GLOBAL CHANGE ECOLOGY - ORIGINAL PAPER

Phenotypic and genetic differences in a perennial herbacross a natural gradient of CO2 concentration

Ito Nakamura • Yusuke Onoda • Noe Matsushima •

Jun Yokoyama • Masakado Kawata •

Kouki Hikosaka

Received: 1 June 2010 / Accepted: 23 December 2010 / Published online: 14 January 2011

� Springer-Verlag 2011

Abstract The atmospheric CO2 concentration [CO2] has

been increasing markedly since the industrial revolution

and is predicted to reach 500–1,000 lmol mol-1 by the

end of this century. Although the short-term and acclima-

tory responses to elevated [CO2] have been well studied,

much less is understood about evolutionary responses to

high [CO2]. We studied phenotypic and genetic differences

in Plantago asiatica populations around a natural CO2

spring, where [CO2] has been consistently high over an

evolutionary time scale. Our common-garden experiment

revealed that plants transferred from habitats with higher

[CO2] had higher relative growth rates, greater leaf to root

ratios, lower photosynthetic rates, and lower stomatal

conductance. The habitat-dependent differences were

partly heritable because a similar trend of leaf to root ratio

was found among their offsprings. Genetic analyses indi-

cated that selfing or biparental inbreeding might promote

local adaptation in areas with high [CO2] despite sub-

stantial gene flow across the [CO2] gradient. These results

indicate that phenotypic and genetic differences have

occurred between high and normal [CO2] populations.

Keywords Adaptation to elevated CO2 concentration �CO2 spring � Evolutionary response � Plant function �Plantago

Introduction

Since plant functions, such as photosynthesis, transpiration,

and biomass production, are sensitive to atmospheric CO2

concentration [CO2], plant responses to elevated [CO2] are

critical for predicting functions of future ecosystems and

have attracted considerable interest. However, most pre-

vious studies have implicitly assumed that, in the future,

plants will respond to [CO2] in the same manner as they

currently do, and relatively little attention has been paid to

the effects of [CO2] on genetic changes, i.e., evolution.

Significant genotypic variations have been found in [CO2]

responses (Korner and Bazzaz 1996; Andalo et al. 2001;

Wineneke et al. 2004; Steinger et al. 2007), which will be

subject to natural selection in high [CO2] environments.

Previous selection experiments have revealed that [CO2]

can act as a selective agent for C3 plants (Ward et al. 2000).

If increasing [CO2] affects the genetic structure of wild

plants and alters plant traits, plant functions in the future

may substantially deviate from predictions made based on

previously conducted experiments using current plants

(Wieneke et al. 2004; Ward and Kelly 2004; Lau et al.

2008).

Evolutionary responses to elevated [CO2] in wild plants

are, in general, difficult to detect using growth experiments

because the duration of experiments is often too short

compared to the time required for evolution. Natural CO2

Communicated by Evan DeLucia.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00442-010-1900-1) contains supplementarymaterial, which is available to authorized users.

I. Nakamura � N. Matsushima � M. Kawata � K. Hikosaka (&)

Graduate School of Life Sciences, Tohoku University,

Aoba, Sendai 980-8578, Japan

e-mail: hikosaka@m.tohoku.ac.jp

Y. Onoda

Department of Biological Sciences, Faculty of Science,

Kyushu University, Fukuoka 812-8581, Japan

J. Yokoyama

Faculty of Science, Yamagata University, Koshirakawa,

Yamagata 990-8560, Japan

123

Oecologia (2011) 165:809–818

DOI 10.1007/s00442-010-1900-1

springs represent a unique model ecosystem for the study

of the evolutionary effects of elevated [CO2] because

plants in these areas have been exposed to high [CO2] over

an evolutionary time scale (Miglietta et al. 1993; Raschi

et al. 1999). Since 1993, many researchers have studied the

long-term effects of high [CO2] on physiological and

morphological characteristics of plants growing around

natural CO2 springs (see Pfanz et al. 2004 for review).

However, such field studies do not necessarily provide

knowledge on the adaptations occurring in plants, because

the differences may include both acclimatory responses

and genetic differences. In order to clarify genetic differ-

ences in plant phenotypic traits, plants originating from an

area with high [CO2] and those from a nearby control area

should be compared in a common-garden experiment. Thus

far, only a few researchers have conducted common-garden

experiments, from which they have found that plants from

CO2 springs differ in some traits from those from areas

with normal [CO2] (Fordham et al. 1997; Polle et al. 2001;

Onoda et al. 2009). For example, Agrostis canina from

high [CO2] habitat had higher relative growth rates than

those from habitats with normal [CO2] (Fordham et al.

1997). Quercus ilex from a habitat with high [CO2] had a

higher shoot to root ratio than the control (Polle et al.

2001). Recently, we found variations in leaf traits of a few

herbaceous species between spring and control populations

(Onoda et al. 2009).

Although the previous findings suggested that genetic

differentiation occurred in plants in the area with high

[CO2] around CO2 springs, no study has investigated

whether the phenotypic differences are heritable. For

example, maternal growth [CO2] is known to affect the

growth of offspring plants in Poa annua even when they

were grown in the same environment (Bezemer et al.

1998). Furthermore, to our knowledge, no study has

investigated whether the differences in phenotypic traits

are linked with the genetic structure of plant populations.

Adaptation is influenced by the spatial structure of a pop-

ulation. Local adaptation to a particular environment can

occur only when the selective pressure is sufficiently strong

to counteract the effect of gene flow across the environ-

mental gradient (Linhart and Grant 1996; Kawecki and

Ebert 2004). In CO2 spring ecosystems, areas where [CO2]

is maintained at high levels are spatially restricted. The

presence of substantial gene flow may prevent local

adaptation if these areas are not sufficiently large. It is

important to employ genetic analysis to examine the

manner in which populations are genetically isolated from

each other.

We investigated whether local adaptation occurred in

plants growing around natural CO2 springs. Our study

consisted of three steps. Firstly, we transplanted Plantago

asiatica plants from several plots around a CO2 spring to

our experimental garden and analyzed their growth char-

acteristics under two CO2 concentrations. Secondly, we

tested whether the differences in growth characteristics

were heritable to their offsprings. Finally, we studied the

genetic structure of P. asiatica populations with micro-

satellite makers to examine possible causes of genetic

differentiation.

Materials and methods

Site and transplantation

The Nibu (Nyuu) spring is situated at the foot of Mount

Gassan, Japan (38�320N, 139�590E, 1,000 m above sea

level). This is a CO2-enriched water spring in a Fagus

crenata forest, and small streams run from the spring

which maintain the atmospheric [CO2] in an adjoining

open land at high levels. This open land has a small path,

along which a large gradient of [CO2] is formed. The

light environment and nutrient availability were consid-

ered to be similar across the gradient. We determined

concentrations of H2S and SO2 but did not detect.

Detailed information of the study site has been described

previously (Onoda et al. 2007). We used P. asiatica L., a

C3 rosette perennial herb, which was dominant along the

path. This species is distributed across a wide range of

habitats and exhibits large genotypic and phenotypic

variations across environmental gradients (Ishikawa et al.

2006, 2007).

On May 21, 2004, several days after the disappearance

of snow from the site, five plots were set across a gradient

of [CO2]. The plots, L1, L2, L3, L4 and L5, were situated

at distances of 1, 8, 13, 20 and 30 m from the stream,

respectively. Each plot was 30 9 30 or 50 9 50 cm in size

to include more than 24 individuals (older than 1 year)

except for L5. Since L5 contained only 16 individuals, we

considered an additional plot (L50) located 7 m away from

it. We collected the individuals with soil to avoid damaging

the roots. The plants were brought to the laboratory and

were washed with water to remove soil and dead parts.

The atmospheric [CO2] in the six plots was determined

from August 31 to September 1, 2005 and from September

6 to 7, 2006. For plots L1–L4, we used an infrared analyzer

with an air sampling system (see Onoda et al. 2007). Tube

inlets for sampling air were positioned at a height of 5 cm

from the ground, where P. asiatica leaves developed and

air was taken 10 min for each plot. For plots L5 and L50,we used CO2 monitors (Telaire 7001; TELAIRE, Goleta,

CA, USA; Osada et al. 2010), in which [CO2] was recorded

every 5 min. Both systems were calibrated with the same

standard gas. Due to the effects of wind direction and

speed, there were large fluctuations in atmospheric [CO2]

810 Oecologia (2011) 165:809–818

123

near the CO2 spring. The means and standard errors of the

daytime atmospheric [CO2] in plots L1, L2, L3, L4, L5,

and L50 were 5,339 ± 2,098, 1,044 ± 289, 771 ± 128,

726 ± 109, 364 ± 7, and 380 ± 21 lmol mol-1, respec-

tively (n = 6; hereafter referred as habitat [CO2]).

Ecophysiological analysis

We grew plants in four open top chambers (OTC) in the

experimental garden of Tohoku University (38�390N,

140�570E). The atmospheric [CO2] in two OTCs was

adjusted to 700 lmol mol-1, while that in the other two

was not regulated, and was around 370 lmol mol-1.

Details of the OTC system have been described previously

(Nagashima et al. 2003).

Hydroponic culture experiment

A hydroponic culture experiment was conducted to analyze

plant growth without nutrient limitation. On May 22, 2004,

12 pairs of similar-sized plants were selected from each

plot except for L5 and L50, from which 8 and 4 pairs of

plants were selected, respectively. Since the atmospheric

[CO2] in the habitat was similar between L5 and L50, we

regarded the plants from plots L5 and L50 as one group

(hereafter referred to as L5 plants). One from each pair

was grown at 370 lmol mol-1 [CO2] and the other at

700 lmol mol-1 [CO2] (12 individuals per growth [CO2]

per plot). On May 25, 2004, the fresh mass of every indi-

vidual was determined. The means and standard deviations

of the plant mass for L1, L2, L3, L4, and L5 were

1.05 ± 0.70, 1.45 ± 0.66, 1.00 ± 0.68, 1.14 ± 1.09, and

2.39 ± 1.81 g, respectively. Four plants per container

(36 9 27 9 12.5 cm) were allotted so that the sum of the

plant mass was similar among the containers. Plants were

floated on half-strength Hoagland nutrient solution (8 L;

Hewitt and Smith 1975) with a polystyrene foam plate. The

solution was continuously aerated and replaced every

week. The containers were swapped between the two OTCs

in each [CO2] treatment every week. Furthermore, the

positions of the containers in the OTCs were randomized

every week. Plants were grown for 2 weeks to acclimate to

growth [CO2]. Plant mass increased on average more than

3.4-fold during this period, indicating that more than 2/3 of

biomass was produced after transplanting. We therefore

judged that plants had acclimated to their respective growth

[CO2] when the growth analysis started.

The fresh mass and leaf blade size were determined

weekly for each individual on June 4, 11 and 18, 2004. The

length and width of all leaves were measured, and indi-

vidual and total leaf areas were estimated. The hydroponic

solution was carefully removed with paper towels and the

fresh mass was then determined. The relative growth rate

(RGR, growth rate per unit total fresh mass), net assimi-

lation rate (NAR, growth rate per unit leaf area), and leaf

area ratio (LAR, leaf area per unit total fresh mass) were

calculated for every individual as described previously

(Ishizaki et al. 2003). On June 30, 2004, 4 out of 12

individuals per plot per growth [CO2] treatment were

harvested. Their leaf blades, roots, reproductive organs and

petioles were separated and weighed after drying at 70�C.

Their nitrogen concentration was determined with an ele-

mental analyzer (NC-80; Shimadzu, Kyoto). To examine

whether nitrogen availability limited plant growth, we

calculated the potential requirement of nitrogen absorption

by plants (the highest plant nitrogen concentration multi-

plied by plant mass). It was smaller than the amount of

nitrogen supplied until June 18 in all individuals studied,

suggesting no nitrogen limitation. After June 18, however,

it was believed that weak nitrogen limitation might occur

in some large individuals.

Sand culture experiment

A sand culture experiment was conducted to study leaf gas

exchange characteristics. On June 30, 2004, the remaining

eight plants per plot were transplanted into 1.5 L pots (one

plant per pot) filled with washed river sand. Each indi-

vidual was grown at the same [CO2] as in the hydroponic

experiment. All plants received 100 mL of the same

nutrient solution per week and were watered when neces-

sary. From August 16 to 20, gas exchange measurements

were conducted with a portable photosynthesis measure-

ment system (Li-6400; LiCor, Lincoln, NE, USA) for

leaves emerged after transplanting. Steady-state gas

exchange rates were determined at a photosynthetically

active photon flux density of 2,000 lmol m-2 s-1, leaf

temperature of 25�C, and [CO2] of 370 and 700 lmol

mol-1. Following measurements, four leaf discs of 1-cm

diameter were punched out from each measured leaf.

Chlorophyll was extracted from one of the discs with

dimethylformamide and its content was spectrophotomet-

rically determined (Porra et al. 1989). Other discs were

dried, and the dry mass and nitrogen concentration were

determined as above. Plants were further grown in order to

collect seeds. Their flowers were covered with a thin

plastic mesh to avoid cross-pollination between individuals

and self-fertilized seeds were collected in September 2004.

Offspring experiment

On May 21, 2007, seeds obtained from L1 and L5 plants in

OTCs were sown in a 1.3 L pot filled with washed river

sand. On June 22, germinated plants were transplanted and

grown hydroponically, similar to the experiment conducted

in 2004. We used three offsprings per parent, which were

Oecologia (2011) 165:809–818 811

123

allotted to three pairs of OTCs (3 OTCs 9 8 parents 9 2

original plots 9 2 growth [CO2]). The offspring plants

were grown at the same [CO2] as their parents. Initiation of

flowering was checked every week and the date when half

the individuals flowered was calculated for each plot in an

OTC with logistic regression. We harvested the plants on

July 16 and determined their leaf area using a leaf area

meter (Li-3000; LiCor) and dry mass as above.

Statistical analyses were performed with R (version

2.6.2; R Development Core Team, Vienna, Austria). The

sample size per plot per growth [CO2] was 12 for growth

analysis in the hydroponic culture experiment, 4 for bio-

mass and nitrogen in the hydroponic-culture experiment, 8

for leaf traits in the sand culture experiment, and 8 for data

obtained from the offspring experiment (mean for three

offsprings per parent plant). A generalized linear model

was applied to assess the effects of habitat [CO2] and

growth [CO2]. The values of habitat [CO2] were log-

transformed prior to the analyses. The functions of distri-

bution were selected according to the Akaike information

criterion. The link function was ‘‘identity’’ in all cases.

Genetic analyses

Genetic analyses were performed for the individuals that

were sampled from plots L1–L5. The number of individ-

uals sampled from L1, L2, L3, L4, and L5 was 40, 42, 31,

39 and 28, respectively. In addition, 40 and 44 individuals

were sampled from the plots located at distances of 50 (L6)

and 100 m (L7) from L5, respectively. Total genomic

DNA was isolated from leaf tissues using the method

described previously (Edwards et al. 1991). Seven previ-

ously described microsatellite loci (Pm3, JPi11, JPi7, JPi4,

Pi3, JPm3, and JPm11; Squirrel and Wolff 2001) were used

for the analyses. The primers used were those that were

previously described (Squirrel and Wolff 2001), except for

the forward primer of Pi3 (50-TCACACACTGCACAC-

ACT-30). The number of alleles and the allele sizes of each

locus are represented in Table S1. The microsatellites were

amplified in five multiplexes using the Multiplex PCR Kit

(Qiagen, Japan). PCR conditions were employed according

to the manufacturer’s instructions, at an annealing tem-

perature of 58�C. An internal standard marker (Gene-

scan500 ROX; Applied Biosystems) was used to determine

allele sizes. The amplified DNA was analyzed by per-

forming fluorescence imaging using an ABI PRISM

3130-Avant Genetic Analyzer (Applied Biosystems).

Genotyping was conducted using the GeneMapper soft-

ware ver. 3.7 (Applied Biosystems).

The number of alleles per locus (A), percentage of

polymorphic loci (P), allelic frequencies, and expected and

observed heterozygosity (He and Ho) were calculated for

all the microsatellite loci over all the plots (Table S1) and

for each plot (Table S2) using the GENEPOP 3.4 software

program (Raymond and Rousset 1995). The pairwise FST

and FIS values were calculated for the microsatellites (Weir

and Cockerham 1984) using ARLEQUIN ver. 3.0 (Excof-

fier et al. 2005). The level of significance was estimated by

performing a permutation test (1,000 permutations) using

ARLEQUIN ver. 3.0.

To explore the possibility of genetic discontinuities from

microsatellites without a priori notion of groups, spatial

Bayesian analyses and individual assignment tests were

conducted using STRUCTURE (Pritchard et al. 2000;

Falush et al. 2003). We assumed the admixture model and

performed the analyses based on the correlated allele fre-

quency model. Burn-in length and length of simulation

were set at 500,000 and 1,000,000 repetitions, respectively.

To test the convergence of the priors and the appropriate-

ness of the chosen burn-in length and simulation length, we

ran a series of independent runs (20 repeats) for each value

of K (from L1 to L7). To obtain the true number of clusters

(K), we used the ad hoc stastitic DK (the rate of change in

the log probability of data between successive K values)

and ln P(D) (the posterior probability of the data for a given

K) (Evanno et al. 2005).

To estimate selfing rates of each plant, 13, 15, 6, 18,

and 8 individuals that bore seeds were sampled from

areas near plots L1, L2, L3, L4, and L5, respectively, in

September 2005, and 16–20 seeds were collected from

each individual. The paternity of each seed was then

assigned based on even microsatellite loci using CERVUS

2.0 software (Marshall et al. 1998). Paternity assignment

by CERVUS requires gene frequencies and candidate

male. All individuals in plots L1–L5 were considered for

the gene frequencies (n = 240), 60 seed-bearing individ-

uals that were collected in September 2005 were con-

sidered as candidate male. Standard simulation parameters

were used with 10,000 cycles. In cases where the pater-

nity of a seed was unambiguously assigned to the indi-

viduals from which it had been obtained with [95%

confidence, the seed was considered to have been pro-

duced by selfing. The selfing rate was calculated as the

ratio of the number of seeds obtained in this manner to

the total number of seeds sampled from each individual.

The exclusion probabilities for the microsatellite loci are

shown in Table S1. Although many individuals were

found in study areas, most of them did not produce seeds

in September 2005. For example, we found only eight

seed-bearing individuals in and around plot L5. This

indicated that the number of reproducing individuals in

these areas was small. Therefore, we consider that the

number of candidate males that we sampled was not too

low and that the genotypes of sampled males adequately

represented the genotypes of the unsampled males, as

required for paternity determination.

812 Oecologia (2011) 165:809–818

123

Results

Plantago asiatica plants transplanted from plots around the

Nibu CO2 spring were hydroponically grown at normal

(370 lmol mol-1) or elevated (700 lmol mol-1) [CO2] in

OTCs. Since there was a large variation in the initial bio-

mass both within and among plots, we compared only traits

expressed as relative values. Elevated growth [CO2] sig-

nificantly enhanced RGR (Fig. 1a; Table S3). RGR mea-

sured in the first period (4–11 June) was significantly

higher in plants from the plots with higher habitat [CO2]

than in those from plots with lower habitat [CO2] (Fig. 1a;

P \ 0.001). A similar trend was found in the second period

(11–18 June), but the correlation was slightly weaker

(Table S3; P \ 0.1). The effect of habitat [CO2] on RGR

was also significant when the analysis incorporated initial

size as an independent (P \ 0.01 and P \ 0.05 for the first

and second period, respectively), suggesting that size-

dependence of RGR did not affect the result. RGR mea-

sured on the two periods (RGR1 and RGR2) were not dif-

ferent from the 1:1 relationship across the individuals

(RGR1 = 0.005 ? 0.99 RGR2; r2 = 0.50; intercept and

slope were not significantly different from 0 and 1,

respectively), indicating that RGR did not change during

these periods. Analysis of the RGR as a product of NAR

and LAR showed that the higher RGR in plants from high

habitat [CO2] was mainly attributable to a significantly

higher LAR, while NAR did not change across habitat

[CO2] (Fig. 1b, c). Greater LAR was mainly due to sig-

nificantly greater biomass allocation to the leaves than to

0.05

0.10

0.15

0.20

100 1000 10000

Rel

ativ

e gr

owth

rat

e(d

-1)

140

120

100

80

60

Net

ass

imila

tion

rate

(g m

-2 d

-1)

16

12

8

Leaf

are

a ra

tio

(cm

2 g-1

)

4

3

2

1

Leaf

to r

oot r

atio

(g g

-1)

20

15

10

5

Pho

tosy

nthe

tic r

ate

(µm

ol m

-2 s

-1)

Habitat: P < 0.05Growth: P < 0.001H x G: NS

1.2

0.9

0.6

0.3

0

Sto

mat

al c

ondu

ctan

ce(m

ol m

-2 s

-1)

100 1000 10000

Habitat: P < 0.01Growth: P < 0.01H x G: NS

a b

c d

e f

Habitat: P < 0.001Growth: P < 0.001H x G: NS

Habitat: NSGrowth: P < 0.001H x G: NS

Habitat: P < 0.01Growth: P < 0.1H x G: NS

Habitat: P < 0.01Growth: NSH x G: NS

Habitat CO2 concentration (µmol mol

-1)

Fig. 1 Traits of parent plants

transplanted from different plots

(L1–L5) as a function of habitat

[CO2]. Relative growth rate

(RGR) between June 4 and 11

(n = 12; a), net assimilation

rate (NAR) between June 4 and

11 (n = 12; b), leaf area ratio

(LAR) on June 11 (n = 12; c),

leaf to root ratio for harvested

plants (n = 4; d),

photosynthetic rate (n = 8; e),

and stomatal conductance

(n = 8; f) determined at

700 lmol mol-1 CO2. The

closed and open circles denote

the means with standard

deviations (bars) for individuals

that were grown under elevated

and ambient [CO2] conditions,

respectively. The results of the

analysis of deviance with the

generalized linear model are

indicated. Lines are linear

regression of variables on log-

transformed habitat [CO2],

which are shown when the

effect of habitat [CO2] was

significant

Oecologia (2011) 165:809–818 813

123

the roots (Fig. 1d). The leaf and whole-plant nitrogen

concentration per unit dry mass were independent from

habitat [CO2] (Table S3).

Elevated growth [CO2] enhanced the photosynthetic rate

and decreased stomatal conductance when determined at a

common [CO2] (Fig. 1e, f; Table S3). There were signifi-

cant effects of habitat [CO2]: plants from higher habitat

[CO2] had lower photosynthetic rates and stomatal con-

ductance determined at elevated [CO2] (Fig. 1e, f). Similar

results were also obtained when the gas exchange was

determined at ambient [CO2] (Table S3). Nitrogen and

chlorophyll content per leaf area were lower in plants from

high habitat [CO2] (Table S3).

To examine whether phenotypic differences between

populations were heritable, we collected seeds from the

transplanted plants following artificial self-fertilization.

Offsprings from L1 started flowering 5–6 days earlier than

those from L5 at elevated (15.1 and 21.1 days after trans-

planting for L1 and L5, respectively) and at ambient

growth [CO2] (17.4 and 23.2 days, respectively). Similar to

trends found in their parents, the leaf to root ratio was

significantly greater in offsprings from L1 than in those

from L5 (Fig. 2a). While the total biomass was not sig-

nificantly different at the time of harvest (Table S3), the

reproductive mass was significantly greater in offsprings

from L1 than in those from L5 (Fig. 2b). There was a

significant interactive effect of habitat and growth [CO2]

on the reproductive mass (Fig. 2b), implying a non-parallel

norm of reaction to growth [CO2]. While the effect of

habitat [CO2] on the LAR’ (leaf area per vegetative dry

mass) was not significant (Table S3), LAR’ was signifi-

cantly correlated with the LAR of their parents (r = 0.45,

P \ 0.01, n = 32), suggesting that biomass allocation was

partly heritable and conservative within plots.

We examined the genetic structures of our plants by

using microsatellite makers. The pairwise FST values,

which represented an estimate of the genetic divergence

and gene flow between the plots, ranged from 0 to 0.121

(Fig. 3a). These values indicate substantial gene flow

among the plots, suggesting that there was no strong

genetic barrier among them. Although the distance between

the plots with high [CO2] (L1–L4;\20 m) was shorter than

that between plots with normal [CO2] (L5–L7;[50 m), the

FST values were higher in the former than in the latter

group. Furthermore, the FST values were relatively high

between the high and normal [CO2] populations. A Bayes-

ian assignment test indicates that there was some genetic

structure between the plots with high (L1–L4) and those

with normal [CO2] (L5–L7), although this structure was

not very distinct (Fig. 4). These results indicate that dis-

tinct genetic differentiation among plots was not evident

owing to substantial gene flow, but some level of gene

flow was prevented between high and normal [CO2]

populations.

In the field, more than 30% of the seeds yielded by

reproducing plants were produced by selfing across study

plots regardless of habitat [CO2] (Fig. 3b). However, the

FIS values, the inbreeding coefficient within the plot pop-

ulations, decreased with decreasing habitat [CO2] (Fig. 3b),

suggesting that in areas with normal [CO2], plants pro-

duced by selfing had been eliminated before maturation.

Discussion

Adaptation is expected to occur when selection acts on a

trait that has a heritable basis of phenotypic variation. In

the present study, we found phenotypic differences

between populations in areas with high and normal [CO2]

(Fig. 1), some of which were heritable (Fig. 2). These

0

1

2

3

Growth CO2

Elevated Ambient

0.3

0.2

0.1

0L1 L5 L1 L5

Habitat: P < 0.001Growth: P < 0.001H x G: NS

Habitat: P < 0.001Growth: P < 0.001H x G: P < 0.05

Leaf

to r

oot r

atio

(g

g-1)

Plot

Rep

rodu

ctiv

e m

ass

(g)

a

b

Fig. 2 Growth analysis of offspring plants. Leaf to root ratio (a) and

total reproductive mass (b) with standard deviation (n = 8) for plants

from L1 and L5. The results of the analysis of deviance with the

generalized linear model are shown

814 Oecologia (2011) 165:809–818

123

results indicate that an evolutionary differentiation occur-

red in the P. asiatica population across a [CO2] gradient.

We found that RGR was higher in parent plants that

originated in areas with higher [CO2], suggesting that

plants from higher [CO2] populations had an inherent

potential for higher productivity. In the offspring experi-

ment, at the time of harvest, a similar final biomass but a

greater reproductive mass was found in L1 than in L5

plants. Because an increase in biomass investment in

reproductive organs reduces allocation to leaves, repro-

duction is costly for vegetative growth (Reekie and Bazzaz

1992; Obeso 2002). Similar biomasses irrespective of

greater reproductive allocation in L1 offsprings imply that

they also had a potential of higher productivity. Therefore,

our results suggest that the potential of higher productivity

was a heritable trait in plants from habitats with high

[CO2]. It should be noted that higher productivity in these

experiments does not necessarily mean that plants are also

productive in the native habitats because the growth con-

ditions in the original habitat are different from those in our

experiments (no limitation of water and nutrients) and

because the lifespan of P. asiatica individuals in the field is

much longer than that in our experiment (2 months).

However, it is likely that a higher potential of biomass

production contributes to fitness and has selective advan-

tages in field. These results are consistent with those of

previous experiments, wherein artificial selection increased

seed production under the respective [CO2] condition

compared to non-selected plants (Ward et al. 2000; Ward

and Kelly 2004).

How does [CO2] act as a selective agent? There is a

functional trade-off in biomass allocation between the roots

and leaves (Wilson 1988; Ishizaki et al. 2003). Larger root

size can increase the acquisition of water and nutrients,

while larger leaf area can increase light capture and carbon

assimilation. From the viewpoint of the carbon–nutrient

balance, the greater allocation to roots in high-[CO2] plants

should be advantageous, because higher carbon availability

results in a relative decrease in nutrient availability (Ber-

nacchi et al. 2000). This hypothesis contrasts with our

results that plants from habitats with higher [CO2] exhib-

ited a greater biomass allocation to leaves (Figs. 1, 2).

However, a high LAR may have a competitive advantage

because light competition becomes more intense under

high [CO2] conditions (Nagashima et al. 2003; Hikosaka

et al. 2003). If a competitive advantage due to higher LAR

at high [CO2] overrides an advantage of increased nutrient

acquisition due to higher allocation to roots, a higher LAR

may be evolutionarily favorable at high [CO2]. Alterna-

tively, as high [CO2] enhances water- and nitrogen-use

efficiency in photosynthesis (Saxe et al. 1998; Onoda et al.

2009), limitation by water or nitrogen availability may

become less important in areas with high [CO2], which

might allow a reduction in biomass investment in the roots.

Similar habitat [CO2]-dependent variation in the bio-

mass allocation was also observed in other studies. Quer-

cus ilex seedlings collected from a CO2 spring had higher

shoot to root ratios that those from a control site (Polle

et al. 2001). Data shown by Wieneke et al. (2004) suggest

that plants with higher shoot to root ratios were selected by

exposure to elevated [CO2] for 6 years. These results

suggest that high [CO2] may favor plants that invest more

biomass in shoots than in roots.

In the present study, plants from areas with high [CO2]

had lower gas exchange rates and a lower nitrogen con-

centration per unit mass (Fig. 1; Table S3). In our previous

study, decreased stomatal conductance was observed in

Polygonam sachalinense, which originated from habitats

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

10 100

Pai

rwis

e F

ST v

alue

Spatial distance (m)

a

b

FIS

( )

See

d se

lfing

rat

e (

)

L1 L2 L3 L4 L5 L6 L7

0

-0.1

0.1

0.2

0.3

0.4

Plot

Fig. 3 Genetic analyses. Pairwise FST values between the plots (a).

The closed, open and gray circles indicate the FST values determined

for the plots within plots with high [CO2] (L1–L4), within plots with

normal [CO2] (L5–L7), and between plots with high and normal

[CO2], respectively. Self-fertilization rates and FIS values (b). FIS

values were significantly higher than zero (P \ 0.01), except for those

determined in L6

Oecologia (2011) 165:809–818 815

123

with high [CO2] (Onoda et al. 2009). In the same study,

however, we did not find such a habitat [CO2]-dependent

decrease in stomatal conductance and photosynthetic

capacities in P. asiatica, though we used the same species

from the same CO2 spring as in the present study. The

reasons for this discrepancy are unclear. There were some

differences in growth conditions, measuring times and

individuals. Furthermore, ontogenic shift could also be

important. As observed in the offspring experiment, L1

plants started reproductive growth earlier than L5 plants,

which might have accelerated leaf senescence and re-

translocation of nitrogen from leaves (Shitaka and Hirose

1998). This possibility cannot be excluded because the

present study measured photosynthesis in plants that had

reproductive parts while the previous study used non-

flowering plants.

Genetic analyses using neutral makers indicated that

there was substantial gene flow among the examined plots.

Local adaptation in a small area with high [CO2] can occur

only when the selective pressure is sufficiently strong to

counteract the effect of gene flow across the environmental

gradient (Linhart and Grant 1996; Kawecki and Ebert

2004). In the present study, FST values between high and

normal [CO2] populations were higher even though they

were spatially close to each other (Fig. 3a). This contrasted

with smaller FST values among normal [CO2] plots even

though they were spatially separated by more than 100 m.

These results suggest that some level of gene flow was

prevented between high and normal [CO2] populations.

What factors prevent gene flow and facilitate local adap-

tation in areas with high [CO2]? We found that more than

30% of the seeds were produced by selfing across study

plots regardless of habitat [CO2] in the field (Fig. 3b).

However, the FIS values, the inbreeding coefficient within

the plot, decreased with decreasing habitat [CO2] (Fig. 3b).

This suggests that most of the individuals produced by

selfing could not survive in normal [CO2] areas, but a

significant number survived in high [CO2] areas. Selfing

may promote local adaptation by reducing the effects of

migration load from other areas (Hu and Li 2003). This is

analogous to the local adaptation to heavy metal contam-

ination, where increased self-fertility might act as a barrier

to gene flow between tolerant and non-tolerant populations

(Antonovics 1968). However, there is also another possi-

bility that biparental inbreeding increased with increasing

habitat [CO2]. Regardless of the precise mechanisms, our

results suggest that inbreeding was likely to promote local

adaptation in a high [CO2] environment.

In recent years, heritable epigenetic changes have been

revealed to play important roles in various aspects of plant

growth and development (Grant-Downton and Dickinson

2005). It has been shown that the epigenetic modifications

are induced environmentally in some instances (Grant-

Downton and Dickinson 2006; Bossdorf et al. 2008;

Fig. 4 Structure analysis

results. Bayesian assignment

probabilities of individuals to

three putative lineages (a). The

vertical line represents an

individual and colors indicate

the proportion of an individual’s

genotype assigned to a

particular linage. The values of

DK and ln P(D) are a function of

K based on 20 runs (b). The

maximum value of DK could be

obtained when K was 3,

although an increasing trend in

ln P(D) did not clearly indicate

a correct K. Thus, K = 3 was

the most probable number of

putative clusters that best

explains the pattern of genetic

variability (color figure online)

816 Oecologia (2011) 165:809–818

123

Chinnusamy and Zhu 2009). Although the feature of bio-

mass allocation in our study plants was, at least partly,

inherited to the next generation in our preliminary obser-

vations, we could not reject a hypothesis that epigenetic

remodeling could play a role in driving multi-generational

responses to habitat of high [CO2]. Further studies are

needed to test the hypothesis.

In summary, our study clearly shows that phenotypic

and genetic differences have occurred between high and

normal [CO2] populations. Some of the differences were

consistent with those found in previous selection studies.

However, we have to be careful to conclude that high

[CO2] has acted as a selective agent because other factors

might also affect plant evolution. Further studies using

other CO2 spring ecosystems may be needed to confirm

generality of local evolution to high [CO2].

Acknowledgments We thank Yudonosan-shrine, the landowner of

the CO2 spring, for permission for the study. We thank Yayoi Ko-

izumi, Risako Akita, Tomomi Kojima and Soichiro Nagano for

technical support and Roger Butlin for comments on an early draft.

The study was supported in part by KAKENHI (16687001, 19370008,

20677001, 21114009), Global Environment Research Fund (D-0904)

and Global COE program (J03).

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