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Odonatologica32(4):375-380 December I,2003 Theuse ofOdonatamuseumspecimens in questions of molecular evolution R.J.LodgeandJ.R.Freeland* DepartmentofBiologicalSciences, OpenUniversity,Walton Hall, MiltonKeynes, MK76AA,UnitedKingdom Received July8, 2002/RevisedandAcceptedJanuary20,2003 Studies ofpopulationgenetics andphylogeneticsrequiresamplesfromindividuals representingavarietyofspp.andpopulations.Collectingthenecessary individuals may beproblematic,particularlyforseasonal,rare,orgeographicallyremoteorganisms.Museum collections therefore provideapotentially valuableresource, andthe widespreaduseof polymerase chainreactions (PCR) meansthattargetregionsofDNAcan beamplified from very smallamountsoftissue.Here modifications toDNAextraction techniquesare described thathave allowed theauthors to extract, amplify, and sequence aportionof mitochondrial DNAfrompartsofsingledragonflylegstakenfrommuseumspecimensup to80yrsold.Itisanticipatedthatinfuture thesetechniqueswillbeappliedtoarange of odon. studies,includingquestionsofconservation genetics. INTRODUCTION Successful extractionof DNA from museum samples can beofgreatbenefitto a wide rangeof studies. First, museums often house samples from much broader geographical rangesthancanbe visitedby individualresearcherson grantsoflimited duration,andthey maythereforeallowbiogeographical studiesto cover muchlarger areas thanwouldotherwisebeinvestigated. Second,access tosamples collectedover varyingtimescales willaddanimportant dimensiontostudiesthatare concernedwith alteredspecies distributions,conservationgenetics,andotherquestions thatmayinvolve changes overtime.Third,becausemuseumsamples already exist,theymayprovidean alternativeto capturing and sampling live individuals, and therefore in some circumstancesprovideamorehumanealternativeto denovosampling. Anumberofstudiesonbirdsandmammals(e.g. ROYetal., 1994;TAYLORetal„ 1994;MUNDY etal., 1997)haveused geneticinformationfrommuseumsamples to *Authorforcorrespondence:[email protected] 376 R.J.Lodge&J.R. Freeland address questionsofconservationgenetics (seealsoLANDWEBER,1999forreview). However, insect museum samples have not yetbeenused in acomparable manner (CATERING etal., 2000).Herewereportonoursuccessfulextractionandamplification ofDNA fromdragonfly museumsamples upto80 yearsold, taken fromAnaxjunius andAeshnamixta. METHODS ANDRESULTS Indevelopingtheseprotocols,weusedAnaxjuniusandAeshnamixtasamplesthatwereprovidedbya number ofmuseumsand othercollections (seeacknowledgements).The samplesincluded airoracetone dried,adultsamplesandsmall,upto80yearsold,(<10mmlong)larvalsamplesstoredinethanol.Inorder tominimisecontamination risk,extractionsfromtissuewith arelativelylow DNA yield(e.g.partiallegs) were doneinalaminarflowhood inasmall,enclosed roomonadifferent floortothelab in whichfresh material wasprocessed. Inaddition, dedicated pipetteswere used,and otherequipment such as the microcentrifugeandvortex werespecifictothisseparate ‘clean’ area. Twomethodsofextractionwereused.Thefirstmethodwasappliedtosamples upto ten yearsold,andis amodificationofthesingle legextractionprotocol by WATTS et al.(2001), whichis a salting-outextraction methodusing DTABand CTABbuffers andGeneclean (BiolOl) kit components (seeWATTS etal., 2001 fordetails). The modificationstothisexisting protocol were necessarybecause museumsamples are generally dried,andareseldomifeverstoredinDTABbuffer.Single legswereplaced in 1.5mleppendorf tubes and crushedwithforceps, then ground using amotorised pelletpestle withmultipleadditionsofliquid nitrogen,untilatleast50%ofthesample hadformed afine powder. DTAB (prepared as forWATTS etal. (2001)shortterm storagebuffer),acationicdetergent,wasthenaddedtothehomogenate tolysethecell componentsand denatureproteins, thereby releasing theDNA. Thesecond cationic detergent CTAB hadtheaddedadvantage that itselectively bindsDNA inhigh salt concentrationsaiding intheseparationofothercellcomponents(SAUNDERS, 1999). When required, a proteinase K digestion was included(for details see later in this section), withincubationinDTABwhichwasextendedfrom20to48hoursifproteinase K was added.Theadditionalproteinase Kdigestion stepwas requiredforsmalllegs, whereonlysmallquantitiesofmusclemayhavebeenpreserved. ProteinaseKremoves nucleasesfromDNA,aidsinthedigestionofadhering proteins andthustheseparation ofDNA fromothercellularcomponents(SAUNDERS, 1999),thereby increasingthe DNA yield fromthesesamples. Thisproved tobe moreeffectivethan theovernight incubationusedby WATTS etal.(2001) onnewersamples. The final modificationthat improved yield was torotate with glassmilk atroom temperaturefortwo hours, not 30minutes as outlinedin the original protocol. The incubationtimewas extended hereto increasethe yieldofDNA by providing more opportunities forDNA moleculestocollideandbindwiththesuspended silicamatrix (BiolOl, 2001).The selectivebinding ofDNA to silica inhigh concentrationsofa chaotrophic salt allowsthe separation ofDNA fromotherproteins, most forms of RNA, and substances inhibitory to PCR, such as chitin andotherpolysaccharides DNA extraction techniqueformuseumspecimens 377 (SAUNDERS, 1999),whichmayformasubstantialproportion ofthesesamples. Thesecondmethodofextractionwas usedforsamples morethantenyearsold.For thesesamples (oftenas smallashalfasingleleg) weusedtheGenecleankitforAncient DNA(BiolOl), whichisdesignedforisolationofDNAfromsamplesofbone,preserved tissue,oranimalby-products. ThiskitcontainsinstructionsforDNAextractionusing ultra-pure,ultra-cleansolutions(tofurtherminimisecontaminationrisk),butnevertheless required someexperimentation withtheprotocol. First, inadditiontoincubationwith chemicaldenaturantsinthekit’s dehybemationsolutions,themanufacturerssuggested apre-incubation ofsamples withproteinase K toaid intheseparation of DNAfrom cellcomponentsandadhering proteins. However,wehadmuchgreatersuccess when we dida48 hourproteinase K digestion (as opposed to the maximumof 15 hours recommendedby themanufacturer)inasoaking solutionconsistingof2pi0.5MEDTA, 20 pi 10% SDS, 10pi 20 mg/ml proteinase K and 168 pi distilleddeionisedwater. EDTA wasaddedtothesoaking solutiontoinhibitDNasesbychelating divalentcations such as calciumand magnesium andSDS, ananionic detergent, to solubilize cell membranesand denatureproteins (MILLIGAN, 1998). Our second modification, echoing an alterationof the aboveprotocol foryoungermuseum samples, was an extensionoftheincubationwith glassmilk to 1-2hours atroom temperature,as this improvedthelikelihoodofDNAbinding.Thethirdmodificationwastowardstheend Fig.1.Cytochromeoxidase1mtDNAPCRproducts(354basepairslong)fromAeshnamixta,runona1% agarosegel.Lanelabels showtheextractionbufferused(dehybemationA,A2,BorDTAB)andadditions to standard protocols,which included proteinase K digestion (prot. K.), and/oran additional 10 min. heatingstepafterinitial incubation (80°C).Sample7 (usingprotocolasforsample 1,withdehybemation AandproteinaseK)representstheoldest 4eshnamixtasample(80yearsold)forwhichmtDNAsequence wasobtained.Samples1-6 were 1year old driedA.mixta. 378 R.J.Lodge&J.R.Freeland oftheprotocol. Atthisstage, manufacturerssuggestresuspending theDNA pellet in 50-100pielutionsolutionbyvortexingfor1-2seconds,butwehadhigher DNAyields whenweusedonly 35piofelutionsolution,andallowedthissolutiontosoak intothe glassmilkandthenmixedbygentle pipetting. Theotherpoint worthnotingabouttheGenecleankit forAncientDNA is thatthe manufacturersinclude two dehybemation solutions forthe initiallysis/denaturation step. Oneofthese, dehybemation solutionA,isbasedon guanidinewhichis astrong chemical denaturantof proteins, capable of dissolving cytoplasmic and nuclear membranesand inhibiting nuclease activity (SAUNDERS, 1999). Dehybemation solutionB is anaqueousEDTAbasedsolution,whichwillinhibitDNases, andinthe case ofspecific samples demineralisebonetissue,combinedwitha detergent tolyse cellmembranesandothercomponents(MILLIGAN,1998;Bio101,2001).Theefficacy ofthesetwo solutionsvariesbetweensamples,particularlywithrespect tothemethod ofsample preservation and the environmentin which samples were kept. For the dragonfly legs, weobtainedthebestresults from dehybemation solutionA (Fig. 1), withoutthesuggested additionaldetergent (A2)provided(BiolOl, 2001). SuccessofDNAextractionswasconfirmedbyamplifyingaregion ofmitochondrial (mtDNA) cytochrome oxidase I using ‘universal’ COI primers Cl-J-1751 (5’GGATCACCTGATATAGCATTCCC3’)andCl-N-2191(5’CCCGGTAAAAT- TAAAATATAAACTTC3’)(PALUMBI etal., 1991).Amplificationreactionsincluded 2-6piDNA, IXPCRbufferwith1.5 mMMgCl (Promega), 200pM dNTPs,0.5pM 2 ofeach primer, and 1U Taq DNA polymerase (Promega) in a50 pireaction. The amplification programproceeded with2minutesat95°C,followedby 40 cycles of 95°Cfor30seconds,50°Cfor30secondsand72°Cfor30seconds,andafinalstepof 72°C for5 minutes.Anaeshnid-specific primerpair, internaltoCl-J-1751andCl-N- -2191,wasalsousedinnestedPCR ondifficult/lowyielding samples(J.R.Freelandet al., unpublished data).A large proportion ofthesewere subsequently sequenced in ordertoverifythattheDNAwasfromA.junius orA.mixta.Analignment of92Anax juniusindividualsplus 5 confamilials(including Aeshnasubpupilla fromthestudyof Wishartetal., GenBankaccession no. AF429284)verifiedthe sequencesas aeshnid COLThesesequenceshavebeensubmittedtoGenBank(accession numbersAF550515 to AF550581).Themaximumsequencedivergencewas 3.65%forA.junius, increasing to 14%whenthe5 confamilialswereincluded.Itisworthnoting thatthefragmentswe amplified were354-490bp long,andare thereforeasomewhatconservativeestimate ofoursuccess becauserelatively smallfragmentsare generally easiertoamplify from old, degradedDNA thanarerelativelylarge fragments. Todate,wehavehadreasonably highlevelsofsuccess,butthis variedwiththeage ofthesamplese.g.only42%of7 samples aged80-40yearsold,75%ofthe8 samples aged 20-39yearsold, 100%ofthe6samplesaged 10-19yearsand74%of65samples aged2-9 yearsold.Thedropinsuccess rateforthelastagecategorymaybe duetothe lackofmuscletissueinverysmalllarvalsamples(especially comparedtothetoughened exoskeleton), although as most ofthe recent samples were preserved inethanolas DNA extraction techniqueformuseumspecimens 379 opposed toairdried,preservation technique mayalsoaffectthe qualityofthesample. Oftheremaining driedadultsamplestheexactmethodofpreservation andstoragehad noeffectonthepercentageofsuccessful amplifications,witheithertheCTABorAncient DNAkitmethods,although theadditionoftheproteinaseKdigestiondidyieldbrighter bandsuponPCR withsamples over20 yearsold(Fig. 1). Somesamples fromallage ranges did not yield any DNA upon PCR amplification, for reasons that remained largely unclear,although thesewereintheminority.Forexample, a 144bp fragment mayamplify fromasample thatdidnot generatea345bp fragment (HANDT etal., 1994). DISCUSSION Theseresults suggestthatmuseumsamples are a viablesource ofDNA forfuture research onodonates, and shouldbeparticularly usefulfor studiesinwhich current sampling ishighlyrestricted.Although thereisno guaranteethatanyparticular sample willyieldDNA,by followingtheprotocolsoutlinedabove,thislikelihoodcanbekept reasonably high. Museumcurators canbeassured that dryingsamples (withacetone) orkeeping samples in envelopes shouldnot preclude possible DNArecovery. The ease withwhich usefulsequencewas obtainedfromvery smallquantitiesof sample e.g.partsofasingleinsectleg,allowstheretentionofalarge proportionofsamples at therespective museumsforfurtherstudy(CATERINGetal., 2000).Researchersshould beaware that, although they mayhavetofactorinthelikelihoodthatnot allsamples willyieldDNA, by obtainingapproximately a 35%excess ofsamples (in the 10-80 yearsample agerange) they should beable to reach theirtarget numberofdesired sequences from museum specimens. Degradation of DNA over timeregardless of preservation methodmaylimitthemoleculartechniquesavailable,butas manystudies have shown, the informationobtained canbe invaluableto studies of conservation genetics (ROYetal., 1994,CATERING etal.,2000). ACKNOWLEDGEMENTS K.F.CONRAD,M.MAY,K.SIMPSON,B.LANDWER(WilburR.EnnsEntomologyMuseum),D. GOODGER(NaturalHistoryMuseum,London), C.BELLAMY(LosAngelesNaturalHistoryMuseum), P. BRUNELLE(NovaScotiaMuseum), V. SCOTT(University ofColoradoMuseum),R.LARSEN (BitterLakeNational Wildlife Refuge),J.ACORN,F.SPERLING,S.SHAW(UniversityofWyoming InsectMuseum),B.KONDRATIEFF(ColoradoStateUniversity),N.VONELLENRIEDER,R.DUBOIS, M.McPEEK,R.ORR,G.SPRANDEL. REFERENCES BiolOl,2001.GENECLEANkitforancientDNAprotocol. CATERING,M.S.,S.CHO&F.A.H.SPERLING,2000.Thecurrentstateofinsectmolecularsystematics: athrivingtowerofBabel. Amu.Rev. Enl.45: 1-54. 380 R.J.Lodge&J.R.Freeland HANDT, O.,M. HOSS,M.KRINGS & S.PAABO, 1994. Ancient DNA: methodologicalchallenges. Experientia50: 524-529. LANDWEBER,L.F., 1999. Somethingoldforsomethingnew:ThefutureofancientDNA inconservation biology.In:L.F. Landweber & A.P. Dobson, [Eds],Genetics andthe extinction ofspecies,pp. 163-186.Princeton Univ. Press,Princeton. MILLIGAN,B.G., 1998.Total DNA isolation. In:A.R. Hoezel,[Ed.],Moleculargeneticanalysisof populations,pp.29-62.OxfordUniv. Press. MUNDY, N.I.,C.S. WINCHELL,T. BURR & D.S.WOODRUFF, 1997. 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