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Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 © J. Hong and B. Gu, Published by EDP Sciences 2020 https://doi.org/10.1051/kmae/2020033 Knowledge & Management of Aquatic Ecosystems www.kmae-journal.org Journal fully supported by Office français de la biodiversité RESEARCH PAPER Responses of nitrogen stable isotopes in ﬁsh to phosphorus limitation in freshwater wetlands Jianming Hong1 and Binhe Gu2,* 1 2 College of Life Sciences, Capital Normal University, Beijing, 100048, China Soil and Water Science Department, University of Florida, 106 Newell Hall, Gainesville, FL 32611, USA Received: 28 May 2020 / Accepted: 30 September 2020 Abstract – Human-induced eutrophication has altered ecological processes in aquatic ecosystems. Detection of ecological changes is a prerequisite for protecting ecosystems from degradation. In this study, nitrogen stable isotopes (d15N) in ﬁsh are evaluated as indicators of environmental changes in south Florida wetlands. Stable nitrogen isotope (d15N) data of select ﬁsh species and water quality collected from the Florida Everglades between the 1990s and 2000s were used to assess the relationship between total phosphorus concentrations and d15N ratios. The d15N ratios in nine of ten select ﬁsh species increase signiﬁcantly as total phosphorus concentration in the surface water increases. There were signiﬁcant relationships between total nitrogen concentration in the surface water and d15N ratios in several ﬁsh species. The pattern of changes in d15N ratios along nutrient gradients suggests that increased eutrophication is recorded as the d15N ratios in ﬁsh. The lack of human wastewater loading, the dominance in agricultural runoff and the high TN:TP ratio suggest that phosphorus is the limiting factor driving ecosystem productivity and the changes of d15N ratios in ﬁsh. Results from this analysis demonstrate that d15N ratios in ﬁsh integrate biotic responses to eutrophic process over time and could be a robust indicator for early ecological changes. Keywords: Eutrophication / Everglades / Fish / Nitrogen / Nutrient gradient / Phosphorus / Stable nitrogen isotopes / Wetlands Résumé – Réponses des isotopes stables de l’azote chez les poissons à la limitation du phosphore dans les zones humides d’eau douce. L’eutrophisation d’origine humaine a altéré les processus écologiques des écosystèmes aquatiques. La détection des changements écologiques est une condition préalable à la protection des écosystèmes contre la dégradation. Dans cette étude, les isotopes stables de l’azote (d15N) chez les poissons sont évalués comme indicateurs des changements environnementaux dans les zones humides du sud de la Floride. Les données sur les isotopes stables de l’azote (d15N) de certaines espèces de poissons et sur la qualité de l’eau recueillies dans les Everglades de Floride entre les années 1990 et 2000 ont été utilisées pour évaluer la relation entre les concentrations de phosphore total et les rapports d15N. Les rapports d15N de neuf des dix espèces de poissons sélectionnées augmentent de manière signiﬁcative lorsque la concentration de phosphore total dans les eaux de surface augmente. On a constaté des relations signiﬁcatives entre la concentration d’azote total dans les eaux de surface et les rapports d15N chez plusieurs espèces de poissons. Le schéma des changements des rapports d15N le long des gradients de nutriments suggère qu’une eutrophisation accrue est enregistrée dans les rapports d15N chez les poissons. L'absence de charge en eaux usées, la prédominance du ruissellement agricole et le rapport élevé TN:TP suggèrent que le phosphore est le facteur limitant qui détermine la productivité des écosystèmes et les changements des rapports d15N chez les poissons. Les résultats de cette analyse montrent que les rapports d15N chez les poissons intègrent les réponses biotiques au processus d’eutrophisation au ﬁl du temps et pourraient être un indicateur robuste des premiers changements écologiques. Mots-clés : Eutrophisation, Everglades, Poissons / Azote, Gradient de nutriments / Phosphore / Isotopes d’azote stables / Zones humides *Corresponding author: gubinhe@gmail.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY-ND (https://creativecommons.org/licenses/by-nd/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. If you remix, transform, or build upon the material, you may not distribute the modiﬁed material. J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 1 Introduction A number of environmental indicators have been used to assess ecological processes in aquatic ecosystems. Current indicators of environmental changes for freshwater include nutrient loading such as soil and water total phosphorus (TP), total nitrogen (TN) concentrations, biodiversity and primary productivity (Jeppesen and Sammalkorpi, 2002). These indicators often reveal the changes in the impacted systems at the basal resource levels (nutrients and primary producers). Identiﬁcation of signs of environmental change along higher trophic levels in aquatic food webs is critical for the restoration of disturbed systems and wildlife protection (Vander Zanden et al., 2005). Nitrogen stable isotope compositions of organic matter may be a complementary means to detect environmental changes in aquatic ecosystems. The ratios of 15 N/14N (deﬁned as d15N) may provide insight into the sources, sinks and cycling of nitrogen in biota that interact with their physical and chemical environments (Peterson and Fry, 1987). The use of d15N as an indicator of aquatic eutrophication is based on the fact that increases in ecosystem productivity controlled by nutrient enrichments will lead to decreases in isotope fractionation by primary producers and the transfers of organic matter from one trophic level to another will result in predictable isotope enrichment along food chain (Post, 2002; Vander Zanden et al., 2015). At present, the use of consumer d15N largely focuses on the source of nitrogen contaminations (Lake et al., 2001; Vander Zanden et al., 2005; Schlacher et al., 2005; Robinson et al., 2016; Souza et al., 2018) and trophic interactions (Post, 2002; Vander Zanden et al., 2015; Wang et al., 2018). Very few studies link consumer isotope composition to primary productivity in freshwater ecosystems (Woodland et al., 2012; Hou et al., 2013). In this study, we compared d15N ratios in 10 species of freshwater ﬁsh collected along a nutrient gradient in the Everglades, Florida, USA. The purposes of this study were (1) to understand the responses and mechanisms controlling the isotope variations along the nutrient gradient, and (2) to evaluate if ﬁsh d15N is a reliable and feasible candidate for human-induced eutrophication in freshwater wetlands. This study provides insight into isotopic responses to changing water quality. 2 Materials and methods 2.1 Site description The Everglades is the largest subtropical peatland in the United States with its historic geochemistry and biological community characterizing an oligotrophic ecosystem (Wright et al., 2008; Richardson, 2010). Since human settlement, a large portion of the Everglades peatland immediately south of Lake Okeechobee was converted into farmlands, i.e., Everglades Agricultural Area (EAA). The remaining Everglades has been divided by drainage canals, levees and water control structures into three Water Conservation Areas (WCA-1, WCA-2 and WCA-3), and Everglades National Park (ENP). Discharge of EAA runoff which contains high concentrations of total phosphorus (TP) and total nitrogen (TN) has led to cattail (Typha spp.) invasion and replacement of the native macrophytes and periphyton (Sklar et al., 2005). Increased P loads in surface water runoff have shifted portions of the ecosystem from oligotrophic to eutrophic states. As a result, TP and TN concentrations in the water column and soil near the inﬂow regions are elevated (Wright et al., 2008). 2.2 Sources of data Stable isotope data on ﬁsh collected from 1994 to 1999 were downloaded from the United States Geological Survey South Florida Information Access (Appendix Tab. A1). A total of 16 sites, with three sites in Stormwater Treatment Area-1 West (STA-1W) and 13 sites in the WCAs and ENP (Fig. 1) were sampled, often on multiple ﬁeld trips. These sites include canals, near levee inﬂow and outﬂow structures and interior marshes. Fish were caught randomly and brought to a laboratory where muscle tissue from each ﬁsh was removed, dried and ground to ﬁne powder for stable isotopes analysis. Additional samples of mosquitoﬁsh were collected along the nutrient gradient in WCA-2A in 2007 (Fig. 1 and Tab. 1). Muscle tissue from 3 to 5 mosquitoﬁsh was composed into a single sample per site and processed as above prior to stable isotope analysis. Select environmental data were downloaded from DBHYDRO, a hydrometeorologic, water quality, and hydrogeologic data retrieval system managed by the South Florida Water Management District (West Palm Beach Florida, USA). Water quality data recorded one year before ﬁsh collection date were averaged to reﬂect environmental conditions of each habitat. When environmental data were not available from the same ﬁsh collection site, data from closely located sites were used. Fish samples collected in the 1990s were analyzed for stable isotopic composition (d13C and d15N) using a Micromass Optima continuous ﬂow mass spectrometer coupled to a Carlo Erba elemental analyzer at US Geological Survey. Results are reported in the usual delta notation relative to V-PDB for 13C and air for 15N. Analytical precision (1s) based on repeat analysis of both samples was generally in the range of 0.1−0.2‰ for both C and N, but for some samples replication was no better than ± 0.5 ‰ due to sample heterogeneity (Kendall et al., 2005). Mosquitoﬁsh samples collected in 2007 were analyzed using a Carlo Erba Elemental Analyzer interfaced to a Finnigan MAT Delta Plus XP stable isotope ratio mass spectrometer (IRMS) at Florida State University. The precision of the C and N isotope analysis was ±0.2‰ (1s) or better on the basis of repeated analysis of different laboratory standards. 2.3 Statistics Because not all ﬁsh were found in each site, only ﬁsh found in at least 6 sites were used in this analysis. Ten species belonging to different trophic levels met this criterion. The d15N ratios of select ﬁsh species from different years of collection were pooled from a given site (Appendix Tab. A1). Spearman Rank Correlation analysis was used to establish relationships between TP, TN and molar TN:TP ratios and d15N of each ﬁsh species. All statistics were performed using SAS JMP (Version 7, SAS Institute). Statistics were considered signiﬁcant at P < 0.05. Page 2 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Fig. 1. Map showing sampling sites in Stormwater Management Area 1 West (STA-1W), Water Conservation Areas (WCAs) and Everglades National Park (ENP) of south Florida. The insert indicates location of the study site in the USA. 3 Results 3.1 Environmental conditions The 16 study sites received water with considerably different concentrations of nutrients and other chemical compounds (Tab. 1). Study sites at Cell 3 and Cell 4 of the STA-1W, and E0 and F1 of WCA-2A, which received direct EAA discharges are highly enriched with TP (>40 mg L−1). Study sites located immediately downstream of STA-1W, WCAs or close to interior canals are moderately enriched with TP (>10 and <30 mg L−1). Interior marshes in WCAs and ENP typically maintain the oligotrophic state indicated by a low TP concentration (<10 mg L−1). Total nitrogen (TN) and dissolved inorganic nitrogen (DIN) concentrations were also higher at the near inﬂow sites than the marsh interior sites, except for Page 3 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table 1. Averages of environmental variables for each study site during the study period. Sites are generally listed from north to south. Site pH SU DO mg L1 TP mg L1 SRP mg L1 NH4þ mg L1 NOx mg L1 TN mg L1 TN:TP Molar ratio STA-W Cell 3* STA-W Cell 4* ENR outﬂow* L7 LOX* E0 F1 U3* L35B 2BS L67* 3A15* 3ATH TS-7 TS-9 North Prong Creek 7.4 7.1 7.4 7.4 6.4 7.1 7.2 7.5 7.2 7.3 7.0 7.1 7.2 7.7 7.7 7.4 2.8 1.5 3.6 3.6 3.1 2.0 1.7 4.8 4.1 3.9 2.5 2.9 3.6 7.0 7.0 4.0 42 46 24 24 8 74 70 5 13 21 15 6 13 4 4 9 16 12 8 8 1 41 24 2 5 4 5 1 4 3 3 3 0.13 0.169 0.047 0.047 0.023 0.186 0.575 0.269 0.017 0.049 0.34 0.036 0.032 0.058 0.058 0.029 0.051 0.026 0.061 0.061 0.007 0.29 0.061 0.006 0.038 0.058 0.086 0.008 0.063 0.02 0.02 0.043 2.049 2.071 2.271 2.271 1.208 2.97 2.861 2.177 1.748 1.541 1.648 1.016 1.377 0.775 0.775 1.327 108 99 206 206 322 89 91 964 298 162 238 401 242 446 446 326 Note: DO: dissolved oxygen. TP: total phosphorus. SRP: soluble reactive phosphorus; TN: total nitrogen. Averages are calculated from weekly to biweekly samples taken between 1990 and 1999. Table 2. List of ﬁsh species and mean d15N ratios used in this analysis. Common name Scientiﬁc name Mean SD N Sailﬁn molly Golden topminnow Mosquitoﬁsh Blueﬁn killiﬁsh Least killiﬁsh Spotted sunﬁsh Redear sunﬁsh Bluegill Largemouth bass Florida gar Poecilia latipinna Fundulus chrysotus Gambusia holbrooki. Lucania goodei Heterandria formosa Lepomis punctatus Lepomis microlophus Lepomis macrochirus Micropterus salmoides Lepisosteus platyrhincus 8.4 9.3 10.6 10.6 10.8 9.3 8.8 9.8 11.1 11.6 2.4 2.9 2.8 2.8 3.0 1.1 1.5 1.5 1.5 1.3 49 43 473 72 171 81 76 88 576 56 U3, the interior site of WCA-2A, which is enriched with ammonium and TN. Theses study sites are also generally characterized by above-neutral pH, high alkalinity, and low dissolved oxygen (DO) concentrations, with an exception of an interior site in WCA-1. This is a rain-driven system, where pH values are low (Tab. 1). 3.2 Fish ecology and d15N ratios Species described in Table 2 represent major ﬁsh assemblage in the subtropical wetlands. They are either omnivorous, feeding on both algae, aquatic macrophytes and invertebrates (killiﬁsh, golden topminnow, mosquitoﬁsh and sailﬁn molly), primary consumers, feeding on aquatic insects (bluegill and spotted sunﬁsh), snails (redear sunﬁsh) or piscivores (largemouth bass and Florida gar). Average d15N ratios for each species collected from multiple sites and years ranged from 8.4‰ in sailﬁn molly to 11.6‰ in Florida gar. In general, d15N ratios reﬂect the trophic position of each species. For example, both the Florida gar and largemouth bass which are piscivores displayed higher d15N ratios than all other species preying on lower trophic levels. Omnivorous species depending on both primary producers and invertebrates typically show low d15N ratios. It is surprising that the least killiﬁsh, blueﬁn killiﬁsh and mosquitoﬁsh which are reported depending partially on primary producers had higher d15N ratios than those reportedly true primary consumers such as the three sunﬁsh species. 3.3 Patterns of ﬁsh d15N along the nutrient gradient Fish d15N ratios selected in this analysis generally increased with the increases in TP concentrations (Figs. 2 and 3). Except for the golden topminnow, all other ﬁsh had signiﬁcant correlation between water column TP concentrations and d15N ratios (Tab. 3). Signiﬁcant correlation between TN concentrations and ﬁsh d15N ratios were found in ﬁve species (Tab. 3). Nearly all ﬁsh displayed a decline in d15N ratios with increases in molar TN/TP ratio although only seven species displayed signiﬁcant correlation (Tab. 3). Highly signiﬁcant correlations (p < 0.001) between TP, TN, TN/TP ratio and d15N ratios were found in the sailﬁn molly, Page 4 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Fig. 2. d15N ratios (mean ± SD) of ten ﬁsh species along the TP concentration gradient in the Everglades wetlands. Each dot represents data from a speciﬁc date of sample collection in the 1990s. mosquitoﬁsh, least killiﬁsh, largemouth bass and Florida gar (Tab. 3). The d15N ratios of mosquitoﬁsh samples collected from seven study sites in 2007 were plotted against TP concentrations (Fig. 4). A highly signiﬁcant relationship between nutrients and d15N ratios (p < 0.001) was also found in these samples. The d15N ratios of mosquitoﬁsh and least killiﬁsh were available in all monitoring sites from WCA-2 (Fig. 1) and plotted along with TP concentrations collected for each site (Fig. 5). The d15N ratios of both ﬁsh and TP concentrations were considerably higher in both inﬂow (E0) and near inﬂow (F1) sites than those in the interior site (U3) of WCA-2A and near a canal site (L35B) and interior (2BS) WCA-2B which displayed similar d15N ratios but variously low TP concentrations. 4 Discussion Findings from this analysis are consistent with previous studies, which reveal positive relationship between nutrient concentrations and biota d15N ratios (Cole et al., 2004; Inglett and Reddy, 2006; Gu et al., 2009). However, the results from this analysis may be complicated by several factors, including study design and variability of nutrient data selected for this analysis. For example, biological characteristics of ﬁsh including age, size, gender, growth rate and feeding habits at each site will certainly introduce additional variations. Without data for ﬁsh age and tissue turnover time, we used the average TP concentrations measured one year prior to ﬁsh collection, which may not accurately reﬂect the growth condition of ﬁsh. Page 5 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Fig. 3. d15N values (mean ± SD) of ten ﬁsh species along the TN concentration gradient in the Everglades wetlands. Each dot represents data from a speciﬁc date of sample collection in the 1990s. Half of the species also had signiﬁcant relationship between TN concentrations and d15N ratios. Nitrogen from human and animal wastes is often enriched in 15N (McClelland et al., 1997). Therefore, the increasing pattern of d15N ratios along the nutrient gradient may also be caused by increases in wastewater loading. Enriched d15N of various ﬂora and fauna has been used as an indicator for sewage inﬂuence in the freshwater and coastal marine environments (Cole et al., 2004; Rožič et al., 2014; Souza et al., 2018; de Carvalho et al., 2019). There have been no reports of any signiﬁcant wastewater contributions from human or animal sources to the Everglades. Inglett et al. (2005) reported that the d15N ratios of porewater NH4þ (the dominant N species in reduced soils) is similar at both the eutrophic and nonaffected WCA-2A sites. The high d15N at the impacted sites is unlikely the result of the uptake of wastewater enriched with 15N and subsequent transfers to the consumer community (Cabana and Rasmussen, 1995). Other nitrogen cycling processes (nitriﬁcation, denitriﬁcation and volatilization) may impact on the isotope composition of DIN in natural wetlands. However, ammonium, not nitrate, was the dominant species of DIN in this region (Tab. 1). Under low DO concentrations in this shallow wetland, nitriﬁcation is not likely the main process. Denitriﬁcation occurs under low DO concentrations and may result in signiﬁcant changes in isotope composition the substate and products. However, nitrate was not the major form of DIN in the Everglades. Finally, volatilization occurs only under high pH and the nearly neutral pH found in the south Florida wetlands (Tab. 1) makes this process highly unlikely. Along with the ﬁndings from a previous study showing the similar N signatures in both Page 6 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table 3. Result of Spearman Rank Correlation between ﬁsh d15N ratios, water-column TP and TN concentrations and molar TN/TP ratios in this study. Species name TP TN TN:TP N Sailﬁn molly Golden topminnow Mosquitoﬁsh Blueﬁn killiﬁsh Least killiﬁsh Spotted sunﬁsh Redear sunﬁsh Bluegill Largemouth bass Florida gar 0.76*** 0.46 0.76*** 0.77*** 0.72*** 0.69*** 0.52* 0.53* 0.69*** 0.80*** 0.55 0.29 0.55*** 0.50* 0.64*** 0.23 0.44 0.38 0.46*** 0.63*** −0.50** −0.31 −0.72*** −0.72*** −0.73*** −0.12 0.17 −0.51* −0.57*** −0.64*** 12 16 56 21 33 17 16 21 40 21 *p < 0.05; ** p < 0.01; *** p < 0.001. Fig. 4. Relationship between total P (TP) concentration and d15N ratios in mosquitoﬁsh collected from the Everglades Protection Area in 2007. affected and unaffected area of Everglades (Inglett et al., 2006), site-speciﬁc N transformation would not likely be the major process leading to the differences in ﬁsh d15N. Nitrogen concentration may also inﬂuence biota d15N ratios through a substrate-mediated isotope effect (Peterson and Fry, 1987). When N is not a limiting nutrient in a system, an increase in N concentration will normally cause an increase in isotopic fractionation and therefore a decrease in d15N ratios. This would be evidenced by a negative correlation between TN concentration and ﬁsh d15N ratios in this study. Instead, the majority of the ﬁsh in this study displayed positive relationship between TN and d15N ratios. This implies that N is not a limiting nutrient to plant growth in the Everglades. This is also supported by the high molar ratios of TN:TP ratios. Some negative correlation between the water column TN:TP ratio and the ﬁsh d15N ratios also indicate that P, not N, is the limiting nutrient in the Everglades. The major external source of N in the Everglades is agricultural runoff (Richardson, 2010). Because manufactured fertilizers are depleted in 15N (Kohl et al., 1971), assimilation of this 15N-depleted N will not result in 15N enrichment in the impacted sites. We conclude that the 15N enrichment in the Fig. 5. Changes in the d15N ratios of mosquitoﬁsh and least killiﬁsh and TP concentrations along the nutrient gradient in the WCA-2. Everglades is the result of increased primary production stimulated by P availability. The average d15N ratios for ﬁsh increase progressively along the TP gradient. The low d15N ratios in ﬁsh at the unimpacted sites was the result of low TP concentration and large 15N fractionation by primary producers during DIN uptake under P stress. The high N availability at the unimpacted sites also allowed selective assimilation of 14N by aquatic plants. In contrast, the high d15N ratios at the impacted sites were due to P enrichment which leads to high N demand and low 15N fractionation. Many studies have demonstrated that P is the limiting nutrient in the Everglades (e.g., Newman et al., 1996). Recent studies using stable isotopes found a positive relationship between the TP and d15N ratios of periphyton, sawgrass and cattail in WCA-1 and WCA-2A, which was attributed to P-driven plant growth and reduced isotope fractionation (Inglett and Reddy, 2006; Chang et al., 2009; Wang et al., 2015). The widespread signiﬁcant correlation between TP concentrations and ﬁsh d15N ratios is the consequence of the transfers of plant protein and the associated 15N/14N signal to the consumers, with 15N enrichment along the food chain. The consumer d15N ratios increases along the TP gradient, which is consistent with the pattern of increases in the primary Page 7 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 producers. Furthermore, the relationship between TP concentration and ﬁsh d15N ratios seems to improve as the trophic level increases. For example, Florida gar, which is positioned at the highest trophic level in our samples, had the highest correlation coefﬁcient (r = 0.80) with a moderate sample size (n = 21) among ﬁsh selected for this study. In general, ﬁsh d15N is a better indicator of the eutrophication process because they integrate temporal and spatial variations in source d15N ratios over longer time periods (Cabana and Rasmussen, 1996; Vander Zanden et al., 2005). 5 Conclusions The eutrophication process resulting from excessive P loading from the agricultural runoff to the Everglades is demonstrated using the nitrogen stable isotopic ratios of ﬁsh. The d15N ratios of nearly all ﬁsh species responded positively to the increases in TP concentration. This is considered to be caused by increasing the N uptake and decreasing the 15N fractionation by primary producers stimulated by P enrichment. The 15N enrichment along the nutrient gradient is evident in ﬁsh that reliably transfer the isotope signals from primary producers along the trophic level. The signiﬁcant correlations between TP concentration and d15N ratios in mosquitoﬁsh in the 1990s and 2007 suggest that the eutrophication trend along the nutrient gradient persisted after almost two decades. Results from this study indicate that aquatic consumers such as ﬁsh are the better environmental indicators because they are capable of integrating biogeochemical changes over time. Compliance with ethical standards Conﬂict of interest: On behalf of all authors, the corresponding author states that there is no conﬂict of interest. Acknowledgements. We appreciate the United States Geological Survey and South Florida Water Management District for providing stable isotope and water quality data. References Cabana G, Rasmussen JB. 1996. Comparison of Aquatic Food Chains Using Nitrogen Isotopes. 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Page 8 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Wang J, Gu B, Ewe SM, Wang Y, Li Y. 2015. Stable isotope compositions of aquatic ﬂora as indicators of wetland eutrophication. Ecol Eng 83:13–18. Wang J, Chapman D, Xu J, Wang Y, Gu B. 2018. Isotope niche dimension and trophic overlap between bigheaded carps and native ﬁlter-feeding ﬁsh in the lower Missouri River, USA. PloS one, 13: e0197584. Woodland RJ, Magnan P, Glémet H, et al. 2012. Variability and directionality of temporal changes in d 13C and d 15 N of aquatic invertebrate primary consumers. Oecologia 169: 199–209. Wright AL, Reddy KR, Newman S. 2008. Biogeochemical response of the Everglades landscape to eutrophication. Int J Environ Res 2: 102–109. Cite this article as: Hong J, Gu B. 2020. Responses of nitrogen stable isotopes in ﬁsh to phosphorus limitation in freshwater wetlands. Knowl. Manag. Aquat. Ecosyst., 421, 41. Page 9 of 15 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Appendix Table A1. Stable isotope data used in this analysis. 1. Data from 1990s Site DATE Common Name d15N mean d15N SD N TS-7 TS-9 3A15 U3 3A15 3A15 U3 L35B 2BS L67 2BS ENR-OUT L67 Cell 3 E0 F1 3A15 U3 3A15 L35B L35B 2BS L67 L67 Cell 3 Cell 4 Cell 3 Cell 3 Cell 3 Cell 3 Cell 3 LOX E0 E0 E0 E0 F1 F1 F1 F1 F1 F1 U3 U3 L35B 2BS L67 L67 L67 Jan-1998 Jan-1998 Jan-1998 Sep-1997 Sep-1997 Jun-1998 Jul-1997 Sep-1997 Jul-1997 Jan-1998 Jan-1998 Jan-1998 Sep-1997 Jun-1997 Jul-1997 Sep-1997 Jan-1998 Jun-1998 Sep-1997 Jan-1998 Sep-1997 Jan-1998 Jan-1998 Jun-1998 Jun-1997 Jun-1998 Jun-1998 Jan-1998 Jun-1997 Jan-1998 Jun-1998 Jan-1998 Dec-1995 Jul-1995 Jun-1998 Sep-1997 Mar-1995 Jul-1995 Jun-1996 Dec-1996 Sep-1997 Jan-1998 Jul-1997 Sep-1997 Sep-1997 Jan-1998 Jun-1996 Dec-1996 Sep-1997 Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Golden Topminnow Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Sailﬁn Molly Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh 9.92 9.22 7.29 8.39 7.58 8.40 8.10 5.36 8.68 11.68 7.61 9.86 10.20 10.38 18.66 7.87 6.52 7.23 6.39 7.59 4.02 6.52 11.08 10.49 9.28 8.66 10.95 11.99 11.04 13.44 13.02 8.63 11.66 14.51 15.97 16.77 17.64 11.35 15.54 13.71 11.01 12.64 8.43 8.27 6.71 7.68 9.10 10.25 12.57 0.26 0.45 0.27 0.19 0.65 0.12 4 3 7 3 2 2 1 3 1 3 9 1 1 1 1 1 4 4 1 2 4 5 5 1 6 7 6 4 12 9 10 5 2 1 9 5 1 1 1 2 4 6 2 2 5 11 1 1 1 Page 10 of 15 0.36 0.46 0.42 0.85 0.46 0.56 0.84 0.34 1.10 0.55 1.37 0.19 1.09 0.62 0.54 0.59 0.34 6.89 0.62 1.57 0.57 0.33 0.47 0.06 0.60 1.07 0.47 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table A1. (continued). 1. Data from 1990s Site DATE Common Name d15N mean d15N SD N L67 L67 3A15 3A15 3A15 3A15 3A15 3A-Th 3A-Th TS-7 TS-9 TS-9 2BS 2BS 2BS 2BS 3A15 3A15 3A15 3A15 3A15 3A15 3A-Th 3A-Th 3A-Th Cell 3 Cell 3 Cell 3 Cell 4 E0 E0 E0 E0 E0 E0 E0 ENR-OUT ENR-OUT F1 F1 F1 F1 F1 F1 F1 L35B L35B L35B L67 L67 L67 L67 Jan-1998 Jun-1998 Jun-1996 Jul-1997 Sep-1997 Jan-1998 Jun-1998 Sep-1997 Jan-1998 Jan-1998 Jan-1998 Jun-1998 Jul-1997 Jan-1998 Apr-1997 Jun-1998 Jan-1998 Dec-1996 Jul-1997 Sep-1997 Apr-1997 Jun-1996 Jan-1998 Sep-1997 Jul-1997 Jun-1998 Jun-1997 Jan-1998 Jun-1998 Dec-1996 Jun-1998 Mar-1995 Sep-1997 Jul-1995 Jul-1997 Jun-1996 Jan-1998 Jun-1998 Feb-1998 Jun-1998 Dec-1996 Jun-1996 Sep-1997 Jan-1998 Jul-1995 Jan-1998 Sep-1997 Jun-1998 Jan-1998 Jul-1997 Jun-1998 Sep-1997 Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Least Killiﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh 12.28 12.00 8.90 8.39 7.88 7.98 7.80 7.38 7.87 9.49 9.63 8.30 7.95 8.30 6.53 7.93 7.64 8.97 8.31 7.16 9.05 10.86 7.28 7.69 7.12 12.77 11.68 12.73 11.09 13.20 15.12 17.52 16.77 13.89 16.61 15.89 11.89 10.71 12.20 12.71 13.55 16.64 11.94 11.77 11.62 8.22 8.96 11.45 10.91 10.88 12.35 10.98 0.93 2.03 8 8 1 3 5 15 12 3 15 2 4 4 5 11 1 12 14 5 5 5 9 1 15 5 1 20 21 15 8 5 18 2 5 1 5 1 9 9 1 14 2 1 5 10 1 19 4 20 20 8 40 10 Page 11 of 15 0.55 0.54 0.33 0.43 0.33 0.37 1.04 0.30 0.56 0.35 0.69 0.54 0.38 0.68 1.40 0.52 0.81 0.25 0.19 1.27 0.77 0.71 1.75 0.48 1.92 0.51 1.90 1.03 0.67 0.76 0.74 0.11 0.35 1.09 1.44 0.43 1.08 1.25 1.25 0.98 0.72 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table A1. (continued). 1. Data from 1990s Site DATE Common Name d15N mean d15N SD N L67 L67 L67 LOX TS-7 TS-7 TS-7 TS-9 TS-9 TS-9 U3 U3 U3 U3 U3 U3 Cell 3 Cell 3 Cell 3 ENR-OUT E0 E0 E0 F1 U3 U3 L35B L35B 2BS L67 L67 L67 L67 3A15 3A15 TS-7 TS-9 Cell 3 Cell 3 U3 U3 U3 U3 U3 L35B L35B L67 L67 L67 3A15 3A15 3A15 Jul-1995 Dec-1996 Jun-1996 Jan-1998 Jul-1997 Jan-1998 Jun-1998 Jul-1997 Jan-1998 Jun-1998 Dec-1996 Jun-1998 Sep-1997 Mar-1995 Jul-1995 Jul-1997 Jun-1997 Jan-1998 Jun-1998 Jan-1998 Dec-1995 Dec-1996 Sep-1997 Sep-1997 Dec-1996 Sep-1997 Sep-1997 Jan-1998 Jan-1998 Jun-1996 Sep-1997 Jan-1998 Jun-1998 Sep-1997 Jan-1998 Jan-1998 Jan-1998 Apr-1997 Oct-1997 Oct-1996 Sep-1997 Oct-1997 Nov-1997 Jan-1998 Oct-1996 Jan-1998 Feb-1997 Jun-1997 Jan-1998 Sep-1997 Oct-1997 Jan-1998 Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Mosquitoﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Blueﬁn Killiﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh Spotted Sunﬁsh 11.68 10.92 9.86 8.18 8.92 9.71 9.94 7.35 8.88 7.78 8.93 8.40 8.48 9.98 6.49 8.50 11.41 12.60 13.14 11.83 13.97 15.00 17.61 11.74 8.06 6.93 8.76 9.45 7.90 10.06 8.59 10.77 11.87 8.14 7.47 8.85 8.98 11.12 12.21 8.36 8.86 9.36 9.09 9.29 9.18 9.90 8.60 8.28 10.51 8.62 8.80 9.42 0.76 0.96 4 12 2 6 2 10 10 1 20 16 2 13 5 1 1 5 12 4 5 2 1 1 7 1 1 1 3 1 1 1 3 8 6 2 5 2 5 6 1 5 5 2 1 3 15 14 4 3 4 5 5 4 Page 12 of 15 0.35 0.13 0.36 0.41 0.46 1.95 0.15 1.29 0.33 0.40 1.16 1.72 0.71 0.00 2.47 0.35 2.77 0.83 2.04 0.10 0.11 0.26 0.38 0.97 0.47 0.24 0.28 0.42 1.12 2.03 1.67 1.25 0.52 0.56 0.48 0.45 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table A1. (continued). 1. Data from 1990s Site DATE Common Name d15N mean d15N SD N 3A15 3A15 Cell 3 ENR-OUT U3 U3 U3 U3 U3 L35B L35B L35B 2BS L67 L67 3A15 3A15 3A15 3A15 U3 U3 3A15 3A15 U3 U3 3A15 L35B L67 L35B L35B L67 L35B L35B L67 L67 L67 Cell 3 Cell 3 Cell 3 LOX LOX LOX Cell 3 Cell 3 Cell 3 Cell 3 Cell 3 Cell 4 L-7 L-7 L-7 U3 Mar-1998 Jan-1999 Jan-1998 Jan-1998 Oct-1996 Sep-1997 Oct-1997 Nov-1997 Jan-1998 Oct-1996 Jun-1997 Jan-1998 Jan-1998 Feb-1997 Jan-1998 Dec-1996 Sep-1997 Mar-1998 Jan-1999 Oct-1996 Jan-1998 Oct-1997 Nov-1997 Oct-1997 Sep-1997 Dec-1996 Jan-1998 Jun-1997 Feb-1997 Jun-1998 Jan-1998 Sep-1998 Oct-1996 Nov-1997 Sep-1998 Feb-1997 Jun-1998 Jun-1997 Jan-1998 Dec-1996 Sep-1997 Oct-1998 Oct-1996 Apr-1997 Oct-1997 Jan-1998 Aug-1998 Jan-1999 Oct-1996 Sep-1997 Sep-1998 Oct-1996 Spotted Sunﬁsh Spotted Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Redeared Sunﬁsh Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Bluegill Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass 8.88 8.22 12.40 11.51 7.88 9.70 10.53 8.66 8.70 8.07 7.36 8.04 7.17 7.70 8.21 8.52 7.84 8.70 7.24 8.46 9.91 9.06 9.77 8.77 9.70 9.86 8.83 11.36 8.85 8.97 9.81 10.41 9.74 9.70 9.96 8.06 13.38 12.01 12.53 9.70 9.28 8.76 13.13 12.84 12.74 11.98 13.61 12.61 14.17 15.05 14.32 9.62 0.34 3 1 4 9 5 6 1 3 6 9 2 5 2 10 8 1 4 1 1 2 1 2 1 4 9 1 5 1 8 4 2 9 11 3 2 9 5 2 6 36 6 20 34 8 26 2 20 6 32 20 20 12 Page 13 of 15 1.18 0.97 0.53 4.94 0.06 0.43 0.52 0.38 0.41 0.86 0.69 0.97 0.19 0.03 1.07 0.27 2.99 0.99 1.03 1.62 0.63 1.64 0.93 0.19 1.53 0.93 1.04 0.62 0.73 0.36 0.77 0.63 0.58 0.41 1.24 1.25 0.49 0.70 1.16 1.47 1.70 0.68 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table A1. (continued). 1. Data from 1990s Site DATE Common Name d15N mean d15N SD N U3 U3 U3 U3 U3 L35B L35B L35B 2BS L67 L67 L67 L67 L67 L67 L67 3A15 3A15 3A15 3A15 3A15 LMC LMC LMC NPC NPC NPC Cell 3 Cell 3 Cell 3 ENRout ENRout E0 U3 U3 U3 U3 U3 L35B 2BS L67 L67 3A15 3A15 3A15 3A15 3A15 3A15 Sep-1997 Oct-1997 Nov-1997 Jan-1998 Mar-1998 Oct-1996 Jan-1998 Sep-1998 Jan-1998 Dec-1996 Jun-1997 Sep-1997 Nov-1997 Jan-1998 Sep-1998 Oct-1998 Dec-1996 Sep-1997 Oct-1997 Nov-1997 Mar-1998 Dec-1996 Aug-1997 Mar-1998 Dec-1996 Aug-1997 Mar-1998 Oct-1997 Jan-1998 Aug-1998 Oct-1997 Jan-1998 Sep-1997 Oct-1997 Nov-1997 Jan-1998 Mar-1998 Jan-1999 Jan-1998 Jan-1998 Nov-1997 Jan-1998 Oct-1997 Nov-1997 Jan-1998 Mar-1998 May-1998 Jan-1999 Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Laregmouth bass Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar Florida Gar 10.43 10.21 10.33 10.77 9.86 10.64 10.54 11.49 10.11 10.30 10.47 11.75 10.67 10.61 11.57 11.47 10.47 9.76 8.94 10.55 9.60 9.92 10.64 9.84 11.68 12.46 11.80 12.63 13.67 13.32 12.60 12.06 14.99 11.17 10.95 10.69 11.09 11.00 11.88 10.95 11.75 12.21 9.97 10.74 10.91 10.88 10.57 9.95 0.27 0.30 0.48 0.11 0.83 0.98 0.54 0.93 0.50 0.46 1.82 1.18 1.24 1.36 1.14 0.97 0.52 0.50 1.31 6 13 4 2 7 35 11 20 10 35 5 20 8 15 10 10 12 18 5 1 6 33 2 10 5 14 16 1 2 4 2 2 3 2 2 2 2 3 5 1 5 6 1 1 4 3 4 1 Page 14 of 15 0.95 0.51 0.16 0.63 0.11 0.38 2.66 0.54 0.71 0.16 0.05 1.34 0.64 0.97 0.20 0.38 0.77 0.78 1.05 0.91 0.33 0.90 0.92 J. Hong and B. Gu: Knowl. Manag. Aquat. Ecosyst. 2020, 421, 41 Table A1. (continued). 1. Data from 1990s Site DATE 2. Data from 2007 TP (ug/L) 4.9 7 8.5 15 24.5 42.1 46.5 d15N (‰) 10.2 8 9.1 11.3 13.1 14.2 14.5 Common Name Page 15 of 15 d15N mean d15N SD N