Submitted 29 January 2018Accepted 1 May 2018Published 25 May 2018

Corresponding authorDiego Paacuteez-Rosas dpaezusfqeduec

Academic editorD Ross Robertson

Additional Information andDeclarations can be found onpage 14

DOI 107717peerj4818

Copyright2018 Paacuteez-Rosas et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Feeding behavior and trophic interactionof three shark species in the GalapagosMarine ReserveDiego Paacuteez-Rosas12 Paul Insuasti-Zarate3 Marjorie Riofriacuteo-Lazo14 andFelipe Galvaacuten-Magantildea4

1Galapagos Science Center Universidad San Francisco de Quito Galaacutepagos Ecuador2Unidad Teacutecnica San Cristoacutebal Direccioacuten del Parque Nacional Galaacutepagos Galaacutepagos Ecuador3Programa de Maestriacutea en Manejo Sustentable de Biorecursos y Medio Ambiente Universidad de GuayaquilGuayaquil Ecuador

4Centro Interdisciplinario de Ciencias Marinas Instituto Politeacutecnico Nacional La Paz Meacutexico

ABSTRACTThere is great concern about the future of sharks in Ecuador because of the lack ofbiological knowledge of most species that inhabit the region This paper analyzesthe feeding behavior of the pelagic thresher shark (Alopias pelagicus) the blue shark(Prionace glauca) and the silky shark (Carcharhinus falciformis) through the use of stableisotopes of carbon andnitrogen (δ13Cand δ15N)with the aimof determining the degreeof interaction between these species in the Galapagos Marine Reserve No interspecificdifferences were found in use of oceanic vs inshore feeding areas (δ13C KruskalndashWallistest p = 009) The position in the hierarchy of the food web where A pelagicus feedsdiffered from that of the other species (δ15N KruskalndashWallis test p = 001) Therewere no significant differences in δ13C and δ15N values between males and femalesof the three species (Studentrsquos t -test pgt 005) which suggests that both sexes have asimilar feeding behavior A specialist strategy was observed in P glauca (trophic nichebreadth TNB = 069) while the other species were found to be generalist (A pelagicusTNB = 150 and C falciformis TNB = 109) The estimated trophic level (TL) variedbetween the three species C falciformis occupied the highest trophic level (TL = 44)making it a quaternary predator in the region The results of this study coincide with theidentified behavior in these predators in other areas of the tropical Pacific (Colombiaand Mexico) and suggest a pelagic foraging strategy with differential consumption ofprey between the three species These ecological aspects can provide timely informationwhen implementing in conservation measures for these shark species in the TropicalPacific and Galapagos Marine Reserve

Subjects Aquaculture Fisheries and Fish Science Biodiversity Conservation Biology MarineBiologyKeywords Feeding behavior Stable isotopes Trophic niche breadth Galapagos islands Sharks

INTRODUCTIONGlobal increase in fishing effort has led to a decline of nearly 90 inoceanic fish populationswith elasmobranchs being one of the most affected groups (Stevens et al 2000 Myers ampWorm 2003) Of the worldrsquos 400 shark species 40 are found in Ecuadorian waters and 30are caught in both commercial and artisanal fisheries (Jacquet et al 2008Martiacutenez-Ortiacutez et

How to cite this article Paacuteez-Rosas et al (2018) Feeding behavior and trophic interaction of three shark species in the Galapagos MarineReserve PeerJ 6e4818 DOI 107717peerj4818

al 2011) The lack of biological knowledge supporting the regulation and conservation ofthese resources has led to several species of sharks being listed as endangered or vulnerableby the IUCN (Clarke et al 2006)

Most sharks are top predators controlling trophic relationships and energy flows withinthe ecosystems they inhabit (Myers et al 2007 Heithaus et al 2008) These predators aretypically considered as generalist consumers and many have adopted strategies to exploitpersistent and profitable resource regions (Au 1991 Compagno Dando amp Fowler 2005)However the trophic interactions of elasmobranchs are sometimes difficult to determineusing traditional methods (diet through stomach content or behavior using tagging anddirect observations) so the use of alternative techniques such as stable isotopes become anopportunity to infer from another perpestive the trophic ecology of these species

The analysis of stable isotopes of carbon and nitrogen (δ13C and δ15N) is based on thepremise that these natural chemical tracers are retained in the tissues of consumer allowingresearchers to identify energy flows and characterize the resources use (Newsome et al2007 Martiacutenez del Rio et al 2009) This biogeochemical method provides informationregarding general of habitat used by their prey coastaloceanic (δ13C) as well as thetrophic strategies of a species (δ15N) (Boecklen et al 2011 Kim et al 2012) So the useof the isotopic niche makes it possible to infer about the foraging patterns over a spatialrange (using δ13C) and the level and trophic breadth of a predator (using δ15N) (Boecklenet al 2011 Kim et al 2012) Differences in δ13C are determined by physicochemicaloceanographic and biological factors which influence the taxonomic composition ofphytoplankton concentration of dissolved CO2 in primary consumers (Goericke amp Fry1994 France 1995) and the influence of carbon derived from benthic macrophytes incoastal zones that are 13C enriched compared to phytoplankton in pelagic environments(Michener amp Schell 1994) On the basis of this application lower values of δ13C frompredator and its prey are expected in offshore environments For the nitrogen valuesthere is a strongly fractionated of δ15N values from prey to predator resulting in isotopicenrichment from one trophic level to the next (DeNiro amp Epstein 1981 Post 2002)

Polyspecific associations include different species that migrate interact and foragetogether for different periods of time It seems that one of the main reasons for formingsuch associations is the search for food (Au 1991) The pelagic thresher shark (Alopiaspelagicus) the blue shark (Prionace glauca) and the silky shark (Carcharhinus falciformis)are pelagic species that inhabit Equadorian waters and the tropical zones of several oceans(Markaida amp Sosa-Nishizaki 2010 Polo-Silva et al 2013 Duffy et al 2015) These speciesare mainly found in the epipelagic zone and are very active predators so their abundanceand distribution is determined by food availability (Compagno Dando amp Fowler 2005Rabehagasoa et al 2012 Klarian et al 2018) Several studies have shown that the pelagicthresher shark and blue sharks have a broad diet (eg cephalopods crustaceans pelagicand benthic fish) and their feeding behavior can vary depending on seasonal conditionsmarine productivity and abundance of resources (Polo-Silva et al 2013Hernaacutendez-Aguilaret al 2015 Klarian et al 2018) While the silky shark is considered a piscivorous predatorconsuming mainly fishes of the Scombridae family (Duffy et al 2015 Estupintildeaacuten Montantildeoet al 2017a)

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 221

The foraging success of sharks is linked to the diversity of their diet which places limitson their behavior and is a decisive factor in determining the feeding strategy of thesepredators (Yunkai et al 2014 Duffy et al 2015) Species of similar evolutionary origin aresusceptible to overlap in their trophic niches whichmay eventually lead to the displacementor extinction of one or more populations (Hardin 1960 Page Mckenzie amp Goldsworthy2005) For this reason reducing the level of competition over food resources becomesa determining factor in facilitating the coexistence of these species thus maintainingcommunity structure (Bolnick et al 2003 Pinaud amp Weimerskirch 2007) However at theinterspecific level the partition of resources is more frequent and occurs in response tohigh trophic competition allowing these populations to use different food resources toeffectively reduce competition and facilitate the survival of individuals (Bolnick et al 2003Cherel et al 2007 Paacuteez-Rosas et al 2012) In spite of this understanding the conditionsunder which varying degrees of resource portioning could affect sharks remains relativelypoorly understood

In Ecuador very little is known about the ecology population structure and demographyof sharks including in the Galapagos Islands an area that is a biodiversity hotspot whereseveral species of sharks and other pelagic fish congregate (Hearn et al 2010 Salinas deLeoacuten et al 2016) Therefore as protection and conservation measures the commercialfishing and shark fishing are prohibited in the area but illegal fishing boats enter theGalapagos Marine Reserve from the Ecuadorian coast (Jacquet et al 2008 Carr et al2013)

Several studies have been conducted in recent years referent to habitat use andmigratorypatterns of different sharks in Galapagos (Hearn et al 2010 Ketchum et al 2014 AcuntildeaMarrero et al 2018) However there is no information available on the role of thesepredators in the regional food web This paper thus compares the isotopic niches of threecommercially important shark species caught illegally in the Galapagos Marine Reserveand calculates the degree of niche overlap between them with the aim of creating baselineknowledge about the trophic ecology of these species

METHODSStudy areaThe Galapagos Islands are located 960 km from mainland Ecuador in the Pacific OceanThe waters associated with this island complex form a marine reserve delineated by alsquolsquobaselinersquorsquo linking the outer edges of the islands to a distance of 74 km creating a protectedarea of about 138000 km2 (Heylings Bensted-Smith amp Altamirano 2002) (Fig 1) Thearchipelagorsquos unique oceanographic setting is believed to be largely responsible for thesporadic colonization of the islands which led to the evolution of the divergent species thatcan be observed today in the archipelagorsquos ecosystems (Ryan et al 2006 Nims et al 2008)It is the Cromwell and Humboldt ocean currents that bring most nutrients to the regiongenerating around the islands and seamounts areas of continuous upwelling that inducephytoplankton and zooplankton blooms (Palacios et al 2006 Schaeffer et al 2008) henceincreasing species richness and diversity in the region

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 321

Figure 1 Map of the Galapagos Islands showing the boundaries of the Galapagos Marine Reserve andlocation where the illegal fishing boat was seized

Full-size DOI 107717peerj4818fig-1

Sample collectionThis research was performed under the research permits PC-13-12 PC-52-13 and PC-38-16 and was carried out following the protocols of ethics and animal handling approved bythe Galapagos National Park and the Ecuadorian laws

Sampling was done on board a boat seized in July 2011 by the Galapagos National Parkauthorities and the Ecuadorian Navy while illegally fishing sharks in the northwest regionof the Galapagos Marine Reserve (010prime18primeprimeN 8921prime58primeprimeW) (Fig 1) The fishing boat lsquolsquoFerMary Irsquorsquo from Manta Ecuador was equipped with a 370-hook longline fishing gear and six8-m fiberglass boats with outboard motor to check the longline for sharks A total of 380sharks found in the boatrsquos hold were seized For each specimen the species and sex wereidentified and age group was determined based on body size Samples of muscle tissuewere taken from a total of 91 adult sharks belonging to the three species mentioned above(A pelagicus P glauca or C falciformis) (Table 1) All remaining shark material was thendestroyed as required by Ecuadorian laws

The samples were washed with distilled water placed into 20-ml Eppendorf tubesand then frozen to minus20 C To confirm that all sharks were adult the total length (TL)and precaudal length (PCL) of each specimen were measured Total length could not bemeasured in A pelagicus however because the upper lobes of caudal fins had been cut offit was thus calculated using the allometric equations proposed by Carr et al (2013)

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 421

Table 1 Values of δ13C and δ15N in the muscle tissue of three sharks species Total length and values ofδ13C and δ15N (expressed in h meanplusmn SD) in the muscle tissue of three sharks species A pelagicus Pglauca and C falciformis in the Galapagos Marine Reserve

Species Sex n Length (cm) δ13Cplusmn SD δ15Nplusmn SD CN

A pelagicus Male 16 2699 minus1654plusmn 043 1265plusmn 158 308 Female 23 2711 minus1672plusmn 045 1204plusmn 110 314P glauca Male 9 1748 minus1662plusmn 030 1313plusmn 071 296 Female 11 1766 minus1665plusmn 020 1376plusmn 100 299C falciformis Male 20 1717 minus1682plusmn 030 1426plusmn 143 317 Female 12 1689 minus1677plusmn 016 1409plusmn 149 305

Sample processingAll muscle tissue samples were rinsed with deionized water to eliminate residues that couldalter their isotopic signature and placed in glass vials previously treated for 24 h with achromic acid mixture prepared from sulfuric acid and potassium dichromate They werethen dried in a desiccator at 80 C for 12 h to remove all moisture A microwave-assistedextraction protocol (MAE) was applied (Microwave oven model 1000-WMARS 5x CEMMatthews USA) using 25 ml of a 11 chloroformmethanol solution (Tieszen et al 1983)and dried again The samples were homogenized with an agate mortar to obtain a veryfine powder of which sim1 mg was weighed by means of an analytical microbalance with aprecision of 0001 mg and transferred into a tin capsule for isotopic analysisδ13Cand δ15Nstable isotope ratios were determined by a PDZEuropa 20ndash20 continuous-

flow isotope-ratio mass spectrometer (Sercon Ltd Cheshire UK) at the Stable IsotopeFacility of the University of California at Davis (CA USA) The results expressed inparts per thousand (h) were obtained using the following equation δ13C or δ15N =1000([RsampleRstandard]minus1) where Rsample and Rstandard are the 13C12C or 15N14N ratiosof the sample and the standard respectively The standards used were Pee Dee Belemnite(PDB) for δ13C and atmospheric N2 for δ15N

Data analysisData were tested for normality and homoscedasticity using the ShapirondashWilk and Levenetest respectively The statistical significance of differences in δ13C and δ15N values wasdetermined using parametric or non-parametric tests and reported when P lt 005 Allstatistical analyses were performed using the software Statistica 80

The bayesian method SIBER (Stable Isotope Bayesian Ellipses in R) was used to definethe isotopic niche space among the three species as a measure of their isotopic resourceuse area at the population level This method is based on the two-dimensional isotopicspace of δ13C and δ15N and assessed using Bayesian analysis of standard ellipses thatunlike the Euclidean methods (eg convex hulls) can incorporate uncertainties such assampling biases and small sample sizes into niche metrics (Layman et al 2007) We usedMonte Carlo simulations to correct the bivariate ellipses (δ13C and δ15N) surroundingthe data points in the 95 confidence interval for the distributions of both stable isotopes(Jackson et al 2011) These corrected standard ellipse areas (SEAc) represent the isotopic

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 521

niche width and the overlap parameters (Jackson et al 2011) Furthermore we calculatedthe magnitude of the isotopic overlap among the three species of sharks based on 100000posterior draws of the SEAc parameters (Jackson et al 2011)

The trophic position of each shark species was estimated by the formula proposed byPost (2002) TL= λ+((δ15Npredatorδ

15Nbase)1N) assuming that the δ15N of a consumerand other components of the food web provides information on a speciesrsquo trophic level Thevalues used were the trophic position of the base species (λ) the δ15N value of zooplanktonoccurring in the region (as base) value previously reported by Paacuteez-Rosas et al (2012)and the isotopic fractionation factor (1N) for marine predators in general established byHobson et al (1996)

RESULTSIsotopic comparison of species and sexesThe mean estimated δ13C and δ15N values in the muscle tissue of A pelagicus were minus1659plusmn 044h and 1189 plusmn 132h in the tissue of P glauca minus1666 plusmn 005h and 1346 plusmn092h and in the tissue ofC falciformisminus1682plusmn 025h and 1399plusmn 142h respectively(Table 1) The CN ratios of the samples ranged from 28 to 32 and were thus withinthe theoretical range established for the assimilation of protein from a predatorrsquos diet(McConnaughey amp McRoy 1979) therefore the isotopic values reflects the diet of thesepredators (Table 1) There were no significant interspecific differences in δ13C values(KruskalndashWallis test p= 009) The δ15N values in contrast were significantly differentbetween shark species (KruskalndashWallis test p= 001) the δ15N of A pelagicus differingfrom those of P glauca andC falciformis (multiple comparisons of median ranks plt 005)(Fig 2)

The mean δ13C and δ15N values of A pelagicus P glauca and C falciformis are shownby sex in Table 1 None of the three species showed significant differences in δ13C andδ15N values between the sexes (A pelagicus MannndashWhitney U test p= 022 and 023respectively P glauca paired t -test p= 081 and 013 respectively and C falciformisMannndashWhitney U test p= 049 and 076 respectively) (Fig 3) The comparison by sexof δ13C values between the species showed that the females of P glauca (mean minus1665h)had higher δ13C than those of A pelagicus (minus1672h) and C falciformis (minus1677h)but these differences were not significant (KruskalndashWallis test p= 009) (Table 2) Theδ15N values of the females of A pelagicus (1204h) in contrast significantly differed fromthose of the females of P glauca (1376h) andC falciformis (1409h) (KruskalndashWallis testp= 001) (Table 2)With regards tomales the only significant difference in δ15N values wasbetween A pelagicus (1265h) and C falciformis (1426h) (KruskalndashWallis test p= 001)(Table 2)

Trophic level niche breadth and niche overlapThe average estimated trophic level was 38 for A pelagicus 41 for P glauca and 44 for Cfalciformis making these shark species tertiary or quaternary predators in the food web

The corrected standard ellipse area (SEAc) in SIBER showed that A pelagicus and Cfalciformi s could be exploiting different types of habitat unlike P glauca (Table 3) These

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 621

Figure 2 δ15N values in the muscle tissue of three shark species in the Galapagos Marine Reserve Thewhiskers represents the minimum and maximum value in h the black square contains the 50 75 per-centiles and black dot is the median value

Full-size DOI 107717peerj4818fig-2

Figure 3 Values of δ13C and δ15N in the muscle tissue of males and females of three shark species Val-ues of δ13C and δ15N (expressed in h meanplusmn SD) in the muscle tissue of males and females of A pelagi-cus P glauca and C falciformis in the Galapagos Marine Reserve

Full-size DOI 107717peerj4818fig-3

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 721

Table 2 δ15N values in the muscle tissue of males and females of A pelagicus P glauca and C falci-formis in the Galapagos Marine Reserve Significant differences (Tukeyrsquos HSD test p lt 005) are shownin bold

A pelagicus P glauca C falciformis

FemalesA pelagicus XP glauca 0001 XC falciformis 0001 0785 XMalesA pelagicus XP glauca 1000 XC falciformis 0001 0211 X

Table 3 Basic Standard Ellipse Area (SEA) and Corrected standard ellipse area (SEAc) measured usingStable Isotope Bayesian Ellipses in R as an estimate of the trophic niche breadth (TNB) of A pelagicusP glauca and C falciformis

Species SEA SEAc TNB

A pelagicus 150 155 467P glauca 069 073 219C falciformis 109 114 419

results suggest that feeding habitat of P glaucamay be limited to a specific area as opposedto the other species The Bayesian ellipse of C falciformis (SEAc = 114h 95 credibilityinterval of 082ndash146h) and A pelagicus (SEAc = 155h 95 credibility interval of093ndash217h) have a minimal overlap (Table 3 and Fig 4) confirming different resourceuse patterns for these two groups of sharks In contrast the Bayesian ellipse of P glauca(SEAc = 073h 95 credibility interval of 028ndash118h) is overlapped in large part withthe ellipses of the other two species (Fig 4) The overlap area (033) of the Bayesianellipses from C falciformis and P glauca represented the 301 of the ellipse surface ofthe former and the 465 of the ellipse surface of the latter Conversely the overlap area(026) of the Bayesian ellipses from A pelagicus and P glauca represented only the 197of the former and the 483 of the ellipse surface of the P glauca (Fig 4)

When taking sex into account a significant isotopic overlap was observed betweenmalesand females in A pelagicus (100) and C falciformis (069) While males and females of Pglauca showed only a small isotopic overlap (036) (Table 4 and Fig 5) In all cases withthe exception of P glauca the Bayesian ellipses of males are larger and encompass most ofthe Bayesian ellipses of females of the same species (Fig 5) suggesting a higher diversity offoraging strategies in males compared to females in A pelagicus and C falciformis

DISCUSSIONInterspecific differencesIt is widely known that resource use by wildlife species may vary according to a number offactors eg body size sex or age Depending on the availability of resources (eg space

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Figure 4 Values of δ13C and δ15N trophic niche breadth and degree of trophic niche overlap betweenthree shark species A pelagicus P glauca and C falciformis in the Galapagos Marine Reserve Dottedlines represent the Convex Hull areas (polygons) while the subgroups within are formed by the standardellipse areas corrected (SEAc) provided by SIBER analysis

Full-size DOI 107717peerj4818fig-4

Table 4 Degree of isotopic niche overlap betweenmales and females of A pelagicus P glauca and C falciformis in the Galapagos Marine Re-serve The degree of trophic niche overlap between shark populations was estimated with the overlap index of the SIBER model where a valuesclose to 1 (shown in bold) indicating a large overlap between their trophic niches

Species and sex FemaleA pelagicus

MaleA pelagicus

FemaleP glauca

MaleP glauca

FemaleC falciformis

MaleC falciformis

Female A pelagicus XMale A pelagicus 100 XFemale P glauca 005 032 XMale P glauca 030 036 062 XFemale C falciformis 005 019 039 031 XMale C falciformis 001 009 039 023 069 X

food) in the ecosystem this may lead to interspecies or within-species resource segregation(Bolnick et al 2003 Matich Heithaus amp Layman 2011)

In this study no differences were observed in the foraging habitats among the three sharkspecies The δ13C values (between minus172 and minus162) indicate an oceanicpelagic foragingstrategy consistent with what was previously reported in various studies conducted in thecoasts of Ecuador Colombia Chile and Mexico (Polo-Silva et al 2013 Hernaacutendez-Aguilaret al 2015 Estupintildeaacuten Montantildeo et al 2017a Estupintildeaacuten Montantildeo et al 2017b Klarianet al 2018) Spatial variation in δ13C values of predators can be partly explained bydifferences in the isotopic composition of primary producers which are the main energysuppliers for the food web (Goericke amp Fry 1994 Pancost et al 1997) Indeed differentlevels of primary production concentration of dissolved CO2 macro and microalgaecomposition and phytoplankton growth rate among other factors create a coastndashocean

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 921

Figure 5 Qualitative description of the trophic niche overlap betweenmales and females of A pelag-icus P glauca and C falciformis Dotted lines represent the Convex Hull areas (polygons) while the el-lipse represents the isotopic niche breadth of a group of sharks

Full-size DOI 107717peerj4818fig-5

gradient in C-isotopic composition with a decrease in δ13C from the coastal to the pelagiczone (France 1995 Newsome et al 2007)

These variability factors are present in the marine ecosystem of the Galapagos Islandswhich despite being located in tropical (oligotrophic) waters are characterized byhighly productive local conditions due to the lsquolsquoisland-mass effectrsquorsquo (Palacios 2002) thecombination of oceanographic currents flowing there generating high productivity aroundthe islands (Feldman 1986 Palacios et al 2006) A δ13C-enriched isotopic signature whichwas not observed in this study would thus only be expected if the three shark species hadforaged close to the insular shelf

The δ15N values revealed differences in the trophic position occupied by the three sharkspecies A pelagicus being a lower trophic-level predator compared with the other twospecies The consumption of prey from different environments (epipelagic or mesopelagic)and different trophic levels is reflected by δ15N values that differ between populationsexploiting similar habitats (Takai et al 2000) These differences in δ15N values may bedue not only to differential consumption of prey but also to isotopic differences at thebase of the food web (Newsome et al 2007) Such isotopic differences manifest themselvesat higher trophic links through bioaccumulation of δ15N from prey to consumer and theresulting isotopic enrichment along the food chain (Minagawa ampWada 1984 NewsomeClementz amp Koch 2010)

In Ecuadorian waters Alopias pelagicus has been shown to feed on squid and small fish(Polo-Silva Rendoacuten amp Galvaacuten-Magantildea 2009 Polo-Silva et al 2013) with a preference forepipelagic squids like Sthenoteuthis oualaniensis (Galvaacuten-Magantildea et al 2013) The reportedprevalence of these preys suggests that A pelagicus forages in epipelagic waters possibly atnight when squid are more likely to be caught (Markaida amp Sosa-Nishizaki 2010) While

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 1021

P glauca is known to be mainly teuthophagous in the North Pacific (Kubodera Watanabeamp Ichii 2007 Hernaacutendez-Aguilar et al 2015) recent studies carried out in South Pacificmention that its diet is based in pelagic fish reaching even to consumemarinemammals thatare dead as dolphins (Klarian et al 2018) This shark has a greater diving capacity than theother two species allowing it to explore various habitats along the water column (CareyScharold amp Kalmijn 1990 Roper Sweeney amp Hochberg 1995) It even undertakes dailyvertical migrations to depths of over 600 m in order to feed on mesopelagic cephalopodsoccupying a high trophic level like Vampyroteuthis infernalis and Ancistrocheirus lesueurii(Kubodera Watanabe amp Ichii 2007 Galvaacuten-Magantildea et al 2013)

The highest δ15N values were found in Carcharinus falciformis making it the highesttrophic-level predator of the three species This result agrees with other trophic studies thatfound this shark to have a certain preference for high-energy oceanic prey like fish of theScombridae family (Duffy et al 2015 Estupintildeaacuten Montantildeo et al 2017b) Consistent withthese references and the isotopic values observed in this study it could be assumed that Cfalciformis spend most of its time in surface waters at a depth of sim50 m conditions thatare usually favorable for the aforementioned preys (Filmalter et al 2010)

Intersexual differencesA dietary study of populations of A pelagicus occurring along the coasts of Ecuador foundthat their diet varies with stage of sexual maturity but that male and female adults feed onthe same prey mostly juvenile squids of the species D gigas and S oualaniensis (Polo-SilvaRendoacuten amp Galvaacuten-Magantildea 2009) Isotopic analyses of adult specimens caught by thefishing fleet at the Ecuadorian ports of Manta and Santa Rosa showed that male adults havehigher δ15N values than females Generally in most shark species the females are biggerin size compared to males this condition allows them explore more feeding zones Whilethe males normally stay close the bottom to search protection and easier prey to catch(Compagno 1990 Polo-Silva et al 2013) These characteristics could influence that themales consume larger prey associated with the bottom habitats that are enriched in 15Nso this behavior can explain this difference

The females of A pelagicus had δ13C and δ15N values that were slightly more negativeand with smaller variance than those of males This suggests that females adopt aforaging strategy that is more oceanic and focused on specific prey while males exploitdifferent ecosystems (oceanicndashcoastal) with a higher diversity of foraging strategiesIsotopic variability within a population is known to reflect up to a certain point dietaryheterogeneity among individuals their high mobility among feeding areas allowing themto consume different prey (Estrada et al 2003 Jaeger et al 2009)

The populations of P glauca in theNorth Atlantic is known to exhibit spatial segregationof the sexes the males tending to frequent coastal waters more intensively than femaleswhich are completely oceanic (Carey Scharold amp Kalmijn 1990 Vandeperre et al 2014)This may influence the level of consumptionabundance of their main prey (HendersonFlannery amp Dunne 2001 Hernaacutendez-Aguilar et al 2015) This differential behavior ofmales and females could explain what was observed in this study since despite notobserving statistical differences the females had higher δ15N values than males In light of

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 1121

the above studies the spatial heterogeneity in δ15N values between sexes can be explainedto some extent by the Galapagos Islandsrsquo oceanographic setting which allows high levelsof primary productivitymdashnot characteristic of the regionmdashto be maintained throughoutthe year (Banks 2002 Palacios et al 2006)

With regards to the feeding behavior of C falciformis both isotopes showed nointersexual difference which suggests that both sexes exploit similar prey and foraginghabitats Dietary studies conducted on the eastern Pacific Ocean has reported fish ofthe Scombridae family and squid as the speciesrsquo main prey (Duffy et al 2015 EstupintildeaacutenMontantildeo et al 2017a Estupintildeaacuten Montantildeo et al 2017b) Other authors have found malesof C falciformis to be more active than females undertaking vertical migrations in theevening to complement their diet with squid and rest there at night (Compagno Dando ampFowler 2005) Such a behavior may explain the high variability observed here in the δ13Cand δ15N values of males

Trophic level niche breadth and niche overlapOne method to estimate trophic levels is to use the isotope fractionation occurring in thefood chain The formula proposed by Post (2002) used in this study allowed A pelagicusand P glauca to be classified as tertiary predators and C falciformis as a quaternarypredator (Mearns et al 1981) The trophic levels (TLs) obtained for A pelagicus (TL= 38)and P glauca (TL = 41) agree with the diet previously reported for both species basedmainly on small or juveniles squid and fishes of intermediate trophic levels (Polo-Silva etal 2013 Hernaacutendez-Aguilar et al 2015) A higher trophic level was estimated for malesin A pelagicus which may be associated with them having a greater ability to approachcoastal areas where they can feed on fish that are larger and occupy a higher trophic level(Rosas-Luis et al 2017)

The highest trophic-level predator was C falciformis (TL = 44) which agrees with thefeeding habits previously reported for this species consisting in targeting large carnivorousfish such as tuna black skipjack and supplement their diet with other prey like cephalopods(Duffy et al 2015Estupintildeaacuten Montantildeo et al 2017b) Such as feeding behavior could explainthe speciesrsquo heavy isotopic signature and allows to put forward the hypothesis that thesesharks feed on other high trophic-level predators occurring in the region eg Galapagos sealions which have δ15N valuessim15h lower (equivalent to prey-to-predator fractionation)than those of C falciformis (Paacuteez-Rosas amp Aurioles-Gamboa 2014) This would explain theorigin of the attack marks (shark bites) that are observed in the pinnipeds of GalapagosIslands in different regions of the archipelago

Sharks have long been classified as opportunistic predators feeding on the resourcesavailable at a given place and time (Calow amp Tytler 1985) In recent years it has been shownfor some sharkrsquos species that although their food spectrum includes a large variety of preythe largest part of their diet is made up of three or four prey species which may change withthe seasons (Polo-Silva et al 2013 Loor-Andrade et al 2015) The trophic breadth of thethree shark species was calculated based on isotopic niche breadth resulting in a generalisttype of feeding strategy for A pelagicus and C falciformis and a specialist strategy for Pglauca It is possible that given the great diving capacity that P glauca (Roper Sweeney amp

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 1221

Hochberg 1995) this species is only using a specific habitat being more selective in the useof the resources that exist in that space

This result about A pelagicus differs from those of other studies that reported thisspecies as a specialist predator based on stomach content analysis (Polo-Silva Rendoacuten ampGalvaacuten-Magantildea 2009 Polo-Silva et al 2013) Several studies mention that A pelagicusshows some variability in trophic niche breadth over its life depending on its stage ofsexual maturity and the resources available in the environment (Compagno 1990 Gerking1994) It is thus likely that these sharks tend towards a specialist feeding strategy as theirenergy requirements increase (eg for reproduction gestation etc)

The narrower trophic niche breadth estimated for P glauca suggests that this speciesexploits specific prey and environments consistent with the results ofHernaacutendez-Aguilar etal (2015) and Klarian et al (2018)who found that populations of P glauca off the coasts ofBaja California and Chile show a high degree of specialization as evidenced by the reducednumber of food components that are well represented in their diet

This study allowed C falciformis to be classified as a generalist predator in the GalapagosMarine Reserve It has been shown however that when there is a lower diversity of prey inthe environment these sharks tend to bemore selective in choosing them (Cabrera-Chaacutevez-Costa Galvaacuten-Magantildea amp Escobar-Saacutenchez 2010 Filmalter et al 2010) Other authors havedescribed this shark as a specialist consumer because despite feeding on a wide foodspectrum a large proportion of its diet is made up of a limited number of prey types (Duffyet al 2015 Estupintildeaacuten Montantildeo et al 2017a) This may explain to some extent the greatvariability in δ15N values observed in this study

Population-level approaches have often used trophic position and niche breadth tounderstand interactions within the food web and the role of predators in the ecosystem(Bearhop et al 2004) Isotopic niche analysis showed have a minimal overlap between Apelagicus and C falciformis which suggests that both species feed on prey from differenttrophic levels and different feeding areas (Jackson et al 2011) In contrast the small (butnon-significant) overlap between the isotopic ellipse of P glauca and those of the othertwo shark species reveals the consumption of a similar combination of prey and use ofthe same feeding areas (Matich Heithaus amp Layman 2010) P glauca would thus be moresusceptible to competition for food resources (eg squid) and even feeding areas (Newsomeet al 2007 Graham Spalding amp Sheppard 2010)

CONCLUSIONSThe highly selective foraging behavior in P glauca and the existence of interespecificvariation in feeding strategies of the three populations may give rise to particular energyneeds depending on the availability of the different types of prey that occur in its habitatThis would lead to some degree of specialization for certain prey which could also be partof the food spectrum of other sharks with similar feeding strategies Our work is the firststudy that uses this methodological approach to provide novel insights into the trophicecology of sharks in the Galapagos Islands In any case it should be reminded that theisotopic niche is only a proxy for the trophic niche and that the absence of significant

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 1321

differences between isotopic ellipses does not necessarily mean that trophic niches areidentical Further research based on stomach content analysis is thus needed to obtainmore accurate information on the prey consumed by these three shark species in theGalapagos Marine Reserve

ACKNOWLEDGEMENTSWe thank the Galapagos National Park (GNP) for logistical support and granting uspermission to collect the samples used in this study We are grateful to Leandro Vaca JuanCarlos Murillo Ignasi Montero Colombo Estupintildean and the GNP rangers for their help incollecting processing and analyzing the biological material used in this study Finally wethank the Galapagos Science Center in Ecuador for providing the facilities for informationprocessing and analysis

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was supported by Universidad San Francisco de Quito (USFQ) in Ecuador andthe Centro Interdisciplinario de Ciencias Marinas (CICIMAR) in Mexico The funders hadno role in study design data collection and analysis decision to publish or preparation ofthe manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsUniversidad San Francisco de Quito (USFQ) in EcuadorCentro Interdisciplinario de Ciencias Marinas (CICIMAR) in Mexico

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Diego Paacuteez-Rosas conceived and designed the experiments performed the experimentsanalyzed the data contributed reagentsmaterialsanalysis tools prepared figures andortables authored or reviewed drafts of the paper approved the final draftbull Paul Insuasti-Zarate performed the experiments analyzed the data prepared figuresandor tables authored or reviewed drafts of the paper approved the final draftbull Marjorie Riofriacuteo-Lazo analyzed the data contributed reagentsmaterialsanalysis toolsauthored or reviewed drafts of the paper approved the final draftbull Felipe Galvaacuten-Magantildea conceived and designed the experiments authored or revieweddrafts of the paper approved the final draft

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

This research was carried out following the protocols of ethics and animal handlingapproved by the Galapagos National Park and the Ecuadorian laws

Paacuteez-Rosas et al (2018) PeerJ DOI 107717peerj4818 1421

Data AvailabilityThe following information was supplied regarding data availability

The raw data have been uploaded as Supplemental File and are available athttpwwwdatagalapagosorg

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4818supplemental-information

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Estrada J Rice A Lutkavage M Skomal G 2003 Predicting trophic position insharks of the northndashwest Atlantic Ocean using stable isotope analysis Jour-nal of the Marine Biological Association of the United Kingdom 831347ndash1350DOI 101017S0025315403008798

EstupintildeaacutenMontantildeo C Galvaacuten-Magantildea F Tamburin E Saacutenchez-Gonzaacutelez AVillalobos-Ramiacuterez D Murillo-Bohoacuterquez N Bessudo-Lion S Estupintildeaacuten OrtiacutezJ 2017a Trophic inference in two sympatric sharks Sphyrna lewini and Car-charhinus falciformis (elasmobranchii carcharhiniformes) based on stable isotopeanalysis at Malpelo Island Colombia Acta Ichthyologica et Piscatoria 47357ndash364DOI 103750AIEP02177

EstupintildeaacutenMontantildeo C Pacheco-Trivintildeo F Cedentildeo Figueroa L Galvaacuten-Magantildea FEstupintildeaacuten Ortiacutez J 2017b Diet of three shark species in the Ecuadorian PacificCarcharhinus falciformis Carcharhinus limbatus and Nasolamia velox Journal of theMarine Biological Association of the United Kingdom 1ndash9DOI 101017S002531541600179X

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Jackson A Inger R Parnell A Bearhop S 2011 Comparing isotopic niche widths amongand within communities SIBERmdashStable Isotope Bayesian Ellipses Journal of AnimalEcology 80595ndash602 DOI 101111j1365-2656201101806x

Jacquet J Alava J Pramod G Henderson S Zeller D 2008 In hot soup sharks capturedin Ecuadorrsquos waters Environmental Sciences 5269ndash283DOI 10108015693430802466325

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Ketchum J Hearn A Klimley A Pentildeaherrera C Espinoza E Bessudo S Soler G ArauzR 2014 Inter-island movements of scalloped hammerhead sharks (Sphyrna lewini)

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Kubodera TWatanabe H Ichii T 2007 Feeding habits of the blue shark Pri-onace glauca and salmon shark Lamna ditropis in the transition region ofthe Western North Pacific Reviews in Fish Biology and Fisheries 17111ndash124DOI 101007s11160-006-9020-z

Layman C Arrington D Montantildea C Post D 2007 Can stable isotope ratios pro-vide for community-wide measures of trophic structure Ecology 8842ndash48DOI 1018900012-9658(2007)88[42CSIRPF]20CO2

Loor-Andrade P Galvaacuten-Magantildea F Elorriaga-Verplancken F Polo-Silva C Delgado-Huertas A 2015 Population and individual foraging patterns of two hammerheadsharks using carbon and nitrogen stable isotopes Rapid Communications in MassSpectrometry 291ndash19 DOI 101002rcm7075

Markaida U Sosa-Nishizaki O 2010 Food and feeding habits of the blue shark Prionaceglauca caught off Ensenada Baja California Mexico with a review on its feedingJournal of the Marine Biological Association of the United Kingdom 90977ndash994DOI 101017S0025315409991597

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Myers R Baum J Shepherd T Powers S Peterson C 2007 Cascading effects ofthe loss of apex predatory sharks from a coastal ocean Science 3151846ndash1850DOI 101126science1138657

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Newsome S Clementz M Koch P 2010 Using stable isotope biogeochemistryto study marine mammal ecologyMarine Mammal Science 26509ndash572DOI 101111j1748-7692200900354x

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Paacuteez-Rosas D Aurioles-Gamboa D 2014 Spatial variation in foraging behavior of theGalapagos sea lions (Zalophus wollebaeki) assessed using scat collections and stableisotope analysis Journal of the Marine Biological Association of the United Kingdom941099ndash1107 DOI 101017S002531541300163X

Paacuteez-Rosas D Aurioles-Gamboa D Alava J Palacios D 2012 Stable isotopesindicate differing foraging strategies in two sympatric otariids of the Galapa-gos Islands Journal of Experimental Marine Biology and Ecology 42544ndash52DOI 101016jjembe201205001

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Palacios D Bograd S Foley D Schwing F 2006 Oceanographic characteris-tics of biological hot spots in the North Pacific a remote sensing perspec-tive Deep-Sea Research Part II Topical Studies in Oceanography 53250ndash269DOI 101016jdsr2200603004

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Post DM 2002 Using stable isotopes to estimate trophic position models methods andassumptions Ecology 83703ndash718DOI 1018900012-9658(2002)083[0703USITET]20CO2

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