has not always been found reduce tissue N concentration and N uptake (mea- 15 (Polley et al., 2011). Elevated CO2 and defoliation may sured by N) in all functional groups, particularly have opposing as well as additive effects on plant without N addition. Defoliation and N addition will productivity, species composition and allocation of counter the effect of elevated CO2 on these N resources (Morgan et al., 2001a; Harmens et al., 2004; parameters.

Wand and Midgley, 2004; Lau and Tifﬁn, 2009; H3: Elevated CO2 will increase volumetric soil-water Augustine et al., 2010). Consequently, it is presently content (VWC) that will contribute to increased unclear how defoliation interacts with rising atmo- biomass of all functional groups. Defoliation will spheric CO2 to affect rangeland productivity and ecol- have minimal effect on VWC. ogy, particularly in a mixed-species ecosystem. In many arid and semi-arid rangelands, nitrogen (N) Materials and methods dynamics are second only to precipitation in impor- tance for plant–ecosystem processes (Burke et al., Site characteristics 1997). Responses of plant productivity to elevated

CO2 and defoliation, and the resulting changes in Plant–soil cylinders for this experiment were extracted ecosystem functioning, are co-dependent on the cycling from the USDA-ARS High Plains Grasslands Research of N through the plant–soil–atmosphere. In the long Station (HPGRS), near Cheyenne, WY, USA (4111¢ N term, plants will not sustain increased productivity lat, 10454¢ W long). The HPGRS is within the under elevated CO2 when other factors become growth northern mixed-grass prairie, with elevations ranging limiting (De Graaff et al., 2006). Nitrogen limitation is from 1910 to 1950 m, an average 127-d growing

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 352 D. R. LeCain et al.

season, and annual precipitation of 384 mm. Site Growth chamber conditions vegetation is mostly comprised of grasses, averaging

55% C3 grasses and 23% C4 grasses plus a variety of Environmental conditions in the growth chambers forbs, sedges and sub-shrubs. In our cylinders, there simulated the growing season of SE Wyoming. Sodium were typically four C3 grasses dominated by Pascopy- vapour and metal halide lamps provided a realistic )2 )1 rum smithii [Rybd.] A. Love, two C4 grasses dominated spectrum of light at 600 lmol m s photosynthetic by Bouteloua gracilis [H.B.K.] Lag. and five forbs photon flux density at plant height. Photoperiod and dominated by Sphaeralcea coccinea [Nutt.] Rydb. Soils day ⁄ night temperature were adjusted weekly to mimic are Ascalon sandy loams. The pasture from which the seasonal climate in SE Wyoming. Long-term cylinders were extracted had been lightly grazed by precipitation data were used to calculate weekly water cattle (annual removal of 30% vegetation) since 1982 additions similar to what occurs in the field, with most (LeCain et al., 2000). precipitation occurring in May, June and early July followed by late season drought. All cylinders were Experimental treatments weighed weekly (prior to irrigations) to calculate VWC (m3 m)3). In early April of year one (plants dormant), forty PVC cylinders measuring 30 cm tall by 25 cm diameter were Plant sampling pushed into the soil, then extracted with intact plant– soil cores, capped at the bottom and transported to the On the harvest dates, all plants were defoliated to 1 cm

USDA-ARS Crops Research Lab (Fort Collins, CO, height and separated into C3 grasses, C4 grasses and USA). Eight cylinders were immediately harvested for forbs. The soil cores were pushed out of cylinders, and plant and soil analyses (T0). The soil of each of the all visible roots were hand picked and washed. Sub- remaining thirty-two cylinders was injected with samples of all plant fractions were frozen in liquid N, )215 15 1gm N (as KNO3 99 atom% N). The cylinders lyophilized and ground to 0Æ5 mm for tissue analysis. were randomly separated into four groups of eight; each The remaining shoot and root material was oven-dried group was then placed into four growth chambers at 60C, weighed and sub-sampled for ash analysis: all (EGC, Chagrin Falls, OH, USA), two at present atmo- data were ash-corrected. Above-ground plant biomass spheric CO2 concentration (ACO2) and two others at and nutrients collected from the June defoliation )1 720 lLL (ECO2). treatment were added (for biomass) and weighted (for The eight plant–soil cylinders within each of the four nutrients) to the July and September data. Therefore, growth chambers were randomly partitioned into two these data are cumulative for the +D treatment. groups of four, one receiving supplemental N in the amount of 10 g m)2 as KNO (+N treatment), the other 3 Shoot and root tissue analyses with no supplemental N ()N treatment). The ﬁrst week of June, all vegetation in two of the cylinders from the Total N and 15N analyses were performed by combust- +N and )N groups was defoliated to 2Æ5 cm height (+D ing samples in a Carlo Erba Series II automated N ⁄ C treatment), which corresponds with typical late spring analyzer interfaced with a Europa isotope-ratio mass cattle grazing on this rangeland. The other two cylin- spectrometer (Knowles and Blackburn, 1993). ders were not defoliated ()D treatment). The cylinders We calculated 15N recovery (% of applied 15N) in the 15 were destructively sampled for plant attributes at different plant N pools ( Nrec, plant) by: ecologically signiﬁcant times: the time of typical peak ÀÁ 15N ¼ N =N 15N 15 N green biomass, (15 July; T1), and at the end of the rec; plantÀÁplant label plant plant; T0 15 15 growing season but before leaf loss, (28 September; T2). Nlabel Nplant; T0 100 15 Clipped tissue from the June defoliation treatment (+D) where Nplant and Nplant are the total amount of N and 15 15 was dried and weighed and analysed with the same N atom% in the plants at T1 and T2, Nplant, T0 is the 15 protocol as the two complete harvests (see below). To average N atom% in the plants at T0, and Nlabel and 15 15 summarize, the experimental treatments consisted of Nlabel are the total amount of N and the N atom% of two CO2 · two defoliation · two nitrogen with two the label applied. replications each, sampled on two harvest dates. The entire experiment was repeated the following year, Statistics extracting new plant–soil cylinders from the same pasture in early April. The T0 assessment showed that The experiment had three measured or calculated the soil texture and nutrients and initial species com- parameters in both above- and below-ground tissues: position were similar between the 2 years (data not biomass, %N and 15N recovery. Interactions with the shown). experimental treatments (CO2, nitrogen, defoliation)

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 CO2, defoliation and N interactions on rangeland plant species 353

and both ‘Harvest Date’ and ‘Year’ were rare for all Table 1 Probabilities from Proc Mixed analysis of CO2, experimental parameters; therefore, a conservative (elevated and ambient), nitrogen (addition and control) and statistical analysis was made by pooling the data over defoliation (defoliated and control), and their interactions, on Harvest Date and Year, using the separate growth plant biomass and nitrogen resources of C3 grasses, C4 – chambers as replicates (n = 2). The data were analysed grasses, forbs and roots from the semi-arid prairie of NE using Proc Mixed (SAS Institute Inc., Cary, NC USA) as Wyoming, USA a split-split plot design, with CO as main plots, N as a 2 Biomass %Nitrogen 15N recovery split plot and defoliation as a second split plot within N. Also, there were signiﬁcant treatment by functional C3 grass group interactions for all the experimental parameters CO2 0Æ0001 0Æ0538 0Æ4100

(for biomass the functional group* CO2 P =<0Æ0001); NIT 0Æ0003 0Æ0107 0Æ3626 therefore, each functional group was analysed inde- CO2*NIT 0Æ0198 0Æ0357 0Æ9324 pendently to investigate the unique responses of each. DEFOL 0Æ9521 0Æ0009 0Æ0100 CO2*DEFOL 0Æ4118 0Æ6330 0Æ0697 NIT*DEFOL 0Æ6428 0Æ8049 0Æ8782 Results CO2*NIT*DEFOL 0Æ1654 0Æ5742 0Æ0835

C grass Biomass responses to CO2, defoliation and 4 nitrogen in three functional groups CO2 0Æ5509 0Æ0366 0Æ3177 NIT 0Æ0313 <0.0001 0Æ0399 Elevated CO2 significantly increased shoot biomass only CO2*NIT 0Æ3405 0Æ0772 0Æ1291 in C3 grasses (P = 0.0001) and particularly when com- DEFOL 0Æ2047 <0.0001 0Æ4113 bined with the +N treatment (CO2 *N interaction CO2*DEFOL 0Æ8913 0Æ6961 0Æ3624 (P =0Æ02); Table 1 and Figure 1). C4 grass and forb NIT*DEFOL 0Æ6314 0Æ7580 0Æ5581 shoot biomass did not respond to ECO2 (P =0Æ55 and CO2*NIT*DEFOL 0Æ1040 0Æ8806 0Æ1437 0Æ98). The absence of a CO2 effect on forb shoot biomass in particular was surprising (H1). However, forb biomass Forbs was low, and quite variable between cylinders (0–20%) CO2 0Æ9894 0Æ5825 0Æ9508 suggesting that our sampling scheme may have been NIT 0Æ2547 0Æ0460 0Æ3419 inadequate to detect treatment effects in this functional CO2*NIT 0Æ3841 0Æ3696 0Æ3219 group. Contrary to our hypothesis (H1), there was no DEFOL 0Æ1424 0Æ0598 0Æ0761 significant effect of the defoliation treatment on the total CO2*DEFOL 0Æ7072 0Æ3732 0Æ9451 amount of shoot biomass that accrued over the growing NIT*DEFOL 0Æ5598 0Æ5030 0Æ2976 season in forbs, or any functional group (Table 1); CO2*NIT*DEFOL 0Æ2514 0Æ3320 0Æ2619 ) biomass was nearly equal in +D and D. We observed Roots no significant interaction between the CO and defoli- 2 CO2 0Æ2198 0Æ1762 0Æ1405 ation treatments on above-ground biomass in any NIT 0Æ0072 0Æ1184 0Æ0089 functional group. Nitrogen addition increased shoot CO2*NIT 0Æ0010 0Æ1345 0Æ1011 biomass in both grass groups (C4 grasses P =0Æ03; DEFOL 0Æ0020 0Æ6311 0Æ0002 Figure 1), but for C grasses, this was significant only 3 CO2*DEFOL 0Æ0521 0Æ5762 0Æ2154 in combination with ECO2 (CO2*Nit P =0Æ02: Figure 1). NIT*DEFOL 0Æ1471 0Æ8499 0Æ3290 In contrast, +N did not increase forb biomass (P =0Æ25) CO2*NIT*DEFOL 0Æ8432 0Æ6888 0Æ6810 but again forb variability was very high. Roots could not be separated by functional group in Bold values are significant at P <0Æ054. these cylinders. Interactions between treatments were common with root biomass. Root biomass was not root turnover in this ecosystem is about 5 years (Mil- significantly increased by the main effect of ECO2 chunas and Lauenroth, 2001). Therefore, our data show (P =0Æ22) but was significantly reduced with defolia- a short-term view of root responses and not a measure tion (P =0Æ002; Table 1 and Figure 1). Unlike shoots, of actual root productivity (Milchunas, 2009). the effects of defoliation on root biomass interacted with CO (P = 0.052). Defoliation reduced root biomass 2 Nitrogen resource responses to CO , overall, but to a larger extent under ECO (Figure 1). 2 2 defoliation and nitrogen Root biomass increased under +N, but only in combination with ECO2 (CO2*Nit P =0Æ001; Table 1 and Defoliation and CO2 treatments influenced shoot %N Figure 1). It is important to note that many of the completely independently (Table 1). Supporting our collected roots had formed prior to the experiment as hypothesis (H2), defoliation increased shoot %N in all

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 354 D. R. LeCain et al.

20 (a) E + N E – N 15 A + N A – N

5 grass AGB (g per pot) 3 C

0 (b) 8 + N – N

2 grass AGB (g per pot) 4 C E + N – D 0 E + N + D E – N – D (c) 100 E – N + D A + N – D A + N + D A – N – D 80 A – N + D

Root biomass (g per pot) 20

Figure 1 Statistically signiﬁcant effects of CO2 (elevated and ambient: E and A), nitrogen (addition and control: +N and )N) and defoliation (defoliated and control: +D and )D) treatments on C3 grass (a) and C4 grass (b) above-ground biomass (AGB) and root biomass (c) in soil–plant cylinders extracted from a Wyoming, USA rangeland.

functional groups regardless of CO2 or N treatment (C3 Similar to %N, CO2 and defoliation treatments acted 15 grass, C4 grass, forbs P =0Æ0009, 0Æ0001, 0Æ059: Tables 1 independently on N recovery (Table 1). Contrary to 15 and 2). Also supporting H2, shoot N concentration H2, the recovery of N was not reduced by CO2 (%N) declined in both ECO2-grown C3 and C4 grasses treatment in any of the functional groups or in roots particularly in the +N treatment (CO2 *N interaction in (Table 1). However, defoliation reduced C3 grass shoot 15 C3 grasses P =0Æ036; Tables 1 and 2). The +N treatment N recovery (P =0Æ01) (Tables 1 and 2). The +N 15 increased %N in all functional groups, but in C3 grasses, treatment increased N recovery in C4 grasses, but only signiﬁcantly when grown under ACO2 (CO2 *Nit not in the other groups (P =0Æ04, Tables 1 and 2). P = 0.036; Table 2). In contrast to our hypothesis, forb Compared with controls, root 15N recovery was higher

%N was not reduced by ECO2 (P =0Æ58). There were under +N (P =0Æ009), but lower under +D (P =0Æ0002; no signiﬁcant treatment effects on root %N (Table 1). Tables 1 and 2).

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 CO2, defoliation and N interactions on rangeland plant species 355

Table 2 Means of significant CO2 (elevated and ambient: E are shown in Figure 2, with averages in the figure and A), nitrogen (addition and control: +N and )N) and legend. Supporting our hypothesis (H3), after an initial defoliation (defoliated and control: +D and )D) treatment dry-down from winter-stored soil moisture, it was clear effects (P <0Æ05) for nitrogen resource parameters of func- that the ECO2 ⁄ )N treatment was the most conservative tional groups and roots of the semi-arid Wyoming prairie: with soil water. The ACO2 ⁄ +N treatment had the non-significant means in parenthesis lowest VWC during much of the experiment. Therefore, ECO promoted water conservation, while +N pro- Treatment C3 grass C4 grass Forb Roots 2 moted water usage. Note that ECO2 improves growth, %N while conserving water [improved water-use efficiency A )N1Æ03 b 1Æ07 b 1Æ20 b (1Æ44) – water-use efficiency (WUE)], while +N improves A+N 1Æ48 a 1Æ32 a 1Æ43 a (1Æ56) growth while using more water (reduced WUE). E )N0Æ97 b 0Æ92 c 1Æ15 b (1Æ39) Largely supporting our hypothesis (H3), defoliation E+N 1Æ04 b 1Æ08 b 1Æ51 a (1Æ39) treatment had only a small impact on VWC: average ) D0Æ92 1Æ01Æ21 (1Æ46) VWC was improved by +D in the ACO2 ⁄ )N treatment +D 1Æ35 1Æ21Æ51 (1Æ44) (18Æ4% vs. 16Æ1% in +D vs. )D) but not in the other 15 N recovery (%) CO2 ⁄ N combinations (Figure 2). )N (10Æ6) 3Æ5(2Æ1) 25Æ7 +N (11Æ2) 5Æ5(3Æ5) 30Æ3 )D12(4Æ8) (1Æ45) 32Æ1 Discussion +D 9Æ69 (4Æ1) (3Æ62) 23Æ9 Above- and below-ground biomass responses of When significant interactions occurred, Tukey’s means com- three functional groups to CO2 and defoliation parison test was used; means with different letters are significantly different at P <0Æ05. Our most important finding was that CO2 and defoliation treatments appeared to have independent influences on shoot growth (Table 1). Elevated CO Soil water content responses to CO , 2 2 improved above-ground biomass only in the C grass defoliation and nitrogen 3 group, while there was no effect of defoliation on As water is the primary driver of productivity in this above-ground biomass of any functional group (defo- ecosystem, soil water was carefully monitored. Treat- liated and non-defoliated plants had equal biomass). ment differences in VWC were most apparent at certain This was contrary to our hypothesis that ECO2 would times in the growing season. Therefore, all of the data increase forb growth and that ECO2 would improve

0·40 E + N – D (18·5) E – N – D (22·9)

) 0·35 E + N + D (18·4)

–3 E – N + D (22·7) m

3 A + N – D (15·4) 0·30 A – N – D (16·1) A + N + D (14·8) A – N + D (18·4)

0·25

0·20

0·15 Volumetric water content (m

Standard deviation 0·10 115 122 128 136 142 150 157 164 171 174 178 183 188 192 195 198 206 213 220 227 234 241 248 255 262 269 Day of the year

Figure 2 Effects of CO2 (elevated and ambient: E and A), nitrogen (addition and control: +N and )N) and defoliation (defoliated and control: +D and )D) on volumetric soil water content in soil–plant cylinders extracted from a Wyoming, USA rangeland. The average volumetric soil water content (VWC) from day of year 142–269 for each treatment is shown in parenthesis in the legend.

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 356 D. R. LeCain et al.

recovery from defoliation. Our results differ from reports Root biomass was not significantly increased under that forb production can be stimulated more than ECO2. While increased root productivity under elevated grasses by elevated CO2 (Reich et al., 2001; Tey- CO2 has been observed in other experiments (Hebeisen ssonneyre et al., 2002; Polley et al., 2003). However, et al., 1997; Wilsey et al., 1997; Morgan et al., 2001b; De forbs averaged <10% of the shoot biomass and were Graaff et al., 2006), our results are consistent with other extremely variable between pots, potentially making reports from near by semi-arid grasslands in which detection of treatment effects difficult. Although the 5 years of CO2 enrichment caused little change in root lack of a forb response was unexpected, it is not unique; biomass in shortgrass steppe (LeCain et al., 2006), and in a California grassland, where annual plants dominate, 4 years of CO2 enrichment were required to detect ECO2 ‘alone’ also had no effect on forb productivity. significant changes in root biomass of a northern This was speculated to result from increased shading by mixed-grass prairie (Morgan et al., 2011). While expo- the dominant grasses. However, the combination of the sure of grasslands to CO2-enriched atmospheres tends global changes ECO2 + warming + precipitation in- to enhance productivity and root growth, it also speeds creased the relative abundance of forbs (Zavaleta et al., up root turnover (Allard et al., 2005; Milchunas et al.,

2003a,b). A 9-year Free Air CO2 Enrichment (FACE) 2005b). Thus, the biomass of perennial root systems study in Minnesota grassland, with highly mixed species may not change much over time, and multiple years of composition, reported a strong reduction in photosyn- ECO2 might be needed to observe any signiﬁcant thetic capacity of forbs under ECO2 but not in C3 grasses, change. suggesting the potential for major shifts in species Reduced root biomass, but not shoot biomass, with composition and diversity in mixed functional group defoliation suggests resource translocation from roots to grasslands (Crous et al., 2010). More work is needed to shoots after defoliation. This translocation may have establish these treatment responses of forbs in mixed- been stronger under ECO2, where we observed greater species grasslands. It is likely that grouping forbs into a reductions in root biomass with defoliation (CO2*D single functional group is not appropriate, but future P =0Æ05; Figure 1). We suspect the domination of this research might consider multiple ‘forb’ groups, such as grassland community by C3 grasses may be involved in annual forbs, perennial forbs, woody forbs, invasive this interaction. Remobilization of below-ground re- forbs, etc. (Suding et al., 2005). serves to shoots after defoliation appears to be a more Contrary to this study, we previously reported important response mechanism of Pascopyrum smithii, improved growth in C4 grasses in controlled environ- one of the dominant C3 grasses in the mixed-grass ment elevated CO2 studies (Read and Morgan, 1996; prairie, compared with the C4 dominant Bouteloua LeCain and Morgan, 1998; Morgan et al., 1998). gracilis (Skinner et al., 1999; Augustine et al. 2010). However, those earlier experiments were performed Greater remobilization of root reserves plus stronger in monoculture. The more realistic prairie microcosms responses of C3 plants in general to CO2 may tend to used in this experiment represent natural plant amplify this apparent defoliation response of plant communities and soils where muted responses to community root biomass.

CO2 sometimes occur because of competition for soil and climate resources (Campbell et al., 1997; Owensby The influence of available soil N on biomass et al., 1999; Korner, 2000; Morgan et al., 2004b, responses to CO2 and defoliation 2011). Further, the sensitivity of C4 grasses as well as intact grassland ecosystems to CO2 is inversely CO2 by N interactions on above-ground biomass only proportional to the supply of soil water available to occurred in C3 grasses. Our result of a stronger C3 grass plants (Wand et al., 1999; Joel et al., 2001; Morgan biomass response to CO2 when more N is available et al., 2004b; Wand and Midgley, 2004). Although agree with many reports that CO2-induced increases in CO2 did induce water savings in this experiment and plant productivity are often constrained by soil-N realistic field ‘precipitation’ resulted in terminal water availability (Hebeisen et al., 1997; Joel et al., 2001; stress in our microcosms (Figure 2), the potential Grunzweig and Korner, 2003; De Graaff et al., 2006). 15 benefit to the C4 grasses was apparently insufficient In our study, the amount of N uptake ( N) did not to elicit a water relations-growth response. We con- increase in conjunction with increased C resources clude that the indirect effects of water savings on (ECO2), resulting in reduced tissue %N in plants under elevated CO2-improved plant growth depend on elevated CO2 (see below). We conclude that while CO2- threshold-dependent soil moisture level as well as enhanced water relations is a major factor behind the complicated interactions of multiple species with positive production effects of elevated CO2 in dry other climate and nutrient factors (Campbell et al., ecosystems (Morgan et al., 2004b, 2011), soil N may 1997; Owensby et al., 1999; Korner, 2000; Morgan constrain those responses. There were no defoliation by et al., 2004b, 2011). N interactions in any of the experimental parameters,

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 CO2, defoliation and N interactions on rangeland plant species 357

and defoliation did not reduced above-ground biomass. depleted N in the entire root zone, thereby reducing

We therefore conclude that above-ground productivity %N in C4 grasses too (Table 2). Our results agree with is not reduced by defoliation even under naturally low an OTC study with similar species composition, which soil N availability. reported reduced %N in the dominant grass species

Although not directly investigated in this study, under elevated CO2 (King et al., 2004). As with increasing N deposition because of human activity is biomass, there were no interactions between CO2 and another important global change. Reports from other defoliation on important N resources. Elevated CO2 grasslands show that increased plant productivity, quite consistently reduced shoot tissue ‘quality’ of whether through ECO2 or increased N deposition, can grasses (reduced %N). This will have negative conse- reduce plant diversity, primarily through elimination of quences for indigenous and domestic grazing animals in rare species (Zavaleta et al., 2003b; Suding et al., 2005). world-wide grasslands. In this and other rangelands, Although our forb results were variable, the lack of a grasses are the major forage for grazing animals. In

ECO2 and +N response in forb biomass suggests that, N-limited soils, increased grass biomass may not com- similar to a native California grassland, forbs will be pensate for reduced grass protein contents, and there- losers under multiple global changes as the dominant fore, animal nutrition and landscape carrying capacity

C3 grasses out-compete them (Zavaleta et al., 2003b). will suffer (Milchunas et al., 2005a). Many studies have investigated how ‘global change’ might alter nutrient cycling. Increased atmospheric The inﬂuence of CO and defoliation on plant N 2 CO can alter soil N availability, potentially in several dynamics 2 ways. Improved soil water content can improve Shoot %N was higher after defoliation in all functional microbial activity and N mineralization rates (Hungate groups, while ECO2 reduced %N in grasses (Table 2), et al., 1997; Dijkstra and Cheng, 2008). Soil microbial similar to results reported in an earlier open-top- activity could be improved by ‘priming’ with more chamber (OTC) experiment on the Colorado short- abundant C from roots (Reich et al., 2006). However, grass steppe (Milchunas et al., 2005a). Thus, these De Graaff et al. (2006) performed a meta-analysis on observations support our second hypothesis that defo- ﬁeld studies (FACE and OTC) and concluded that liation may ameliorate the ECO2 effects on tissue N. gross and net N mineralization were not affected by Defoliation can improve tissue N through remobiliza- ECO2, but that gross N immobilization increased by tion of N resources from roots and crowns and ⁄ or 22%. In a recent report from our same growth physiological signals to roots that temporarily improve chamber study, we showed increased N immobiliza- root N uptake (Skinner et al., 1999). Leaf tissue N may tion into the soil under elevated CO2, which may also be higher owing to younger leaves after defoliation further have reduced %N in both C3 and C4 plants (Milchunas et al., 1995). 15N recovery was reduced with (Dijkstra et al., 2011). defoliation in roots and C3 grasses, suggesting that N From our study, it appears that ‘grazing’ is likely to uptake from the soil did not improve with defoliation have as much impact on some grassland ecosystem and implying that improved shoot tissue %N was functions as increasing atmospheric CO2. Defoliation primarily from N remobilization after defoliation. consistently increased shoot %N but reduced below- 15 CO2 by N interactions on shoot %N also only ground biomass. Also, defoliation reduced N recovery occurred in C3 grasses (although a trend was seen for by roots without improving recovery in shoots. Of C4 grasses: P =0Æ077). Reduced %N in plant tissues is a course our experiment was short-term and long-term common response to elevated CO2 (Morgan et al., speculation should be made cautiously. 2001b; Reich et al., 2001; King et al., 2004), particularly In this semi-arid and typically grazed ecosystem, under limited soil-N availability and especially in C3 plant processes are very much driven by soil water plants, but we observed that ECO2 reduced %N in C3 availability. Therefore, it is important to note that there plants only in the +N treatment (Tables 1 and 2), which were no apparent interactions between CO2 and defo- was unexpected. Forb %N was not reduced by ECO2, liation treatments on VWC. Overall, defoliation had which was also unexpected. Our results differ from little effect on VWC (Figure 2). Elevated CO2 improved those of a Minnesota prairie FACE study, where leaf VWC an average of 27% over the growing season,

Nmass was strongly reduced in forbs but not in C3 grasses while N addition reduced VWC by an average of 17%. (Crous et al., 2010). The forb response was attributed to Elevated CO2 increased biomass (in C3 grasses) while lesser roots and consequent nutrient foraging. It seems reducing water use; therefore, WUE was improved clear that comparisons between studies based on func- (23%). In the field, we expect water savings owing to tional groups may be too simplistic (Suding et al., 2005). ECO2 to significantly impact plant growth and nutrient C4 grasses did not have a significant growth response to cycling, primarily during below-normal precipitation ECO2, but improved growth in the C3 grasses likely years (Morgan et al., 2004b).

2012 Blackwell Publishing Ltd. Grass and Forage Science, 67, 350–360 358 D. R. LeCain et al.

Conclusions project was funding by the United States Department of Agriculture-Agricultural Research Service. From our study, we conclude that in mixed-grass prairie ecosystems of the world, there will be little interaction References between appropriate grazing practices and increasing A V. N P.C.D. L M. S atmospheric CO2 on many important ecosystem pro- LLARD , EWTON , IEFFERING , OUSSANA cesses. However, over the long-term, increasing atmo- J.F., CARRAN R.A. and MATTHEW C. (2005) Increased quantity and quality of coarse soil organic matter fraction spheric CO2 will cause changes in ecosystem functions. Our results support other reports that show a shift in at elevated CO2 in a grazed grassland are a consequence of enhanced root growth rate and turnover. Plant and species composition to more C grass dominance as 3 Soil, 276, 49–60. atmospheric CO increases (Korner, 2000; Reich et al., 2 AUGUSTINE D.J., DIJKSTRA F.A., WILLIAM HAMILTON E.S. 2001; Polley , 2003; Morgan , 2004a, 2007; et al. et al. and MORGAN J.A. (2010) Rhizosphere interactions, Crous et al., 2010). Increased competition with C4 carbon allocation, and nitrogen acquisition of Bouteloua grasses would have negative consequences on avail- gracilis and Pascopyrum smithii in response to defoliation ability of forage late in the growing season when C3 and elevated atmospheric CO2. Oecologia, 165, 755–770. grasses are dormant. We also show limited evidence BURKE I.C., LAUENROTH W.K. and PARTON W.J. (1997) that forbs will be out-competed in this grassland under Regional and temporal variation in net primary global change. The resulting reduced plant diversity will production and nitrogen mineralization in grasslands. reduce ecosystem adaptability to global changes (Reich Ecology, 78, 1330–1340. CAMPBELL B.D. and STAFFORD SMITH D.M. (2000) A et al., 2004), suggesting the potential for a - decline synthesis of recent global change research on pasture and in the health and adaptability of mixed-species grass- rangeland production: reduced uncertainties and their lands around the world. Improved available soil mois- management implications. Agriculture, Ecosystems & ture under elevated CO2 should increase plant Environment, 82, 39–55. productivity, but only near a critically low soil moisture CAMPBELL B.D., STAFFORD SMITH D.M. and MCKEON threshold. Morgan et al. (2011) reported that water G.M. (1997) Elevated CO2 and water supply interactions conservation under ECO2 can compensate for desicca- in grasslands: a pastures and rangelands management tion under global warming and that combined ECO2 perspective. Global Change Biology, 3, 177–187. and warming might favour some C grasses. Therefore, CROUS K.Y., REICH P.B., HUNTER M.D. and ELLSWORTH 4 D.S. ultimate species composition will depend on complex (2010) Maintenance of leaf N controls the photosynthetic CO response of grassland species interactions of multiple biotic and abiotic factors. 2 exposed to 9 years of free-air CO enrichment. Global Elevated CO quite consistently reduced forage ‘quality’ 2 2 Change Biology, 16, 2076–2088. in this study. As it is impractical to add fertilizer to most DE GRAAFF M., GROENIGEN J., SIX J., HUNGATE B. and rangelands, changes in grazing management strategies VAN KESSEL C. (2006) Interactions between plant or supplemental feed may eventually be needed to cope growth and soil nutrient cycling under elevated CO2:a with reduced forage quality and changing species meta-analysis. Global Change Biology, 12, 2077–2091. composition. DIJKSTRA F.A. and CHENG W. (2008) Increased soil This ecosystem has a long evolutionary history of moisture content increases plant N uptake and the 15 grazing by large mammals and has been proven to be abundance of N in plant biomass. Plant and Soil, 302, well adapted to grazing (Milchunas and Lauenroth, 263–271. DIJKSTRA F.A., HUTCHINSON G.L., REEDER J.D., LE CAIN 1993). This and other rangeland ecosystems are less D.R. and MORGAN J.A. (2011) Elevated CO , not well adapted to global change induced perturbations 2 defoliation, enhances N cycling and increases short-term on ecosystem processes. Understanding and planning soil N immobilization regardless of N addition in a for these perturbations remains a challenge for scien- semiarid grassland. Soil Biology and Biochemistry, 43, tists and land managers. Not surprisingly, plant and 2247–2256. ecosystem responses to atmospheric CO2, defoliation GRUNZWEIG J. and KORNER C. 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