Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72
DOI: 10.17581/bp.2020.09207

Interspecific variation in foliar
nutrients and isotopes of submerged
macrophytes in the Cau Hai Lagoon,
the typical brackish lagoon in Vietnam

Dang Thi Nhu Y1,2*, Nguyen Tien Hoang7, Pham Khac Lieu3,
Hidenori Harada5, Keisuke Koba4, Natacha Brion6, Duong Van Hieu8,
Nguyen Van Hop8, Tim Sierens1 & Harry Olde Venterink1,

Dang Thi Nhu Y1,2*
e-mail: y.dang@vnuk.edu.vn
Nguyen Tien Hoang7
e-mail: nguyenhoanggis@gmail.com
Pham Khac Lieu3
e-mail: pklieu@hueuni.edu.vn
Hidenori Harada5
e-mail: harada.hidenori.8v@kyoto-u.ac.jp
Keisuke Koba4
e-mail: keikoba@ecology.kyoto-u.ac.jp
Natacha Brion6
e-mail: nnbrion@vub.beDuong Van Hieu8
e-mail: dvhieu@hueuni.edu.vn
Nguyen Van Hop8e-mail: nvanhop@hueuni.edu.vn
Tim Sierens1
e-mail: Timothy.Sierens@vub.beHarry Olde Venterink
e-mail: Harry.Olde.Venterink@vub.be1

Department of Biology, Vrije Universiteit
Brussel (VUB), Brussels, Belgium
2
VNUK Institute for Research and Execu­
tive Education, The University of Danang,
158A Le Loi Street, Hai Chau District,
Danang, Vietnam
3
Department of Science, Technology
and Environment, Hue University, Hue,
Vietnam
4
Center for Ecological Research, Kyoto
University, Otsu, Shiga 520–2113, Japan
5
Graduate School of Global
Environmental Studies, Kyoto
University, Kyoto 606–8501, Japan
6
Analytical, Environmental andGeochemistry, Vrije Universiteit
Brussel (VUB), Brussels, Belgium
7
Laboratory of Environmental Geosphere
Engineering, Department of Urban
Management, Graduate School ofEngineering, Kyoto University, Kyoto
615–8540, Japan
8
College of Sciences, Hue University, Hue,
Vietnam1

ABSTRACT

Submersed macrophytes are key elements of shallow coastal ecosystems but glob­
ally declined due to anthropogenic disturbances and climate change. Foliar ele­men­tal and isotopic compositions of macrophytes are being increasingly used toiden­tify nutritional status and environmental conditions including potential stressfac­tors, thereto insight is required in its interspecific variation. In this paper, we
present interspecific variations in foliar nutrients (C, N, P) and stable isotopes (δ13Cand δ15N) of four macrophyte species (Najas indica, Halophila beccarii, Halodule uni­
nervis, Halophila ovalis) in Cau Hai lagoon (Vietnam) in relation to key environmen­tal
variables. The foliar C, N, δ13C and δ15N values varied among species and related
to salinity, water depth and/or DIN concentrations. Foliar N and C:N values werecor­re­lated to DIN concentration in the water (N. indica and H. beccarii, respectively),
whereas foliar C:P was correlated to P concentrations in sediment (N. indica) or
water depth (H. uninervis). Foliar δ13C of H. beccarii and H. uninervis increased withsa­li­nity and δ15N of N. indica with both salinity and DIN concentration in the wa­
ter. These aquatic plants likely varied either in resource acquisition strategies (e.g.
uptake of CO2 or HCO3–, N and P from different sources) or in their usage (e.g.
C in­vest­ment in structural tissue) and in their response to changes in salinity, water
depth and DIN concentration in the water. The studied macrophytes likely experi­
enced relatively high N and P availability and another factor than P or N probably
inhibited C-fixation and/or carbon storage of these plants.
K e y w o r d s : Najas, Halophila, Halodule, salinity, depth, DIN, phosphorusРЕЗЮМЕ

Данг Ти Нху И, Нгуен Тиен Хоанг, Фам Хак Лию, Харада Х., Коба К.,Брион Н., Ван Хью Д., Ван Хоп Н., Сиренз Т., Вентеринк Г.О. Межви­до­вая изменчивость листовых питательных веществ и изотопов по­гру­женных видов макрофитов в типичной солоноватой лагуне. По­гру­жен­ные макрофиты являются ключевыми компонентами мелководных
при­бреж­ных экосистем. В глобальном масштабе их сообщества существенно
сократились из-за антропогенных нарушений и изменения климата. Эле­мент­ные и изотопные составы листьев макрофитов все чаще используются дляопределения их пищевого статуса и условий окружающей среды, включая по­тенциальные стрессовые факторы, для чего необходимо понимание их меж­ви­довой изменчивости. В настоящей работе мы представляем результаты ис­сле­дования межвидовых вариаций внекорневых питательных веществ (C, N,P) и стабильных изотопов (δ13C и δ15N) четырех видов макрофитов: Najas in­di­ca, Halophila beccarii, Halodule uninervis, Halophila ovalis в лагуне Кау-Хай (Вьетнам)в за­висимости от ключевых переменных окружающей среды. Содержания C,N, значения δ13C и δ15N варьировали в зависимости от вида и были связаны ссо­ле­ностью, глубиной воды и/или концентрацией DIN. Содержание N и зна­че­ния C:N коррелировали с концентрацией DIN в воде у N. indica и H. beccarii,тог­да как значения C:P коррелировали с концентрацией P в детрите (N. indica)или в толще воды (H. uninervis). δ13C у H. beccarii и H. uninervis уве­ли­чи­валась спо­вы­шением солености, а δ15N у N. indica – как с повы­шением солености, таки с кон­центрацией DIN в воде. Макрофиты различались в стра­тегиях потреб­ле­ния ресурсов (например, поглощение CO2 или HCO3–, N и P из различных
источников), в их использовании (например, инвестиции C в структурные
ткани), а также по их реакции на изменения солености, глу­би­ны воды и кон­цен­трации DIN в воде. Изученные макрофиты находились в ус­ло­ви­ях до­ступности N и P, а фиксация С ингибировалась другими факторами.
Ключевые слова: Najas, Halophila, Halodule, соленость, глубина, DIN, фосфор
(переведено редколлегией)* corresponding author
Manuscript received: 04.05.2020
Review completed: 15.07.2020
Accepted for publication: 22.07.2020
Published online: 25.07.2020©Botanical Garden-Institute FEB RAS. 2020

Submersed macrophytes constitute key elements of shallow coastal eco­sys­
tems, but they are currently declining at an alarming rate (globally 7 % per year
accor­ding to Waycott et al. 2009). This decline is partly due to nutrient enrichment
in aquatic eco­systems worldwide (Paerl 2009, Van Katwijk et al. 2011). Elemental
61

Nhu Y et al.

and isotopic compositions of macrophyte leaves provide in­sight into the en­vi­ronmental conditions and availability ofessential re­sources (N, P and light) for growth of aquaticplants and/or to detect stressors (shading, high salinity andhigh temperature) that predict an upcoming macrophyte de­cline (Fourqurean et al. 2007, Walton et al. 2016). There­fore,
it is important to have insight in the variation in foliar con­cen­trations of the key elements such as carbon (C), ni­tro­gen(N), phosphorus (P) and their ratios – among spe­cies, as wellas in relation to important environmental factors.
Nutrient (N and P) concentrations in water and sedi­ment as well as water depth, salinity, temperature and lightcon­di­tions are considered to be key factors in controllingsub­mer­ged macrophyte distribution, growth and foliar C,
N and P concentrations (Hoyer et al. 2004, Ferdie & Four­qurean 2004, Campbell & Fourqurean 2009). Foliar con­cen­
tra­tions of C, N, P and their ratios depend on the relative
avai­lability of N and P in water and sediment and on themac­rophyte growth, which also depend on C assimilation
du­ring photosynthesis and hence on light and other growth
limiting resources (Four­qu­rean et al. 2007). The benthic
mac­rophyte Redfield Ratio C:N:P of 550:30:1 (molar base)has been used as an in­di­ca­tor for light, N or P limitation ofmac­ro­phyte growth (At­kin­son & Smith 1983, Johnson etal. 2006), although some authors have debated the use ofthis ratio (Ferdie & Fourqurean 2004, Walton et al. 2016).
Plants growing in nutrient poor waters display significantly
higher C:N and/or C:P ratios than those from nutrient richconditions, provided they receive sufficient light (Atkinson& Smith 1983).
Stable isotope signatures of C and N in macrophyte tis­sues have been used to elucidate the sources and relative
avai­lability of C and N (Anderson & Fourqurean 2003, Le­point et al. 2004, Campbell & Fourqurean 2009) as well as
light condition in their habitats (Gorman et al. 2017). Ge­ne­rally, the 13C values in seagrasses range from -23 ‰ to -3 ‰(An­derson & Fourqurean 2003). This large variation can beas­cribed to interspecific differences in discrimination against
the heavier 13C isotope during pho­to­synthetic fixation of
CO2 (Lepoint et al. 2004, West et al. 2006) or variationsin δ13C of the available carbon sources (CO2 and HCO3–)(Fal­kow­ski et al. 1989). Hence, the variation in foliar δ13C
also depends on light conditions and ambient temperature
which influence the photosynthetic rate (Campbell & Four­qu­rean 2013, Fourqurean et al. 2015), or factors which affect
HCO3– availability, such as salinity (Fry 2002). Plants thatas­si­milate HCO3– for photosynthesis are more enriched inδ13C compared to those that assimilate CO2, because HCO3–
has a less negative δ13C than CO2 (0 ‰ vs. -9 ‰) (Ra­ven
et al. 2002, Chappuis et al. 2017). Thus, submerged plants
have enriched values of δ13C (-16.7 ‰ to -10.5 ‰) in com­
parison to aquatic plants with leaves in contact with the at­mo­sphere (-34.8 ‰ to -14.6 ‰) (Chappuis et al. 2017). The
δ15N values in macrophytes range from -5.2 ‰ to 20.1 ‰(Cha­ppuis et al. 2017), with tropical macrophyte genera in­clu­ding Halodule spp., Halophila spp. and Syringodium spp.
be­ing among the most depleted (Fourqurean et al. 2015,
Wal­ton et al. 2016). Foliar δ15N is affected by N sources
and plant acquisition mechanisms (Sun et al. 2011, Peipoch62

et al. 2014, Walton et al. 2016). The use of δ15N in aquaticplants to distinguish between natural and sewage derived
N is well documented (Risk et al. 2009). A high foliar δ15N
(above 5 ‰) in macrophytes indicates that the acquired N
as­so­ciated to human and animal waste inputs (Yamamuro
et al. 2003, Connolly et al. 2013, Fourqurean et al. 2015).
In­stead, low foliar δ15N values (~0 ‰) in macrophytes do­
cu­ments that N was derived from nitrate (fertilizer), fromre-mineralised N from locally produced organic matter, orfrom N derived from atmospheric N2 fixation (Peipoch etal. 2013, Fourqurean et al. 2015, Walton et al. 2016). Ne­ga­tive δ15N values in plants can indicate that N was de­rived
from nitrification (Yu et al. 2015) or to mycorrhizal asso­
ciations (Hobbie et al. 2005). Hence, proportion of C andN isotopes in macrophytes can reflect the availability ofessential resources and provide insight in their growth con­ditions (Campbell & Fourqurean 2009). However, know­
ledge about interspecific differences and the effects ofen­vi­ronmental factors on isotope ratios is still limited formany seagrasses and macrophytes.
Although there are many studies that have evaluated  
variation in foliar nutrients of submerged macrophyte
species such as Halodule uninervis, Halophila ovalis (Yamamuro
et al. 2003, Camp­bell & Fourqurean 2009, Walton et al.
2016), there is still limited information about elemental andisotopic com­positions of Najas indica and Halophila beccarii
so far. Ad­di­tionally, the effects of environmental factors intropical lagoons on such differences are still limited. In thisstudy, we examined interspecific variation in foliar nut­ri­ents(C, N, and P) and stable isotopes (δ13C and δ15N) of fourmac­ro­phyte species in Cau Hai lagoon (Vietnam) in relation
to key environmental variables (such as salinity, water depth
or nutrient concentrations in water and sediment). This is
the first attempt to characterize the sources of variationin ele­men­
tal and isotope ratios of these submerged
macrophyte species and discuss role of ecological and/or physiological characteristics in a tropical monsoon
lagoon. We focused on the four macrophyte species Najas
indica (Willd.) Cham., Halophila beccarii Aschers., Halodule
uninervis (Forssk.) Boiss. and Halophila ovalis (R. Br.) Hook. f.
which are common in Cau Hai lagoon, but internationallyconsidered as threatened spe­cies (Short et al. 2011, Odelu
et al. 2014). Although N. indica is generally classified as a
freshwater mac­ro­phyte, it is quite abundant in the Cau Hai
lagoon and usu­al­ly forms mixed beds with H. beccarii.
M AT E R I A L A N D M E T H O D S

S tu d y site

The Cau Hai lagoon (16°19'22"N 107°50'59"E) is thesouthern part of the large Tam Giang-Cau Hai lagoon
system, loca­ted on the central coast of Vietnam (Fig.
1). It has an area of approximately 11 200 ha. Besides
precipitation, there are three main sources of water flows
joining into the la­goon: freshwater from Dai Giang andTruoi Rivers in the west of the lagoon, brackish waterfrom the connected Thuy Tu lagoon in the north and saline
water from the sea through the Tu Hien inlet in the east.
The shallo­west areas close to the lagoon edge are used
for aquaculture practices (Disperati & Virdis 2015). The

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

Interspecific variation in foliar nutrients and isotopes of submerged macrophytes in Vietnam

area close to the river mouths and theadjacent lagoon are almost completelyco­vered by shrimp farms, whereas thearea close to the inlet contains somefish cages (small scale). In general, thestron­gest aquaculture activity is in thenorth-western region of the lagoon.
Therefore, this region is considered to
be a main source of nutrient enrichment
for the Cau Hai lagoon (Nguyen & Yabe
2014, Disperati & Virdis 2015).
The benthic macrophyte commu­ni­ties in the Cau Hai lagoon are lar­ge­lydo­mi­na­ted by both freshwater mac­ro­­phytes (Najas indica, Vallisneria spi­ra­lis L.
and Myriophyllum spi­ca­tum L.) and sea­grassspe­cies (Zostera japonica Asch. & Graebn.,Ha­lo­phila beccarii, Ha­lo­phi­la ova­lis, Ha­lo­dule
pinifolia (Miki) Har­tog, Ha­lo­du­le uni­ner­vis
and Ruppia bre­vi­pe­dun­cu­lata ShuoYu &Har­tog) (Phan et al. 2018). N. indica andH. beccarii are the most dominant spe­
cies (Phan et al. 2018). Du­ring the pastde­cades, the area co­ve­red with mac­ro­phytes has declined by more than 50 %compared to that in 1990–2000 (Caoet al. 2012). Changes in en­vi­ron­men­tal
variables such as salinity, water depth andnutrient availability re­la­ted to na­tu­ral andanthropogenic dis­tur­ban­ces, may havecaused ne­ga­tive ef­fects on mac­ro­phyte
growth within the lagoon (Thanh et al.
2004, Le 2012, Phan et al. 2018).

Figure 1 Location of sampling points for water, sediment and macrophytes in 2015
in the Cau Hai lagoon, central Vietnam. The black dots indicate coordinators of the
25 permanent sampling sites; the colour dots indicate the occurrence of the four
macrophyte species in sampling events

Sa mp lin g a n d me asure me nts
Samples of water, sediment and plants were collected si­
mul­taneously at 25 sites (located with Garmin GPS MAP®
78) in Cau Hai lagoon in March, May, July and September
2015 (Fig. 1). Water and sediment were sampled during all
four events (n=100 for water and sediment samples; n=50
for chlorophyll a (Chl-a) which was only measured in theJuly and Sep­tember), and macrophytes of the four focus
spe­cies were collected when present (n=23 for N. in­di­ca; n=28 for H. beccarii; n=18 for H. univervis; n=8 for H.
ovalis). Samples of water, sediment and macrophytes weretaken together in four events and formed a gradient ofenvironmental and fo­liar variables to interpreter correlation
among foliar nut­rients and environmental variables. All
glassware and con­tai­ners were cleaned with a 5 % H2SO4
solution and distilled water prior to use. All samples wereplaced in ice boxes after col­lection and transported to thelaboratory. The details of sampling and measurements aredescribed as follows.
Water. During each sampling event, in-situ measure­ments were done about 30 cm below the water surface forwater temperature, pH, dissolved oxygen, turbidity and sa­li­nity, using a water quality monitoring unit with specificsen­sors (HORIBA U–5000, Horiba, Japan). Water depth
was measured with a depth sounder (Hondex PS–7, Honda

Elec­tro­nics, Japan). Surface water samples were collectedwith a Van Dorn sampler at 50 cm below the surface. The
water samples were transferred into glass bottles and trans­por­ted to the lab in an icebox. Samples for Chl-a analysis
were obtained by filtration on glass fibre fil­ters (Whatman
GF/F) and were stored in the dark at -20°C until analyses.
Unfiltered water samples were stored in glass bottles
and frozen until they were submitted to a digestion pro­cedure for total N (TN) and total P (TP) analyses. Fil­te­redsamples were stored in glass bottles and frozen until they
were analyzed for nitrate + nitrite (NOX) and ammonium(NH4+) spectrophotometrically. All nutrient measurements
followed standard protocols (APHA 1998). The dissolvedin­organic nitrogen (DIN) was calculated by summation of
NOX–N and NH4–N. Trophic state indices (TSI) of thewa­ter were calculated using equations described by Carlson(1977), Kratzer & Brezonik (1981) for Chl-a, TN and TP,
respectively.
Sediment. At each site three replicate sediment cores
were taken to a depth of 10 cm, using a sediment coresampler (4.5 cm diameter). The fresh sediments were sto­
red in aluminium foil and transported refrigerated to thelaboratory. Extractable N (NOX– and NH4+) and dissolved
inorganic phosphorus (DIP) were determined by extracting
fresh sediment with 2M KCl or 0.5M NaHCO3, respectively(Estefan et al. 2014). Concentrations of NOX–, NH4+ andBotanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

63

Nhu Y et al.

DIP were measured spect­ro­photometrically. Fresh sediment
samples were dried at 50°C for 48 h, ground and analysed for
TN using an elemental analyser coupled to an isotope ratio
mass spectrometer (EA–IRMS) as described below. TP wasana­lysed using a modification of the combustion (500°C for
2 hours) and hot 1M HCl extraction procedure of Andersen(1976) according to Johengen (1997). Subsequently, phos­
phate was measured spectrophotometrically using a molyb­
date/ascorbic acid procedure (Murphy & Riley 1962) with
an automatic segmented flow nutrient analyser (QuAAtro,
Seal Analytical).
Macrophytes. Four focus species (Najas indica, Halo­phi­la beccarii, Halodule uninervis and Halophila ovalis) werecollected at each site when present. The four macrophytes
showed a dis­tinc­tive spatial distribution in the Cau Hai
lagoon (Fig. 1). Green leaves were scraped to remove epi­
phytes and then rinsed again using distilled water. Oncedried (50°C for 48 h in the oven), leaf samples were groundand analysed for C, N, δ13C and δ15N values using an ele­men­tal analyser (Flash1120 series EA, Thermo) coupled to
an isotope ratio mass spectrometer (DeltaV, Thermo). Ana­ly­ses were ca­lib­ra­ted against reference materials: IAEA–
CH6 (C12H22O11) and IAEA–N2 (NH4)2SO4) for C and
N respectively. Samples’ isotopic ratios (R) are repor­ted
in the standard delta notation (δ) of the heavy to the lightisotope (13C/12C or 15N/14N in either sample or stan­dard)
as follows: δ (‰) = [(RSample/RStandard)–1] × 1000 (West et al.
2006). TP was determined as described above for sediment
samples. C:N:P elemental ratios of studied mac­ro­phytes
were compared with the benthic macrophyte Red­field Ratio(SRR) of 550:30:1 (Atkinson & Smith 1983) to evaluate
potential growth limiting factors.

Data a n a lys is
One-way ANOVA and Tukey’s post hoc test were used
to examine which species were different from each other.
Linear regressions were used – per species – to determine
the (correlative) influence of water and sediment parameters
on fo­liar nutrients concentrations or isotopic ratios. The ef­
fects of environmental variables (explanatory variables) onfo­liar nutrients of macrophytes (response variables) werevi­sua­lised with redundancy analysis (RDA) using the veganpa­ckage in R (Oksanen et al. 2013). Multicollinear variables
were discarded using the Variance Inflation Factor (VIF<4),
if not already excluded by step-wise selection (Oksanen etal. 2013). For three of the studied plant species, datasets
were large enough to calculate a RDA with foliar nutrientsand isotopes as well as environmental variables; however,for H. ovalis the dataset was too limited. This had to be done
per species because of interspecific differences in foliar
nutrient and isotopes. To meet test assumptions of pa­ra­met­ric statistics, data were log-transformed if needed. Allanalyses were carried out with R version 3.4.0 on R-Studio(Logan 2010, R Core Team 2017).
The geostatistical kriging technique was used to inter­po­late the spatial distribution of key water variables in thela­goon that have significant spatial variation (saliity, depth
and DIN in the water). Kriging assumes that the distance or
direction between sample points reflects a spatial corre­la­64tion that can be used to predict unknown values for any geo­
graphic point data (Goovaerts 1997, Daya & Bejari 2015).
The method with smaller prediction errors will be selec­
ted for the spatial interpolation. The model type, nugget,
sill and range were modified to fit each of the vario­gram
models by using ISATIS Software (Geovariances 2016).
R E S U LT S

Distr ib u tion of key env iron m ental
factors an d m acrop hy tes
Water and sediment variables showed strong variationin space or time (Table 1). There was a clear west-east gra­dient in salinity (Fig. 2A; Fig. 3A), as well as a north-south
gradient in water depth (Fig. 2B). There were also spatial
gra­dients in DIN concentrations in the water with higherva­lues in the western part of the lagoon (Fig. 2C; Fig. 3C),
whereas, the P concentrations did not show spatial variation(Table 1; Fig. 3D). Additionally, salinity, concentrations of
N and P in the water and sediment varied in time (Table 1).
The four macrophytes showed a distinctive spatial dis­tri­bu­tion in the Cau Hai lagoon (Fig. 1, 3). N. indica andH. beccarii occurred in the western and central parts of thela­goon with lower salinity and higher DIN concentrations(Fig. 3A, C). Whereas, H. uninervis occurred in the central
and eastern part and H. ovalis occurred only at two sites inthe eastern part with higher salinity and lower DIN con­cen­
tra­tions) (Fig. 3A, C). Furthermore, H. uninervis and H. ovalis
dis­tri­buted in the narrow range of salinity, water depth,
DIN and DIP concentrations (Fig. 3).

Intersp ecif ic var iation in m acrop hy tefoliar C, N, P, δ 1 3 C an d δ 1 5 N
There were significant differences in foliar C, N and
C:N among the four studied species (averaged over timeand space), but not in foliar P, C:P or N:P (Fig. 4). H. beccarii
had a relatively high foliar N (3.1±0.1 %) and a low foliar
C (32.0±0.8 %) concentration and therefore a lower foliar
C:N (12.7±0.5) than the three other species (Fig. 4D),
whe­reas H. ovalis had the lowest foliar N concentration(2.3±0.1 %) (Fig. 4B).
The macrophyte species clearly differed in foliar δ13C,
with most negative values for N. indica (-18.4±0.3 ‰) and
H. beccarii (-17.3±0.5 ‰) and least negative for H. ovalis(-10.7±0.5 ‰) (Fig. 4G). Foliar δ15N of H. uninervis(1.3±0.5 ‰) was significantly lower than that of the threeother species (Fig. 4H).

Cor relation s am on g m acrop hy te fol i a r
n u tr ients an d env iron m ental var iab l e s
The foliar N of N. indica and foliar C:N of H. beccarii
were significant correlated to DIN concentrations in thewater (Fig. 5A, B; 6A, C). Correlations of foliar P and C:Pand DIP concentrations in the sediment were observed in
N. indica (Fig. 5A, 6B). For H. uninervis, foliar P and C:P
were correlated to water depth (Fig. 5C, 6D).
Foliar δ13C values of H. beccarii and H. uninervis were po­si­tively correlated to salinity (Fig. 5B, C; 6E). Noteworthy, thedif­ference in foliar δ13C among studied species did not de­pend on salinity; i.e., the interaction between species and sa­li­
ni­ty was not significant (F = 1.0 and p = 0.39). A significant

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

Interspecific variation in foliar nutrients and isotopes of submerged macrophytes in Vietnam

Table 1. Summary statistics for water and sediment parameters (means ± S.E) and one-way ANOVA results for effectsof sampling time and sampling site. The number of observations for each quantity=100; for Chl-a:  n=50. The significant
statistics are shown in bold. Levels of significance are as follows: *p<0.05; **p<0.01; ***p<0.001.
Parameter
Water quality parameters
Temperature (°C )pH
DO (mg/L)
Salinity (‰)
Turbidity (NTU)
Water depth (m)NOx–N (µg/L)
NH4–N (µg/L)
DIN (µg/L)
TN (µg/L)
TP (µg/L
TN:TP (Molar base)Chl-a (µg/L)
TSI
Sediment quality parameters
NOx–N (mg/Kg)
NH4–N (mg/Kg)
DIN (mg/Kg)
PO4–P (mg/Kg)
DIN:DIP (Molar base)
TN (%dw)
TP (g/Kg)
TN:TP (Molar base)

ANOVA (F ratio)
Site

Min

Max

Mean ± S.E

26.2
5.4
3.8
0.2
0.0
0.5
7.1
17.4
31.4
114.2
7.0
8.6
0.4
31.3

35.2
11.6
11.2
29.9
50.8
2.4
54.9
241.2
269.7
770.6
132.8
79.5
105.9
79.0

31.38 ± 0.20
8.52 ± 0.10
7.22 ± 0.15
13.65 ± 0.61
4.93 ± 0.68
1.51 ± 0.42
18.97 ± 0.81
58.48 ± 3.81
77.45 ± 4.20
403.77 ± 12.91
29.07 ± 2.03
39.51 ± 1.79
6.3 ± 2.1
44.1 ± 0.6

170.99***9.35***
18.46***
15.96***
41.38
0.50
2.82*
3.06*
1.52
0.36
7.89**
2.65
1.90
1.04

0.25
0.96
0.46
4.64***  
0.89
26.46***  
1.18
0.82
3.83*
9.67**
0.95
1.18
3.39*
4.96**

0.3
4.3
4.7
1.1
1.2
0.5
0.05
7.4

19.2
30.7
32.5
17.0
33.2
2.9
0.5
63.1

1.96 ± 0.22
15.46 ± 0.53
17.03 ± 0.53
4.07 ± 0.26
12.69 ± 0.75
1.53 ± 0.05
0.20 ± 0.01  
23.02 ± 1.13

14.57***
1.51
0.56
2.40*
1.78*
1.87*
6.16*
1.21

1.22
3.85*
3.39*
0.87
1.74
0.99
0.88
0.65

cor­re­la­tion between foliar δ15N and environmental variables
was only found for N. indica, foliar δ15N was negatively
correlated to sa­li­ni­ty and positively correlated to DIN inwater (Fig. 5A, 6H).
DISCUSSION

Variation in foliar C, N and P and their ratios
Foliar C and N concentrations differed among species,
whe­reas P did not. Foliar C and C:N were relatively low in
H. beccarii. This species might have less structural carbon(as in­di­cated by depleted C:N and C:P ratios) than theother three spe­cies, as was also found for Cymodocea nodosa(Ucria) Asch. near the Balearic Islands (Fourqurean et al.
2007). Foliar N was low in H. ovalis, but this difference was
likely a consequence of the limited spatial occurrence in thefar eastern part of the lagoon where N concentrations in
the water and sediment were relatively low, rather than an
inherent difference between species. At two other sites in
Aus­tra­lia and Fiji where H. ovalis co-occurred with one ofTime

the other species (H. uninervis), H. ovalis had a higher N con­cen­tration (Yamamuro et al. 2003), which supports that thelower N concentration of H. ovalis in the Cau Hai lagoon
was likely not due to inherent interspecific differences and/or insignificant fluctuation of DIN concentration in its
distributed areas (Fig. 3C). Noteworthy, numbers of re­pli­
cation was too low for statistical comparisons among spe­
cies that occurred at the same sites.
Variation in foliar N and C:N was associated with varia­
tion in DIN concentration in the water, although onlyob­ser­ved for N. indica and H. beccarii. This suggests that
DIN concentration in the water column was an importantsource of N for these macrophyte species, particularly inthe north western part of the lagoon. In this region, a re­la­ti­vely high DIN concentration in the water compared toplant demand might have led to excess uptake of DIN or‘lu­xu­ry consumption’ by the macrophytes, as observed inother studies (cf. Gordon et al. 1981, Stapel & Hemminga
1997, Ferdie & Fourqurean 2004, Apostolaki et al. 2011,
Vizzini et al. 2004, Walton et al. 2016). Similarly, Zostera

Figure 2 Spatial variation in mean salinity, depth, DIN concentration in the water collected between March and September 2015 in the Cau
Hai lagoon. Black dots indicate locations of the 25 permanent sampling sites used for spatial interpolations. The darkness of the shading
increases with the value of variables
Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

65

Nhu Y et al.

Figure 3 Distribution of key environmental variables and four macrophyte species at 25 sampling sites in Cau Hai lagoon. Bars charts indicateen­vi­ron­mental variables: salinity (A), depth (B), DIN concentration in the water (C) and DIP concentration in the sediment (D). Error bars are
the standard error of the means of environmental variables. Dots inside the bar charts indicate the presence of macrophyte species: Najas indica(Willd.) Cham. (n=23); Halophila beccarii Aschers. (n=28), Halodule uninervis (Forssk.) Boiss. (n=18) and Halophila ovalis (R. Br.) Hook. f. (n=8)

marina L. in Atlantic Canada showed an increasing foliar
N concentration with N enrichment although its biomassde­clined due to shading (Schmidt et al. 2018). Variation in
foliar P and C:P could not be ascribed to differences in P in
the water, but for N. indica foliar P was positively correlated
to va­riation in DIP in the sediment. This indicates that forthis species P-acquisition by the roots from the sediment
may be an important factor as observed for Najas marina
in Myall Lake, Australia (Shilla et al. 2006). Foliar P of
H. uninervis was not correlated to P in water or sediment,
but it increased with increasing water depth. There is no
good explanation for these correlations, at this point.
The average concentrations of C, N and P and theirratios in the macrophytes of the Cau Hai lagoon indicatethat these plants likely experienced relatively high N and P
availability. The average C:N (15) and C:P ratios (464) of all
species were clearly below the Redfield Ratio for benthic
macrophytes (C:N≈18, C:P≈550) (Atkinson & Smith 1983,
Duarte 1990, Johnson et al. 2006). Foliar N and P con­cen­
tra­tions in this study (2.3–3.1 % for N, 0.19–0.21 % for P)
were in the range of the data reported in other coastal areas(1.6–3.1 % for N and 0.14–0.44 % for P; Yamamuro et al.
2003, Walton et al. 2016). Plants growing in excess nutrient
conditions reflect this in high N and P concentrations com­pared to C (Atkinson & Smith 1983, Duarte 1990, Johnson
et al. 2006). A moderate to high nutrient availability as in­di­cated by the foliar nutrients in the plants agreed withtro­phic state indices in Cau Hai lagoon. Hence, it appears
that another factor than P or N inhibited C-fixation and/or carbon storage of macrophytes (Burkholder et al. 2007).

Va riation in fol i ar δ 1 3 C and δ 1 5 N
On average, foliar δ13C values differed strongly among thefour macrophyte species of this study reflecting interspecific66

variation in bicarbonate acquisition mechanisms (Invers etal. 1999, Schwarz et al. 2000, Uku et al. 2005, Borum et al.
2016). The difference was also partly due to variation in thespatial distribution and salinity of the macrophyte spe­cies’
habitats, although the difference among studied spe­cies
did not depend on salinity (the species × salinity in­ter­ac­tion was not significant). The observed interspecific trendin δ13C (H. ovalis > H. uninervis > H. beccarii > N. indica) was
consistent with the limited available information about
δ13C values of macrophytes from previous studies (Wet­zel
1969 in Keeley & Sandquist 1992). Averaged per spe­cies,
we observed the most negative values for N. indica (-18 ‰)which were in the range of previously observed fresh­water macrophytes (-11 to -50 ‰; Wetzel 1969 in Keeley& Sandquist 1992, Chappuis et al. 2017). Moreover, Wet­zel (1969) observed that the foliar δ13C value of the closely
related species Najas flexilis (Willd.) Rostk. & Schmidt wasquite low (-22 ‰) because it did not take up HCO3– (Wetzel
1969 in Keeley & Sandquist 1992). This may also count for
N. indica in the Cau Hai la­goon given its low foliar δ13C
value. Also, the relatively low δ13C value of H. beccarii (17 ‰)in comparison to the other Halophila species is consistent
with previous studies (McMillan et al. 1980 in Hemminga &Mateo 1996). Highest δ13C values (-10.7 ‰) were observedin H. ovalis which occurred at the shallowest depth (Fig. 3B)
and highest salinity suggesting that photosynthesis of thisspecies may depend more on HCO3– as a carbon source(Borum et al. 2016) than the three other species. Foliar
δ13C of H. beccarii and H. uninervis in the Cau Hai la­goon
increased with salinity, as was previously observed in othercoastal waters (Fry 2002, Hitchcock et al. 2017). Ge­ne­rally,
δ13C in seagrasses ranges from -24 ‰ to -3 ‰ (Hem­min­ga& Mateo 1996, Anderson & Fourqurean 2003), where theleast negative values in marine primary producers indicate

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

Interspecific variation in foliar nutrients and isotopes of submerged macrophytes in Vietnam

Figure 4 Mean foliar carbon, C (A) , nitrogen, N (B), phosphorus, P (C) concentrations, ratios of C:N (D), C:P (E) and N:P (F) as well as stable
isotopes δ13C (G) and δ15N (H) of four macrophyte species collected in 2015 in the Cau Hai lagoon. Error bars are the standard error of themeans. Significant differences are indicated by different letters at p<0.05 (Tukey’s test after Anova Type II, taking into account the repetitive
measurements). Freshwater species: Najas indica (Willd.) Cham. (●, purple, n=23); Seagrass species: Halophila beccarii Aschers. (*, green, n=28),
Halodule uninervis (Forssk.) Boiss. (▲, red, n=18) and Halophila ovalis (R. Br.) Hook. f. (■, blue, n=8)

a relatively high assimilation of HCO3– in­stead of CO2(Hemminga & Mateo 1996) to adapt to marine life (Beeret al. 2002). These foliar δ13C values were much lower thanthose observed in the saline habitats such as Shark Bay, Aus­
tralia (δ13C = -13.2 ‰ in Halodule uninervis, salinity > 42 ‰,Fraser et al. 2012) and Bermuda (δ13C = -9.6 ‰ in Halodulesp., salinity > 36 ‰, Fourqurean et al. 2015). The positive
correlation between foliar δ13C and salinity may also reflectthat the isotopic signature of dissolved inorganic C varies
with salinity, from close to 0‰ in ocean waters to -10 ‰to -15 ‰ in freshwaters (Mook 2000). Indeed, according

to Henry’s law, dissolution of CO2 in water is a function oftemperature and salinity. Under the high pH conditions in
the brackish water of the Cau Hai lagoon (8.5±0.1), DIC
may be comprised for more than 80 % of HCO3− (Michener& Lajtha 2007, Schmidt et al. 2012). Macrophytes species
vary in their ability to use HCO3−. In Cau Hai lagoon,
H. beccarii and H. uninervis appeared to acquire relatively highamounts of C from bicarbonate.
H. uninervis had a consistently lower foliar δ15N value
compared to the other three species in the Cau Hai lagoon,
which supported previous observations for this species

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

67

Nhu Y et al.

ammonium in water or sediment), or that the relationshipbetween the N demand and N availability was different forthis species (Peipoch et al. 2014, Fourqurean et al. 2015,
Walton et al. 2016). Although foliar δ15N of H. uninervis wascorrelated differently to the measured environmental va­
riables than that of N. indica (cf. temperature, pH; Table 2),
we could not identify a different source of N for H. uni­
nervis. With a lower demand for nitrogen, plants dis­cri­mi­nate more against the heavier isotope, as was describedfor Halodule uninervis in Al Khor bay (Qatar) (Walton et al.
2016) and Moreton Bay or in an experiment in Green Is­land(Australia) with an excess supply of nitrogen (Udy & Den­ni­son 1997). Generally, foliar δ15N values of macrophytesgro­wing in anthropogenically disturbed water are higher
than those of macrophytes from undisturbed habitats(Four­qurean et al. 1997, Yamamuro et al. 2003, Lepoint etal. 2008, Herbeck et al. 2014, Hitchcock et al. 2017). In ourstu­dy, foliar δ15N values were highest in the north-westernpart of the lagoon as observed in foliar δ15N of N. indica(3.9±0.8 ‰), where aquaculture activity is most intensive
and where also DIN concentrations are at the highest.
Foliar δ15N of macrophytes in the Cau Hai lagoon wherehigh compared to values from undisturbed waters, such as
Dravuni (Fiji) and Green Island (Australia) (Yamamuro etal. 2003). This indicates that the Cau Hai lagoon suffersfrom N enrichment, at least in the north-western part as
illust­rated by the spatial patterns of DIN concentrations in
the water (Fig. 2C). The lagoon on average would be clas­si­fied as mesotrophic (ranging 40–50) according trophic
state indices (TSI) of Carlson (1977), Kratzer and Brezonik(1981), but locally it could vary from oligotrophic (TSI<40)
to eutrophic (TSI=50–70) (Table 1).

CONCLUSION

Figure 5 Biplots of the first two axes of the redundancy analysis
showing the association of macrophyte occurrence (R2 = 0.38,
p = 0.02), foliar nutrients and environmental variables: Najas indica(Willd.) Cham. (R2 = 0.52, p = 0.04), H. beccarii (R2=0.39, p=0.011),
Halodule uninervis (Forssk.) Boiss. (R2 = 0.56, p = 0.012). Abbreviation:
DINW, DINSed: DIN concentrations in the water and sediment,
respectively; TPW: TP concentration in the water; DIPSed: DIP
concentration in the sedimentin other regions (Yamamuro et al. 2003, Frase et al. 2012,
Walton et al. 2016). This indicates that this species either
differed from the other species in the proportions of N
that it acquires from the various N sources (e.g. nitrate and68

There was a considerable interspecific variation in fo­
liar nutrient concentrations and isotopic composition in thestudied macrophytes in the Cau Hai lagoon. This varia­tion
was associated to intrinsic interspecific differences (dif­fe­rent N source of H. uninervis), natural variation in ha­bitat
conditions among species (salinity, depth), and anthro­po­genic influences (N enrichment). Variations in foliar
N and P could be explained by the mea­sured variation inenvironmental conditions only to a limited extent. Variations
in foliar δ13C and δ15N among species were distinct and werelikely related to variation in C and N acquisition and in DIN
availability. The high nutrient concentrations in the studied
mac­ro­phytes in Cau Hai lagoon indicates that possibly ano­
ther factor was limiting growth of these plants such as ex­
cess nutrients affecting their nutrient uptake and growthrate (toxicity or inhibited C-fixation) or shading from algae(phy­to­plankton, macroalgae and epiphyte) (Whalen et al.
2013).
Competing Interests Statement: The authors declareno competing interests.
Author Contributions Statement: D.T.N.Y. and
H.O.V. designed the study. D.T.N.Y. collected field samples,ana­lysed water, sediment and macrophyte samples; N.B.D.V.,
P.C. and T.S. contributed to the chemical analyses. N.T.H

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

Interspecific variation in foliar nutrients and isotopes of submerged macrophytes in Vietnam

Figure 6 Correlations between foliar nutrients and environmental parameters in Cau Hai lagoon. Only significant regression lines are drawn.
Levels of significance are as follows: *p<0.05; **p<0.01. Freshwater species: Najas indica (Willd.) Cham. (●, purple, n=23); Seagrass species:
Halophila beccarii Aschers. (*, green, n=28), Halodule uninervis (Forssk.) Boiss. (▲, red, n=18) and Halophila ovalis (R. Br.) Hook. f. (■, blue, n=8)

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

69

Nhu Y et al.

Table 2. Pearson correlation coefficients (r) of plant, water and sediment quality parameters. Levels of significance are as
follows: *p<0.05; **p<0.01. N. indica: n=23, H. beccarii: n=28, H. uninervis: n=18, H. ovalis: n=8
Temp
.02
.17
-.33
-.11

Water parameters
pH Salinity Depth DIN TN
-.25
.31
-.08
.43* -.38-.09 -.05
-.18
-.18
.17
-.20 -.25
-.12
.37
-.29
.26 -.21
-.37
.63
-.19

.00
-.09
-.34
-.63

.11
-.07
-.04
-.02

-.22
-.31
-.47
-.15

-.12
-.34
.35
-.50

.49*
.24
-.12
.40

-.33
.15
-.20
-.38

-.48*
.21
-.24
-.14

.40
-.04
.17
-.02

-.02
-.15
.11
.47

.09
.05
.20
.20

.05
.02
.40
-.78*

-.36
.07
.15
-.71*

.30
-.03
-.04
.58

.28
.14
.03
.18

.12
-.10
.39
-.08

-.18
.22
-.24
.33

-.21
.04
.61**
.07

.23
-.06
-.27
-.06

.19
.24
-.05
.05

-.16
.13
-.19
.09

.26
.06
.20
-.08

-.15
.03
-.03
.31

.53**
.09
.02
-.04

-.14
-.42*
.47*
-.14

-.10
.04
.25
.27

-.06
-.36
-.10
-.54

Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

-.06
.22
Foliar C:N .25
.77*

.01
-.04
-.01
.17

.07
.40*
.41
.16

-.01
.25
-.38
.45

-.38
-.44*
.24
-.25

.25
-.06
.12
.47

.43*
-.01
.16
.22

-.38
-.13
-.10
.01

.06
.10
-.18
-.50

-.07
-.16
-.23
-.30

-.10
.00
-.37
.66

.43*
.13
-.16
.64

-.39
-.12
.07
-.54

Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

Foliar C:P

-.28
-.13
-.18
-.32

.08
.06
-.46
.10

.01
-.22
.17
-.38

.12
-.06
-.60**
-.20

-.12
.01
.32
.20

-.25
-.19
-.04
-.14

.15
-.03
.09
-.12

-.26
-.13
-.12
.08

.20
-.13
.01
-.23

-.49*
-.12
-.06
.09

.12
.38
-.36
-.11

.18
.01
-.25
-.47

-.03
.30
.15
.69

Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

Foliar N:P

-.13
-.25
-.39
-.58

.04
.07
-.48*
.01

-.05
-.43*
-.16
-.38

.08
-.20
-.32
-.34

.23
.25
.15
.26

-.36
-.15
-.14
-.31

-.25
-.03
.03
-.19

.14
-.05
-.05
.06

.08
-.17
.15
.01

-.26
-.03
.13
.20

.15
.36
-.08
-.36

-.22
-.06
-.14
-.65

.29
.35
.11
.79**

Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

.15
-.20
Foliar δ13C -.53
*
-.03

-.34
.03
-.67**
.08

.37
.50**
.57*
.62

.32
.07
-.34
.05

.01
-.33
-.06
-.14

.20
-.01
-.29
.35

.06
-.16
-.25
-.61

.00
.13
.14
.91**

.06
-.11
-.38
-.10

.06
-.27
.29
.21

-.04
-.15
-.17
.55

.05
-.09
-.18
.48

-.11
.05
.13
-.49

Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

.10
-.08
Foliar δ N -.55
*
-.33

.16
-.11
-.51*
.19

-.58*
.02
.23
-.31

-.54**
-.42*
-.42
-.70

.48*
.08
.20
.38

-.42*
.03
-.27
-.01

-.50*
-.09
-.14
.16

.38
.14
.03
-.13

-.16
.03
-.24
.28

.25
.04
.35
-.09

-.09
-.08
-.40
-.41

-.30
-.25
-.08
-.78*

.21
.22
-.07
.88

Plant parameters
Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis
Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis
Najas indica
Halophila beccarii
Halodule uninervis
Halophila ovalis

Foliar C

Foliar N

Foliar P

15

made maps of sampling sites and spatial distribution of key
en­vi­ronmental variables. K.K. contributed to the evaluationof the variation in δ13C and δ15N of macrophytes. D.T.N.Y.
and H.O.V. wrote the manuscript with contributions fromall co-authors.
ACKNOWLEDGEMENTS
The study was funded by grants of the Belgian Govern­
ment (VLIR–UOS) and the Graduate School of GlobalEn­vironmental Studies (GSGES Seeds, Kyoto University,
Japan). I am grateful to various students of the Faculty ofEn­vironmental Sciences of Hue University of Sciences(Viet­nam) for their assistance with the field and lab acti­vi­
ties. Stable isotope facilities were supported by grants fromFWO Hercules (HERC46) and VUB SRP2: Tracing andmodelling of present and ancient global changes.

L I T E R AT U R E C I T E DAndersen, J.M. 1976. An ignition method for determination ofto­tal phosphorus in lake sediments. Water Research 10: 329–331.
Anderson, W.T. & J.W. Fourqurean 2003. Intra- and inter­an­nual variability in seagrass carbon and nitrogen stableiso­topes from south Florida, a preliminary study. Organic
Geochemistry 34: 185–194.
70

TP TN:TP DIN
-.08
.07
.13
.32
-.21
-.14
-.25
.18
-.03
.07
-.13
.17

Sediment parameters
DIPTN
TP TN:TP
.12
-.05
.24
-.25
-.13
.03
.26
-.22
-.07
.11
-.04
.14
*
-.01
-.81
-.64
.47

APHA: American Public Health Association 1999. Standard
Me­thods for the Examination of Water and Wastewater, 20thedition. American Public Health Association, AmericanWa­ter Works Association, Water Environment Federa­tion, Washington DC.
Apostolaki, E.T., M. Holmer, N. Marbà & I. Karakassis
2011. Reduced carbon sequestration in a Mediterraneansea­grass (Posidonia oceanica) ecosystem impacted by fish
far­ming. Aquaculture Environment Interactions 2:49–59.
Atkinson, M. & S. Smith 1983. C:N:P ratios of benthic ma­rine plants. Limnology and Oceanography 28: 568–574.
Beer, S., M. Bjork, F. Hellblom & L. Axelsson 2002. In­or­ga­
nic carbon utilization in marine angiosperms (seagrasses).
Functional Plant Biology 29:349–354.
Borum, J., O. Pedersen, L. Kotula, M.W. Fraser, J. Statton, T.D.
Colmer & G.A. Kendrick 2016. Photosynthetic res­ponse to
globally increasing CO2 of co-occurring tem­pe­rate seagrass
species. Plant, Cell and Environment 39:1240–1250.
Burkholder, J.M., D.A. Tomasko & B.W. Touchette 2007.
Sea­grasses and eutrophication. Experimental Marine Biology
and Ecology 350:46–72.
Campbell, J.E. & J.W. Fourqurean 2009. Interspecific va­ria­tionin the elemental and stable isotope content of sea­grasses inSouth Florida. Marine Ecology Progress Series 387:109–123.

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

Interspecific variation in foliar nutrients and isotopes of submerged macrophytes in Vietnam

Campbell, J.E. & J.W. Fourqurean 2013. Mechanisms ofbi­car­bonate use influence the photosynthetic carbondi­oxide sensitivity of tropical seagrasses. Limnology andOceanography 58:839–848.
Cao, V.L., V.T. Nguyen, T. Komatsu, D.V. Nguyen & D.T.
Dam 2012. Status and threats on seagrass beds using GIS in
Vietnam. SPIE–Remote Sensing of the Marine En­vi­ron­ment II.http://dx.doi.org/10.1117/12.977277. p. 8525
12. Last accessed 20.09.2019.
Carlson, R.E. 1977. A trophic state index for lakes. Limnology
and Oceanography 22:361–369.
Chappuis, E., V. Seriñá, E. Martí, E. Ballesteros & E. Ga­cia
2017. Decrypting stable isotope (δ13C and δ15N) varia­bi­lity in aquatic plants. Freshwater Biology 62(11):1–12.
Connolly, R.M., D. Gorman, J.S. Hindell, T.N. Kildea &T.A. Schlacher 2013. High congruence of isotope sewage
signals in multiple marine taxa. Marine Pollution Bulletin 71:
152–158.
Daya A.A. & H. Bejari 2015. A comparative study between
simple kriging and ordinary kriging for estimating andmodeling the Cu concentration in Chehlkureh deposit,
SE Iran. Arabian Journal of Geosciences 8:6003–6020.
Disperati, L. & S.G.P. Virdis 2015. Assessment of land-use
and land-cover changes from 1965 to 2014 in Tam GiangCau Hai Lagoon, central Vietnam. Applied Geography 58:
48–64.
Duarte, C.M. 1990. Seagrass nutrient content. Marine Ecology
Progress Series 67:201–201.
Estefan, G., R. Sommer & J. Ryan 2014. Analytical Methods
for Soil, Plant and Water in the Dry Areas: A manual of Rele­vance to the West Asia and North Africa Region, Third Edition.
International Center for Agriculture research in the Dry
Areas, Aleppo, 255 pp.
Falkowski, G.D. Farquhar, J.R. Ehleringer & K.T. Hubick 1989.
Carbon isotope discrimination and photosynthesis. Annual
Review of Plant Physiology and Plant Molecular Biology 40:503–537.
Ferdie, M. & J.W. Fourqurean 2004. Responses of seagrass
communities to fertilization along a gradient of relative
availability of nitrogen and phosphorus in a carbonate
environment. Limnology and Oceanography 49:2082–2094.
Fourqurean, J.W., S.A. Manuel, K.A. Coates, W.J. Ken­wor­
thy & J.N. Boyer 2015. Water quality, isoscapes and stoi­chio­scapes of seagrasses indicate general P limitation andunique N cycling in shallow water benthos of Bermuda.
Biogeosciences 12:6235–6249.
Fourqurean, J.W., N. Marba, C.M. Duarte, E. Diaz-Almela& S. Ruiz-Halpern 2007. Spatial and temporal variationin the elemental and stable isotopic content of theseagrasses Posidonia oceanica and Cymodocea nodosa from theIlles Balears, Spain. Marine Biology 151:219–232.
Fourqurean, J.F., T.O. Moore, B. Fry & J.T. Hollibaugh 1997.
Spatial and temporal variation in C:N:P ratios, δ15N, andδ13C of eelgrass Zostera marina as indicators of ecosystemprocesses, Tomales Bay, California, USA. Marine Ecology
Progress Series 157:147–157.
Fraser, M.W., G.A. Kendrick, P.F. Grierson, J.W. Fourqurean,
M.A. Vanderklift & D.I. Walker 2012. Nutrient status ofsea­grasses cannot be inferred from system-scale dist­ri­
bu­tion of phosphorus in Shark Bay, Western Australia.
Marine and Freshwater Research 63:1015–1026.
Fry, B. 2002. Conservative mixing of stable isotopes acrossestuarine salinity gradients: a conceptual framework formonitoring watershed influences on downstream fishe­ries production. Estuaries and Coasts 25:264–271.
Geovariances 2016. ISATIS Software: Technical References
Release 2016.1. Geovariances & Ecole des Mines de Paris,
Paris, France.
Goovaerts P (1997) Geostatistics for Natural Resources
Evaluation. Oxford University Press, New York.
Gordon, D.M., P.B. Birch & A.J. McComb 1981. Effects ofinorganic phosphorus and nitrogen on the growth of anestuarine Cladophora in culture. Botanica Marina 24: 93–106.
Gorman, D., A. Turra, R.M. Connolly, A.D. Olds &T.A. Schla­cher 2017. Monitoring nitrogen pollution insea­so­nal­ly-pulsed coastal waters requires judicious choiceof indicator species. Marine Pollution Bulletin 122:149–155.
Hemminga, M.A. & M.A. Mateo 1996. Stable carbon iso­
topes in seagrass: variability in ratios and use in ecologicalstudies. Marine Ecology 140:285–298.
Herbeck, L.S., M. Sollich, D. Unger, M. Holmer & T.C.
Jen­nerjahn 2014. Impact of pond aquaculture effluentson sea­grass performance in NE Hainan, tropical China.
Marine Pollution Bulletin 85:190–203.
Hitchcock, J.K., S.C. Courtenay, M.R.S. Coffin, C.C. Pater &M.R. van Den Heuvel 2017. Eelgrass bed structure, leaf
nutrient, and leaf isotope responses to natural and an­th­ro­pogenic gradients in estuaries of the Southern Gulf of
St. Lawrence, Canada. Estuaries and Coasts 40:1653–1665.
Hobbie, E., A. Jumpponen & J. Trappe 2005. Foliar andfun­gal 15N:14N ratios reflect development of mycorrhizae
and nitrogen supply during primary succession: testingana­lytical models. Oecologia 146:258–268.
Hoyer, M.V., T.K Frazer., S.K. Notestein & D.E. Canfield
2004. Vegetative characteristics of three low-lying Flo­ri­dacoastal rivers in relation to flow, light, salinity and nut­ri­
ents. Hydrobiologia 528:31–43.
Invers, O., M. Perez & J. Romero 1999. Bicarbonate uti­li­za­tion in seagrass photosynthesis: role of carbonic
anhydrase in Posidonia oceanica (L) Delile and Cymodocea
nodosa (Ucria) Ascherson. Journal of Experimental Marine
Biology and Ecology 235:125–133.
Johengen, T. 1997. Standard operating procedures for de­ter­mining total phosphorus, available phosphorus, andbiogenic silica concentrations of Lake Michigan se­di­ments and sediment trap material, in Lake Michigan massbalance study (LMMB) methods compendium. In: Me­tals,
conventionals, radiochemistry and biomonitoring sample ana­ly­sis
techniques, vol. 3, (L. Blume, ed.), pp. 305–312, Uni­ted States
Environmental Protection Agency (EPA 905R97012c).
Johnson, M.W., K.L Heck & J.W. Fourqurean 2006. Nutrient
content of seagrasses and epiphytes in the northern
Gulf of Mexico: Evidence of phosphorus and nitrogen
limitation. Aquatic Botany 85:103–111.
Keeley, J.E. & D.R. Sandquist 1992. Carbon: freshwater
plants. Plant, Cell and Environment 15:1021–1035.
Kratzer, C.R. & P.L. Brezonik 1981. A carlson-type trophic
state index for nitrogen in florida lakes. Water Resources
Bulletin 17:713–715.
Le, X.T. 2012. Preliminary assessment of sea level rise im­pacts to coastal ecosystems in Thua Thien-Hue. VNU
Journal of Science, Earth Sciences 28:140–151.
Lepoint, G., P. Dauby & S. Gobert 2004. Applications of C andN stable isotopes to ecological and environmental studies inseagrass ecosystems. Marine Pollution Bulletin 49: 887–891.

Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72

71

Nhu Y et al.

Lepoint, G., B. Frederich, S. Gobert & E. Parmentier 2008.
Isotopic ratios and elemental contents as indicators ofseagrass C processing and sewage influence in a tropicalmac­ro­tidal ecosystem (Madagascar, Mozambique Chan­nel). Scientia Marina 72:109–117.
Logan, M. 2010. Biostatistical design and analysis using R: A
prac­tical guide. Wiley & Blackwell, England, 557 pp.
Michener, R.H. & K. Lajtha 2007. Stable isotopes in ecology
and environmental science. Blackwell Scientific Publications,
Oxford, 592 pp.
Mook, W.G. 2000. Environmental isotopes in the hyd­ro­lo­gical cycle:
principles and applications, vol. 1: Introduction; theory, methods,
review. IHP–V Technical documents in hydrology. No 39,
Vol. I. UNESCO, Paris.
Murphy, J. & J.P. Riley 1962. A modified single solution
method for the determination of phosphate in natural
waters. Analytica Chimica Acta 27:31–36.
Nguyen, Q.C.T. & M. Yabe 2014. Shrimp poly-culture de­ve­lopment and local livelihoods in Tam Giang-Cau Hai
Lagoon, Vietnam. Agricultural Science 6:1–14.
Odelu, G., N.M. Kumar, N. Siddulu & K. Raghu 2014.
Enu­me­ration of macrophytes of eutrophicated and noneutrophicated lakes of two tahasils of Karim Nagar Dist­
rict. Biolife 2:1170–1180.
Oksanen, J., G.F. Blanchet, R. Kindt, P. Legendre, P.R. Min­chin, R.B. O’Hara, G.L. Simpson, P. Solymos, M.H.H.
Ste­vens & H. Wagner 2013. vegan: Community Ecology Pa­
ckage. R package version 2.0-9. Available at :http://cran.rproject.org/package=vegan.
Paerl, H 2009. Controlling eutrophication along the fresh­water – marine continuum: dual nutrient (N and P) reduc­tions are essential. Estuaries and Coasts 32:593–601.

Short, F.T., B. Polidoro, S.R. Livingstone, K.E. Carpenter, S.
Ban­deira, J.S. Bujang, H.P. Calumpong, T.J.B. Carruthers,
R.G. Coles, W.C. Dennison, P.L.A Erftemeijer, M.D.
For­tes, A.S. Freeman, T.G. Jagtap, A.H.M. Kamal, G.A.
Kend­rick, W. Judson Kenworthy , Y.A. La Nafie, I.M.
Na­su­tion, R.J. Orth, A. Prathep, J.C. Sanciangco, B. vanTus­senbroek, S.G. Vergara, M. Waycott & J.C. Zieman
2011. Extinction risk assessment of the world’s seagrass
species. Biological Conservation 144:1961–1971.
Schmidt, M., H.S. Windisch, K.U. Ludwichowski, S.L.L. See­gert, H.O. Portner, D. Storch & C. Bock 2018. Seawatercar­bonate chemistry and neurochemical profiles of two
gadid species. PANGAEA. https://doi.org/10.1594/
PANGAEA.892312.
Stapel, J. & M.A. Hemminga 1997. Nutrient resorption
from seagrass leaves. Marine Biology 128:197–206.
Sun, Z., X. Mou, X. Li, L. Wang, H. Song & H. Jiang 2011.
Ap­plication of stable isotope techniques in studies ofcar­bon and nitrogen biogeochemical cycles of ecosystem.
Chinese Geographical Science 21:129–148.
Thanh, T.D., Y. Saito, V.D. Huy, V.L. Nguyen, T.K.O. Ta &M. Tateishi 2004. Regimes of human and climate changes
in Vietnam. Regional Environmental Change 4:49–62.
Udy, U.W & W.C. Dennison 1997. Growth and physiologicalresponses of three seagrass species to elevated sedimentnutrients in Moreton Bay, Australia. Experimental Marine
Biology and Ecology 217:253–277.
Uku, J., S. Beer & M. Bjork 2005. Buffer sensitivity of pho­to­synthetic carbon utilisation in eight tropical seagrasses.
Marine Biology 147:1085–1090.

Peipoch, M., E. Gacia, A. Blesa, M. Ribot, J.L. Riera & E.
Mar­tí 2014. Contrasts among macrophyte riparian species
in their use of stream water nitrate and ammonium: in­sightsfrom 15N natural abundance. Aquatic Sciences 76:203–215.

Van Katwijk, M.M., M.E.W. Van der Welle, E.C.H.E.T. Lu­
cas­sen, J.A. Vonk, M.J.A. Christianen, K.W. HakimI, A.
Ari­fin, T.J. Bouma, J.G.M. Roelofs & L.P.M. Lamers 2011.
Ear­ly warning indicators for river nutrient and sedimentloads in tropical seagrass beds: a benchmark from a nearpristine archipelago in Indonesia. Marine Pollution Bulletin
62:1512–1520.

Phan, T.T.H. 2018. Submerged aquatic vegetation in a tro­picallagoon environment: Dynamics and resilience strategy. PhD thesis,
Vrije Universiteit Brussels, Belgium. 193 pp.

Vizzini, S. & A. Mazzola 2004. Stable isotope evidence forthe environmental impact of a land-based fish farm in thewestern Mediterranean. Marine Pollution Bulletin 49: 61–70.  

R Core Team 2017. R: A language and environment for statisticalcomputing. R Foundation for Statistical Computing,
Vienna, Austria.

Walton, M.E.M., I. Al-Maslamani, N. Haddaway, H. Ken­ne­dy & A. Castillo 2016. Extreme 15N depletion in sea­gras­ses. Estuaries and Coasts 39:1709–1723.

Raven, J.A., A.M Johnston, J.E Kübler, R. Korb, S.G. McIn­
roy, L.L. Handley, C.M. Scrimgeour, D.I. Walker, J. Bear­dall,
M. Vanderklift, S. Fredriksen & K.H. Dunton 2002. Me­cha­nistic interpretation on carbon isotope by ma­rine mac­ro­algae and seagrasses. Functional Plant Biology 29:355–378.

Waycott, M., C.M. Duarte, T.J.B. Carruthers, R.J. Orth,  W.C.
Den­nison, S. Olyarnik, A. Calladine, J.W. Fourqurean,
K.L. Heck Jr., A. Randall Hughes, G.A. Kendrick, W.J.
Ken­worthy, F.T. Short & S.L. Williams 2006. Accelerating
loss of seagrasses across the Globe threatens coastal
ecosystems. PNAS 106(30):12377–12381.

Risk, M.J., B.E. Lapointe, O.A. Sherwood & B.J. Bedford
2009. The use of δ15N in assessing sewage stress on coral
reefs. Marine Pollution Bulletin 58:793–802.
Schmidt, A.L., J.K.C. Wysmyk, S.E. Craig & H.K. Lotze
2012. Regional-scale effects of eutrophication on eco­sys­
tem structure and services of seagrass beds. Limnology andOceanography 57:1389–1402.

West, J.B., G.J. Bowen, T.E. Cerling & J.R. Ehleringer 2006.
Stable isotopes as one of nature’s ecological recorders.
Trends in Ecology and Evolution 21:408–414.
Whalen, M.A., J.E. Duffy & J.B. Grace 2013. Temporal
shifts in top-down vs. bottom-up control of epiphytic
algae in a seagrass ecosystem. Ecology 94:510–520.

Schwarz, A.M., M. Björk, T. Buluda, H. Mtolera & S. Beer
2000. Photosynthetic utilisation of carbon and light by
two tropical seagrass species as measured in situ. Marine
Biology 137:755–761.

Yamamuro, M., H. Kayanne & H. Yamano 2003. δ15N ofsea­grass leaves for monitoring anthropogenic nutrient in­creases in coral reef ecosystems. Marine Pollution Bulletin
46:452–458.

Shilla, D.A., T. Asaeda, S. Kian, R. Lalith & J. Manatunge
2006. Phosphorus concentration in sediment, water andtissues of three submerged macrophytes of Myall Lake,
Australia. Wetlands Ecology and Management 14:549–558.

Yu, H., Z. Yu, X. Song, X. Cao, Y. Yuan & G. Lu 2015. Sea­so­nal variations in the nitrogen isotopic composition ofdissolved nitrate in the Changjiang River estuary, China.
Estuarine, Coastal and Shelf Science 155:148–155.

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Botanica Pacifica. A journal of plant science and conservation. 2020. 9(2): 61–72