The striped marlin Kajikia audax (Philippi, 1887), the skipjack tuna Katsuwonus pelamis (Linnaeus, 1758) and the yellowfin tuna Thunnus albacares (Bonnaterre, 1788) are pelagic fishes widely distributed in the oceans (Smith, Brown, 2002) and are the most important fishing sources for local and international fishing fleets in Ecuador (Schaefer et al., 2009; Martinez-Ortiz et al., 2015; Tanabe et al., 2017). The capture of these species has an economic value of approximately 73 million US dollars per year for Ecuador (Martínez-Ortiz et al., 2015). These economic gains have promoted the study and development of fisheries, mainly for T. albacares (Martinez-Ortiz et al., 2015). For K. audax, K. pelamis and the small tunas Auxis spp., there is a lack of biological and ecological knowledge for Ecuador. Hence, it is necessary to assess the trophic web to detect shifts or impacts in the ecosystem resulting from the extraction of these species and to establish relationships or differences in their trophic strategies.

The pelagic fishes K. audax, T. albacares, K. pelamis and Auxis spp. are important components in the ecosystem and facilitate energy transfer between low and top trophic levels because they are preyed on by sharks, fishes, seabirds, and marine mammals (Wang et al., 2003; Arizmendi-Rodríguez et al., 2006; Galván-Magaña et al., 2013; Rosas-Luis et al., 2016; Diop et al., 2018). They are also active predators of fishes, cephalopods, and crustaceans (Alverson, 1963; Loor-Andrade et al., 2017; Rosas-Luis et al., 2017; Varela et al., 2017). In addition, these species are efficient transfers of biomass to other areas and water depths since they are fast-moving species that perform horizontal and vertical movements (Holland et al., 1990).

The study of the trophic ecology of sympatric species in marine environments is achieved by using traditional stomach content analysis, and more recently the analysis of stable isotopes of carbon (denoted as δ¹³C) and nitrogen (denoted as δ¹⁵N) (Peterson, Fry, 1987). Stable isotope analysis allows the characterization of migratory movements (Wunder, 2012; Segers, Broders, 2015) and is useful for obtaining information about sympatric species (Vanderklift et al., 2006; Cabanillas-Terán et al., 2016). δ¹⁵N is an indicator of a consumer’s trophic position, as the value in consumer tissues becomes higher compared to their prey (McCutchan et al., 2003; Vanderklift, Ponsard, 2003). δ¹³C values can indicate primary sources in a trophic network (McCutchan et al., 2003). In marine environments, δ¹³C values indicate the inshore/pelagic versus offshore/benthic contribution to food intake, indicating areas with low and high primary production respectively (Hobson et al., 1994; Cherel, Hobson, 2007; Navarro et al., 2013). The stable isotopes of δ¹⁵N and δ¹³C have been used to study the feeding behavior of large pelagic fishes in the central and north Pacific Ocean (Graham et al., 2007; Acosta-Pachón et al., 2015; Li et al., 2016; Young et al., 2018). In Ecuadorian waters, isotope values showed that sympatric species, such as the billfish Istiophorus platypterus (Shaw, 1792), the blue marlin Makaira nigricans Lacepède, 1802, and the swordfish Xiphias gladius Linnaeus, 1758, do not compet for food sources (Rosas-Luis et al., 2017). The isotope analysis and stomach contents demonstrated that X. gladius consumed prey from deeper waters, while I. platypterus and M. nigricans fed mainly in upper waters (Rosas-Luis et al., 2017). For Thunnus albacares, Varela et al., (2017) found that stable isotope ellipses had no overlap among size classes and suggested that the prey size increases as the tuna grow. These studies allowed the understanding of the food web; however, it is necessary to include K. audax, K. pelamis, and Auxis spp. in the analysis, to better explain the trophic structure of the ecosystem.

Pelagic fishes are important and abundant components in the marine ecosystem of Ecuador (Martínez-Ortiz et al., 2015), but there is a lack of knowledge related to the trophic role of the sympatric species K. audax, K. pelamis, and Auxis spp. Therefore, this study represents an effort to identify the trophic relationships that these species have in the marine ecosystem off the coast of Ecuador, with a main objective to compare the δ¹⁵N and δ¹³C values of each species found in their muscle tissue. Moreover, we aim to explore the hypothesis that medium-sized pelagic fish species have higher δ¹⁵N values than those of the prey they consumed, and that the prey consumed are different for each predator. Our results represent the first attempt to study tissue samples of the fishes K. audax, K. pelamis, and Auxis spp. collected in the fishing ports of Ecuador and analyzed by isotopic analysis.

Study area. The marine environment off the coast of Ecuador is characterized by warm waters coming from the Equatorial Current System, with the influence of cold waters from the Humboldt Current System (Bendix, Bendix, 2006). High primary production areas are promoted by the convergence of the two current systems off the coast of Ecuador (Bendix, Bendix, 2006; Rincón-Martínez et al., 2010). Fisheries in Ecuador are characterized by two main groups the longline fishery, targeting large and medium pelagic fishes, and a fishery that uses gillnets to capture cephalopods and other fishes (Martínez-Ortiz et al., 2015). The longline fishery works in areas between 37 and 130 km off the Ecuadorian coast in the pelagic environment of oceanic waters and the gillnets from the shore to 130 km off the Ecuadorian coast (Rosas-Luis et al., 2017).

Samples. Kajikia audax, T. albacares, K. pelamis and the group Auxis spp. were collected from catches brought to the fishing ports of Playita Mía, Manta, Ecuador and Santa Rosa, Salinas, Ecuador during June 2014 and May 2015 (Fig. 1). The total body length (TL) was recorded to the nearest 10 mm. Auxis spp. grouped the frigate tuna, Auxis thazard (Lacepède, 1800), and the bullet tuna, Auxis rochei (Risso, 1810) since separation by morphological characteristics was not possible. Additionally, the Patagonian squid Doryteuthis gahi (d’Orbigny, 1835) and the dart squid Lolliguncula diomedae (Hoyle, 1904) were collected in the same fishing ports. A small portion of the dorsal muscle of the caudal peduncle of fishes and the mantle of squids was extracted and stored at -20°C in the laboratory of trophic ecology at the Universidad Laica Eloy Alfaro de Manabí until lipid extraction and isotopic procedures. Furthermore, prey items, collected from the stomach contents of predators reported by Rosas-Luis et al., (2017), were taken to characterize their values. Samples included complete individuals of the Peruvian anchovy Engraulis ringens Jenyns, 1842, the Peruvian hake Merluccius gayi (Guichenot, 1848), the Reinhardt’s cranch squid Liochranchia reinhardti (Steenstrup, 1856), and the pelagic octopod Japetella sp. (Tab. 1).

FIGURE 1 |

Lipid extraction and isotopic analysis. To avoid biases in the δ¹³C values, lipid extraction was applied to all tissue samples (Post et al., 2007). Lipids were extracted from all muscle samples with chloroform and methanol following the protocol of Bligh, Dyer, (1959). All samples were then freeze-dried and powdered, with 0.3 to 0.4 mg of each sample packed into tin capsules. Isotopic analyses were performed at the Estación Biológica de Doñana, Spain. Samples were combusted at 1,020°C using a continuous flow isotope-ratio mass spectrometer (Thermo Electron) by means of a Flash HT Plus elemental analyzer interfaced with a Delta V Advantage mass spectrometer. Stable isotope ratios were expressed in the standard δ-notation (‰) relative to Vienna Pee Dee Belemnite (δ¹³C) and atmospheric N₂ (δ¹⁵N). Based on laboratory standards, the measurement error was ±0.1‰ and ±0.2‰ for δ¹³C and δ¹⁵N, respectively. The standards used were EBD-23 (cow horn, internal standard), LIE-BB (whale baleen, internal standard) and LIE-PA (razorbill feathers, internal standard). These laboratory standards were previously calibrated with international standards supplied by the International Atomic Energy Agency.

Diet. The diet of K. audax was taken from Loor-Andrade et al., (2017) and for T. albacares, diet information was based on Varela et al., (2017). These previous reports used samples from the same area and the same sampling time of this work (Tab. 2). Unfortunately, there were no reports on the feeding habits of Auxis spp. and K. pelamis in the study area; thus, for increased clarity, fish diets from outside the study area were used for K. pelamis, based on Tanabe, (2001) (Tropical Western Pacific Ocean), and for Auxis spp. based on Siraimeetan, (1985) (Tuticorin coast, Gulf of Mannar).

Trophic width. As a measure of trophic width (Jackson et al., 2011), we calculated the corrected standard ellipse area (SEAc) for K. audax, T. albacares, K. pelamis, and Auxis spp. This metric represents a measure of the total amount of isotopic niche exploited by a predator and is thus a proxy for the extent of the trophic niche exploited by the studied species (high values of SEAc indicate high trophic width) (Jackson et al., 2011). The corrected standard ellipse area (SEAc) based on the Bayesian ellipse area was proposed as an unbiased metric with respect to the sample size, particularly for the Bayesian method, which incorporates a robust comparison considering uncertainty with smaller sample sizes, resulting in larger ellipse areas. The SEAc was calculated by a covariance matrix of the samples. The sample variance provides an unbiased estimate of the population variance for data x and y, that defines their shape and area (Jackson et al., 2011). The SEAc was fitted using R 3.1.0 for Windows (R Development Core Team, 2017). Isotopic standard ellipse areas were calculated using the SIBER package (Jackson et al., 2011) included in the SIAR library, with R 3.1.0 for Windows (R Development Core Team, 2017). The Niche Overlap Metric was calculated as the probability that an individual from the predator species will be found within the niche of the other predator species with an alpha=0.95 using the nicheROVER routine in R (Swanson et al., 2015).

C and N contributions. Kajikia audax, T. albacares, K. pelamis, and the Auxis spp. were used as single species in the isotopic analysis because the number of tissue samples was greater than 7 for each species (Tab. 1). A sample number greater than 7 is considered adequate for posteriori statistical analysis (Jackson et al., 2011).

The Stable Isotope Analysis in R (SIAR) was used to calculate the proportion of δ¹³C and δ¹⁵N isotopes in the diets of the predators (Parnell et al., 2010). The prey species of K. audax and T. albacares were mixed into composite groups to obtain a high number of isotope values despite their different body size (Tab. 1). Groups were established considering that they were consumed by predators (according to previous reports for the area, Tab. 2). The fish group for K. audax and T. albacares was composed of K. pelamis, Auxis spp., O. libertate, Scomber japonicus Houttuyn, 1782, E. ringens, L. lagocephalus, M. gayi, and P. serrula. The cephalopod group for K. audax and T. albacares was composed of L. reinhardti, Japetella sp., Dosidicus gigas, T. rhombus, and A. lesueurii. Unfortunately, we had no muscle tissue of prey consumed by K. pelamis and Auxis spp., thus the contribution of δ¹³C and δ¹⁵N for these species was not calculated. The Trophic Discrimination Factor (TDF) used was 1.9 ± 0.4 for δ¹⁵N and 1.8 ± 0.3 for δ¹³C related to Pacific bluefin tuna (Madigan et al., 2012), which were the most appropriate for the species in this work.

Statistical analysis. Size, δ¹³C and δ¹⁵N differences between species were tested using one-way ANOVA tests, and significant differences (p≤ 0.05) between pairs of species were identified with a post hoc Tukey test. All tests were performed in the IBM SPSS statistics software v.19 (IBM, 2010).

Isotope values. The mean δ¹³C values of K. audax, T. albacares. K. pelamis and Auxis spp. ranged between -17.9‰ and -16.6‰ (Tab. 1). The mean δ¹³C value of K. audax was -16.6‰, higher than those of T. albacares, K. pelamis, and Auxis spp. (F_3,59 = 25.69, p < 0.05). T. albacares and K. pelamis had similar δ¹³C values (Tabs. 1–3). The mean δ¹⁵N values ranged between 11.1‰ and 14.9‰ (Tab. 1). Significant differences in the δ¹⁵N values were found between species (F_3,59 = 35.73, p < 0.05) (Tabs. 1–3) with K. audax showing the highest values. The post hoc Tukey test showed similar δ¹⁵N values for K. pelamis and Auxis spp. (Tab. 3).

Trophic width. The broadest SEAc was observed for K. pelamis (1.6), followed by Auxis spp. (1.4) (Fig. 2). Narrow SEAcS were recorded for K. audax (0.51) and T. albacares (0.7). A high overlap probability was found between T. albacares and K. pelamis (71.8%) (Tab. 3; Fig. 2). Moderate overlap probability was found among K. audax, T. albacares and K. pelamis, T. albacares and Auxis spp., and K. pelamis and Auxis spp. (Tab. 3; Fig. 2). Low overlap probability was found between K. audax and Auxis spp. (0.4%) (Tab. 3; Fig. 2).

FIGURE 2 |

δ13C and δ15N contribution of prey groups in the diet. The results of the SIAR analysis showed that fishes were the most important δ¹³C and δ¹⁵N contributors (up to 87%) for T. albacares, while cephalopods were the most important contributors for K. audax (up to 53%) (Fig. 3). The summary of the diet reports based on stomach contents indicated that fishes represent 95.8% of the diet of T. albacares and 83.9% of the diet of K. audax, with cephalopods being the second most represented group, but significantly less important (4.2 and 16.1%, respectively; Fig. 3).

FIGURE 3 |

In this study, isotope analysis allowed the identification of the trophic width and overlap between Auxis spp., K. pelamis, T. albacares, and K. audax. The ellipse metrics provided quantitative and integrated information about sources and niche breadth (Boecklen et al., 2011), contributing to the ecological knowledge of pelagic and commercial species in the marine ecosystem of Ecuador. The δ¹³C values suggest that K. audax has a different trophic strategy, probably consuming prey from a trophic chain based in high productivity areas, while Auxis spp. may be moving to low productivity areas and consuming different prey sources. T. albacares and K. pelamis had similar δ¹³C values, thus indicating that they coexist in the same areas. Based on these results, the discussion is focused on explaining the trophic strategy and interactions of these sympatric species.

The highest δ¹³C values were recorded for K. audax, followed by T. albacares and K. pelamis. On the one hand, the highest δ¹³C values were related to high productivity ocean areas (France, Peters, 1997; Ménard et al., 2007; Carlisle et al., 2014), coinciding with marine areas where fishing activity in Ecuador occurs (Martinez-Ortiz et al., 2015). These species have been described as fishery sources with high abundances in waters where upwelling events favor the enrichment of primary production, such as the Humboldt Current and the Gulf of California (Stock et al., 2017). For the Ecuadorian waters, these species are usually found in catches close to the coast (Martinez-Ortiz et al., 2015). On the other hand, low values of δ¹³C in Auxis spp., compared to those of the other species, could be the result of feeding habits related to pelagic and open waters, affecting the signal in the muscle samples. Auxis spp. and other scombrid fishes, including T. albacares and K. pelamis, are fast-moving species in interior as well as more distant coastal waters (Holland et al., 1990; Schaefer et al., 2009), resulting in a different feeding strategy consuming small pelagic fishes, such as S. japonicus and myctophids, and pelagic crustaceans (Varela et al., 2017), that could be available in and outside of the studied area. The trophic width as indicated by the SEAc showed that the fishes Auxis spp. and K. pelamis had broader isotopic ellipse areas, and that K. audax had the narrowest area. The consumption of similar prey by predators was confirmed by the isotope values and niche overlap probability between Auxis spp. and K. pelamis (46%). Nevertheless, it is necessary to identify the potential prey of these species. If they are voracious and active predators, the results will show a wide range of prey as observed in squids of similar size (Rosas-Luis et al., 2014).

The highest δ¹⁵N values were recorded for K. audax and T. albacares, and the lowest were found in K. pelamis and Auxis spp. These values agree with the assumption that the increase in δ¹⁵N values results from prey ingestion, because the type and size of prey consumed affect the δ¹⁵N values in the predator tissue. The consumption of large prey increases the δ¹⁵N values (Post, 2002; Hussey et al., 2014). The largest predator in this study was K. audax, which consumes large prey such as K. pelamis and Auxis spp. (Loor-Andrade et al., 2017). The δ¹⁵N values allowed the comparison of K. audax with top predators and T. albacares with mid-level predators, which corresponds to the trophic position calculated for T. albacares in Ecuadorian waters (Varela et al., 2017) and for K. audax in the north Pacific Ocean (Torres-Rojas et al., 2013). The lowest level position was found in K. pelamis and Auxis spp.

As top predators, K. audax and T. albacares segregate from the other species, as suggested by the stomach contents and isotope results. They share food resources, with K. audax feeding mainly on Auxis spp. and other fishes and cephalopods, and T. albacares feeding mainly on Auxis spp. (Varela et al., 2017). For T. albacares and K. pelamis, a higher overlap probability was recorded; thus, it can be suggested that these species also share food resources in Ecuadorian waters. However, a comparison of the δ¹⁵N values indicates that T. albacares had higher δ¹⁵N values than K. pelamis. Thus, they could consume prey that are located in the same area, but of different sizes (large prey for T. albacares), as was reported for these species in the Gulf of California (Alatorre-Ramírez et al., 2017). The isotope and stomach content results are complementary because the isotope values support the evidence of prey contribution, taking into account the turnover rate of muscle tissue (several months) (Madigan et al., 2012; Vander-Zanden et al., 2015) and the estimated diet with stomach content identification, hours or days depending on the prey tissue (Olson, Boggs, 1986; Acosta-Pachón, Ortega-García, 2019). Thus, these results highlight the importance of Auxis spp. in the diets of T. albacares and K. audax, and cephalopods in the diet of K. audax. In Ecuadorian waters, K. audax and T. albacares seem to be opportunistic predators that feed on available and abundant species. The fishes Auxis spp. could be abundant in the area because they have been reported as components in the diet of top predators (Loor-Andrade et al., 2017; Rosas-Luis et al., 2017; Varela et al., 2017). More descriptions of the feeding habits of marine species are needed to corroborate the trophic relationships in the ecosystem, and other species such as Auxis spp. and K. pelamis should be included in the analysis.

In conclusion, our results suggest that the δ¹³C values of T. albacares and K. pelamis overlap, indicating that they share similar foraging areas or a similar trophic strategy. Their δ¹⁵N values allowed the categorization of the food web; the highest position in the food web was occupied for the large species K. audax and middle trophic positions for T. albacares, K. pelamis, and Auxis spp. confirming the hypothesis that medium-sized pelagic fish species accumulate δ¹⁵N isotopes according to the size of prey consumed (large predators consumed larger prey than medium-sized predators). In addition, the different predator size allows the use of the same habitat by partitioning in the prey consumed by each predator. Considering these results and the fact that the fishes K. audax, T. albacares and K. pelamis are important for fisheries in Ecuador, it is necessary to identify the impact that fisheries have on natural populations. The SEAc of Auxis spp. could be related to our analysis of two species as a single group, which likely do not have similar feeding habits. Unfortunately, it was not possible to segregate the two species during the morphological identification, and no stomach content samples were taken. Thus, future research will require the use of genetic and morphological methods to separate the two species and to continue trophic ecology studies of all species caught in fisheries to better understand the food web of the marine ecosystem.