Aquatic Botany xxx (xxxx) xxxxxx

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Aquatic Botany
journal homepage: www.elsevier.com/locate/aquabot

Stable carbon isotopic composition of submerged plants living in karst water

and its eco-environmental importance

Pei Wanga,b,c, Gang Hua,b,d, Jianhua Caoa,b,
a
Karst Dynamics Laboratory (MLR and GZAR), Institute of Karst Geology (CAGS), Guilin 541004, China
b
International Research Center on Karst, UNESCO, Guilin 541004, China
c
China University of Geosciences, Beijing 100083, China
d
Guilin University of Technology, Guilin 541004, China

A R T I C L E I N F O A B S T R A C T

Keywords: The stable carbon isotopic composition of submerged plants (13CP) can be controlled by physiological and
Zhaidi river environmental factors. Herein, we took advantage of a short, natural karst river with an annual mean
Submerged plants bicarbonate (HCO3) value of 3.8 mmol L1 to study the stable carbon isotopic composition of submerged
Stable carbon isotope plants along the river and the inuence of environmental conditions on the 13CP values. The 13CP values of
Dissolved inorganic carbon
Ottelia auminata, Potamogeton wrightii, Vallisneria natans, and Hydrilla verticillata from upstream to downstream
Karst water environment
show a gradient distribution and ranged from 34.78 to 27.83, 36.56 to 23.70, 35.06 to
25.29, and 38.56 to 26.32, respectively and even more depleted values for the rst two species at
the uppermost site. Diurnal variation of water chemistry and concentration of the dissolved inorganic carbon
(DIC) and the stable carbon isotopic composition of DIC (13CD) indicate that the river has a very high net
photosynthetic rate. The gradient distribution of 13CP values was consistent with CO2 being a declining source
of inorganic carbon for photosynthesis in the downstream transect. The results demonstrate that the high DIC
concentration with lower negative 13C value, particularly in karst water environment has a signicant role in
controlling the stable carbon isotopic composition of submerged plants living in it.

1. Introduction boundary layer around submerged plant surfaces, CO2 is generally less
available in water than in air. In addition, photosynthesis can also be
Much recent work has focused on the inter-relationships between further limited by the intermittent depletion of CO2 produced when the
the ecological, hydrological, and physico-chemical processes in ground- rates of photosynthetic demand exceed those of replenishment and by
water/surface water interactions (Sophocleous, 2002; Hancock et al., the generation of high concentrations of oxygen that promotes photo-
2009; Bork et al., 2009). One of the most important interactions respiration (Maberly and Madsen, 2002a; Pedersen et al., 2013).
between surface water and groundwater occurs in spring-fed rivers in Therefore, submerged plants require high concentrations of DIC to
which groundwater chemistry controls solute inputs to surface water saturate their photosynthesis. A number of submerged macrophytes
and represents the initial control on river ecology (Holmes, 2000; possess physiological and biochemical features that ameliorate the
Harvey and McCormick, 2009). Surface water function and biodiversity eect of low carbon availability and minimize the eects of its potential
are controlled by interactions between the physical and chemical limitation (Spence and Maberly, 1985). Klavsen et al. (2011) summar-
environments, in addition to the physiological and biochemical accli- ized that avoidance and exploitation strategies are eective ap-
mation and adaptation of organisms as well as their short-term proaches to obtain sucient CO2 for photosynthesis. In addition,
behavioral responses (Maberly et al., 2015). amelioration strategies based on carbon dioxide concentrating me-
Submerged plants are important primary producers, maintaining chanisms (CCMs) can be present, including the ability to use HCO3,
the ecological balance of aquatic systems and taking part in biogeo- crassulacean acid metabolism (CAM) and C4-like photosynthesis
chemical cycling. However, unlike terrestrial plants, it is a common (Maberly and Madsen, 2002b; Bowes, 2011; Dou et al., 2013). The
phenomenon that photosynthesis and growth are strongly restricted to ability of HCO3 utilization is thus particularly advantageous under
DIC supply (Maberly and Spence, 1989). Because of the low diusion alkaline conditions, and the most widespread CCM involves the use of
rates of gases in water and the existence of a well-developed diusive HCO3 as an alternative carbon source. In terrestrial plants, the 13CP

Corresponding author at: Karst Dynamics Laboratory (MLR and GZAR), Institute of Karst Geology (CAGS), Guilin 541004, China.
E-mail address: ecogene_wp@cugb.edu.cn (J. Cao).

http://dx.doi.org/10.1016/j.aquabot.2017.03.002
Received 1 May 2015; Received in revised form 19 December 2016; Accepted 4 March 2017
0304-3770/ 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Pei, W., Aquatic Botany (2017), http://dx.doi.org/10.1016/j.aquabot.2017.03.002
P. Wang et al. Aquatic Botany xxx (xxxx) xxxxxx

value is closely related to the photosynthetic pathway used in carbon respectively. Dominant algae in Zhaidi River are Synedra sp., Navicula
xation. The 13CP value of freshwater aquatic plant is aected by the sp. and Pinnularia sp., all belonging to Bacillariophyta, with an average
type and extent of the CCM and also by the stable isotope signature of density of 0.34 105 ind./L. At the inlet of the Zhaidi River, the
the carbon source and has been found to vary from 50 to 11 chemical composition is dominated by Ca2+ and HCO3, with annual
(Keeley and Sandquist, 1992). mean concentrations of 1.9 mmol L1 and 3.8 mmol L1 respectively
Ecological processes, particularly the photosynthesis of macro- (Pei, 2012). The annual discharge at the outlet ranges from 33 to
phytes, can signicantly impact the hydrochemical characteristic of 13000 L s1.
surface water fed by underground water (de Montety et al., 2011).
Photosynthesis of macrophytes is considered to be a crucial biochemical 2.2. Field methods
process in controlling the DIC diurnal cycling in spring-fed surface
water (Liu et al., 2008). The 13CD value has been used to improve the Temporal variation in water chemistry was assessed at the source of
understanding of the carbon cycle and diel process by macrophytes in the Zhaidi River and the point upstream from the conuence with the
the catchment (Clarke, 2002; Heernan and Cohen, 2010; Parker et al., Chaotian River (Fig. 1C). The sampling survey started from 11:00 am on
2010; Poulson and Sullivan, 2010). The 13CD value in the upstream is September 10 and lasted to 15:00 pm on September 12, 2014. Water
mainly controlled by geochemical processes, while in the downstream, temperature, dissolved oxygen (DO) and pH were monitored and
it value is mainly aected by photosynthesis and respiration of recorded by an oxygen meter (YSI6400, YSI, USA) at 5-min interval
macrophytes (Parker et al., 2007; Poulson and Sullivan, 2010). A at the two locations. The optical DO sensor was calibrated to atmo-
variety of studies focused on the diurnal variation and the utilization spheric oxygen concentrations before deployment and veried in the
of DIC by macrophytes in spring-fed rivers, particularly on the laboratory after deployment to be within 3% of 100% saturation. The
calculation of submerged macrophyte capacity for a karst carbon sink pH sensor was calibrated using pH 7 and pH 4 standard solutions in the
(Neal et al., 2002). However, it is still unclear for certain submerged laboratory the day before deployment, the drift in pH electrodes after
plants what the 13CP value is and which carbon sources they tend to deployment was 0.01 pH unit.
use in photosynthetic processes in karst water environment. Therefore, At each site, water samples for stable carbon isotope measurement
the 13CP values at various sites along the karst river, along with diurnal were collected with a 100 mL disposable sterile syringe at 1-h intervals
variations of DIC species and concentration, as well as the 13CD values, during the daytime from 5:00 am to 8:00 pm and 3-h intervals
were monitored to assess the photosynthetic carbon source and karst overnight. Each water sample was ltered through a Millipore lter
impact on the stable carbon isotopic composition of submerged plants. of 0.45 m pore size and preserved in a 50 mL polyethylene bottle
without any air-space after injecting three drops of a saturated solution
2. Materials and methods of HgCl2 to prevent microbial alteration. At 6 hourly intervals, water
samples were collected at the upstream and downstream site for the
2.1. Site description analysis of major water elemental components (K+, Na+, Ca2+, Mg2+,
SO42, Cl, HCO3, OH, and CO32). The water was ltered through
The Zhaidi karst underground river system is located in eastern a Millipore lter of 0.45 m pore size and stored in 596 mL plastic
Guilin Haiyang village Lingchuan County, Guangxi Zhuang bottles without any air-space. The water samples were stored in
Autonomous Region, China (Fig. 1). Its geographic coordinates are portable ice boxes until the evening when they were sent back to the
1103236 to 1103722 E, 251359 to 251819 N, with approxi- laboratory and kept in a refrigerator at 4 C until analysis.
mately 32.7 km2 of recharge area. The recharge area is mainly The submerged plant samples from sites A to F were collected by
comprised of Devonian limestone, which covers 89.5% of the total hand and washed repeatedly with a soft brush to remove adhering
catchment. In the catchment, underground rivers, karst caves, karst material and epiphytic algae from the surface of the leaves. Plant shoots
sink holes, underground river skylights, and karst depressions are fully were transferred to ziplock polythene bags and stored in portable ice
developed (Chen et al., 2013). Meanwhile, the main geomorphology is boxes.
peak cluster with thin soil; scrub and grass are the dominant vegetation.
The main land-use types are farmland and orchard in the depression, 2.3. Laboratory analyses
whereas in the middle and western regions, the rock desertication is
very serious. The area has a subtropical monsoon climate, hot and The water samples were analyzed for major cations (K+, Na+, Ca2+
rainy; the annual mean temperature is about 18 C19 C, and it and Mg2+) with an IRIS Intrepid II XSP (Thermo Scientic, USA). The
receives an annual rainfall of about 1650 mm. The rainy season begins anions Cl and SO42 were analyzed with an 861 Advanced Compact
in April and ends in August, and this period accounts for approximately Ion Chromatograph (Metrohm, Switzerland). The analytical precision
60% of the annual precipitation total. The rainfall is collected in the was better than 5% based on duplicate measurements of internal
depression and charges the underground water through underground standards. Water pH was measured with SevenMulti pH meter
river skylights, karst funnels, karst sink-holes, karst mountain foot (Mettler Toledo, USA) with a precision of better than 0.01 unit and
holes. The karst aquifer medium is characterized by two structures, the concentration of HCO3, OH and CO32 in a 50 mL sample was
enormous underground pipelines and karst ssures. The groundwater titrated within two days of collection by using 0.05 mol L1 HCl that
ows through the underground pipeline from north to south and is had been standardized against 0.05 mol L1 Na2CO3. The error calcu-
discharged into the Chaotian River via the Zhaidi River, which is the lated by averaging numerous duplicate samples was 0.03 mg L1.
site of this study (Fig. 1B). The concentration of free CO2 was calculated by the geochemical
The Zhaidi River has a total length of 512 m and is mostly 26 m modeling program PHREEQC (Parkhurst and Appelo, 1999).
wide and 0.62.2 m deep. The river has been channelized on both sides In the laboratory, all plant samples were removed from the
with a wall. The river sediments, which range in pH value from 8.23 to refrigerator, and ultrapure water (Milli-Q, Millipore, Germany) was
8.88, are mainly composed of sand grains with a diameter of 0.075 used to carefully rinse the sample twice. Plants were then dried at
2 mm, and the organic matter content is between 0.12 and 0.95% 105 C for 12 h to deactivate enzyme and then dried at 50 C to a
(unpublished data). The Zhaidi River is colonized by eight species of constant weight. Dried samples were ground in an agate mortar and
submerged plants aliated to four families and six genera (Wang et al., passed through a 100 sieve mesh. A small amount of the powdered
2015). Among them, Ottelia acuminata, Potamogeton wrightii, Vallisneria plant sample was put, together with a copper oxide wire, into a 6 mm
natans and Hydrilla verticillata are the dominant species, and these diameter silica tube. The tube was sealed under vacuum and combusted
species have an average fresh biomass of 526, 357, 977, and 193 g m2, for 5 h at 850 C in a mue furnace. Liquid N2 and dry ice were used to

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Fig. 1. Location of the study area in China (A), showing the catchment area (dashed line), the source and the downstream ow to the Zhaidi river (B) and six plants sampling sites A-F
distribution in Zhaidi river (C).

Table 1
Stable carbon isotope composition of four species at dierent sites.

Species A B C D E F

Ottelia acuminata 40.48 34.78 34.95 30.61 34.80 27.83

Vallisneria natans 35.06 34.82. 33.32 34.92 25.29
Potamogeton wrightii 39.16 36.56 35.83 33.09 34.40 23.70
Hydrilla verticillata 38.56 34.55 34.00 26.32
Distance (m) 0 85 128 260 332 512

Species not present at the site. The distances are from the source to each sampling site.

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extract and purify the sample, and the liberated CO2 was analyzed with (Fig. 2). The diurnal change of DIC in water demonstrated that
a Stable Isotope Mass Spectrometer (MAT253, Thermo Scientic, USA). submerged plants utilized the DIC as carbon sources for photosynthesis,
The instrument precision is better than 0.2 based on three replica- simultaneously leading to a diurnal pH change of about 0.26 units (pH
tions. The isotope data are reported in the conventional delta notion 7.467.72). The DIC together with the DO variation exhibited that
() versus Vienna Pee Dee Belemnite (V-PDB). strong photosynthesis occurred in the river. This is consistent with
signicant photosynthesis by the submerged plants causing the diel
3. Results variation of dissolved inorganic carbon isotope in the river by consum-
ing HCO3 and CO2.
3.1. Stable carbon isotopic composition of submerged plants

4. Discussion
The stable carbon isotope composition of 21 plant samples belong-
ing to four species is shown in Table 1. The 13CP value of each species
4.1. Inuencing factors for measured 13CP values
became less depleted from upstream to downstream of the Zhaidi River.
The most negative 13CP value appeared at the upstream with a value of
The values of 13CP values reported here for a karst river ranging
40.48, and the least negative 13CP value appeared at the down-
from 40.48 to 23.70 are consistent with the range reported by
stream with a value of 23.70. At the rst site where all species
Keeley and Sandquist (1992). The two factors inuencing 13CP value
were found (site B, 85 m from the source, Fig. 1C), there was a 3.78
are the value of the source (complicated by whether or not CO2 or
range in 13CP values and at the downstream site there was a 4.13
HCO3 was used and in what proportion) and the extent of discrimina-
range of values among the four species.
tion which will depend largely on the extent of carbon limitation which
is controlled by the concentration of CO2 at the active site of Rubisco
3.2. DIC species and concentrations (Jasper et al., 1994; Lin and Wang, 2001). In Zhaidi River, the 13C
value of the carbon source is the reason that led to a gradient in 13CP
The discharge of the spring-fed river was moderate and stable values for a certain species at dierent sites. In this study, the 13CCO2
during the monitoring periods, with a ow of 130 L s1. At the and 13CHCO3- are respectively 22.21 and 13.73 at the up-
upstream site, the mean temperature and pH were respectively stream and 21.66 and 13.36 at the downstream sites, calcu-
1.68 C and 0.13 lower than at the downstream site. At both sites, the lated after Mook et al. (1974) by using the mean temperature,
main cations and anions were Ca2+ and HCO3, and DIC consisted of concentration of CO2, HCO3 and CO32, the 13CD, and 13Cp at both
HCO3 and CO2 (Tables 2a and 2b). At the upstream site the mean monitoring sites. From upstream to downstream, not only the 13C
concentrations of HCO3 and CO2 were 3.90 and 0.24 mmol L1, while values of CO2 and HCO3 become more depleted but also the ratio of
at the downstream site they had decreased to 3.78 mmol L1 and HCO3/CO2 become larger, which may cause the variation of carbon
0.12 mmol L1, respectively. Consequently, the molar ratio of HCO3 source. For terrestrial plants as well as submerged plants, CO2 is the
to CO2 declined from 16 at the upstream site to 32 at the downstream optimal inorganic carbon for photosynthesis, however many kinds of
site. submerged plants have the ability of using HCO3 (Madsen, 1993).
The results here showed that at night the 13CD values were nearly
3.3. Diurnal variation of DIC isotopic composition the same upstream and downstream, implying that the dierences
during the day were caused by photosynthesis and not by other
The 13CD values in the river showed a signicant spatial and processes such as degassing of CO2. The lesser depletion in 13CD
temporal variation (Fig. 2). Compared with upstream, diurnal variation values during the day is consistent with a greater photosynthetic
of 13CD at the downstream indicated that rapid photosynthesis had removal of CO2 compared to HCO3 since the former is more depleted
taken place, which was proven by the diurnal variation of the dissolved in 13C (Mook et al., 1974). The downstream reduction in depletion of
oxygen (Fig. 3). The strong photosynthetic processes of submerged 13CP will be partly caused by this lower depletion, but this only
plants consumed abundant inorganic carbon, which is consistent with accounts for a maximum of about 1 while there was downstream
the variation in HCO3 and CO2 concentration and 13CD. The mean dierence of about 10 in 13CP in the plants. This is likely to be
13CD value was 0.52 less negative at the downstream site compared caused by altered reliance on dierent forms of inorganic carbon for the
to 14.24 at the upstream site. The diurnal variation value was less upstream vs the downstream plants. Unlike terrestrial plants, certain
than 1, with the highest values occurring between 3:00 p.m. and 4:00 submerged plants may use HCO3 in addition to CO2 (Allen and
p.m. and the lowest values occurring during the night between 2:00 Spence, 1981; Maberly and Spence, 1989). Previous studies have shown
a.m. and 3:00 a.m. at which time the carbon isotope composition were that Ottelia acuminata, Potamogeton wrightii, Vallisneria natans, and
nearly the same at both sites. The DIC in the water, which consisted of Hydrilla verticillata have the ability to utilize both HCO3 and CO2 as
HCO3 and CO2, decreased during the day but rose during the night carbon sources (Prins et al., 1979; Prins et al., 1980; Bowes, 2011; Dou

Table 2a
Main physico-chemical conditions at site A.

Time Temperature pH K+ Na+ Ca2+ Mg2+ Cl SO42 HCO3 CO2

11:18 19.85 7.22 0.80 1.00 74.36 3.98 2.42 9.26 232.75 16.72
17:18 19.84 7.43 0.86 0.99 73.96 3.97 2.42 9.25 238.96 10.56
23:18 19.83 7.44 0.82 1.00 74.45 3.99 2.42 9.26 234.31 10.12
5:18 19.83 7.45 0.80 1.03 75.5 4.07 2.42 9.22 245.17 10.12
11:18 19.83 7.45 0.83 1.01 74.84 4.06 2.45 9.24 232.76 9.68
17:18 19.82 7.45 0.81 1.01 73.57 4.00 2.42 9.15 235.86 9.68
23:18 19.82 7.45 0.82 1.02 74.18 4.05 2.44 9.19 237.41 9.68
5:18 19.81 7.45 0.76 1.15 81.59 4.26 2.43 9.29 235.86 9.68
11:18 19.82 7.46 0.81 1.04 77.99 4.06 2.54 9.26 238.96 9.68
15:18 19.82 7.46 0.82 1.02 77.30 4.04 2.48 9.29 246.72 10.12

In the Table, the unit for Temperature is C, for cations and anions the units are mg L1.

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P. Wang et al. Aquatic Botany xxx (xxxx) xxxxxx

Table 2b
Main physico-chemical conditions at site F.

Time Temperature pH K+ Na+ Ca2+ Mg2+ Cl SO42 HCO3 CO2

11:18 21.32 7.46 0.86 1.01 75.91 4.05 2.4 9.03 237.41 9.24
17:18 22.05 7.64 0.83 1.01 75.00 4.04 2.41 9.20 229.65 6.16
23:18 21.05 7.47 0.97 1.06 76.59 4.09 2.46 9.18 232.75 9.24
5:18 20.91 7.47 0.83 1.01 76.52 4.08 2.39 9.11 234.31 9.24
11:18 21.41 7.58 0.83 1.00 74.41 4.00 2.43 9.15 226.55 7.04
17:18 22.35 7.68 0.81 1.02 73.91 4.02 2.42 9.09 223.44 5.72
23:18 21.03 7.49 0.81 1.04 74.52 4.02 2.45 9.10 235.86 9.24
5:18 20.89 7.48 0.8 1.01 74.96 4.06 2.42 9.20 234.31 9.24
11:18 21.41 7.58 0.83 1.02 73.49 3.99 2.43 9.21 232.76 7.04
15:18 22.70 7.72 0.81 1.02 73.18 3.97 2.42 9.20 221.27 5.28

In the Table, the unit for Temperature is C, for cations and anions the units are mg L1.

Fig. 2. Diel variation of concentrations of HCO3 and CO2, and of the 13C value of Fig. 3. Diel variation of temperature, pH and dissolved oxygen at the upstream (solid
dissolved inorganic carbon at the upstream (circle) and downstream (triangle) sites. Night line) and downstream (dashed line) sites. Night time is shown by hatched shading.
time is shown by hatched shading.
4.2. Environmental importance
et al., 2013; Zhang et al., 2014). The reduced depletion is consistent
with a greater reliance on HCO3 at the downstream site where the The CO2-HCO3-CO32 system in water may be described by the
concentrations of CO2 are lower during the day. It is also possible that equation:
the more depleted values downstream result from carbon limitation.
CO2 (aq) + H2O HCO3 + H+ CO32 + H+
Madsen and Maberly (1991) found carbon limitation at similar CO2
concentrations to those reported here in macrophytes from a Danish which establishes an equilibrium mixture of H2CO3, HCO3, and
stream. A similar downstream change in 13CP was reported down a CO32 that make up the DIC fraction. At a pH between 7 and 9,
longer transect at a French karst system, although in this case it was approximately 95% of the carbon in the water is in the form of HCO3,
caused by changing species composition away from species dependent and at a pH higher than 10.1, CO32 predominates (Dreybrodt, 1988; Li
on CO2 near the source to species able to use both CO2 and HCO3 and Yin, 2008; Manahan, 2000; Stumm and Morgan, 2012). In our
downstream (Maberly et al., 2015). In non-karst water, the carbon study, 95.73% of DIC existed in the form of HCO3 and the dissolved
isotopic composition of submerged plants ranged from 27.91 to CO2 only contributed 4.27% at the upstream site. Downstream, the
approximately 17.16 with 22.32 for Vallisneria natans and proportions of HCO3 and CO2 were 97.77% and 2.23%, respectively.
17.16 for Hydrilla verticillata, which is less negative than in this In the karst catchment, limestone dissolution can remove atmo-
study (Huang et al., 2003). Therefore, we suggest that in Zhaidi River sphere/soil CO2, directly giving rise to high HCO3 concentrations,
the 13CP values of the four species can be explained by the utilization generally 35 mmol L1, which are several-fold higher than in non-
of HCO3 as a carbon source in a karst water environment. karst water (Cao et al., 2011; Cao et al., 2012), and the alkalinity is

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P. Wang et al. Aquatic Botany xxx (xxxx) xxxxxx

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