! ! ! The!Influence!of!Genetic!Variation!on!Susceptibility!of!Common!Bottlenose!Dolphins! (Tursiops)truncatus)!to!Harmful!Algal!Blooms! !by! !Kristina!Marstrand!Cammen! University!Program!in!Ecology! Duke!University! ! Date:_______________________! Approved:! ! ___________________________! ! ! Andrew!J.!Read,!Supervisor! ! ___________________________! Richard!T.!Di!Giulio! ! ___________________________! Eric!P.!Palkovacs! ! ___________________________! Patricia!E.!Rosel! ! ___________________________! Randall!S.!Wells! ! Dissertation!submitted!in!partial!fulfillment!! of!the!requirements!for!the!degree!of!! Doctor!of!Philosophy!in!the!University!Program!in!Ecology! in!the!Graduate!School!of!Duke!University! ! 2014! ! ! ! ! ABSTRACT! The!Influence!of!Genetic!Variation!on!Susceptibility!of!Common!Bottlenose!Dolphins! (Tursiops)truncatus)!to!Harmful!Algal!Blooms! by! Kristina!Marstrand!Cammen! University!Program!in!Ecology! Duke!University! ! Date:_______________________! Approved:! ! ___________________________! Andrew!J.!Read,!Supervisor! ! ___________________________! Richard!T.!Di!Giulio! ! ___________________________! Eric!P.!Palkovacs! ! ___________________________! Patricia!E.!Rosel! ! ___________________________! Randall!S.!Wells! ! An!abstract!of!a!dissertation!submitted!in!partial! fulfillment!of!the!requirements!for!the!degree!of!! Doctor!of!Philosophy!in!the!University!Program!in!Ecology! in!the!Graduate!School!of!Duke!University! ! 2014! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Copyright!by! Kristina!Marstrand!Cammen! 2014! ! ! ! ABSTRACT! The!capacity!of!marine!organisms!to!adapt!to!natural!and!anthropogenic! stressors!is!an!integral!component!of!ocean!health.!Harmful!algal!blooms!(HABs),!which! are!one!of!many!growing!threats!in!coastal!marine!ecosystems,!represent!a!historically! present!natural!stressor!that!has!recently!intensified!and!expanded!in!geographic! distribution!partially!due!to!anthropogenic!activities.!In!the!Gulf!of!Mexico,!HABs!of! Karenia)brevis!occur!almost!annually!and!produce!neurotoxic!brevetoxins!that!have!been! associated!with!largeVscale!mortality!events!of!many!marine!species,!including!the! common!bottlenose!dolphin!(Tursiops)truncatus).!The!factors!resulting!in!largeVscale! dolphin!mortality!associated!with!HABs!are!not!well!understood,!particularly!in!regards! to!the!seemingly!different!impacts!of!HABs!in!geographically!disjunct!dolphin! populations.!My!dissertation!investigates!a!genetic!basis!for!resistance!to!HABs!in! bottlenose!dolphins!in!centralVwest!Florida!and!the!Florida!Panhandle.!I!used!both! genomeVwide!and!candidate!gene!approaches!to!analyze!genetic!variation!in!dolphins! that!died!putatively!due!to!brevetoxicosis!and!live!dolphins!from!the!same!geographic! areas!that!survived!HAB!events.!Using!restriction!siteVassociated!DNA!sequencing,!I! identified!genetic!variation!that!suggested!both!a!common!genetic!basis!for!resistance!to! HABs!in!bottlenose!dolphins!across!the!Gulf!coast!of!Florida!and!regionally!specific! resistance.!Many!candidate!genes!involved!in!the!immune,!nervous,!and!detoxification! systems!were!found!in!close!genomic!proximity!to!survivalVassociated!polymorphisms! throughout!the!bottlenose!dolphin!genome.!I!further!investigated!two!groups!of! ! iv! ! candidate!genes,!nine!voltageVgated!sodium!channel!genes!selected!because!of!their! putative!role!in!brevetoxin!binding!and!four!major!histocompatibility!complex!(MHC)! loci!selected!because!of!their!genomic!proximity!to!a!polymorphism!exhibiting!a!strong! association!with!survival.!I!found!little!variation!in!the!sodium!channel!genes!and! conclude!that!bottlenose!dolphins!have!not!evolved!resistance!to!HABs!via!mutations!in! the!toxin!binding!site.!The!immunologically!relevant!MHC!loci!were!highly!variable!and! exhibited!patterns!of!genetic!differentiation!among!geographic!regions!that!differed! from!neutral!loci;!however,!genetic!variation!at!the!MHC!also!could!not!fully!explain! variation!in!survival!of!bottlenose!dolphins!exposed!to!HABs.!In!my!final!chapter,!I! consider!the!advantages!and!drawbacks!of!the!genomeVwide!approach!in!comparison!to! a!candidate!gene!approach!and,!as!laid!out!in!my!dissertation,!I!recommend!using!both! complementary!approaches!in!future!investigations!of!adaptation!in!genomeVenabled! nonVmodel!organisms.!! ! v! ! CONTENTS! Abstract!..........................................................................................................................................!iv! List!of!Tables!..................................................................................................................................!x! List!of!Figures!..............................................................................................................................!xii! Acknowledgements!...................................................................................................................!xvi! Introduction!...................................................................................................................................!1! HAB!occurrence!and!toxicity!................................................................................................!3! Bottlenose!dolphin!mortality!................................................................................................!5! Research!objectives!...............................................................................................................!11! Chapter!I:!GenomeVwide!investigation!of!adaptation!to!harmful!algal!blooms!in!common! bottlenose!dolphins!(Tursiops)truncatus)!..................................................................................!15! Introduction!...........................................................................................................................!15! Methods!.................................................................................................................................!18! Samples!................................................................................................................................!18! RAD!library!preparation!...................................................................................................!19! SNP!identification!and!genotyping!.................................................................................!21! LiveVdead!comparisons!.....................................................................................................!22! Candidate!gene!identification!...........................................................................................!22! Results!....................................................................................................................................!23! Discussion!..............................................................................................................................!30! SurvivalVassociated!genetic!variation!..............................................................................!30! Candidate!genes!.................................................................................................................!31! Immune!system!..............................................................................................................!31! ! vi! ! Nervous!system!..............................................................................................................!32! Detoxification!system!....................................................................................................!33! Conclusion!.............................................................................................................................!34! Chapter!II:!Lack!of!variation!in!voltageVgated!sodium!channels!of!common!bottlenose! dolphins!(Tursiops)truncatus)!exposed!to!neurotoxic!algal!blooms!......................................!35! Introduction!...........................................................................................................................!35! Methods!.................................................................................................................................!40! Samples!................................................................................................................................!40! Genetic!analyses!.................................................................................................................!42! Results!and!Discussion!........................................................................................................!46! Sodium!channel!isoforms!..................................................................................................!46! Sodium!channel!variation!.................................................................................................!47! Effects!of!geographic!region!and!survival!......................................................................!51! Discussion!of!lack!of!survivalVassociated!SCN!variation!.............................................!52! Conclusion!.............................................................................................................................!54! Chapter!III:!From!genomeVwide!to!candidate!gene:!An!investigation!of!variation!at!the! major!histocompatibility!complex!in!common!bottlenose!dolphins!exposed!to!harmful! algal!blooms!.................................................................................................................................!56! Introduction!...........................................................................................................................!56!