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698 New Phytologist (2009) 182: 698–709 © The Authors (2009)698 www.newphytologist.org Journal compilation © New Phytologist (2009)

Blackwell Publishing LtdOxford, UKNPHNew Phytologist0028-646X1469-8137© The Authors (2009). Journal compilation © New Phytologist (2009)278610.1111/j.1469-8137.2009.02786.xFebruary 200900698???709???Original ArticleXX XX

Does leaf photosynthesis adapt to CO2-enriched environments? An experiment on plants originating from three natural CO2 springs

Yusuke Onoda1,3, Tadaki Hirose1,2 and Kouki Hikosaka1

1Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan; 2Department of International Agriculture Development, Tokyo

University of Agriculture, Sakuragaoka 1-1-1, Setagaya, Tokyo 156-8502, Japan; 3Present address: Department of Biological Sciences, Macquarie University,

Sydney, NSW 2109, Australia

Summary

• Atmospheric CO2 elevation may act as a selective agent, which consequently mayalter plant traits in the future. We investigated the adaptation to high CO2 usingtransplant experiments with plants originating from natural CO2 springs and fromrespective control sites.• We tested three hypotheses for adaptation to high-CO2 conditions: a higherphotosynthetic nitrogen use efficiency (PNUE); a higher photosynthetic water useefficiency (WUE); and a higher capacity for carbohydrate transport from leaves.• Although elevated growth CO2 enhanced both PNUE and WUE, there was nogenotypic improvement in PNUE. However, some spring plants had a higher WUE,as a result of a significant reduction in stomatal conductance, and also a lower starchconcentration. Higher natural variation (assessed by the coefficient of variation)within populations in WUE and starch concentration, compared with PNUE, mightbe responsible for the observed population differentiation.• These results support the concept that atmospheric CO2 elevation can act as aselective agent on some plant traits in natural plant communities. Reduced stomatalconductance and reduced starch accumulation are highlighted for possible adaptationto high CO2.Abbreviations: CV, coefficient of variation; IPCC, Intergovernmental Panel onClimate Change; OTC, open-top chamber; PNUE, photosynthetic nitrogen useefficiency; RuBP, ribulose-1,5-bisphosphate; WUE, water use efficiency.

Author for correspondence:Yusuke OnodaTel: +61 2 98506270Email: yonoda@bio.mq.edu.au

Received: 12 January 2009Accepted: 16 January 2009

New Phytologist (2009) 182: 698–709 doi: 10.1111/j.1469-8137.2009.02786.x

Key words: acclimation, adaptation, nitrogen use efficiency, Rubisco, RuBP regeneration, natural selection, starch accumulation, water use efficiency.

Introduction

The atmospheric CO2 concentration has been lower than thecurrent level for the past 650 000 yr. Currently, it is c. 380 µmolmol−1 and is predicted to reach 700 µmol mol−1 by the end ofthis century [Intergovernmental Panel on Climate Change(IPCC), 2007]. As CO2 is a substrate for photosynthesis,the increase in CO2 concentration generally enhances thephotosynthesis and growth rates of plants. Many researchershave conducted elevated CO2 experiments using ‘currentplants’ to simulate plant growth in the future environment.The prediction based on these data is valid if ‘future plants’respond to elevated CO2 in the same manner as current plants.However, it has been repeatedly discussed that atmosphericCO2 concentration is likely to act as a selective agent (e.g.

Woodward et al., 1991; Thomas & Jasienski, 1996; Wardet al., 2000; Kohut, 2003; Ward & Kelly, 2004; Lau et al.,2007). The extent to which plant carbon assimilation isenhanced by elevated CO2 is significantly different bothwithin and between species (Wulff & Alexander, 1985;Poorter, 1993; Thomas & Jasienski, 1996; Ward & Strain,1997; Klus et al., 2001; Körner et al., 2005). Different growthenhancements influence competition and fitness betweenindividuals and, consequently, cause evolutionary shifts inpopulations (Farnsworth & Bazzaz, 1995; Thomas & Jasienski,1996). Such evolutionary responses have been found in selectionexperiments with short-lived organisms, such as Arabidopsisthaliana (e.g. development rate and biomass production;Ward et al., 2000) and Chlamydomonas reinhardtii (e.g.photosynthesis and cell size; Collins & Bell, 2004). However,

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the evolutionary response of wild plants (especially long-lived plants) is, in general, difficult to evaluate using growthexperiments, because of the fundamental discrepanciesbetween the time scales of experiments and the time scalesrelevant in an evolutionary context (Fordham et al., 1997).

Genetic differentiation in plant communities occursacross natural environmental gradients, such as light, water,temperature, nutrients and heavy metals (Linhart & Grant,1996). Such differentiation can be found even on a small scale(a few metres), although gene flow between plant communitiessomewhat counteracts genetic differentiation (Linhart & Grant,1996; Kelly et al., 2003; Jump & Peñuelas, 2005). By thesame token, plant communities around natural CO2 springsprovide us with a unique opportunity to explore the micro-evolutionary response of wild plants to elevated CO2 (e.g.Miglietta & Raschi, 1993; Körner & Miglietta, 1994; Raschiet al., 1997; Kohut, 2003). At these sites, plant communitieshave been exposed to a CO2-enriched atmosphere over manygenerations (e.g. Miglietta & Raschi, 1993; Onoda et al.,2007); therefore, some genotypes that have a higher fitness atelevated CO2 could have been selected around natural CO2springs (Cook et al., 1998). Several studies have found geno-typic differences in plants from CO2 springs: for example,plants originating from high-CO2 areas show a lower sugaraccumulation (Schulte et al., 2002), higher relative growthrate (Fordham et al., 1997), higher shoot/root ratio (Polleet al., 2001) and a large number of small-sized seeds (Andaloet al., 1999). These results suggest that some variation intraits can be selected under CO2 enrichment. However, eachof these studies used a single CO2 spring, and therefore it isnot clear whether such differences represent general trendsfor CO2 adaptation. Furthermore, there have been few studieson photosynthetic traits, which have potentially large influenceson whole plant growth and fitness. As photosynthesis andgrowth are limited by resource availability, the efficient use ofresources is important for higher growth and survival in thenatural environment. This study therefore employs three CO2springs and focuses on photosynthetic traits, particularly withrespect to nitrogen use, water use and starch accumulation.

The photosynthetic apparatus requires a large amount ofnitrogen, whose availability is limited in most natural ecosystems(Chapin, 1980). Efficient allocation of proteins in the apparatusis necessary to maximize photosynthetic rates (Evans &Seemann, 1989; Hikosaka & Terashima, 1995). Rubisco,which constitutes 10–40% of leaf nitrogen, limits photosynthesisat ambient CO2, whereas, at double ambient CO2, photosyn-thesis is often limited by ribulose-1,5-bisphosphate (RuBP)regeneration (Stitt, 1991). In the latter case, Rubisco nolonger limits photosynthetic rate, and its amount is in excess(Sage, 1994; Woodrow, 1994). Theoretical studies havesuggested that a large increase in the ratio of RuBP regenerationcapacity (Jmax) to RuBP carboxylation capacity (Vcmax) (i.e.selective reduction in Rubisco or reallocation of nitrogen fromRubisco to the RuBP regeneration process) will increase

carbon assimilation for a given amount of nitrogen in elevatedCO2 (Sage, 1994; Medlyn, 1996; Drake et al., 1997; Hikosaka& Hirose, 1998). This prediction is partly supported byevidence that transgenic plants with a reduced Rubisco contentachieve higher growth rates than wild types at elevated CO2(Makino et al., 2000). However, contrary to theoreticalpredictions, most current plants grown at elevated CO2 donot or only slightly increase the ratio of Jmax to Vcmax (e.g.Medlyn et al., 1999; Long et al., 2004). Therefore, it has beenargued that current plants are inefficient in nitrogen use atelevated CO2, and therefore they may have substantial roomfor improving photosynthetic nitrogen use efficiency (PNUE)(Sage & Coleman, 2001). If plants originating from naturalCO2 springs have adapted to elevated CO2, higher PNUEthrough higher ratios of Jmax/Vcmax would be expected.

As water is lost from leaves via the same pathway as CO2enters, CO2 assimilation is inevitably accompanied by waterloss (Mooney & Gulmon, 1979). Because water availability isnot infinite for plants, excessive use of water can lead to thedesiccation of soil and hence to plant death (Cowan, 1986).Elevated CO2 allows plants to save water via a reduction instomatal conductance as a result of an increased entry of CO2into leaves, which leads to a higher carbon assimilation perunit transpiration (water use efficiency, WUE) (Drake et al.,1997). It is therefore hypothesized that plants originatingfrom natural CO2 springs should show a reduction in stomatalconductance and a higher WUE. A reduction in stomatalconductance has been observed in plants growing at naturalCO2 springs in situ (Tognetti et al., 1996; Bettarini et al.,1998; Paoletti et al., 1998; Onoda et al., 2007). Part of thisreduction might be attributed to adaptation to elevatedCO2.

Increased photosynthetic rates with elevated CO2 enhancecarbohydrate production. This often leads to an overaccumu-lation of carbohydrate in the leaf (Curtis & Wang, 1998;Wand et al., 1999), causing negative effects on photosynthesisthrough the suppressed expression of genes related to photo-synthesis (e.g. Sheen, 1990; Webber et al., 1994; Moore et al.,1998), disruption of chloroplast structures (Cave et al., 1981)and obstacles to CO2 diffusion in chloroplasts (Stitt, 1991;Nakano et al., 2000). The excessive accumulation of carbohy-drates in leaves is caused by a lower sink strength comparedwith source strength (Stitt, 1991) or by lower carbohydratetransport capacity (Grimmer & Komor, 1999). The adaptationto elevated CO2 therefore should involve an increase in sinkstrength (e.g. higher root/shoot ratio or higher relativegrowth rate) or higher carbohydrate transport capacity, whichconsequently reduces carbohydrate accumulation and helpsto maintain high photosynthetic rates without the down-regulation of photosynthesis.

In this study, the adaptation of leaf photosynthesis toelevated CO2 was tested by a common garden experimentwith herbaceous species originating from three different naturalCO2 springs in Japan: Nibu, Ryuzin-numa and Yuno-kawa.

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Several genotypes were collected from each high-CO2 area(spring population) and nearby control areas (control popu-lation), and each genotype was propagated or divided into tworamets, and grown in pots at 370 and 700 µmol CO2 mol−1.We tested the following three possible adaptive scenarios toelevated CO2:• higher PNUE through increase in Jmax/Vcmax;• higher WUE with lower stomatal conductance;• lower carbohydrate accumulation which mitigates the down-regulation of photosynthesis.

Materials and Methods

Plant materials and growth conditions

We used one dominant herbaceous species from each of thethree natural CO2 springs: Plantago asiatica from Nibu (alsoknown as Nyuu), and Polygonum sachalinense from Ryuzin-numaand Yuno-kawa. These species were selected for two reasons:they are known to adapt to a wide range of environments withgenetic differentiation (i.e. cDNA and morphology; Sawadaet al., 1994; Inamura et al., 2000); they were dominant speciesand probably had been subject to intense competition, inwhich genotypes that grew better at elevated CO2 might havebeen selected.

All of these sites were located in National Parks and thevegetation in the surrounding area was generally well preserved.Nibu is located at 1000 m above sea level (asl) in YamagataPrefecture, Japan (38°32′N, 139°59′E), and Ryuzin-numaand Yuno-kawa are located at 570–575 m asl in AomoriPrefecture, Japan (40°41′N, 140°55′E, c. 300 km fromNibu). The distance between Ryuzin-numa and Yuno-kawa isc. 1 km. At these CO2 springs, the surrounding air has beenmaintained at high CO2 levels for more than 100 yr withouttoxic gas contaminations (H2S, SO2) (< 0.03 µmol mol−1 evenat the highest CO2 concentration spot). The areas in whichthe mean daily CO2 concentration was above 500 µmol mol−1

were approximately 600, 300 and 400 m2 at Nibu, Ryuzin-numa and Yuno-kawa, respectively. A detailed description ofthese CO2 springs is available in Onoda et al. (2007).

Plantago asiatica, a small perennial rosette (0.1–0.3 m), grewpredominantly along a path at Nibu where above-groundvegetation was mown annually. There were hundreds ofmature individuals with large numbers of seedlings (c. 5000plants m−2 in May 2003). Pollen dispersal has been reportedto be very limited (0.1–0.4 m) in a closely related species(Plantago major; Kuiper & Bos, 1992). Seeds are dispersed bygravity or by attachment to animals’ feet via the sticky seedcoat. About 20 genets of P. asiatica were collected at the high-CO2 area near the spring (abbreviated as ‘S’) and the controlarea (abbreviated as ‘C’) (40 m distant from the high-CO2area) on 20 May 2003. The two areas had a similar lightenvironment (S, 75–100% of full sun; C, 75–100% of fullsun) and soil pH (S, 3.9 ± 0.3; C, 4.2 ± 0.2). The mean

daytime CO2 concentrations determined by monthlymeasurements from July to October in 2003 were 750 ±199 µmol mol−1 (S) and 370 µmol mol−1 (C).

Polygonum sachalinense is a relatively tall stature (0.5–1.5 min our study site) perennial herb, and grew predominantly ina semi-open place (relative light intensity, 30–50% of full sun)at Ryuzin-numa. This species is a clonal plant, and a genetproduces many ramets. Some ramets have flowers in summer,which attract small insects (e.g. beetles, hoverflies and bees),and seeds are normally dispersed by wind (most seeds appearto drop within a few metres). In the high-CO2 area, there were> 500 shoots in which > 20 genets were included (each genetwas recognized by differences in the phenology in 2002).Eight pieces of rhizomes that had more than two buds werecollected from different genets in the high-CO2 area andnearby control areas (50–100 m distant from the high-CO2area) on 6 May 2003. The two areas had a similar lightenvironment (S, 30–50% of full sun; C, 30–60% of full sun)and soil pH (S, 3.6 ± 0.1; C, 3.1 ± 0.3). The mean daytimeCO2 concentrations evaluated by monthly measurements fromJune to October in 2002 were 670 ± 123 µmol mol−1 (S) and375 µmol mol−1 (C).

Polyponum sachalinense also grew predominantly at Yuno-kawa and was located under an oak–beech forest. There were> 20 genets and 400 shoots in the high-CO2 area. Eight piecesof rhizomes with more than two buds were collected from dif-ferent genets in the high-CO2 area and nearby control areas(500 m distant from the high-CO2 area) on 6 May 2003. Thelight environment was 2–20% of full sun for both areas, andthe soil pH values were 4.1 ± 0.4 (S) and 4.1 ± 0.4 (C). Themean daytime CO2 concentrations from monthly measure-ments from June to October in 2003 were 526 ± 58 µmolmol−1 (S) and 370 µmol mol−1 (C).

Plants originating from the CO2 springs (spring plants) andcontrol areas (control plants) were grown in the ExperimentalGarden of Tohoku University (38°15′N, 140°50′E). Eachgenet of P. asiatica was grown separately for 1 yr to produceseveral ramets. Two ramets from each genet were plantedseparately in two 1.5-l pots filled with washed river sand,and assigned to one of the two CO2 treatments [ambient(c. 370 µmol mol–1) and elevated (targeted at 700 µmol mol−1)CO2 concentrations] with four open-top chambers (OTCs;2 × 2 × 2 m3; two chambers for each CO2 treatment) on 12May 2004. The CO2 concentration in the chamber was 360–400 µmol mol−1 for the ambient treatment, and was regulatedto c. 700 µmol mol−1 for the elevated CO2 treatment (fordetails, including the OTC structure, see Nagashima et al.,2003). During the experiment, each pot received sufficientwater every day and 90 ml of nutrient solution (7 mM [N];Hyponex NPK = 6 : 10 : 5; M. Scott & Sons Co., Marysville,OH, USA) every week, and the positions of the pots wererandomized within each CO2 treatment every week.

Each rhizome piece of P. sachalinense was split into twosimilar-sized pieces, both of which had one bud, and was

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grown in peat moss in 1.5-l pots for 0.5 month. On 23 May 2003,these plants were transplanted to 4-l pots filled with washedriver sand, and one of each pair was assigned to elevatedCO2 treatment and the other to ambient CO2 treatment.These plants were grown in the same manner as for P. asiatica.

Gas exchange measurements

The CO2 dependence of the net CO2 assimilation rate wasdetermined on attached, fully expanded young leaves with anopen gas-exchange system (LI-6400, Li-Cor, Lincoln, NE,USA) on 16–18 August 2003 for P. sachalinense (c. 85 d aftertransfer to OTC) and on 8–10 July 2004 for P. asiatica(c. 60 d after transfer to OTC). During the measurements, theleaf temperature in the chamber was kept at 25°C, the vapourpressure deficit at < 1 kPa and the photon flux density at1500 µmol m−2 s−1 with an artificial light source (LI-6400-02B, Li-Cor). The measurements were performed on seven toeight pairs of genotypes (one ramet of each genet grown inambient CO2 treatment and another grown in elevated CO2treatment) for each origin (control and spring) and foreach site (Nibu, Ryuzin-numa and Yuno-kawa). The totalnumber of samples was therefore 7–8 replications × 2 growthCO2 × 2 origins × 3 sites.

Photosynthesis plotted against intercellular CO2 concentra-tion (A/Ci curve) was analysed to estimate the in vivo maximumrate of RuBP carboxylation (Vcmax) and the in vivo maximumrate of electron transport driving RuBP regeneration (Jmax).Vcmax was calculated by fitting the following equation (Farquharet al., 1980) to the initial slope of the A/Ci curves (Ci <300 µmol mol−1) using the least-squares method with KaleidaGraph™ (Synergy Software, Reading, PA, USA):

Eqn 1

(Ac, photosynthetic rate limited by Rubisco activity; Ci,intercellular CO2 concentration; Γ*, CO2 compensation pointin the absence of day respiration (Rd); Kc and Ko, Michaelis–Menten constants of Rubisco activity for CO2 and O2,respectively.) Γ*, Kc and Ko are assumed to be 42.8 µmol mol−1,405 µmol mol−1 and 278 mmol mol−1, respectively, accordingto Bernacchi et al. (2001). Rd was assumed to be 0.01 of Vcmax(von Caemmerer, 2000). Jmax was calculated by fitting thefollowing equation (Farquhar et al., 1980) to a near-plateau ofA/Ci curves (Ci > 600 µmol mol−1):

Eqn 2

(Aj, photosynthetic rate limited by RuBP regeneration.)

Biochemical measurements

All leaves used for photosynthesis measurements were sampledat around noon, and seven leaf discs of 1 cm in diameter were

punched out from each leaf. Three leaf discs per leaf wereweighed after drying in an oven at 75°C for more than3 d, and their total nitrogen content was determined withan NC analyser (NC-80, Shimadzu, Kyoto, Japan). Theother leaf discs were frozen in liquid nitrogen and storedat −80°C.

The starch content was determined according to Rufty &Huber (1983). Two leaf discs from each leaf were homogenizedin liquid nitrogen and suspended in 1.5 ml of 80% ethanol.The suspension was heated at 80°C for 30 min and centrifugedat 10 000 g for 10 min, and the supernatant was removed.This extraction procedure was repeated three times. The pelletwas dried in an oven for 1 d, and then suspended in 1.0 ml of0.2 M KOH and heated at 90°C for 30 min. After cooling,0.2 ml of 1.0 M acetic acid and 1.0 ml of amyloglucosidase(35 units ml−1 in sodium acetate buffer, pH 4.5; Rhizopusmould; Sigma, St Louis, MO, USA) were added, and incubatedat 55°C for 30 min. The suspension was centrifuged at10 000 g for 10 min and the supernatant was used for thedetermination of glucose using the glucose B test (Wako,Osaka, Japan).

Calculations and statistics

The WUE value was calculated as the photosynthetic ratedivided by the transpiration rate, which was measured atgrowth CO2 concentration (370 or 700 µmol mol−1). Similarly,PNUE was calculated as the photosynthetic rate at growthCO2 concentration (Agrowth) divided by the leaf nitrogenconcentration per unit area (Narea). For statistical analysis, amixed-model ANOVA (within and between subject factors)was used. As each genotype was grown at two different CO2concentrations (ambient and elevated CO2), the response tothese two CO2 concentrations can be regarded as repeatedmeasures. Therefore, growth CO2 was treated as a within-subject factor, and origins (spring and control populations)and sites (Nibu, Ryuzin-numa and Yuno-kawa) were treatedas between-subject factors. The effects of origin and site weretested by two-way ANOVA, which allowed us to use a singleANOVA for all data without losing statistical power. Forpractical reasons, the effect of site in our analysis also includesthe effect of species, as different species were used for Nibuand the other sites. Traits were log-transformed before analysisif equality of the covariance matrices was rejected (P < 0.05)with Box’s test. The coefficient of variation (CV) was calculatedas the standard deviation divided by the mean of eachtrait × 100 (%) as an index of the natural variation of thetrait. CV can also be influenced by measurement errors orheterogeneity of traits within a plant, but we assume that themain source of variation was provided by genotypicdifferences in the community. CV was calculated for each site,origin and growth CO2 (3 sites × 2 origins × 2 growth CO2).Standardized effect size (η2, sum of squares of an effect as aproportion of the total sum of squares) was calculated for

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each explanatory factor. All statistical analyses were performedusing SPSS 15 (SPSS Inc, Chicago, IL, USA).

Results

The photosynthetic rate at growth CO2 concentration(Agrowth) increased significantly with elevated CO2 treatmentby 20–40% (on average 31%), but the effect of origin onAgrowth was not significant (Fig. 1a–c, Table 1). Growth CO2concentration significantly reduced nitrogen concentrationper unit area (Narea) on average (−11%) (Fig. 1d–f, Table 1).There was a significant interaction of growth CO2 treatment ×site × origin. The spring plants from Nibu showed a smallerreduction in Narea with elevated CO2 treatment (−10%) thandid control plants (−22%) (Fig. 1d; ANOVA, P = 0.028 forNibu site).

The maximum rates of RuBP carboxylation (Vcmax) andelectron transport (Jmax) were lower in plants grown at elevatedCO2 treatment (on average −17% for Vcmax and −11% forJmax). There was no significant difference in these reductionsbetween spring and control plants (Table 2). However, similarto Narea, the spring plants from Nibu showed a trend ofreduced down-regulation of Jmax with elevated CO2 (−7%)compared with their respective control plants (−14%), althoughthis interaction was insignificant (P = 0.11; Table 2). Yuno-kawa plants, however, showed an opposite trend (−16% and−5%). The Jmax/Vcmax ratio, an indicator of protein allocation,was slightly higher in all plants grown at elevated CO2treatment (on average 8%; Fig. 2a–c). However, the ratio wasnot different between spring and control plants (Table 1), whichdid not support the first hypothesis. Higher Agrowth with lowerNarea at elevated CO2 treatment greatly increased PNUE (on

Fig. 1 Box plots of leaf traits of plants originating from three natural CO2 springs (filled boxes; mean spring CO2 was 500–1000 µmol mol−1) and nearby control areas (open boxes; c. 370 µmol mol−1): Plantago asiatica from Nibu, and Polygonum sachalinense from Ryuzin-numa and Yuno-kawa. (a–c) Photosynthetic rate at growth CO2 concentrations (Agrowth) (370 or 700 µmol mol−1); (d–f) leaf nitrogen concentration per unit area (Narea). The central box in each box plot shows the interquartile range and median; whiskers indicate the 10th and 90th percentiles.

Table 1 Statistical analysis of leaf traits shown in Figs 1 and 2

Source d.f.

Agrowth Narea Jmax/Vcmax gs1 Starch

MS F MS F MS F MS F MS F

Between-subject effectsOrigin 1 7.804 0.71ns 0.001 0.06ns 0 0.001ns 0.002 1.81ns 0.857 0.05nsSite 2 457.203 41.82**** 0.387 15.49**** 0.048 1.25ns 0.003 3.32** 266.189 16.14****Origin × site 2 4.056 0.37ns 0.013 0.53ns 0.006 0.15ns 0.003 3.71** 59.852 3.63*Error 37 10.933 0.025 0.038 0.001 16.496Within-subject effectsGrowth CO2 1 372.055 175.00**** 0.324 23.21**** 0.459 48.79**** 0 0.01ns 470.698 25.15****Growth CO2 × origin 1 0.039 0.02ns 0.009 0.62ns 0 0.05ns 0 0ns 2.197 0.12nsGrowth CO2 × site 2 2.097 0.99ns 0.034 2.46* 0.009 0.92ns 0.001 1.78ns 2.031 0.11nsGrowth CO2 × site × origin 2 3.225 1.52ns 0.057 4.11** 0 0.01ns 0.001 1.75ns 3.643 0.19nsError 37 2.126 0.014 0.009 0.001 18.714

Mixed-model ANOVA was used. Growth CO2 is a within-subject factor, and site and origin are between-subject factors (two-way ANOVA). Mean square (MS), F value and levels of significance are presented. Significance levels: *, P < 0.1; **, P < 0.05; ***, P < 0.01; ****, P < 0.001;ns, not significant.1Log-transformed before analysis to satisfy equality of covariance matrices (Box’s test).Agrowth, photosynthetic rate at growth CO2 concentration; gs, stomatal conductance; Jmax, maximum electron transport rate driving ribulose-1,5-bisphosphate (RuBP) regeneration; Narea, leaf nitrogen concentration per unit area; Vcmax, maximum rate of RuBP carboxylation.

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Table 2 Effects of CO2 concentration on leaf traits of plants originating from three natural CO2 springs (mean spring CO2 was 500–1000 µmol mol−1) and nearby control areas (c. 370 µmol mol−1)

Site Growth CO2 Origin Ci/Ca

Vcmax Jmax Nmass Cmass LMA

(µmol m–2 s–1) (µmol m–2 s–1) (%) (%) (g m−2)

Nibu LC Control 0.83 ± 0.06 74.3 ± 9.9 136.7 ± 14.6 2.32 ± 0.58 47.4 ± 1.0 57.7 ± 13.6Spring 0.83 ± 0.05 71.0 ± 11.1 129.6 ± 17.1 2.40 ± 0.64 46.6 ± 1.7 53.0 ± 9.1

HC Control 0.86 ± 0.05 58.8 ± 9.2 114.3 ± 11.1 1.49 ± 0.22 45.9 ± 1.6 69.0 ± 11.7Spring 0.84 ± 0.06 61.7 ± 9.4 120.2 ± 11.2 1.90 ± 0.37 46.7 ± 1.4 59.9 ± 10.4

Ryuzin-numa LC Control 0.79 ± 0.03 48.8 ± 11.5 90.9 ± 21.9 2.17 ± 0.25 48.1 ± 2.1 46.5 ± 9.2Spring 0.81 ± 0.05 47.1 ± 8.1 86.8 ± 17.3 2.16 ± 0.26 48.4 ± 1.0 46.9 ± 7.9

HC Control 0.80 ± 0.05 39.5 ± 7.4 80.8 ± 14.8 1.77 ± 0.21 49.1 ± 1.1 56.6 ± 5.4Spring 0.81 ± 0.05 38.5 ± 7.4 77.3 ± 11.4 1.59 ± 0.28 48.7 ± 0.6 54.9 ± 6.1

Yuno-kawa LC Control 0.83 ± 0.06 48.0 ± 8.7 91.2 ± 14.6 2.24 ± 0.26 49.2 ± 1.6 43.4 ± 7.1Spring 0.75 ± 0.06 48.4 ± 7.2 93.0 ± 13.8 1.99 ± 0.28 49.2 ± 1.1 53.3 ± 4.4

HC Control 0.85 ± 0.05 43.5 ± 11.2 86.8 ± 14.0 1.74 ± 0.24 48.8 ± 2.6 54.1 ± 7.2Spring 0.81 ± 0.03 38.4 ± 7.2 78.3 ± 11.4 1.67 ± 0.53 48.6 ± 2.0 56.7 ± 14.3

Ci/Ca Vcmax Jmax Nmass Cmass LMA

Mixed-model ANOVA d.f. MS F MS F MS F MS F MS F MS F

Between-subject effectsOrigin 1 0.006 2.06ns 37.597 0.27ns 145.37 0.45ns 0 0.03ns 0.102 0.03ns 37.308 0.29nsSite 2 0.012 4.04** 5139.879 37.26**** 15997.28 49.78**** 0.002 0.16ns 44.954 13.67**** 829.208 6.47***Origin × site 2 0.009 2.80* 8.932 0.06ns 23.24 0.07ns 0.015 1.43ns 0.019 0.01ns 212.817 1.66nsError 37 0.003 137.935 321.38 0.011 3.289 128.198Within-subject effectsGrowth CO2 1 0.010 6.20** 1985.278 63.92**** 3027.12 27.30**** 0.238 41.00**** 0.519 0.34ns 1236.18 16.74****Growth CO2 × origin 1 0 0ns 0.893 0.03ns 6.65 0.06ns 0.008 1.44ns 0.321 0.21ns 215.311 2.92*Growth CO2 × site 2 0.002 1.15ns 52.485 1.69ns 97.44 0.88ns 0.011 1.86ns 3.919 2.57* 51.116 0.69nsGrowth CO2 × site × origin 2 0.002 1.16ns 63.153 2.03ns 256.89 2.32ns 0.009 1.63ns 2.899 1.9ns 50.313 0.68nsError 37 0.002 31.061 110.89 0.006 1.525 73.84

Plants were grown at ambient CO2 (LC, 370 µmol mol−1) and elevated CO2 (HC, 700 µmol mol−1). Mean ± 1SD (n = 7–8). Mixed-model ANOVA was used. Growth CO2 concentration is a within-subject factor and fixed between-subject factors are site and origin (two-way ANOVA). Mean square (MS), F value and significance are presented. Significance levels: *, P < 0.1; **, P < 0.05; ***, P < 0.01; ****, P < 0.001; ns, not significant.Ci/Ca, ratio of intercellular and ambient partial pressures of CO2; Cmass, leaf carbon on a mass basis; Jmax, maximum electron transport rate driving ribulose-1,5-bisphosphate (RuBP) regeneration; LMA, leaf mass per unit area; Nmass, leaf nitrogen on a mass basis; Vcmax, maximum rate of RuBP carboxylation.

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Fig. 2 Box plots of leaf traits of plants originating from three CO2 springs (filled boxes; mean spring CO2 was 500–1000 µmol mol−1) and nearby control areas (open boxes; 370 µmol mol−1): Plantago asiatica from Nibu, and Polygonum sachalinense from Ryuzin-numa and Yuno-kawa. (a–c) Ratio of the maximum electron transport rate driving ribulose-1,5-bisphosphate (RuBP) regeneration (Jmax) to the maximum rate of RuBP carboxylation (Vcmax); (d–f) stomatal conductance (gs) for water vapour at growth CO2 concentrations; (g–i) leaf starch concentration. The central box in each box plot shows the interquartile range and median; whiskers indicate the 10th and 90th percentiles.

Site Growth CO2 OriginWUE (mmol mol–1)

PNUE (µmol g–1 s–1)

Nibu LC Control 3.36 ± 0.71 14.3 ± 3.0Spring 3.30 ± 0.82 14.4 ± 2.4

HC Control 4.77 ± 1.32 21.9 ± 2.8Spring 5.31 ± 1.53 20.9 ± 2.9

Ryuzin-numa LC Control 4.97 ± 0.67 11.8 ± 1.7Spring 4.26 ± 0.85 11.7 ± 2.4

HC Control 7.62 ± 1.63 15.5 ± 2.2Spring 7.51 ± 1.86 17.2 ± 2.6

Yuno-kawa LC Control 4.19 ± 1.53 12.5 ± 1.5Spring 5.89 ± 1.37 10.4 ± 1.6

HC Control 6.64 ± 2.38 17.8 ± 2.8Spring 8.46 ± 1.42 16.2 ± 2.2

Mixed-model ANOVA Source d.f.

WUE PNUE

MS F MS F

Between-subject effectsOrigin 1 5.414 1.86ns 5.643 0.62nsSite 2 40.237 13.84**** 133.779 14.74****Origin × site 2 7.935 2.73* 11.186 1.23nsError 37 2.908 9.076Within-subject effectsGrowth CO2 1 121.811 113.43**** 729.401 248.19****Growth CO2 × origin 1 0.778 0.72ns 1.232 0.42nsGrowth CO2 × site 2 2.882 2.68* 10.426 3.55**Growth CO2 × site × origin 2 0.269 0.25ns 3.84 1.31nsError 37 1.074 2.939

Plants were grown at ambient CO2 (LC, 370 µmol mol−1) and elevated CO2 (HC, 700 µmol mol−1). Mean ± 1SD (n = 7–8). Mixed-model ANOVA was used. Growth CO2 concentration is a within-subject factor and fixed between-subject factors are site and origin (two-way ANOVA). Mean square (MS), F value and levels of significance are presented. Significance levels: *, P < 0.1; **, P < 0.05; ***, P < 0.01; ****, P < 0.001; ns, not significant.PNUE, photosynthetic nitrogen use efficiency; WUE, water use efficiency.

Table 3 Effects of CO2 concentration on leaf-level resource use efficiencies of plants originating from three natural CO2 springs (mean growth CO2 was 500–1000 µmol mol−1) and nearby control areas (c. 370 µmol mol−1)

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average 45%; Table 3). However, there was no furtherincrease in PNUE in spring plants when compared withcontrol plants. The spring plants from Yuno-kawa showedlower PNUE than control plants (−13%), which was partlyascribed to their lower stomatal conductance (see below).

Stomatal conductance tended to be lower in plants grownin elevated CO2 (average reduction of −23%; Fig. 2d–f,Table 1), although the difference was not significant. Therewas a significant interaction between site and origin (Table 1).In particular, spring plants from Yuno-kawa showed markedlylower stomatal conductance (−40%) than control plants. Thelower stomatal conductance was also accompanied by lowerCi/Ca (Table 2). The lower stomatal conductance with higherphotosynthetic rate (Fig. 1a–c) resulted in significantly higherWUE with elevated CO2 treatment (on average 55%; Table 3).The effect of origin was not significant, but there was amarginally significant interaction between site and origin, inwhich the spring plants from Yuno-kawa showed higherWUE (+32%) than control plants. The latter is partly inagreement with our second hypothesis that spring plants havea higher WUE.

Starch concentration per unit mass was sharply andsignificantly increased by elevated CO2 treatment (21–90%; onaverage 57%; Fig. 2g–i). The increases were more pronouncedwhen starch was expressed per unit area (on average 85%increase; data not shown) because of the higher leaf mass perunit area (LMA) at elevated CO2 treatment. The effect oforigin was not significant, but there was a significant interac-tion between site and origin (Table 1). The spring plants fromNibu showed, on overage, 15% lower starch concentrationper unit mass (and 22% lower per unit area) than controlplants. The third hypothesis, that spring plants would havelower starch accumulation, was supported in this case (Fig. 2g),but was not supported by plants from Ryuzin-numa.

The CVs of the measured traits ranged widely, dependingon the trait, from c. 3% in carbon concentration to c. 40% instarch concentration (Fig. 3, Table S1). CV is considered toreflect genotypic variation within a population. If high CO2acts as a very strong selective agent, causing a bottleneckeffect, it could be expected that CV of the selected trait wouldbe smaller in spring than control populations because ofreduced genetic variation. However, no such trends wereobserved (Fig. 3a), suggesting that high CO2 acted as a mildselective agent. The presence of gene flow between springand control populations may also increase genetic variabilityand, consequently, counteract the possible reduction ingenetic variability.

To investigate a possible reason why some traits were changedand others were not, CVs were compared between measuredtraits. Traits that were different between some spring andcontrol populations (e.g. stomatal conductance, WUE andstarch) had generally high CVs; however, Jmax/Vcmax had asmall CV (Fig. 3a). Although there was no correlationbetween CV and effect size of origin (sum of squares of the

origin effect as a proportion of the total sum of squares), therewas a positive trend between CV and effect size of origin × site,suggesting that traits with larger natural variation might bemore susceptible to long-term elevated CO2, depending onspecies or site environments (Fig. 3b).

Discussion

Our study is the first work to use multiple CO2 springs andto investigate plant adaptation to high CO2 concentration.Although significant interactions between site and origin werefound in several traits, no consistent change across the threeCO2 springs was found (Tables 1–3). General trends of plantadaptation to high CO2 therefore could not be found in this

Fig. 3 (a) Coefficient of variation (CV) for measured traits. CV was calculated for each growth CO2 treatment (370 and 700 µmol mol−1) and the mean is shown for each site and origin. (b) Relationship between effect sizes (proportion of the total sum of squares which can be explained by origin or origin × site) and mean CV across measured traits. (See text for details of the calculation.) The numbers correspond to the traits in (a). There is a positive correlation between CV and effect size of origin × site (y = 0.0038x + 0.0069, R2 = 0.539, P = 0.0018).

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study. However, significant site × origin interactions suggestthat high CO2 acted as a selective agent in a different manner,depending on the species and other environmental conditions.A recent molecular study also found relatively large geneticdifferentiation across the CO2 gradient in these plants (I.Nakamura et al., unpublished). Several previous studies, thatused a single CO2 spring, also found significant differences inplant traits between spring and control plants (Fordham et al.,1997; Polle et al., 2001; Schulte et al., 2002). Together withthese studies, we support the concept that high CO2 will actas a selective agent, but that the effects of selection will varyacross sites and species.

The spring plants, especially those from Yuno-kawa,showed a lower stomatal conductance than did control plants(Fig. 2f). The lower stomatal conductance reduced water lossand resulted in a higher WUE (Table 3). Theory predicts thata decrease in stomatal conductance under high CO2 should beadaptive, because plants can save water while keeping photo-synthesis at relatively high levels (Cowan, 1986). Field studiesat natural CO2 springs have found that the water saving at ele-vated CO2 extends the photosynthetically active time in thedry period (Jones et al., 1995; Hättenschwiler et al., 1997)and mitigates the midday depression of stomatal conductance(Tognetti et al., 1999). These advantages may be particularlyimportant for P. sachalinense as it has a relatively large leaf area,which is sensitive to drought. Furthermore, the decrease intranspiration rates may affect the hydraulic balance at thewhole plant level. Nakamura et al. found that P. asiatica fromthe Nibu CO2 spring invested relatively less biomass in rootsand more in leaves compared with P. asiatica from the controlarea (I. Nakamura et al., unpublished), which may contributeto increased fitness in the high-CO2 environment.

It is well known that plants decrease stomatal conductanceunder short-term elevation of CO2 concentration (up toseveral years) (−20% at 550 µmol mol−1; Long et al., 2004).Our results suggest that further decreases in stomatal con-ductance would be favoured under long-term elevated CO2.Our previous study also found that Yuno-kawa spring plantsshowed very low stomatal conductance compared with controlplants in their original locations (−41% with 530 µmol mol−1

[CO2]; Onoda et al., 2007), suggesting that the in situdifference was caused by the combined effects of acclimationand adaptation. As lower stomatal conductance would leadto lower transpiration from ecosystems and, consequently, anincrease in soil moisture (Field et al., 1995), a further changein stomatal conductance as a result of adaptation may affectthe ecosystem function.

Spring plants from Nibu showed a smaller reduction inNarea (and, similarly, in Jmax) with high growth CO2 concentra-tion than did control plants (Fig. 1d, Table 1). This suggeststhat the spring plants maintained a high photosyntheticcapacity at elevated CO2 concentration without serious down-regulation, and thus utilized CO2 more efficiently than didcontrol plants. This was associated with lower carbohydrate

accumulation (Fig. 1). The spring plants showed reducedstarch accumulation for a given Agrowth (Fig. S1a,b, seeSupporting Information), suggesting that lower starchaccumulation was probably caused by higher carbohydratetransport. A reduction in carbohydrate accumulation was alsofound in Quercus ilex from Italian CO2 springs (Schulte et al.,2002). These results suggest that CO2 adaptation mayinclude an alteration in sink–source balance or an increase incarbohydrate transport capacity.

The Jmax/Vcmax ratio reflects the protein allocation betweenRubisco and the RuBP regeneration process (Onoda et al.,2005; Hikosaka, 2005). An increase in this ratio by 5–10%(Fig. 1) suggests that the plants seem to respond positively tohigh CO2 with slightly more efficient allocation of nitrogen.However, this change may not be sufficiently large to track theCO2 elevation (from 370 to 700 µmol mol−1), because itincreased the co-limiting Ci, where nitrogen was optimallyallocated between Rubisco and RuBP regeneration pro-cesses (Medlyn, 1996; Hikosaka & Hirose, 1998), by 60–120 µmol mol−1 only. Therefore, a further increase in theJmax/Vcmax ratio should be expected with adaptation to highCO2. However, we did not find any difference in the Jmax/Vcmax ratio between spring and control plants (Table 1),suggesting that the photosynthesis machinery is quite con-servative against long-term elevated CO2.

Why were some traits changed, but others were not, inspring plants? As evolution is the interaction of naturalselection and genetic variability, one explanation could be thatgenetic variation in some traits was limited. For example, inthe case of Jmax/Vcmax, CV was only 7.7% on average (Fig. 3a),whereas the theoretical study suggests that Jmax/Vcmax shouldincrease by 20–30% following adaptation to high CO2 (from350 to 700 µmol m−2 s−1; Hikosaka & Hirose, 1998), sug-gesting that the genetic variability in this trait constrains theadaptive response to high CO2 concentration. Consideringthe fact that atmospheric CO2 concentration has been moreor less constant over the past 650 000 yr (IPCC, 2007), theJmax/Vcmax ratio might have been strongly selected for the paststable CO2 concentration. If so, the optimization in the pastmay constrain the optimization in the near future (i.e. ~100 yr).By contrast, stomatal conductance and starch concentrationshowed relatively large CVs. A large CV implies that: (1) suchtraits may have been neutral or contributed little to fitness atthe current or past CO2 concentration; or (2) such traits mayhave been selected for various environmental heterogeneities(light, soil nutrients, etc). Regardless of the cause, if such largevariations interact with fitness at high CO2 concentration,traits with more variation are more likely to be affected byselective pressure of elevated CO2 (Tonsor & Scheiner, 2007).

The evolutionary response to CO2 has not received asmuch attention as the short-term response to CO2, but theincreasing number of studies on CO2 springs (e.g. Fordhamet al., 1997; Polle et al., 2001; Schulte et al., 2002) and selec-tion experiments (Ward et al., 2000; Collins & Bell, 2004)

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suggest that high CO2 will act as a selective agent. As theatmospheric CO2 concentration is more or less homogeneousover the world, all terrestrial plants have been exposed toincreasing CO2 concentration, and thus have been subject toselection pressure of elevated CO2. This could be an importantissue in plant evolution, when compared with global warmingin which plants may escape (migrate or be translocated) fromwarming towards high latitude or altitude without evolution(Hoegh-Guldberg et al., 2008). Evolutionary responses tohigh CO2 may therefore have important effects on ecosystemfunction and species’ interactions all over the world.

Conclusion

High CO2 concentration directly and greatly increased PNUEand WUE (Table 2), suggesting that plants will show highergrowth rates at a given resource availability and, consequently,that plants will be subject to more intense competition.However, a further improvement in PNUE with a selectivereduction in Rubisco relative to the RuBP regenerationprocess (e.g. Sage & Coleman, 2001) was not observed,probably because of the limited genetic variation in thephotosynthetic machinery. However, a significant reductionin stomatal conductance, which contributed to higher WUE,and a trend of reduced down-regulation of photosynthesiswith a lower starch accumulation were found in some springplants. These results suggest that there is substantial room forplant evolution in high-CO2 environments, and such evolutioncould have important implications for species’ interactionsand ecosystem functioning in the future.

Acknowledgements

The landowners of the CO2 springs (Hakkoda-Onsen Co.,Tashiro Bokuya-Chikusan Kumiai and Yudonosan shrine)kindly gave us permission to collect plants growing in theirlands for this study. We thank Ken-ichi Sato, ToshihikoKinugasa, Yuko Yasumura, Kay-May Miyagi, Ito Nakamuraand Noriyuki Osada for their help with fieldwork and growingplants, and Masakado Kawata, Shin-ichi Morinaga, NielsAnten, Belinda Medlyn, David Ackerly and anonymousreviewers for critical and constructive comments on the draftmanuscript. This study was partly supported by a Grant-in-Aid from the Japan Society for the Promotion of Sciencefor Young Research Fellows (Y.O.), and from the JapanMinistry of Education, Culture, Sports, Science and Technologyfor K.H. and T.H.

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Supporting Information

Additional supporting information may be found in theonline version of this article.

Fig. S1 Relationships between maximum electron transportrate driving ribulose-1,5-bisphosphate (RuBP) regeneration(Jmax) and leaf starch concentration (a) and between leaf starchconcentration and photosynthetic rate at growth CO2concentrations (b) of Plantago asiatica originating from aCO2 spring and nearby control area.

Table S1 Mean, standard deviation and coefficient of varia-tion of all leaf traits in this study (n = 7–8)

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