Revealing the trophic role of the invasive African clawed frog Xenopus laevis through combined analysis of stable isotopes and heavy metals in a Mediterranean stream from central Chile

495 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 Revealing the trophic role of the invasive African clawed frog Xenopus laevis through combined analysis of stable isotopes and heavy metals in a Mediterranean stream from central Chile Gabriel Lobos1,2, Gianina Tapia2, Alejandra Alzamora2, Nicolás Rebolledo2, Hugo Salinas2, Juan Carlos Trujillo2, Juan Sánchez3, Victoria Gómez-Aburto4, Cristóbal Galbán-Malagón4,5,6 1 Centro de Gestión Ambiental y Biodiversidad, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile 2 Ecodiversidad Consultores, Santiago, Chile 3 Gerencia SHE, Operación El Soldado, Anglo American, Santiago, Chile 4 Centro de Genómica, Ecología y Medio Ambiente (GEMA), Universidad Mayor, Santiago, Chile 5 Institute of Environment, Florida International University, Miami, Florida, USA 6 Data Observatory Foundation, Santiago, Chile Corresponding author: Gabriel Lobos ([email protected]) Aquatic Invasions 2025 Volume 20, Issue 4: 495–511 Copyright: © Gabriel Lobos et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Academic editor: Jaclyn Hill Received: 17 June 2024 Accepted: 20 June 2025 Published: 12 November 2025 Citation: Lobos G, Tapia G, Alzamora A, Rebolledo N, Salinas H, Trujillo JC, Sánchez J, Gómez-Aburto V, Galbán-Malagón C (2025) Revealing the trophic role of the invasive African clawed frog Xenopus laevis through combined analysis of stable isotopes and heavy metals in a Mediterranean stream from central Chile. Aquatic Invasions 20(4): 495–511. https:// doi.org/10.3391/ai.2025.20.4.164197 Abstract The African clawed frog Xenopus laevis is invasive on four continents, and is recognized as one of the invasive amphibians that generates the greatest impacts in the ecosystems it invades. Although its diet has been studied in its native habitat and invaded areas, its trophic role is still unclear, especially in the communities it invades. We studied the diet of X. laevis, and looked at its stable isotope signatures and its bioaccumulation of heavy metals, to gain a better understanding of its trophic role. The diet was found to consist mainly of aquatic invertebrates, with some consumption of the native fish Cheirodon pisciculus. The isotope analysis revealed that the assimilation of prey by X. laevis is unrelated to the most-consumed item. Xenopus laevis occupied a high trophic position in its own stream and was segregated from fish in by its use of trophic resources. Despite its high trophic position, only biomagnification of copper and zinc was found in relation to some prey, but not manganese or arsenic. Key words: Bioaccumulation, biomagnification, diet, 13C/12C, 15N/14N Introduction Invasive herpetofauna can cause significant trophic disruptions in the ecosystems they invade, and predation is one of their main mechanisms, as it reduces the abundance of native species, causing population extinctions or the change in nutrient cycles (Kraus 2015). Understanding the trophic position of a species is important in such circumstances since the introduction of top predators can have major ecosystem impacts (Fritts and Rodda 1998). Organisms at intermediate trophic levels, like amphibians, can also have an effect through top-down or bottom-up mechanisms (Kraus 2015). For instance, on a global scale, freshwater fish invasions Trophic role of Xenopus laevis from a Mediterranean stream 496 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 significantly disturb the trophic structure of invaded ecosystems. In this context, Sagouis et al. (2015), in a global evaluation, reported that in lotic systems, the main changes are driven by the introduction of non-native top predators at the top of the food chain, which can expand the total isotopic niche without altering the trophic niche of native species. Conversely, in lentic systems, changes are primarily associated with introducing mesopredators, which reduce the size of the trophic niche, the range of consumed resources, and the trophic niche breadth of the native fish community, thereby increasing direct and indirect competition. The African clawed frog Xenopus laevis (Daudin, 1802) has invaded an extensive geographic area on several continents, with established populations in the USA (McCoid and Fritts 1980), France (Fouquet and Measey 2006), Italy (Lillo et al. 2011), Portugal (Rebelo et al. 2010), Japan (Kokuryo 2009), China (Wang et al. 2019), Mexico (Peralta-García et al. 2014), and Chile (Lobos and Measey 2002; Lobos and Jaksic 2005). Xenopus laevis has demonstrated great success in aquatic systems with a Mediterranean climate (Measey et al. 2012), except in Australia where the species has not yet invaded, despite ecological niche models which predict favorable environmental conditions for the species (Rödder et al. 2017). Records of X. laevis have been reported in Chile since the 1970s, with a considerable expansion in the country’s Mediterranean region (Mora et al. 2019). From a trophic perspective, X. laevis has been characterized mainly as a predator of zooplankton invertebrates in lentic environments and of zoobenthos in lotic environments in both its native and invasive populations (see Courant et al. 2017). The same review shows that predation on fish (USA, France, and Portugal) is generally low, similar to that reported by Lafferty and Page (1997) in California. Predation on amphibian eggs and larvae has been observed in South Africa, France, and Portugal. Additionally, in France, cannibalism accounts for 19% of the total predation on eggs (Courant et al. 2017), with lower rates reported in South Africa, Wales, Portugal, and the USA. Although there are extensive data on the diet of X. laevis in the literature (e.g., Courant et al. 2017), its trophic role (trophic level, range of trophic resources used, nutrient assimilation, and niche overlap with other vertebrates) remains unclear. In this context, stable isotope analysis (SIA) is a tool to determine the trophic positions of organisms (e.g., Davis et al. 2012). Isotopic signatures of carbon (δ13C) have been used to identify sources of primary energy production of consumers, and nitrogen (δ15N) is used to estimate the trophic levels of the consumers (Baeta 2019). SIA studies permit the evaluation of trophic dynamics and help quantify the use of resources among native and invasive species (McCue et al. 2019). Estimating the isotope niche (breadth of δ13C and the δ15N) is widely used to represent the range of resources a consumer uses and the overlap between species’ ecological niches (Davis et al. 2012). In biological invasions, the use of SIA has made remarkable progress in recent years (McCue et al. 2019), enabling in-depth analyses of trophic relationships (Pujol-Buxó et al. 2018), effects on ecosystem functioning (Sagouis et al. 2015), competition among invasive species (Jackson et al. 2012), plasticity and adaptation (Pérez-Diz et al. 2023), and interactions among invasive species (Yelenik and D’Antonio 2013). Heavy metals are typically found with minerals (such as carbonates and sulfates) and organic substances. They accumulate in aquatic sediments, which serves as their primary reservoir (Hua et al. 2016). Metals can remain stored for long periods, particularly in Mediterranean watercourses, where the climate is characterized by long periods of drought. These metals can bioaccumulate (reaching concentrations greater than in their environment) and/or biomagnify through trophic chains (Barra et al. 2021), generating lethal or sub-lethal effects on local populations (Shahjahan et Trophic role of Xenopus laevis from a Mediterranean stream 497 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 al. 2022). Chile has important metal reserves and is one of the global producers of copper, which has been linked to surface water pollution issues (Copaja et al. 2016; Copaja et al. 2017; Ali et al. 2020; Barra et al. 2021). However, due to a volcanic history and general plate tectonics, Chilean soils are also naturally high in heavy metals (Oyarzún and Oyarzún 2011). The use of SIA combined with bioaccumulation studies may provide insight into how these heavy metals move through the trophic levels in relation to a species’ diet (Marambio-Alfaro et al. 2021). Xenopus laevis has been categorized as an aquatic top predator due to its large size, generalist diet, and fully aquatic life history (Courant et al. 2017; Secondi and Raux 2020). If these assumptions are correct, X. laevis will occupy the highest trophic level (δ15N) in the food web of an aquatic community in a stream impacted by mining activities, a predominant land use across much of Chile. Furthermore, these mining activities are expected to result in metal bioaccumulation and/or biomagnification of heavy metals within the frog’s tissues. Consequently, this study aimed to investigate the trophic role of X. laevis by analyzing its diet, stable isotope signatures, and concentrations of heavy metals (Mn, As, Zn, and Cu) in a Chilean stream associated with mining activity. Methods Study area and sample collection The study was performed in El Cobre stream, located in the Valparaiso administrative region of Chile and forms part of the Río Aconcagua watershed (33°34.027'S, -70°37.896'W; 623 m elevation). The stream has an intermediate flow, depending on winter precipitation, although some pools persist during the dry season. The high part of the system is drained by the El Gallo and El Sauce streams, in whose watersheds are located “El Soldado” and “Los Navíos” copper mines. The vegetation is dominated by spiny and sclerophyllous shrubs typical of the semi-arid Mediterranean region of Chile (Di Castri 1968). This water system is one of the few that retains surface water during the dry season (summer), and it is common for it to flow for approximately 5 km before infiltrating the subsoil. It connects with the rest of the hydrological network only in heavy rainfall. In October 2021 (austral spring), two transects of 100 m length and 10 m width in the stream were performed, separated by 3400 m (called sites CO-1 and CO-2). Fish and anurans were captured at both sites using a 12 V battery-operated SAMUS 725 MD electrofisher unit (600 W), which generates a DC pulse of up to 1000 V, with a current intensity of 20 to 60 A, and a pulse duration of 10 seconds (Pottier et al. 2020). Captured individuals were examined for visible injuries prior to release, confirming that the procedure did not harm the collected animals. Individuals were identified by species – fish according to Dyer (2000) and amphibians following Correa et al. (2011) – and weighed to the nearest gram. Calipers were used to measure the snout ventral length (SVL) of amphibians and total length of fish. We collected aquatic invertebrates with a Surber net, sampling an area of 0.09 m2 with three replicates at each site (Ramírez 2010). We sampled the native macrophyte Myriophyllum aquaticum, the only species in CO-1 (no macrophytes were found at CO-2). To analyze heavy metals in the environment, surface sediments (a compound sample from 10 sampling points in each site, taken from the top 2 cm) and surface water from the stream (a compound sample of 3 sampling points at each site) were examined. Additionally, the results of 15 previous sampling sessions conducted between 2018 and 2022 are reported here to assess the historical diversity of aquatic vertebrates in this stream. These campaigns were part of the Chilean Environmental Assessment System. Trophic role of Xenopus laevis from a Mediterranean stream 498 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 Diet Ten individuals of X. laevis (all from site CO-1) were collected and used for diet analysis (stomach contents); animals were euthanized using immersion in a 0.2% tricaine methanesulfonate (MS-222) bath. Stomach contents were identified under a stereoscopic microscope to the minimum possible taxonomic level (Fernández and Domínguez 2001). The length and width (mm) of each prey item were measured to estimate the volume and percentage contribution of each prey taxon (Barreto-Lima 2009). Other estimations included the percentage of each prey category, i.e., the proportion (%N) of each prey of the total prey found. The prey percentage, frequency, and volume data were integrated into a relative importance index of prey RII (Pinkas et al. 1971). Analysis of stable isotopes Two samples of M. aquaticum were used to analyze the isotopes of macrophytes. For invertebrates, a pooled compound sample of each taxon was used due to the small size of these organisms after removing hard tissues (mollusk shells, exoskeletons). For fish and amphibians (adults only), a 4 mg sample of muscle tissue (dorsal muscle in fish and thigh muscle in anurans) was obtained. Samples were ground, dried, and lipids were extracted in petroleum ether using a Soxhlet extractor (Lobos et al. 2022). The isotopic composition of carbon and nitrogen (δ13C and δ15N) was determined with a continuous flow isotope ratio mass spectrometer (Thermo Finnigan Delta V Advantage) coupled to a Carlo Erba NC 2500 elemental analyzer via continuous flow in the Laboratory of Biogeochemistry and Applied Stable Isotopes (LABASI) at the Pontifical Catholic University of Chile. Stable isotope ratios are reported in δ notation, expressed as deviations from international standards: Pee Dee Belemnite for δ13C and atmospheric N2 for δ15N. Values were normalized to internal standards calibrated against International Atomic Energy Agency (IAEA) reference materials (IAEA-N1, IAEA-N2, and IAEA-NO3 for nitrogen; NBS22, CH6, and NBS19 for carbon, respectively). The ratio of 13C/12C and 15N/14N was expressed as relative difference per thousand (‰) using the equation: Equation 1 Rsample and Rstandard are the corresponding ratios of heavy to light isotopes (13C/12C and 15N/14N) in the sample and the standard, respectively. Typical precision of the analyses was ± 0.23‰ for δ15N and 0.25‰ for δ13C. To perform niche comparisons between X. laevis and the collected fishes, analyses were conducted using R (SIBER package; Jackson et al. 2011) to obtain four quantitative Bayesian metrics (mean and 95% confidence intervals) adapted from Layman et al. (2007): nitrogen range (NR), carbon range (CR), Euclidean distance of each individual to the centroid (CD), and standard deviation of the nearest neighbor distance (SDNND). A fifth metric was the corrected standard ellipse area (SEAc expressed in ‰2). SEAs are comparable to the univariate SD and contain ca. 40% of the data, providing a better description of the isotope niche of a population. For a posteriori comparison of the ellipses, the Bayesian standard ellipse areas (SEAb) were estimated according to Jackson et al. (2011). Then, the proportions of prey that contributed to the assimilated diet of X. laevis were determined using the SIAR mixing model (Parnell and Jackson 2013). To minimize the parameters to be estimated in the model (Phillips et al. 2014), prey were grouped into three trophic groups: carnivores, detritivores, and herbivores, an approach Trophic role of Xenopus laevis from a Mediterranean stream 499 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 previously used by Molina-Burgos et al. (2018). In this study, these three groups are isotopically distinct in δ15N (ANOVA, F2,5 = 9.43; p = 0.04), but not in δ13C (F2,5 = 1.81; p = 0.31). The parameters of the model were estimated with 300,000 Markov chain iterations, and 1.3 ± 0.3 δ13C and 2.3 ± 0.18 δ15N were used as the trophic enrichment factors (McCutchan et al. 2003). The trophic level of organisms was estimated as follows: Equation 2 (Post 2002). Where NT is the trophic position n of an organism, δ15N consumer is the value of the organism evaluated, and δ15N base is the nitrogen value of a basal organism (in this case the invertebrate with the lowest position; Baetidae). It was assumed that the NT base in this case occupies position 2 (over the producers of the trophic web). For the trophic discrimination factor (TDF), the reference value 2.3‰ was used (McCutchan et al. 2003). Analytical procedures for heavy metals Cu, Mn, As, and Zn levels were evaluated in sediments, water, and aquatic biota. For the aquatic biota, a variable number of replicates for each species collected was obtained (Appendix 1: Table A1); these were homogenized to obtain a humid paste of 0.5–1 mg. Samples were dried (60 °C for 48 h) before metal analysis. The US EPA 3050B protocol (US EPA 1996) was used, which involves repeated digestion with HNO3 and hydrogen peroxide (H2O2) to recover the metals present in the organic fraction (where metals are bioavailable). Metal concentrations for biological samples and sediments were determined using an atomic absorption spectrophotometer (ICP-OES) by the flame technique (Thermo Scientific X series 2, at the Laboratory of Agronomy and Natural Systems of the Pontifical Catholic University of Chile). The analytic procedure was verified using the Dorm-3 reference material (biological samples) and Mess-3 (sediments) obtained from the National Research Council Canada (NRC). The analytical error in both cases was less than 5%. Concentrations were expressed as mg Kg-1 of dry weight. The reference values used for sediments were those of the “interim sediment quality guideline” (ISQG) and “probable effect level” (PEL); the former is the concentration below which adverse effects are not expected, while the latter is the concentration over which adverse biological effects are expected to appear (CEQG 2003). The levels of heavy metals in water samples (mg L−¹) were measured after storage in cold conditions, followed by laboratory analysis using the flame technique (APHA 2017). Estimation of bioaccumulation and trophic magnification factor To integrate the information on isotopes and metals, the biomagnification factor (BMF) for X. laevis and the bioaccumulation factor (BAF) for the community (Dehn et al. 2006) were calculated as follows: Equation (3) Equation (4) Trophic role of Xenopus laevis from a Mediterranean stream 500 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 Where CPredator and CPrey are the concentrations of metals in the predator and the prey, respectively, in mg K-1, and the δ15NPredator and δ15NPrey are the ‰ of the 15N content, respectively. Values of BMF greater than 1 are interpreted as a signal of a possible biomagnification process of the metal. In contrast, values lower than 1 are considered as biodilution of the metal in the predator. In the case of BAF, CBiota and CSediment represent the metal concentrations in biota and sediment in mg K-1. BAF values greater than 1 could be interpreted as a signal of bioaccumulation (Dehn et al. 2006; Mortuza and Al-Misned 2015). Results Captures Three fish species (Cnesterodon decenmaculatus invasive; Basilichthys microlepidotus and Cheirodon pisciculus native) and one invasive amphibian (Xenopus laevis) were collected from the two sampling sites (Table 1). Sixteen aquatic invertebrate taxa were found, 13 of which (81%) were of the class Insect. Richness was 14 taxa for CO-1 and 7 for CO-2 (Table 2). The most represented families were Chironomidae (larvae, 46.63% in CO-1 and 92.07% in CO-2), Coenagrionidae nymphs (15.4% in CO-1), and Physidae (14.9%) in CO-1. Diet The diet of X. laevis was composed of 41 prey items from seven taxa, including invertebrates (insects and mollusks) and fish (Table 3). The relative importance index RII shows that the diet was mainly snails of the genus Physa (44.78%), dragonfly nymphs of Coenagrionidae (19.06%), and the aquatic coleoptera Dytiscidae (17.16%). The native fish (Cheirodon pisciculus) was important in the percentage of volume consumed (28.50%; due to their size), but less important in terms of its low RII value (present in only two of the 10 frogs analyzed). Analysis of stable isotopes The isotope comparison of the community analyzed is shown in Fig. 1 and Table 4. Nitrogen varied from a basal value of 2.60 (macrophyte) to 13.52 (the fish B. microlepidotus). Carbon ranged from -16.36 (B. microlepidotus) to -32.46 Table 1. Fish and amphibians by sampling site (n = number of individuals). Size (cm), mass (g) are presented as mean ± SD. F indicates number of historical records for the stream between 2018–2022 (15 sampling events). Sites Species n Size (cm) Mass (g) F CO-1-Fishes Cheirodon pisciculus 7 4.44 ± 0.70 1.77 ± 0.59 14/15 Cnesterodon decemmaculatus 53 3.18 ± 0.39 0.29 ± 0.10 14/15 CO-1-Anurans Xenopus laevis 10 7.54 ± 0.91 41.31 ± 16.94 3/15 CO-2-Fishes Basilichthys microlepidotus 5 8.92 ± 2.17 5.38 ± 2.95 8/15 Cheirodon pisciculus 1 3.7 0.46 CO-2-Anurans Xenopus laevis 1 7 38 Other species seen (but not collected) during the 2018–2022 sampling events Fishes Gambusia holbrooki (invasive, absent since 2021) 7/15 Trichomycterus areolatus (native) 5/15 Anurans Rhinella arunco (terrestrial, tadpoles with short aquatic development) 10/15 Trophic role of Xenopus laevis from a Mediterranean stream 501 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 (Hydrophilidae). The trophic grouping of invertebrates is well supported in terms of isotope ratios for δ15N (ANOVA, F2,5 = 9.43; p = 0.04), not for δ13C (F2,5 = 1.81; p = 0.31). There were at least five trophic levels; the highest of which were occupied by the native fish (C. pisciculus, B. microlepidotus) and the invasive vertebrates (X. laevis, C. decenmaculatus). The nitrogen range in the community was 1.86‰ (Cis: 1.84–1.88), and the carbon range was 13.64‰ (Cis: 13.62–13.65). The trophic diversity, measured as the mean distance to the centroid CD, was 4.87‰ (Cis: 4.86–4.87), and the trophic evenness SDNND was 3.27‰ (Cis: 3.25–3.28). SEAc was different between species evaluated (SEAc C. decenmaculatus = 1.02‰2, SEAc B. microlepidotus = Table 2. Density of aquatic invertebrates (individuals/m2 and percentage of total invertebrates) by sampling site (CO-1 and CO-2) collected during Surber net sampling. Class Order Family CO-1 CO-2 Density % Density % Gastropoda Hygrophila Physidae 114.81 14.90 0.00 0.00 Clitellata Oligochaeta 37.04 4.81 5.56 0.61 Arachnida Acariformes Hydrachnidia 14.81 1.92 0.00 0.00 Insecta Coleoptera Dytiscidae 25.93 3.37 44.44 4.88 Hydrophilidae 11.11 1.44 5.56 0.61 Gyrinidae 40.74 5.29 0.00 0.00 Diptera Ceratopogonidae 3.70 0.48 0.00 0.00 Chironomidae 359.26 46.63 838.89 92.07 Simuliidae 3.70 0.48 0.00 0.00 Ephemeroptera Baetidae 3.70 0.48 0.00 0.00 Leptophlebiidae 3.70 0.48 0.00 0.00 Hemiptera Belostomatidae 0.00 0.00 5.56 0.61 Notonectidae 0.00 0.00 5.56 0.61 Odonata Aeschnidae 29.63 3.85 5.56 0.61 Coenagrionidae 118.52 15.38 0.00 0.00 Protoneuridae 3.70 0.48 0.00 0.00 Total density (ind/m²) 770.37 911.11 Total number of families 14 7 Table 3. Diet of Xenopus laevis determined via stomach content analysis. Ni = number of prey, %N = percentage of total prey, Vi = volume of prey (mm3), %Vi = percentage of total volume, Oi = number of animals that consumed this prey and %RII = relative importance index, in percentages. The three highest RII values are shown in bold. Order Family Ni %N Vi %V Oi % RII Odonata Coenagrionidae 6 14.63 77.35 21.54 4 19.06 Diptera Chironomidae 1 2.44 0.09 0.02 1 0.32 Coleoptera Dytiscidae 5 12.20 73.11 20.36 4 17.16 Hydrophilidae 3 7.32 45.84 12.76 3 7.94 Ephemeroptera Baetidae 3 7.32 0.27 0.07 2 1.95 Gastropoda Physidae 21 51.22 60.16 16.75 5 44.78 Fish C. pisciculus 2 4.88 102.36 28.50 2 8.80 Total 41 100.00 359.17 100.00 10 100.00 Trophic role of Xenopus laevis from a Mediterranean stream 502 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 0.79‰2, SEAc C. pisciculus = 4.49‰2, SEAc X. laevis = 6.10‰2), and position in the δ 13C-δ 15N biplot (Fig. 2). The only overlap seen between vertebrate species was between C. decenmaculatus and C. pisciculus at 4.61%. The probability that C. decenmaculatus had lower SEAb than B. microlepidotus was 42% and was 99% for C. pisciculus and X. laevis, respectively. The likelihood that B. microlepidotus had lower SEAb than C. pisciculus and X. laevis was 99%; the probability that C. pisciculus had lower SEAb than X. laevis was 73%. Despite the similarity in Figure 1. Stable isotope signatures of δ13C and δ15N of the community of El Cobre stream in central Chile (mean ± SD). The figure shows the isotopic space of the aquatic community. Table 4. δ 13C and δ 15N values of food web components in El Cobre stream. SD = standard deviation. Trophic groups are H herbivores, D detritivores and C carnivores. Composite samples of invertebrates were used due to their small body mass (only soft tissues). Taxon Samples δ15Nδ13C DS N DS C Trophic position Trophic groups Macrophyte M. aquaticum 2 2.60 -31.64 0.07 3.97 0.7 H Invertebrates Baetidae 1 2.96 -28.47 0 0 1.0 H Physidae 1 4.24 -26.55 0 0 1.4 H Dytiscidae 2 5.83 -29.87 1.17 1.76 2.0 D Hydrophilidae 1 6.57 -32.45 0 0 2.4 D Coenagrionidae 2 8.45 -29.03 2.47 3.19 3.5 C Vertebrates X. laevis 8 11.85 -23.96 1.22 1.45 4.7 C C. decenmaculatus 8 12.17 -29.96 0.38 0.96 4.9 C C. pisciculus 8 13.08 -29.37 0.79 1.55 5.25 C B. microlepidotus 4 13.52 -16.36 0.24 1.58 5.5 C Trophic role of Xenopus laevis from a Mediterranean stream 503 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 carbon values between food resource groups (herbivores, detritivores and carnivores), the distinct nitrogen values between groups permitted the determination that the greatest dietary contribution of prey by X. laevis was detritivore invertebrates (62.36±2.96%), followed by carnivorous organisms (36.54±2.82) and herbivorous invertebrates (1.1±0.99) (Fig. 3). Biomagnification and bioaccumulation The concentrations of heavy metals varied among the taxa analyzed (Appendix 1: Table A1). All the metals present in the sediments of the El Cobre stream were above the ISQG and PEL reference norms. In contrast, the metal concentrations in water had values under the detection limits. As shown in Fig. 4, all the analyzed elements, except Zn, decreased as the trophic level of the organisms increased (except the Baetidae). Biomagnification was found for Cu and Zn in X. laevis (Appendix 1: Table A2). The increase in Cu was noted for the prey C. pisciculus, and in Zn for the Physidae and the odonate Coenagrionidae. Only Baetidae was near the threshold (≥1) of the bioaccumulation factor (Appendix 1: Table A3) for Zn. Discussion Invasive species may affect ecosystem functioning by modifying the trophic structure and dynamics (Sagouis et al. 2015). Understanding the trophic roles of species is necessary to evaluate their impact. This is highly relevant for X. laevis given that it is the Figure 2. Stable isotope composition from El Cobre stream. Solid lines enclose the standard ellipse areas (SEAc) containing c. 40% of the data, showing the core isotope niche of each species (3 fish, 1 frog). Dotted lines are the convex hull areas, which are the areas encompassed by each species in the δ 13C-δ 15N plot. Trophic role of Xenopus laevis from a Mediterranean stream 510 Gabriel Lobos et al. 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Nature 503: 517. https://doi.org/10.1038/nature12798 Trophic role of Xenopus laevis from a Mediterranean stream 511 Gabriel Lobos et al. (2025), Aquatic Invasions 20(4): 495–511, 10.3391/ai.2025.20.4.164197 Appendix 1 Table A1. Concentration of heavy metals in mg/K (except stream water which is in mg/L) in El Cobre stream (mean ± SD). Samples represent the n used for each analysis. Reference norms corresponded to the Interim Sediment Quality Guidelines (ISQG) and Probable effect levels (PEL). Samples Cu Mn As Zn Sediments 2 3024±530 2208±858 26±7.6 323±57 Stream water 2 <0.01 <0.01 <0.01 <0.01 Macrophytes 2 189±66 278±169 2.13±1.20 56.3±9.40 Physidae 1 261.55 176.89 2.61 26.36 Coenagrionidae 3 124±63 122±38 1.48±1.17 99±20.30 Baetidae 1 458.32 964.53 4.88 309.69 Dytiscidae 1 116.47 63.74 2.06 109.87 Hydrophilidae 1 160.54 102.49 2.50 153.70 Cheirodon pisciculus 2 19.8±3.30 30.9±5.70 1.7±0.20 228±112 Xenopus laevis 5 80.9±25.30 18.42±4.80 0.53±0.20 135.9±29 C. decenmaculatus 1 66.60 58.30 2.06 110 B. microlepidotus 1 39.88 28.50 1.46 155.80 ISQG 35.70 500 5.90 123 PEL 197 17 315 Table A2. Biomagnification factor BMF of heavy metals in Xenopus laevis in relation to its prey. Significant biomagnification is highlighted in bold. BMF Cu Mn As Zn Physidae 0.11 0.04 0.07 1.84 Coenagrionidae 0.50 0.12 0.27 1.05 Baetidae 0.14 0.01 0.08 0.34 Dytiscidae 0.32 0.13 0.12 0.57 Hydrophilidae 0.23 0.08 0.10 0.41 Cheirodon pisciculus 4.50 0.66 0.34 0.66 Table A3. Bioaccumulation factor BAF in El Cobre stream community. BAF Cu Mn As Zn Physidae 0.09 0.08 0.10 0.08 Coenagrionidae 0.04 0.06 0.06 0.31 Baetidae 0.15 0.46 0.19 0.96 Dytiscidae 0.04 0.03 0.08 0.34 Hydrophilidae 0.05 0.05 0.10 0.47 Cheirodon pisciculus 0.01 0.01 0.07 0.70 Xenopus laevis 0.03 0.01 0.02 0.42 C. decenmaculatus 0.02 0.03 0.08 0.34 B. microlepidotus 0.01 0.01 0.06 0.48