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Identifying hidden biocomplexity and genomic diversity in Chinook salmon, an imperiled species with a history of
anthropogenic influence
Journal: Canadian Journal of Fisheries and Aquatic Sciences
Manuscript ID cjfas-2019-0171.R1
Manuscript Type: Article
Date Submitted by the Author: 13-Aug-2019
Complete List of Authors: Meek, Mariah; Michigan State University, Department of Integrative Biology, and AgBio ResearchStephens, Molly; University of California MercedGoodbla, Alisha; University of California DavisMay, Bernie; University of California DavisBaerwald, Melinda; University of California Davis; California Department of Water Resources, Division of Environmental Services
Keyword: portfolio effect, RAD-seq, GENOMICS < General, POPULATION STRUCTURE < General, SALMON < Organisms
Is the invited manuscript for consideration in a Special
Issue? :Not applicable (regular submission)
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IdentifyinghiddenbiocomplexityandgenomicdiversityinChinooksalmon,animperiled1specieswithahistoryofanthropogenicinfluence2MariahH.Meek1*,MollyR.Stephens2,AlishaGoodbla3,BernieMay3,MelindaR.Baerwald43
4
1.DepartmentofIntegrativeBiologyandAgBioResearch,MichiganStateUniversity,EastLansing,MI.Email:5
[email protected]
2.SchoolofNaturalScience,UniversityofCalifornia,Merced,Merced,CA.Email:[email protected]
3.DepartmentofAnimalScience,UniversityofCalifornia,Davis,Davis,CA.Email:AGoodbla:8
[email protected] ,BMay:[email protected]
4.CaliforniaDepartmentofWaterResources,DivisionofEnvironmentalServices,3500IndustrialBoulevard,10
WestSacramento,California95691,USA.Email:[email protected]
*correspondingauthor12
13
RunningHead:usinggenomicstoidentifybiocomplexity14
15
16
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ABSTRACT17
Biocomplexityisanimportantmechanismforpopulationresilienceinchanging18
environments.However,wearejustbeginningtounderstandhowtoidentify19
biocomplexitysowecanmanagespeciestopromoteresilienceandstability.Genomic20
techniquesareemergingasanimportantmethodforidentifyingbiocomplexity.Central21
Valley(CV)Chinooksalmonareanexampleofaspeciesatriskofextinctionifbetter22
methodsforidentifyingandprotectingbiocomplexityarenotemployed.Toaddressthis23
knowledgegap,weemployedrestriction-siteassociatedDNAsequencingtoconductthe24
firstgenomicstudyofallmajorpopulationsofCVChinooksalmon.Wefoundgreater25
populationstructureacrosstheCentralValleythanpreviouslydescribed.Additionally,we26
showevidencefordifferentiationandadaptationwithinmigratoryphenotypesdespite27
highlevelsofgeneflow.Toassistinmanagementpractices,wealsofindthatgenomicdata28
canvastlyimproveourabilitytoassignindividualstotheirnatalpopulations,evenasthey29
mixduringmigration.Theseresultsdemonstratehowgenomicstudycangreatlyimprove30
ourabilitytoidentifyandconservebiocomplexity.31
32
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INTRODUCTION33
Biocomplexityreflectsthelevelofintraspecificdiversitywithinandamongpopulations,34
andisimportantforthestabilityandresilienceofmetapopulations(Hilbornetal.2003).It35
isnecessarytoidentifythefactorsthatcontributetobiocomplexity,suchasgenetic36
diversity,forincreasedunderstandingoftherolesuchfactorsplayinspeciesresilienceand37
persistence.Additionally,thisisimportantfordevelopingsustainablemanagement38
practicesthatpromotelong-termstability(DedrickandBaskett2018).However,fine-scale39
informationongeneticdiversitycanbelackingforspeciesofconservationormanagement40
concern,compromisingtheabilityofresourcemanagerstomonitorandenhancegenetic41
variationessentialtothebiocomplexityofmetapopulations(Angelonietal.2012;Ouborg42
etal.2010).43
44
Pacificsalmonidsareanexcellentexampleoftheimportanceofbiocomplexityfor45
persistenceandharvestproduction.PacificsalmonidstocksinAlaskahavebeenshownto46
displayhighbiocomplexity,allowingforstabilityinannualreturns(Schindleretal.2010).47
Thisisoftenreferredtoastheportfolioeffect,wheredifferencesamongindividual48
populationsinresponsetoenvironmentalconditionscandrivegreaterstabilityofthe49
wholepopulationcomplex,whencomparedtothestabilityofeachpopulationindividually50
(Hilbornetal.2003;Schindleretal.2010).Forexample,variabilityinannualreturnsof51
sockeyesalmon(Oncorhynchusnerka)intheBristolBayofAlaskaisovertwotimeslower52
thanreturnswouldbewithoutthepopulationandlifehistorydiversitypresentinthe53
system(Schindleretal.2010).Incontrast,demographicsynchronyhasincreasedoverpast54
decadesinChinooksalmon(O.tshawytscha)fromtheSnakeRiver,decreasingtheportfolio55
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effectandincreasingextinctionrisk(Mooreetal.2010).Thisincreaseinsynchronyhas56
beenattributedtohumanactivitiesthathavedecreasedgeneticdiversityandhomogenized57
habitats,suchashatcheryproductionanddamconstruction(Mooreetal.2010).58
59
Ourabilitytomonitorchangesinbiocomplexityislimitedbyourabilitytoidentify60
componentsofbiocomplexityandmanageforfinescaledifferencesinthesecomponents61
amongpopulations(Hilbornetal.2003;Mooreetal.2010).Advancesingenomic62
techniques,however,providethemeanstoaddressthisproblemthroughmuchfinerscale63
resolutionintheidentificationofbiocomplexity(e.g.Larsonetal.2017;Princeetal.2017).64
Byapplyinggenomicstoidentifyfinescalebiocomplexity,wecanthenmanagetoprotect65
allthediversityfoundwithinspecies,boostingtheportfolioeffectanddecreasing66
extinctionrisk.67
68
Inthisstudy,weapplygenomictoolstobetterunderstandthebiocomplexitycontained69
withinCentralValley(CV)Chinooksalmon.TheCentralValleyofCaliforniaishometofour70
runsofChinooksalmon,eachnamedforthetimingoftheirfreshwaterspawningmigration71
(Fall,Late-fall,Spring,andWinterruns).Theserunsaredifferentiatedbythetimingof72
majorlifehistorytransitions,andtheCentralValleyistheonlyareainthespeciesrange73
wherethesefourrunsco-occur(Williams2006).Giventhisdiversityinlifehistory,74
ChinooksalmonintheCVshouldhavearobustportfolio.However,CVChinookhave75
amongtheweakestportfoliointhespeciesrange(Griffithsetal.2014).76
77
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CVChinookwildpopulationabundanceshavebeeninseriousdeclineinrecentdecades78
(Yoshiyamaetal.1998),andseveralofthesepopulationsarepredictedtogoextinctin79
Californiain0-100yearsifnonewactionsaretaken(Moyleetal.2017).SacramentoRiver80
WinterrunandCentralValleySpringrunChinooksalmonarelistedundertheUnited81
StatesEndangeredSpeciesAct(ESA)asendangeredandthreatened,respectively(Federal82
Register1999,2005),whiletheFallandLate-fallrunsarefederalspeciesofconcern83
(Myersetal.1998).SpringandWinterrunsareeachdesignatedastheirownEvolutionary84
SignificantUnit(ESU)andgiventheprotectionof“species”undertheESA,whileFalland85
Late-fallaregroupedtogetherinasingleESU(Myersetal.1998).86
87
Apopulationcollapseintheearly2000sresultedintheclosingofthefisheryin2008and88
2009.Lossoflifehistoryandgeneticdiversityarecitedasmakingthestockssusceptibleto89
collapse(Lindleyetal.2009).Previousworkhasshownthatmuchofthebiocomplexityin90
theCentralValleyhasbeenlost(CarlsonandSatterthwaite2011).Thisincludesdecreased91
portfolioeffectsandincreasesinsynchronyamongpopulationsandresultingincreasesin92
varianceinpopulationabundances(CarlsonandSatterthwaite2011;Griffithsetal.2014).93
ThislossinbiocomplexityincludesgenetichomogenizationoftheFallrun(Williamsonand94
May2005)andintrogressionbetweenrunsduetohatcherypractices(CaliforniaHatchery95
ScientificReviewGroup2012).96
97
Inordertoprotectallthediversityfoundinaportfolioofstocksandpromotepopulation98
buffering,itisvitaltobeabletoaccuratelyassignindividualstotheirpopulationoforigin.99
Thiscanbeverydifficultwhenmultiplepopulationsinterminglealongtheirmigration100
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paths,asisthecasewithCVChinooksalmon.CVChinookrunscanco-occuronthe101
spawninggrounds,butevenmorecommonly,theyco-occuralongmigrationroutes,as102
juvenilesinrearinggrounds,andintheopenocean(Williams2006;2012).Forexample,103
allfourrunsofCVChinookusethelimitedfloodplainhabitatintheSanFranciscoEstuary104
tofeedandgrowbeforetheymoveouttotheoceanandtheycanoccurinthishabitatat105
overlappingtimes(Sommeretal.2001a;b).Currently,mucheffortisbeinginvestedin106
understandinghowdifferentpopulationsofjuvenilesusetheSanFranciscoBay-Delta107
system(Johnsonetal.2017).Becausetherearenomorphologicaldifferencesamongthe108
differentruns,thishasmadeaccuratelymonitoringandmanagingthedifferentpopulations109
veryerrorprone(Harveyetal.2014).110
111
Hatcherymanagementpracticescanalsodisruptthenaturalprocessesthatcreateand112
maintainpopulationdiversificationandbiocomplexity(HuberandCarlson2015;113
SatterthwaiteandCarlson2015).IntheCV,hatcherieswerecreatedtomitigateforthe114
negativeeffectsofthedamsonChinookpopulations,yetwenowknowhatcheriescanhave115
strongnegativeeffectsonwildpopulations(Arakietal.2007;butalsoseeHessetal.2012).116
TherearefivehatcheriesthroughouttheCVproducingFallrunpopulationsandoneeach117
fortheremainingruns.Hatcherypracticeshaveincludedtruckingandreleasingjuveniles118
downstreamofthehatcheryorintheSanFranciscoEstuarytodecreasejuvenilemortality,119
whichhindershomingduringspawningmigrations(CaliforniaHatcheryScientificReview120
Group2012).Additionally,theonlyCVhatcherytoproducebothspringandfallrun121
Chinooksalmon(theFeatherRiverHatchery)hascausedhybridizationbetweenthetwo122
runsintheirhatcherybreedingprogrambynotproperlyseparatingthebroodstockforthe123
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tworuns(CaliforniaHatcheryScientificReviewGroup2012).Thesepracticeshaveledto124
increasedstrayingamongpopulationsandincreasedsynchronyviademographiccoupling125
(HuberandCarlson2015;SatterthwaiteandCarlson2015).Thisinturnhasresultedinthe126
mixingofgenepoolsamongrunsandpopulationsthatwerepreviouslydistinct,and127
ultimatelycomplicatestheidentificationofmanagementunitsandassignmentof128
individualstothoseunits.129
130
PreviousgeneticstudiesofCVChinookpopulationshavereportedvaryinglevelsofgenetic131
distinctivenessamongtherunsandlocations(reviewedin:Lindleyetal.,2004;Williams,132
2006).Studieshaverangedfromthosethatreportdivergenceamongallfourruntypes133
(Banksetal.2000),tostudiesthatfindevidenceforandagainstintrogressionbetweenthe134
runs(Banksetal.2000;Garzaetal.2008;O’Malleyetal.2013;Clementoetal.2014).These135
conflictinggeneticreportsmakeitdifficulttodesignmanagementactionstoprotect136
biocomplexityinCVChinookpopulations.Allofthesegeneticstudiesusedlimitedmarker137
sets(range:10-95markers).Studiesthattakeadvantageofadvancednext-generation138
sequencingtechniques,samplingagreaterproportionofthegenome,areneededtoclarify139
therelationshipsamongCVChinooksalmonandelucidatehowgenomicinformationcan140
assistinconservingbiocomplexityinthisspecies.141
142
Wetakearestriction-siteassociatedDNAsequencing(RAD-seq,Bairdetal.2008;Etteret143
al.2011)approachtoconductthefirstcomprehensivegenome-widegeneticstudyofallthe144
majorpopulationsofChinooksalmonfoundintheCentralValley.Ofprimaryimportanceis145
identifyingtheuniquegeneticdiversitycontainedwithinandamongtheCVChinook146
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salmonpopulationcomplex.Weaimedtodothisbyinvestigatingthepatternsof147
diversificationshownacrosstheCVChinooksalmongenome.Specifically,weaskthe148
followingquestions:149
i. WhatisthepopulationstructureofCVChinooksalmonandhowis150
diversitypartitionedamongmigratoryphenotypes?151
ii. AretheresignalsoffinescalepopulationstructurewithinFalland152
Springruns,despitehighlevelsofhumanmediatedgeneflow?153
iii. Canweassignindividualstotheirmigratoryphenotypes(akarun154
type)andpopulationsusinggenomicdata?155
Informationresultingfromthesequestionswillbegreatlybeneficialforidentifyingthe156
biocomplexityfoundwithinCVChinooksalmon,andforenablingresourcemanagersto157
developstrategiesforprotectinggeneticvariation.158
159
MATERIALSANDMETHODS160
161
Samplecollection162
163
WeobtainedfintissuesamplesofadultChinooksalmonfromtheCaliforniaDept.ofFish164
andWildlifeAnadromousResourcesTissueArchive.Sampleswereoriginallycollected165
duringspawningmigrationsandcomefromallmajorpopulationswithinthefourChinook166
salmonruns(Fall,Late-fall,Spring,Winter)(Table1,Figure1).Weanalyzed28-32167
individualsperpopulation.Previousworkhasshownthissamplesizetobemorethan168
adequateforcapturinggeneticvariationwithgenomicdata(Nazarenoetal.2017).169
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Populationswererepresentedbysamplescollectedfromtwoormoreyears,tocapture170
temporalvariation.However,previousworkhasshowntemporalvariationwithin171
populationsofChinooksalmontoberelativelysmallcomparedtovariationamong172
populations(Banksetal.2000;Beachametal.2006;Narumetal.2008).173
174
Molecularbiology175
176
WeusedthemolecularmethodsoutlinedinMeeketal.(2016).Briefly,weextracted177
genomicDNAandconstructedRADlibrariesusingtheSbfIrestrictionenzymefollowingthe178
protocolfromMilleretal.(2012).Eachsamplewasligatedwithauniquecustomsixbase179
pairbarcodeandwemultiplexed30–47individualsperlibrary.Wesequencedthelibraries180
as100basepairsingle-endreadsonanIlluminaHiSeq2000(VincentJ.CoatesGenomics181
SequencingLaboratory,Berkeley,CA),runningasinglelibraryperlane.182
183
SNPgenotyping184
185
WealignedthesequencesfromeachindividualtotheRADlociinMeeketal.(2016)using186
Bowtie(Langmeadetal.2009)andperformedgenotypingandqualityfilteringfollowing187
themethodsdetailedinMeeketal.(2016).Insummary,wetrimmedthereadsfromthe3’188
endto92bpandeliminatedthosewitha>20%probabilityofsequencingerrorbasedon189
PHREDscores.Wealsoeliminatedthosethathadoneormoreambiguousbasecalls.We190
usedthegenotypingmethodofLewetal.(2015).Aftergenotyping,weremovedindividuals191
thatweregenotypedatfewerthanthelowerconfidenceintervalofgenotypedlociper192
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individual(<6,621loci).Wethenremovedlocithatweregenotypedatfewerthan70%of193
theremainingindividuals,followedbyremovingindividualsthatweregenotypedatfewer194
than70%ofremainingloci.195
196
AlignmenttoChinooklinkagemap197
WealignedourRADlocitotheintegratedChinooklinkagemap,usingtheconsensusfemale198
map(McKinneyetal.2016).Weusedblast+(Camachoetal.2009)withdefaultsettingsto199
makethealignments.Wefurtherfilteredtheblast+outputinR(RDevelopmentCoreTeam200
2005),onlyretainingalignmentswherethelengthwas≥78basepairs,1orfewer201
mismatches,percentidentitywas≥95,andtherewerenogaps.202
203
PopulationStructureandGeneticDiversityAnalyses204
205
Weanalyzedthedatasetforpopulationstructureusingtwomethods.First,weusedthe206
programSTRUCTURE(Pritchardetal.2000)toidentifygeneticallydistinctpopulationsin207
ourdataset.Weranfiveiterationsofeachmodelwith3-8clusters,withaburn-inof20,000208
stepsfollowedby750,000steps.WethenemployedtheprogramsCLUMPP(Jakobssonand209
Rosenberg2007),ingreedymode,andStructureHarvester(EarlandVonHoldt2011)to210
averageoverreplicates.WevisualizedourplotsusingcustomcodeinR(RDevelopment211
CoreTeam2005),availableuponrequest.212
213
Tofurtherexplorepopulationstructure,weconductedadiscriminantanalysisofprincipal214
components(DAPC),asimplementedintheRpackageadegenet(JombartandAhmed215
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2011).ThismethodallowsanalysisofgroupclusteringsimilartoBayesianmethods,such216
asSTRUCTURE(Pritchardetal.2000),withouttheassumptionsofHardy-Weinbergand217
linkageequilibria.DAPChasbeenshowntobebetteratfindingfinescalestructurethanthe218
programSTRUCTURE(Jombartetal.2010;Benestanetal.2015).WerantheDAPConceto219
optimizethenumberofprincipalcomponents(PCs)thatwereretained,testingupto25220
clustersandretaining250PCs.Inthisrun,thealphascoreshowedthatretaining11-14PCs221
providestheoptimalnumberwithoutoverfittingthedata.InthefinalDAPCanalysis,we222
testeduptotenclusters,retainingalldiscriminantfunctionsandtheoptimalnumberof223
principalcomponentsforeachnumberofclusters(K).ResultsoftheDAPCanalysis224
identifiedseveralindividualsthatappearedtobemislabeled,misidentifiedinthefield,or225
werestrays,astheyhadhighprobabilitiesofclusteringwithadifferentrun.Weremoved226
anyindividualwhosemembershipprobabilitytoanalternatepopulationwasgreaterthan227
0.85(Individualsremoved:1F_BUT,2F_MKH,2L_USR,and1S_DER--seelocation228
abbreviationsinTable1).WethencalculatedWeirandCockerham’sunbiasedestimatorof229
FST(WeirandCockerham1984)asimplementedinGENODIVE(MeirmansandVan230
Tienderen2004),using999permutationsanddeterminingsignificancewithaBenjamini231
andYekutieli(2001)FalseDiscoveryRate-correctedvalue(p<0.015),asperNarum(2006).232
Wealsocalculatedallelicrichness,observedandexpectedheterozygosity(Nei1987),and233
inbreedingcoefficientsusingGENODIVE.Weconductedananalysisofmolecularvariance234
(AMOVA),usingtheinfiniteallelesmodeland1000permutations,toinvestigatethe235
partitioningofgeneticvariationacrosspopulationsandrunsinGENODIVE.Wecalculated236
effectivepopulationsize(Ne)withNeEstimator(Doetal.2014)usingthefollowing237
parameters:thelinkagedisequilibriummodelwithrandommating,amiminumallele238
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frequencycutoffof0.02,andcalculatingtheparametric95%confidenceintervalforeach239
estimate.Weremovedlocifromthedatasetthatwerefoundtobepotentialoutliers(see240
methodsbelow)andonlyincludedthoseSNPsthatcouldbelocatedonthelinkagemap.We241
incorporatedchromosomeinformationinthemodelsolinkagedisequilibriumcalculations242
wereonlymadeinpairwisecomparisonsbetweenlociondifferentchromosomes(the“LD243
locuspairingacrosschromosomes”optioninNeEstimator).244
245
WedeterminedpresenceofisolationbydistancebytestingforcorrelationbetweenFST246
(transformedtoFST/(1-FST))andriverdistanceusingapartialManteltestinRusingthe247
veganpackage(Oksanenetal.2017),accountingforruntypeinadissimilaritymatrixand248
usingruntypeasthestrata.Wecalculatedriverdistancebymeasuringtheriverdistance249
betweenrivermouthsinGoogleEarth.Wealsotestedforcorrelationsbetweenriver250
distanceandFSTwithinFallandSpringrunsusingaManteltest.251
252
AlignmenttotheGREB1Llocus253
WealignedourRADlocitotheGREB1Lscaffoldshowntobeassociatedwithpremature254
andmatureruntiminginChinookandsteelheadsalmoninotherpartsoftherange(Prince255
et.al2017;Thompsonetal.2018).Weusedblast+(Camachoetal.2009)usingthedefault256
settingstomakethealignmentsandthenlookedforalignmentsintheregionsidentifiedby257
Princeet.al(2017)andThompsonetal.(2019).Usingthesamemethods,wealsoaligned258
ourlocitotheGREB1LtoROCK1regionidentifiedbyNarumetal.(2018)asbeingrelated259
tomigrationphenotypes.260
261
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AssignmentTesting262
WeevaluatedtheabilityoftheSNPdatasettoassignindividualstothedifferentlyidentified263
groups.Wefirstmadeadatasetofonlylociwithlessthan10%missingdata.Wethen264
conductedleave-one-outtestsinGENODIVE(settings:0.005inplaceofzerofrequencies,265
likelihoodratioteststatistic,alphalevelof0.001,and500permutations)toassessthe266
dataset’sabilitytocorrectlyassignindividualstotheirpopulationoforigin.Wetested267
severalconfigurationsofuniquepopulations:1.Eachtributaryorhatcheryasaunique268
population,2.AllFallrunindividualsincludedinonepopulation,and3.CombiningSpring269
runfromMillandDeerCreeksinonepopulation,butleavingtheotherSpringrun270
populationsasindividualpopulations.Thisallowedustoevaluatewhattributariesand271
hatcheriescouldbeuniquelyidentifiedwiththisdataset.272
273
IdentifyingCandidateLociUnderSelection274
FallrunChinooksalmonistheonlyrunthatstilloccursinboththeSacramentoRiverbasin275
andtheSanJoaquinRiverbasin.PreviousworkhasshownthatFallrunaregenetically276
indistinguishableacrosstheirrangeintheCentralValley(Banksetal.2000;Williamson277
andMay2005).WewantedtoexplorethisfindingfurtherusingoursetofdiscoveredSNPs.278
ToseeifthereishiddengeneticdifferentiationamongFallrunpopulations,weconducted279
anoutliertesttoidentifylocithatshowsignalsofbeingunderselection.Ourgoalwasto280
comparethepopulationstructurefoundusingtheentireSNPdatasetwiththepopulation281
structurefoundamongFallrunusingjusttheFallrunoutlierSNPs.282
283
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Toidentifyoutliers,weemployedtheFDIST2approachimplementedinArlequinv3.5284
(Excoffieretal.2009;ExcoffierandLischer2010).Afterrunning20,000coalescent285
simulations,locithatwereoutsidethe99%quantilewereidentifiedasbeingcandidatesfor286
selection.Wethencreatedadatasetofjustthelocithatwereidentifiedascandidatesfor287
positiveselection.Weevaluatedpopulationclusteringbasedonthesetoflociunder288
positiveselectionusingthesameDAPCmethodsasdescribedabove.Wealignedthe289
outlierstotheSalmosalar(Davidsonetal.2010;ICSASG_v2:Genebankaccession290
GCA_000233375.4)andO.mykissgenomes(GenBankAssemblyAccession291
#GCA_002163495.1),usingBowtie2(LangmeadandSalzberg2012)anddefaultsettings,to292
investigatefunctionalannotationsoftheoutliers.TheS.salargenomeprovidedthemost293
annotations,soweproceededwiththisgenomeforannotatingoutliersfortheSpringand294
Fallruns.Weusedthefollowingalignmentcriteriaforannotation:alignmentlength>75295
basepairs,percentidentity>90%,e-value>0.0001.296
297
Tofurtherinvestigatethevalidityofouroutlierresultsandthepotentialforfalsepositives,298
wecreatedanulldatasetofFallrunindividuals,randomlyreorganizingindividualsinto299
populations.Werandomlyassignedindividuals(usingtherandomnumbergenerator300
functionrunifinR)tothesamenumberofpopulationswiththesamenumberof301
individualsperpopulationastherealdataset.Wethenconductedthesameanalysesfor302
detectingoutliersasdescribedabove.Giventhatthereisnobiologicalmeaningbehindour303
randomlycreatedpopulations,weexpectedthatthereshouldbenooutliersdetected.If304
outliersweredetected,itwouldindicatethattheoutlierdetectionmethodispronetoa305
highnumberoffalsepositivesandlesscertaintycanbeappliedtotheresults.306
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307
WeconductedthesameoutlieranalysesontheSpringrunpopulations,toseeifthereis308
anysignalofselectiondrivingdifferentiationamongthedifferentSpringrunpopulations,309
despiteallextantpopulationsbeinglocatedintheSacramentoRiverbasin.310
311
RESULTS312
313
SequencingandSNPgenotyping314
Weobtainedanaverageof248,595,544readspersequencinglibrary(range:136,417,117-315
460,215,599).Thenumberofuniquereadsperindividualrangedfrom15,822-316
24,137,958,withanaverageof2,985,730perindividual.Themeannumberofgenotyped317
lociforeachindividualwas18,041(CI:6,621–22,969).Weremovedsixindividualsthat318
weretypedatfewerthanthelowerconfidenceinterval(<6,621loci).Weusedthefinalset319
of11,783SNPloci,asdescribedinMeeketal.(2016)forgenotyping.ThisSNPdataset320
excludedlociwithaminorallelefrequencyof<0.01andobservedheterozygosity>0.55in321
ordertoremovepotentialparalogs.Table1showsthenumberofindividualsper322
populationthatremainedafteralsofilteringoutindividualsthatwerenotgenotypedinat323
least70%ofthe11,783SNPloci(12-30individualsperpopulation).Themeanreaddepth324
perlocuswas22.4(95%CI:22.3–22.6).Wealigned6,666ofourlocitotheconsensus325
Chinooksalmonlinkagemap.326
327
GeneticDiversityandPopulationStructure328
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Meanexpectedheterozygocity(He)acrossallpopulationswas0.24(S.D.:0.08),withWinter329
runhavingthelowestHeof0.19andallothergroupsbeingbetween0.24-0.25(Table1).330
Theinbreedingcoefficientforallpopulationswasslightlynegative(range:-0.072---0.03),331
withall95%confidenceintervalsbelow0.332
333
TheAMOVAanalysisshowedthehighestamountofbetweengroupgeneticvariationis334
partitionedamongruns,followedbyamongpopulationsnestedwithinruns(Table2).335
STRUCTUREanalysesshowedthatpopulationstructuringwith6clusters(K=6)hadboth336
thehighestlikelihoodandthehighestdeltaK(FigureS1,TableS1,Evanno,Regnaut,&337
Goudet,2005),butthelikelihoodsplateauedbetweenK=3andK=7.The6-clustermodel338
groupedindividualsbyruntype,withsomesubstructurewithinSpringrunpopulations339
(FigureS1).SpringruninButteCreekclustereduniquely,whereasMillandDeerCreek340
clusteredtogether.SpringrunfromtheFeatherRiverHatcheryshowedadmixturebetween341
auniquegeneticlineageandtheFallruncluster.AllofFallrundisplayedprimary342
membershipinthesameuniquecluster.FallrunfromtheFeatherRiverHatcheryshowed343
someadmixturewiththeuniqueFeatherRiverHatcherySpringruncluster.Thereisalsoa344
signatureofauniqueLate-fallrunlineage,howeveritisadmixedwiththeFallruncluster.345
TheDAPCanalysisshowedsimilarresults,howevereachrunandSpringrunpopulation346
showedgreaterdistinctionandlessindividualadmixturethanintheSTRUCTUREanalysis347
(Figure2andFiguresS2-S3).IntheDAPCanalysisforK=6,Late-fallclustereduniquely,as348
didtheFeatherRiverHatcherySpringrun.Additionally,intheK=7model,Fallruninthe349
ColemanHatcherydisplayeduniqueclustermembership.350
351
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Wefoundsignificantdifferentiationwithinandamongthedifferentruns,basedonFst352
values(range:0-0.161,Table3).ThehighestvalueswerebetweenWinterrunandallother353
populations(range:0.135-0.161).WithinSpring-run,Fstvaluesrangedfrom0.004-0.033,354
withallvaluesbeingsignificant.FstwithinFallrunrangedfrom0-0.005.Fallrunfromthe355
ColemanFishHatcherywassignificantlydifferentfromallotherFallrunpopulations,as356
wasFallrunfromtheFeatherRiverHatchery.FallrunfromMillCreekwasalso357
significantlydifferentfromtheNimbusHatcherypopulationandtheStanislausandMerced358
Rivers.359
360
Usingadatasetof4689locithatwereidentifiedneutralmarkersandcouldbeplacedon361
theChinooklinkagemap,wecalculatedNeforthesixgroupingsidentifiedintheDAPC362
analysis.TheconfidenceintervalfortheNeestimateincludedinfinityforallgroups,with363
theexceptionofSpringrunfromMill/DeerCreeksandWinterrun.TheNeestimatefor364
SpringrunfromMill/DeerCreekswas591.1(CI:558.3-628.0)andforWinterrunwas365
376.4(CI:352.6-403.5).EstimatesofinfinityforNearelikelyduetosamplingerror366
outweighingtheeffectsofgeneticdrift(i.e.samplesizetoosmalltocapturetheeffectsof367
geneticdriftinlargepopulations,seeWaplesandDo(2010)forfurtherdiscussionofthis368
phenomenon).369
370
Therewassignificantisolationbydistancewhenallrunswereusedinthemodelandrunis371
accountedforinthepartialManteltest(P=0.007).Isolationbydistancewasnotsignificant372
whenlimitedtoonlySpringrun(P=0.125),butwashighlysignificantwithinFallrun373
populations(P=0.007,FigureS4).374
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375
AlignmenttoGREB1Lloci376
WefoundoneSNPinourfinaldatasetthatalignedwiththeruntimingassociatedGREB1L377
locus.Thislocus(SNP#R008612)alignedtoposition595079-595161ofthescaffold378
79929edescribedinPrinceet.al(2017).Thegenotypesatthislocuswerenearlyperfectly379
associatedwithpre-mature(SpringandWinterrun)andmature(FallandLate-fall)run380
timings(Figure3).Interestingly,allSpringrunindividualswerehomozygousforoneallele,381
withtheexceptionofsixindividualsfromtheFeatherRiverHatchery(representing7.6%382
oftheSpringrun),whichwereheterozygous.WithinFallrun,90%(170individuals)were383
homozygousforthealternateallele,with8.5%(16individuals)beingheterozygous,and384
1.6%(3individuals)beinghomozygousfortheSpring/Winterassociatedallele.Twoofthe385
individualsthatwerehomozygousfortheSpring/WinterallelewerefromtheFeather386
RiverHatcheryandonewasfromMillCreek.Wefound87SNPlocithatalignedtothe203387
KbregioncontainingtheGREB1LandROCK1genesidentifiedbyNarumetal.(2018)on388
Ots28between11.022and11.225Mb.Wecalculatedtheallelefrequencydifferencesfor389
eachrunatthese87locitoevaluatethepatternsamongthedifferentruns(Figure4).The390
samelocusidentifiedbyaligningtothePrinceetal.(2017)scaffold(SNP#R008612)391
alignedtothisregionandclearlyshowsdifferencesamongthepre-matureandmaturerun392
timings.Nootherlocusshowedasclearofapattern.393
394
Assignmenttesting395
Assignmenttestingshowedhighprobabilityofcorrectassignmentusingthedatasetofloci396
withlessthan10%missingdata(7829loci).WhenFallrunpopulationsweregrouped397
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togetherinonegeneral“FallRun”reportinggroup,assignmentprobabilitieswerehighfor398
Fallrun(100%),butalsoFallrundrewalotofnon-Fallrunassignments(e.g.individuals399
fromLate-fallandSpringrun).Thevastmajority(96%)oftheLate-fallrunassignedtothe400
FallrunpopulationusingthisdatasetandassignmentofSpringrunpopulationsranged401
from40-100%(TableS2).However,whenweusedeachindividualFall-runpopulationasa402
uniquereportinggroup,assignmentforindividualFallrunpopulationsrangedwildly(0-403
100%),however,95%oftheassignmentsforFallrunindividualswereassignedtoaFall404
runpopulation.Notably,FallrunfromtheMercedRiver,ColemanHatchery,andNimbus405
HatcherydrewalmostallofassignmentsfromotherFallrundrainageswhenusing406
individualreportinggroups(TableS3).Basedonthesedata,wecannotdistinguish407
individualswhohadgeneticmis-assignmentstothewrongnatalstreamfromthosethat408
wereassignedcorrectlybutreturnedtonon-natalstreamstospawn(“strayed”),making409
themappeartobemis-assigned.Notably,weachieved100%accuracyofassignmentfor410
Late-fallwhenusingindividualFallrunreportinggroups.Assignmentaccuracywas100%411
whenwecombinedtheSpringrunfromMillandDeerCreeksintoasinglegroup(Figure5).412
ThesedatashowthatusingthefullSNPdatasetenableshighassignmentaccuracyof413
populations,withhighaccuracyofFallrunwhenusingageneralFallrunreportinggroup.414
415
LociUnderSelection416
OutlieranalysesperformedwithinFallrunfound829locithatweresignificantforshowing417
signaturesofdiversifyingselectionusingArlequin.654oftheoutliersalignedtothe418
Chinooksalmonlinkagemap,withthelocispreadthroughoutthegenome(between10-38419
outliersalignedtoeachchromosome)(Figure6).Twenty-threelocimappedtolinkage420
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groupsbutwerenotpositionedonachromosome.Forty-fiveoutliershadfunctional421
annotationsassociatedwiththem(TableS4).Wefound0outliersintherandomized422
dataset,providingreassurancethatourresultsfromanalysisoftherealdatasetare423
meaningful.424
425
TheDAPCmodelusingjusttheselociandretaining70principlecomponentsfoundthat426
K=1-3hadthelowestBIC,withK=4and5slightlyhigherbutwithclusteringthatshows427
biologicalrelevance(Figure7,FigureS5).AtK=2,auniqueclusterwasformedbyseveral428
individualsfromMill,Deer,andButteCreeks,theTuolumneandMercedRiverHatchery,429
andoneindividualfromtheMercedRiverpopulation.WhentheSpringandWinterrun430
populationsarealsoincluded,itbecomesclearthatthisuniqueclusterisformedwith431
SpringrunfromtheFeatherRiverHatchery(datanotshown).Therefore,itisverylikely432
thatthisclusterisdrivenbyintrogressionwithstraysfromtheSpringrunintheFeather433
RiverHatchery,ratherthanbeingauniqueFallrunlineage.TheFallrunfromColeman434
HatcheryshoweduniqueclustermembershipstartingatK=3.AtK=4,twoadditional435
groupsemergedwithDeer,Butte,andMillCreeksclusteringtogetheralongwiththe436
MercedRiverandMercedRiverHatcherypopulations,andtheNimbusandMokelumne437
HatcheriesandStanislausandTuolumneRiversclusteringtogether.AtK=5,theMerced438
Riverpopulationwasresolvedasitsownuniquecluster,withsomeindividualsinButte439
Creekdrawingmembershipfromthiscluster.Theseclusteringpatternsshowageneral440
geographicpatternofclustering,withmanyofthewildSacramentoRiverbasin441
populationsclusteringtogetherandmanyoftheSanJoaquinRiverbasinpopulationsin442
anothercluster.443
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444
TheoutlieranalyseswithinSpringrunfound940locithatweresignificantforsignaturesof445
diversifyingselection.776oftheoutliersalignedtotheChinooksalmonlinkagemap,with446
between12-46outliersalignedtoeachchromosome.Twenty-ninelocimappedtolinkage447
groupsbutwerenotpositionedonachromosome.Fifty-sevenoutlierlocihadannotations448
associatedwiththem(TableS4).Nooutliersweredetectedwhenanalyzingthe449
randomizeddataset.450
451
TheDAPCmodelusingjusttheselocifoundK=3-4hadthelowestBIC(Figure8,FigureS6).452
AtK=3,DeerandMillCreeksgroupedtogether,whileatK=4,eachtributarypopulation453
groupedseparately.101lociwereidentifiedasbeingpotentialoutliersinboththeFalland454
Springrunanalyses.455
456
Fstvaluesweremuchhigheramongpopulationswhenusingtheoutlierdataset,andall457
pairwisecomparisonsweresignificant(Fallrunmean=0.035andrange:0.008–0.061,458
Springrunmean=0.102andrange:0.036–0.166;TablesS5andS6).459
460
DISCUSSION461
462
Chinooksalmonexhibitsomeofthehighestgeneticandlifehistorytraitdiversityofthe463
Pacificsalmonids(Waples2001).Inparticular,thepresenceoffourdistinctspawningruns464
intheCentralValleyofCaliforniarepresentsthegreatestlifehistorytraitdiversity465
observedforChinooksalmon,andpresumablycorrespondswiththehighestbiocomplexity466
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andportfolioforthisspecies.Inreality,CVChinooksalmondisplaytheweakestportfolioin467
thespeciesrange(Griffithsetal.2014);however,ourresultsindicatethatthereisgreater468
geneticbiocomplexitythanpreviouslydescribed.Protectionofthisremaining469
biocomplexitymaybeimportantforsustainablemanagementoftheCVChinooksalmon470
stockcomplexandrestorationoftheportfolio.471
472
Thisstudyisthefirstgenomicinvestigationintothepopulationstructureandgenomic473
diversityofCentralValleyChinooksalmon.Bygenotypinghundredsofindividualsat474
thousandsofSNPsdistributedacrossthegenome,wefoundevidenceforgreater475
populationstructuringbeyondthelevelofrun,includingfine-scalepopulationstructurefor476
Springrunfishandapatternofisolation-by-distanceamongFallrunpopulations.Wealso477
findgeneticdistinctivenessofFallandLate-fallruns,despiteconclusionsfromprevious478
scientificstudyandcurrentmanagementasasingleESU(Lindleyetal.2004).Ourresults479
providemanagerswithinformationnecessarytonotonlyconservethediversityofrunsin480
theCentralValley,butalsotomaintainandenhancelifehistorydiversitywithinrunsfor481
maximalbufferingfromenvironmentalvariationandoverallportfolioperformance482
(Satterthwaiteetal.2017).483
484
WeshowthatSpringruncontainsfinerscalesub-structure,withevidenceforthreedistinct485
populations:1)MillandDeerCreek,2)ButteCreek,and3)theSpringrunintheFeather486
RiverHatchery.Thislatterfindingisquiteremarkable,aspreviousworkhasshownthat487
SpringrunintheFeatherRiverHatcheryisgeneticallyindistinguishablefromFallrun488
(Lindleyetal.2004;Garzaetal.2008).Pasthatcherypracticesincludeinadequate489
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separationofthebroodstockforFallandSpringrun,leadingtointrogressionbetweenthe490
tworuns,andtheprevailingwisdomhasbeenthattheserunsaregenetically491
indistinguishable(CaliforniaHatcheryScientificReviewGroup2012).O’Malleyetal.(2007;492
2013)foundnodifferentiationbetweenFallandSpringrunintheFeatherRiverHatchery493
basedonnineneutralmicrosatelliteloci,buttheydidfindcleardistinctionatthree494
circadianclockgenesthoughttocontrolruntiming.Ourresultsshowthatifthefull495
genomeissurveyed,additionaldistinctionsbetweentheFeatherRiverHatcheryFalland496
Springrunareobserved.WedoseeevidenceofpastintrogressionattheGREB1L497
associatedlocus,withtheSpringrunfromtheFeatherRiverHatcherybeingtheonlySpring498
runpopulationsthatcontainedheterozygousindividuals.Theseresultsshowthatsomeof499
thegeneticdiversityfoundintheFeatherRiverHatcherySpringrunisuniqueandhasnot500
beeneliminatedfromintrogressionwithFallrun.Additionally,theseresultssupportthe501
useofrevisedhatcherypracticesthataimtobetterseparatethetworunsinthehatchery502
(Baerwaldetal.2011).503
504
Ourresultsdemonstratethatwiththefull~12KSNPdataset,notonlycanwedistinguish505
SpringrunintheFeatherRiverHatcheryfromotherSpringrunpopulationsandfromFall506
run,wecanalsodistinguishtheLate-fallfromFallrunpopulations.Ourdemonstrated507
abilitytodistinguishindividualsfromtheserunsisasignificantleapforwardinourability508
tomonitorandmanagethesepopulationsseparately,enablingprotectionofbiocomplexity509
inthesystem.Itisvitalthatallofthegeneticdiversitypresentinthissystemisprotectedin510
ordertomaintainahealthyportfoliooflifehistoriesandgeneticdiversity(Schindleretal.511
2010;CarlsonandSatterthwaite2011).512
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513
Recently,ahigh-throughputCentralValleyspecific80-SNPpanelwasdevelopedthatgoesa514
longwaytowardimprovingourabilitytodistinguishthedifferentruns(Meeketal.2016).515
ThebenefitofthispanelisitisavailableasFluidigmSNPtypeassays(FluidigmCorp.San516
Francisco,CA)allowingveryfastturnaroundfromtissuesampletogenotypeandrun517
assignment.Thisfastturnaroundisrequiredforsomemanagementquestions(e.g.real-518
timetakedeterminations).Thetrade-off,however,isthisSNPpanelisunabletodistinguish519
theSpringrunintheFeatherRiverHatcheryfromtheFallrunpopulationsandithaslower520
assignmentaccuracies.Weshowthat,withalmost12,000SNPs,wecandistinguishthe521
differentpopulationswithhighaccuracy.Therefore,whenrunassignmentisnotneededin522
real-time,genotypingindividualsviaasequencebasedapproachwillallowfinerscaleand523
moreaccuratemanagementofthedifferentpopulations(MeekandLarson2019).Our524
workalsoshowsthatbylookingattheGREB1Lassociatedlocus,wecaneasilydistinguish525
theESAlisted(SpringandWinterrun)fromtheunlisted(FallandLate-fall)individuals.526
FutureworkwillinvolveexploringthisGREB1Llocusfurtheranddevelopingtheseloci527
intoRapturebaits(Alietal.2016),toallowveryhighthroughputandcosteffective528
genotypingviasequencingofthousandsofsamples.529
530
OurworkalsoshowsthattheWinterrunisthemostgeneticallydistinctpopulationinthe531
CentralValley,butitalsohasthelowestgeneticdiversity(e.g.observedandexpected532
heterozygosity,SeeTable1).WeshowthattheWinterrunhasaloweffectivepopulation533
size(NE=376)andmaybeexperiencinghighlevelsofgeneticdriftassociatedwith534
decreasedpopulationsizes.Frankhametal.(2014)suggestthatanNEbelow500istoolow535
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toretainevolutionarypotentialinperpetuity,andthatatargetofNE=1000forwild536
populationsismoreappropriate.Followingthisguideline,SpringrunfromtheMill/Deer537
Creekcomplexarealsoatriskofreducedlong-termviabilitygiventheirNE=591.These538
resultsarenotsurprising,especiallygiventheEndangeredandThreatenedstatusof539
WinterandSpringrun,butithighlightstheimportanceofmanagementeffortsaimedat540
protectingtheportfolioofdiversitypresentintheseruns,andCentralValleyChinookasa541
whole.542
543
Dispersalamongpopulationsisoneofthemainmechanismsforincreasedsynchrony544
amongpopulationsandweakeningoftheportfolioeffect(Liebholdetal.2004).Human545
activities,suchasartificialproduction,canincreasedispersalamongpopulations,546
decreasinglocallyadaptedlifehistoryvariationandincreasingsynchrony(McClureetal.547
2008;Mooreetal.2010).ItisestimatedthatstrayinginhatcheryrearedChinookcanbe548
above70%(CaliforniaHSRG2012;Palmer-Zwahlen,&Kormos,2015).Thisisadrastic549
increasecomparedtotheoftenlessthan5%strayingthatoccursinwildsalmonid550
populations(Quinn2005).Itisthoughtthatthisincreasedstrayingduetohatchery551
productionandthelackofmigrationpathimprintingfromtruckinghasledtothegenetic552
homogenizationanddecreasedportfolioeffectamongFallrunintheCV(Huberand553
Carlson2015;SatterthwaiteandCarlson2015).Infact,DedrickandBaskett(2018)found554
thatgenetichomogenizationintheCentralValleyFallrunplaysalargerroleindecreasing555
theportfolioeffectthandemographicsynchronization.Theroleoftruckingandincreased556
strayingcouldbeplayingalargeroleinthegenetichomogenizationweseeinFallrun.Our557
workhere,however,showsthatdespiteincreasedstraying,therearestillsomeremaining558
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geneticdifferencesamongpopulations.Thisisevidentinthestructuringweseewiththe559
putativelociunderselection.Thisworkalsodemonstratestheimportanceofaltering560
hatcheryandmanagementpracticesnowtoprotectextantdiversity,whileitstillexistsin561
thesystem.Maintenanceofbiocomplexityinasystemcancounteracthomogenizingforces562
andpromotepopulationasynchrony(Mooreetal.2014).563
564
Ourworkshowshowinsightsaboutpopulationdifferentiationcanbegainedbyevaluating565
genomiclocipresumablyunderselection.Whenweevaluatedpopulationstructureusing566
onlylocithatarecandidatesforselection,weobservedevenfinerscalestructuringofboth567
theSpringandFallrunthanwasobservedusingthefulldataset.AlloftheSpringrun568
populationsshowdifferentiation,anddifferencesamonggroupsofFallrunpopulationsare569
apparent.TheFallrunpopulationintheColemanHatcheryisveryclearlydistinctfromthe570
restofFallrunbasedontheoutlierdataset,suggestingthatsomeaspectofColeman571
Hatcherypracticesmaybedrivingdifferentiation.Additionally,theFallrunfromthe572
NimbusHatchery,MokolumneHatchery,StanislausRiver,andTuolumneRiveralso573
separateintoonegroupthatisdifferentiatedfromtherestoftheFallrunpopulations.Itis574
possiblethatfishinthesetributariesarefacingsimilarselectionpressuresdrivingthis575
pattern.AtK=5,wealsostarttoseetheFallrunspawningintheMercedRivershow576
distinctionfromtherestofFallrun.Interestingly,theFallrunspawningintheMerced577
RiverHatcheryshowverymixedclustermembership,withrepresentationfromother578
clustersfoundintheCV.Itispossiblethatthereisuniqueselectionpressureexperienced579
byfishspawningintheriverhabitatversusinthehatchery,whichdrivesthisdifference580
(Vasemägietal.2016),orthattheMercedRiverHatcheryreceivesalotofstraysfrom581
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othertributaries.Indeed,a2012studyofhatcheryreturnsfoundonly12.7%ofreturnsto582
theMercedRiverHatcherywerefromMercedRiverHatcheryraisedfish(Palmer-Zwahlen583
andKormos2015).584
585
Thereissomedebateregardingtheuseofadaptivelociinconservationplanning.Allendorf586
etal.(2010)cautionagainstfocusingonadaptivelociforconservation,statingthatloci587
identifiedasbeingadaptivemightnotbethoselocithatarecrucialforfutureadaptation.588
Additionally,theauthorswarnthatfocusingondetectableadaptivedifferencesmayresult589
inoverlooking,andpotentiallylosing,importantgeneticdiversityinotherregionsofthe590
genome.Funketal.(2012),however,highlighttheimportanceofusinginformationfrom591
bothneutralandnon-neutralgeneticdatatoidentifyanappropriatemanagement592
approachforspecies.WeagreewithbothFunketal.andAllendorfetal.andbelievethere593
isvalueinincorporatingbothneutralandnon-neutralgeneticinformationinconservation594
strategies.Webelieveboththesecautionshighlighttheneedtoconserveasmuchgenetic595
diversityasisfeasible,becauseitisverydifficulttopredictwhatdiversitywillbe596
“important”bothnowandintothefuture.Webelievethistobeparticularlytruefor597
migratoryspecies,asmigratoryspeciesaresubjecttoselectiveforcesacrossthemigratory598
pathway,itcanbeverydifficulttodecipherwhatisdrivingselectivedifferences,and599
selectiveforceswillcertainlychangeovertime(Dingle2014).Forexample,thedifferences600
weseeinselectedlociamongCVChinooksalmonpopulationsmaybeduetoselection601
differencesamongthespawninggrounds,seasonaldifferencesamongmigrationlife602
histories,ordifferencesinselectiveforcesinseparateregionsoftheocean.Additionally,603
ourstudyisnotanexhaustivestudyofallpossibleformsofbiocomplexityinthissystem.604
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Forexample,weknowthatthereisalsodiversityintimingofjuvenileout-migrationthatis605
certainlyimportantforbiocomplexity.Ourstudyhighlightstheimportanceofidentifying606
andprotectingasmuchdiversityaspossiblesothereisafullportfolioofgeneticdiversity607
presentinthesystem.608
609
CONCLUSION610
Collectively,theseresultshavestrongimplicationsforthemanagementofCVChinook611
salmonandprovideimportantlessonsforusinggenomicstoidentifybiocomplexityand612
applyresultinginferencestotheconservationofmigratoryspecies.Genomicsarean613
increasinglyimportanttoolformanagingspecies.Theyprovidetheabilitytoidentify614
biologicaldiversitypresentinasystem,andtoassignindividualstopopulationoforigin615
despitespatiotemporalmixing,suchasonmigratorypathways.Itisvitalthatweimprove616
ourabilitytoidentifyandprotectbiocomplexityinmanagementandconservationsowe617
canimprovespeciesandmetapopulationstabilityandresilience.618
619
ACKNOWLEDGEMENTS620
WewouldliketothankDr.MortenLimborgandDr.WesLarsonforhelpfuldiscussionsthat621
improvedthemanuscriptandDr.NadyaMamoozadehandanonymousreviewersfor622
suggestionsandedits.WethankDr.MikeMillerandDr.DanPrinceforsharingtheirdata623
andinsightsontheGREB1Llocus.WealsothankLeaKoerberandtheCaliforniaDept.of624
FishandWildlifeTissueArchiveforsupplyingChinooktissuesamples.Alltissueswere625
heldunderauthorizationofESASection10(a)(1)(A)permit(Pacificfish)#15926,a626
MemorandumofUnderstandingwithCaliforniaDepartmentofFishandWildlife,and627
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CaliforniaDepartmentofFishandWildlifeScientificCollectingPermits#8871,3561,8862,628
11381,and1751.ThisworkwasmadepossiblebyfundingfromtheCaliforniaDept.ofFish629
andWildlife,contractnumberP0740017,andMHMwassupportedbytheDavidH.Smith630
ConservationResearchFellowshipProgram.ThisworkusedtheVincentJ.CoatesGenomics631
SequencingLaboratoryatUCBerkeley,supportedbyNIHS10InstrumentationGrants632
S10RR029668andS10RR027303.633
634
DataArchivingStatement:635
Dataforthisstudyareavailableat:tobecompletedaftermanuscriptisacceptedfor636
publication.637
638
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847
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TABLES848
849
Table1:Sampleinformation.SamplesizecolumngivestotalnumberusedinRAD-850
sequencing/Totalnumberusedinanalysesafterfiltering.He=expectedheterozygosity,851
Ho=observedheterozygosity,Ar=averagenumberofalleles,Fis=inbreedingcoefficient852
(95%confidenceinterval)853
854
Location Years Sampled
Location Abbreviation
Sample Size
He Ho Ar Fis
Fall Run
Butte Cr. 2002-2004 F_BUT 32/16 0.246 0.259 1.844 -0.054 (-0.063 - -0.044)
Coleman Hatchery (Battle Creek)
2002 F_COL 30/30 0.245 0.261 1.915 -0.064 (-0.072 - -0.056)
Deer Cr. 2002-2004 F_DER 30/13 0.245 0.259 1.826 -0.056 (-0.065 - -0.046)
Feather R. Fish Hatchery
2001, 2007-2010
F_FRH 28/21 0.244 0.259 1.870 -0.061 (-0.069 - -0.052)
Merced R. 2001, 2004, 2008
F_MER 34/27 0.245 0.262 1.910 -0.072 (-0.08 - -0.063)
Mill Cr. 2001-2004 F_MIL 35/14 0.249 0.264 1.847 -0.061 (-0.07 - -0.051)
Mokelumne R. Fish Hatchery
2004-2005, 2008
F_MKH 30/18 0.244 0.258 1.854 -0.058 (-0.068 - -0.049)
Merced R. Fish Hatchery
2001-2004 F_MRH 30/21 0.245 0.258 1.877 -0.05 (-0.059 - -0.041)
Nimbus Fish Hatchery
(American River)
2002-2005 F_NIM 30/25 0.243 0.259 1.886 -0.068 (-0.076 - -0.06)
Stanislaus R. 2001, 2002, 2004
F_STN 30/12 0.243 0.259 1.791 -0.067 (-0.077 - -0.058)
Tuolumne R. 2004, 2008 F_TOU 33/18 0.246 0.260 1.862 -0.061 (-0.07 - -0.052)
Late Fall Run
Upper Sacramento R.
2003-2005 L_USR 30/24 0.245 0.26 1.881 -0.062 (-0.071 - -0.054)
Spring Run
Butte Cr. 2008-2009 S_BUT 30/22 0.241 0.250 1.827 -0.034
(-0.043 - -0.026)
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Deer Cr. 2002, 2005 S_DER 31/20 0.246 0.254 1.864 -0.031 (-0.039 - -0.022)
Mill Cr. 2002, 2004, 2005
S_MIL 32/18 0.247 0.261 1.858 -0.056 (-0.065 - -0.048)
Feather R. Fish Hatchery
2009-2010 S_FRH 30/30 0.247 0.263 1.910 -0.068 (-0.076 - -0.06)
Winter Run
Upper Sacramento
R. 2001, 2002 W_USR 30/30 0.194 0.199 1.717 -0.03
(-0.04 - -0.02) 855
Table2:ResultsofAMOVAanalysis856
Source of Variation Nested in %variance F-stat P-value Within Individual -- 1.006 F_it -- Among Individual Population -0.051 F_is 1 Among Population Run 0.005 F_sc 0.001 Among Run -- 0.039 F_ct 0.002 857
858
859
860
861
862
863
864
865
866
867
868
869
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Table3:Fstvaluesamongpopulationsusingfull,filtereddataset.Valuesbelowthediagonal870
areFst.Valuesabovethediagonalarethesignificancevalue.Bolded,italicizedFstvalues871
aresignificantatp<0.015.872
873
F_COL F_MIL F_DER F_BUT F_FRH F_NIM F_MKH F_STN F_TOU F_MER F_MRH L_USR S_BUT S_DER S_MIL S_FRH W_USR
F_COL -- 0.002 0.009 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
F_MIL 0.002 -- 0.441 0.309 0.013 0.002 0.039 0.007 0.038 0.002 0.22 0.001 0.001 0.001 0.001 0.001 0.001
F_DER 0.002 0 -- 0.864 0.01 0.022 0.112 0.037 0.033 0.024 0.106 0.001 0.001 0.001 0.001 0.001 0.001
F_BUT 0.003 0 -0.001 -- 0.299 0.306 0.589 0.977 0.528 0.534 0.489 0.001 0.001 0.001 0.001 0.001 0.001
F_FRH 0.003 0.001 0.001 0 -- 0.001 0.001 0.001 0.003 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
F_NIM 0.004 0.002 0.001 0 0.002 -- 0.713 0.558 0.231 0.234 0.029 0.001 0.001 0.001 0.001 0.001 0.001
F_MKH 0.004 0.001 0.001 0 0.002 0 -- 0.524 0.37 0.442 0.038 0.001 0.001 0.001 0.001 0.001 0.001
F_STN 0.004 0.002 0.002 -0.002 0.003 0 0 -- 0.703 0.593 0.276 0.001 0.001 0.001 0.001 0.001 0.001
F_TOU 0.004 0.001 0.001 0 0.002 0 0 0 -- 0.231 0.16 0.001 0.001 0.001 0.001 0.001 0.001
F_MER 0.004 0.002 0.001 0 0.002 0 0 0 0 -- 0.037 0.001 0.001 0.001 0.001 0.001 0.001
F_MRH 0.005 0.001 0.001 0 0.003 0.001 0.001 0.001 0.001 0.001 -- 0.001 0.001 0.001 0.001 0.001 0.001
L_USR 0.009 0.007 0.007 0.006 0.009 0.008 0.007 0.007 0.008 0.007 0.008 -- 0.001 0.001 0.001 0.001 0.001
S_BUT 0.037 0.035 0.038 0.038 0.035 0.041 0.04 0.041 0.04 0.04 0.041 0.042 -- 0.001 0.001 0.001 0.001
S_DER 0.02 0.019 0.02 0.021 0.018 0.024 0.023 0.024 0.023 0.023 0.024 0.025 0.017 -- 0.002 0.001 0.001
S_MIL 0.021 0.019 0.021 0.021 0.019 0.024 0.024 0.024 0.024 0.023 0.024 0.026 0.023 0.004 -- 0.001 0.001
S_FRH 0.01 0.007 0.008 0.009 0.005 0.012 0.011 0.012 0.01 0.01 0.011 0.015 0.033 0.019 0.019 -- 0.001
W_USR 0.14 0.148 0.152 0.15 0.145 0.152 0.153 0.161 0.154 0.15 0.154 0.151 0.151 0.14 0.146 0.135 --
874
875
876
877
878
879
880
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FIGURELEGENDS:
Figure1:CentralValleyChinooksalmonsamplinglocations.Thepointsrepresenttheriverssamples
werecollectedfrom,nottheexactsamplinglocation.FiguremodifiedfromMeeketal.(2016).
Figure2:DiscriminantAnalysisofPrincipleComponents(DAPC)clusteringresults,testingdifferent
numberofgroups(K=3-8)usingthefullSNPdataset.Eachbarrepresentsanindividualandthex-axis
showssampledlocation(locationabbreviationsdefinedinTable1).Thecolordistinguishesclustersand
they-axisquantifiesclustermembership.
Figure3:ProportionofindividualsshowingeachgenotypeattheGREB1LassociatedSNP,byruntiming.
Figure4:Heatmapofallelefrequenciesfor87SNPsalignedtotheGREB1LtoROCK1regionofOmy28
identifiedinNarumetal.(2018).Eachrowisoneofthe87SNPs.ThearrowpointstoSNPR008612
showninFigure3thatalignstotheGREB1LregionidentifiedinPrinceetal.(2017).Thecolorscalebar
denotesthefrequencyofthefirstalleleateachlocus.
Figure5:Assignmentaccuraciesusing7892SNPswith<10%missingdata.Pointsarescaledtothe
assignmentvalue.Thevalueontopofeachpointisthepercentassigningtoeachgroup,thevalueunder
eachpointisthenumberofindividualsthatwereassignedtoeachgroup.
Figure6:ManhattanplotofoutliersofSNPsmappedtotheChinooklinkagemap(McKinneyetal.2016).
EachpointrepresentsaSNP.X-axisshowsthepositionoftheSNPonthelinkagemap.Alternating
chromosomesarecoloredinblackorgrey.ThetoppanelhastheFallrunoutlierlocihighlightedinred,
thebottompanelhastheSpringrunoutlierlocihighlightedinred.Colorfigurefoundonline.
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Figure7:DiscriminantAnalysisofPrincipleComponents(DAPC)clusteringresultsforFallrun
populationsusing852candidateoutlierlocifordiversifyingselection.Eachbarrepresentsanindividual
andthex-axisshowssampledlocation(locationabbreviationsdefinedinTable1).Thecolor
distinguishesclustersandthey-axisquantifiesclustermembership.
Figure8:DiscriminantAnalysisofPrincipleComponents(DAPC)clusteringresultsforSpringrun
populationsusing940candidateoutlierlocifordiversifyingselection.Eachbarrepresentsanindividual
andthex-axisshowssampledlocation(locationabbreviationsdefinedinTable1).Thecolor
distinguishesclustersandthey-axisquantifiesclustermembership.
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Figure1:
H
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Figure2:
K=3
Mem
bers
hip
prob
abili
ty
F-CO
L
F-M
IL
F-DE
R
F-N
IM
F-M
KH
F-BU
T
F-FR
H
F-M
ER
F-M
RH
F-TO
U
L-U
SR
S-BU
T
S-DE
R
S-FR
H
W-U
SR
S-M
IL
POPULATIONS
F-ST
N
K=3
K=4
K=5
K=6
K=7
K=8
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Figure3:
Fall Late-fall Spring Winter
Homozygous (T/T)Heterozygous (C/T)Homozygous (C/C)
SNP R008612
Run
Proportion
0.0
0.2
0.4
0.6
0.8
1.0
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Figure4:
Fall Late-fall Spring WinterRUNS
LOCI
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Figure5:
Fall
Late_fall
Sp_Butte
Sp_FRH
Sp_MillDeer
Winter
Fall Late_fall Sp_Butte Sp_FRH Sp_MillDeer WinterActual
Assigned
Value0.00
0.05
0.50
0.75
0.90
1.00
94.9%
100%
100%
96.7%
100%
100%
0.05%
4.7%
3.3%
204
1
10
24
38
22
1
29
30
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Figure6:
Chromosome
Fst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Chromosome
Fst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
0.0
0.1
0.2
0.3
0.4
0.5
0.6
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Figure7:
F-CO
L
F-MIL
F-DE
R
F-NIM
F-MKH
F-BU
T
F-FRH
F-MER
F-MRH
F-TO
U
F-STN
POPULATIONS
K=2
K=3
K=4
K=5
Mem
bershipprob
ability
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Figure8:
S-MIL
S-DE
R
S-BU
T
S-FRH
POPULATIONS
Mem
bershipprob
ability
K=4
K=3
K=5
K=6
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