Foraminifera as bioindicators of water quality: The FoRAM Index revisited

EnvironmentalPollution257(2020)113612

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journalhomepage: www.elsevier.com /locate/envpol

Foraminiferaasbioindicatorsofwaterquality:TheFoRAMIndex revisited*

MartinaPrazeres a, *,MichaelMartínez-Colon b,PamelaHallock c

a MarineBiodiversityGroup,NaturalisBiodiversityCenter,Leiden,Netherlands

b SchooloftheEnvironment,FloridaA&MUniversity,Tallahassee,FL,USA

c CollegeofMarineScience,UniversityofSouthFlorida,St.Petersburg,FL,USA

articleinfo

Articlehistory: Received31July2019

Receivedinrevisedform 10November2019

Accepted10November2019

Availableonline11November2019

Keywords: Benthicforaminifera Coralreefs Monitoring Environmentalassessment abstract

1.Introduction

Coralreefsworldwidearedegradingatalarmingratesduetolocalandglobalstressors.Thereare ongoingneedsforbioindicatorsystemsthatcanbeusedtoassessreefhealthstatus,thepotentialfor recoveryfollowingdestructiveeventssuchastropicalstorms,andforthesuccessofcoraltransplants. Benthicforaminiferalshellsareubiquitouscomponentsofcarbonatesedimentinreefenvironmentsthat canbesampledatminimalcostandenvironmentalimpact.Herewereviewthedevelopmentand applicationoftheFoRAMIndex(FI),whichprovidesabioindicatormetricforwaterqualitythatsupports reefaccretion.Weoutlinethestrengthsandlimitationsofthe FI,andproposehowitcanbeappliedmore effectivelyacrossdifferentgeographicalregions.

© 2019ElsevierLtd.Allrightsreserved.

Acrosstheglobe,coralpopulationsandreefaccretionhave declinedastheenvironmentalconditionsuponwhichtheydepend havebeendegradedbyanthropogenicactivities.Asaresult,reef managementagenciesarefacedwiththechallengeofmanaging resourcestoincreasetheresilienceoflocalreefstothebroaderscaleenvironmentalchanges.Becauselong-termtrendsinphysicalandchemicalparametersofreefenvironmentscanbesubtle andmaskedbydiurnal,seasonalandinterannualvariability,the necessityforbioindicatorsthatintegrateenvironmentalconditions waswidelyrecognisedbythemid-1990s(e.g. Crosbyetal.,1995; Jacksonetal.,2000).

Evidenceforbothsubtlechangesanddramaticlossesincoral reefcommunitiesemergedinthe1970s.AroundtheCaribbean, growinghumanpopulationswereclearinguplandsforagriculture andcoastlinesforurbanandtouristdevelopment(e.g. Rogers, 1990;Ogden,1996).Withincreased fishingpressureonreef fish, theseaurchin(Diademaantillarum)populationsexploded, increasingreefbioerosion(e.g. Hay,1984;Sammarco,1982),while minimisingmacroalgalgrowth;followedbythediseaseoutbreak

* ThispaperhasbeenrecommendedforacceptancebyDr.SarahHarmon.

* Correspondingauthor.

E-mailaddress: martina.prazeres@naturalis.nl (M.Prazeres).

https://doi.org/10.1016/j.envpol.2019.113612 0269-7491/© 2019ElsevierLtd.Allrightsreserved.

andmassivedieoffoftheurchinsin1983,thathasalloweduncheckedmacroalgalproliferation(Lessios,2016).Ontheclearest offshorereefs,white-banddiseasewasdestroyingstandsof Acropora spp.(Antonius,1977;Bruckner,2016).InthePacific, Crown-of-Thornsstarfish(Acanthasterplanci)outbreakswerebeing documented(e.g. Sapp,1999,andreferencestherein).InKaneohe Bay,Oahu,Hawai’i,inthe1960sand1970s,coralreefswerebeing takenoverbymacroalgaesuchas Dictyospheria.Concernforthe reefspromptedaground-breakingstudyoftheinfluenceof nutrientpollutiononthereefcommunities(e.g. LawsandRedalje, 1979,1982;Smithetal.,1981).Thesestudiesfound,whilemonitoringsewagedischargeintothebay,dissolvedinorganicnitrogen wasrapidlytakenupbyphytoplankton,andthatchlorophyll a,and particulatenitrogenweremuchmoresensitiveandwidelyapplicableindicesofnutrientenrichmentthaninorganicnutrientconcentrations.Moreover,becausethetimeframeforinfluenceon benthicbiomassandcommunitystructureismuchlongerthanfor responseswithinthewatercolumn, LawsandRedalje(1982) concludedthatthebenthosmustbeconsideredwhenassessing theimpactofsewagepollution.

Ataboutthesametime,changesinassemblagesofbenthic foraminiferalshellsinsedimentswererecognisedasindicativeof nutrientpollutionintropicalcoastalwaters(e.g. Hirshfieldetal., 1968;Seiglie,1968,1971).Benthicforaminiferaareprotists,many ofwhichbuildcalciumcarbonateshells,andareacrucial

componentofthebenthos(Murray,2014).Whileassessingimpacts ofatomictestingatEnewetakAtollinthecentralPacific, Hirshfield etal.(1968) observedthatforaminiferalassemblagesweredominatedbylargerbenthicforaminifera,exceptinthevicinityofthe researchlaboratoryoutfall,whereother,fast-growing,smaller, heterotrophicforaminiferaltaxadominated.Insubtropicaland tropicalreefenvironments,reef-associatedlargerbenthicforaminiferaaregenerallydominant,astheyutilisealgalendosymbionts, andhaveenvironmentalrequirementssimilartothoseofreefbuildingcorals(e.g. Hallock,1999;PrazeresandRenema,2019). Moreintenseanthropogenicactivities,suchasagriculturaland sewagepollutionininshorebaysandlagoons,canproducemore extremeconditionsthatbenefitafewstress-tolerantforaminiferal taxa(e.g. Seiglie,1968,1971;Culver,1990;CulverandBuzas,1995). Therefore,foraminiferalabundances,diversityandassemblagelevelsensitivitytoshiftsinwatertransparencyandfoodsources, combinedwiththeirproductionofsand-sizedshellsthataccumulateinthesediments,makeforaminiferanaturalbioindicators ofwaterqualityincoastalenvironments(Alve,1995;Schaffer, 2000).

Theseobservationsofchangesinforaminiferalassemblages withthegradientofavailableorganicmatterprovidedthebasisof the ForaminiferainReefAssessmentandMonitoring (FoRAM)Index of Hallocketal.(2003).TheFoRAMIndex(FI)wasbasedonstudies ofnorthwestAtlanticandCaribbeanforaminiferalassemblagesin sedimentsamples,especiallyPuertoRicoandtheFloridaReefTract. The FI wasdesignedtoprovidearelativelysimple,low-costmetric toassessthepotentialforassociatedhard-bottombenthicenvironmentstosupportcalcifyingorganismsthathostalgalsymbionts,includingreef-buildingcorals.Thisindexisbasedon sedimentsamples,inwhichproportionsofshellsofthreefunctionalgroupsofforaminiferaaredetermined:(1)thelargerbenthic foraminifera(LBF),(2)fast-growing,smallerheterotrophictaxathat requirewell-oxygenatedconditions(OSF),and(3)stress-tolerant taxa(STF)thatcantolerateintermittenthypoxia.The FI was developedtobeacoral-independentmeasuretoprovideresource managerswithameansofassessingwhetherwaterqualityissufficienttosupportreefgrowthorrecoveryaftermajorstressor coral-mortalityevent.Therefore,the FI isnotapplicableinenvironmentsdominatedbymudorvery finesandparticles(<125 mm), suchasestuaries,whereSTFwouldlikelydominate(Hallocketal., 2003).

Sinceitspublicationin2003,the FI hasbeenappliedtoreef environmentsworldwideandhasalsobeenutilisedinmarginal andnon-reefalenvironments(e.g. Hallock,2012,andreferences therein).However,modificationsoftheoriginalmethodsmake comparisonsacrossstudieschallengingand,insomecases, impossible.Moreover,the FI thresholdsproposedforsediments fromtheCaribbeanregioncannotbeassumedtobeappropriate undermodifiedmethodsorinotherbiogeographicregions,suchas thePacificOceanorsouthwestAtlantic.Therefore,wepropose furtherstandardisationofthe FI,andproviderecommendations thatwillallowthebroadapplicationofthisindexasapowerfultool inassessmentandmonitoringprogramsinreefenvironments worldwide.Atthesametime,wenotethatsomevariationsin methodscanprovideusefuldataforlocalstudies,andwefurther explainwhythe FI isnotappropriateinsomesubregionsandreef environments.

Theobjectiveofthispaperistoclarifyandfacilitatetheuseof the FI inreefassessmentprogramsworldwide.Ourgoalsareto:(1) discussexamplesoftheuseandapplicationofthe FI,(2)address importantconsiderationsontheuseofthe FI suchassizerangeof specimensexaminedasafunctionofsieve-meshsizeusedin sampleprocessing,replicatesamplingandnumbersofspecimens counted,live(stained)versustotalassemblages,sedimenttextures,

depthofsamplecollection,andultimately,interpretationof resultingdata;and(3)proposestandardisedprotocolsforthe FI withthegoaltoimproveitsapplicabilityforcoralreefsinthePacific Oceanandotherreefareas,andtoproducecomparabledatathat willleadtowiderapplicabilityofthe FI inassessmentofwater qualityandmonitoringprograms.

2.Theneedforbioindicatorsinshallowcoralreef environments

Inthe1960sintothe1990s,localimpactssuchasincreased terrigenoussedimentationassociatedwithlandclearing,and nutrientinputfromagriculturalrunoffandever-increasinghuman populationsincoastalregions,aswellasoverfishing,were consideredthemajorcausesofreefdegradation(e.g. Pandolfi etal., 2003;Ramos-Scharron,2010;Burkeetal.,2011).Widespreadcoral bleachingeventsassociatedwithstrongElNinoconditions occurredin1982 83and1987,buttheconsequencesofocean warmingwerenotfullyrecogniseduntil1997‒98,whenanElNinoassociatedwarmingofsurfacewatersproducednearlycircumtropicalbleachingandwidespreadcoralmortality.Eventhough oceanwarmingisnowconsideredthemajorthreatfacingcoral reefsworldwide(Hughesetal.,2017),improvementsinwater qualityareconsideredparamounttotheresiliencyandrecovery ratesofreef-buildingcoralsafterbleachingevents(Wooldridgeand Done,2009;MacNeiletal.,2019).

Reefassessmentandmonitoringprogramsgenerallyare designedtoidentifythecausesandconsequencesofthelossof coralcoveronreefs.Thepotentialtodistinguishbetweenlocal(e.g. terrestrialrunoffandnutrification)andglobal(e.g.oceanwarming andacidification)stressors,aswellaspossiblewaystoreducelocal impactstoincreaseresiliencetoglobalstressors,havebecome increasinglyimperative(e.g. Bellwoodetal.,2004;Marshalland Schuttenberg,2006).Interestintheapplicabilityofspecificmarineorganismsasbioindicatorsinresourcemanagementemerged inthe1990s(e.g. Crosbyetal.,1995),withthegoaltoidentifyorganismsthatrespondtoenvironmentalstressorsinarelatively predictablemanner.Samplecollectionshouldberelativelylowcost andhaveminimalimpactonreefresources(Crosbyetal.,1995; Jacksonetal.,2000).Whiledirectmeasurementofwaterquality canprovideinformationabouttheconditionofthewatercolumnat thetimeofsampling,bioindicatorscanprovideatime-integrated measure(daystoyears)oftheeffectsofenvironmentalchanges (Cooperetal.,2009).

‘Waterquality’ isatermcommonlyusedtodescribethe chemical,physical,andbiologicalcharacteristicsofwater,usually concerningitssuitabilityforaparticularpurpose(e.g.drinking, swimming, fishing).However,whattheterm ‘waterquality’ indicatescanbedependentuponavarietyofenvironmentalparameters(e.g.coastalproximity,seasonality,andweather/climate). Incoastalsurfacewaters,waterqualityisrelatedtonutrient flux, whichlargelycontrolsthebiomassofplanktoninthewatercolumn andthereforelightpenetration,foodsupply,benthiccommunities, andbiological-oxygendemandatthesediment-waterinterface. Inspiredbytheobservationsof Hirshfieldetal.(1968) fromEnewetak,resultsfromKaneoheBayreportedby Smithetal.(1981) and LawsandRedalje(1979,1982),studiesofbioeroderabundancesby Highsmith(1981),andpersonalobservationsonboth Indo-PacificandWesternAtlanticreefs,Hallockproposedthat benthic-communitycompositionandcarbonate-sedimentconstituentscanprovideusefulintegratedsignalsofwaterquality(e.g. HallockandSchlager,1986;Hallock,1987,1988a).

Forcoralreefs,appropriatebioindicatorscanprovidespatialand temporalmeasuresoftheeffectsofwaterqualitychangesfrom

periodsofdaystoyears,eventodecadalandmillennialscaleswith theuseofsedimentcores.Asnotedthreedecadesagoby Hallock (1987,1988a) and WilkinsonandCheshire(1988),pre-impact baselinescandifferwidely,evenamongwhatwerethrivingreef systems.Coralreefecosystemsoccuracrossgradientsoftemperatureandsalinity,aswellasvaryinginfluencesofterrestrialrunoff orupwelling,bothofwhichinfluencewatertransparencyand benthiccommunitystructure(Hallock,1987;1988a).Althoughno singlebioindicatorcanbe “ideal” inallcircumstances, Dauvinetal. (2010) explainedthatanenvironmentalindicatorgathersabiotic informationbasedonthebioticandecologicalresponsesofthe organismsstudiedfromauniqueecosystemsetting.Similarly, Hu etal.(2019) arguedthatbioindicatorsareeffective insitu tools neededbyresourcemanagerstoprovidekeyinformationonthe causeandeffectofpollutantsandothermarinestressors.Thus, effectiveenvironmentalmanagementrequiresresearch-based monitoringprogramsthatusebothdirectmeasurementsandbioindicators,thelatterlinkingchangesinwaterqualityparameters withtheconditionofcoralreefecosystems(e.g. Hallocketal.,2004; Cooperetal.,2009;Oliveretal.,2014).

3.Challengesindevelopingsuitablebioindicators

Akeycategoricalaspectofamarinebioindicatorisitsresilience and fitnesstowardsshortand/orlongtermsenvironmental changes. BonannoandOrlando-Bonaca(2018) clearlyarticulated thatabioindicatormustbeabletocapturetheaccumulatedeffects ofpertinentstressorsbeforebiologicalfunctionalityisaltered, whichultimatelywouldhinderanorganismscapacitytorespond predictablytoenvironmentaldisturbances.Thisresonateswith majorchallengesindevelopingabioindicatorinreefecosystems giventhecomplexityofhabitatsandabioticconditionsfoundin theseenvironments.Asnotedby Adey(1978),Kinsey(1981), WilkinsonandCheshire(1988),and Birkeland(1988,1997),reefbuildingcoralsoccuracrossbroadregionalnutrientgradients, fromextremelynutrientdeplete,oligotrophicsubtropicalPacific gyres,totheintermediateconditionsofthesubtropical/tropical northwesternAtlantic/Caribbean/GulfofMexico(henceforth referredtoastheCaribbeanregion),totheupwelling-influenced easterntropicalPacificand fluviallyinfluencedIndonesianand Brazilianreefs. Hallock(1988a) discussedthisgradientsemiquantitativelybasedonwatertransparency,estimatedbythe depthtowhich~1%ofsurfacesolarirradiancecanpenetratesufficientlyandconsistentlytosupportsomezooxanthellatecorals: oligotrophic(>100m),mildlyoligotrophic(~50 100m),mesotrophic(~30 50m),mildlyeutrophic(~10 30m),andeutrophic (<10m).

Fullrecognitionofthesignificanceofregional-scalegradients washistoricallyimpededbyhighcoralcoveracrosssuchregions, especiallyatwaterdepths <10m.Recognitionwasalsohinderedby thepotentialofafewspeciesofcorals,undersuitableenvironmentalconditions,toproducereefswithveryhighcoralcover, thoughwhentheyexperiencedsignificantmortality,bio-erosion andchemicaldissolutiondestroyedreefframeworkatratesthat farexceededaccretionrates(e.g. Smithetal.,1981;Glynn,1988). Highsmith(1981),examiningmuseumspecimensfromdifferent regions,notedsuchgradientsintheabundanceofbio-erodingbivalvesincoralspecimens. Hubbard(1985) suggestedthatbioerosionratescouldexplaindifferencesinaccretionratesbetween PacificandAtlanticreefs.

Directproductionandbioerosionratescanchangealongthose gradients,andalsoalongmorelocalonshore-offshoregradients (Hallock,1988a,2001).Othermembersofthereefcommunities respondtothesamegradients,andassociatedsedimentscan reflectthosedifferences.Incoralreefscharacterisedbyveryclear,

nutrient-poorwaters,LBFarecommonlythedominantproducers ofcarbonatesandandgravel-sizedsediments(e.g. Chapman,1900; Wells,1957;Hallock,1988a).Asnutrientinfluxincreases,bioerodedcoralfragments,calcareousalgalandmolluscandebris (LidzandHallock,2000),andsmallerherbivorousanddetritivorous foraminifera(OSF)becomemorecommoninthesediment (Hirshfieldetal.,1968;Cockeyetal.,1996;Ebrahim,2000;Renema, 2002,2008).Onlyinquiterestrictedlagoonalorestuarineenvironments,inorganic-richanddysoxicconditions,willSTFtaxa thrive;fewifanystonycoralscansurviveinsuchconditions(e.g. Hallocketal.,2003;Carnahanetal.,2009;Cardinietal.,2014; Martínez-Colonetal.,2018).Asaresult,shiftsinbenthicforaminiferalassemblagesfromLBF-dominatedtoOSF-dominatedhave beendetectedandcorrelatedtoincreasesinnutrientinputtocoral reefenvironments.Somecommonreef-dwellingbenthicforaminiferalformsareillustratedin Fig.1

Mostreefmonitoringprogramsinvolveintensive fieldwork, measurementofphysicalandchemicalparameters,andphotographyofbenthictransects,followedbyequallyintensivevisualand digitalanalysesofcommunitycomposition.Unfortunately,while reefmacrobenthoscanrespondquicklytomajorevents,theymay respondslowlytoadeclineinwaterquality,aprocessnow commonlyreferredtoas “phaseshift” (e.g. Done,1992).For example,adultcoralscanpersistunderconditionsthatpreclude recruitment,asbenthicalgaeandspongesproliferateonhard substrata(e.g. Birkeland,1977,1988). Lidzetal.(1985) notedthat thesedimentconstituentsoftheFloridaReefTractreflectmicroandmeiobenthiccommunitystructure,and LidzandHallock (2000) observedincreasedproportionsoffragmentsof “reef rock”,coral, Halimeda,andmolluscshellsconsistentwiththe declineincoralcover. Cockeyetal.(1996) similarlynotedthat proportionsofOSFshellsinFloridareefsedimentshadincreased comparedtoLBF,whichprompted Hallocketal.(2003) topropose thatforaminiferalshellsinthesedimentscanbequantifiedtoyield acommunity-levelbioindicatorofaccretionpotentialinreefenvironments.Relativelysmallsamplescanbecollectedinconjunctionwithestablishedmonitoringprogramswithminimal additionalexpenseandenvironmentalimpact.However,processingandfullyevaluatingsedimentconstituentsorforaminiferal assemblagesinsamplesinthelaboratoryrequirestimeand expertisecomparabletootherassessmentprotocols(e.g. Hallock etal.,2004).Thus,theobjectiveofthe FI wastoprovidean approachthatwouldberelativelyrobustyetreducetheamountof timeandtaxonomicexpertiserequiredforroutineevaluationof foraminiferalassemblagesinreefsediments(Hallocketal.,2003).

4.ThedevelopmentoftheFoRAMIndex

Inreefecosystems,benthicforaminiferaareparticularlyuseful becausetheirlifespans(afewweeksupto~2years)arerelatively shortwhencomparedtoreef-buildingcorals.Assuch,theresponse tochangesinnutrient flux,forexample,canbedetectedinshorter timeframes(Crevisonetal.,2006)whencomparedtothepotential multi-yearresponseofcoralpopulations.Forexample,achangein waterqualitythatisnotdetectableinadultreef-buildingcoralsbut thatreduceslarvalrecruitmentsuccesscanresultinrapidincreases inthepopulationsofshorter-lived,purelyheterotrophicspeciesof benthicforaminifera,resultinginreductionintheproportionsof LBFshells(Hallock,1988b;Cockeyetal.,1996;KelmoandHallock, 2013).Additionally,foraminiferalpopulationscanrecoverwithina fewgenerations,asdemonstratedby Amphisteginagibbosa populationsalongtheFloridareeftractaftertheysufferedamassive bleaching-mortalityeventin1991(Hallocketal.,1995).TheLBFare immunetocoral-specificdiseases(Cockeyetal.,1996),and

Fig.1. Commonshapesoflargebenthicforaminifera(LBF),othersmallheterotrophictaxa(SFT),andstress-tolerantforaminifera(STF)typicallyfoundinreefenvironments,and usedtocalculatethe FI.EachshapecorrespondstoacommonfamilyofLBF(A E),OSF(F J)andSTF(K O)trophicgroup.LBFspecimensusuallyrangebetween150 mmuptoafew centimetres,whileOSFandSTFarerarelylargerthan500 mm.(A)Peneroplidae,(B)Amphisteginidae,(C)Calcarinidae,(D)Nummulitidae,(E)Soritidae,(F)Hauerinidae,(G) Rosalinidae,(H)Planorbulinidae,(I)Spiroloculinidae,(J)Lagenidae,(K)Elphidiidae,(L)Nonionidae,(M)Haynesinidae,(N)Ammoniidaeand(O)Bolivinitidae.

assemblagecompositionsareminimallyaffectedbycycloneevents oroutbreaksofthecorallivorousCrown-of-Thornsstarfish Acanthasterplanci (Uthickeetal.,2010).Therefore,foraminiferal assemblagescanindicateifambientwaterqualitycansupportreef recoveryviacoralrecruitmentornurserytransplants.

TheSTFincludeforaminiferaltaxathatareadaptedtonatural sourcesofnutrientsfromterrestrialrunoffandaretypically tolerantoflowsalinityanddysoxia(e.g. Carnahanetal.,2009). Thesetaxaareespeciallywell-suitedtoutilisingabundantlabile organicmatteravailableinbothfreshphytoplanktonproduction andsewage.Atnon-reefalsiteswithlocallycompromisedwater quality,relativeabundancesofOSFtaxadeclinerelativetothoseof STF,asshownby Carnahanetal.(2009) and Koukousiouraetal. (2011).Inmostreefsystemsstudiedtodate(e.g. Hallock,2012), theSTFfunctionalgroupisonlyaminorcomponent,ifpresentat all.Thus,themajortrendsareintheproportionsofLBFcompared totheOSF.Theindexasoriginallyproposedisalsosimpleto calculate:

FI ¼ð10 Ps Þþð2 Ph Þþ P0

where Ps ¼ theproportionofLBFshellsinthetotalcountofbenthic foraminiferaltaxainasedimentsample, Ph ¼ theproportionofOSF inthesample,and Po ¼ theproportionofSTFinthesample.The originalabbreviationswere s forsymbiont-bearing(¼LBF), h for otherheterotrophictaxa(¼OSF),and o foropportunistic,later termed “stress-tolerant” (¼STF)by Carnahanetal.(2009). Basedonthisformula, FI valuesrangefrom1 10(Hallocketal., 2003).Thebasicpremiseofthe FI isthatanassemblageof100%OSF givesan FI ¼ 2(Carnahanetal.,2009).PresenceofLBFtaxaraises the FI,whilethepresenceofSTFlowersthe FI fromthatreference value.AnassemblagedominatedbyOSFimpliesthereisabundant food(i.e.autotrophicandheterotrophicprocessespredominate), butnotsomuch(orthereissufficientcirculation)thatbiological oxygendemandintheuppersedimentsbecomeslimiting.Asfresh, labileorganiccarbonbecomesmoreplentifulandoxygenisintermittentlylimitingatthesediment-waterinterface,STFcanthrive suchthat FI valuesdropbelow2.For FI valuestoexceed2,dissolved

nutrientsandorganicmattermustbesufficientlylimitingthatalgal symbiosisissomewhatadvantageous.Giventhefasterturnover ratesofsmallertaxa,waterqualitymustbesufficientlynutrientpooronaveragefortheshellsofsymbiont-bearingtaxatobe relativelyabundant.Thisisthebasisfortheoriginallyproposed FI thresholdsof >4tobeinterpretedaswaterqualitythatsupports reefgrowth,2 4asindicatinganenvironmentmarginalforreef growthandunsuitableforrecovery,and FI < 2indicatingwater qualityconditionsunsuitableforreefgrowth. Hallocketal.(2003) alsonotedthatthe FI insamplesfromhabitatsexperiencing nutrificationoftenvarybetween3and5.Thiscaveatisimportant becauserecognizingtheactualbenthicforaminiferalassemblage representingeachfunctionalgroup(e.g.LBF,OSF)willassistinthe interpretationandassessingthefeasibilityofthe FI outsidethe Caribbeanregion.

Sincethepublicationsby Hallocketal.(2003,2004),widespread assemblageshiftsfromdominancebyLBFtaxainreefsedimentsto anincreasingprevalenceofsmaller,faster-growingOSFhavebeen reported.Althoughthisformulawasdesignedforreefalsediments whereSTFarerelativelyrare,itnonethelessperformedwellasan indicatorofassemblagechangesinnon-reefalsampleswhereLBF occur,includingsamplesfrombaysinFlorida(Carnahanetal., 2009),Australia(NarayanandPandolfi,2010),Greece (Koukousiouraetal.,2011),andBrazil(Barbosaetal.,2016).Insuch environments,trendsin FI valuescanbeusefulindicatorsof nutrification,evenwheretheyneverexceed4.Forexample,as anthropogenicactivitiesinduceeutrophicationthatexpandsthe rangesofthenearshorestress-toleranttaxaattheexpenseofthe othersmallertaxa,theregionofnutrificationexpandssothatthe abundancesofOSFoverwhelmLBFfurtheroffshore(e.g. Carnahan etal.,2009).

5.ApplicationoftheFoRAMIndex

Sinceitspublicationin2003,the FI hasbeenappliedtoreef environmentsinbiogeographicregionsnearlyworldwide.The FI hasalsobeenutilisedinmarginalandnon-reefalenvironmentsin thesubtropicalnorth-andsouthwesternAtlantic,the

Mediterranean,andIndo-Pacific(e.g. Barbosaetal.,2009;Dimiza etal.,2016).Notesonmethodsutilisedandnotableobservations inavarietyofpertinentpapersaresummarisedin Table1. Asnotedby Hallock(2012),becauseLBFtaxadifferbetweenthe AtlanticandIndo-Pacific,andacrossregionswithinthoseoceans, keytaxaand FI thresholdsalsocanbeexpectedtodiffer(Fig.2). Additionally,insomeinstances,the FI isinappropriate.For

Table1

example,inthesouthwestAtlanticoffBrazil,theoccurrenceof uniquemushroom-shapedreefstructures(‘chapeiroes’)occurin somereefareas(e.g. Barbosaetal.,2009,2012).Thesestructures severelylimittheoccurrenceofLBF,asthe finesedimentsthat accumulatebeneaththesestructuresfavoursmallerheterotrophic species(Barbosaetal.,2012).Incontrast,the FI hasbeensuccessfullyappliedinotherareasoftheSouthAtlantic.Intheoffshore

Summaryofmain findingsandmethodologicaldetailsofstudiesthatappliedtheFoRAMIndexsinceitspublicationin2003. #individualsMeshsize(mm)DepthLocationReferenceNotes

150 20063Upto20mFloridaandCaribbean Hallocketal.(2003) ThisstudyproposedtheFI

120þ 637 9mGBR,PacificOcean UthickeandNobes(2008) TestedtheapplicabilityoftheFI formonitoringpurposes;used rarefactionanalyses

200þ 2500.5 3.5mGBR,PacificOcean SchuethandFrank(2008) Usedmediumandcoarser sand-sizefractions,which skewedthedatatowardlarger specimens;detectedlocal trends

150þ 632 20mBrazilianreefs,southwest Atlantic Barbosaetal.,(2009),(2012) FIvaluesinthe ‘chapeiroes’ do notreflecttruestateofcoral coverinthosereefs;very shallowreefsstructures

150þ 63 <10mBiscayneBay,Florida,USA, Caribbean Carnahanetal.(2009) Studiedsubtropicalbay:FI values < 2reflectedareasof pollutionandFIof2 4inopen bayandprotectedestuary

200þ 635mGBR,PacificOcean Uthickeetal.(2010) FIshowedsignificant correlationwithwater-quality parameters

3001250.2 6mEasternMediterranean Koukousiouraetal.(2011) Basedonlivespecimensinalgal samples,whichwasthe predominantsubstratum; locallyusefulcomparisons

AllindividualsNotusedNotgivenColombia, Caribbean

150þ 632 20mBrazilianreefs,southwest Atlantic

Velasquezetal.(2011) FoundhigherFIsatoffshore islandthanincoastalMPAs;no grain-sizeanalysisbutFI > 8 indicateshydrodynamic influence

Oliveira-Silvaetal.(2012) Foundsomeseasonal differencesinFIbetween summerandwintersamples, indicatingseasonal hydrodynamicinfluence

Notgiven63NotgivenGBR,PacificOcean Fabriciusetal.(2012) Reportedsignificantcorrelation ofFIwithwaterqualityon inshorereefs,GBR

60þ 6310 12mKirimati,PacificOcean CarilliandWalsh(2012) AuthorscomparedFI(including rarefactioncurves)forliveand deadassemblages,withhighly significantdifferences.

AllstainedindividualsNotusedSubtidalNorthernBahia,Brazil, southwestAtlantic

KelmoandHallock(2013) Basedonlivespecimens,10 replicates/reef,numbersof specimenscountedpersample notreported,butdataindicate typically >100speciesper replicate Producedlocally/interannually usefulcomparisons

150þ 63 <10mPuertoRico,Caribbean Oliveretal.(2014) Comparedtocoralcoverand sedimentquality;providedbest indicatorofhumanimpact

Allindividuals6313and26mFloridareeftractwestern Atlantic Stephensonetal.(2015) ComparedFIoftotal assemblagesfromrubble(mean 3.6)andsediment(mean5.6) samples

200 500630 40mFrenchPolynesia, PacificOcean Fajemilaetal.(2015) Examinedsedimentsamples acrossabroaddepthrange; grain-sizeanalysiswasnot reported

200 3001255 15mSouthChinaSea,PacificOcean ChenandLin(2017) SamplesdominatedbyOSF, onlytwosamplesFI > 4 (continuedonnextpage)

Table1 (continued )

#individualsMeshsize(mm)DepthLocationReferenceNotes

Allin1gor200 300632 5mBelize,Caribbean Emrichetal.(2017) Sedimentsamplescollected fromthebackreefenvironment. Assessed fixedamountof sediment;numberof specimenspersamplevaried overtwoordersofmagnitude

150 200Notused10mMaldives,IndianOcean Pisapiaetal.(2017) ComparedFIfromreefswith differentmanagementschemes

150þ 635 9mVirginIslands,Caribbean Oliveretal.(2018) FI2.8 6.5,highestoffshore awayfromland-useindicators

Notgiven631 46mGalapagos,EastPacific Humphreysetal.(2018) FIbetween1.2and4.2,most <2

NotavailableNotused3.5 4mNorthernPersianGulf Parsaianetal.(2018) OnlyLBFwasAmphistegina; comparedindustrial(FI > 4) andaquaculturepollution;FI 2 4

150þ Notused6 12mSerranaandRoncadorBanks, southwestCaribbean Sanchezetal.(2019) Reporteddeclineofcoral.FI averaged2.5 3

NotavailableN/A <3mEstuary,TamilNanduCoast, India,IndianOcean NagendraandNallapaReddy (2019) Assessedavarietyof assemblageparameters;FI1 1.45

Fig.2. Differencesin FI valuesandinterpretationsofwaterqualitybetweentheCaribbeanandthePacificOcean.Thekindandsizeoftheforaminiferaoutline(picturedin Fig.1) representtrophicgroupsandabundanceofspecies,respectively,expectedtooccuratagiven FI rangeandequivalentwaterquality.ShiftsfromLBFtoSTFdominated-sedimentscan beobservedwhennutrientconcentrationincreasestoeutrophicconditions.However,mesotrophicconditionscanstillsupportabundantLBFcommunitiesinthePacificOcean, skewing FI valuesandmaskingdeclinesinwaterquality.

coralcommunitiesoftheFernandodeNoronhaarchipelago (Barbosaetal.,2012),the FI reflectswaterqualityandiscorrelated withcoralcover.OtherinshorereefsinBrazilshowedaconsistent correlationbetweenwaterqualityandsedimenttexture,andthe FI (Barbosaetal.,2009).Finally,changesinbenthicforaminiferalassemblageshavebeenlinkedtoorganicmatterinputinthelast3kyr,withaconsistentdeclineofLBFinrecentsamples(Almeida etal.,2013).Thisimpliesthat,exceptforreefareaswherethe mushroom-shapedreefstructuresareprevalent,the FI canbe appliedinfringingreefsoftheSouthAtlantic.

IncontrasttotheAtlanticOcean,intheIndo-westernPacific region,LBFspeciesofthefamilyCalcarinidaecandominateshallow reef flats.Forexample, Renema(2010) notedthatcalcarinidsthrive inenvironmentsinIndonesiawheremesotrophicconditionsand algalovergrowthprecludecoralrecruitment.Itispossiblethatthe abundanceofcalcarinidsinthesereefsmightskewthe FI values, howeverthishasnotbeentestedyetforthatregion.Whenthe purposeofdetermining FI valuesisonlyforcomparisonamong samplestoassesstrendsforaspecificarea(e.g.asappliedby Koukousiouraetal.,2011),consistencyinsamplepreparationand

analysesareofprimaryimportance.Insuchcases, FI datacan simplybeusedtoassesstrendsalonggradients.However,to compareresultswithstudieselsewhere,astandardprotocolis necessary.Someoftheconsiderationsthatweaddresshereinclude sizerangeofspecimensexaminedasafunctionofmeshsizeofthe sievesusedinsampleprocessing,replicationandinterpretationof individualsamples,thenumberofspecimenscounted,live (stained)versustotalassemblageanalysis,anddepthofsample collection.

6.Technicalissueswhileapplyingthe FI

Samplingdesign: The FI wasoriginallydevelopedusingsamples fromdepthsof~3 15m.Fordetailedcomparisonsofreefswithin anarea,thestrategyemployedby Pisapiaetal.(2017) canberecommended.Additionally, Pisapiaetal.(2017) successfullyintegratedtheutilisationofthe FI withpre-existingmonitoring programswithoutanycompromise.Theyselectedlocationsofinterestbasedonthemanagementstatusoftheadjacentisland,and thencollectedthreesamplesat~50mintervalsfrom10mdepth

alongthereefslopeateachlocation.Theycouldhavesampledat perhaps5mdepthinsteadof10m,likelywithsimilarresults,given thattheirsamplingdepthisstillwithintherangeproposedbythe FI.If noprior knowledgeofreefsitesorenvironmentaldatais readilyavailable,researcherscanutilisealine-intersecttransect, andsamplescanberandomlyorhaphazardlycollectedalongthe transect,asdescribedin Stephensonetal.(2015)

Samplingsedimentsanyshallowerthan~3mdepthrisksbias towardsveryhighlysortedsedimentsinhigh-energyenvironments,andverymuddysedimentsinshelteredenvironments(e.g. Mosesetal.,2017).Also,itisimportanttoconsiderthereef’s rugosity.Forexample,insouthernPuertoRico,thefringingreefsof JobosBayaresmall,andwithina100-mtransectthewaterdepth canrangefrom2to10m.Thehighrugosityofthesereefsandthe associatedhigh-energyenvironmentproducescoarse-grained sedimentswhereforaminiferaldensityanddiversityarelowand LBFarerelativelyabundantandabraded(MM-C,per.observations) yieldingbiased FI values.Similarly,samplingdeeperthan~10m, sometimesevenshallower,inlagoonalenvironmentsmayyield samplesdominatedby finesedimentsandOSF.Moreover,reef flats andshallownearshoreenvironmentscanbeproblematic.Evenin quiteturbidwaters,sufficientlightcanpenetrate,atleastduring lowertideorcalmconditions,forsomeLBFtothrive,including A.angulatus intheCaribbeanregion,and Neorotaliagaimardi and Calcarinahispida inthewesternPacificregion.

Replication: Thenumberofreplicatesexaminedhasalsovaried amongstudies.Samplereplicationallowsresearcherstocontrolfor stochasticfactors,whichcaninfluenceresultsandsubsequent interpretation(Hurlbert,1984).Replicationisalsocrucialto determinevariabilityamongsamplesandaccountforpatchiness (Murray,2006).Higherlevelsofreplicationincreasesampling sensitivity,allowinginterpretationsofrandomvariationsor ‘noise’ , increasingtheprecisionofanestimateofthemeanofatreatment orthedifferencebetweentwotreatments(Hurlbert,1984).Samplingdesignsarealsocriticaltoanalyses.Forexample,ifcomparing samplesfromatransectinapotentiallyimpactedareawithsamplesfromareferencetransect,thesetofsamplesfromeachtransect canrepresentreplicates.Moreover,nosinglesampleshouldbe interpretedinisolation;outliersshouldbeidentifiedandcanbe useful.Forexample, Emrichetal.(2017) detected “hotspots” of pollutantsandrecognisedlikelygroundwaterseepageonareef flat inBelize.Asderiveddatasuchas FI valuesaregenerallynonnormallydistributed,mediansofreplicatesmaybemoreappropriatethanmeansforcomparisonsamongsitesorstudies.

Howareplicateisdefined,andthenumberofreplicatesused canbeproblematic. Hallocketal.(2003) didnotaddresstheissueof replicatesamplesdirectly,astheywerecomparingtrendsinmultiplesamplescollectedoverseveraldecadesandalongonshoreoffshoretransects.FortheFOBIMO, Schoenfeldetal.(2012) recommendedamandatorythreereplicatespersamplingsite. Oliver etal.(2014),assessingresponsestosedimentcontaminationand humandisturbance,surveyed24sitesforavarietyofresponse parameters.Threesedimentsamplesforgrain-sizeand FI analyses werecollectedalongtransectsateachsite.The FI producedthe strongestnegativecorrelationswithproxiesforanthropogenic disturbancesamongthereefbiotaassessed(Oliveretal.,2014).

Thequestionofthenumberofreplicatesiscloselyrelatedtothe numberofspecimenstocount.Threereplicatesamplesof~100 specimenspersampleprovidemorestatisticalpowerthan300 specimensfromonesample(e.g. HayekandBuzas,2013).Similarly, counting100specimensfrom fivereplicatesamplesshouldprovide morepowerthan150 200specimensfromeachofthethree replicates.However,becausesamplepreparationisrequiredfor bothgrain-sizeanalysisand FI analysis,inacost/benefit

consideration,thebenefitofcounting fivereplicatesamplesof~100 specimensversus~150fromthreereplicateswouldlikelybelostin theadditionalsamplepreparationtimefor fivesamplesversus three.Additionally,giventhat FI studiesshouldbebasedonthe interpretationoftheproportionaldistributionofthedominanttaxa withintheLBF,OSF,andSTF,countsaslowas100specimensper samplecanbesufficient(e.g. FatelaandTaborda,2002).

Meshsizeandsedimenttexture:Standardproceduresforpreparingsedimentsamplesforanalysisofforaminiferalassemblages includetheremovalof finesedimentsanddebrisusinga fine-mesh sieve.Meshsizeisamajorcontroversy,asdiscussedby SenGupta etal.(1987),subsequentlyby Schonfeldetal.(2012) regardingthe developmentoftheFOBIMOprotocols(softandmuddysediments), andinapplicationsofforaminiferalindicessuchasForam-AMBI (Alveetal.,2016;Jorissenetal.,2018).Meshsizescansignificantlyinfluenceresultsbecauseforaminiferarangefrom~40 mmto asmuchasafewcentimetresindiameter.Theoriginalstudies, uponwhichthe FI thresholdinterpretationswerebased,used 63 mmmeshsieves,whichremovesilt-andclay-sizedparticles(i.e. mud)whileretainingallsand-sizedparticles.Useofa125 mmmesh sieve,whichalsoremovesvery finesand-sizedparticles(e.g. Koukousiouraetal.,2011),ismoretime-efficient,asiteliminates small “juveniles” thatareoftendifficulttoidentify(Schoenfeld etal.,2012).Moreover, Hallocketal.(2003) recommended includinganalysesofsedimenttexture,assamplesthatarepredominantlycomposedofvery finesedimentscanexhibitasorting biasthatskewsthe FI valuesto3orless(Hallocketal.,2003).This approachiscriticalsincesedimenttextureplaysaroleinforaminiferalassemblagedistributions(e.g. Emrichetal.,2017;MartínezColonetal.,2018).

Useofcoarsersievesizescanskew FI datatoanomalouslyhigh values,asLBFspeciestendtobelarger(>500 mm)thanmostOSF andSTFtaxa.AdultLBFspeciescanreachsizesthatareanorderof magnitudelargerthansomesmallerspeciesthatoccurinreefenvironments(Hallock,1999). SchuethandFrank(2008),usingasize fraction >250 mm(mediumandcoarsesand),reportednosupport foroveralllowwaterqualityintheLowIsles,intheGreatBarrier Reef(GBR),basedonthe FI,detectingonlylocalpollutionsources. Thisreefareaishistoricallyimpactedbyterrestrialrunoffand presentshighturbidity,whichincreasedfollowingEuropean colonisationofthecoastofQueensland(WolanskiandSpagnol, 2000)andischaracterisedbyalowhard-coralcoverandsubstantialphytalsubstrata(FrankandJell,2006).Examiningonly mediumandcoarsersandfractionscanresultinunderrepresentationofsmallerheterotrophicspecies(Schonfeldetal.,2012; Hallock,2012).

Anotherconsiderationforwhatsizefractiontoexamineisthe intendedanalysesofthesamples.Iftheyareonlytobeassessedfor the FI,thenwerecommendtheuseofmeshsize >125 mm.However, ifdetailedassemblageanalysesareplannedinadditiontodetermining FI (e.g. Carnahanetal.,2009;Koukousiouraetal.,2011), considerationofthesizerangesusedinotherstudiesisessential,as speciesdiversityandothercommonassessmentmetricsalsoare stronglysize-dependent(Murray,2006).Thedifferencesbetween theuseof63and125 mmmeshsievesshouldnotproduceserious discrepanciesinthe FI,asthe125 mmfractionwillretainmost smallertaxainadditiontoLBFspecies.However,additionalworkis neededtoassesshowthe FI differsbetweenstudiesusing63 mm and125 mmmeshsieves.Suchstudiesmayrecommendmodified FI thresholds,correctionfactors,orevenamodifiedformula,to accommodatereducednumbersofsmallertaxainanalysesusing 125 mmmeshsieves.

Somestudiesofforaminiferalassemblagesalsosieveoutthe coarsersedimentfraction.Forexample,someresearchershave

examinedonlythe fineandmediumsizefraction(125 500 mm). Thatsize-rangecanbeusefulforlocalcomparisonsoftrends,but cannotbecomparedmorewidely.Forsamplesthatmaycontain foraminiferaltaxahostingalgalsymbionts(orthatencrustphytal substrata),anuppersievesizeshouldbeatleast2mm(transition fromsandtogravel),andtheresultingcoarsefractionshouldbe scannedforlargeforaminiferalshells.

Samplesizeandprocessing:Anothertypicaldiscrepancythatcan limitcomparisonsacrossstudiesisthenumberofspecimens identifiedpersample. Hallocketal.(2003) suggestedtheuseofa1gsedimentsample,andsubsamplingbetween150and200specimens,orallwell-preservedforaminiferalshellsinthe1gsample,if necessary.Reportingthenumbersofspecimenspergramofsample,toprovideinformationonabsoluteabundance,isessential.Ifa 1gsamplecontainsfewerthan50identifiableforaminiferalshells, thesampleshouldnotbeincludedinthe FI assessmentbutshould benotedasverylowdensity.Incontrast,ifsamplescontainover 1000individualsper1-gofsediment,thentheycouldbesubsampled,tomaintaintheapplicationofthe FI astime-efficient butstillrobust.

Moststudieshaveexaminedbetween150and300specimens persample(Table1). UthickeandNobes(2008) and Carilliand Walsh(2012) countedfewerspecimenspersample,basedupon rarefactioncurvesofspeciesaccumulation(e.g. Ludwigand Reynolds,1988;Colwelletal.,2004).Moreover,themaingoalof thestudyby UthickeandNobes(2008) wastodeveloparapidindicatormethodtoassesswaterqualityatawholereeflevelinthe GBR.Somestudiesdidnotreportthetotalnumberofspecimens countedpersample(e.g. Fabriciusetal.,2012;Velasquezetal., 2011),thuslimitingthepotentialforcomparisonwithother studies.

Hallocketal.(2003) notedthatidentificationtogenuslevel requirescountingfewerindividualspersamplethanstudiesidentifyingtospecies(e.g. Buzas,1990).Thenumberofspecimens necessarytocapturediversityvariesregionallyandamongreef environments.Thus,ifdiversityindicesarebeingassessedalong withotherparameters,apreliminarysurveyonthetotalnumberof individualstocapturediversityshouldbeconductedusingrarefactioncurves.However,capturingdiversityisnotessentialfor routineuseofthe FI asabioindicator,socountingfewerspecimens canbemorecost-effective,asnotedby UthickeandNobes(2008)

Live versus totalassemblage: Thespecimensthatwerealiveina samplewhencollected,asindicatedbyavitalstainoralgalsymbiontcolour,reflectstheforaminiferalresponsetolocal-scale environmentalconditionsinthedaysorweeksimmediatelyprecedingsamplecollection(e.g. Buzasetal.,2002).Thedead assemblagerepresentsthesuccessiveaccumulationofgenerations offoraminiferalshellsoversometimedependingonspeciesproductionandtaphonomicfactors,suchaspost-mortemdestruction, sortingandredeposition(Schonfeldetal.,2012).Whilemost studieshaveanalysedtheabundanceofbenthicforaminiferaand calculated FI fromtotalassemblages(Table1),somestudies assessedlivespecimensandcalculated FI valuesbasedonthose datasets(e.g. CarilliandWalsh,2012;KelmoandHallock,2013). Thetotalassemblage(dead þ live)datareflecttheoverallenvironmentalconditions,whiletheliveassemblagedataprimarily revealtheresponseofforaminiferatorecentlocalconditions (Buzasetal.,2002). CarilliandWalsh(2012) foundmajordifferencesbetweentheaverage FI valuecalculatedforlive(4.6)and dead(9.1)assemblagesatreefsitesinKiritimatiIslandaffectedby anthropogenicimpacts.Theyattributedthedifferencestoincreases innutrientpollutionand fishingpressureinrecentdecades. Kelmo andHallock(2013) examinedonlylive(stained)specimensin sedimentscollectedannuallyover17years.Althoughthecalculated

FI valueswereconsistently <4,theinter-annualchangesinlive foraminiferalassemblagesandcorresponding FI valueswere consistentwithchangesinrainfalland fluvialrunoffassociated withElNino-SouthernOscillationcycles.Bothofthesestudies revealedthatuseofthe FI conceptprovidedimportantinsightinto environmentalchanges,despitethedeviationfromoriginallyproposedprotocols(i.e.evaluationofliveassemblages).

Intheory,assessingliveassemblagesshouldbeidealtoassess ongoingenvironmentalconditions.However,sincelivingspecimenstypicallymakeuponlyasmallpercentageoftheassemblage inreef-sedimentsamples,distinguishingandidentifyingadequate numbersrequiresasmuchas20timeslonger,greatlyincreasing thecostofsampleanalysis.Nevertheless,stainingofsamplescan stillbeconductedifresearcherswishtoidentifyliveindividualsin thetotalassemblage,andiftheabundanceoflivespecimens changesovertime,forexample.Inpractice,assessmentofliveassemblagesrequiresnotonlystainingofsamples,butacompletely differentsamplingstrategy.Relativelyfewshallow-water (e.g. < 20m)reef-associatedforaminiferaliveinsediments,but ratherliveonhardorphytalsubstrata,asnotedabove. Stephenson etal.(2015) comparedforaminiferalassemblagesfromsediment andreef-rubblesubstrata,concludingthatdiversityofheterotrophicgeneraishigheron firmsubstratethaninsediments.Moreover,becausesedimentscanbeintermittentlystabilisedby filamentousalgalmats,theforaminiferathatcanrecruitand completetheirlifecyclesinsuchenvironmentstendtobesmall, fast-growingspecies. Donnelly(1993) studiedthelive(stained)and totalassemblagesfromreef-rubbleandsedimentsamplescollected offsouthwestPuertoRicoin1985,documentingthatliveindividualsaccountedfor~15%ofspecimensfromrubblesamples and <3%ofspecimensfromsedimentsamples.Andwhilethe numberofspeciesidentifiedfromrubblesamples(211)wasstatisticallyidenticaltothenumberfromsedimentsamples(215),the numberofspeciesidentifiedaslivefromrubblesamples(124)was 55%morethanthenumberofspeciesfoundliveinsediment samples(80).

Inanidealtotalassemblage,theproportionsofallspecieswould bethesameasinthelivingassemblage.However,avarietyof environmentalandlife-historyprocessesproducethedisparities commonlyobservedbetweenthelivingandtotalassemblages,and betweentherubbleandsedimentassemblages(e.g. Donnelly, 1993;Stephensonetal.,2015). Renemaetal.(2013) notedthat short-livedtaxacanproducefarmoreshellswithinagivenperiod thanlong-livedtaxa(i.e. r versus k-strategists). Crevisonetal. (2006) observedseasonaldifferencesintheproportionsoftaxain sedimentsamples,withahigherproportionofLBFshellsinspring samples,followingwinterandspringstormconditionscompared withsamplescollectedinlatesummer/earlyautumnfollowing summerconditionsunderminimalstormactivityandmaximum algal-matstabilisation. Barbosaetal.(2016) alsofounddifferences betweensummerandwintersamplesfromBrazilianreefs.Thus, seasonalitymustbeconsideredindevelopingsamplingstrategies; studiesthatspanmultipleyearsshouldcollectsamplesduringthe sameseasoneachyear.

Additionally,insedimentsthathavebeenhistoricallydominatedbyLBF,theretentionofforaminiferalshellsissuchthateven wherewaterqualityisobviouslyindecline,thesedimentscanstill bedominatedbythesespecies(e.g. CarilliandWalsh,2012).This issuecanbeespeciallyproblematicinhigh-energyenvironments suchasbeaches(Hallock,2012).Moreover,suchsedimentscanbe transportedintootherhabitats,especiallydownslopeonshelf margins(BriguglioandHohenegger,2011).Thus,assemblagescan becomprisedofamixofmodernandrelictmaterial(Renemaetal., 2013),andlong-termstorageofsedimentsinareassuchasreef flats

canoccur(DawsonandSmithers,2014;Nageletal.,2016).Allof thesecomplexitiesfurtherindicatethatbaselinestudiesusing multivariateanalysesapproachesshouldbecarriedoutinpreparationforwider-scaleortemporalimplementationofassessments focusingonamoresimplifiedstrategyutilisingthe FI

Whethertoassessliveortotalassemblagesshouldbedeterminedbythegoalsofthestudyandresourcesavailable.Ifthe purposeistoassessongoingenvironmentalconditionssuchas seasonalorinter-annualcycles,theliveassemblageshouldbeused. However,theinterpretationthresholdsmustberecalibrated because FI valuesassessedfromliveassemblagesarelowerthan valuesfromtotalassemblages(e.g. CarilliandWalsh,2012;Kelmo andHallock,2013;Stephensonetal.,2015).Ifthegoalistodecipher theintegratedsignaloverrecentyearsordecades,thetotal assemblagecanprovidethatinformation.Wecanfurtherrecommendthatassessingtheliveassemblagesonreefrubbleorphytal substratescanindicateconditionspresentatthetimeandimmediatelybeforesampling,whilethe FI basedonthetotalassemblage fromsedimentsprovidesatooltoassessthecombinedeffectsof naturalandanthropogenicstressorsovertime.

Classificationlevel:Indesigningaprojectinwhichthe FI willbe assessed,investigatorsmustdecideonthetaxonomicorfunctional leveltowhichforaminiferalspecimensareidentified.Theoriginal intentionofusingthreefunctionallevels(i.e.LBF,STF,andOSF)was toprovidealowcost,rapid-assessmentmethodforwhichtechnicianscouldbereadilytrainedtoidentifyforaminiferalspecimens foreachofthosecategories.Withtheuseofacountingdeviceanda stereo-microscope,atechniciancouldassessmultiplesamplesper day.However,theapplicationofthe FI asalow-cost,low-technologyassessmenttool,asoriginallyintendedbasedonfunctional groups,hasnotyetbecomereality,withtheexceptionofasingle study(e.g. Pisapiaetal.,2017).Instead,mostapplicationsofthe FI todatehaveincludedbroaderfaunalassessmentsandmultivariate analysisofassemblages.Thus,theforaminiferalspecimensin sampleshavebeenidentifiedeithertogenusorspecieslevel.The advantageofthebroaderassessmentsistheextractionofmore detailedinformationregardingforaminiferalassemblages.The disadvantageisthatthetimerequiredtoassesseachsampleto genuscanbeatleastthreetimeslonger,andtospeciesupto five timeslonger.Theclassificationofbenthicforaminiferalshellsto genuslevelefficientlygeneratesreliabledata.

Studiestodatethathaveutilisedmultivariateanalysesofforaminiferalassemblageshaveprovidedinsightintowhattaxatendto occurtogetherinalocalityorregion.InareassuchastheCaribbean, BrazilianandAustraliareefs,thereef-associatedforaminiferalassemblageshavebeenreasonablywellstudied(e.g. Cockeyetal., 1996;UthickeandNobes,2008;Barbosaetal.,2009,2012;Oliver etal.,2014;Emrichetal.,2017).Thus,taxathataresufficiently abundanttosignificantlyinfluencethe FI arewellknownand anomaliesusuallycanberecognised.Ideally,beforeapplyingthe FI asanassessmenttoolinanewarea,foraminiferalassemblages shouldbewelldefinedusingmultivariateanalysesthatgroupby taxa(e.g.r-modeclusteranalyses,multidimensionalscaling,or someformoffactor-basedmultivariateanalysis),whichcanpromoteunderstandingoftheforaminiferalfaunaandthemagnitude oftheinterrelationshipsamonggenera/species.Ifassessmentsare beingcarriedoutbytechniciansjustlearningaboutforaminiferal assemblages,theyshouldbesupervisedoratleast,aspecialist consultantshouldbeavailabletodiscussanomalies.However,an advantageofusingsedimentsamplesisthattheyarereadily archivedandcanbequalitycheckedatspecificintervalsandif anomalousresultsarenoted.

AmajorconcernregardsthepresenceofcalcarinidsintheIndoPacificregion,wherespeciescanreachhighdensitiesevenin mesotrophicreefs(e.g. Renema,2010).Thisisbecausesomespecies

ofthefamilyCalcarinidaethriveepiphyticallyonavarietyof macrophytes,especiallycorallineredalgae(bothcrustoseandnoncrustosetaxa),calcareousgreenalgae, filamentousalgae,some macroalgae,andseagrasses(e.g. Renema,2010).Inareaswhere bleaching,disease,overfishingofherbivorous fish,orotherimpacts havereducedcoralcover,aslongasthereissufficientsunlight, somecalcariniddensitiesincreaseasalgalcoverincreases.Therefore,calibrationofthe FI fortheIndo-westPacificregionwherethe Calcarinidaeoccurisneeded,especiallywherealgalcoverisvisuallyhigherthancoralcover.

7.Towardsastandardisedprotocol

Theoriginalgoalofthe FI wastoprovidearapidandcosteffectiveassessmenttoolofwaterqualityincoralreefecosystems (Figs.2and3).Modifiedapproachesappliedbysomeresearchers havedemonstratedhowthe FI canbeusedtoreveallocalgradients ortemporalchanges(e.g. Koukousiouraetal.,2011;Carilliand Walsh,2012;KelmoandHallock,2013),whicharelegitimateapplications.Inothercases,regionalcharacteristicsofthereefscan resultin FI valuesthatarenotuseful,evenlocally.Thesuccessofan environmentalindicatorisbasednotonlyonitsapplicabilitybuton howwellprotocolsaredesignedandcommunicated.Withinthe Caribbeanregion,becausetheforaminiferalassemblagesarewell knownandrelativelypredictable,the FI shouldbedirectlyapplicabletototalassemblagesfromsedimentsinmostareas.However, ifthegoalisforspecificindexvaluestobecomparableacross studiesandregions,futureapplicationsofthe FI shouldconsider thefollowing:

(1)Theprocessofcollectionofsmallsamplesofsurficialsedimentsintosmallvials,bagsorminicoresissimpleand straightforward.However,wheresamplesarecollectedcan limittheirusefulness.The firstconsiderationissampling depthandexposuretowavesandcurrents. Hallock(2012) recommendeddepthsnoshallowerthan~3m,especiallyin veryhighenergyorverylowenergyenvironments,andno deeperthan~15m,withthe5 10mrangepreferred.

(2)Aminimumofthreeindependentreplicatesamplesshould beused,preferablyalongatransectasdescribedby Pisapia etal.(2017)

(3)Sedimenttextureplaysaroleinthepatchinessofforaminiferaldistributions,sothatsortingisaninherentproblem (e.g. Ramirez,2008).Applicationofthe FI shouldinclude grain-sizeanalysisandsamplesthatarewell-sorted,very coarse(>2mm)sedimentsshouldbeexcludedfrom FI analysis,asshouldsamplesthatarepredominantlymud (<63 mm).Also,formulti-yearmonitoringprojects,samples shouldbetakenwithinthesameseasoneachyearandnot immediatelyaftermajorstormevents.Aroundthesummer solsticecanbeoptimuminsubtropicalregionssuchas Florida,asitisafterthedirectinfluenceofwinterstormsand beforelatesummerwheneitherprolongedcalmweatheror tropicalstormstendtooccur.

(4)Towashoutmudand finesandsbeforeforaminiferal assessment,sievemeshof125 mmcangeneratesreliableand time-efficientresults.Useof125 mmmeshalsocanreduce theproblemofsamplesdominatedbyvery finesandsand muds.Thismeshsizeisalsorecommendedforotherprotocols(e.g.FOBIMO,Foram-AMBI)utilisingbenthicforaminiferaasbioindicatorsofenvironmentalchange(Schonfeld etal.,2012;Alveetal.,2016;Jorissenetal.,2018).However,becausetheoriginal FI thresholdswerebasedonsamples sievedover63 mmmesh,previouslypublishedassessment thresholdslikelywillneedtobemodified.

Fig.3. Quickguideforthecollectionandprocessingofsedimentsamplesforthecalculationofthe FI.Redarrowsdenoteimpossibilityof FI calculation,whereasbluearrowsmean thatresearchercanproceedtonextstep.Thegreenarrowstandsforthealternativepossibilityof FI calculation.

(5)Recordingthenumberofforaminiferalshellspergramof sedimentisessentialforinter-samplecomparisons,atminimumtoanorderofmagnitudeestimate(e.g.rare~101/g, common~102/g,abundant~103/g),especiallyincoarser samplesorsampleswithterrigenouscomponents(e.g. quartzsand).

(6)Assessmentofallrelativelyfresh-appearingforaminiferal shellsinsedimentsamples(i.e.totalassemblage)isrecommendedforbasicanalysesasresultingassemblagescan providereliableinformationonbothshort-andlong-term impacts,andshouldberegionallycomparable.Badly brokenorpossiblyreworkedspecimensshouldnotbe includedin FI calculations.

(7)Ifpreviousforaminiferalassemblagestudiesareunavailable fortheareaorspecifichabitatofinterest,apreliminarystudy comparingreferenceandimpactedsites,usingfullassemblageidentificationfollowedbymultivariateanalyses,is recommendedtodeterminespeciesthattendtoco-occur andunderwhatenvironmentalconditions,todetermine whattaxatocountineachofthethree FI categories.Alternatively,thecollectionofsedimentorreef-matrixcores couldalsoprovidebaselineinformationtoanalyselocal changesinforaminiferalassemblages(e.g. Reymondetal., 2013).

(8)Arecommendedminimumof150foraminiferalspecimens shouldbeidentifiedandclassifiedtoafunctionalgroupin eachsample.Foreachsamplingstation,the final FI willbe calculatedastheaverageamongreplicatesamples.Ifless than100individualsarefoundin1gofsedimentsamples, theFIcanstillbeapplied,butrarefactiontestsshouldbe carriedouttoverifythereliabilityofassessing100specimens(orpossiblyfewer,see CarilliandWalsh,2012).This approachcanreducethephysicalandmonetaryinvestments bystakeholdersandend-userswhoareinterestedin implementingthe FI intotheirshortorlong-termenvironmentalassessmentefforts.

(9)WhenworkingintheIndo-westPacificregion,wherecalcarinidsaccountforasubstantialproportionofthesediment,

theyshouldberemovedfromthe final FI calculationifthere isno a-priori historicalinformationavailableforthesitetobe studied.Thisisparticularlyimportantinveryshallow, mesotrophic(i.e.inshore)reefs,whereturbiditycanbehigh, thoughthisissuemaynotbeaconcernifsamplesaretaken attherecommendeddepthsof5 15m.Inoligotrophicareas, suchastheMaldives,calcarinidsshouldstillbeincludedin theFIcalculationsastheyrepresentasignificantproportion ofthesediment(Pisapiaetal.,2017).Alternatively,calcarinidssuchas Neorotaliagaimardi and Calcarinahispida may beusefulindicatorsofintermediatenutrification,asthey replace Amphistegina andmoresensitivecalcarinidsandincreaseinparallelwithsomeheterotrophictaxaand Elphidium (e.g. Renema,2010;Reymondetal.,2013).In oligotrophicreefareas,wheretheabundanceofalgaeisnot significant,butcalcarindsareabundantlypresent,they shouldstillbeincludedinthe FI count.

(10)FoRAMIndexvaluesfromnon-standardisedprotocolscanbe comparedlocallywithinterpretationsbasedontrends, ratherthanontheoriginallyproposedthresholds;they shouldnotbedirectlycomparedtodatacollectedusingthe recommendedprotocols.Forexample:

(a)Samplesfrombeachsandsandotherhighenergyenvironments <3mcanbeexamined,buttherelativeabundanceofLBFshellstonon-foraminiferalconstituentscan bemoreindicativeofwaterqualitythanisthepresence ofOSF,becausethelatterwillhavebeenselectively removedbybreakageandsorting(e.g. LidzandHallock, 2000).

(b)Live(stained)assemblagescanprovideinsightsonrecent stresseventsandinter-annualorseasonaltrends.

(c)Valuesobtainedfromsamplesforwhicha63 mmmesh sievehasbeenusedtoremovemuds-sizedsediments canbecomparedwitheachotherandwithprevious studiesinwhichthatsievemeshwasused.However,in futureapplicationsofthe FI,useofsieveswith125 mm meshisrecommendedtoincreaseprocessingefficiency.

8.Assessmentofforaminiferalpopulationsandglobal climatechange

Anapproachmoresuitabletomonitoringonseasonalscalesisto assesstheresponsesofkeytaxa,withrespecttoabsoluteabundancesand “condition”,throughvisualandmoleculartoolsusing the “omics” approach,ifavailable.Forexample, Hallock(1996) and Hallocketal.(2006) proposedthepossibilityofdifferentiating betweenlong-termwaterqualitydeclineandtheeffectsofphotooxidativestress(i.e.fromelevatedwatertemperatureorphotic stress)byanalysingpopulationsof A.gibbosa.Thisspeciesoccurs throughouttheCaribbeanregion;itscloserelative, A.lessonii,is foundthroughoutmostoftheIndo-Pacificregion.Thesetaxa typicallycanbecollectedalivefromreefrubbleatdepthsbetween 5and30m.Theassessmentstrategyisbaseduponsizedistributions(i.e.theabundanceofadultsand “juveniles”)andbleaching prevalenceandintensity,whichcanbequantifieduponreturnfrom the field(e.g. Hallocketal.,1995;Prazeres,2018).

In1991,thediscoveryofbleachingin A.gibbosa populationsin thereefsofFloridaKeysledtomonitoringofreef-tractpopulations, samplingofpopulationselsewhereasresourcespermitted,and laboratoryobservationsandexperiments,revealingnotonlysymbiontloss,butimpairedorfailedasexualreproduction,shellmalformationsandbreakage,andmicrobialinfestationonannual cyclesreflectingsolarirradianceratherthanhabitattemperature (e.g. Hallocketal.,2006,andreferencestherein).SamplingatHeron Island,GBR,inMarch1992,confirmedthatbleachingwasoccurring in A.lessonii and A.lobifera populations,andalsorevealedunusual shellbreakageandrepair(HallockandTalge,1994).Since1992, bleachinghasbeenobservedin Amphistegina populationsinall threeoceans(Hallock,2000;Hallocketal.,2006;Spezzaferrietal., 2018).Thediscoveryandintensivestudyofbleachingintheubiquitous Amphistegina spp.havecontributedtounderstandingthe cascadeofenvironmentalconsequencesofstratosphericozone depletion,whichhavecontributedtothecontinuedphotooxidativestressoncoralreefsworldwidesincethe1980s(Hallock etal.,2006),includingroughly20%oftheincreaseinglobaltemperature(WorldMeteorologicalOrganization,2018).

Amphistegina respondtoacutephoto-inhibitionwithinhoursto daysandtochronicstressoverseveraldaystoweeks(e.g. Talgeand Hallock,2003;Prazeresetal.,2016).Consequently,thisgenus providesabioindicatorofdamagingintensitiesofphoto-oxidative stressonthereefthatcanprecedeelevatedtemperature(Hallock etal.,2006). Spezzaferrietal.(2018) appliedthisstrategy,which theycalledthe Amphistegina BleachingIndex(ABI),tosamplesof Amphistegina from10-mdepthsinNorthAriAtollintheMaldives, IndianOcean,whichwerecollectedjustpriortoamasscoralbleachingevent.Theirresultsshowedthattheanalysisofbleachingprevalencein Amphistegina populationshasthepotentialto predicttheonsetofcoralbleaching.Temperature-induced bleachinghasalsobeenobserved(Prazeresetal.,2017;Prazeres, 2018),andhighlightlevelscombinedwithheatstresscanleadto severebleachingandimpairmentofreproduction(Prazeresetal., 2017).Therefore,in Amphistegina,bleachingcanbeinducedby photo-inhibitorystressassociatedwithhightemperatures(usually exceeding30 C)duringpeakseasonalsolarirradianceandwater transparency(e.g. Prazeres,2018;Spezzaferrietal.,2018).The observedbleachingin Amphistegina demonstratesthepotentialof thisgenustoidentifystressorsincoralreefs.Whenmonitoredin conjunctionwithexistingglobaltemperaturenetworks, Amphistegina providesthepotentialforforecastingnotonlymasscoral bleachingeventsbutalsoelevatedsusceptibilitytodisease(Hallock etal.,2004,2006).

9.Conclusions

Managinglocalwaterqualityincoastal-marineenvironmentsis fundamentalforthelong-termprotectionofdiversityandto maintaincarbonateaccretioninreefecosystems(Hughesetal., 2003;WooldridgeandDone,2009).Establishingbiologicaltime seriesbasedonforaminiferaisarelativelystraightforwardandcosteffectivewaytoevaluatechangesintheenvironmentovertime (Hallocketal.,2003).Theuseofbenthicforaminiferaprovidesa simpletoolforassessmentoflocalandglobalenvironmental stressors,byinvestigatingforaminiferalassemblagesthroughthe applicationofthe FI andassessmentofpopulationsof Amphistegina throughtheapplicationoftheABI(Spezzaferrietal.,2018).Thus, benthicforaminiferaaretheidealbioindicatortodistinguishbetweenlocalimpacts,whichcanbelocallymanaged,andregionalto globalstresses,whichrequireregionaltoglobalactiontoameliorate.

The FI wasdesignedtobearelativelysimpleindicatorof whetherwaterqualitysupportscalcifyingorganismsthathostalgal endosymbionts.Aswithanygroupoforganisms,somespeciesmay notbehaveexactlyaspredictedby a-priori assignmenttoafunctionalgroup.Moreover,regionaltaxonomicdifferencesmayprecludetheestablishmentofgloballyrelevantmetricthresholdsfor the FI.Nevertheless,thevarietyofapplicationsreportedtodate, includingcomparisonswithspecificbiologicalandgeochemical parameters(e.g. Fabriciusetal.,2012;Oliveretal.,2014),comparisonsacrossspecificgradientsofimpact(e.g. Carnahanetal.,2009; Koukousiouraetal.,2011),andeventheinter-annualtrendsina multi-yeardatasetbasedonspecimenscollectedlive(Kelmoand Hallock,2013),alldemonstratethatthebasicpremiseofthe FoRAMIndexissoundandreadilyapplicable.

Finally,additionalstudiesareneededtodeterminehowrelevant proposed FI thresholdsareandtodeterminewherethe FI should notbeused.Thisknowledgecanprovidelocalresourcemanagementagencieswithrecommendationsastowhetherthe routineapplicationofthe FI bytechnicianstrainedtoenumerate thosespecificfunctionalgroupscanbepractical,cost-effective,and worthincorporatinginmulti-yearevaluationsofreefresourcesor sitesofconcern.

Acknowledgements

Dr.T.EdwardRobertsforillustratingthespecimensofforaminiferain Figs.1and2.Dr.WillemRenemaforhiscommentsonearly versionsofthemanuscript.Thisworkwaspartiallysupportedby thePuertoRicoSeaGrant(grantnumberR/104-1-18)awardedto MM-C.

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