TROPHIC INFERENCE IN TWO SYMPATRIC SHARKS , SPHYRNA LEWINI AND CARCHARHINUS FALCIFORMIS ( ELASMOBRANCHII : CARCHARHINIFORMES ) , BASED ON STABLE ISOTOPE ANALYSIS AT MALPELO ISLAND , COLOMBIA

Background. Elasmobranchs can play important roles in marine communities. But, relatively little is known about their diet, and movement. Sphyrna lewini (Griffith et Smith, 1834) consumes fishes, cephalopods, rays, and crustaceans. Carcharhinus falciformis (Müller et Henle, 1839) feed on fishes, cephalopods, crustaceans and sea turtles. To date, there are no studies available on the trophic ecology of sharks in Malpelo Island. The aim of this study was to describe the trophic ecology of S. lewini and C. falciformis, using stable isotope analysis of δ13C and δ15N, to better understand the role of both shark species in the Malpelo Island ecosystem. Material and methods. In January, February, and November 2013, specimens of Sphyrna lewini and Carcharhinus falciformis illegally caught at Malpelo Island were confiscated at the port of Buenaventura, Colombia. For each shark specimen, total length and sex were registered. Samples of muscle tissue were taken from the nape of all specimens. Each muscle sample was lyophilized for 24 h and analysed with lipid and urea extraction and without extraction. For each shark specimen, a subsample of ~1.0 mg was used for isotopic analysis. Results. A total of 14 Sphyrna lewini (Griffith et Smith, 1834) and 12 Carcharhinus falciformis (Müller et Henle, 1839) were analysed. δ13C values were similar between S. lewini (–16.3 ± 0.1‰) and C. falciformis (–16.5 ± 0.1‰). Sphyrna lewini showed a wider trophic niche than C. falciformis, with low trophic overlap (5%) between the two species. The δ15N values of S. lewini (15.9 ± 0.11‰) were higher than those of C. falciformis (14.9 ± 0.09‰). In C. falciformis, δ13C values were similar in both sexes (–16.5 ± 0.1‰), while δ15N values were significantly different between males (14.6 ± 0.1‰) and females (15.0 ± 0.1‰). The trophic position of S. lewini was 5.25 ± 0.12, and that of C. falciformis, 5.48 ± 0.18, which suggests that both shark species occupy a high position in the marine food chain. Conclusion. Both shark species co-occur at Malpelo Island, but they do not share food resources and feeding areas, and they probably feed far from the island, using it as a resting and cleaning area. This indicates the need for more research to increase biological and ecological knowledge of both species, particularly within marine protected areas and their influence areas throughout the Colombian Pacific.


INTRODUCTION
Elasmobranchs can play important roles in marine communities, occupying a wide range of habitats as apex predators.However, relatively little is known about their abundance, diet, and movement (Baum et al. 2003, 2005, Heithaus et al. 2008, Hussey et al. 2012).Many shark species are in decline, mainly due to overfishing, bycatch, pollution, and habitat degradation (Baum et al. 2003, 2005, Dulvy et al. 2008).
Knowledge of diet, trophic position, movement patterns and habitat use of species have been recognized as critical factors in implementing successful conservation and management strategies of species and the environment (Hussey et al. 2012).The diets and trophic interactions of elasmobranchs are sometimes difficult to determine using traditional methods alone (e.g., gut content analysis, direct observations, tagging), however, a complementary technique is stable isotope analysis (SIA).SIA is an important, effective tool that can be applied to elasmobranch conservation research (Kim et al. 2012, Shiffman et al. 2012).This technique is based on the premise that heavy isotopes of an element are preferentially retained (e.g., nitrogen isotopes via protein amination/deamination, carbon isotopes via respiration) and that specific ratios of heavy to light isotopes are indicative of specific resource use (e.g., diet and habitat) (Wolf et al. 2009) and trophic position (Post 2002).This biogeochemical method allows quantitative analysis of dietary composition and foraging patterns over a range of spatial and temporal scales (MacNeil et al. 2005), as well as the examination of more complex questions pertaining to community dynamics, feeding strategies or diet, trophic position, and movement in aquatic organisms (Hussey et al. 2012).
This is the first study of the trophic ecology of the sharks, Sphyrna lewini and Carcharhinus falciformis, in an area of sympatry, Malpelo Island.Malpelo Island is a marine reserve located ~490 km off the coast of Buenaventura, Colombia (Fig. 1).The species under study are the most abundant sharks at Malpelo Island.Thus, the aim of this study was to describe the trophic ecology of these two shark species, using stable isotope analysis of carbon (δ 13 C) and nitrogen (δ 15 N), to better understand the role of both shark species in the Malpelo Island ecosystem.

MATERIAL AND METHODS
In January, February, and November 2013, a total of 14 Sphyrna lewini and 12 Carcharhinus falciformis illegally caught at Malpelo Island were confiscated at the port of Buenaventura, Colombia.For each shark specimen, total length (TL, in cm) was measured and sex was determined.Samples of muscle tissue were taken from the nape of all specimens, stored in plastic bags, and frozen for transport to the laboratory.
Each muscle sample was lyophilized for 24 h and analysed with lipid and urea extraction (lipid-free) and without extraction (Bulk).Lipid and urea extraction was performed following the procedure described by Kim and Koch (2012).After drying, each sample was homogenized using an agate mortar to produce a very fine powder.For each shark specimen, a subsample of 0.3-1.0mg of powder was obtained and loaded into a tin capsule for isotopic analysis.To know if lipid extraction was necessary, the C ÷ N ratios of both sets of samples (Free-lipid and Bulk) were measured.A C ÷ N mass ratio < 3.5 indicated there was no effect of lipid content (Post et al. 2007).
Isotope ratios were measured using a Thermo Scientific™ Delta V Plus™ isotope-ratio mass spectrometer (IRMS), with ConFlo IV interface and a Costech™ elemental analyser, in the chemistry laboratory of the Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional (CICIMAR-IPN) in La Paz, Mexico.Isotope ratios are presented using δ notation: where X is the element, h is the high mass number, R sample is the heavy-to-light isotope ratio, and R standard is Vienna Pee Dee Belemnite for carbon and AIR for nitrogen.Units are parts per thousand (‰).The precision of the method was ± 0.3‰ and ± 0.1‰ for C and N stable isotopes, respectively.
The trophic position (TP) was calculated using the equation proposed by Post (2002): where λ is the TP of the base (i.e., the most important prey species for each predator); δ 15 N predator and δ 15 N base are the isotopic signatures of the predator and the base, respectively; and Δ n is the trophic discrimination factor (TDF), 3.7 ± 0.4‰ (Kim et al. 2012) Isotopic niche breadth and trophic overlap between species and sexes were estimated using SIBER (Stable Isotope Bayesian Ellipses in R) (Jackson et al. 2011) from the SIAR package (Stable Isotope Analysis in R) (Parnell and Jackson 2013).This analysis uses measurements based on ellipses calculated by a covariance matrix that defines their shape and area (Jackson et al. 2011) to show the trophic niche breadth (standard ellipse corrected area, SEA c ).With this method it is possible to obtain the overlap between ellipses, where values close to 1 represent high trophic overlap (Jackson et al. 2011).
The normality and homoscedasticity of isotope data were tested using the Shapiro-Wilk and Levene's test, respectively.The parametric Student's t-test and the nonparametric Wilcoxon signed-rank test were used to determine statistical differences in C ÷ N ratio among shark species and sexes.All tests were performed using a significance level (α) of 0.05.Statistical analyses and figures were obtained using the R statistical package (R Core Team 2014).
For Sphyrna lewini, the C ÷ N ratios of the bulk and lipid-free samples ranged from 2.71 to 3.19 and from 2.99 to 3.19, respectively.For Carcharhinus falciformis, the C ÷ N ratio ranged from 2.81 to 3.10 (bulk samples) and from 2.81 to 3.07 (lipid-free samples).We found significant statistical differences in C ÷ N ratio between bulk and lipid-free samples (Table 1), likely an effect of the lipid extraction process (Hussey et al. 2012).However, since C ÷ N values were lower than 3.5 (Post et al. 2007), all the analyses were performed using the δ 13 C and δ 15 N values of bulk samples.
The TP estimated for Sphyrna lewini was in the range of 3.83-5.94,and that of Carcharhinus falciformis was in the range of 5.10-5.81.For C. falciformis, the estimated TP was between 5.36 and 5.65 (mean ± SE = 5.51 ± 0.03) for males, and between 5.31 and 5.81 (5.63 ± 0.04) for females.The relative TP of S. lewini and C. falciformis suggests that both species occupy a high position in the marine food chain (Table 3).There was little isotopic overlap between Sphyrna lewini and Carcharhinus falciformis (5%; Fig. 3), and the isotopic niche of C. falciformis (SEA c = 0.20, n = 12) was narrower than that of S. lewini (SEA c = 0.64, n = 14; Fig. 3).
The silky shark, Carcharhinus falciformis, feeds mainly on teleosts but also on cephalopods and crustaceans (Cabrera-Chávez et al. 2010, Duffy et al. 2015, Estupiñán-Montaño et al. 2017) and, occasionally, on sea turtles (Estupiñán-Montaño et al. 2017, Acevedo unpublished * ).This species is considered to be a selective predator in the ETP, with a preference for the scombrid genus Thunnus (see Duffy et al. 2015, Estupiñán-Montaño et al. 2017), which normally inhabits oceanic zones and forms schools near seamounts.Previous results are in line with those of the present study, as the depleted 13 C values obtained here (Tables 1 and 2) are typical of oceanic areas.The narrow isotopic niche observed in this study suggests that C. falciformis has a reduced habitat use, with narrow depth (0 to 85 m) (Kohin et al. 2006, Filmalter et al. 2010) and temperature range (26 to 30ºC) (Kohin et al. 2006).
The enriched values of δ 15 N in its muscle tissue suggest that Sphyrna lewini spans primary to tertiary consumer roles (TP Range: 3.4-6.6)(Hussey et al. 2015) (Table 3), and that Carcharhinus falciformis has a tertiary consumer role (Hussey et al. 2015) (Table 3), occupying high trophic levels in the marine ecosystem of Malpelo Island and the ETP.These results agree with other studies that have estimated a high trophic position for sharks (Cortés 1999, Bornatowski et al. 2014, Li et al. 2014, Hussey et al. 2015, Estupiñán-Montaño et al. 2017).
Our results suggest that Sphyrna lewini and Carcharhinus falciformis co-occur around of Malpelo Island, with resource partitioning, similar to other reports for sympatric species (Bethea et al. 2004, Papastamatiou et al. 2006, Vaudo and Heithaus 2011).Our results further support the theoretical prediction that the diets of cooccurring species of sharks differ and that sharks with very similar diets do not co-occur (Papastamatiou et al. 2006).
These sympatric sharks co-occur in Malpelo Island, but they do not share their isotopic niche in a significant way.This result may be caused by: (1) the trophic specialization of both species, which reduces interspecific competition; or (2) different habitat use, with Sphyrna lewini showing greater horizontal and vertical movements (Bessudo et al. 2011a(Bessudo et al. , 2011b)), and Carcharhinus falciformis being more limited in its movements by its physiological characteristics, which restrict it to narrower niches (Kohin et al. 2006, Filmalter et al. 2010).In conclusion, this study suggests that Sphyrna lewini and Carcharhinus falciformis do not feed around Malpelo Island, but they may use Malpelo Island as a resting and cleaning area (Klimley andNelson 1984, Klimley et al. 1993).There is a need for more research to increase biological and ecological knowledge of both species, which will allow us to understand their role, particularly within marine protected areas throughout the Eastern Pacific Ocean.This study contributes to the knowledge of the trophic ecology of S. lewini and C. falciformis in the largest marine protected area of the Colombian Pacific, the Malpelo Fauna and Flora Sanctuary (Malpelo FFS), and the Eastern Tropical Pacific (ETP).This information helps to understand the role of the two most abundant shark species at Malpelo Island.Our results will help to develop management and conservation plans for these sympatric species in this marine protected area.

Table 1 δ 13
C and δ 15 N values, isotopic range and C ÷ N ratio of Sphyrna lewini and Carcharhinus falciformis samples, with (bulk) and without (lipid-free) lipid and urea extraction Values are mean ± standard error of the mean (SE).

Table 2 δ
13C and δ 15 N values, isotopes range and estimated trophic position (TP) of Sphyrna lewini and Carcharhinus falciformis at Malpelo Island, Colombia ND = not determined, SE = standard error of the mean.

Table 3
Trophic position estimated for Sphyrna lewini and Carcharhinus falciformis, based on important prey species (mixing model and stomach contents studies)