基于COⅡ基因的我国海域4个长蛸群体的遗传变异分析研究 计算机科学与技术专业

摘要采用PCR扩增技术对中国4个不同海域（山东青岛、浙江温州、浙江舟山、福建东山）的长蛸群体的COII基因片段进行了扩增和测序，获得了长度为560bp长度的同源序列，分析了不同海域群体内和群体间的遗传变异。结果表明，89个个体共有28个单倍型，其中青岛群体8个，舟山群体7个，温州群体6个，东山群体7个。通过序列比对，变异均匀地分布于序列各个区域，但密码子偏好不明显。总群体单倍型多样性指数（H）0.884±0.018，核苷酸多样性指数（Pi）0.10640±0.00656，平均核苷酸差异数（K）57.348，显示出一定的遗传多样性。采用MEGA3.1对4个海域的长蛸群体进行聚类分析，构建系统树，结果表明：青岛、舟山2个群体由于遗传距离较近首先聚为一支，而与东山群体遗传关系较远，与温州群体亲缘关系最远而最后聚类。Tajima’sD和Fu’sFS检验显示温州群体可能经历过种群扩张，而其它3个群体可能未经历过历史扩张事件。我国海域长蛸的这种遗传结构的形成原因还有待于进一步证实。通过本次研究，从线粒体DNA水平分析我国不同海域长蛸群体的遗传变异情况，期望为开展我国长蛸群体的资源评价、物种保护及分子标记育种提供基础资料。关键词：长蛸；线粒体基因；COⅡ基因；遗传变异AbstractFragmentsofCOIIgenewereamplifiedandsequenced.inOctopusvariabilissamplesfrom4ourdifferentseaareasinChina(Qingdao,Wenzhou,Zhoushan,Fujian)A560bpalignedsequencewereobtainedandanalyzedtodetectgeneticvariationwithinandamongthegroups.Theresultsshowedthat89individualsproduceatotalof28haplotypes,witheightinQingdaogroup,seveninZhoushangroup,sixinWenzhougroup,seveninDongshangroup.Bysequencealignment,thevariationisevenlydistributedinthevariousregionsofthesequence,butthecodonbiasisnotobvious.Thetotalpopulationhaplotypediversityindex(H)is0.884±0.018,nucleotidediversityindex(Pi)is0.10640±.00656,theaveragenumberofnucleotidedifferences(K)is57.348,showingacertaindegreeofgeneticdiversity.Aphylogenetictreeamongthe4OctopusvariabilisbyadoptingMEGA3.1softwareshowedthat:thegeneticdistanceamongQingdaoandZhoushangroupwasminimum.thegeneticdistancebetweenthosetwoandDongshanorWenzhougrouparemuchbig.Wenzhougroupsmayhaveexperiencedapopulationexpansion,whiletheotherthreegroupsmaynothaveexperiencedahistoricalexpansionevents.Thephylogenetictreereflectsthegeneticdifferencesbetweenthegeographicaldistributionofthesegroups.Throughthisstudy,thelevelofmitochondrialDNAanalysisofgeneticvariationindifferentwatersOctopusvariabilisgroupsexpecttocarryoutthegroupofChina'sOctopusvariabilisresourceassessment,protectionofspeciesandmolecularmarkerbreedinggeneticdata.Keywords：Octopusvariabilis;mitochondrialgenes;COIIgene;Geneticvariation目录TOC\o"1-3"\h\u32555摘要 III基因进化率，实验所用的序列如“Appendix1”所示。结果本实验得到了23个COⅠ序列和24个16SrRNA序列，均提交给了GenBank(基因号DQ683205–DQ683251)，发现了18个16SrRNA(去掉gap的话是7个)和6个COⅠ独特的基因型，16SrRNA平均长度为508bp（真蛸），COⅠ平均长度为526bp。实验结果显示，南非5个地区采集的真蛸均具有1个独特的构型区（如图2所示）图2综合南非、特里斯坦库尼亚、塞内加尔和西班牙的章鱼的COⅠ和16SrRNA序列，建立的基因网络图，小黑圈表示的是以上样本没有体现出的内部节点，缩略语意义如下：HB,豪特湾；SB，struisbaai；PE,伊丽莎白港；DB，德班；SE,特里斯坦达库尼亚；GA,加利西亚（大西洋海岸的西班牙）；ME，地中海沿岸的西班牙。讨论本实验所得到的南亚真蛸的COⅠ基因和16SrRNA的基因多态性，表明早前的实验所得到的COⅢ序列并不是假基因的扩增，并且也没有充分数据表明真蛸的线粒体基因比此地域的头足纲动物（其展现了很高的基因多态性）突变率低，这表明南非真蛸群体遇到了基因瓶颈或是新近才形成的。然而，这也很难解释为什么在南非有两个真蛸群系的出现。1个全系占绝对优势沿着海岸线广泛分布，而另1个只有在德班才有发现，这一现象表明后者有可能是最近才形成的。它在南非最繁忙的港口附近才发现显示其可能是港口轮船所排出的压舱水中带有的，而且它和另一群系基因上紧密联系，地域上的远距离表明此亚种是最近才形成的。需要更进一步的研究才能确定德班的真蛸群系是从它处引入的（如果是此种情况，那么其他地域的真蛸应和其基因型一致）亦或是它是别的种属新近运动到德班的（如果是此种情况，它应和其他区域的真蛸基因型有差异）。BLASTN2.2.14分析结果显示德班附近真蛸的COⅠ基因和16SrRNA和其余地方（欧洲、塞内加尔、南非、特里斯坦·达库尼亚、美国、日本）均有显著差异，但仍需在另外地区采集更多的样品以进一步分析，例如东部非洲、印度和东南亚。参考文献AltschulSF,MaddenTL,SchäVerAA,ZhangJ,ZhangZ,MillerW,LipmanDJ(1997)GappedBLASTandPSI-BLAST:anewgenerationofproteindatabasesearchprograms.NucleicAcidsRes25:3389–3402ExcoYerL,LavalG,SchneiderS(2005)Arlequinver.3.0:anintegratedsoftwarepackageforpopulationgeneticsdataanalysis.EvolBioinformOnline1:47–50FolmerO,BlackM,HoehW,LutzR,VrijenhoekR(1994)DNAprimersforampliWcationofmitochondrialcytochromecoxidasesubunitIfromdiversemetazoaninvertebrates.MolMarBiolBiotech3:294–299GopalK,TolleyKA,GroeneveldJC,MattheeCA(2006)MitochondrialDNAvariationinspinylobsterPalinurusdelagoaesuggestsgeneticallystructuredpopulationsinthesouthwesternIndianOcean.MarEcolProgSer319:191–198HerkeSW,FoltzDW(2002)Phylogeographyoftwosquid(LoligopealeiandL.plei)intheGulfofMexicoandnorthwesternAtlanticOcean.MarBiol140:103–115KassahnK,DonnellanS,FowlerA,HallK,AdamsM,ShawP(2003)MolecularandmorphologicalanalysesofthecuttleWshSepiaapamaindicateacomplexpopulationstructure.MarBiol143:947–962KlopperAW(2005)IntraspeciWcgeneticvariationinthepercoidteleostsArgyrosomusjaponicus(TemminckandSchlegel,1843)andPomadasyscommersonnii(Lacepède,1801)asinferredfromthemitochondrialcontrolregion.M.Sc.thesis,UniversityofPretoria,SouthAfricaKumarS,TamuraK,NeiM(2004)MEGA3:integratedsoftwareformolecularevolutionarygeneticsanalysisandsequencealignment.BriefBioinform5:150–163MangoldK,BoletzkyS(1973)NewdataonthereproductivebiologyandgrowthofOctopusvulgaris.MarBiol19:7–12NortonO(2006)ThepopulationstructureoftwoestuarineWshspeciesalongthesouthernAfricancoastline.M.Sc.Thesis,RhodesUniversity,SouthAfricaOosthuizenA,JiwajiM,ShawP(2004)GeneticanalysisoftheOctopusvulgarispopulationonthecoastofSouthAfrica.SAfrJSci100:603–607PalumbiSR(1994)Geneticdivergence,reproductiveisolationandmarinespeciation.AnnuRevEcolSyst25:547–572PalumbiSR(1996)NucleicacidsII:thepolymerasechainreaction.In:HillisDM,MoritzC,MableBK(eds)Molecularsystematics.SinauerAssociates,Sunderland,pp205–247RidgwayTM,StewardBA,BranchGM,HodgsonAN(1998)MorphologicalandgeneticdiVerentiationofPatellagranularis(Gastropoda:Patellidae):recognitionoftwosiblingspeciesalongthecoastofsouthernAfrica.JZoolLond245:317–333Robinson-RechaviM,HuchonD(2000)RRTree:relative-ratetestsbetweengroupsofsequencesonaphylogenetictree.Bioinformatics16:296–297SmaleM,BuchanP(1981)BiologyofOctopusvulgarisoVtheeastcoastofSouthAfrica.MarBiol65:1–12TeskePR,CherryMI,MattheeCA(2004)Theevolutionaryhistoryofseahorses(Syngnathidae:Hippocampus):moleculardatasuggestaWestPaciWcoriginandtwoinvasionsoftheAtlanticOcean.MolPhylogenetEvol30:273–286TeskePR,McQuaidCD,FronemanPW,BarkerNP(2006)ImpactsofmarinebiogeographicboundariesonphylogeographicpatternsofthreeSouthAfricanestuarinecrustaceans.MarEcolProgSer314:283–293TolleyKA,GroeneveldJC,GopalK,MattheeCA(2005)MitochondrialDNApanmixiainspinylobsterPalinurusgilchristisuggestsapopulationexpansion.MarEcolProgSer297:225–231VillanuevaR(1995)ExperimentalrearingandgrowthofplanktonicOctopusvulgarisfromhatchingtosettlement.CanJFishAquatSci52:2639–2650WarnkeK(1999)DiversitätdesArtenkomplexesOctopuscf.VulgarisCuvier,1797inBeziehungzuseinerVerbreitunganderOst-undWestküsteLateinamerikas.PhDthesis,UniversitätBremen,ShakerVerlag,AachenZardiGI,McQuaidCD,TeskePR,BarkerNP(2007)Unexpectedgeneticstructureinindigenous(Pernaperna)andinvasive(Mytilusgalloprovincialis)musselpopulationsinSouthAfrica.MarEcolProgSer(inpress)PhylogeographicstructureofOctopusvulgarisinSouthAfricarevisited:identiWcationofasecondlineagenearDurbanharbourP.R.Teske·A.Oosthuizen·I.Papadopoulos·N.P.BarkerReceived:13June2006/Accepted:8February2007/Publishedonline:8March2007AbstractInapreviousstudythatinvestigatedgeneticstructureofOctopusvulgarisalongtheSouthAfricancoastbysequencingthemitochondrialcytochromeoxidaseIIIgene(COIII),allsequencesgeneratedwereidentical.SuchaWndingisunusual,becausemitochondrialDNAmutatesquickly,andseveralmarineinvertebratespresentinsouthernAfricashowconsiderablegeneticvariationandstructure.WereanalysedthesamplesusingtwodiVerentmitochondrialmarkers,namelycytochromeoxidaseI(COI)andthelargeribosomalsubunit(16SrRNA).Sequencesofboththesemarkersshowedvariation.Theconclusionofthepreviousstudy,thatSouthAfrica’sO.vulgarispopulationischaracterisedbyalackofgeneticstructurealongthecoast,isrejected.SomespecimensfromDurban(southeastAfrica)weregeneticallymorediverentfromthosefoundintheremainderofthecountrythanwerespecimensfromotherregions(TristandaCunhaandSenegal).WesuggestthatthelineageinDurbanmayhavebeenrecentlyintroduced.IntroductionTheamountofgeneticstructuringinmarineorganismsisgenerallyconsideredtobenegativelycorrelatedwithdispersalability(Palumbi1994).RecentgeneticstudiesonpassivelydispersingsouthernAfricanmarineinvertebrateshaveidentiWedconsiderablephylogeographicsubdivisionalongthecoast,withseveralwidelydistributedspeciescomprisingtwoormoreregionallineages(Ridgway1998;Teskeetal.2006;Zardietal.2007).Incontrast,mostspeciesthatareabletodisperseactivelyarecharacterisedbylittleornogeneticstructure(Tolleyetal.2005;Klopper2005;Norton2006;butseeGopaletal.2006),suggestingthataspecies’degreeofgeneticstructuringalongtheSouthAfricancoastdependstoalargeextentonitsmodeofdispersal.InapreviousstudyontheSouthAfricanpopulationofOctopusvulgaris,Oosthuizenetal.(2004)identiWedonlyasinglemitochondrialCOIIIhaplotypeamong35samplescollectedbetweenHoutBay(westcoast)andPortAlfred(southcoast)andconcludedthatasinglegeneticpopulationexistsinSouthAfrica.SuchaconclusionisreasonablegiventhatnewlyhatchedO.vulgarisareplanktonicforapproximately50–60days(Villanueva1995)andadultsareknowntomigrate(MangoldandBoletzky1973;SmaleandBuchan1981).However,thefactthatallsequenceswereidenticalisproblematic,asmitochondrialDNAmutatesquicklyandalong-establishedpopulationshouldshowsomevariation.Thereareseveralpossibleexplanationsforthelackofgeneticdiversity:(a)thesouthernAfricanO.vulgarispopulationhasundergonearecentgeneticbottleneckorfounderevent;(b)mitochondrialDNAinO.vulgarisevolvesmoreslowlythaninothermarineorganisms;(c)thelowamountofgeneticdiversityisasamplingartifact(thisisunlikely,becausethenumberofsamplescollectedwassimilartothoseinotherstudies);(d)therehasbeenacontaminationproblemduringtheampliWcationprocess(thisisalsounlikely,becausethesequencegeneratedwasidenticaltooneindependentlygeneratedbyanotherresearcher[Warnke1999])and(e)theCOIIIsequencesgeneratedpreviouslyarenotactuallyofmitochondrialorigin,butareampliconsofanumt(i.e.anuclearmitochondrialpseudogenethathasarisenfrommitochondrialDNAbeingintegratedintothenucleus),whichexhibitsaconsiderablylowermutationratethanthehomologousmitochondrialmarker.TheCOIIIsequencepreviouslygenerated(AccessionNo.AJ250487)doesnotcontainanystopcodonsorshiftsofthereadingframe,whichwouldindicatethatitcouldbefromanon-functionalnumt.However,iftheinsertionintothenucleushastakenplacerecently,theninsuYcienttimehaspassedforsuchmutationstotakeplace.Inthisstudy,wereanalysedsomeofthesamplesusedbyOosthuizenetal.(2004)todeterminewhichofthepossiblereasonsforthelackofgeneticvariationisthemostlikely,byamplifyingpartialsequencesofCOIand16SrRNAanddeterminingwhetherthesealternativemarkersshowedhigherlevelsofgeneticvariation.WealsosequencedsomeadditionalsamplesfromSouthAfricaandelsewhere.MaterialsandmethodsSamplingandlaboratoryworkWesequencedaportionoftheCOIgeneandofthe16SrRNAofthreerandomlyselectedsampleseachfromthreelocalitiesintheregionpreviouslysampledbyOosthuizenetal.(2004):HoutBay,StruisbaaiandPortElizabeth(southwestandsouthcoast;Fig.1).Inaddition,wesequencedeightsamplesfromSouthAfrica’seastcoast(sevenfromDurbanandonefromUmhlanga),aswellasanumberofspecimensfrompopulationsthatarecloselyrelatedtotheSouthAfricanlineageofOctopusvulgaris(Oosthuizenetal.2004).TheseoriginatedfromtheSouthAtlanticislandofTristandaCunha(threespecimens),Senegal(WestAfrica;threespecimens)andSpain(AtlanticcoastandMediterraneancoast;threeandonespecimens,respectively).Extraction,ampliWcationandsequencingofmolecularmarkersfollowedprotocolsdescribedpreviously(Teskeetal.2004,2006).TheCOIgenewasampliWedusingprimersLCO1490(5
-GGTCAACAAATCATAAAGATATTGG-3
)andHCO2198(5
-TAAACTTCAGGGTGACCAAAAAATCA-3
;Folmeretal.1994)Fig.1AmapoftheeasternAtlanticOceanshowingsamplingsites.Acronymsrepresentthefollowinglocalities:GA,Galicia(AtlanticOceancoastofSpain);ME,MediterraneancoastofSpain;SE,Senegal;TC,TristandaCunha;HB,HoutBay;SB,Struisbaai;PE,PortElizabeth;DB,Durban;UM,Umhlangaand16SrRNAwasampliWedusingprimers16SarL(5
-CGCCTGTTTATCAAAAACAT-3
)and16SbrH(5
-CGGGTCTGAACTCAGATCACGT-3
;Palumbi1996).DataanalysesSequenceswerealignedbyeyeinMEGAversion3.1(Kumaretal.2004).The16SrRNAsequencesincludedashortmicrosatellite,whichwasexcludedfromtheanalysesbecauseitwasnotphylogeneticallyinformative(whenthemicrosatellitewasexcluded,sequencesweregroupedaccordingtogeographicalregion;whenitwasincluded,allgeographicalstructurewaslost).AminimumspanninghaplotypenetworkofcombinedCOIand16SrRNAsequenceswasconstructedusingArlequin3.1(ExcoYeretal.2005).TodeterminewhethermitochondrialDNAinOctopusvulgarisevolvesmoreslowlythaninothermarineinvertebrates,wecarriedoutpairwiserelativeratetestsbetweenCOIandCOIIIsequencesofOctopuswiththoseofothercephalopodsusingtheprogrammeRRTree(Robinson-RechaviandHuchon2000).Testswereperformedseparatelyforsynonymousandnon-synonymoussubstitutionsbycomputingtheparametersB4(numberofsynonymoustransversionsperfourfolddegeneratesite)andKa(numberofnon-synonymoussubstitutionspernon-synonymoussite).Cephalopodspeciesforcomparisonwereselectedonthebasisthattheyoriginatedfromregionallineagesthatshowedgeneticvariation.TheevolutionaryrateoftheCOIsequencesofallOctopusvulgarisspecimensgeneratedinthisstudyweresimultaneouslycomparedwiththatoftwosquidspecies,LoligopealeiandL.plei(HerkeandFoltz2002).InthecaseoftheCOIIIsequences,wecomparedOctopusvulgarissequencesavailableonGenBankwiththoseofSepiaapama(Kassahnetal.2003).Inbothcases,NautilusmacromphaluswasspeciWedasoutgroup.Accessionnumbersofallsequencesusedfortherelativeratetestsarelistedin“Appendix1”.ResultsAtotalof23COIsequencesand2416SrRNAweregenerated.TheseweresubmittedtoGenBank(accessionnumbersDQ683205–DQ683251).Eighteenunique16SrRNAhaplotypes(or7whengapswereexcluded)andsixuniqueCOIhaplotypeswererecovered.Thelengthofaligned16SrRNAsequencesofO.vulgariswas508basepairs,andtheCOIsequenceswere526basepairsinlength.TheSouthAfricanpopulationwascharacterisedbyadominanthaplotypethatwasfoundatallWvesamplinglocalities(Fig.2).TwootherSouthAfricanhaplotypesdiVeredfromitbyasinglenucleotidesubstitution,asdidthespecimensfromTristandaCunhaandthespecimensfromSenegal.TwooftheindividualsfromDurbanhadcombinedCOI/16SrRNAsequencesthatwereasdiVerentfromthemainSouthAfricancladeaswerethespecimensfromSpain.ThemaximumnumberofpairwisediVerencesbetweensequencesofSouthAfricansampleswasseven(1.3%)forCOIandtwo(0.4%)forcollectedinDurban(AccessionNo.DQ683251)revealedthatthisindividualhadbeenmisidentiWedandwasactuallyamemberofthetropicalspeciesO.cyanea(itsCOIgenewasnotsequenced).The16SrRNAsequenceofthissamplediVeredfromthatofapreviouslypublishedsequenceofaspecimenfromtheMarianaIslands(AccessionNo.AJ252779)byasinglenucleotidesubstitution.RelativeratetestscomparingCOIsequencesofOctopusvulgarisgeneratedinthisstudywiththesquidspeciesLoligopealeiandL.plei,aswellasthosecomparingCOIIIsequencesofOctopusvulgarisandSepiaapama,werenon-signiWcant(COI,B4:P=1.0;Ka:P=0.5;COIII,B4:P=1.0;Ka:P=0.7).Fig.2Aminimum-spanninghaplotypenetworkconstructedfromcombinedCOIand16SrRNAsequencesofOctopusvulgarisfromSouthAfrica,TristandaCunha,SenegalandSpain.Smallblackcirclesareinteriornodehaplotypesnotpresentinthesamples.Acronymsrepresentthefollowingpopulations:HB,HoutBay;SB,Struisbaai;PE,PortElizabeth;DB,Durban;SE,Senegal;TC,TristandaCunha;GA,Galicia(AtlanticcoastofSpain);ME,MediterraneancoastofSpainDiscussionandconclusionsThelowamountofgeneticdiVerentiationamongcombinedCOIand16SrRNAsequencesofthemajorityofSouthAfricansamplesindicatesthatthelackofvariationfoundinthepreviousstudyisunlikelytohavebeenduetotheampliWcationofanumt.Morever,thereisnosupportforthehypothesisthatmitochondrialDNAevolvesmoreslowlyinOctopusvulgaristhaninregionallineagesofothercephalopodsthatshowedhigherlevelsofgeneticvariation.ThissuggeststhatSouthAfrica’sOctopusvulgarispopulationhaseitherexperiencedarecentgeneticbottleneckorhasrecentlybeenfounded.ItisasyetdiYculttointerpretthepresenceoftwogeneticallydiVerentlineagesofO.vulgarisinsouthernAfrica.Thefactthatonelineagewasveryabundantalongmostofthecoast,whereastheotherhassofaronlybeenfoundinDurban,suggeststhatthelattermayhavebeenrecentlyintroduced.ItspresenceinthevicinityofoneofAfrica’sbusiestharbourscouldbeanindicationthatitarrivedthereinaship’sballastwater.Ontheotherhand,thelowgeneticvariationinthemorecommonlineage,anditscloseassociationwithpopulationsfromfairlydistantlocalities,couldbeanindicationofarecentfounderevent.MoresamplingisnecessarytodeterminewhetherthelesscommonlineagefoundinDurbanhasrecentlybeenintroduced(inwhichcaseidenticalmitochondrialhaplotypesshouldbefoundinotherregions),orwhetheritcouldbealong-establishedlineagethathasbeenreplacedinmostofitssouthernAfricanhabitatbyamorerecentarrival(inwhichcaseitshouldbegeneticallydistinctfromotherpopulations).BLASTsearchesofGenBankusingBLASTN2.2.14(Altschuletal.1997)revealedthattheCOIand16SrRNAsequencesofthetwospecimensfoundnearDurbanharbouraredistinctfromalloftheO.vulgarissequencesthathavesofarbeensubmitted(generatedfromsamplescollectedinEurope,Senegal,SouthAfrica,TristandaCunha,theAmericasandJapan).ThissuggeststhatfurthersamplingneedstobeconductedinregionsfromwhichnogeneticdataonO.Vulgarisareasyetavailable,suchasEastAfrica,IndiaandSoutheastAsia.AcknowledgmentsWearegratefultoFranciscoSantaclara(Vigo,Spain)forprovidingtissuesamplesfromSpainandSenegal,toJamesGlassforprovidingsamplesfromTristandaCunha,andtoPierrePradervandforprovidingsamplesfromDurbanandUmhlanga.Wealsothankthreeanonymousrefereesforsomeusefulcommentsthatconsiderablyimprovedthequalityofthismanuscript.ThisstudywassupportedbyaClaudeHarrisLeonFoundationpostdoctoralresearchfellowshipawardedtoPRT,RhodesUniversityandanNRFgranttoNPB(GUN2069119).TheexperimentsconductedinthisstudycomplywithcurrentSouthAfricanlaws.ReferencesAltschulSF,MaddenTL,SchäVerAA,ZhangJ,ZhangZ,MillerW,LipmanDJ(1997)GappedBLASTandPSI-BLAST:anewgenerationofproteindatabasesearchprograms.NucleicAcidsRes25:3389–3402ExcoYerL,LavalG,SchneiderS(2005)Arlequinver.3.0:anintegratedsoftwarepackageforpopulationgeneticsdataanalysis.EvolBioinformOnline1:47–50FolmerO,BlackM,HoehW,LutzR,VrijenhoekR(1994)DNAprimersforampliWcationofmitochondrialcytochromecoxidasesubunitIfromdiversemetazoaninvertebrates.MolMarBiolBiotech3:294–299GopalK,TolleyKA,GroeneveldJC,MattheeCA(2006)MitochondrialDNAvariationinspinylobsterPalinurusdelagoaesuggestsgeneticallystructuredpopulationsinthesouthwesternIndianOcean.MarEcolProgSer319:191–198HerkeSW,FoltzDW(2002)Phylogeographyoftwosquid(LoligopealeiandL.plei)intheGulfofMexicoandnorthwesternAtlanticOcean.MarBiol140:103–115KassahnK,DonnellanS,FowlerA,HallK,AdamsM,ShawP(2003)MolecularandmorphologicalanalysesofthecuttleWshSepiaapamaindicateacomplexpopulationstructure.MarBiol143:947–962KlopperAW(2005)IntraspeciWcgeneticvariationinthepercoidteleostsArgyrosomusjaponicus(TemminckandSchlegel,1843)andPomadasyscommersonnii(Lacepède,1801)asinferredfromthemitochondrialcontrolregion.M.Sc.thesis,UniversityofPretoria,SouthAfricaKumarS,TamuraK,NeiM(2004)MEGA3:integratedsoftwareformolecularevolutionarygeneticsanalysisandsequencealignment.BriefBio