Bull Mar Sci. 96(3):469–486.

2020 research paper

https://doi.org/10.5343/bms.2018.0093

Feeding habitats of juvenile reef fishes in a

tropical mangrove–seagrass continuum along a
Malaysian shallow-water coastal lagoon

1
Institute of Oceanography
and Environment, Universiti
Malaysia Terengganu, 21030
Kuala Nerus, Terengganu,
Malaysia
2
Atmosphere and Ocean
Research Institute, The
University of Tokyo, 5-1-5,
Kashiwanoha, Kashiwa-shi,
Chiba 277-8564 Japan
* Corresponding author email:
<lqdungimer@gmail.com>, <le.
dung@umt.edu.my>
Guest Editor: Amy Y Then
Date Submitted: 11 December, 2018.
Date Accepted: 6 September, 2019.
Available Online: 11 September, 2019.
Dung Quang Le 1 *
Siau Yin Fui 1
Kentaro Tanaka 2
Suhaimi Suratman 1
Yuji Sano 2
Kotaro Shirai 2
ABSTRACT.—We conducted stable isotope (δ13C and
δ15N) and gut content analyses to understand habitat use
of juvenile reef fishes (Lethrinus lentjan, Lutjanus russellii,
and Epinephelus coioides), particularly the influence of
spatial habitat structure along seagrass–mangrove continua.
Sampling was conducted in Setiu Lagoon, located in the
southern waters of the South China Sea (Malaysia). Gut
content analysis indicated that the focal fishes preyed mainly
on benthic invertebrates and other smaller fishes; these were
used as potential prey items for isotopic tracers to track fish
habitat use. Stable isotope analyses highlighted that the
juvenile reef fishes were highly associated with seagrass beds
as feeding grounds; however, site-specific analyses suggest
differences in fish feeding habitats. The juvenile fishes,
particularly large juveniles, preyed in both mangrove and
seagrass areas from the central lagoon, whereas all juveniles
showed preferential foraging within seagrass meadows in the
nearby lagoon mouth. Furthermore, the mean stable isotope
values of muscle tissue differed among fish size classes,
indicating their ontogenetic habitat/diet shifts, especially
for L. lentjan and E. coioides. This study provides important
information to support current efforts in protecting and
preserving mangroves and seagrasses as crucial nursery
habitats for juvenile reef fishes from the anthropogenic
activities influencing Setiu Lagoon.

Mangrove and seagrass habitats in tropical shallow-water coastal lagoons are con-
sidered crucial nursery and feeding grounds for numerous fish species including reef
fishes (Blaber 2000, Sheaves and Molony 2000, Beck et al. 2001) because they provide
protection from predators and rich food resources, which maximize growth during
the juvenile stage (Sheaves et al. 2015). They also serve to replenish and sustain adult
populations on coral reefs (Beck et al. 2001, Nagelkerken et al. 2008, Sheaves et al.

Bulletin of Marine Science 469

© 2020 Rosenstiel School of Marine & Atmospheric Science
of the University of Miami
470 Bulletin of Marine Science. Vol 96, No 3. 2020

2015). The interlinked mangrove and seagrass habitats have been intensively studied
due to their importance as feeding habitats for juvenile reef fishes, particularly in
the Caribbean region (Nagelkerken and van der Velde 2004, Vaslet et al. 2015). In
this region, substantial areas exist where mangrove prop-roots are inundated during
low tide, thus juvenile reef fishes can shelter in mangrove habitats and make feeding
forays into nearby seagrass beds (Rooker and Dennis 1991, Nagelkerken and van der
Velde 2004). Additionally, some studies conducted on size-related dietary shifts of
reef fishes suggested that small juveniles utilize mainly seagrass beds and then in-
crease their range of foraging grounds (a mangrove–seagrass continua) as they grow
before migrating to coral reefs (Nagelkerken and van der Velde 2004, Mumby 2006,
Vaslet et al. 2015). Fish feeding activities can also be site-specific and depend on
geographic regions as well as tropical estuary environments (Barletta et al. 2005,
Lugendo et al. 2007). In the Indo-Pacific region, mangroves and seagrasses also form
continua of intertidal mosaic habitats in lagoons and estuaries; however, the man-
grove habitats are alternately inundated and exposed by the high-tide/low-tide cycle.
During low tidal periods, such habitats are often not continuously available for most
fishes, thus seagrass meadows provide food and shelter while mangroves are unavail-
able (Sheaves 2005, Nagelkerken 2009). Several studies suggest that mangroves may
not be the main sources of carbon for fish assemblages in mangrove estuaries, as
the isotopic ratios in fish species were more enriched in 13C than mangrove-derived
carbon sources (Kieckbusch et al. 2004, Lugendo et al. 2007, Nyunja et al. 2009, Tue
et al. 2014). However, other studies have measured stable isotope ratios in benthic
invertebrates to trace the origins or ontogenetic movements of their consumers, par-
ticularly fish species (Nakamura et al. 2008, Berkström et al. 2013, Connolly and
Waltham 2015, Escalas et al. 2015) because, given that benthic invertebrates have
lower mobility or are habitat-specific, the isotopic signatures of their tissues reflect
the relative changes in the isotopic values of local dietary sources, seagrasses or man-
groves (Fry 1984, Nagelkerken et al. 2001, Bouillon et al. 2002, Vaslet et al. 2011,
2012, Berkström et al. 2013, Le et al. 2018). While juvenile reef fishes can access
mangroves for feeding during tidal inundation, they mainly prey on benthic inver-
tebrates that rely on mangrove carbon sources, such as sesarmid crabs (Sheaves and
Molony 2000, Le et al. 2018). Such feeding activities of the juveniles reflect complex
functional webs integrated across interlinked, intertidal vegetated habitats in tropi-
cal regions (Nagelkerken and van der Velde 2004, Igulu et al. 2013). Furthermore,
the nursery functions of vegetated habitats are structurally complex and dependent
on habitat configuration and environmental conditions (Blaber 2000, Nagelkerken
et al. 2008, Taylor et al. 2017). Thus, the loss or damage of one nursery habitat can-
not only alter ecological functioning of entire coastal systems, but also profoundly
affect juvenile fish biomass and sustainable fishery stocks (Honda et al. 2013). Very
little information is available regarding juvenile reef fishes feeding habits in coastal
nursery habitats of the Pacific Ocean, particularly around Malaysia. Therefore, such
research is urgently needed to improve our understanding of these habitats and the
food webs they support in order to successfully design marine reserves and resource
management systems to preserve mangrove and seagrass habitats.
In recent studies, gut content and stable isotope analyses have been combined as
an effective approach to study food web structure and the movement and feeding ac-
tivities of juvenile fishes (Cocheret de la Morinière et al. 2003, Vaslet et al. 2011, Igulu
et al. 2014, Escalas et al. 2015, Le et al. 2018). Gut contents provide a 1–2 hr snapshot
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 471

of information on fish diets (George and Hadley 1979) and can help determine the
available prey items within habitats (Vaslet et al. 2011). Stable isotopic values in fish
tissue provide information relating to the isotopic signature of fish diets during the
preceding days, weeks, or months (Gearing 1991, Fry 2008). According to Post (2002)
and McCutchan et al. (2003), the degree of enrichment of stable isotopes between
fishes and their diets are slightly enriched (<1‰) or conserved for δ13C but vary from
2.0‰ to 3.5‰ for δ15N. Furthermore, mangroves often show distinct δ13C signatures
(δ13C generally between −34‰ and −27‰) compared to seagrasses (δ13C > −18‰; Fry
and Sherr 1984, Bouillon et al. 2008). Thus, stable isotope signatures of fish tissue
can reflect the corresponding isotopic signatures of local vegetation-based food webs
in which the fish has grown (Nagelkerken and van der Velde 2004, Vaslet et al. 2011,
Le et al. 2018). Recently, Bayesian mixing models, such as Stable Isotope Analysis in
R (SIAR), have been widely applied for their utility in analyzing isotope data (Parnell
et al. 2010, Phillips et al. 2014). SIAR was developed to estimate distributions of con-
sumers’ possible diets (Vaslet et al. 2011, Abrantes et al. 2015, Connolly and Waltham
2015), to elucidate species-specific ontogenetic shifts, or to characterize broader food
web structure (Cocheret de la Morinière et al. 2003, Nakamura et al. 2008, Vinagre
et al. 2012, Abrantes et al. 2015, Artero et al. 2015, Le et al. 2017).
In the present study, we used gut content and stable isotope analysis (δ13C and δ15N)
to determine the feeding grounds of juvenile reef fishes, especially of emperor fish
(Lethrinus lentjan), snapper (Lutjanus russellii), and grouper (Epinephelus coioides)
in structurally complex nursery habitats in a shallow-water lagoon.

Materials and Methods

Study Area.—Setiu Lagoon (Fig. 1) is a critical nursery wetland for the juveniles
of many fish species along the eastern coast of Peninsular Malaysia (Azmi 2014). An
earlier study indicated that the mangrove and seagrass continua that form along the
lagoon are utilized by a number of juvenile fishes, particularly reef fishes (Le et al.
2018). Approximately 14 km long, the lagoon contains several habitat types with a
gradient of salinity from the center to the mouth (Nakisah and Fauziah 2003, Azmi
2014). In the central lagoon, where the water is influenced by the Ular rivulet and
mangroves are dominated by Rhizophora spp. and Avicennia spp., the salinity varies
from 24‰ to 27‰. In contrast, waters which are in the narrow mouth, connected to
the open sea, are strongly influenced by tidal and riverine effects, hence salinity is
related to whether there is a predominance of marine or riverine input. Due to less
precipitation and therefore less freshwater discharging from rivers into the lagoon
during the dry season (March to October), the water salinity is relatively high and
uniform, varying within mean 25.1‰ (SD 0.5) in the lagoon center and with mean
29.6‰ (SD 0.9) in the mouth.

Sampling.—Fieldwork was conducted in two seagrass areas adjacent to man-

groves during a 6-d trip during the dry season, July 2017 (Fig. 1). The first seagrass
site in the center of the lagoon (STA1) has been used as a sampling site in a previous
study (Le et al. 2018) and is 7–10 m from nearby mangroves (Rhizophora apiculata).
The second seagrass area (STA2) is approximately 10 km away from STA1 and is
close to the mouth of the lagoon. The seagrass beds are about 150–200 m from a
mixed mangrove forest (R. apiculata and Nypa fruticans). The lagoon was subjected
472 Bulletin of Marine Science. Vol 96, No 3. 2020

Figure 1. The location of the study areas and sampling sites in Setiu Lagoon.

to semidiurnal tides with a tidal range of 1.7 m and the water depth varied from 0.2
to 2 m above the seagrass beds at low and high tides, respectively. The measured wa-
ter salinity [mean (SD)] at STA1 [25.1 (0.5)] was lower than that in STA2 [29.6 (0.9)].
A small fishing boat with a 30-hp (22-kW) engine, designed to operate a trawl-
ing net (2 cm mesh size), was used to collect the target juvenile fishes L. lentjan (95
specimens), L. russellii (27 specimens), and E. coioides (29 specimens) during high
tidal periods at both sites. Trawl duration was 10 min at a stable pulling speed for
approximately 250 m along seagrass meadow. Sampling was repeated three times
per day at each site and sampling was conducted over 3 d. Bycatch was released af-
ter target species and potential prey items (crustacean and juvenile fishes) were col-
lected. The focal fishes feed primarily on benthic invertebrates, although small fishes
have been observed in their gut contents (Nuraini et al. 2007, Carpenter et al. 2016).
Potential food items including benthic invertebrates and small fishes were also col-
lected during trawl sampling. Benthic invertebrates, considered less-mobile species,
were collected from both mangroves and seagrass meadows and included crusta-
ceans, bivalves, gastropods, and peanut and annelid worms. Of these, bivalves, gas-
tropods, and annelid worms were collected by sieving sediments. Crustaceans and
prey fishes were collected from the seagrass target fish trawl sampling or by using a
plankton net at night illuminated by flashlight. Gillnets (2-cm and 3-cm mesh sizes,
50 m long, and 2 m deep) were deployed along the edges of the mangrove fringe to
collect the fishes that foraged in the mangroves. Gillnets were deployed for 3 d and
were checked every 6 hrs.
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 473

To obtain triplicate particulate organic matter (POM) samples, two filter steps
were applied to 1.0 L surface water samples that were collected at 20 cm depth from
the water surface. The water samples were first filtered through 200-μm mesh plank-
ton nets to remove zooplankton, then filtered through precombusted (500 °C), 47-μm
Whatman GF/F glass fiber filters. Zooplankton (ZP) were sampled with a 200-μm
mesh zooplankton net by towing the net horizontally at the surface for about 5 min
at a uniform speed. Mangrove leaves and seagrass were collected to determine their
role as autotrophic, end-member food sources. All samples were packed in labeled
polyethylene bags and immediately stored in coolers, transported to the laboratory
for further processing, and frozen at −20 °C until analysis.

Sample Preparation.—All fish samples were cleaned with deionized water be-
fore identification and then sorted by species. The total length (TL; nearest mm) and
body weight (nearest g) of each fish were measured. The dorsal white muscle tissue
of the fish was dissected for stable isotope analysis. Benthic invertebrate tissues were
separated from shells, and the heads and fins of juvenile fishes (potential fish prey
items) were removed before the samples were dried to minimize the effects of cal-
cium carbonate in the samples. Small gastropods and bivalves (2.0–10.0 mm) were
pooled as 5–20 individuals to a single sample depending on body size. All samples
were dried at 60 °C for 48 hrs before grinding into a fine powder using a mortar
and pestle. To achieve optimum results of both δ13C and δ15N in a measurement,
we used dilution methods using CO2 gas to measure δ13C. Powdered samples of all
animal tissues were weighed to approximately 2 mg in silver capsules, treated with
three drops of 10% HCl to remove calcium carbonate from broken shells or bones,
and then dried at 60 °C overnight (10 hrs) before being transferred into tin capsules.
Similarly, 5- or 10-mg powder samples of seagrass and mangrove litter, respectively,
were transferred into silver and tin capsules for stable isotope analysis.

Gut Content Analyses.—Gut content analyses were conducted based on the

index of relative importance (RI; George and Hadley 1979). In brief, after fishes
were dissected at the laboratory at the Institute of Oceanography and Environment,
Universiti Malaysia Terengganu, digestive tracts were removed and transferred into
a petri dish. All gut contents were examined under a stereo microscope. The cat-
egories of gut contents were determined as crustaceans (crab, shrimp), polychaetes,
gastropods, bivalves, and fishes. Prey items were identified, counted, and weighed as
described by George and Hadley (1979). RI values were calculated for comparison
among species and body size classes of each species as follows:

AIa = % frequency of occurrence + % total numbers + % total weight for food item a

, where n is the number of different food types.

In which RIa is the relative importance index for food item a, and AIa is the absolute
importance index for a.

The results of gut content analysis provided amended information to choose poten-
tial prey items for the stable isotope analysis. For example, Faunus sp. was frequently
474 Bulletin of Marine Science. Vol 96, No 3. 2020

found in seagrass meadows from both sampling sites; however, this species has not
been observed in focal fishes’ gut contents in the previous and current studies (Siti et
al. 2017, Le et al. 2018). Due to its thick and hard shell, the species is not considered a
realistic food item and was excluded in the analysis.

Stable Isotopic and Mixing Model Analyses.—The stable isotope (δ13C and
δ N) composition of the samples was measured using an IsoPrime100 mass spec-
15

trometer (IsoPrime, UK) coupled with a vario MICRO cube combustion device
(Elementar, Germany) at the Atmosphere and Ocean Research Institute, University
of Tokyo, Japan. The measurements of stable isotope composition were conducted as
described in Le et al. (2017).
The sample output was calibrated to δ values using the traceable standards ammo-
nium sulphate (IAEA-N-1) for δ15N [0.4‰ (0.2)] and sucrose (IAEA-CH-6) for δ13C
[−10.449‰ (0.033)]. Repeated analyses of muscle tissue standard reference material
(n = 18, SRM2976, National Institute of Standards and Technology, USA) gave a re-
producibility (SD) of 0.2‰ for both δ15N and δ13C. As the fishes were considered at
the juvenile stage and C:N ratios in their tissue were below 3.5, no lipid correction
was conducted on the samples (Post et al. 2007).
The Bayesian stable isotope mixing model SIAR v3.4.2 developed by Parnell et al.
(2010) was used to determine the extent of the food sources contributing to fish diets
and to compare food sources among fish species and size classes. Due to the wide
ranges in body sizes of L. lentjan (STA1) and E. coioides (STA2) that were collected,
they were sorted into size classes as small (S), medium (M), and large (L) juveniles to
evaluate their dietary shifts (Table 1).
The potential prey sources used for the SIAR modelling consisted of benthic in-
vertebrates from seagrass beds (seagrass prey items), benthic invertebrates from
mangroves (mangrove prey items), and small abundant juvenile fishes collected in
seagrass beds (seagrass fish prey items (Siganus spp., Leiognathus spp., and Pelates
spp.). The three studied fish species are considered demersal carnivorous fishes (in-
vertebrate and fish feeders; Carpenter and Allen 1989, Matsunuma et al. 2011) and
the gut contents identified in this study mainly included invertebrates. Therefore,
POM, ZP, seagrasses, and mangrove litter were excluded as food sources in the
mixing model. The trophic enrichment factors (TEFs) examined in this study had
a ΔN of 2.4‰ (0.22) and ΔC of −0.2‰ (0.21), as recommended for acidified samples
(McCutchan et al. 2003). Concentration dependences were set to zero.

Statistical Analyses.—Values are expressed as mean (SD). Prior to statisti-

cal analyses, all data were tested for normality using the Kolmogorov–Smirnov
and Shapiro–Wilk tests. As the isotopic values of POM and ZP were not normal-
ly distributed, nonparametric Mann–Whitney U-tests were conducted to detect
significant differences between sampling sites and habitats. One-way analysis of
variance (ANOVA) and post hoc Tukey’s honest significant difference (HSD) tests
were performed to detect differences in stable isotopic signatures among fish food
source species within each site. Student’s t-tests were conducted to compare stable
isotopic signatures among fishes and their dietary sources between sampling sites.
Relationships between TLs and dual stable isotope signatures of the fishes were ana-
lyzed by simple linear regression prior to analysis of covariance (ANCOVA) to de-
tect significant differences in the stable isotopic signatures of the fishes (dependent
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 475

Table 1. Index of relative importance (RI) values (%) of taxa identified in gut contents of juvenile fish species.

Species Sites Size classes ACR N % RI

LT (cm) Crustaceans Polychaetes Gastropods Bivalves Fishes
mean (SD)
L. lentjan STA1 S: 5.1 (0.8) L-S1 7 20.9 39.0 40.0 0.0 0.0
L. lentjan STA1 M: 7.6 (0.7) L-M1 16 2.9 91.7 5.4 0.0 0.0
L. lentjan STA1 L: 10.6 (0.6) L-L1 8 74.6 0.0 1.8 10.2 13.3
L. lentjan STA2 M: 8.5 (2.2) L-M1 8 76.6 0.0 3.1 20.3 0.0
L. russellii STA1 S: 6.3 (1.8) R-S1 13 92.5 4.6 0.0 0.0 2.9
L. russellii STA2 M: 9.7 (2.2) R-S2 3 90.7 9.3 0.0 0.0 0.0
E. coioides STA1 L: 16.7 (2.0) E-L1 0 - - - - -
E. coioides STA2 S: 8.8 (0.8) E-S2 3 85.5 0.0 0.0 0.0 14.5
E. coioides STA2 M: 11.6 (0.7) E-M2 5 0.0 0.0 0.0 0.0 100.0
E. coioides STA2 L: 15.5 (0.9) E-L2 2 86.9 0.0 13.1 0.0 0.0

variables) between samples (fixed factor) using TL as the covariate. A significance

level of 0.05 was used in all tests and statistical analyses were performed using SPSS
for Windows software (v25; IBM Corp., USA).

Results

Fish Sizes, Gut Contents, and Potential Diets.—The stomachs of L. lentjan

(n = 39), L. russellii (n = 16), and E. coioides (n = 10) contained food that was sorted
into five prey categories: crustaceans, polychaetes, gastropods, bivalves, and fishes
(Table 1). The diets of all fish species contained a high proportion of crustaceans,
mainly juvenile penaeid shrimp (Penaeus spp.), whereas the other categories varied
proportionally according to species and body sizes. Lethrinus lentjan had the most
varied diet among the focal species. Its gut contents contained all prey categories;
however, prey items differed among body size classes and sampling sites. The snap-
per L. russellii fed mainly on crustaceans and polychaetes in all size classes, although
small proportions of fishes were found in the stomach contents of those from STA1.
In contrast to L. lentjan and L. russellii, crustaceans and fishes were the most com-
mon prey items in the stomachs of E. coioides, including penaeid shrimp (Penaeus
spp.) and juvenile siganid fishes (Siganus spp.) and ponyfishes (Leiognathus spp). A
few gastropods were also found in stomachs of E. coioides.

Stable Isotopic Signatures of Basal Sources.—Stable isotope signatures

varied widely among the basal resources: mangroves, seagrasses, POM, and ZP
(Table 2). Discrimination of isotopic signatures between mangrove litter and sea-
grasses was evident at both sampling sites (Mann–Whitney U-test, P < 0.001). POM

Table 2. Basal resource δ13C and δ15N signatures (‰) between sampling sites [mean (SD)].

Basal sources STA1 STA2

N δ15N δ13C N δ15N δ13C
POM 5 5.0 (0.4) −24.8 (0.8) 4 5.4 (0.8) −22.1 (0.8)
ZP 3 7.3 (0.3) −22.8 (0.5) 4 6.2 (1.0) −20.9 (2.4)
Mangrove litter 6 2.9 (1.0) −29.3 (1.3) 6 2.6 (0.8) −28.6 (1.0)
Fresh seagrass 5 7.4 (0.3) −19.0 (0.4) 5 7.2 (0.3) −12.2 (0.2)
476 Bulletin of Marine Science. Vol 96, No 3. 2020

Table 3. Results of one-way analysis of variance (ANOVA) performed on stable isotope signatures among fish
food sources in the sampling sites. Mg = mangrove sources; Sg = seagrass sources.

STA1 STA2
df Mean F P Sources Tukey df Mean F P Sources Tukey
squares HSD squares HSD
tests tests
δ15N
Between groups 2 54.60 74.81 0.000 Sg-Mg <0.001 2 24.35 34.41 0.000 Sg-Mg 0.001
Within groups 57 0.73 Mg-Fish <0.001 49 0.71 Mg-Fish <0.001
Total 59 Fish-Sg <0.001 51 Fish-Sg <0.001
δ13C
Between groups 2 92.87 62.71 0.000 Sg-Mg <0.001 2 173.96 67.73 0.000 Sg-Mg <0.001
Within groups 57 1.48 Mg-Fish <0.001 49 2.57 Mg-Fish <0.001
Total 59 Fish-Sg 0.001 51 Fish-Sg 0.135

and ZP showed intermediate isotopic values between mangroves and seagrasses. The
δ13C values of the basal food sources were lower at STA1 than STA2 (Mann–Whitney
U-test, P < 0.001), but no significant difference in δ15N values was found between
sites.

Stable Isotopic Signatures of Potential Prey Sources.—Mangrove prey

items generally had lower mean isotopic carbon values [δ13Cmean; STA1 = −23.6‰
(1.2), STA2 = −22.5‰ (1.7)] compared with seagrass prey [δ13Cmean; STA1 = −19.5‰
(1.4), STA2 = −17.0‰ (1.7)] at both sites (Table 3, Fig. 2). For fish prey items, the mean
δ13C values were −21.1‰ (0.8) from STA1 and −18.3‰ (0.7) from STA2. A similar
trend for δ15N values among potential prey sources at the sites emerged—the highest
values were found in fish prey, followed by seagrass prey, and finally mangrove prey
items.
The δ13C values of both mangrove and seagrass prey items from STA1 were signifi-
cantly lower than those from STA2 (P < 0.05 and P < 0.0001, respectively), whereas
δ15N values showed the opposite trend (P < 0.001 and P < 0.0001, respectively). The
mean δ13C values of fish prey items from STA1 [–21.1‰ (0.6)] were lower than those
from STA2 [–18.3‰ (0.7)] (t-test, P < 0.0001), whereas the mean δ15N values of fish
prey items from STA1 [10.7‰ (0.6)] were more enriched in 15N compared to those
from STA2 [9.0‰ (0.6)] (t-test, P < 0.0001).

Stable Isotopic Signatures of Juvenile Reef Fishes.—Stable isotope signa-

tures in the juvenile fishes had narrow ranges of δ15N but wide ranges of δ13C values
among species at STA1. The δ13C values were found to be lower in E. coioides (range
from −24.6‰ to −20.5‰) than those in L. lentjan (range from −22.5‰ to −19.7‰)
and in L. russellii (range from −20.3‰ to −18.9‰; Figs. 2 and 3). Unlike at STA1,
small variations of stable isotope signatures were found among species at STA2 (Fig.
3); δ13C mean and SD values were −17.2‰ (0.3) in E. coioides, −17.1‰ (0.8) in L. rus-
sellii, and −17.4‰ (0.7) in L. lentjan. The δ15N values of all three fish species varied
narrowly, ranging between 8.8‰ and 10.2‰ (Fig. 3).
ANCOVA results indicated significant differences between sites in the stable iso-
tope signatures of juveniles of the three fish species. Fish species generally had higher
δ15N values at STA1 than STA2. In contrast, fishes had lower δ13C values at STA1
than STA2 (Table 4, Fig. 3).
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 477

Figure 2. Stable isotope signatures of potential prey sources from sites STA1 and STA2. Body
sizes: L-S = L. lentjan small, L-M = L. lentjan medium, L-L = L. lentjan large; R-S = L. russellii
small, R-L = L. russellii large; E-S = E. coioides small, E-M = E. coioides medium, E-L = E.
coioides large. Horizontal and vertical bars are standard deviations of δ13C and δ15N values,
respectively.

Contribution of Food Sources to the Focal Fish Species (Isotopic

Mixing Model).—The mean (95% Bayesian credible interval) proportion of prey
sources for the juvenile fishes showed a high probability of the contribution from
seagrasses at both sampling sites; however, the prey source contributions were de-
pendent on species, size class, and location (Fig. 4).
For L. lentjan at STA1, a negative proportional trend in seagrass prey items with
increasing body size [65.9%, 50.1%, and 32.1% for small (L-S1), medium (L-M1), and
large (L-L1) size classes, respectively] was observed, with concurrent increase in
mangrove (28.1%, 36.4%, and 41.0%, respectively) and fish prey items (6.1%, 13.5%,
and 26.6%, respectively; Fig. 4, left column). However, the fishes may have foraged
generally in seagrass habitats at STA2 where the contributions of the seagrass prey
(69.5%) and seagrass fish prey (22.2%) were over 80% in the focal fishes (Fig. 4, right
column).
Lutjanus russellii (R-S1) was reliant mainly on seagrass prey items (82.7%), whereas
both mangrove and seagrass fish prey items were minor contributors to this species’
478 Bulletin of Marine Science. Vol 96, No 3. 2020

Figure 3. Isotopic compositions of juvenile reef fish species from the studied sites.

diet (10.1% and 7.2%, respectively) at STA1 (Fig. 4, left column). A high proportion
of seagrass food items was also found in the L. russellii diet; however, the juveniles
tended to rely more on seagrass fish prey (12.5%) at STA2 than STA1 (Fig. 4, right
column).
Epinephelus coioides was equally dependent upon all prey sources in STA1, with
proportions of 32.8%, 39.2%, and 28% from prey items of seagrass, mangroves, and
seagrass fishes, respectively (Fig. 4, left column). However, high proportions of sea-
grass food sources were estimated for fishes from STA2. Interestingly, the mixing
model also suggested that the proportions of seagrass prey in E. coioides decreased
sharply from 70.1% at E-M2 [11.6 cm (0.7)] to 54.7% in E-L2 [15.5 cm (0.9)], concur-
rent with an abrupt increase in its reliance on seagrass fish prey from 20.9% to 40.0%
(Fig. 4, right column), suggesting an ontogenetic diet shift to piscivory at 15.5 cm
(0.9).

Discussion

Benthic invertebrates, particularly less mobile species, have been successfully used
as tracers to study juvenile carnivorous reef fish feeding habits and movements in
mangrove–seagrass continua, as their tissue isotopic signatures distinctly reflect the
isotopic composition of their local dietary sources (Nagelkerken and van der Velde

Table 4. Results from analysis of covariance (ANCOVA) performed on stable isotope values of fish species
between sites using fish total length (TL) as the covariate.

N δ15N δ13C
F P F P
L. lentjan 44 137.798 <0.0001 501.411 <0.0001
L. russellii 21 98.088 <0.0001 8.388 0.0100
E. coioides 29 56.766 <0.0001 116.869 <0.0001
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 479

Figure 4. Mean proportions (Bayesian 95% low and high values) of food sources in the diets of
fish species in sites STA1 (left) and STA2 (right). Body sizes: L-S = L. lentjan small, L-M = L.
lentjan medium, L-L = L. lentjan large, R-S = L. russellii small, E-S = E. coioides small, E-M =
E. coioides medium, and E-L = E. coioides large.

2004, Vaslet et al. 2011, 2015, Le et al. 2018). In this study, the isotopic signatures of
benthic invertebrates also confirmed the isotopically distinct sources between man-
grove and seagrass habitats along the lagoon (Fig. 2). Gut content analysis indicated
that the juvenile reef fishes ingested considerably more seagrass fish prey, which also
had differing isotopic signatures from mangrove and seagrass prey. Hence, seagrass
fish prey and mangrove and seagrass invertebrates were considered reliable diet
sources to reflect habitat use and dietary shifts of juvenile reef fishes in the lagoon.
Seagrass can be the primary foraging habitat for juvenile fishes, while mangroves
are considered to play a limited role as nursery habitats due to frequent exposure
during low tide in the Indo-Pacific region (Nagelkerken et al. 2008). As expected,
480 Bulletin of Marine Science. Vol 96, No 3. 2020

the isotopic data in the juvenile reef fishes indicated enriched 13C associated with
seagrass food sources. Furthermore, the SIAR mixing model suggested a higher pro-
portion of seagrass sources compared to mangrove sources contributed to fish diets
from both sites. However, the probabilities of their dietary proportions varied spa-
tially in relation to the habitat types.
For L. lentjan, a habitat/diet shift in juveniles was observed, as their isotopic sig-
natures in tissues reflected the gradual switch from seagrass to mangrove foraging
habitats associated with fish growth in STA1 (Fig. 4, left column). This finding is
somewhat similar to a study by Le et al. (2018), suggesting large juvenile fishes forage
more broadly within their nursery home range. The large juveniles could access the
mangrove fringes during inundation to prey on sesarmid crabs, a leaf-eating crab
species, and return to seagrass meadows to complement their diets during low tide
(Nagelkerken and van der Velde 2004, Vaslet et al. 2015, Le et al. 2018). At STA2,
δ13C isotopic signatures suggested that L. lentjan predominantly relied on seagrass
prey items without a trophic connection between mangrove and seagrass habitats.
This may indicate a trade-off in juvenile fishes between foraging behavior and pre-
dation risk in relation to the habitat structure. Crossing the distance separating the
different habitats could expose fishes to higher predation and energetic cost (Blaber
2000, Sheaves 2005, Snover 2008, Hammerschlag et al. 2010, Kimirei et al. 2013). The
seagrasses at STA2 are located close to the lagoon inlet, where they may be highly
favored by new recruits, such as juvenile fishes and invertebrates, for presettlement
before spreading further up the lagoon. These seagrasses often host high abundances
of fish and decapod larvae as prey items, increasing foraging opportunities for car-
nivorous juvenile fishes and enhancing growth (Bell et al. 1988, Beck et al. 2001,
Baker and Sheaves 2009, Taylor et al., 2017).
Unlike L. lentjan, isotopic signatures suggested that L. russelli appears to forage
predominantly in seagrass meadows at both sites. Fish habitat selection may be size-
dependent, as refuge from predation is likely to be of greater importance to smaller
fish juveniles (Werner and Gilliam 1984, Post 2003, Unsworth et al. 2009). With the
smallest sizes among fish species collected, L. russelli juvenile can be vulnerable to
predation in the lagoon, while the mangroves are only a temporary habitat. Hence,
it is likely that these fishes may prefer to remain in the seagrass beds for shelter and
feeding habitat in the lagoon.
The grouper, E. coioides, is an opportunistic carnivore and considered close to the
top of the mangrove food chain; they preferentially feed on various prey items in-
cluding fishes, shrimps, and crabs (Nuraini et al. 2007). Thus, it was not surprising to
find the greatest proportion of fish prey sources in their diet when using the mixing
model to elucidate dietary ontogeny. The roughly equal proportions of mangrove
and seagrass prey sources suggested that these fishes foraged over a broader range
of interlinked habitats at STA1. However, like the feeding strategies of L. lentjan and
L. russelli at STA2, E. coioides were nutritionally reliant on seagrass habitats since
their narrow variation of isotope values were strongly associated with seagrass prey
items. Interestingly, the result of the mixing model revealed an ontogenetic diet shift
in juvenile E. coioides from invertebrate to piscivorous feeder, implying an increase
in new potential predation to the faunal juveniles in the seagrass meadows. With the
presence of active predators as well as cannibalism, the large grouper juveniles influ-
ence not only the survival of other fish juveniles, including L. lentjan and L. russelli,
but may also threaten the smaller groupers. This scenario raised a query about the
 Le et al.: Feeding habitats of juvenile reef fishes in interlinked habitats 481

possibility of an apparent trade-off in habitat selection. Do the juvenile fishes move

to more complex structured habitats for shelter in the central lagoon or emigrate to
nearby reefs due to predator-prey interactions? This issue requires consideration in
future studies. Furthermore, this study was conducted entirely in the dry season, as
seasonal changes may act as important drivers of the fish habitat use and foraging
behavior (Barletta et al. 2003, Faye et al. 2011, Olin et al. 2012). During the peak of
the monsoon season (December and January), the lagoon was strongly influenced
by rainfall and freshwater runoff, which can cause alterations to trophic interac-
tions between primary producers and consumers, resulting in the variation of their
isotopic composition (Faye et al. 2011, Olin et al. 2012). Hence, further studies are
recommended to understand seasonal variability in habitat use and feeding grounds
of focal juvenile reef fishes during monsoon and postmonsoon seasons.
In conclusion, stable isotope signatures indicated the overall reliance of three juve-
nile reef fish species on seagrass prey items. The mixing model revealed that the large
juveniles of L. lentjan and E. coioides may derive a part of their diets from mangroves
during their ontogeny. However, their feeding habits may be influenced by spatial
habitat configuration. This study highlights the importance of interlinked habitats
as feeding grounds of juvenile reef fishes with possible implications for the marine
reserve design in the lagoon.

Acknowledgments

The authors are grateful to the staff at Universiti Malaysia Terengganu for their kind as-
sistance in field work. We very much appreciate the constructive comments and suggestions
from the editors and reviewers for improving the manuscript. We also sincerely appreciate
Dr. R Norman for the English language review. This work was supported by the Ministry of
Higher Education Malaysia, under the Fundamental Research Grant Scheme (FRGS) proj-
ect number FRGS/1/2016 (No. 59425) and Higher Institution Centre of Excellence Research
Grant (No. 66928). This study was partly supported by a Grant-in-Aid for Scientific Research
(A) from the Japan Society for the Promotion of Science (JSPS; No. JP26252027).

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