Dual isotope study of food sources of a fish assemblage in the red river mangrove ecosystem, vietnam

Hydrobiologia DOI 10.1007/s10750-013-1737-9 BIODIVERSITY IN ASIAN COASTAL WATERS Dual isotope study of food sources of a fish assemblage in the Red River mangrove ecosystem, Vietnam Nguyen Tai Tue • Hideki Hamaoka • Tran Dang Quy • Mai Trong Nhuan • Atsushi Sogabe • Nguyen Thanh Nam Koji Omori • Received: 24 February 2013 / Accepted: 27 October 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract The food source utilization and trophic relationship of the fish assemblage in the Red River mangrove ecosystem, Vietnam were examined using dual isotope analysis The carbon and nitrogen stable isotope signatures of 23 fish species ranged from -24.0 to -15.7% and from 8.8 to 15.5%, respectively Cluster analysis based on the d13C and d15N signatures clearly separated the mangrove fish into five feeding groups, representing detritivores, omnivores, piscivores, zoobenthivores, and zooplanktivores, which Guest editors: M Tokeshi & H T Yap / Biodiversity in Changing Coastal Waters of Tropical and Subtropical Asia N T Tue Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama, Japan N T Tue (&) Á H Hamaoka Á K Omori Center for Marine Environmental Studies, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan e-mail: tuenguyentai@gmail.com T D Quy Á M T Nhuan Faculty of Geology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam A Sogabe Research Center for Marine Biology, Asamushi, Tohoku University, Sakamoto, Asamushi, Aomori 039-3501, Japan N T Nam Faculty of Biology, VNU University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam concurred with the dietary information The results suggested that mangrove carbon contributed a small proportion in the diets of the mangrove fish, with dominant food sources coming from benthic invertebrates, including ocypodid and grapsid crabs, penaeid shrimps, bivalves, gastropods, and polychaetes The d15N values showed that the food web structure may be divided into different trophic levels (TLs) The lowest TLs associated with Liza macrolepis, Mugil cephalus, and Periophthalmus modestus; 18 fish species had TLs between 3.0 and 3.8; and Pennahia argentata had the highest TL (c 4.0) Keywords Mangrove ecosystem Á Stable isotopes Á Fish Á Food sources Á Trophic level Á Vietnam Introduction Mangrove ecosystems have often been considered as hot spots of fish diversity (Nagelkerken et al., 2008) The hypotheses of the high diversity of the fish in the mangrove ecosystem include reduced predation, increased living space, and dominated food supply (Nagelkerken et al., 2008) The last of these hypotheses stated that the mangrove ecosystem produces greater food densities such as mangrove detritus, benthic microalgae (BMA), sediment organic matter, infauna, and invertebrates that form the basal food sources of the fish in the mangrove ecosystem 123 Hydrobiologia The linkage between the mangroves and the fish has been the focus of numerous studies (e.g., Odum & Heald, 1972; Blaber, 2007; Layman, 2007; Nagelkerken et al., 2008) Based on the stomach content analysis, Odum & Heald (1972) demonstrated that the mangrove detritus was a predominant food source of the fish in the estuarine habitats Nevertheless, isotopic studies have failed to confirm the contribution of the mangrove detritus in the diets of the fish in the estuarine food web (Rodelli et al., 1984), apparently because the refractory mangrove detritus is not easily digested by the gut system of the fish (Fry & Ewel, 2003) Subsequently, numerous studies have focused on determining the functional relationship between the mangroves and the fish through examining the food source utilization (Sheaves & Molony, 2000; Nanjo et al., 2008), and the trophic relationship (Abrantes & Sheaves, 2009; Giarrizzo et al., 2011) The results would be useful for ecosystem-based fishery management and mangrove conservation practices (Nagelkerken et al., 2008) The stable isotope ratios of carbon (d13C) and of nitrogen (d15N) are useful in determining the timeaveraged relative importance of the ingested food sources and the relative trophic level (TL) of a consumer (Michener & Lajtha, 2007) The mean (±1SD) trophic enrichment factor (TEF) between an animal and its diet for d13C and d15N is 0.4 ± 1.3 and 3.4 ± 1%, respectively (Post, 2002) Therefore, the d13C values can be used to trace the carbon utilization by an organism when the stable isotope signatures of the food sources are different (Bouillon et al., 2008) In addition, the d15N values can be used to estimate the relative TL of the organism (Zanden & Rasmussen, 1999; Post, 2002) The d13C and d15N values have been frequently used to examine the food sources (Sheaves & Molony, 2000) and the TL of the fish (Abrantes & Sheaves, 2009) in the mangrove ecosystem, and the ecological connectivity between mangrove forests and other coastal ecosystems (Layman, 2007) The isotopic studies have shown that the contribution of the mangrove carbon in the diets of the fish varies by landscape characteristics (Thimdee et al., 2004; Lugendo et al., 2007) and tidal water levels (Sheaves & Molony, 2000) of the mangrove ecosystem However, the application of the stable isotope methods in understanding ecological functions of the mangrove ecosystem in Vietnam has been few, and there remains 123 a significant gap in knowledge concerning the importance of horizontal and vertical trophic dynamics of the fish assemblage within and between adjacent systems These problems certainly constrain our understanding of the importance of the mangroves to fisheries, the valuation of ecological services of mangroves, and the planning and implementation effective conservation In the present study, we analyzed the stable isotope signatures of carbon (d13C) and nitrogen (d15N) of 183 individuals from 23 fish species, and used the isotopic data of primary production (mangroves, BMA, and marine phytoplankton), mangrove creek particulate organic matter (POM), sediments, and major groups of the benthic invertebrates for testing the hypothesis of whether the mangrove carbon was a major food source of the fish assemblage in the Red River mangrove ecosystem of Vietnam To test our hypothesis, two objectives were investigated: (1) to determine the utilization of food sources by the fish assemblage and (2) to determine the relative trophic relationship of the fish assemblage in the Red River mangrove ecosystem of Vietnam Materials and methods Study area The present study was conducted in the Red River Delta Biosphere Reserve (RRBR) in northern Vietnam The RRBR has two primary mangrove forests, the Xuan Thuy National Park and the Tien Hai Natural Reserve (Fig 1) (http://www.unesco.org) The characteristics of the mangrove forests are earlier described in Tue et al (2011, 2012a, c) Briefly, the mangrove forests are predominated by Sonneratia caseolaris (L.) Engl., Bruguiera gymnorrhiza (L.) Lamk., Kandelia candel (L.) Druce, and Aegiceras corniculatum (L.) Blanco, and consist of several major creek systems (Fig 1), which remain inundated throughout the tidal regimes Moreover, mangroves are an important source of the POM (Tue et al., 2012b), and important sinks to organic carbon and fine sediment particles (Tue et al., 2012d) As a result, the mangrove forests are thought to provide productive food sources for the benthic invertebrates (Tue et al., 2012c) and the fish in the estuarine habitats (Cuong & Khoa, 2004) The mangrove forests also play important roles in the filtering and containment of various Hydrobiologia pollutants (i.e., trace elements; Tue et al., 2012a), as well as a physical buffer against erosion and surge from major storm events Moreover, the mangrove forests are of great importance as major feeding, breeding, and stopover grounds for migratory birds, including several highly threatened species Platalea minor (Temminck & Schlegel, 1849), Larus ichthyaetus (Pallas, 1773), Tringa orchropus (Linnaeus, 1758), and Egretta eulophotes (Swinhoe, 1860) (Nhuan et al., 2009) Field sampling Fish samples were collected by a gill net during spring and ebb tides in two major tidal creeks of the Xuan Thuy National Park and the Tien Hai Nature Reserve (Fig 1) in January–February 2008 The sampling was designed to collect most predominant fish species and those of high economic values (Cuong & Khoa, 2004; Than, 2004) Fish samples were placed in labeled polyethylene bags, immediately stored in ice, and transported to the laboratory where they were frozen at -20°C until processing and analysis Sample preparation and analysis In the laboratory, the fish samples were first rinsed with distilled water, wiped with paper towel, then identified to species level, measured for total body length, and categorized into feeding groups based on the literature (Balan, 1967; Koslow, 1981; Elliott et al., 2007; Platell et al., 2007; Baeck et al., 2008; Nanjo et al., 2008; Salameh et al., 2010; Froese & Pauly, 2011) The list of the mangrove fish observed in the RRBR and their feeding ecology is shown in Table These feeding groups consisted of detritivores (detritus and/or microphytobenthos feeders), omnivores (filamentous algae, macrophytes, Fig Sampling sites within the Red River mangrove ecosystem, Vietnam 123 Hydrobiologia Table The list of fishes observed in the Red River mangrove ecosystem, Vietnam Order Taxa Major food items Feeding ecology References Anguilliformes Moringua sp NA Aulopiformes Harpadon nehereus (Hamilton, 1822) Muraenesox cinereus (Forsska˚l, 1775) Nekton and fishes PV Froese & Pauly (2011) Fishes and crustaceans PV Froese & Pauly (2011) Escualosa thoracata (Valenciennes, 1847) Zooplankton (copepods, crab zoea, larvae of bivalves, and fish eggs) and phytoplankton ZP Froese & Pauly (2011) Coilia mystus (Linnaeus, 1758) Zooplankton and phytoplankton ZP Koslow (1981) Acanthopagrus latus (Houttuyn, 1782) Mangrove detritus, sesarmid crabs, small gastropods, worms, crustaceans, and mollusks ZB Platell et al (2007) Bostrychus sinensis (Lacepe`de, 1801) Crustaceans and small fishes PV Froese & Pauly (2011) Clupeiformes Perciformes Butis butis (Hamilton, 1822) Small fishes and crustaceans PV Froese & Pauly (2011) Gerres limbatus (Cuvier, 1830) Glossogobius biocellatus (Valenciennes, 1837) Small benthic animals Fishes, detritus, and gammaridean amphipods ZB PV Froese & Pauly (2011) Nanjo et al (2008) Gobiomorphus sp NA Leiognathus bindus (Valenciennes, 1835) Copepods, phytoplankton, detritus, and zoobenthos ZP Balan (1967) Liza macrolepis (Smith, 1846) Algae, diatoms, forams, benthic polychaete, crustaceans, mollusks, organic matter, and detritus; copepods and floating algae DV Froese & Pauly (2011) Lutjanus russellii (Bleeker, 1849) Crabs, shrimps, fishes, crustaceans, and insects PV Nanjo et al (2008) Mugil cephalus (Linnaeus, 1758) Zooplankton as larvae; detritus, microalgae, and benthic organisms DV Nanjo et al (2008) Oxyeleotris marmorata (Bleeker, 1852) Small fishes, shrimps, aquatic insects, mollusks, and crabs PV Froese & Pauly (2011) Parapercis sp NA ZB Pennahia argentata (Houttuyn, 1782) Small fishes and invertebrates PV Froese & Pauly (2011) Periophthalmus modestus (Cantor, 1842) Gammarids, crabs, and other crustaceans OV Baeck et al (2008) Sillago sihama (Forsska˚l, 1775) Polychaete worms, small prawns (penaeus), shrimps, and amphipods ZB Froese & Pauly (2011) Terapon theraps (Cuvier, 1829) Animals ZB Froese & Pauly (2011) Trypauchen vagina (Bloch & Schneider, 1801) Small invertebrates and crustaceans ZB Salameh et al (2010) NA NA Scorpaeniformes Onigocia sp General feeding ecology and reference are shown for each fish species NA Not available, DV detritivores, OV omnivores, PV piscivores, ZB zoobenthivores, and ZP zooplanktivores 123 Hydrobiologia periphyton, epifauna and infauna feeders), zooplanktivores (zooplankton, hydroids, planktonic crustacean, and fish eggs/larval feeders), zoobenthivores (benthic invertebrate feeders), and piscivores (finfish and nektonic invertebrate feeders) (Elliott et al., 2007) The processing of fish for the stable isotope analysis involved the extraction of white muscle tissue from the anterior dorsal region The white tissue is more isotopically homogenous than other tissues (Michener & Lajtha, 2007) The fish tissues were then placed in Eppendorf tubes, dried in an electric oven at 60°C for 24 h, and ground to fine powder by an agate mortar and pestle The lipids were extracted from the fish tissues prior to the stable isotope analysis following methods described in Tue et al (2012c) Briefly, the pulverized fish tissues were placed in the Eppendorf tubes, immersed in a 2:1 chloroform:methanol (by volume) solution, and left at room temperature for 24 h to extract the lipids The samples were then rinsed with distilled water, and dried in an electric oven at 60°C for 24 h For all samples, 1.0 ± 0.1 mg of the pulverized fish tissues was packed in a tin capsule The carbon and nitrogen stable isotope signatures were measured using an isotope ratio mass spectrometer (ANCAGSL; Sercon Inc, UK) and expressed in d notion as parts per thousand (permil, %) as shown in Eq (1): ÂÀ  Á Ã d13 C or d15 N ¼ Rsample Rstd À Â 1000 ð1Þ where R is isotope ratios 13C/12C or 15N/14N Rsample is the isotope ratio of the sample, and Rstd is the isotope ratio of a standard referenced to Pee Dee Belemnite limestone carbonate (PDB) for d13C, and to atmospheric nitrogen for d15N During analysis processes, 13 15 L-histidine (d C = -11.4% and d N = -7.6%) was used for quantifying the analyzed results Analytical errors were 0.1% for d13C and 0.2% for d15N, respectively Background data for potential food sources of the mangrove fish The ranges of the d13C and d15N values of the mangrove leaves, the marine phytoplankton, the BMA, the POM, the sediments, and the benthic invertebrates are shown in Fig The respective means of the d13C values increased in the order of the mangrove leaves, mangrove sediments, the tidal flat, Fig Dual isotope plot of mean d13C and d15N signatures (±1 SD) of the different food sources, and the fish in the Red River mangrove ecosystem, Vietnam Acronyms of fish taxa are shown in Table 2, and DV, OV, PV, ZB, and ZP denotes the detritivores, omnivores, piscivores, zoobenthivores, and zooplanktivores, respectively Mang, Mang sed, Adj sed, POM, Phyto, and BMA denotes mangrove leaves, mangrove sediments, adjacent habitat sediments, creek particulate organic matter, marine phytoplankton, and benthic microalgae, respectively The stable isotope data of mangrove leaves, POM, and phytoplankton; benthic microalgae and invertebrates; and mangrove, creek bank, tidal flat and bottom sediments are presented in Tue et al (2012a, b, c), respectively the creek bank and creek bottom sediments, the POM, the marine phytoplankton, and the BMA (Tue et al., 2012b, c, d) The benthic invertebrates (e.g., grapsid crabs) have been shown to be important food sources for the mangrove fish (Sheaves & Molony, 2000) The ranges of the d13C and d15N values of the major benthic invertebrate groups in the mangrove ecosystem of the RRBR are shown in Fig Tue et al (2012c) reported that the gastropods, bivalves, grapsid crabs, and polychaetes inhabiting the mangrove forests directly relied on the mangrove detritus The ocypodid crabs inhabiting the land–water ecotone showed preference for the BMA and other food sources (i.e., bacteria, ciliate protozoa, and nematodes) over the mangrove detritus The diets of tidal flat bivalve Ensis magnus (Ensis Schumacher, 1817) was a mixture of the marine phytoplankton and the BMA The penaeid prawns 123 Hydrobiologia were opportunistic omnivorous, feeding on the BMA, the marine phytoplankton, the POM, and juvenile invertebrates (i.e., crabs, gastropods, and bivalves), with the latter being predominant (Tue et al., 2012c) Estimation of the relative trophic level and contribution of the food sources in diets of the mangrove fish The diversity of the food sources generates difficulties in establishing an isotopic baseline for estimating the relative TL of the mangrove fish (Layman, 2007) Instead, the d15N values of the primary consumers (invertebrates potentially eaten by fish) were used as an index of nitrogen isotope compositions entering the base of the food web This method reduces the temporal and spatial variations in the d15N values of the primary producers (Post, 2002), and provides a more temporally integrated measurement of the relative TL of the mangrove fish (Zanden & Rasmussen, 1999) In the present study, the relative TL of the mangrove fish was estimated from the d15Nbase values of the bivalve E magnus based on the Eq (2) (Post, 2002) The d15N values of the E magnus were used as the isotopic baseline, because this species is a true suspension feeder, feeding on the marine phytoplankton and the BMA (Tue et al., 2012c)  TL ẳ d15 Nfish d15 Nbase ị 3:4 ỵ 2ị where TL is the relative trophic level of the mangrove fish; d15Nfish and d15Nbase are the nitrogen stable isotope values of the mangrove fish and the bivalve E magnus, respectively; the mean trophic enrichment factor between the mangrove fish and the bivalve E magnus for d15N is 3.4; and the TL of the bivalve E magnus is The contribution of the mangroves, the marine phytoplankton, the BMA, the POM, the sediment organic matters, grapsid crabs, ocypodid crabs, E magnus, gastropods, and polychaetes to the diets of the mangrove fish was estimated using a Stable Isotope Analysis in R (SIAR) package (Parnell et al., 2010) on R software (R Core Team, 2012) The SIAR package is based on a Bayesian framework that can be used to find a solution for an isotope mixing model (Parnell et al., 2010) The SIAR package requires the d13C and d15N values of the mangrove fish, the mean and SD of the d13C and d15N values for the food 123 sources, and the mean and SD of the trophic enrichment factors In the present study, the mean trophic enrichment factors (±1SD) for d13C and d15N were 0.4 ± 1.3 and 3.4 ± 1.0%, respectively (Post, 2002) The stable isotope values of the sediments from mangrove forests, and the adjacent habitats (tidal flats, bank and bottom creeks) were pooled, representing the sediment organic carbon source The isotope mixing model was run 106 iterations with an elimination of the first 104 The contributions of the food sources in the diets of the mangrove fish were reported as means and lower and upper ranges (5th and 95th percentiles) Statistical analysis The d13C and d15N values of the mangrove fish were used to perform hierarchical cluster analysis, which can be used to categorize the mangrove fish into feeding groups based on their similarity (Mazumder et al., 2011) and their dietary information (Table 1) The distance metric was based on the Euclidean distance completed linkage method The statistical analysis was performed using the SPSS statistical software package 17 (SPSS 17.0) Results Stable isotope values of the mangrove fish The d13C and d15N values of the mangrove fish ranged from -24.0 to -15.7% and from 8.8 to 15.5%, respectively (Table 2; Fig 2) The lowest and highest d13C values were expressed in Periophthalmus modestus and Lutjanus russellii, respectively (Fig 2) Pennahia argentata showed the highest mean d15N values, while the lowest mean d15N values were expressed in Mugil cephalus and Liza macrolepis (Fig 2) Feeding groups and the contribution of different food sources to diets of the mangrove fish Five groups of the mangrove fish were categorized at a similar index level of 15 (Fig 3) Based on the dietary information (Table 1), the mangrove fish were classified into five feeding groups, consisting of detritivores, omnivores, piscivores, zoobenthivores, and zooplanktivores Detritivorous fish were M cephalus Hydrobiologia Table d13C and d15N values of the fish collected from the Red River mangrove ecosystem, Vietnam Order Taxa ACR n L (cm) d13C (%) d15N (%) 24.9 -20.8 ± 0.5 11.3 ± 0.3 3.11 24.8 ± 3.0 -17.1 ± 0.3 13.0 ± 0.3 3.6 ± 0.1 -19.6 ± 1.1 12.4 ± 0.7 3.45 ± 0.2 -18.7 ± 0.1 13.0 ± 0.4 3.63 ± 0.13 -20.1 ± 0.3 13.4 ± 0.1 3.73 ± 0.04 18.2 ± 3.1 -20.8 ± 1.1 12.3 ± 0.7 3.42 ± 0.2 (13.3 - 22.5) 8.7 ± 1.0 -20.0 ± 0.3 11.5 ± 0.2 3.17 ± 0.06 -20.4 ± 0.6 11.9 ± 0.2 3.30 ± 0.05 -19.1 ± 0.7 13.3 ± 0.7 3.69 ± 0.21 -20.3 ± 0.8 11.3 ± 0.4 3.11 ± 0.13 -19.5 ± 1.3 11.7 ± 0.7 3.23 ± 0.2 -19.5 ± 1.5 13.7 ± 0.1 3.81 ± 0.03 -18.6 ± 1.2 10.6 ± 0.9 2.91 ± 0.25 11 ± 0.7 (10.2 - 11.7) -17.2 ± 1.1 12.0 ± 0.2 3.34 ± 0.05 -17.4 ± 0.8 10.5 ± 0.8 2.88 ± 0.24 -19.8 ± 1.3 11.9 ± 0.7 3.28 ± 0.21 -19.5 ± 0.8 12.4 ± 0.4 3.44 ± 0.11 -17.3 ± 0.5 14.2 ± 0.5 3.97 ± 0.15 -21.8 ± 1.7 10.7 ± 0.8 2.95 ± 0.22 -17.9 ± 0.2 13.4 ± 0.2 3.74 ± 0.05 Trophic level Anguilliformes Moringua sp Mor Hn Aulopiformes Harpadon nehereus (20.7 - 28.6) Muraenesox cinereus Mc 30.9 ± 7.6 (23.5 - 41.4) Clupeiformes Escualosa thoracata Et 5.3 ± 0.5 (4.7 - 5.7) Coilia mystus Cm 12.9 ± 1.6 (11.9 - 15.8) Perciformes Acanthopagrus latus Al 12 Bostrychus sinensis Bs Butis butis Bb (7.7 - 10) 7.1 ± 0.6 (6.7 - 8.1) Gerres limbatus Gl 10 10.8 ± 1.6 (8.9 - 13.9) Glossogobius biocellatus Gb 19 11.6 ± 4.6 Gobiomorphus sp Gob 23.8 ± 2.9 (5.0 - 26.5) (20.7 - 26.6) Leiognathus bindus Lb 8.2 ± 0.6 (7.4 - 8.6) Liza macrolepis Lm 18.1 ± 5.5 (13.8 - 27.3) Lutjanus russellii Lr Mugil cephalus Mcl 18 15.8 ± 3.1 Oxyeleotris marmorata Om 28 8.2 ± 4.8 (11.1 - 21.3) (3.5 - 18) Parapercis sp Ps 11 12.2 ± 2.6 (7.0 - 16.4) Pennahia argentata Pa 24.5 ± 1.4 Periophthalmus modestus Pm 10 3.6 ± 1.7 (23.4 - 26.8) (1.8 - 6.6) Sillago sihama Ss 8.4 ± 0.7 (7.4 - 9.1) 123 Hydrobiologia Table continued Order Taxa ACR Terapon theraps Tt Trypauchen vagina Tv L (cm) d13C (%) d15N (%) Trophic level 11.7 ± 1.0 -18.8 ± 0.4 13.2 ± 0.6 3.67 ± 0.17 14.2 ± 4.0 -20.4 ± 1.3 10.9 ± 1.5 3.00 ± 0.43 -19.0 ± 0.8 11.6 ± 0.4 3.21 ± 0.11 n (10.6 - 12.4) (8.2 - 18.7) Scorpaeniformes Onigocia sp On 11.5 ± 4.5 (8.1 - 19.4) ACR acronym; n is number of the samples; mean, and mean ± 1SD values are given where n = 2, and C3, respectively; L total body length (mean ± 1SD (min - max)); Trophic levels of the mangrove fishes are estimated by the d15N values and L macrolepis The P modestus could be distinguishable with other fish groups and fed on the filamentous algae, small invertebrates, and infauna (Table 1), representing the omnivorous fishes The piscivorous fishes were P argentata, Sillago sihama, Harpadon nehereus, and L russellii, and may consume other fish and the invertebrates (Table 1) The zoobenthivorous fishes consisted of Acanthopagrus latus, Butis butis, Bostrychus sinensis, Gobiomorphus sp., Glossogobius biocellatus, Moringua sp., Muraenesox cinereus, Oxyeleotris marmorata, Onigocia sp., Parapercis sp., and Trypauchen vagina whose prey included invertebrates (polychaetes, crabs, and mollusks) and prawns (Table 1) The zooplanktivorous fishes were Escualosa thoracata, Coilia mystus, Leiognathus bindus, Gerres limbatus, and Terapon theraps, feeding predominantly on copepods, crab zoera, bivalve larvae, and fish eggs (Table 1) The isotope mixing model results showed that the mangrove carbon was a minor contributor to the diets of the mangrove fish (Table 3) The major carbon food sources of the mangrove fish were the benthic invertebrates, consisting of the panaeid prawns, the ocypodid and grapsid crabs, gastropods, the E magnus, and polychaetes (Table 3) 3.97 ± 0.15), and followed by L bindus (mean 3.81 ± 0.03) The relative trophic level of the mangrove fish Discussion The mean relative TL (±SD) of the mangrove fish ranged between 2.88 ± 0.24 and 3.97 ± 0.15 (Fig 2; Table 2) The relative TLs for M cephalus, L macrolepis, and P modestus were below 3.0 The relative TLs of 18 fish species ranged between 3.0 and 3.8 The highest TL was observed from P argentata (mean The mangrove leaves, the marine phytoplankton, the BMA, the POM, and the sediments had different d13C signatures, but all had low d15N values The d15N values of the mangrove fish concurrently increased with the TLs in the food web (Fig 2) The d13C values were, therefore, a good indicator of the origin of the 123 Fig Results of hierarchical cluster analysis of 23 fish species based on d13C and d15N signatures DV, OV, PV, ZB, and ZP denotes the detritivores, omnivores, piscivores, zoobenthivores, and zooplanktivores, respectively 3.2 (0–26.7) 4.7 (0–27) Pennahia argentata Sillago sihama 7.5 (0–33.2) 6.2 (0–33.6) 7.9 (0–34.3) (0–37.7) 7.9 (0–37.7) 2.9 (0–18.4) 3.5 (0–22.3) 4.8 (0–25.3) 8.4 (0–31.7) 8.5 (0–42.1) (0–35.3) 5.6 (0–24.7) 6.3 (0–37) 5.6 (0–27.7) 3.3 (0–22.2) 8.6 (0–33.6) 8.7 (0–34.5) 3.1 (0–2.9) 8.6 (0–39.5) 7.9 (0–35.4) 6.6 (0–33.9) 7.8 (0–36.4) 6.1 (0–35) Phytoplankton (0–37.1) 4.5 (0–27.3) 7.2 (0–35.2) 4.7 (0–30) 6.1 (0–30.1) 2.1 (0–20.4) 2.6 (0–18.8) 3.1 (0–18.5) 7.4 (0–35.1) 7.6 (0–35) 5.6 (0–31.7) 3.9 (0–21.6) 4.8 (0–28.4) 3.9 (0–21.6) 2.8 (0–23.3) 7.6 (0–35.3) 7.9 (0–32.2) 2.9 (0–22.9) 7.5 (0–34.6) 5.4 (0–30) 3.1 (0–22.9) (0–32.7) 5.7 (0–32.5) POM 7.5 (0–36) 6.2 (0–33.1) 7.6 (0–34.3) 5.8 (0–10) 7.6 (0–36.6) 2.5 (0–20) (0–18.5) 4.5 (0–25.8) 8.3 (0–36.2) 8.3 (0–36.2) 6.6 (0–34.7) 4.8 (0–22.8) 5.8 (0–35.8) 4.8 (0–29.9) 2.6 (0–21.9) 8.7 (0–36.3) 8.7 (0–36) (0–31) 8.6 (0–32.8) 8.2 (0–43.6) 10.8 (0–36.7) 7.6 (0–32.6) (0–25.3) BMA (0–35.6) 4.5 (0–32.7) 7.5 (0–35.5) (0–32.3) (0–28.3) 2.4 (0–16.3) (0–18) (0–20.4) 7.1 (0–30.4) 7.6 (0–31.2) 5.8 (0–33.1) 4.5 (0–23.5) 5.1 (0–29.3) 4.2 (0–26.6) 3.6 (0–25.6) 7.4 (0–37.4) 7.7 (0–33.1) 2.3 (0–17.9) 7.3 (0–41.4) 4.7 (0–26.1) 2.1 (0–21.6) (0–30.5) 7.6 (0–37.4) SOM 10.7 (0–41) 11 (0–50.6) 9.8 (0–39.7) 10.3 (0–46.6) 11.5 (0–48) 6.3 (0–37.2) 8.4 (0–32) 8.6 (0–43.7) 10.3 (0–44.1) 10 (0–37.2) 10.8 (0–48.5) 6.7 (0–27.9) 10 (0–43.2) 8.3 (0–38.8) 7.5 (0–38.7) 14.3 (0–48.5) 10 (0–36) 7.7 (0–45) 10 (0–42.9) 10.6 (0–42.7) 11.1 (0–44.4) 11.3 (0–46.1) 9.5 (0–37) Grapsid crabs 14.8 (0–64) 19.6 (0–77.7) 11.3 (0–58.2) 15.7 (0–64.8) 13.6 (0–52.6) 22.4 (0–55.6) 20.6 (0–59.5) 26 (0–61.6) 11.8 (0–53) 11.1 (0–41.6) 14.2 (0–53.8) 20.1 (0–50.5) 14.8 (0–57.5) 18.6 (0–52.9) 13.1 (0–46.6) 11.8 (0–55.2) 11 (0–52.6) 22.9 (0–61.4) 11.8 (0–47.4) 16.1 (0–49.3) 41.7 (0–75.5) 14.3 (0–53.5) 10.5 (0–37.4) Ocypodid crabs 10 (0–42.3) 9.6 (0–51) 9.8 (0–41.8) 9.6 (0–40.7) 10.7 (0–54.5) 6.4 (0–30.9) 8.2 (0–32.4) 7.6 (0–40.3) 10 (0–39.4) 9.8 (0–37.8) 10.2 (0–53.4) 6.7 (0–28.6) 9.8 (0–46.7) 8.5 (0–43.6) 8.2 (0–44.4) 9.8 (0–38.9) 9.7 (0–39.8) 7.1 (0–41.2) 9.9 (0–44.1) 10 (0–46.2) (0–39.4) 10.7 (0–53.1) 10.2 (0–39.8) Gastropods 9.2 (0–39.7) 8.3 (0–41.2) 9.5 (0–41.1) 8.8 (0–46.7) 9.7 (0–46.7) 7.7 (0–38.3) (0–43.3) (0–41.2) 9.5 (0–36.4) 9.6 (0–41.6) 9.5 (0–38) 10.9 (0–44.7) 9.8 (0–47.7) 10.6 (0–47.7) (0–42.4) 9.3 (0–35.8) 9.3 (0–35.5) 6.2 (0–34.8) 9.3 (0–38.6) 9.8 (0–46.2) 4.5 (0–37.7) 9.5 (0–40.8) 10.3 (0–43) E magnus 14.9 (0–67.3) 19.6 (0–73.4) 12.6 (0–54.1) 20.8 (0–73) 13.9 (0–52.6) 37 (1–67) 32.4 (0–63.7) 27.1 (0–62.2) 12.1 (0–59.3) 11.5 (0–45.5) 16 (0–60.1) 25.3 (0–52.5) 19.1 (0–56.3) 24.3 (0–57.2) 29.5 (0–69) 11.5 (0–46.7) 11 (0–50.5) 38.5 (0–78.9) 11.7 (0–52.6) 17 (0–67.6) 9.4 (0–45) 13.8 (0–51.6) 13.9 (0–44) Prawns 8.4 (0–45.7) 7.1 (0–55) 10.3 (0–41.2) 9.7 (0–55.6) 8.6 (0–38.4) 8.6 (0–27) 8.1 (0–28.6) (0–33.9) 8.9 (0–38.3) 9.4 (0–42.1) 10 (0–48) 8.7 (0–24.2) 10.8 (0–46.6) 8.5 (0–35) 18.9 (0–47.5) 8.5 (0–48.2) 8.9 (0–38) 5.3 (0–26.8) 8.6 (0–35.3) 6.8 (0–35.2) 1.9 (0–22.6) 8.3 (0–33.7) 15.9 (0–40.7) Polychaetes BMA, POM, and SOM denotes the organic carbon sources of benthic microalgae, particulate organic matter, and sediment organic matter; DV, OV, PV, ZB, and ZP denotes the detritivores, omnivores, piscivores, zoobenthivores, and zooplanktivores, respectively 6.4 (0–30.5) Lutjanus russellii 3.7 (0–28.2) (0–13) Oxyeleotris marmorata PV (0–17.8) Onigocia sp Harpadon nehereus 6.2 (0–30.2) Muraenesox cinereus 16 (0–11.3) 6.4 (0–31.5) Moringua sp 4.4 (0–24.5) 4.4 (0–28.5) Gobiomorphus sp Trypauchen vagina 2.8 (0–15.3) Glossogobius biocellatus Parapercis sp 3.6 (0–24.3) Butis butis 2.3 (0–20) Acanthopagrus latus 2.7 (0–18.8) 6.4 (0–32.5) Terapon theraps Bostrychus sinensis 6.9 (0–34.2) Leiognathus bindus ZB 1.5 (0–13.7) Gerres limbatus 3.4 (0–21) 6.4 (0–32) Escualosa thoracata ZP 4.5 (0–28) Coilia mystus DV Liza macrolepis 5.3 (0–25) 1.7 (0–16.9) OV Periophthalmus modestus Mangroves Mugil cephalus Group Dietary source Species Table The proportional contribution of the food sources in the diets of the mangrove fish based on the Bayesian stable isotope mixing model Hydrobiologia 123 Hydrobiologia food sources (Bouillon et al., 2008), while d15N values were indicative of the relative TLs (Michener & Lajtha, 2007) The wide variations in the d13C and d15N values of the mangrove fish (Fig 2; Table 2) indicated that they utilized heterogeneous diets Moreover, the fish tissues were much more enriched in 13C composition relative to the mangrove leaves (Fig 2), suggesting that the fish had little reliance on the mangrove carbon sources (Table 3) This pattern was consistent with findings in the mangrove ecosystems in the Tanzanian coastal waters (Lugendo et al., 2007) and Gazi Bay, Kenya (Nyunja et al., 2009) Among fish taxa analyzed, the detritivorous fishes had the lowest d15N values (Fig 2) and high BMA proportion in their diets (Table 3) The results indicated that they fed on lower trophic order sources, such as the BMA, the sediment organic matter, and the POM (Tables 1, 3; Fig 2) This finding was consistent with the observation of Lin et al (2007), who showed that the preferred food sources of the L macrolepis and other detritivorous fishes were the BMA and the POM Moreover, the d13C values of the detritivorous fish in the present study were much higher than those of the BMA, the POM, and the sediments (Fig 2), suggesting that they also fed on other 13C-enriched food sources such as the benthic invertebrates The mixing model results showed that the ocypodid crabs contributed up to 41.7% in the diet of M cephalus (Table 3) The benthic invertebrates could be incidentally ingested while the detritivorous fishes were feeding on the detritus, placing them at higher TLs than the secondary consumers in the mangrove food web (Nanjo et al., 2008) The d13C values of the P modestus were higher than those of polychaetes, the POM, the sediments, and overlapped with the d13C values of E magnus and gastropods (Fig 2) The mixing model results showed that the major food sources of the P modestus were polychaetes and other invertebrates (Table 3) This is consistent with Baeck et al.’s (2008) observation that the major food items of the Periophthalmus species were gammarid amphipods, crabs, other crustaceans, and benthic organisms The zooplanktivorous fishes C mystus, E thoracata, G limbatus, L bindus, and T theraps were closely positioned in the food web (Fig 2) and clustered in the same group (Fig 3), indicating that they had similar feeding behaviors The mixing model results showed that the major prey items of the zooplankton fishes were the grapsid and ocypodid crabs, the penaeid 123 prawns, gastropods, and bivalves (Table 3) The diets of the zooplanktivorous fishes in the present study were in reasonable agreement with information on their feeding ecology from the literature (Balan, 1967; Koslow, 1981) In which, the anchovy C mystus and sardine E thoracata are suspension feeders, feeding on a diversity of available zooplankton, fish eggs, and the invertebrate larvae, rather than selecting specific species (Koslow, 1981) In addition, L bindus is reported to feed on copepods, bivalve larvae, crustaceans, and marine phytoplankton (Balan, 1967) Despite the wide feeding preferences of the zoobenthivorous fishes (Nanjo et al., 2008; Froese & Pauly, 2011), their d13C and d15N values varied slightly (Table 2; Fig 2), indicating that they could feed on similar food sources, consisting of the penaeid prawns, the ocypodid and grapsid crabs, and the bivalve (E magnus) (Table 3) The food sources of the zoobenthivorous fishes were consistent with the dietary information from the literature (Platell et al., 2007; Froese & Pauly, 2011) For example, the A latus collected from the mangrove ecosystem from Shark Bay (Australia) fed predominantly on the sesarmid crabs, small gastropods, and the mangrove materials (Platell et al., 2007) The present study showed that the d13C values of the A latus were higher than those of the bivalve E magnus, and overlapped with the d13C values of the gastropods and grapsid crabs (Fig 2) In addition, the bivalve E magnus, ocypodid crabs, polychaetes, and prawns were predominant food items of the A latus (Table 3) The low contribution of the mangrove detritus in their diets could be interpreted by either the selective feeding mechanisms or assimilation efficiency of the A latus The A latus could predominantly ingest the benthic invertebrates selectively and reject the mangrove detritus In that case both the benthic invertebrates and the mangrove detritus were simultaneously ingested by the fish, yet the mangrove detritus was too refractory for assimilation (Fry & Ewel, 2003) The d13C values of the piscivorous fishes were overlapped, and higher than those of other fish groups from 1.3 to 4.1% (Table 2; Fig 2); hence, they may not extensively feed upon other fishes The mixing model results showed that the grapsid and ocypodid crabs, and the penaeid prawns were their major food sources (Table 3) Furthermore, the benthic invertebrates in the mangrove ecosystem of the RRBR are known to consume the mangrove detritus, the BMA, marine Hydrobiologia phytoplankton, the POM, and the sediment organic carbon (Tue et al., 2012c) The high contribution of the benthic invertebrates in the diets of the piscivorous fishes suggested that the carbon pathways from the basal food sources to the piscivorous fishes at/or near the top of the food chain may be shortened in the mangrove ecosystem (Sheaves & Molony, 2000) The pattern suggested that the piscivorous fishes may appear to be a major mechanism of carbon transport from the mangrove ecosystem to adjacent coastal waters (Sheaves & Molony, 2000), and the mangrove forests were important nursery and feeding grounds of the fish (Layman, 2007) However, further studies need to investigate the extent to which the piscivorous fishes prey upon the benthic invertebrates using a combination of stomach content and stable isotope analyses, which would provide a more detailed picture of energy transfer within a mangrove ecosystem The food web structure of the mangrove ecosystem of the RRBR presented four TLs that were similar to the mangrove ecosystems of North Queensland (Abrantes & Sheaves, 2009) and Gazi Bay (Nyunja et al., 2009) The food web structure consisted of primary producers (mangroves, phytoplankton, and the BMA), the POM, and the sedimentary organic matters, which were the basal food sources; the primary consumers were the benthic invertebrates, including of polychaetes, bivalves, gastropods, the ocypodid and grapsid crabs, and the penaeid prawns; the secondary consumers included M cephalus, L macrolepis, and P modestus; the tertiary consumers included zooplanktivorous, zoobenthivorous, and piscivorous fishes; and the piscivorous fish P argentata was positioned at the apex of the food web In the present study, almost all mangrove fishes had TLs [ 3.0, and were one TL higher than that of the primary consumers Moreover, the invertebrate species utilized the food sources from the basal food sources (Tue et al., 2012c) The food web structure clearly showed different carbon pathways from the basal food sources to the primary consumers, to the secondary consumers, and then to the piscivorous fishes at/or near the top of the mangrove food web Conclusions The d13C and d15N signatures were applied to identify the carbon utilization and trophic relationship of the fish assemblage in the Red River mangrove ecosystem, Vietnam The results showed that the fish assemblage had less reliance on the mangrove carbon The major food sources of the mangrove fish were the benthic invertebrates, including the penaeid prawns, the ocypodid and grapsid crabs, bivalves, gastropods, and polychaetes Five feeding groups of the mangrove fish were identified in the cluster analysis, consisting of detritivores, omnivores, zooplanktivores, zoobenthivores, and piscivores The food web structure showed that the carbon energy was transferred from the basal food sources to the piscivorous fishes at/or near the top of the food chain by different trophic pathways These results highlight the need for conservation of mangroves and the preferred habitats of the benthic invertebrates in the mangrove ecosystem The present study has provided baseline information on the food source utilization and the trophic relationship of the mangrove fish, which will be a useful database for future studies to assess the changes in the food sources and the TLs of the fish assemblage in relation to the mangrove and coastal ecosystem management (Wainright et al., 1993) Acknowledgments The authors are grateful to staff of VNU University of Science, and the Xuan Thuy National Park and the Tien Hai Nature Reserve, Vietnam for their help with sampling We express our sincere thanks to anonymous reviewers—Prof Tokeshi and Dr Todd W Miller for their critical reviews and comments which significantly improved the manuscript This work was partially supported by the ‘‘Global COE Program’’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan and the Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) (No 105.09.82.09) The Grant-in-Aid for Scientific Research for Postdoctoral Fellows by the Japan Society for the Promotion of Science (No 24-02386 for NTT) is also acknowledged References Abrantes, K & M Sheaves, 2009 Food web structure in a nearpristine mangrove area of the Australian wet tropics Estuarine, Coastal and Shelf Science 82: 597–607 Baeck, G., T Takita & Y Yoon, 2008 Lifestyle of Korean mudskipper Periophthalmus magnuspinnatus with reference to a congeneric species Periophthalmus modestus Ichthyological Research 55: 43–52 Balan, V., 1967 Biology of the silverbelly, Leiognathus bindus (Val.) of the Calicut coast Indian Journal Fish 10: 118–134 Blaber, S., 2007 Mangroves and fishes: issues of diversity, dependence, and dogma Bulletin of Marine Science 80: 457–472 123 Hydrobiologia Bouillon, S., R M Connolly & S Y Lee, 2008 Organic matter exchange and cycling in mangrove ecosystems: recent insights from stable isotope studies Journal of Sea Research 59: 44–58 Cuong, D N & T M Khoa, 2004 Fish composition in the mangrove of northern communes of Giao Thuy district, Nam Dinh province In Hong, P N (ed.), Mangrove Ecosystems in the Red River Coastal Zone: Biodiversity, Ecology, Socio-economics, Management and Education Agriculture Publishing House, Hanoi: 121–125 Elliott, M., A K Whitfield, I C Potter, S J M Blaber, D P Cyrus, F G Nordlie & T D Harrison, 2007 The guild approach to categorizing estuarine fish assemblages: a global review Fish and Fisheries 8: 241–268 Froese, R & D Pauly, 2011 FishBase World Wide Web electronic publication Version 08/2011 http://www fishbase.org Fry, B & K C Ewel, 2003 Using stable isotopes in mangrove fisheries research - a review and outlook Isotopes in Environmental and Health Studies 39: 191–196 Giarrizzo, T., R Schwamborn & U Saint-Paul, 2011 Utilization of carbon sources in a northern Brazilian mangrove ecosystem Estuarine, Coastal and Shelf Science 95: 447–457 Koslow, J A., 1981 Feeding selectivity of schools of northern anchovy, Engraulis mordax, in the Southern California Bight Fishery Bulletin 79: 131–142 Layman, C A., 2007 What can stable isotope ratios reveal about mangroves as fish habitat? 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consumer d13C and d15N and the trophic position of aquatic consumers Ecology 80: 1395–1404 123 View publication stats ... Matsunaga, 2004 Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove- fringed estuary, Khung Krabaen Bay (Thailand) using dual stable isotope techniques Wetlands... were investigated: (1) to determine the utilization of food sources by the fish assemblage and (2) to determine the relative trophic relationship of the fish assemblage in the Red River mangrove. .. of the relative TL of the mangrove fish (Zanden & Rasmussen, 1999) In the present study, the relative TL of the mangrove fish was estimated from the d15Nbase values of the bivalve E magnus based