TurkJZool 31(2007)69-74 ©TÜB‹TAK KaryologicalAnalysisofTwoEndemicTooth-Carps, Aphaniuspersicus and Aphaniussophiae (Pisces:Cyprinodontidae),fromSouthwestIran H.R.ESMAEILI*,Z.PIRAVARandA.H.SHIVA DepartmentofBiology,CollegeofSciences,ShirazUniversity,Shiraz-IRAN Received:16.01.2006 Abstract: Thekaryotypesof2endemictooth-carpsofIran,Aphaniuspersicus (Jenkis,1910)andAphaniussophiae (Heckel,1849), wereinvestigatedbyexaminingmetaphasechromosomesspreadsobtainedfromgillepithelialandkidneycells.Thediploid chromosomenumbersofbothspecieswere2n=48.Thekaryotypesconsistedof11pairsofsubmetacentricand13pairsof subtelocentricchromosomesin A.persicus and14submetacentricand10subtelocentricchromosomesin A.sophiae .Thearm numbersin A.persicus and A.sophiae wereNF=70andNF=76,respectively.Sexchromosomeswerecytologically indistinguishableinbothtooth-carps. KeyWords: Aphaniuspersicus,Aphaniussophiae,karyotype,chromosome,idiogram Introduction cytogeneticstudiesmayprovideacomplementarydata TheCyprinodontidaearerepresentedinIranby6 sourceformoreaccurateandpreciseidentificationof species(Coad,1988,1995,1996;Scheel,1990): thesefishes.Applicationofthistypeofstudyhasreceived Aphaniusginaonis (Holly,1929); A.mento (Heckel, considerableattentioninrecentyears(Ozouf-Costazand 1843); A.dispar (Rüppell,1828); A.vladykovi Coad, Foresti,1992;Galettietal.,2000).Fishchromosome 1988; A.sophiae (Heckel,1849);and A.persicus datahavegreatimportanceinstudiesconcerning (Jenkins,1910).Theyareverycolorfulfishandcanbe evolutionarysystematics,aquaculture,mutagenesis, keptinaquaria;hence,theymaybecomepartofthe geneticcontrolandtherapidproductionofinbredlines aquariumtrade.Threespeciesofthosetooth-carpsare (Al-Sabti,1991).Theincreasingimportanceof foundinFarsprovince,SouthwestIran.Theyare A. chromosomalstudiesonfishandlackofdataon dispar, A.sophiae and A.persicus . A.dispar isa karyotypeofIranianfishencouragedustoperformthis euryhalinefishthatapparentlyprefersbrackishwatersin firstcytogeneticalanalysis(i.e.diploidchromosome Farsprovince. A.persicus orPersiantooth-carpisan numbers,descriptionofkaryotypes,andidiograms)of2 endemicspeciesfoundintheLakeMaharlubasin.This endemictooth-carps, A.persicus and A.sophiae ,from lakeislocatedinabasininShirazvalleyatanaltitudeof SouthwestIran. about1460m.Thisisachloridelakeandfishless.The fishisfoundinitsincomingstreamsandsprings. A. Materialsandmethods sophiae isfoundintheendorheicKorRiverbasinofthis province.Tooth-carpsofIranhavebeenstudiedmainly A.persicus specimenswerecollectedfromtheBarm- ′ ′ basedontheirmorphologybuttheirmorphologyis e-Shoorspringstreamsystem(29°27 N-52°42 E,alt. conservativeand,duetomorphologicalsimilarities,their 1465m)intheLakeMaharlubasin,andthoseof A. distinctiononthisbasisisnotalwayspossible(Esmaeili sophiae werecollectedfromGhadamgahspringstream ′ ′ andShiva,2006).Theirbiologyisalsounknownand systeminKorbasin(30°15 N-52°25 E,alt.1660m)in thereislittleinformationonthesespecies.The Farsprovince,SouthwestIran,usingadipnet.Thefishes applicationofnon-morphologicalmethodssuchas weretransportedlivetothelaboratory,andkeptina *E-mail:[email protected] 69 KaryologicalAnalysisofTwoEndemicTooth-Carps, Aphaniuspersicus andAphaniussophiae (Pisces:Cyprinodontidae),fromSouthwestIran well-aeratedaquariumat20-25°Cbeforeanalysis.For from3.29to5.95µmbasedonthemeanvaluesofthe karyologicalstudiesthemodifiedmethodofUwa(1986) measurementsofbestmitoticmetaphases.Theresults wasused.Colchicinesolutionwaspreparedwith0.005g arepresentedinTable2. in20mlofphysiologicalserum.Thefishwereinjected Inthecaseof A.sophiae ,thechromosomenumber intraperitoneallywith0.02mlofcolchicinepergramof was2n=48,comprising14submetacentricand10 bodyweightusinganinsulinsyringe,andthenwereput subtelocentricchromosomes(Figure2andTable1). backintheaquariumfor4-5h.Thegillfilamentsand Differentchromosomenumbersinatotalof12.5%of kidneysofthosespecimenswerethenremovedand themetaphaseimageswererecordedrangingfrom47to placedinhypotonic0.36%KClsolutionfor45minat 50(Table1).Thetotallengthofchromosomesvaried roomtemperature(25°C).Thereafter,thesolutions from4.21to7.15µm,beinglongerthan A.persicus werecentrifugedfor10minat1000rpm,adding2-3 chromosomes.Themeasurementsusedtoclassify dropsoffreshandcoldCarnoy’sfixative(1:3,acetic chromosomesandidiogramsaregiveninTable2and acid:methanol)beforecentrifugation.Thesupernatants Figure2. werethendiscardedand5mloffreshandcoldfixative wasaddedtothesediments,whichweremixed Thearmnumbersin A.persicus andA.sophiae were thoroughlyandthenleftfor1h.Thefixationand NF=70andNF=76,respectively.Sexchromosomes centrifugationstageswererepeatedtwice.The werecytologicallyindistinguishableinbothtooth-carps. suspensionswerethentrickledontocoldslides.These slideswerestainedwith10%Giemsafor20min. Discussion Chromosomeswereobserved,selectedandphotographed byOlympuslightmicroscopewithacameramountedon Accordingtoourobservations,thediploid it.Karyotypeswerepreparedbyarrangingchromosomes chromosomenumbersofboth Aphaniuspersicus andA. inpairsbysize.Foreachchromosome,theaverage sophiae were2n=48andthisisinagreementwiththe lengthsoftheshortandlongarmsandarmratio(the chromosomenumberofotherspeciesofthisgenus. ratioofthelongarmlengthtotheshortarmlengthof Klinkhardtetal.(1995)andArkhipchuk(1999)reported chromosomes)werecalculatedandthenthe thechromosomenumberofA.sophiae tobe2n=48and chromosomeswereclassifiedaccordingtothecriteria thatof A.mento tobe2n=48(Vasil'ev,1980; givenbyLevanetal.(1964).Fundamentalnumber(NF) Klinkhardtetal.,1995;Arkhipchuk,1999).The wasexpressedastwicethenumberofatelocentric chromosomenumbersof A.dispar , A.asquamatus,A. chromosomesplusthenumberoftelocentric iberus andA.fasciatus werealso2n=48(Karbe,1961; chromosomes.TheidiogramswerepreparedinExcel GyldenholmandScheel,1974;Arkhipchuk,1999).Itcan 2003software(Microsoft). beconcludedthatthechromosomenumberinthisgenus isconserved.Thenumberofchromosomesinthese tooth-carpsisalsosimilartothatofotherspeciesof Results Cyprinodontidaesuchas Orestiasagassizii,Cyprinodon In82.5%ofthe57observedmetaphaseimages bovines and Cyprinodonmacularius (Klinkhardtetal., obtainedfromthekidneyandgillepithelialcellsof5 A. 1995;Arkhipchuk,1999).Intheorder persicus specimens,thediploidchromosomenumberwas Cyrinodontiformes,themostcommonfishspeciesthat 2n=48.Thetypicalkaryotypeconsistedof11pairsof havesofarbeencytologicallyinvestigated,suchas submetacentricand13pairsofsubtelocentric Gambusiaholbrooki,G.affinis,G.hurtadoi and chromosomes(Figure1).Differentchromosome Girardinusmetallicus;Poecilialatipinna (Poecillidae); numbersinatotalof17.5%ofthemetaphaseimages Fundulusmajalis (Fundulidae); Allotocadiazi,Ameca wererecordedrangingfrom29to46(Table1).There splendens,Goodeaatripinnis,Goodeagracilis,Hubbsina werenoadditionsinthesenumbers(>48).These turneri,Hyodonfurcidenes,Skiffiabilineata,Xenotaenia variationsordeviationsfromvalueswereprobably resolanae,Xenotocaeiseni,X.melanosoma andX.variata causedbylossesduringpreparationorotherartifacts. (Goodeidae),havethediploidchromosomenumber2n= Relativelysmallchromosomeswereobservedin A. 48(Solaetal.,1990;Klinkhardtetal.,1995; persicus.Thesize(totallength)ofchromosomesvaried Arkhipchuk,1999).Yetinafewspeciesof 70 H.R.ESMAEILI,Z.PIRAVAR,A.H.SHIVA Aphaniuspersicus 0.05mm A 0.01mm B 2 Aphaniuspersicus 1 µ 0 -1 -2 -3 -4 C -5 -6 Figure1. Metaphasespreadfromgillepithelialcell(A),giemsastainedkaryotypes(B)andhaploid idiogram(C)ofA.persicus. Table1.Chromosomecomplementsof A.persicus andA.sophiae. Species No.ofmetaphaseplates No.ofchromosomes Frequency Percentage 48 47 82.5 A.persicus 57 46 5 9 31 3 5 29 2 3.5 48 35 87.5 A.sophiae 40 47 3 7.5 19 1 2.5 50 1 2.5 71 KaryologicalAnalysisofTwoEndemicTooth-Carps, Aphaniuspersicus andAphaniussophiae (Pisces:Cyprinodontidae),fromSouthwestIran Aphaniussophiae 0.01mm A Aphaniussophiae B 0.01mm 4 Aphaniussophiae 2 µ 0 -2 -4 -6 -8 C -10 Figure2. Metaphasespreadfromgillepithelialcell(A),giemsastainedkaryotypes(B)andhaploididiogram (C)ofA.sophiae. Cyprinodontiformessuchas Aphyosemionviride and ofallexistingfishformshavebeenderived(Kuda-Bukhsh Fundulopanchaxsjostedti (Aplocheilidae),and etal.,1986)buttheissuestillseemsunresolved.The AllodontichthyshubbsiandAmecasplendens (Goodeidae) discoveryof48ratherlargeacrocentricchromosomesin thediploidchromosomenumberwasreportedtovary thePacifichagfish, Eptatretusstoutii,belongingtothe from2n=26to2n=42(Klinkhardtetal.,1995; orderMyxiniformes(Taylor,1967;Vasil'ev,1980),and Arkhipchuk,1999).Itcouldbesuggestedthatthediploid theoccurrenceof48rodsinthemajorityoffishes chromosomenumber2n=48isthemodalnumberin studiedpriorto1967ledtotheideathattheprimitive cyprinodontfishes.Intheinterpretationofkaryotypic karyotypeofancestralvertebratefreshlyevolvedfrom evolutionitisoftenassumedthattheprimitivefish chrodatemightconsistof48rods.Therefore,mostof karyotypeconsistsof48rodsfromwhichthekaryotypes thesubsequentresearchersassumedthekaryotypic 72 H.R.ESMAEILI,Z.PIRAVAR,A.H.SHIVA Table2. Chromosomemeasurements(inµm)andclassificationof A.persicuseandA.sophiae chromosomes(Ch.No.:Chromosomenumber;LA: Longarm;SA:Shortarm;TL:Totallength;AR:Armratio;CT:Chromosometype;Sm:Submetacentric;St:Subtelocentric). A.persicus A.sophiae Ch.No. LA SA TL AR CT Ch.No. LA SA TL AR CT 1 4.63 1.17 5.80 3.97 St 1 5.65 1.50 7.15 3.77 St 2 4.96 0.99 5.95 5.05 St 2 5.05 1.54 6.59 3.29 St 3 4.54 1.12 5.66 4.07 St 3 5 1.72 6.72 2.91 Sm 4 4.07 0.90 4.97 4.55 St 4 4.86 1.56 6.42 3.12 St 5 3.92 0.89 4.81 4.40 St 5 4.53 1.52 6.05 2.99 Sm 6 3.58 0.84 4.42 4.26 St 6 4.23 1.67 5.9 2.53 Sm 7 3.84 0.89 4.72 4.33 St 7 4.98 1.36 6.34 3.65 St 8 4.51 0.96 4.50 4.58 St 8 4.68