Ecology and Evolution. 2017;7:3773–3783.  | 3773www.ecolevol.org

Received:9January2017  |  Accepted:14March2017DOI: 10.1002/ece3.2957

O R I G I N A L R E S E A R C H

Spatial overlap of shark nursery areas and the salmon farming industry influences the trophic ecology of Squalus acanthias on the southern coast of Chile

Juan Diego Gaitán-Espitia1  | Daniela Gómez2 | Alistair J. Hobday1 | Ross Daley3 |  Julio Lamilla4† | Leyla Cárdenas2

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2017TheAuthors.Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.

1CSIROOceansandAtmosphere,Hobart,TAS,Australia2FacultaddeCiencias,InstitutodeCienciasAmbientalesyEvolutivas,UniversidadAustraldeChile,Valdivia,Chile3InstituteforMarineandAntarcticStudies,UniversityofTasmania,Hobart,TAS,Australia4FacultaddeCiencias,InstitutodeCienciasMarinasyLimnologicas,UniversidadAustraldeChile,Valdivia,Chile

CorrespondenceJuanDiegoGaitán-Espitia,CSIROOceansandAtmosphere,Hobart,TAS,Australia.Email:juadiegaitan@gmail.com

Funding informationCSIRO-OCEpost-doctoralfellowship;MillenniumNucleusCenterfortheStudyofMultipledriversonMarineSocio-EcologicalSystems(MUSELS)-MINECON,Grant/AwardNumber:NC120086

AbstractPotential interactionsbetweenmarinepredators andhumans arise in the southerncoastofChilewherepredatorfeedingandreproductionsitesoverlapwithfisheriesandaquaculture.Here,weassessthepotentialeffectsofintensivesalmonaquacultureonfoodhabits,growth,andreproductionofacommonpredator,thespinydogfish—identifiedasSqualus acanthiasviageneticbarcoding.Atotalof102(89femalesand13males)individualswerecollectedduringwinterandsummerof2013–2014fromtheChiloéSeawheresalmonaquacultureactivitiesareconcentrated.Thelowfrequencyofmalesinourstudysuggestsspatialsegregationofsex,whileimmatureandmaturefemalesspatiallyoverlapped inbothseasons.Femalespinydogfishshoweda func-tionalspecialistbehaviorasindicatedbythesmallnumberofpreyitemsandtherela-tive high importance of the austral hake and salmon pellets in the diet. Immaturesharksfedmoreonpelletsandanchoviesthanthelargerhake-preferringmaturefe-males.Ourresultsalsoindicatethatspinydogfishswitchprey(anchovytohake)totake advantage of seasonal changes in prey availability.Despite differences in thetrophicpatternsofS. acanthiasduetothespatialassociationwith intensivesalmonfarming,inthisregion,thereappearstobenodifferenceinfecundityorsizeatmatu-ritycomparedtootherpopulations.Althoughnodemographiceffectsweredetected,wesuggestthatarangeofadditionalfactorsshouldbeconsideredbeforeconcludingthatintensiveaquaculturedoesnothaveanyimpactonthesemarinepredators.

K E Y W O R D S

artisanalfishery,reproduction,Salmonindustry,sexualmaturity,Squalus acanthias,trophicecology

1  | INTRODUCTION

Inthepastthreedecades,salmonidaquacultureinChilehasexperiencedanexplosivegrowthanddiversificationdrivenbytheuseofintensive

farmingtechniques(Buschmannetal.,2009;Oddone,Paesch,&Norbis,2015)andtheintroductionofseveraldifferentsalmonidspecies(Correa&Gross,2008).AlthoughthisgrowthhasplacedChileasthesecondmostimportantsalmonexporterworldwide(Ellisetal.,2016),ithasalsoraisedcriticismsandconcernsrelatedtoitsnegativeenvironmentalef-fects(Buschmannetal.,2006,2009;Soto&Norambuena,2004).The†DeceasedAuthor.

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marinephaseofsalmonaquacultureisconductedinanopenwaternet-systemthatallowsthedischargeoflargequantitiesofpollutants,mainlyorganicmatter,nitrogen,andphosphorusintowaterbodiesandtheseafloor (Paudeletal.,2015).Discharges fromsalmon farmscan lead toeutrophicationandalterationsoftheseasonalnutrientsupplyofcoastalecosystems(Brooks&Mahnken,2003;Kuttietal.,2007)andthesedi-mentorganiccontent(Carrolletal.,2003;Soto&Norambuena,2004),producing zones of anoxic conditions (Brown, Gowen, & McLusky,1987; Buschmann etal., 2006). All of these environmental changesaroundsalmonfarmsimpactthestructureandcompositionofbenthiccommunitiesbysupportinghighproductionofdeposit-feedinginverte-brates(Guilpartetal.,2012;Kuttietal.,2007)andattractinghighden-sitiesofdemersal fish (Carss,1990;Dempsteretal.,2002)andothermobilecarnivores (Bonizzonietal.,2013;Kemperetal.,2003;Quick,Middlemas,&Armstrong,2004;Ribeiroetal.,2007;Sepúlveda&Oliva,2005;Veríssimo,McDowell,&Graves,2010).Theimmediateoutcomeofthesealterationsisenhancementofbenthic-pelagiccouplingandthepotentialtransferofsediment-associatedcontaminantsintohighertro-phiclevels(Debruynetal.,2006).

InChile,thesalmonindustryisconcentratedaroundtheinnerseaofChiloéIsland(41–43°S,hereafterChiloéSea)(Figure1)(Buschmannetal.,2006,2009).This region isa semi-closedenvironment that ishometorare,uniqueandendangeredmarinespecies,includingcold-watercorals (Försterra&Häussermann,2003),penguinsandmigra-torybirds(Senneretal.,2014;Skewgar,Boersma,&Simeone,2014),otters (Ebensperger & Botto-Mahan, 1997), blue and humpbackwhales (Hucke-Gaete etal., 2004), dolphins and pinnipeds (Ribeiroetal., 2007; Veríssimo etal., 2010), and skates and sharks (Lamillaetal.,2005;Quiroz,Wiff,&Céspedes,2009;Valenzuela,Bustamante,&Lamilla,2008).MostofthesemarineanimalsusetheChiloéSeaasa feeding andnursery area (Hucke-Gaete etal., 2004;Quiroz etal.,2009).However,thissemi-closedenvironmentisundergreatpressurebecausethehighdensityofhuman-relatedactivitiesthatarecarriedoutinthisregionhasbecomeamajorthreatfortheecologicalbalanceandpersistenceoflocalmarinespecies(Oddoneetal.,2015).Forin-stance,differentbiologicalaspects(e.g.,mortalityrates,behavioranddistribution)ofa rangeofmarinepredators, suchasseals,dolphins,andsharks,aresignificantlyaffectedbytheirinteractionswithfisheries

F IGURE  1 Mapofthestudyareawithsalmonfarmsdistributionandactivityareasofsmalltolarge-scalefisheries(e.g.,hake,anchovies,mackerel,cusk-eel,seaurchins)(modifiedfromBustos-Gallardo&Irarrazaval,2016)

0 5 10 20 30 40km

Salmon farmSmall to large-scale fisheries areas

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(e.g.,hake,mackerel,sardine,andanchovy)andintensiveaquaculturesystems(e.g.,salmon,mussels)intheChiloéSea(DeLaTorrienteetal.,2010; Kemper etal., 2003; Ribeiro etal., 2007; Sepúlveda&Oliva,2005;Veríssimoetal.,2010).Someoftheseinteractionshaveledtomortalitiesasaconsequenceofdirecthumanattackorbyaccidentalbycatch(DeLaTorrienteetal.,2010;Ribeiroetal.,2007;Sepulvedaetal.,2007).Oneof thebestexamplesof thissituation is thespinydogfishSqualus acanthias.Thiscoastalsqualoidshark,aswellasotherdogfish species (e.g., Centroscyllium granulatum and Deania calcea),is common bycatch species in many small and large-scale fisheriesin southernChile (De LaTorriente etal., 2010; Lamilla etal., 2005;Valenzuelaetal.,2008).Unfortunately,littleisknownabouttheabun-danceofS. acanthias inChileand theecological implicationsof thepopulationdeclines(~30%)documentedforthisvulnerablespeciesinSouthAmerica(Fordhametal.,2006;Veríssimoetal.,2010).

Giventhepotentialforstronginteractionsbetweenhumanactivi-ties(aquacultureandfisheries)andspinydogfishpopulationsinsouth-ernChile(DeLaTorrienteetal.,2010;Valenzuelaetal.,2008),hereweassessedthepotentialeffectsofsalmonfarmingonthebiologyofS. acanthiasintheChiloéSea.Wealsodiscussthetrophicandreproduc-tivemetricsemployedhereinthecontextofS. acanthiaspopulationselsewhere,whichareunexposedtointensesalmonidaquaculture.

2  | MATERIAL AND METHODS

Spinydogfish(N=102;89femalesand13males)werecollectedfrombycatchofartisanalfisheriesintheinnerseaofChiloeIsland(41°40′S;72°39′W) at the northern part of theChilean Southern fiords area(Figure1)duringMay–August(winter)2013andDecember–February(summer) 2014. These fisheries exploit small pelagic fish speciessuchasjackmackerel,anchoveta,hake,sardineamongothers.Oncecaptured, spiny dogfish specimens were preliminarily identified bymorphologicalexamination,preservedin ice,andtransportedtotheElasmobranchesLaboratoryattheAustralUniversityofChile(UACh).Genderandweight(W)wererecorded,whilematuritystagewasde-terminedfollowingStehmann(2002).Precaudallength(PCL)andtotallength(TL)weremeasuredtothenearestgramandmillimeterforeachanimal.

2.1 | Species identification

Manybycatchspeciesofdogfisharedifficulttoidentifyowingtoex-tensivemorphological similarities. This identification problem is ex-acerbatedbythecommonfisherypracticeofremovingthehead,tail,andmostfinsfromlandedsharkswhilestillatseatoreducerequiredstoragespacefortheanimal. InordertoconfirmtheidentityofthespinydogfishspeciescapturedbytheartisanalfisheryintheinnerseaofChiloé, andassumed tobeS. acanthias,we sequenceda670-bpfragmentof thecytochromecoxidase subunit1gene (Cox1).Totalgenomic DNA (gDNA) was extracted frommuscle of 10 randomlyselected individuals using the standard phenol-chloroformmethod.Cox1sequenceswereamplifiedbypolymerasechainreaction (PCR)

using primers reported elsewhere (Fallabella, 1994; Ward etal.,2007).PCRproductswerepurifiedusingtheENZADNAPurificationKit (OMEGA). Sequencingwas performed at the Departamento deEcología—Universidad Católica de Chile. All sequences were com-pared against the GenBank nonredundant protein database usingBLASTN.Sequencesshowingsignificanthits(Evalue<.001)andhighpercentofidentity(>99%)weredownloadedfromGenBankforpos-terior analysis. Sequences were aligned using theMAFFT platformof the TranslatorX multiple sequence alignment program (Abascal,Zardoya,&Telford,2010).AlignmentswereperformedusingtheL-INS-ioption(accurateforalignmentof≤200sequences)anddefaultsettings.Amedian-joininghaplotypenetworkwasconstructedwiththe PopART program (Leigh & Bryant, 2015) to estimate the “finescaled” intraspecificrelationshipsamongChileansequencesandthereference sequences obtained from GenBank. Haplotype networkanalysesarepowerfulmethodsforintraspecificdatainrevealingmul-tiple connectionsbetweenhaplotypesand thenumberofbase-pairchangesbetweenthem(Mardulyn,2012).

2.2 | Trophic ecology

Toinvestigatehowdietaroundthesalmoncagesmayvarywithsharksize,specimensweredividedintotwoclasses—matureor immature,followingsizecutoffsprovidedinAlonsoetal.(2002).Stomachswerepreservedin10%formalinandstoredfrozenat−20°Cuntilanalyzed.Stomachcontentswerethawedandwashedwithwater.Identificationwas based on intact and remaining hard structures combinedwithgeneral shape and anatomical features of prey. Recognizable preyitems were identified to the lowest possible taxon, following theosteological guide of fishes (Fallabella, 1994) from the BiologicalCollection at theZoology Instituteof theUACh.Because the sam-plesizewasrelativelysmall,cumulativenumberofpreycurveswasused to evaluate adequacy of the sample size in our study. Thesecurveswere estimatedwith asymptoticmodels using twodifferentapproaches: (1) the rarefaction curve and (2) the trophic diversitycurvebasedonthenumberofpreyitemsandthenumberofstomachssampled (Cortés,1997;Mardulyn,2012).Therarefactioncurvewasgeneratedbymeansof theEstimateS8.2 software (Colwell, 2013),randomizingthesampleorder100times.Thereafter,theasymptoticClenchfunctionwasfittedtothesmoothedcurveS(e)=ae/(1+be),whereS(e)isthenumberofspeciesfoundpersampling-effortunit(e);a and barefunctionparameters.ThelatterwereadjustedtothedataofeachcurvebymeansofaSimplexandQuasiNewtonmethodoftheStatisticapackage(StatSoftInc.version6.0,2004)tocalculateregres-sioncoefficientsandasymptote.Thecompositionandrelativeimpor-tanceoffooditemsinthedietwereexaminedusingthethreeindicesdescribedbyHyslop(1980):(1)thenumericalindex(%N);(2)thefre-quencyofoccurrence(%O);and(3)thegravimetricindex(%W).Ifusedseparately,theseindicescanproduceabiastowardhighlyabundantitems (%O), small items (%N), or very large items (%W) (Mardulyn,2012).Toovercometheseindividualbiases,ageneralizedformoftherelativeimportanceindex(%RI)wascalculatedtodeterminethecom-binedeffectoftheseindicesoneachpreycategory(Vögler,Milessi,

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&Duarte,2009).The%RIwasplottedtoestablishthelevelofimpor-tanceofeachpreycategoryaccordingtodiscontinuitiesintheslopeof thecurve.Thecategoriesofpreyweresortedaccordingto levelofimportancewherehigh%RIvalueswereassignedtothefirstlevelofimportanceandsoforth(Vögleretal.,2009).Finally,dietbreadthwascalculatedusingLevin’sstandardizedindex,rangingfrom0to1,wherelowvaluesindicatedietsdominatedbyfewpreyitems(special-istpredators),andhighvaluesindicategeneralistdiets(Krebs,1989).

Forthedataset,asimilaritymatrixwasgeneratedusingtheBray–Curtis similaritymeasure. In addition, thenonparametric analysis ofsimilarities(ANOSIM)wasrunonthematricesusing999permutationswiththePRIMERsoftware(v6.0).Wetestedthenullhypothesisofnodifferencesinthedietcompositionbetweenthematureandimmaturefemale sharks, and betweenwinter and summer (groupingmaturitystages).Percentdataweresquare-root-transformedpriortostatisticalanalysis,whichsatisfiedassumptionsofnormality.

2.3 | Reproductive biology

ReproductionofS. acanthiasintheChiloéSeamayalsobeinfluencedbyassociationwithsalmonaquaculture.Becauseofthelownumberofmalescollectedintheareaofstudy,wefocusedinvestigationonthe89 females.A regressionanalysiswas first conducted todeter-minelength–weightrelationshipsusingthefollowingequation:

whereW is theweight (g),a and b areconstants,andL is the totallengthincm.

Thepresenceofmatureyellowoocytes(diameterover2cm)andembryoswithintheuteriwasconsideredtoindicatematurefemales(Stehmann,2002).Relationshipsbetweenmothersizeandthenumberofeggsandembryoswerethendetermined.Totallengthat50%ma-turity(TL50%)wascalculatedbyalogisticmodelfittedtothematuritydataasfollows:

whereYistheproportionofmatureindividualsandTL50%isgivenby—a/b (Nakano,Hayashi,&Nagamine,2015). Litter sizewas recordedfor pregnant females, along with sex, total length (TLpup), weight(Wpup),andvolumeoftheyolksac(V)foreachembryo.Relationshipsbetweenlength–weight, length–volumeoftheyolksac,andthesexratiooftheembryoswereexploredusingthe lm() function intheRstatisticalpackage.Thedevelopmentalstageoftheembryoswases-timatedfollowingJonesandUgland(2001).Atwo-wayANOVAwasperformedforcomparisonofthenumberofembryosamongmaturityclassesandseasons.

3  | RESULTS

3.1 | Species validation

Spinydogfishsamples(n=10)fromtheChiloéSeaweresuccessfullyamplified,sequenced,anddepositedattheNCBIundertheaccession

numbersGU191146toGU191155.ComparisonofthesesequencesagainstGenBank showed high similarity (>99%) to S. acanthias fol-lowedbyS. suckleyi(98%),S. edmundsi(93%),andS. hemipinnis(92%)(Fig.S1).Consequently,sequencesofS. acanthiasfromdifferentpartsoftheworldwereusedasreferenceinthemedian-joininghaplotypenetwork(Fig.S2).Onlyonehaplotypewassharedamongpopulationsin the Southern (Chile, Argentina, Tasmania, South Africa) and theNorthern(USA,Canada,Europe)Hemisphere.Allotherhaplotypesareinferredtohavebeenderivedfromthishaplotypebyone(e.g.,ChileanandArgentinianpopulations)or>2mutationalsteps(e.g.,UKandUSApopulations)(Fig.S2).

3.2 | Diet composition

Stomachcontentsof26immature(range46–74cmTL;mean±1SD: 57±8.1cm)and63mature(71–98cmTL;85±5.8cm)females,wereexamined,with61(68.5%)stomachscontainingfooditems.Wholeandundigestedprey itemswerefrequentlyfoundinthesamestomachsaswell-digestedprey.Therarefactioncurveandthetrophicdiversitycurveshowedawell-definedasymptote(Figure2),indicatingthatthetotalsamplesizewasadequateindescribingspinydogfishdietintheChiloéSea.Thestomachcontentoffemalesharkswascomposedoffivepreyitems(Table1),wherefishwerethemostrepresentedtaxawiththreeitems:australhake(Merluccius australis),anchovy(Engraulis ringens),andsardine(Sprattus fuegensis).M. australiswasthedominantprey, contributing thehighestvaluesof trophic indices followedbyfoodpelletsfromsalmonfarms (Table1).Accordingtothe%RI, thedietoffemalespinydogfishischaracterizedbythreelevelsofimpor-tance(Figure3a),wherehakeandpelletsaregroupedinthefirstlevel,anchovyinthesecondlevel,andthethirdleveliscomprisedofsardineandunidentifiedpreyitems(e.g.,gelatinousprey).TheLevin’sstand-ardizedindex(Ba)indicatedanarrowtrophicnichebreath(Ba=0.31)forS. acanthias in theChiloé Sea as a result of the lownumber ofpreyitemsandthehigherimportanceofaustralhakeinthedietoffe-malesharks.Therelativeimportanceofpelletsforimmaturefemales

W=a×Lbc

Y=[

1+e−(

a+b×TL

)]

−1

F IGURE  2 CumulativepreycurvesforfemalesofSqualus acanthias,theverticalaxisshowsthenumberofpreyitemsandthehorizontalaxisshowsthenumberofgutsanalyzed

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decreasedinthedietofmaturesharks,inpartduetoincreased%RIoftheaustralhake (Table1,Figure3b).However, theresultsoftheANOSIMtestindicatedthatthedietsofthesetwogroupswerenotsignificantlydifferent(GlobalR=.008,P=.399).Comparisonofstom-achcontentsduringaustralseasonsrevealedsomedifferencesinthe%RIbetweenwinterandsummer(GlobalR=.165,P=.044;Table1,Figure3c). For instance, duringwinter, anchovy and salmon pelletsrepresent 93% of the prey contribution to the %RI, while in sum-mer,hakeandpelletsarethemostimportantitemswith~74%ofRI(Table1,Figure3c).Nodifferences in thenumberof immatureandmaturefemalesweredetectedbetweenseasons(χ2 =1.9,P=.59).

3.3 | Reproductive development and fecundity

FemalesofS. acanthiascollected intheChiloéSearanged in lengthfrom 46 to 98cm (mean±1 SD: 79±11cm). Their weights variedbetween 0.7 and 4.9kg (mean±1 SD: 2.4±1kg). The relationshipbetween these two variables revealed a positive allometric trendfor somatic growth (Figure4a, R2 = .915; P<.001). The estimatedlengthat50%maturitywas72.8cm(r=.998,Figure4b).Thenumberofoocyteswithadiameterover2cmvariedbetweenthreeand17(mean±SD: 7.67±2.8oocytes), andno significant relationshipwasdetectedbetweentheTLofmaturefemalesandthenumberofeggs

TABLE  1 DietcompositionoffemalesSqualus acanthias

Total Stages of development Seasons

Stomach contents %O %N %W %RI %RI mature %RI immature %RI winter %RI summer

Unidentified 13.1 5.2 1.5 0.93 0.7 1.1 0.8 7.3

Pellet 57.4 30.9 26.6 35.5 14.4 35.8 48.6 37.7

Hake 49.2 40.4 52.4 48.9 74.5 43.0 4.5 36.4

Anchovy 37.7 15.7 13.1 11.6 1.0 7.7 44.4 3.2

Sardine 19.6 7.8 6.5 3.1 9.4 12.4 1.7 15.4

F IGURE  3 TrophicecologyinfemalesofSqualus acanthias.(a)Relativeimportance(%RI)offunctionalpreycategories.Verticallinesindicatethethreelevelsofimportanceofthefunctionalpreycategoriesaccordingtodiscontinuitiesintheslopeofthe%RIcurve.Comparativeanalysisoftherelativeimportance(%RI)between(b)maturitystagesand(c)seasons

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(Figure4c,R2 = .002; P<.91).Ontheotherhand,thenumberofem-bryosshoweda lowbutpositive relationshipwith theTLof the fe-malesharks(Figure4d,R2=.348;P<0.012)rangingbetweenoneand12embryosper female (mean±SD:5.52±3.1).A totalof274em-bryoswerefoundwithlengthsbetween3.6and25.8cm(mean±SD: 12.7±5.7cm) and weights between 2.5 and 40.8g (mean±SD: 12.3±11.9g).Therelationshipbetweentotallength(TLpup)andweight(Wpup)ofpupssuggeststhatembryosincreaseinweightexponentially(Figure5a,R2 = .913; P<0.001)andastheygrowtheweightoftheiryolksacdecreases(Figure5b,R2 = .385; P<0.001).Thesexratiooftheembryoswaseven(1:1),andsignificantdifferencesweredetectedinthenumberofembryosatthesmallermaturesizeclassesbetweenseasons(Figure5c,two-wayANOVA;F10,251=1.96,P=.038).

4  | DISCUSSION

Interactionsbetweenmarinepredators andhumans arise in coastalecosystems invariouspartsof theworldwhere feedingand repro-duction sitesoverlapwitheconomic activities suchas fisheries andaquaculture.Theseinteractionsinclude(1)mortalitythroughbycatch;(2) deliberate harming of animals by humans; (3) changes in animalbehavior;(4)competitionforthesameresource;(5)changesinpreysizestructureanddistribution;and(6)changesincommunitycompo-sitionresultingfromfishingorfromaquaculture-inducedalterationsoftheenvironment(DeLaTorrienteetal.,2010;Guilpartetal.,2012;Kemper etal., 2003; Kutti etal., 2007; Papastamatiou etal., 2010;Ribeiroetal.,2007;Sepúlveda&Oliva,2005;Sepulvedaetal.,2007;

F IGURE  4 ReproductivebiologyofSqualus acanthiasfemalesintheChiloeSea.(a)Allometricrelationshipbetweenthetotalweightandtotallengthofspinydogfish.Relationshipsbetweenlengthoffemalesharksand(b)thepercentageofmaturefemales,(c)thenumberofeggsand(d)thenumberofembryos

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Veríssimoetal.,2010).Giventhehighdensityofhuman-relatedac-tivities(e.g.,fisheriesandintensiveaquaculturesystems)intheChiloéSea(Oddoneetal.,2015),stronginteractionswithspinydogfishpopu-lations(DeLaTorrienteetal.,2010;Valenzuelaetal.,2008)werepos-sible.OurresultsshowthatthespatialoverlapofsharknurseryareasandthesalmonindustryinfluencesthetrophicnicheofS. acanthias in thisregionbyaddingnewfooditems(i.e.,pellets;directeffect),andalteringthestructureandcompositionofbenthiccommunities (e.g.,crustaceans;indirecteffect).

4.1 | Genetic validation of Squalus acanthias

AlthoughsharksarenotthetargetspeciesinmanyChileanfisheries,practicessuchasdriftnettingandlonglinefishingresultinhighlev-elsofsharkbycatch(Lamillaetal.,2005;Valenzuelaetal.,2008).Theaccurateidentificationofspeciesofsharksobtainedfrombycatchisdifficultbecausesomespeciesdifferonlyslightlyintheirmorphologyandalsobecauseinsomecases,head,tail,andmostfinsfromlandedsharks are removed at sea tominimize storage needs and prevent

spoiling(Shivjietal.,2002).Therefore,differentmolecularapproacheshave been recommended for shark species identification. One ap-proach isDNAbarcodingusinguniversalprimers toamplifypartoftargetgenes(Holmes,Steinke,&Ward,2009;Wardetal.,2008).Thisapproach is similar to the traditional morphology-based methods,wherespeciesidentificationisbasedonthepresenceorabsenceofadistinctmorphological feature (Viswambharanetal.,2015). Inourstudy,theuseofthecytochromecoxidasesubunit1gene(Cox1)asabarcodingsequenceallowedusforthefirsttimetounambiguouslyidentifyS. acanthiasasthedogfishspeciesinthebycatchofartisanalfisheriesintheregion.Aswithotherreportedbarcodingstudies,theCox1regioniseffectiveindelineatingsharksuptothespecieslevelwithin the genusSqualus (Ward etal., 2007).Despite the relativelylownumberof individualssequencedinthisstudy,wewereabletoidentify10closelyrelatedhaplotypes intheseChileandogfish.OneofthesehaplotypeswassharedmainlywithotherS. acanthiaspopula-tionsintheSouthernHemisphere.Theseresultsareconsistentwithapreviousglobalphylogeographicstudy, inwhichtheSouthPacificandAtlanticlineageofS. acanthiaswascharacterizedbyastar-shaped

F IGURE  5 Relationshipbetweenthetotallengthsofpupswith(a)theirbodyand(b)yolksacweights.(c)Changesindistributionofthetotallengthsofpupsbetweensummerandwinter

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networkwith one very common, central haplotype and several de-rivedandlow-frequencyhaplotypes(Veríssimoetal.,2010).

4.2 | Trophic ecology of Squalus acanthias around salmon farms in Chile

Theoverall dietofS. acanthias females in theChiloéSea consistedmainlyofteleostfishes.However,commercialsalmonfeedswerepre-sentinthestomachsoffemaledogfish.Thisisthefirstrecordofthishigh-energyfooditem(24–30%oil,~19kJg−1)inS. acanthias,whichdiffersfromthenaturaltrophiccompositionforthisspeciesinotherregions of the world (Alonso etal., 2002; Avsar, 2001; Demirhan,Seyhan,&Basusta,2007;Dunnetal.,2013;Hanchet,1991;Holden,1966;Laptikhovsky,Arkhipkin,&Henderson,2001;Tanasichuketal.,1991). Salmon farms are known to affect the surrounding environ-mentand localmarinecommunitiesbytheadditionoforganicmat-terintheformoffecesanduneatenfeed(Kuttietal.,2007;Soto&Norambuena, 2004).Organicwaste from salmon cages produces ahighconcentrationofammoniainthelocalenvironmentthatcanleadtodecreasedgrowth, increasedvulnerability todiseasesandpatho-logicalchangesinanimalsinhabitingsalmonfarmingareas(Piedecausaetal., 2010).Here, the presence of salmon pellets in the diet ofS. acanthiasraisesconcernaboutthepotentialimpactofsalmonfarmson the biology ofmarine predators, their trophic relationships, andecosystem function in this region. For example, the use of corn orsoybeanincommercialpelletsprovidesahighproportionofoleicacid(18:1n-9),linoleicacid(18:2n-6)andlinolenicacid(18:3n-3),thatalterthefattyacidcomposition,physiologyandbehavioroffishandmus-selsfromareasinthevicinityofsalmonfarms(Redmondetal.,2010;Skogetal.,2003).Inaddition,theintensivenatureofsalmonfarminghas resulted in the use of antibiotics (e.g., oxytetracycline), to limitbacterialandprotozoaninfections(Primavera,2006).Theseantibiot-icsareadministeredwiththefeedpelletsandhavethepotentialtobeaccumulatedintissuesofmarineorganismsaroundthesalmoncages(Campbell,Pantazis,&Kelly,2001).Excessiveuseofantibioticsresultsinthedevelopmentofsideeffects,suchasliverdamage,toxiceffects,bacterialresistance,andimmunesuppression,andcanhavenegativeeffects on environmental quality and human health (Nakano etal.,2015;Zounkováetal.,2011).InChile,salmonaquacultureuses8000timesmoreantibioticsthaninothercountries(e.g.,Norway),andthepresenceoftetracyclines(usedforproteininhibition)andquinolones(fastbactericideaction)hasbeendocumentedinnativespeciessur-roundingthesalmonpensoftheChiloéSea(Buschmannetal.,2009;Outeiro&Villasante,2013;Soto&Norambuena,2004).

BeyondthepresenceofsalmonpelletsinthedietofS. acanthias in Chile,thetrophicecologyofthissqualoiddifferscomparedtopopu-lationsinotherregions.Overall,spinydogfisharedescribedasafairlynon-selective(i.e.,generalist)andopportunisticpredatorthatfeedsonmanypelagicandbenthicspecies(Alonsoetal.,2002;Belleggiaetal.,2012).However, female spiny dogfish inChile showed a functionalspecialist behavior as indicated by the small number of prey itemsand the relative high importance of the austral hake and pellets inthediet.ThetrophicnicheofS. acanthiaspopulationsintheNorthern

Hemisphere, theSouthwestAtlantic, andSouthwestPacific is char-acterizedbyagreatdiversityof teleost fishes (e.g.,hakes, sandeels,gobies, herrings, anchovies, andwhiting), crustaceans (e.g., shrimps,crabs,lobsters,andeuphausiids),mollusks(e.g.,squidsandoctopuses),and other invertebrates (e.g., salps, sea anemones, nematodes, andpolychaetes)(Alonsoetal.,2002;Avsar,2001;Belleggiaetal.,2012;Demirhan etal., 2007; Dunn etal., 2013; Hanchet, 1991; Holden,1966;Laptikhovskyetal.,2001;Tanasichuketal.,1991). Inmostofthesestudies,demersalandpelagicfishwerethemaincomponentofthediet,whichisconsistentwithourresult.However,intheseregions,S. acanthiasfeedsonotherimportantpreyitemssuchascrustaceansandmollusksthatareabsentinthedietofsharksintheChiloéSea.One plausible explanation of this discrepancy could be the lowoc-currenceofthesepreysnearsalmonfarmsinSouthernChile.Infact,oneofthemostdrasticenvironmentaleffectsoforganicwastefromsalmonaquacultureisthealterationofthestructureandcompositionofbenthiccommunities (Guilpartetal.,2012;Kuttietal.,2007)andparticularlythereductionsinbiodiversityofcrustaceansinthevicinityof salmon cages (Buschmannetal., 2009;Hall-Spencer etal., 2006;Outeiro&Villasante,2013).

Fromanontogeneticperspective,S. acanthias exhibits substan-tialvariationindietwithbodylength(e.g.,Alonsoetal.,2002;Avsar,2001;Demirhanetal.,2007;Dunnetal.,2013;Hanchet,1991).Thisisconsistentwithour results inwhichsmaller immaturesharks fedmoreonpelletsandanchoviesthanlargermaturefemales.Theonto-genetictransitionfromsmallpreyitems(e.g.,crustaceans,salps,otherinvertebrates)tofishfeedingisatypicalfeatureofthetrophicecologyofS. acanthias (Dunnetal.,2013;Tanasichuketal.,1991).Anothersourceofvariationinthedietcompositionofthespinydogfishissea-sonality (Belleggiaetal.,2012;Demirhanetal.,2007;Laptikhovskyetal.,2001;Tanasichuketal.,1991).Here,despiteofthepresenceofsalmonpelletsinthestomachsoffemalesharksthroughouttheyear,significantdifferencesweredetectedbetween summerandwinter,againconsistentwithother studies that showspinydogfish switchpreytotakeadvantageofspatiotemporalchangesinpreyavailability(Demirhanetal.,2007). IntheChiloéSea,sharkspreymoreonan-choviesduringwinterandhakesduringsummer,whichisconsistentwiththe localavailabilityof thesepreyfishesduetotheirseasonalmigration(Aguayo-Hernández,1995;Lealetal.,2011).

4.3 | Biological parameters

Therelatively largersizeand increasedresidence timeof fishesnearsalmonfarmscomparedwithfishesfromotherareasisexplainedbytheavailabilityofhigh-energypellets fromsalmoncages,asdocumentedinmany species elsewhere (Skog etal., 2003).Hence, differences inbiologicalparametersforspinydogfishintheChiloéSeacomparedtopopulationsnotinfluencedbysalmonidaquaculturemightbeexpected.However,ourresultsdidnotsupportthehypothesisofdifferentrepro-ductiveandgrowthpatternsinspinydogfishinChiloe.ThesizerangeofimmatureandmaturefemaleswassimilartosouthwestAtlanticpopu-lations(Alonsoetal.,2002;Nakanoetal.,2015),andslightlylowerthanNorthernHemisphere populations (Avsar, 2001;Gračan etal., 2015;

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Jones&Ugland,2001;Yigin&Ismen,2013),butwithpositiveallomet-ric trends for somaticgrowth (TL~W) inallof thecases.Thesizeat50%maturityofS. acanthiasintheChiloéSea(72.8cm)washigherthanreported for theMediterranean Sea (51.8–52.8cm) (Chatzispyrou&Megalofonou,2005;Yigin&Ismen,2013),similartoSouthwestAtlantic(70.4cm) (Nakanoetal.,2015)andSouthwestPacific (71.5–74.0cm)populations(Hanchet,1988),andlowerthanintheBlackSea(88cm)(Avsar, 2001), the Northeast (77–78.2cm) (Henderson, Flannery, &Dunne, 2002; Stenberg, 2005) and Northwest (79.1cm) (Sosebee,2005)Atlantic,andBritishColumbiawaters(93.5cm)(Ketchen,1972).Althoughregionaldifferences inTL50%occur, there isnoevidenceofparticulareffectsof localconditions (i.e., salmonfarming)onthispa-rameter of S. acanthias in theChiloé Sea. Fecundity (i.e., number ofeggsandembryos)wasalsowithin thevaluesdescribed forS. acan-thias elsewhere in theworld,with an increasingnumberof embryosin largermaturefemales,andnodifferences intheirsexratio (Avsar,2001;Jones&Ugland,2001;Ketchen,1972;Nakanoetal.,2015).TherangeoftotallengthofembryosexaminedencompassesthetheoreticalcurveproposedbyAvsar(2001)forthisspeciesandfollowstherela-tionshipforembryoweightdescribedinotherregions(Chatzispyrou&Megalofonou,2005;Yigin&Ismen,2013).Thelowfrequencyofmalesinourstudysuggeststhatspatialsegregation(latitudeanddepth)ofsexmayoccur,whileimmatureandmaturefemalesspatiallyoverlappedinallseasons(Nakanoetal.,2015).

Overall,despitedifferencesinthetrophicpatternsinthisregion,from this study, there appears to be no demographic expression intermsof fecundityor sizeatmaturityof these trophic changesdueto the spatial association with intensive salmon farming.We havenotcalculatedthecalorificvalueofthediet,andwesuggestthatthisvariableisneededinoneadditionalcomparativestudywithsamplesfromotherregionsthatarenotinfluencedbysalmonfarming,totestanydietenrichmenthypothesis.Itmayalsobenecessarytoexaminetheeffectofanychemicals in theenvironmenton the reproductiveperformanceofthesharks.Ifthisinnerseaistheonlydogfishnurseryareaintheregion,thenareproductivecomparisonmaynotbepossi-ble,andglobalcomparisonsofdietaryenergyandreproductiveoutputwillbeneeded.Weconcludethatatthisstage,theintensivesalmonaquacultureoftheChiloéSeaisinfluencingthedietofS. acanthias,butwithoutanimpactonsharkreproduction.

ACKNOWLEDGMENTS

ThispaperisdedicatedtothememoryofProfessorJulioLamilla,oneoftheco-authors,whopassedawayduringthedevelopmentofthisarticle,onMarch31,2016.Theauthorsexpresstheirthankstothefishery creek of Anahuac. JDGE was supported by a CSIRO-OCEpostdoctoralfellowship.LCwassupportedbytheMillenniumNucleusCenterfortheStudyofMultipledriversonMarineSocio-EcologicalSystems(MUSELS)-MINECONProjectNC120086.

CONFLICT OF INTEREST

Nonedeclared.

AUTHOR CONTRIBUTION

LC, JDGE,DG, and JL conceivedanddesigned the study.DG sam-pledthebiologicalmaterialandcarriedoutDNAextractions.JD,DG,AH, and RD performed data analyses. JD, AH, and LC drafted themanuscript.Allauthorsread,approved,andcontributedtothefinalmanuscript.

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How to cite this article:Gaitán-EspitiaJD,GómezD,HobdayAJ,DaleyR,LamillaJ,CárdenasL.SpatialoverlapofsharknurseryareasandthesalmonfarmingindustryinfluencesthetrophicecologyofSqualus acanthiasonthesoutherncoastofChile.Ecol Evol.2017;7:3773–3783.https://doi.org/10.1002/ece3.2957