Fromfaeces tofoxes:usinggeneticstomanageaninvasivepredatorforwildlifeconservation

AnnaJMacDonald,AaronAdamack,CatrionaDCampbell,EliseFDewar,BerndGruber,ElodieModave,Sumaiya Quasim &StephenDSarre

anna.macdonald@canberra.edu.au

Photo DPIPWE Photo by Felicity Hatton

FoxeswereintroducedtoAustraliamid-late1800s

• Foxeswereintroducedfrom

Europe- sourcepopulation

uncertainbutthoughttobe

fromtheUK

• Likelyverysmallnumberof

foundersAustralianfoxdistributionin2007

FoxesinTasmania… uncertainty&debate…• 1864-1990s:historicalintroductions,littleevidenceforestablishedpopulation

• 1998:reportoffoxleavingashipinBurnie

• 1999:allegeddeliberateintroductionoffoxes

• 2001:foxprints,foxcarcass(shot)

• 2002:foxscatnearBurnie (IDfromhair),FoxFreeTasmaniataskforce

• 2003-2011:foxcarcasses,sightingreports,foxscats(DNA)…

WhatcangeneticstellusaboutfoxesinTasmania?

Hardevidenceoffoxpresence

2-stageDNAtest:1)PCRtodetectmammal/foxDNA2)SequencingtoconfirmfoxIDs

DetectionoffoxesinTasmaniafromscatDNA

MammalDNA

Fox-specificDNABerryetal. (2007)WildlifeResearch

devil e.quoll s.quoll fox

Sarre,etal (2013)JournalofAppliedEcology

2007-2012• >11,000scats• DNAfrom~80%ofscats• FoxDNAdetectedfrom61scats2014• >2,500scatstested• NofoxDNAdetected

Whatistheriskoffalse+ves using“thefoxtest”?

Gonçalves etal.(2014)ForensicSci.Int.Genet.

…

…

CTGGAAGGGCGTGGTAGTTTATA

AGACTGTGTCGATGACGAAAG

1)“Fox”primerswouldneedtoamplifynon-foxDNA

ThisdependsonprimerspecificityANDthestringencyofthePCRconditions

2)Non-foxDNAsequences wouldneedtobemistakenforfox

MammalDNA

Fox-specificDNABerryetal. (2007)WildlifeResearch

devil e.quoll s.quoll fox???

CannotdistinguishfoxDNAfromnon-specificamplificationusing

gelelectrophoresis

BUTwecanusingDNAsequences!

Blindtrialsincluded208non-foxscats:

• 9putativefoxresultsbasedonPCRtestalone

• 0confirmedfoxresultsbasedonPCR+DNAsequencing

• Nofalsepositivesobserved

149knownfoxscatsalsotested:

• 127scoredasfoxpositive

• 22falsenegatives,i.e.nofoxDNAdetected

How confident can we be in the fox test?

Ramseyetal (2015)JournalofAppliedEcology

How confident can we be in the fox test?

BioinformaticanalysisofspeciesassignmentusingDNAsequencesfrom74Australian(includingintroduced)vertebratespecies:

• Nonon-foxsequencesweremistakenlyassignedtofox=nofalsepositives

• Nogenuinefoxsequencesweremisidentified=nofalsenegatives

• ThissequencingtestcanreliablydistinguishfoxDNAinAustralia

MacDonald&Sarre(2015)JournalofAppliedEcology

• FoxDNAdetectedfrom61Tasmanianscatsfrommultiplelocations

• LimitationsofthisfoxDNAtestarerelativelywellunderstood

• Lowriskoffalsepositives

• Higherriskoffalsenegatives

What we can say from the fox DNA test:

What we can’t say from the fox DNA test:

• Howmanyindividuals?

• Relatedness?

• Sexoffoxes/sexratio?

• Originsoffoxes?

• Provenanceoffoxpositivesamples?We can use other genetic methods to tackle these questions

– but we need appropriate markers and reference data

What do we need to tackle these questions with DNA:population genetics?

• Howmanyindividuals?

• Relatedness

• Sexoffoxes/sexratio

• Originsoffoxes

Populationgeneticmarkersþ

Probabilityofidentity☐

Sex-specificmarkers☐

Populationdiagnosticmarkersý

Needgoodunderstandingofgeneticdiversity&allelefrequenciesofmainlandAustralianfoxpopulation

~

~

What do we need to tackle these questions with DNA:DNA metabarcoding of diet?

• Provenanceoffoxpositivesamples

GoodreferenceDNAdatabase☐Diagnosticgeneticmarkers☐SufficientDNA☐

Needgoodunderstandingofgeographicdistributionofgeneticdiversityineachpotentialpreyspecies

~~

~

MicrosatellitemarkersforindividualID:• 16scats&4carcassessuccessfullygenotyped• Eachhasauniquegenotype=atleast20individualfoxes

How many individual foxes?

• Remnantsofhistoricintroductions?– expectTasmanianfoxestobegeneticallydistinctfrommainlandAustralianfoxes

• Recentintroductionoflitter(s)?- expectTasmanianfoxestobesiblings/havesamemainlandorigin

• Multiplerecentintroductions?- expectTasmanianfoxestonotbecloselyrelated,havedifferentmainlandorigins

What are the origins of Tasmanian foxes?

• Australia-widemicrosat studywith>3000samples(OliverBerry,AaronAdamack)• Microsatellitesproviderelativelypoorresolutionoffoxpopulationstructurein

Australia,butcandistinguisheastfromwest

Tasmaniansamples(fromfoxcarcasses)areindistinguishablefromeasternmainlandAustralian

samples

RulesoutremnanthistoricTasmanianpopulation

Tasmanianfoxesarenotgeneticallydistinct

Basedon30microsatellitemarkers

Canweimproveresolutionusingnewmarkers?

Largernumberofgeneticmarkersmayprovidebetterresolution?160foxsamplesfromacrossAustraliaanalysed withDArTseq

110.0 119.0 128.0 137.0 146.0 155.0

-35

-30

-25

-20

goodinds$long

goodinds$lat

NewSouthWales

Queensland

SouthAustraliaVictoria

WesternAustralia

-5

0

5

-4 0 4PCoAAxis1(2%)

PCoA

Axis2

(1.2%)

SNPsalsoshowlittlegeographicstructureinAustralianfoxes=likelyinsufficientresolutiontoassignoriginsofTas foxes

Basedon5000SNPs

Tasmania

• Needcautionwhenconclusionsinfluencemanagement

• Problemswithincompletereferencedatabases:

• Problemswithpoorgeographiccoverage:

DNA diet analysis may help determine provenance

Conclusions1• Needtounderstandlimitsofdetectionandreal risksoffalsepositives/

negativestomakeeffectiveuseofDNAdetectioninwildlifemanagement.

• Implicationsoffalsepositives/negativeswillvarywithmanagementcontext.

• DNAmayextendourunderstandingofTasmanianfoxorigins– butweneedbetterunderstandingofgeneticdiversityinmainlandfoxpopulationstounderstandwhatispossible.

• DNAdietanalysishaspotentialtodetermineprovenanceofTasmanianscats– butweneedconcertedefforttobuildreferenceDNAdatabasesandIDdiagnosticmarkersforAustralian fauna.

Conclusions2

• Whateveryourinterpretationofrecentevidence,vigilanceisessentialtopreventfutureincursions/establishmentoffoxesinTasmania.

• ThereismuchwecanlearnfromthelastdecadeoffoxDNAdetectiontodevelopmoreeffectivegenetictoolsforbiosecurity&pestmanagement.

• RachelAnnetts• ValCaron• DianneGleeson• MaxPowell• Nur Iylani Ramlee• SamRyan• PiaSalgado• KristinStewardson• Kejun Wei• MattYoung• MikeBunce&theTrEnD LabteamatCurtin• AustralianNationalWildlifeCollection• QueenVictoriaMuseumandArtGallery• SouthAustralianMuseum• TasmanianMuseumandArtGallery• IlluminaMiSeqPilotProjectfunding&AGRF• DiversityArraysTechnologies

• CandidaBarclay• OliverBerry• PeterCremasco• CraigElliot• SimonFearn• StephenHarris• TomPriestley• DavidRamsey• InvasiveSpeciesBranchstaff• Scatsurveyvolunteers

Acknowledgements