Flora 210 (2015) 8086

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Flora
journal homepage: www.elsevier.com/locate/flora

Stable carbon and nitrogen isotope compositions change with leaf age
in two mangrove ferns
Martin Werth a, , Klaus Mehltreter b , Oscar Briones c , Marian Kazda a
a
b
c

Institute of Systematic Botany and Ecology, University of Ulm, 89081 Ulm, Germany
Instituto de Ecologa, A.C. Red de Ecologa Funcional, Xalapa 91070, Veracruz, Mexico
Instituto de Ecologa, A.C. Red de Biologa Evolutiva, Xalapa 91070, Veracruz, Mexico

a r t i c l e

i n f o

Article history:
Received 3 March 2014
Received in revised form 6 November 2014
Accepted 16 November 2014
Edited by Hermann Heilmeier.
Available online 24 November 2014
Keywords:
Isotope discrimination
Translocation
13
C enrichment
15
N depletion
Gas exchange
Sodium concentration

a b s t r a c t
After assimilation, plants often fractionate stable carbon and nitrogen isotopes among different organs
depending on synthesis and transport of metabolites. We investigated stable carbon and nitrogen isotope
compositions among leaves of different age (0 to 6 months) in two mangrove fern species (Acrostichum
danaeifolium Langsd. & Fisch. and Acrostichum aureum L.) from Mexico. Leaves of all ages were analysed
for 13 C, 15 N, carbon and nitrogen concentrations and gas exchange parameters. In both species, 13 C
slightly decreased with leaf age. Leaf salt concentration increased with leaf age, and thus did not decrease
13
C discrimination markedly. Enrichment in 13 C in younger versus older leaves can be explained by stage
of development: Carbon is assimilated and incorporated into autotrophic leaves, but also transported as
13
C-enriched carbohydrates into still expanding and more heterotrophic younger leavesindicated by
lower rates of photosynthesis. Depletion in 13 C in old autotrophic leaves, which export photosynthetic
assimilates, could mainly be explained by respiratory fractionation releasing 13 C-enriched CO2 . In contrast, 15 N values in A. danaeifolium increased with leaf age. This pattern could be related to the transport
of 15 N-depleted amino acids into younger leaves, and leaf construction with these compounds. In conclusion, 13 C depletion and 15 N enrichment with leaf age were described for other plant species earlier
and were explained by different mechanisms of carbon and nitrogen assimilation and the export of these
assimilates from older to younger leaves. These stable isotope patterns were approved for two mangrove
fern species in this study.
2014 Elsevier GmbH. All rights reserved.

Introduction
Fractionation of 13 C during photosynthesis in plants with the
C3 photosynthetic pathway (CalvinBenson cycle) results in 13 C
values between 22 and 32 with an average of 27 (Boutton,
1996; Farquhar et al., 1989; OLeary, 1981). Once plants have assimilated carbon, further 13 C fractionation processes take place. These
are caused by equilibrium reactions or kinetic processes catalysed
by specic enzymes (OLeary, 1981), or by fractionation associated
with transport of metabolites across organ boundaries or by fractionation during heterotrophic metabolism (Badeck et al., 2005).
In the latter, isotope fractionation may occur either due to compartmentation of the source organ or due to a metabolic branching
point or during the export process itself (Badeck et al., 2005).

Corresponding author. Tel.:+49 731 50 23310; fax: +49 731 50 23320.

E-mail addresses: martin.werth@uni-ulm.de (M. Werth),
klaus.mehltreter@inecol.edu.mx (K. Mehltreter), oscar.briones@inecol.edu.mx
(O. Briones), marian.kazda@uni-ulm.de (M. Kazda).
http://dx.doi.org/10.1016/j.ora.2014.11.001
0367-2530/ 2014 Elsevier GmbH. All rights reserved.

Within plants, 13 C values of different compounds vary. It has

been observed that lignin and lipids are usually depleted in 13 C
compared to the bulk plant material, while sugars, amino acids,
and hemicelluloses are enriched in 13 C (Boutton, 1996; Bowling
et al., 2008; Hobbie and Werner, 2004; Wiesenberg et al., 2004,
2008). The specic enrichment of 13 C often observed in transport
compounds like sucrose leads to an enrichment of 13 C in the roots
(Hobbie and Werner, 2004). On average, roots of C3 plants have
been shown to be enriched in 13 C by +1.2 compared to whole
shoots (Werth and Kuzyakov, 2010) and by +2.3 compared to
leaves (Bowling et al., 2008).
Signicant 13 C variation among different plant organs has
been demonstrated for autotrophic shoots and leaves versus
roots as well as among other heterotrophic plant organs, like
young leaves, stems, owers, fruits, and seeds (Badeck et al.,
2005; Bathellier et al., 2008; Bowling et al., 2008; Cernusak
et al., 2005; Damesin et al., 1998; Damesin and Lelarge, 2003;
Ghashghaie and Badeck, 2014; Lamade et al., 2009; OLeary, 1981).
Leaf age is of special interest for studying isotope fractionation,
because younger leaves show different physiological properties

M. Werth et al. / Flora 210 (2015) 8086

compared to older leaves (Medina et al., 2010; Terwilliger et al.,

2001).
Whereas carbon is directly assimilated from the air or remobilized from reserve carbohydrates, nitrogen as key nutrient to build
up the photosynthetic apparatus is translocated either from the
roots, storage organs or mature leaves to growing leaves. Studies on 15 N natural abundance among differently aged leaves are
scarce, but Yoneyama and Kaneko (1989) have shown that 15 N in
roots was lower than in leaves of Brassica campestris plants. They
explained this low 15 N in the roots by a supply of 15 N-depleted
amino acids from the shoots or by an efux of 15 N-enriched nitrate
to the shoots. Exudation of 15 N-enriched rhizodeposits could be a
further explanation.
Stable carbon and nitrogen isotope compositions of different
plant organs have been mainly studied for cultivated crops and
trees; however, there is still a need to conrm these ndings in
a number of wild plant species (Badeck et al., 2005; Yoneyama
et al., 2003). Therefore, we chose two mangrove ferns (Acrostichum
danaeifolium Langsd. & Fisch. and Acrostichum aureum L.) to study
13 C and 15 N natural abundances in autotrophic and heterotrophic
plant organs. Mangrove ferns are known to have a continuous leaf
production in a spiral sequence which makes them suitable to study
isotope fractionation among developing young leaves and fullydeveloped old leaves. It can be expected that expanding leaves are
heterotrophic and import C and N from fully-expanded autotrophic
leaves (Gary et al., 1998; Shishido et al., 1999). At the study site,
A. danaeifolium leaves are developed continuously at a rate of
14.6 0.44 leaves per year and have a mean life span of 7.7 months
(Mehltreter and Palacios-Rios, 2003). While A. danaeifolium is a
neotropical species, A. aureum is distributed pan-tropically. Both
species grow at the study site and follow the same leaf phenology
pattern (K. Mehltreter, pers. obs.).
A typical feature of mangrove soils is the high salt concentration which induces stress to plants growing on the site. Acrostichum
species can tolerate these conditions by increasing salt and sugar
concentrations in their tissues (Medina et al., 1990). When plants
are exposed to high salinity, stomatal closure is induced by physiological drought (Munns and Tester, 2008), gas exchange of the
leaves is reduced (Rawson et al., 1988), and leaf length and leaf
life span decrease (Sharpe, 2010). Additionally, leaf Na+ and C1
concentrations increase with leaf age in plants grown at increased
salt concentrations (Greenway et al., 1965) possibly because older
leaves have transpired for longer time (Rawson et al., 1988). As a
result, we expected that increasing amounts of salt from younger to
older leaves could cause a reduction in gas exchange parameters,
especially a decrease in stomatal conductance and ci , and consequently lead to decreasing 13 C discrimination and increasing 13 C
values according to Farquhar et al. (1989).
In this study we addressed the following questions: (1) Do 13 C
values of A. danaeifolium and A. aureum leaves decrease with leaf
age due to development from heterotrophic to autotrophic organs?
(2) Do 13 C values increase with increasing sodium accumulation
in plant leaves as an indicator of salt stress? (3) Do 15 N values
show a converse pattern to 13 C, i.e. do they increase with leaf
age due to metabolic processes and translocation of 15 N-depleted
compounds?

Materials and methods

Site description
The study was performed at the Biological Station of La Mancha
(19 36 00 N, 96 22 40 W), Veracruz, Mexico. Studied plants
were located at 100 m distance maximum to a brackish-water
lagoon, in the shady understorey of a forest dominated by black

81

mangroves (Avicennia germinans (L.) Stearn, Avicenniaceae) and

red mangroves (Rhizophora mangle L., Rhizophoraceae) as a second
minor contributor. The water level at the study site uctuates
little; the soils are always waterlogged. Climatic conditions are
hot and humid, with a dry season from November to May, when
mean precipitation is less than 45 mm per month. During the
winter (December to March) north winds help to close the lagoon
and disconnect it from the Gulf of Mexico. The water level in the
lagoon rises when the lagoons mouth is closed. Thus, the dry
season in La Mancha coincides with the highest level of the water
table and increased ooding of mangroves and wetlands. Mean
annual temperature was 25.8 C and mean annual precipitation
was 1193 mm for the period between January 1981 and December
2009 (data collected from the Climatological Station of La Mancha, Comision Nacional de Agua, Servicio Meteorolgico Nacional,
http://smn.cna.gob.mx/climatologia/Mensuales/ver/00030353.TXT).

Measurements and samplings

Six large, healthy plants of A. danaeifolium and A. aureum were
selected within an undisturbed mangrove forest fragment of about
1 ha size. Each leaf was tagged and its approximate age was assigned
according to its position on the erect fern rhizome, assuming a
leaf emergence rate of about one leaf per month (Mehltreter and
Palacios-Rios, 2003). Thus the youngest developing leaf crozier was
assigned age 0, the next still expanding leaf located at a circular
distance of about 120 degrees (clock or counter clockwise) was
assigned age 1 and each successive leaf was numbered consecutively up to the approximate age of 6 months.
On March 26, 2009, the net assimilation rate (A), the stomatal conductance (gs ), and the ratio of intercellular to atmospheric
CO2 concentration (ci /ca ) were determined with a portable photosynthesis system (LI-6400; LI-COR Inc., Lincoln, Nebraska, USA)
for three individuals (samples 1, 2, and 3) of each species. Gas
exchange was measured at a photosynthetic photon ux density
(PPFD) of 100 mol m2 s1 , a block temperature of the LI-6400
of 31.5 1.2 C, and a relative air humidity of 56.2 5.7%, corresponding to the general microclimatic conditions in the mangrove
understorey.
After the gas exchange measurements, we sampled about ten
leaets of each leaf and each of the 12 study plants, brought them
immediately to the laboratory of the biological station and dried
them in an oven for 72 h at 60 C. In Germany, dry leaf samples were
ground with an ultra-centrifugal mill (Retsch, Haan, Germany) with
an insert of 0.25 mm mesh size. After grinding of each leaf sample,
the mill was cleaned from remaining powder with compressed air,
and after each species and each leaf age, the mill was wiped out
with ethanol (70%).

Elemental and isotope analyses

For carbon (C), nitrogen (N), and isotope analyses, 0.7 mg ground
sample material was lled into tin capsules (4 mm 6 mm, IVA
Analysentechnik, Meerbusch, Germany). Analyses were performed
at an elemental analyser (Euro EA C/N analyser, EuroVector, Milan,
Italy) coupled with an isotope ratio mass spectrometer (Thermo
Finnigan Delta plus Advantage, Thermo Fisher Scientic Inc.,
Waltham, USA). Laboratory reference gases (CO2 and N2 ) calibrated
against USGS40 were used to obtain 13 C and 15 N values (Qi et al.,
2003). Acetanilide was used as a laboratory standard for isotopic
and elemental drift control. Isotopic differences 13 Creference or
15 Nreference among plant organs or between chemical compounds
and source plant organs cited from literature in the introduction

n.d.
n.d.
0.009**
0.005**

n.d.
n.d.
P

0.121
0.636
2.756
0.813

F
P

0.729
0.030*
0.125
2.106

0.263
0.929
Species
Leaf age (species)

P
F
F-ratios, P-values

Nested design ANOVA

0.617
0.524

1.7A 1.1
1.4A 2.1
2.6A 2.0
2.5A 1.9
2.7A 2.3
3.0A 1.5
1.6A 1.8
43.2A 0.8
41.7A 1.1
41.3A 2.1
40.4A 1.4
40.4A 1.8
41.1A 0.7
39.7A 2.6
30.2A 0.9
30.3A 0.6
30.0A 1.3
31.2A 1.3
30.6A 1.0
31.1A 0.3
30.8A 1.2

0.250
0.061

n.d.
n.d.

5.3A 1.4
8.4A 2.2
7.6A 4.2
8.3A 3.1
9.8A 3.8
10.4A 4.3
10.0A 3.3
10.2A 0.3
19.7AB 7.4
21.7AB 3.9
20.7AB 2.2
21.7B 2.4
22.7AB 3.8
21.9B 3.2
4.2A 0.1
2.4A 1.0
1.9A 0.3
2.0A 0.2
1.9A 0.2
1.8A 0.3
1.8A 0.3

9.0a 0.4
8.5a 1.9
9.6ac 2.0
11.2ab 2.7
12.0ab 2.1
13.5b 1.8
13.3bc 1.9
16.4ab 5.0
11.2b 1.8
14.7ab 4.1
17.2ab 2.3
15.9ab 2.3
16.5ab 3.8
17.3a 1.2
2.8a 1.0
3.9b 0.7
3.1ab 0.9
2.5a 0.3
2.6b 0.3
2.5a 0.5
2.3a 0.2

1.458
1.846

n.d.
3.33A 1.87
3.73A 2.33
4.76A 0.51
3.18A 1.36
4.62A 0.39
4.00A 1.72

n.d.
0.25a 0.13
2.71ab 1.36
4.04ab 0.25
5.30b 0.21
3.77ab 1.16
4.84ab 0.37

n.d.
n.d.

n.d.
0.07A 0.03
0.19AB 0.04
0.15AB 0.04
0.14AB 0.07
0.13AB 0.04
0.23B 0.06

n.d.
0.10ab 0.02
0.05a 0.03
0.05ab 0.01
0.10b 0.02
0.08ab 0.02
0.07ab 0.02

10.543
3.620

n.d.
0.83A 0.02
0.91A 0.05
0.87A 0.03
0.88A 0.08
0.86A 0.02
0.91A 0.04

ci /ca
n=3

A (mol m2 s2 )

gs (mol m2 s2 )

2.3a 1.4
2.0a 0.4
2.3a 0.5
2.8a 0.4
3.1a 1.1
3.3a 0.6
3.2a 1.0
41.2a 1.7
43.2a 0.9
42.8a 3.8
41.8a 1.7
41.1a 1.6
39.3a 2.1
40.1a 2.1
30.0a 0.7
30.2a 0.7
30.0a 1.3
30.6a 1.2
30.9a 1.2
30.7a 0.7
31.0a 1.2

A. danaeifolium
0
1
2
3
4
5
6
A. aureum
0
1
2
3
4
5
6

A highly signicant correlation was found between leaf 13 C values and approximate leaf age of A. danaeifolium: older leaves were
more depleted in 13 C than younger ones (Fig. 1a). Signicant correlations were also found for two specimens of A. aureum (rs = 0.89,
P < 0.01, and rs = 0.90, P < 0.05, data not shown). An overall correlation for all samples of A. aureum, however, was not signicant
(Fig. 1b). Compared to the oldest leaf, the roots were enriched in
13 C for four of six plants of each species (single data not shown).
In contrast to 13 C, 15 N values of A. danaeifolium signicantly
increased with leaf age (Fig. 2a). This could be shown by Spearmans
rank correlation for all plants of A. danaeifolium. For A. aureum, no
signicant correlation could be found, but there was a slight trend
to increased 15 N values of two to ve months old leaves. Even more
pronounced than for 13 C, the roots were enriched in 15 N compared
to the oldest leaf (P < 0.001 for A. danaeifolium).
Nested design ANOVA indicated that A. aureum had a higher stomatal conductance than A. danaeifolium (Table 1). Within species,
however, there was no consistent trend with leaf age. The ci /ca
ratio was mostly higher for A. aureum compared to A. danaeifolium

n=6

Results

c(Na) (g kg1 )

Statistical analyses were performed with Statistica (Version 6.1,

StatSoft Inc. 2002, Tulsa, USA) and SPSS (Version 21.0.0.0, IBM
Corporation, New York, USA). Normality of the data was tested separately for every species with the KolmogorovSmirnov test (for A.
danaeifolium: n = 39 for 13 C and carbon concentration; n = 38 for
15 N, nitrogen concentration, and C/N; n = 37 for sodium concentration; and n = 18 for photosynthetic rate, stomatal conductance,
and ci /ca ; for A. aureum: n = 36 for 13 C, carbon concentration,
15 N, nitrogen concentration, and C/N; n = 37 for sodium concentration; and n = 18 for photosynthetic rate, stomatal conductance,
and ci /ca ) and could be approved for all datasets, except for nitrogen concentration of A. aureum leaves. Homogeneity of variances
was tested with the Levene test and had to be negated for leaf
nitrogen concentration of both species, the C/N ratio, the photosynthetic rate, and the ci /ca ratio of A. aureum leaves. Nested design
ANOVA was used to determine differences between species and
among leaf ages for parametric data (using species as xed factor
and leaf age nested within species). Tukeys HSD test was used for
post hoc testing separately for every species after one-way ANOVA.
The KruskalWallis test was used for non-parametric data among
leaf ages of single species. Spearmans rank correlation coefcients
(rs ) were calculated for isotopic signatures versus leaf age and
linear regressions were calculated for leaf carbon concentrations
versus sodium concentrations. Visualisations were performed using
SigmaPlot (Version 9.0, Systat Software Inc., San Jose, USA).

C/N

Statistics

c(N) (g (100 g)1 )

where 13 Creference or 15 Nreference refer to a source plant organ pool,

and 13 CP or 15 NP refer to a sink pool in another plant organ or a
chemical compound.
For the determination of sodium (Na), 100 mg of each leaf sample was processed with 13.2 M HNO3 in a microwave digester
(MARS Xpress, CEM, Kamp-Lintfort, Germany). Sodium concentrations were determined using an atomic absorption spectrometer
(Vario 6, Analytik Jena, Jena, Germany).

15 N ()

 Nreference = 15 NP 15 Nreference ,

c(C) (g (100 g)1 )

or
15

13 C ()

13 Creference = 13 CP 13 Creference

Species/Leaf age months

and discussion are reported as delta over baseline (Coplen, 2011):

n.d.
0.96a 0.00
0.79a 0.02
0.75a 0.05
0.81a 0.02
0.83a 0.01
0.76a 0.03

M. Werth et al. / Flora 210 (2015) 8086

Table 1
Leaf nutrient, stable isotopic, and photosynthetic characteristics of different leaf ages of two mangrove ferns: Acrostichum danaeifolium and Acrostichum aureum. Results of nested design ANOVAs are given below parametric
data (13 C, carbon concentration, 15 N, sodium concentration, and stomatal conductance gs ). Post hoc testing was done by applying one-way ANOVA followed by Tukeys HSD test to every species. Differences in non-parametric
data (nitrogen concentration, C/N, photosynthetic rate A, and ci /ca ) were tested by KruskalWallis test separately within every species. Means ( standard deviations) followed by the same lowercase letters are not signicantly
different (P > 0.05) among leaf ages within A. danaeifolium, means followed by the same uppercase letters are not signicantly different among leaf ages within A. aureum. n. d.not determined, * P < 0.05, ** P < 0.01.

82

M. Werth et al. / Flora 210 (2015) 8086

83

Fig. 1. Leaf 13 C versus approximate leaf age of Acrostichum danaeifolium (a) and
Acrostichum aureum (b) from a Mexican mangrove forest. Closed symbols represent means standard deviations (4 n 6) of leaves. Open symbols represent 13 C
values of roots not included in the calculations of Spearmans rank correlation
coefcients (rS ).

Fig. 2. Leaf 15 N versus approximate leaf age of Acrostichum danaeifolium (a) and
Acrostichum aureum (b) from a Mexican mangrove forest. Closed symbols represent means standard deviations (4 n 6) of leaves. Open symbols represent 15 N
values of roots not included in the calculations of Spearmans rank correlation
coefcients (rS ).

(Table 1). For both species, the photosynthetic rate and the ci /ca
ratio were almost constant among leaf ages (Table 1). In both
species leaf sodium concentration showed increasing trends with
leaf age, which was signicant for A. danaeifolium (Table 1). The leaf
carbon concentration was negatively correlated with leaf sodium
concentration in both species (Fig. 3).

developmental stages has been shown earlier by 13 CO2 tracer studies (Amziane et al., 1997; Delens et al., 1994; Maillard et al.,
1994a, 1994b). The rst stage is predominantly heterotrophic,
i.e. fuelled by organic carbon imported from elsewhere in the
plant (Terwilliger et al., 2001). The second stage is predominantly
autotrophic, i.e. growth is powered by the products of the leafs own
photosynthesis. In the third stage, organic carbon is exported. From
our data, we could not explicitly distinguish between leaves in the
second stage of pure assimilation and leaves in the third stage of
assimilation and export of carbon. Thus, we discuss our data by a
simplied two-stage model with a rst more heterotrophic stage
and a second more autotrophic stage.
Enrichment in 13 C is most evident during the heterotrophic
growth stage and is supposedly a consequence of the heterotrophic
carbon source for growth. This rst stage was more obvious for A.
danaeifolium, since the 1-month-old leaf was almost not photosynthetically active and had a high 13 C value of 30.2 0.7 (Table 1).
Cernusak et al. (2009) have discussed six hypotheses how to explain
13 C enrichment of heterotrophic versus autotrophic plant organs.
Briey, these hypotheses explaining isotopic variation among plant
organs were (1) a variation in biochemical composition between
autotrophic and heterotrophic organs coupled with export or
import of carbon (older leaves contain more 13 C-depleted lipids
and lignin and export carbon to younger leaves, while younger
leaves contain more 13 C-enriched cellulose and import carbon from
older leaves (Hobbie and Werner, 2004)), (2) seasonal separation
of growth of different plant organs, (3) day versus night sucrose

Discussion
Stable carbon isotope compositions among leaves of different
age
The 13 C enrichment of younger A. danaeifolium and A. aureum
leaves relative to older ones could be related to drought effects
during the dry season. One could argue that leaves 5 and 6 could
probably have grown with reserves produced in the rainy period
and leaves 4 to 0 could have grown with reserves produced progressively more into the dry period, in the months immediately
before leaves were sampled. The former would be relatively more
depleted and the latter more enriched in 13 C due to a reduced
isotopic discrimination related to drought. We can exclude such
drought effects, however, due to the permanently waterlogged soils
at our study site. Another hint to permanently humid conditions
in the understorey can be seen from high ci /ca values measured
immediately before leaf sampling (Table 1).
The isotopic patterns reported in this study can be rather related
to different stages of leaf development. The differentiation of three

84

M. Werth et al. / Flora 210 (2015) 8086

of the leaf (Farquhar et al., 1989; Handley et al., 1997; Pakniyat

et al., 1997; Poss et al., 2000; Winter and Holtum, 2005). In both
Acrostichum species, however, Na accumulated with leaf age and
concentration was highest in the oldest leaves but these were
depleted in 13 C (Table 1). Thus, salinity should have had an opposite effect on 13 C values than observed in our results, negating
our second hypothesis. We may also conclude that compartmentalization is working so well, with increasing Na stored away in the
vacuoles, that it has no effect on 13 C values. Concentration of carbon in the leaves, however, was affected by accumulation of salt for
both species: increasing amounts of Na in the leaves were related to
decreasing carbon concentrations independent of leaf age (Fig. 3).
In conclusion, (1) drought induced by salinity can be excluded in our
study as explanation for different 13 C values among leaf ages and,
furthermore, (2) soil dryness cannot have caused drought due to the
site location in the permanently wet mangrove understorey. The
lower Na concentrations in A. aureum compared to A. danaeifolium
leaves could indicate a better adaptation of the former to salt stress
by controlling Na uptake by the root (Mehltreter and Palacios-Rios,
2003).
Which one of these seven possibilities has led to 13 C-enriched
younger versus 13 C-depleted older leaves cannot be claried in this
study. The most likely reasons, however, are variation in biochemical composition coupled with carbon translocation, day versus night
sucrose utilisation, respiratory fractionation, carbon xation by PEP
carboxylase, or a combination of some or all of them.
Stable nitrogen isotope compositions among leaves of
different age

Fig. 3. Leaf carbon concentration versus leaf sodium concentration of Acrostichum

danaeifolium (a) and Acrostichum aureum (b) from a Mexican mangrove forest. Symbols represent means standard deviations (4 n 6), solid lines show signicant
regression lines. Leaf ages are indicated inside the symbols (0 to 6 months old).

utilisation producing 13 C depletion or enrichment of heterotrophic

tissue, respectively, (4) respiratory fractionation, i.e. respiring
autotrophic leaves would preferably respire 13 C-enriched C compounds leading to a depletion of their tissues, (5) increased xation
of 13 C-enriched HCO3 by PEP carboxylase in heterotrophic plant
organs (Gessler et al., 2009; Tcherkez et al., 2011), and (6) developmental variation in photosynthetic discrimination against 13 C.
The second, autotrophic growth stage, where carbon is assimilated and exported to other plant parts, is most evident for 3- to
6-months-old leaves of both species, because their photosynthetic
rates were higher than for younger leaves (Table 1). In autotrophic
leaves, which are still growing and not completely mature, the
lighter carbon isotope is preferably incorporated at the place of
assimilation and is used for the build-up of lipids and lignin, which
are 13 C depleted (Hobbie and Werner, 2004). The heavier carbon
isotope is transported as 13 C-enriched sucrose to younger predominantly heterotrophic leaves. Additionally, sucrose exported at
night-time from autotrophic to heterotrophic leaves would be relatively enriched in 13 C, because it is associated with transitory starch
degradation (Cernusak et al., 2009; Gessler et al., 2008; Tcherkez
et al., 2004, 2011). As a consequence of these effects, 13 C values
could have become the most negative for older autotrophic leaves
of mangrove ferns studied here.
In relation to our study site, salinity could have an additional
effect on differences in 13 C values among leaves of different age.
Salinity increases Na concentration in the leaf, which has been
shown to be associated with physiological drought inducing stomatal closure and low ci /ca ratios and thus leading to 13 C enrichment

Our third hypothesis was supported: younger leaves were 15 N

depleted compared to older leavesa pattern completely opposite
to stable carbon isotope compositions.
For the 1-month-old leaves of A. danaeifolium, there is strong
evidence that they are mainly heterotrophic, indicated by their
low rates of photosynthesis. The availability of products of photosynthesis, respiration and photorespiration pathways, such as
carbon skeletons, ATP, ferredoxin and NADH, however, are essential for nitrogen assimilation (Masclaux-Daubresse et al., 2010).
Thus, young leaves mainly import amino acids and low-molecular
nitrogen compounds from other parts of the plant, because low
photosynthetic activity in young leaves may not produce enough
carbon skeletons for amino acid synthesis.
Consequently, we assume that nitrogen in young leaves of
both Acrostichum species is imported in the form of amino
acids from older leaves. Ammonium, taken up from the soil, or
coming from nitrate reduction, photorespiration, or amino acid
recycling, is mainly assimilated in the plastid/chloroplast by the
glutamine synthetase/glutamine 2-oxoglutarate amino transferase
cycle (GS/GOGAT cycle) (Lea and Miin, 1974; Lea and Forde,
1994). The glutamine synthetase (GS) xes ammonium on a glutamate molecule to form glutamine. Kinetic 15 N fractionation takes
place during this process of glutamine synthesis (Yoneyama et al.,
1993) leading to enrichment of the educts, i.e. unreacted nitrate
or ammonium (and thus the old bulk leaf) and depletion of the
products (mobile amino acids). Yoneyama et al. (1993) have shown
that glutamine synthetase isolated from spinach leaves caused 15 N
depletion between NH4 + and glutamine of about 16.5 in vitro.
Further discriminations against 15 N take place during build-up of
other amino acids involving glutamate and asparagine synthetase
and amino-transferase enzymes (Macko et al., 1986; Tcherkez,
2010). Our data, therefore, suggest that 15 N-depleted amino acids
such as glutamate, glutamine, and asparagine are transported into
other plant organs such as younger leaves, where they are used for
protein synthesis and thus lead to 15 N depletion of the younger
and 15 N enrichment of the older bulk leaves. Older bulk leaves

M. Werth et al. / Flora 210 (2015) 8086

might especially become 15 N enriched by accumulation of unreacted nitrate in their vacuoles.

In water-logged or ooded soils such as at the study site,
ammonium is the most likely form of inorganic nitrogen (MasclauxDaubresse et al., 2010). Due to temporary low water tables and the
release of oxygen in the rhizosphere by radial oxygen loss of the
roots (Armstrong, 1971), aerobic conditions could occur leading to
nitrication. Like in well-aerated terrestrial soils, nitrate would be
taken up by the plants, transported to the leaves and be reduced
to ammonium,which would then be incorporated into amino acids.
Several studies have shown that nitrate reduction induces large 15 N
depletion of about 15 of ammonia compared to nitrate (Ledgard
et al., 1985; Werner and Schmidt, 2002) or about 19 of amino
acids compared to nitrate (Yoneyama et al., 2003). Hence, 15 N fractionation during nitrate reduction followed by amino acid synthesis
in older leaves could be another reason for 15 N-depleted younger
leaves after amino acid translocation.
In very old leaves, N-containing compounds (like proteins,
chlorophyll, etc.) are degraded during leaf senescence (MasclauxDaubresse et al., 2010). Nitrogen can then be remobilized from
senescing leaves to expanding leaves at the vegetative stage as well
as from senescing leaves to seeds at the reproductive stage (Diaz
et al., 2008; Lematre et al., 2008; Malagoli et al., 2005). During this
degradation, the isotopic lighter part of these compounds might
be easier decomposed, and these 15 N-depleted N species are then
transported into younger leaves. With advancing substrate degradation, also the 15 N-enriched part of decomposing compounds will
be transported to younger leaves. This kind of nitrogen source
might have been used more likely for A. aureum, because, in some
replicates 5- and 6-months-old leaves had lower 15 N values than
2- to 4-months-old leaves (but comparable to 0- to 1-monthsold leaves). This can be explained by the advanced degradation of
chloroplast enzymes, i.e. also the heavier nitrogen is exported to
younger leaves.
Conclusions
Carbon and nitrogen assimilation in mangrove fern species
resulted in two opposite gradients of stable isotope compositions:
13 C enrichment, but 15 N depletion, of younger compared to older
leaves. After assimilation, a part of the carbon is incorporated into
autotrophic older leaves, while the remaining 13 C-enriched sugars are transported into still less photosynthetically active younger
leaves. Alternative explanations could be day versus night sucrose
utilisation or carbon xation by PEP carboxylase in heterotrophic
leaves. Depletion in 13 C in old autotrophic leaves, which export
photosynthetic assimilates, could mainly be explained by respiratory fractionation releasing 13 C-enriched CO2 . The fractionation of
nitrogen occurs during the processes of nitrate and nitrite reduction to ammonium and the following synthesis of amino acids
in autotrophic older leaves. Since the lighter isotope reacts faster
in these reactions, 15 N-depleted amino acids are transported into
younger leaves, where they will be used for building-up of leaf
structures. Compound-specic isotope analyses (e.g. of sucrose or
amino acids) would help for further explanation of the isotopic
composition of differently-aged leaves in mangrove fern species
as well as in other species.
Acknowledgements
We are thankful to Eliana Schacher for stable isotope sample
preparation, Wolfgang Armbruster for stable isotope analyses, as
well as Ellen Salzer and Hans Malchus for the C, N, and Na analyses
of leaf samples. Klaus Mehltreter was supported by the Instituto
de Ecologa, A.C. (20030-10796). We are very grateful to three

85

anonymous reviewers and the editor Prof. Hermann Heilmeier who

gave valuable comments on earlier versions of this manuscript.
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