Title: Physiological and Growth Responses of C 3 and C 4 Plants to Reduced Temperature When Grown at Low CO 2 of the Last Ice Age

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1 Title: Physiological and Growth Responses of C 3 and C 4 Plants to Reduced Temperature When Grown at Low CO 2 of the Last Ice Age Running Title: C 3 and C 4 Responses to Reduced Temperature at Low CO 2 Authors: Joy K. Ward 1 *, David A. Myers 2, and Richard B. Thomas 2 1 Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA; 2 Department of Biology, West Virginia University, Morgantown, WV 26506, USA *Author for Correspondence. Tel: Fax: joyward@ku.edu Supported by the U.S. Department of Energy (DE-FG02-95ER62124), the U.S. National Science Foundation ( and ), and an American Fellowship to J.K.W. from the American Association of University Women Educational Foundation. 1

2 Abstract During the last ice age, atmospheric [CO 2 ] was ppm compared with the modern value of 380 ppm, and temperatures were ~8 C cooler. Relatively little is known about the responses of C 3 and C 4 species to long-term exposure to glacial conditions. Here Abutilon theophrasti (C 3 ) and Amaranthus retroflexus (C 4 ) were grown at 200 ppm CO 2 with current (30/24 C) and glacial (22/16 C) temperatures for 22 d. Overall, the C 4 species exhibited a large growth advantage over the C 3 species at low [CO 2 ]. However, this advantage was reduced at low temperature, where the C 4 species produced 5X the total mass of the C 3 species versus 14X at the high temperature. This difference was due to a reduction in C 4 growth at the low temperature, since the C 3 species exhibited similar growth between temperatures. Physiological differences between temperatures were not detected for either species, although photorespiration/net photosynthesis was reduced in the C 3 species grown at low temperature, suggesting evidence of improved carbon balance at this treatment. This system suggests that C 4 species exhibited a growth advantage over C 3 species during low [CO 2 ] of the last ice age, although concurrent reductions in temperatures may have reduced this advantage. (198 words) Keywords: Abutilon theophrasti; Amaranthus retroflexus; C 3 species; C 4 species; climate change; low CO 2 ; low temperature; photorespiration; Pleistocene 2

3 Studying plant responses to global changes of the past provides valuable insights for predicting future responses to a rapidly changing environment (Ward et al. 2005; Edwards et al. 2007; Jackson 2007). In addition, studies involving treatments that simulate past climates provide a baseline for understanding the physiological functioning of plants prior to anthropogenic influences (Polley et al. 1993a,b; Anderson et al. 2001; Sage and Coleman 2001; Polley et al. 2002; Ward 2005). Atmospheric CO 2 concentration ([CO 2 ]) reached minimum values of 180 ppm during the last ice age, rose to 270 ppm during the recent interglacial period (and just prior to the onset of the Industrial Revolution), and increased to 380 ppm in the modern atmosphere as a result of fossil fuel combustion and deforestation (EPICA 2004). Low [CO 2 ] that occurred during the last ice age is predicted to have produced carbon limitations within C 3 plants, and may have reduced their distribution relative to C 4 species (Polley et al. 1993a; Dippery et al. 1995; Tissue et al. 1995; Ward and Strain 1999; Koch et al. 2004; Vidic and Montanez 2004; Ward et al. 2005; Huang et al. 2006). Over geologic time scales, periods of minimum [CO 2 ] during glacial periods correlate closely with reduced temperatures (~8 ºC reduction relative to modern times on a global average; Petit et al. 1999; Sigman and Boyle 2000; but also see Cowling 1999). Knowledge of the effects of reduced temperature on C 3 and C 4 plants grown at low [CO 2 ] is critical for understanding changes in plant competitive interactions and abundance during the last ice age. C 3 plants are not favored at low [CO 2 ] as a result of increased oxygenase activity of rubisco (ribulose-1,5- bisphosphate carboxylase-oxygenase) and reduced carboxylation activity in response to limiting CO 2 substrate (Pearcy et al. 1987; Tissue et al. 1995; Sage and Cowling 1999; Sage and Coleman 2001). C 4 species, on the other hand, concentrate CO 2 in bundle-sheath cells, and therefore are much less negatively affected by reductions in atmospheric [CO 2 ] (Dippery et al. 1995; Sage and Coleman 2001; Ward 2005). With regard to temperature, C 3 photosynthesis is positively affected by reductions in temperature because oxygenase activity is reduced relative to carboxylation activity, increasing quantum yield (ratio of CO 2 molecules fixed to photons of light absorbed). In contrast, the quantum yield of C 4 photosynthesis is independent of temperature due to the absence of temperature-dependent photorespiration (Ehleringer and Pearcy 1983). Furthermore, low temperatures (especially below 20 ºC) often reduce the photosynthetic rates of C 4 species (Long 1983; Kubien et al. 2003), and low temperatures have been shown to make C 4 plants more susceptible to damage from photoinhibition (Fryer et al. 1995). 3

4 By combining the factors of CO 2 and temperature, Ehleringer et al. (1997) modeled the climate conditions under which C 3 and C 4 plants were favored based on quantum yield. The authors found that at modern [CO 2 ] (~380 ppm) the crossover temperature where C 4 photosynthesis becomes favored over C 3 photosynthesis occurs at C; whereas at ice age [CO 2 ] (180 ppm, 18,000 to 20,000 years ago), the crossover temperature occurs at approximately 15 C. This quantum-yield model has been highly predictive of modern vegetation distributions and clearly indicates that low [CO 2 ] would have favored C 4 species during the last ice age across a wide range of regional temperatures (assuming sufficient moisture was present). The model also predicts that reduced temperatures that occurred in conjunction with low [CO 2 ] may have increased the carbon gain of C 3 species, although this likely did not compensate for the pronounced negative effects of low [CO 2 ]. In addition to physiological models, empirical studies examining the growth and development of C 3 and C 4 species at reduced temperatures and low [CO 2 ] are also needed to better understand the functioning of ice age vegetation (Strain 1991; Sage and Coleman 2001; Ward 2005). Thus, the objective of this study was to determine the physiological and growth responses of co-occurring dicot annuals, Amaranthus retroflexus (C 4 ; hereafter Amaranthus) and Abutilon theophrasti (C 3 ; hereafter Abutilon) to reduced ice age temperatures (-8 C) during growth at low [CO 2 ] (200 ppm) that occurred during the last ice age. Past work has already focused on the responses of these species to current and elevated [CO 2 ] with temperature interactions (see Ackerly et al. 1992; Coleman and Bazzaz 1992; Dippery et al. 1995, Tissue et al. 1995; Ward et al. 1999; Sage and Kubien 2007), and therefore here the focus is on temperature effects when all plants are grown at low [CO 2 ]. We hypothesized that low temperature would enhance the performance of the C 3 species relative to the C 4 species, but that the low [CO 2 ] growth conditions would produce higher performance in the C 4 species overall. Results On an absolute basis, the C 3 (Abutilon theophrasti) and C 4 species (Amaranthus retroflexus) exhibited large differences in total mass when grown at low [CO 2 ] (200 ppm) for 22 d. More specifically, the C 4 species had 5 times the total mass of the C 3 species when grown at the low temperature (22 light/16 dark C), and almost 14 times the total mass of the C 3 species when grown at the high temperature (30/24 C; Table 1). In this case, all plants were grown from seed 4

5 for a total of 22 d, and therefore differences in final biomass reflect differences in relative growth rate (change in biomass per unit biomass per unit time; hereafter RGR). Therefore, the RGR of the C 4 species greatly exceeded that of the C 3 species at low [CO 2 ]. In addition, Amaranthus has inherently small seeds compared with Abutilon, and therefore initial seed size would not have been a factor in providing a relative growth advantage of the C 4 species over the C 3 species when grown at low [CO 2 ]. Furthermore, the C 3 and C 4 species exhibited relative differences in their responses to temperature for total mass and LA (leaf area) (significant species X temperature interactions, P=0.0001, Table 1). The C 3 species had similar total mass and LA at both the high and low temperatures, whereas the C 4 species showed a 65% reduction in total mass and a 55% reduction in LA at the low temperature relative to the high temperature. During growth at low [CO 2 ], the C 4 species allocated proportionally more biomass to roots versus shoots in both temperatures compared with the C 3 species (Table 2). In addition, the C 3 species allocated proportionally more biomass to roots (versus shoots) when grown at the low temperature compared with the high temperature, whereas the C 4 species was unresponsive to temperature for allocation of root mass versus shoot mass (Table 2). Furthermore, the C 3 species had higher LA versus total mass at the high temperature compared with the low temperature. In contrast, the C 4 species had lower LA area versus total mass at the high temperature (Table 2). Overall, g s (stomatal conductance) was higher and SLM (specific leaf mass) was lower in the C 3 species compared to the C 4 species during growth at low [CO 2 ] (Table 1). In response to temperature treatments, the C 3 and C 4 species exhibited similar relative responses for g s (nonsignificant species X temperature interaction at P=0.64), whereby both species were unresponsive to temperature. For SLM, the C 3 and C 4 species exhibited different relative responses (significant species X temperature interaction at P=0.0001), with the C 3 species exhibiting similar SLM at both temperatures, and the C 4 species showing a 20% reduction in SLM between the high and low temperatures. The P r (photorespiration) of the C 4 species was assumed to be negligible because there were no statistical differences between values of A (net photosynthesis) measured at 2% versus 21% O 2 at either temperature treatment (P=0.68 for high temperature, P=0.45 for low temperature; data not shown) when measured at 180 ppm CO 2 and 1400 µmol photons m -2 s -1 PAR. Therefore, P r was only determined for the C 3 species according to the method of Valentini et al. (1995). The slopes of the relationship between photochemical efficiency of PSII 5

6 (photosystem II) and apparent quantum yield of CO 2 assimilation (Φ CO2 ) for the C 3 species were 9.42 (r 2 = 0.83) and (r 2 = 0.93) for the high and low treatments, respectively (data not shown). Overall, the C 3 species exhibited lower rates of A (by 38-45%) than the C 4 species (Fig. 1a) when grown at low CO 2. Within species, reduced temperature did not have a significant effect on physiological responses (A, P r, R (respiration); Fig. 1a,b,c), and there were no significant species X temperature interactions for any of these physiological measurements (P=0.46 for A, P=0.89 for R). Lower A in the C 3 species versus the C 4 species was a result of the effects of P r that reduced net carbon assimilation (Fig. 1a,b), and was not due to R that was similar between both species at both temperature treatments (Fig. 1c). Although a temperature effect was not detected for individual physiological measurements in low [CO 2 ]-grown plants, the ratio of P r /A (measured simultaneously on the same leaf area) for the C 3 species decreased at the low temperature versus the high temperature (Fig. 1b, insert). Furthermore, R/A was similar between temperature treatments within the C 3 (warm = 0.15, cold = 0.15) and within the C 4 species (warm = 0.08, cold = 0.10, data not shown). Discussion This study involved growing C 3 and C 4 plants at low [CO 2 ] (200 ppm) with an 8 C difference in temperature treatments (22/16 C vs. 30/24 C) in order to simulate the average global reduction in temperature that occurred during the last ice age (Petit et al. 1999). Regardless of temperature, the C 4 species (Amaranthus retroflexus) exhibited a large advantage in absolute growth over the C 3 species (Abutilon theophrasti) at low [CO 2 ]. The C 4 species had 5 times the total mass of the C 3 species when grown at the low temperature and almost 14 times the total mass of the C 3 species at the high temperature. In our past work, where the same C 3 and C 4 species were grown at [CO 2 ] near modern values (350 ppm), the C 4 species only exhibited three times the biomass of the C 3 species at 28/22 C (Dippery et al. 1995). Furthermore, in the present study, the C 3 species showed signs of carbon limitations with higher allocation of biomass to shoots versus roots (that enhances total carbon assimilation) and lower photosynthetic rates relative to the C 4 species. Taken together, these results, along with those of other studies (Polley et al. 1993a,b; Sage 1995; Dippery et al. 1995; Ward and Strain 1997; Ward et al. 1999; Ward et al. 2005; Sage and Kubien 6

7 2007), point to the dominant effect of low [CO 2 ] in determining a growth advantage of C 4 over C 3 species during the last ice age. As hypothesized, the relative growth advantage of the C 4 species was reduced at the low temperature treatment in this study. This occurred because the C 4 species exhibited a reduction in growth at low temperature, whereas the C 3 species was unaffected by temperature. This varies from the results of Cowling and Sage (1998) with C 3 Phaseolus vulgaris grown at 200 ppm CO 2, where plants showed a 70% increase in growth between 36 C and 25 C; it is important to note, however, that the Phaseolus study involved a more extreme temperature range and a higher maximum temperature compared with the present study. It was not surprising that the C 4 species exhibited a reduction in growth at the low temperature treatment, although a 65% reduction was greater than anticipated based on physiological predictions of C 4 plants grown at low temperature (Sage and Kubien 2007). This finding suggests that reductions in temperature during the last ice age, when combined with the effects of low [CO 2 ], may have greatly reduced the growth of some C 4 species, and may have limited their distribution. For example, C 4 species were not detected in southern California, a relatively warm region, between 12,000 and 28,000 yr BP, based on carbon isotope measurements of mammal bone collagen that can be used to assess diet (Coltrain et al. 2004). This may have been a result of reduced temperatures during the last ice age that limited the competitive ability of C 4 species in this region. In modern times (when [CO 2 ] has ranged between 270 and 380 ppm), C 4 plants generally occupy regions with warm climates, and occur at low altitudes, and may dominant ecosystems during seasonal peaks in temperature (Rundel 1980; Kemp and Williams 1980; Pearcy et al. 1981), although exceptions have been described (Long 1999). This distribution pattern has been primarily attributed to higher net photosynthesis and quantum yields of C 4 species at warm temperatures relative to C 3 species. In addition, the light-saturated rate of A (net photosynthesis) declines sharply in many C 4 species as temperatures decreases below 20 C (Long 1983). Furthermore, Pitterman and Sage (2000) demonstrated that reduced rubisco activity may limit photosynthesis in C 4 plants grown at low temperatures (<17 C), and Kubien et al. (2003) showed that low rubisco content may limit C 4 photosynthesis at a large range of sub-optimal temperatures (using Flaveria bidentis with an antisense construct for the small subunit of rubisco; see also Sage 2002). C 4 plants may also be more susceptible to photoinhibition at low 7

8 temperatures because the reaction centers of photosystem II may be damaged and the efficiency of energy transfer to these reaction centers may be diminished (Fryer et al. 1995). Despite the findings of these past studies, the lower total mass of the C 4 species grown at the low temperature could not be attributed to any of the physiological measurements in the present study. More specifically, the C 4 species exhibited similar A, R, and g s in response to an 8 C temperature difference when grown at 200 ppm CO 2. It has been shown that A increases sharply between 20 and 36 C in the majority of C 4 species when measured at current and elevated [CO 2 ] (Ghannoum et al. 2000). For example, Sage (2002) showed that the same C 4 species, Amaranthus retroflexus, exhibited large increases in photosynthetic rate between 22 and 30 C (by approximately 25%) at 360 ppm CO 2. However, in the same study, Amaranthus showed very modest increases in photosynthetic rate across these same temperatures when measured at 180 ppm, and did not show any differences in photosynthetic rate at 100 ppm CO 2. Thus, as observed in the present study and in others, modern C 4 plants may be relatively temperature insensitive for photosynthesis when grown at low [CO 2 ] representing the last ice age. This response is driven by the inherent lack of temperature-sensitive oxygenase activity of rubisco (shown to be the case here with no change in A between 2 and 21% O 2 ) that differs from C 3 species, and is further driven by the insensitivity of PEP carboxylase (phosphoenolpyruvate carboxylase) to temperature at low [CO 2 ] conditions that is specific to C 4 species (Sage and Kubien 2007). In the C 4 species grown at low [CO 2 ], partitioning of biomass between roots and shoots was unaffected by temperature when differences in overall plant size were removed (with the linear analysis employed). However, the C 4 species grown at low temperature had greater LA per total mass than plants grown at the high temperature. Despite this growth adjustment (with no change in A per unit leaf area), the C 4 species still produced lower biomass as a result of the stressful effects of low temperature. This result illustrates the importance of considering developmental and growth mechanisms, in addition to physiological responses, when evaluating C 3 and C 4 responses to low [CO 2 ] and temperature of the past. When grown at low [CO 2 ], the C 3 species did not exhibit differences in physiology (A, P r, R, and g s ) or in total mass, total leaf area, or SLM in response to the 8 C difference in temperature treatments. Past studies have indicated that the sensitivity of C 3 photosynthesis to temperature declines as plants become limited by CO 2, much like the patterns exhibited by the C 4 8

9 species (Berry and Björkman 1980; Pearcy and Ehleringer 1984; Sage 2002). In this study, the C 3 plants grown at 200 ppm CO 2 were highly limited by CO 2 availability, and for this reason, they did not exhibit differences in A between temperature treatments. In addition, R of the C 3 species was also unaffected by temperature in the present study. This result varies from the findings of Cowling and Sage (1998) with Phaseolus vulgaris (C 3 ) that exhibited a 68% reduction in R between 36 and 25 C when grown at 200 ppm CO 2. Furthermore, several studies have shown that C 3 species are often conservative in the ratio of R versus A after long-term exposure to increased temperature at current and elevated [CO 2 ] (Dewar et al. 1999), and the present study provides further evidence for this potential acclimation response at low [CO 2 ] as well. Although it did not influence total biomass production, the C 3 species did exhibit differences in the partitioning of biomass in response to temperature, whereby plants grown at the low temperature allocated significantly more biomass to roots versus shoots and had lower LA versus total mass compared with plants grown at the high temperature. This higher investment in root components may have been a response to the early stages of improved carbon balance in the leaves of low temperature-grown plants at low [CO 2 ]. This was evidenced by a reduction in P r versus A from simultaneous measurements on the same leaf area conducted at low temperature. This result suggests that the carbon balance of the C 3 species was beginning to be positively affected by the ice age temperature treatment, but this response was not substantial enough to increase total biomass production between temperature treatments. In summary, this study with a C 3 and C 4 dicot suggests that low [CO 2 ] that occurred during glacial periods was likely a dominant factor that produced higher productivity of C 4 species over C 3 species. However, reduced temperatures in combination with low [CO 2 ] may have reduced the growth advantage of C 4 species in some climates in the past. In addition, this work provides the results of plant growth responses that support previous studies indicating dominance of C 4 species within ancient ecosystems during periods of low [CO 2 ], particularly in relatively warm regions (Street-Perrott et al. 1997, Cerling et al. 1997, Cerling et al. 1998). Furthermore, the results of this study indicate that a combination of both leaf-level physiological studies and growth studies are necessary for better understanding the mechanisms that determined the distribution of C 3 and C 4 species within ancient ecosystems. 9

10 Materials and Methods Growth conditions Amaranthus (C 4 dicot) and Abutilon (C 3 dicot) that originated from old-field populations in Illinois, USA were germinated and grown in monoculture in four growth chambers at the Duke University Phytotron. Plants were grown in a 3:3:1 (v/v) medium of gravel, "Turface" and sterilized topsoil in deep 3.5-L pots that did not restrict root development during the 22 d growth period (Thomas and Strain 1991). Prior to emergence, pots were watered to saturation with deionized water twice each day. Following emergence, pots were watered to saturation with halfstrength Hoagland's solution (Downs and Hellmers 1978) each morning and with de-ionized water each afternoon. Seedlings were thinned to one individual closest to the center of each pot at 5 d after planting (both species emerged at 2 d after planting). Two growth chambers were controlled at day/night temperatures with target values of 30/24 C (Actual: 30.0 ± 0.9 SD/24.0 ± 0.3 C and 30.0 ± 0.4/24.0 ± 0.2 C), and two other chambers were controlled at target values of 22/16 C (Actual: 22.0 ± 0.4/16 ± 0.1 C and 22.0 ± 0.5/16 ± 0.2 C) to simulate current and glacial temperatures, respectively (Petit et al. 1999). [CO 2 ] within all growth chambers was maintained at 200 ± 10 ppm ([CO 2 ] increased for 0.5 h per day while plants were being watered and this time period was not included in the calculation) by passing incoming air over a moist soda lime/vermiculite mixture to scrub CO 2. Light/dark periods were 14 h/10 h and the light level during the day was maintained at 1000 ± 50 µmol photons m -2 s -1 PAR at plant height using metal halide and tungsten/halogen bulbs. Each dark period was interrupted for 1 h with incandescent lighting at 50 µmol photons m -2 s -1 to prevent early initiation of flowering. Growth measurements Amaranthus and Abutilon from both temperature treatments were harvested at 15 d and 22 d after emergence (n = 5-7 plants per chamber per harvest). At each harvest, total leaf area (LA) was measured with a LI-3100 leaf area meter (Li-Cor, Lincoln, Nebraska). Plant material was separated into roots, stems, and leaves and was oven dried (60 C) for 48 h before mass was measured. Specific leaf mass (SLM) was calculated as total leaf mass divided by the total leaf area of individual plants. Partitioning of biomass was determined by comparing linear 10

11 relationships between root versus shoot mass and leaf area versus total mass by inclusion of data from both harvests (see statistical analyses). Physiological Measurements Gas exchange parameters including stomatal conductance (g s ), net photosynthesis (A), and dark respiration (R) were measured at steady state conditions with an open system using a LI-6400 portable photosynthesis system (LI-Cor, Lincoln, Nebraska) at 20 d after planting. Conditions within the leaf cuvette during measurements of g s and A were the same as those within growth chambers during the light period. R was measured under dark conditions with all other conditions in the leaf cuvette being similar to the light period (including temperature). P r of the C 3 species (Abutilon) was measured according to the method of Valentini et al. (1995) at growth CO 2 and temperature conditions and with the following modifications. Gas exchange and chlorophyll fluorescence were measured simultaneously from the most recently mature leaves of Abutilon using the LI-6400 system and a pulse modulated fluorometer (PAM- 2000, Walz, Germany) at each of ten steps of a light response curve. Incoming air to the leaf chamber was delivered from bottled 2% O 2 in balanced nitrogen to provide non-photorespiratory conditions. The incoming air was humidified by bubbling the air stream through distilled water to saturation and then was scrubbed with drierite (W. A. Hammond Drierite Co., Xenia, Ohio) to match growth chamber humidity. Light levels for the response curves were attenuated with fine wire screens in ten steps ranging from a maximum of 1500 µmol photons m -2 s -1 PAR to a light level just below the light compensation point of each measured plant. For each light level, gas exchange and fluorescence parameters were not recorded until CO 2, H 2 O, and flow rate (sample infrared gas analyzer reference infrared gas analyzer) reached a total coefficient of variation of less than 1% and stomatal conductance was stable. The ratio of P r to A of the C 3 species was calculated from simultaneous measurements of these two parameters on the same leaf area. Statistical analyses Analyses of variance (ANOVAs) were conducted on data from individual plants at the second harvest (22 d after emergence) for measurements of total mass, LA, g s, SLM, A, and R. Data were tested for normality and log e transformed when necessary. The main effects of the analyses 11

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17 Figure legends Figure 1. (a) Net photosynthetic rate (A, n=9-12), (b) photorespiration rate (P r, n=5-6), and (c) dark respiration rate (R, n=9-12) of Abutilon theophrasti (C 3 ) and Amaranthus retroflexus (C 4 ) grown at 200 ppm CO 2 for 20 d at either 30/24 C (light/dark, modern) or 22/16 C (glacial). The insert in Figure 1b shows the ratio of photorespiration rate to net photosynthetic rate (P r /A) of the C 3 species. Measurements were made at growth conditions and temperature during the light period. This graph combines data from different chambers of the same treatment because no significant chamber effect was detected. Different letters between temperatures and species indicate significant differences at the P < 0.05 level according to ANOVA. 17

18 Tables Table 1. The effects of modern and glacial temperatures on total mass (n=13-14), leaf area (LA, n=13-14), stomatal conductance (g s, n=9-12), and specific leaf mass (SLM, n=13-14) for Abutilon theophrasti (C 3 ) and Amaranthus retroflexus (C 4 ) grown at low CO 2 (200 ppm) for 22 d. Values are means ± 1 standard error. Data from different chambers within the same treatment were combined because a chamber effect was not detected. Different superscript letters between temperatures and species indicate significant differences at the P < 0.05 level according to ANOVA. Temperature treatment Total mass (g) LA (cm 2 ) g s (mol m -2 s -1 ) SLM (g/m 2 ) Abutilon theophrasti (C 3 ) 30 / 24 C (modern) 0.23 (0.04) c 49 (8) c 1.4 (0.1) a 32 (2) c 22 / 16 C (glacial) 0.22 (0.03) c 39 (5) c 1.2 (0.1) a 34.9 (0.5) c Amaranthus retroflexus (C 4 ) 30 / 24 C (modern) 3.1 (0.2) a 262 (13) a 0.52 (0.07) b 57 (2) a 22 / 16 C (glacial) 1.1 (0.1) b 119 (11) b 0.43 (0.03) b 46 (1) b 18

19 Table 2. Slopes and r 2 values for the regressions of root mass (y) versus shoot mass (x) and leaf area (y) versus total mass (x) for Abutilon theophrasti (C 3 ) and Amaranthus retroflexus (C 4 ) grown at 200 ppm CO 2 for 22 d at modern and glacial temperatures. Different letters within a species and regression type indicate significant differences at the P < 0.05 level according to ANCOVA (n=24). Temperature treatment Root mass (g) vs. shoot mass (g) Leaf area (cm 2 ) vs. total mass (g) Slope r 2 Slope r 2 Abutilon theophrasti (C 3 ) 30 / 24 C (modern) 0.26 b a / 16 C (glacial) 0.41 a b 0.98 Amaranthus retroflexus (C 4 ) 30 / 24 C (modern) 0.74 a b / 16 C (glacial) 0.79 a a

20 Growth at 200 ppm Modern 30 C Glacial 22 C P r / A 30 C 22 C 20