High levels of genetic variability and differentiation in hilsa shad, Tenualosa ilisha (Clupeidae, Clupeiformes) populations revealed by PCR-RFLP analysis of the mitochondrial DNA D-loop region.

0.12 MB·English

by  Mazumder, Sabuj Kanti

GeneticsandMolecularBiology,32,1,190-196(2009)
Copyright©2009,SociedadeBrasileiradeGenética.PrintedinBrazil
www.sbg.org.br
ResearchArticle
High levels of genetic variability and differentiation in hilsa shad,
Tenualosa ilisha(Clupeidae, Clupeiformes) populations revealed
by PCR-RFLP analysis of the mitochondrial DNA D-loop region
SabujKantiMazumderandMd.SamsulAlam
DepartmentofFisheriesBiologyandGenetics,BangladeshAgriculturalUniversity,Mymensingh,
Bangladesh.
Abstract
Thehilsashad,Tenualosailisha(Clupeidae,Clupeiformes)isanimportantanadromousclupeidspeciesfromthe
WesterndivisionoftheIndo-Pacificregion.ItconstitutesthelargestsinglefishablespeciesinBangladesh.Informa-
tion on genetic variability and population structure is very important for both management and conservation pur-
poses.PastreportsonthepopulationstructureofT.ilishainvolvingmorphometric,allozymeandRAPDanalysesare
contradictory.Weexaminedgeneticvariabilityanddivergenceintworiverine(theJamunaandtheMeghna),two
estuarine(KuakataandSundarbans)andonemarine(Cox’sBazar)populationsofT.ilishabyapplyingPCR-RFLP
analysis of the mtDNA D-loop region. The amplified PCR products were restricted with four restriction enzymes
namely,XbaI,EcoRI,EcoRV,andHaeIII.Highlevelsofhaplotypeandgenediversitywithinandsignificantdifferenti-
ations among, populations ofT.ilishawere observed in this study. Significant F values indicated differentiation
ST
amongtheriver,estuaryandmarinepopulations.TheUPGMAdendrogrambasedongeneticdistanceresultedin
twomajorclusters,although,theseweresubsequentlydividedintothree,correspondingtotheriverine,estuarine
andmarinepopulations.ThestudyunderlinestheusefulnessofRFLPofmtDNAD-loopregionasmolecularmark-
ers, and detected at least two differentiated populations ofT. ilishain Bangladesh waters.
Keywords:geneticvariability,PCR-RFLP,mtDNA,Tenualosailisha.
Received:April10,2008;Accepted:July24,2008.
Introduction estimates. Until 1972, hilsa shad fishery was prosperous
upstream in the rivers of Bangladesh, especially in the
The hilsa shad, Tenualosa ilisha, belonging to the
PadmaandMeghna.Fisheryhasenteredintoaseverede-
sub-family Alosinae of the family Clupeidae (Clupeifor-
cline upstream and is nowadays mainly concentrated
mes, Pisces), occurs in foreshore areas, estuaries, brack-
downstream, as well as in estuaries, coastal areas and the
ish-water lakes and freshwater rivers of the western divi-
sea (Nurul Amin et al., 2004). Due to the low water dis-
sion of the Indo-Pacific faunistic region. Its marine
chargefromtheupstreamGangesattheFarakkabarrage(in
distributionextendsfromIranandIraqinthePersianGulf
West Bengal, India) with the consequent heavy siltation,
tothewestcoastofIndiaintheArabianSeaandtheBayof
theindiscriminateexploitationofjuveniles(Jatka),disrup-
Bengal(PillayandRosaJr,1963).
tion of migration routes, loss of spawning, feeding and
Hilsa shad is the largest single fishable species in
nursing grounds, increased fishing pressure, etc., have all
Bangladesh,presentinalmostallthemajorriversystems,
contributedtothisdecline.
estuariesandmarineenvironments(BayofBengal),andat
presentcontributingtoapproximately12%ofthetotalfish
Thehilsashadislargelyananadromousspecies,but
productionand20%offishery-capture(inlandandmarine)
two other ecotypes - a fluvial potamodromous type and a
withabiomassof78,273metrictonsfrominlandfisheries
marine type - have been recognised. The potamodromous
and198,850metrictonsfrommarine(DoF,2006).Hilsais
stocksappeartoremaininthemiddlereachesoftherivers
termedthenationalfishofBangladesh,duetoitspopularity
throughout the year and breed therein. The anadromous
and economic importance. However, the production of
stocks,whosenormalhabitatisthelowerregionoftheestu-
hilsa in Bangladesh has waned when compared to earlier
ariesandtheforeshoreareas,ascendtheriversduringthe
breeding season and return to the original habitat after
spawning(Raja,1985).Theupstreammigrationduringthe
Send correspondence to Md. Samsul Alam. Department of Fish-
eries Biology and Genetics, Bangladesh Agricultural University, main breeding season depends largely on the commence-
Mymensingh, Bangladesh. E-mail: [email protected]. mentofthesouth-westmonsoonandconsequentflooding
MazumderandAlam 191
ofthemajorriversofBangladesh,BurmaandIndia.How- ies),Sundarbans(estuaries)andCox’sBazar(BayofBen-
ever,theexactspawningseasonforthespeciesisstillcon- gal) with the help of artisanal fishers (Figure 1). Immedi-
troversial,asspawningvariesfromafewmonthstoallthe ately after landing the fish on board, a piece of dorsal fin
year round. However, it is not known whether migratory from each was carefully collected and preserved in 95%
populations mix during migration or whether they pass ethanol.Thesampleswerestoredat-20°Cuntilfurtheruse
each other spatially and temporally. Therefore, the exact for the extraction of DNA. Total DNA (nuclear + mito-
stocksarestillindispute. chondrialDNA)ofeachindividualfishwasextractedfrom
Reportsonthestockstructureofthevaluabletropical approximately50mgoffintissuebystandardproteinase-K
shadT.ilisha,basedonsamplescollectedfromBangladesh digestion, phenol:chloroform:isoamyl alcohol extraction
andIndiaandthroughmorphometric,allozymeandRAPD andthealcoholprecipitationmethodasdescribedbyIslam
analyses, are contradictory. Dahle et al.(1997) claimed to and Alam (2004). The quality of the DNA samples was
havediscriminatedthreepopulationsofhilsashadinBan- checked by electrophoresis on 1% agarose gel and the
gladeshwaters,suchasChandpur(Meghnariver),Barguna quantitydeterminedbyusingaspectrophotometer,priorto
(Brackishwater,estuarine)andCox’sBazar(seawater)by PCRamplificationofthemtDNAD-loopregion.
usingRAPDmarkers.Onthebasisofasinglepolymorphic
Amplification of mitochondrial DNA
locus, PGM, Rahman and Naevdal (2000) claimed that
thereexistedtwoseparategenepoolsofhilsashadinBan- The2.2kbD-loopregionofthemtDNA,includinga
gladeshwaters.MiltonandChenery(2001),onthebasisof partofthecytochromeband12SrRNAgenes,wasampli-
otolith chemistry and morphometry, and Salini et al. fiedfromeachofthe90samplesbyusingtheprimer-pairs
(2004),onthebasisofallozymeandmorphometricanaly- L-12260(5’(cid:2)3’,CATATTAAACCCGAATGATAT
ses,inferred,however,thattherewasasinglestockofhilsa TT) and H-1067 (5’ (cid:2) 3’, ATA ATA GGG TAT CTA
in Bangladesh waters, this including the Bay of Bengal. ATCCTAGTTT)(Martinetal.,1992).ThemtDNAfrag-
Brahmaneetal.(2006)identifiedtwogroupsofT.ilishain mentwasamplifiedinareactionvolumeof30(cid:3)Lcontain-
India,onecomprisingtheGangesandYamunariversand ing 75 ng template DNA, 3 (cid:3)L of 10X buffer (100 mM
theothertheHooghlyandNarmadarivers,byusingRAPD Tris-HCl,pH=8.3,15mMMgCl ,500mMKCl),3(cid:3)Lof
2
markers.
2.5mMdNTPs(dATP,dCTP,dGTP,dTTP),1(cid:3)Mofeach
Amongthedifferentmarkersavailableforpopulation
primerand2unitsofTaqDNApolymerase(GENEIBan-
genetic analysis, differences in mitochondrial DNA are
probablythemostwidelyused,sincetheyfollowmaternal
inheritance,donotundergorearrangementsorrecombina-
tionandpresenthighermutationratesthanthoseofnuclear
genes(Avise,2004).RFLPanalysisofthemtDNAcontrol
regionisasimpletechniqueforrevealinggeneticvariation
inthemitochondrialgenomeofanorganism.
Inthisstudy,weanalyzedhaplotypeandgenediver-
sity in five samples of T. ilisha, using PCR-RFLPs of the
mtDNAD-loopregionasgeneticmarkers.Theaimofthe
presentstudywastocomparemtDNAgeneticdiversityin
T. ilisha samples collected from rivers, estuaries and the
sea,andtodelineategeneticdifferentiationamongpopula-
tionsofthethreemajoraquaticenvironments.Wealsore-
portherewhetherthereareanygeneticdifferencesbetween
distant river populations of T. ilisha, as well as between
estuarinepopulations.Thepurposeofthepresentstudyisto
examinetheusefulnessofmitochondrialD-loopregiondi-
versitytosupplementallozymeandRAPDdataonthepop-
ulationgeneticstructureofT.ilisha.
Materials and Methods
Fish samples and extraction of DNA
Ninetyhilsa(T.ilisha),withanaveragebodyweight
Figure1-MapofBangladeshshowingthefivesites((cid:129))fromwherethe
of about 400 g, were collected from five geographic lo-
samples of T. ilisha were collected. The three big rivers, the Padma,
cales:Balashi(Gaibandha,upstreamoftheJamunaRiver)),
JamunaandMeghna,arealsoshown.Thesethreeriversconstitutethema-
Chandpur(middleoftheMeghnaRiver),Kuakata(estuar- jorriverinefisheryofT.ilishainBangladesh.R:river.
192 Geneticvariabilityinhilsashad
galore, India). An oil-free thermal cycler (Master Cycler the hypothesis of no differentiation between populations,
GradientEppendorf,Germany)wasusedforPCRamplifi- weredeterminedbymeansof10000permutationsofhaplo-
cationwiththefollowingcycleparameters:initialdenatur- types between populations. Genetic distance values be-
ation at 94 °C for 3.0 min, followed by 35 cycles of tweenpopulation-pairswerecalculatedfromtheF values
ST
denaturationat94°Cfor45s,annealingat45°Cfor45s of the respective population-pair by using the formula: D
andextensionat72°Cfor2.0min.Afinalextensionstepat (genetic distance) = -log(1 - F ) (Reynolds et al., 1983).
ST
72°Cfor7minfollowedthelastcycle.ThePCRproducts Distance data were used to draw up an unweighted pair-
wereconfirmedbyelectrophoresisonagarosegelandsub- groupmethodofaverages(UPGMA)dendrogrambyusing
jectedtodigestionwithrestrictionendonucleases. MEGAversion4(Tamuraetal.,2007)software.
RFLP analysis Results
Four restriction enzymes (XbaI, EcoRI, EcoRV and
PCRamplificationoftheD-loopregionresultedina
HaeIII) were used for digesting the amplified fragments.
productofapproximately2.2kbwithnodetectablesizedif-
TenmicrolitresofthePCRproductwereusedforeachdi-
ferences between T. ilisha samples. Using this sequence,
gestion following manufacturer’s (Promega) recom-
fourrestrictionenzymes(XbaI,EcoRI,EcoRVandHaeIII)
mended conditions. The restricted PCR products were
were selected to obtain RFLP markers. All enzymes pro-
electrophoresed on 1.4% agarose gel containing ethidium
duced polymorphic banding patterns and restriction sites.
bromideandphotographedunderUVlightusingaGeldoc
Restriction of the PCR fragment with the four restriction
camera (UVP Inc). Two molecular weight markers, (cid:4)- endonucleasesresultedinatotalof35restrictionprofilesin
DNA-HindIIIdigestand100bpDNAladderwererunon the samples collected from five locations in Bangladesh.
eachgelalongwiththedigestedPCRproducts.Thesizesof Thecleavagepatternsandestimatedlengthsoftherestric-
mtDNArestrictionfragmentsweremeasuredbyusingthe tionfragmentsfordifferentrestrictionenzymesareshown
software,DNAfrag(Nash,1991). in Table 1. The cleavage patterns produced due to varia-
tionsinrestrictionsiteswerethreeforXbaIandEcoRI,five
Genetic data analysis and dendrogram construction
forEcoRVandsevenforHaeIII.Insomecases,however,
Restriction patterns generated from each restriction thesumofthefragmentsizesdidnotexactlyequalthetotal
endonucleaseweregivenletterdesignationsintheorderof size of the amplified region, probably due to small frag-
theirfrequencies.HaplotypeAreferstothemostcommon mentsbeinglostorbandsofsimilarsizeco-migrating.The
digestion pattern in the analyzed samples. The remaining 35differenthaplotypes(compositegenotypes)detectedin
alphabeticalprofilenames(B,C,etc.)indicatevariantdi- 90individualsofthefivepopulations,alongwiththeirnu-
gestionpatternsreflectingtheirfrequenciesinorder.Com- mericalfrequencies,arepresentedinTable2.
posite haplotypes were constructed from all the enzymes Outofthe35haplotypes,onlytwo,VIandXI,were
usedandarrangedfromtheenzymegeneratingthefewest distributedrandomlyamongthefivepopulations,theoth-
polymorphic patterns to that generating the most (XbaI, ers being either specific for a particular population or
EcoRI, EcoRV and HaeIII in this order). The presence or sharedbytwoorthree(mostlytwo)populations(Table2).
absence of restriction sites in the control region was in- HaplotypesII,III,IV,V,VII,IXandXwerespecifictothe
ferredforeachofthefourenzymesfromaseriesofrestric- Balashi population, XII and XIII to the Chandpur, XVII,
tionfragmentpatterns.Thesitecodesacrosstheamplified XVIII, XIX, XXI, XXIII, XXIV, XXV and XXVI to the
regionforarestrictionenzymewereconcatenatedandeach Cox’s Bazar, XXVII, XXVIII, and XXIX to the Sundar-
fish was assigned a code of four letters that described its bansandXXXVtotheKuakata.HaplotypeIwassharedby
composite,multi-enzymehaplotype. the Balashi, Chandpur and Sundarbans populations, VIII
Asingledatamatrixcomprisingthecompositehaplo- by the Balashi and Chandpur, XVI by the Chandpur and
typesandtheirfrequenciesinthefivepopulationswascon- Sundarbans and XX by the Cox’s Bazar and Kuakata.
structed.Haplotypeandgenediversity,bothcalculatedas Haplotype I was dominant in the Balashi and Chandpur
per Nei (1987), and a hierarchical analysis of population population, haplotype VI in the Chandpur, haplotypes
subdivisionperformedusingtheanalysisofmolecularvari- XVIIandXXintheCox’sBazarandXXVIIIintheSun-
ancewith1000simulatedsamples(AMOVA,Excoffieret darbans.Noneofthehaplotypeswerefoundtobedominant
al.1992),wereimplementedinARLEQUINv.3.1(Excof- intheKuakatapopulation.
fieretal.,2005).BoththeTajima(1989)D-testandtheFu Polymorphichaplotypeswereobservedinallthefive
(1997)Fs-testwereappliedtotestdeviationsfromneutral populations.Theratesofhaplotypes(numberofhaplotypes
molecular evolution. Significance was assessed in both observed divided by the sample size) ranged from 0.500
casesbygeneratingrandomsamples(numberofsimulated (Chandpur)to0.722(Sundarbans)(Table3).Haplotypedi-
samples:1000)underthehypothesisofselectiveneutrality versity was high in all the five populations, ranging from
and population equilibrium, as implemented in 0.882(Chandpur)to0.960(Sundarbans).Theaveragegene
ARLEQUIN. Significance levels of pair-wise F , under diversityacrossthelociwashighestintheSundarbanspop-
ST
MazumderandAlam 193
Table1-Restrictionpatternsfordifferenthaplotypesinthe2.2kbmtDNAD-loopfragmentfromninetyT.ilishacollectedfromfivelocationsinBangla-
desh(valuesshowrestrictionfragmentinbp).
Haplotypes
Restrictionenzyme Recognitionsequence A B C D E F G
XbaI T/CTAGA 1608 1241 2168
568 930
EcoRI G/AATTC 1439 1566 1529
566 566 566
95
EcoRV GAT/ATC 1432 1296 1296 1375 1432
707 707 675 707 675
HaeIII GG/CC 680 680 680 680 1210 680 849
660 660 660 660 580 660 680
440 541 409 580 200 409 660
425 200 390 200 200
ulation (0.569) and lowest in the Kuakata (0.343). The desh waters, including freshwater, estuarine and marine
overall Fixation Index (F ) across the populations was environments,byPCRmtDNARFLPanalysis.
ST
0.092andthepopulationspecificF indicesrangedfrom Thedetectionof35differenthaplotypesinonly90in-
ST
0.085 (Sundarbans) to 0.105 (Chandpur). Tajima’s D test dividualsoffiveT.ilishasamplesunderlinestheusefulness
(Tajima,1989)andFu’sFtest(Fu,1997)werenotsignifi- ofRFLPsoftheD-loopregionasmolecularmarkersforin-
cant(p>0.05)inallthepopulations. vestigatingthegeographicstructureofthespecies.Highdi-
Variance components and F-statistics analogs (Phi- versityindiceswereobtainedwithineachsample(Table3).
st) were calculated with AMOVA. No significant differ- TheaveragehaplotypicdiversityinT.ilishaobservedinthe
encesamongsampleswerefound.Hierarchicalanalysesin- present study, fell within the upper part of the range
dicated that 90.78% variation was contained within local (0.473-0.998)forsomeotherfishesasreportedbyAviseet
populationswhile9.22%wasdistributedamongthepopu- al.(1989),andreachedashighasthat(0.892)obtainedinred
lations. However, significant differentiation (F ) values seabream collected from four locations of western Japan
ST
werefoundamongthefivepopulationsofT.ilisha.TheF through PCR-RFLP analysis of the mtDNA D-loop region
ST
value between the Chandpur and Sundarbans populations byTabataandMizuta(1997).However,sincetheestimation
wasthehighestandthatbetweentheChandpurandBalashi of haplotype diversity is based on haplotype frequencies
thelowest.TheF valuesbetweentheBalashi-Chandpur, alone,itisdependentonthenumberofrestrictionenzymes
ST
Cox’s Bazar-Kuakata, and Sundarbans-Kuakata popula- used (Graves and McDowell, 1994). These results suggest
tion pairs were insignificant, while these values between thatgeneticvariabilityinT.ilishaisquitehigh.Highlevels
theremainingsevenwerefoundtobesignificant(Table4). ofgeneticdiversityappeartobecommonlyobservedinmi-
Geneticdistancevaluescalculatedfromthecorresponding gratory fishes with large panmictic populations (Santos et
F valuesbetweenthepopulation-pairsareshowninTable al.,2007).Thisisbecauselargeeffectivepopulationsizeand
ST
4.Thehighestgeneticdistancewasobservedbetweenthe highmigrationratesminimizetheeffectofgeneticdriftasa
Chandpur and Sundarbans population, while the lowest forcethatlowersintra-populationgeneticvariability.How-
was observed between the Chandpur and Balashi popula- ever,thepresenceofprivateallelesindicatesthatthesepopu-
tion. The UPGMA dendrogram based on genetic distance lations are not completely panmictic. The non-significant
resulted in two clusters. The first cluster contained four values(p>0.05)obtainedfromTajima’sDandFu’sFssta-
populationsandthesecondcontainedonlytheCox’sBazar tisticaltestsindicatethatthesampledpopulationsofT.ilisha
population.Thefirstclusterwassubsequentlydividedinto are in genetic equilibrium, which means that apparently
two sub-groups. The two river populations, Balashi and there is no pressure of selection on the population with re-
Chandpur,formedonegroupandthetwoestuarinepopula- gardtomitochondrialDNAhaplotypes.
tions,KuakataandSundarbans,thesecond(Figure2). The test for population differentiation gave signifi-
cant p-values (p < 0.05), indicating that composite haplo-
types were not distributed randomly with respect to
Discussion
locality. F-statistics (Weir and Cockerham, 1984) yielded
MitochondrialDNA-RFLPhasbeenprovedtobean an overall F value of 0.092 which indicates that genetic
ST
effective technique for population discrimination. The exchange occurring among the populations was not suffi-
present study was an attempt to reveal genetic variability cienttopreventeithergeneticdifferentiationorstructuring
and stock discrimination of hilsa populations in Bangla- intogeneticallydifferentiatedsubpopulationsinT.ilishain
194 Geneticvariabilityinhilsashad
Table2-Geographicdistributionof35compositemtDNAD-loopregionhaplotypesamongfiveconspecificpopulationsofT.ilishabasedonfourre-
strictionenzymes(XbaI,EcoRI,EcoRV,andHaeIII)(relativefrequenciesareinparentheses).
Compositehaplotypes GeographicdistributionofmtDNAcompositehaplotypes
Balashi Chandpur Cox’sBazar Sundarbans Kuakata
I AAAA 3(0.167) 4(0.222) 0 1(0.056) 0
II AAAB 1(0.055) 0 0 0 0
III ABAC 1(0.055) 0 0 0 0
IV AAAC 2(0.111) 0 0 0 0
V ABCC 2(0.111) 0 0 0 0
VI AABB 2(0.111) 5(0.278) 1(0.055) 1(0.055) 2(0.111)
VII ABBB 1(0.055) 0 0 0 0
VIII AAAE 1(0.055) 1(0.055) 0 0 0
IX AACA 1(0.055) 0 0 0 0
X AABB 2(0.111) 0 0 0 0
XI AADA 2(0.111) 1(0.055) 1(0.055) 1(0.055) 1(.055)
XII ABAD 0 1(0.055) 0 0 0
XIII ACAA 0 1(0.055) 0 0 0
XIV AABC 0 2(0.111) 0 0 2(0.111)
XV AADB 0 1(0.055) 0 0 1(0.055)
XVI AABE 0 2(0.111) 0 1(0.055) 0
XVII AACB 0 0 3(0.167) 0 0
XVIII AACF 0 0 2(0.111) 0 0
XIX AACC 0 0 1(0.055) 0 0
XX CBBA 0 0 4(0.222) 0 2(0.111)
XXI BADA 0 0 1(0.055) 0 0
XXII ABCD 0 0 1(0.055) 1(0.055) 0
XXIII CACA 0 0 1(0.055) 0 0
XXIV BACA 0 0 1(0.055) 0 0
XXV AABG 0 0 1(0.055) 0 0
XXVI AADC 0 0 1(0.055) 0 0
XXVII BACB 0 0 0 2(0.111) 0
XXVIII BADE 0 0 0 3(0.167) 0
XXIX BABA 0 0 0 2(0.111) 0
XXX BABB 0 0 0 2(0.111) 1(0.055
XXXI ABAE 0 0 0 1(0.055) 2(0.111)
XXXII AADG 0 0 0 1(0.055) 2(0.111)
XXXIII BAAE 0 0 0 1(0.055) 2(0.111)
XXXIV BADC 0 0 0 1(0.055) 2(0.111)
XXXV ABDB 0 0 0 0 1(0.055)
Table3-Geneticvariabilitywithinthefivedifferentpopulationsofhilsashad(T.ilisha).
Populations
Balashi Chandpur Cox’sBazar Sundarbans Kuakata
Samplesize 18 18 18 18 18
N.ofhaplotypes 11 9 12 13 11
N.ofpolymorphicsites 20 20 23 24 19
Rateofhaplotype 0.611 0.500 0.667 0.722 0.611
Haplotypediversity 0.948(cid:5)0.05 0.882(cid:5)0.05 0.967(cid:5)0.03 0.960(cid:5)0.03 0.954(cid:5)0.02
Expectedheterozygosity(genediversity) 0.451(cid:5)0.29 0.363(cid:5)0.26 0.531(cid:5)0.16 0.569(cid:5)0.24 0.343(cid:5)0.09
PopulationspecificF indices 0.099 0.105 0.092 0.085 0.079
ST
Tajima’sDtest 0.751 0.150 1.280 0.082 0.565
Fu’sFstest -1.057 0.178 -3-26 -1.899 2.814
MazumderandAlam 195
Table4-EstimatesofpairwiseF (Populationdifferentiation)values(belowdiagonal)andgeneticdistance(abovediagonal)amongfiveT.ilishapopu-
ST
lationsinBangladesh.TheF valueswerecalculatedwith110permutations.
ST
Ba Ch CB Su Ku
Ba 0.00000 0.00243 0.04816 0.07628 0.01959
Ch 0.00563NS 0.00000 0.07252 0.07847 0.03334
CB 0.10497*** 0.15380*** 0.00000 0.05637 0.03402
Su 0.16109*** 0.16531*** 0.12173*** 0.00000 0.00145
Ku 0.044120* 0.07391* 0.07536NS 0.00334NS 0.00000
Note:Ba:Balashi,Ch:Chandpur,CB:Cox’sBazar,Su:Sundarbans,Ku:Kuakata,NS:Notsignificant;*:p<0.05;***:p<0.000.
flectedbythelowernumberofprivatehaplotypesinthese
populations. Higher numbers of private haplotypes have
beenobtainedinthetwomostdistantpopulations,Balashi
andtheCox’sBazar(Table2),whichindicatesthatthelow
numberofsamplesisnotthemaincauseofthehighnumber
of private haplotypes in these populations. Therefore, we
wouldliketoopinethatcertaindifferencesexistamongthe
river populations, perhaps due to preferential movements
Figure2-UPGMAdendrogramsummarisingthegeneticdistancebe- of a fraction of the migratory groups, although the differ-
tweenpairsfromthefivedifferentpopulationsofT.ilisha.
ence is not high enough to distinguish the populations as
separatestocks(sub-populations).
Bangladesh.GeographicdifferentiationofthehilsashadT. We observed significant differentiation between the
ilisha has also been reported previously. Brahmane et al. riverineandmarine(Cox’sBazar)populations,butnotbe-
(2006)identifiedtwogroupsofhilsainIndia,onecompris- tweenthemarineandoneoftheestuarine.Thereisnoevi-
ing the Ganges and Yamuna rivers and the other the Ho- dence of spawning in the sea, but there is evidence of T.
oghlyandNarmadarivers.Dahleetal.(1997)reportedthe ilisha estuarine spawning in Bangladesh (BOBP, 1987).
existence of three stocks of hilsa shad in Bangladesh wa- Thus,fishesfromtheCox’sBazarregionmustmigrateto
ters, namely the Chandpur (Meghna river), Barguna anyoftheriversoratleastuptotheestuaries.Ourresults
(Brackishwater,nearKuakata))andCox’sBazar(saltwa- suggestthatfishesfromtheCox’sBazarregionmaygoup
ter),whenusingRAPDmarkers.Ourresultssuggestthere totheKuakataregion,butnottotheMeghnaandJamuna
are at least two, one marine, and the other riverine- rivers, as significant differentiations between the Cox’s
estuarine,orpossiblythree(riverine,estuarineandmarine), Bazar and the two river populations have been observed,
stocksofhilsainBangladesh.Thesecondpossibilitycom- butnotbetweentheCox’sBazarandKuakata(Table4).
plieswiththehypothesisofthreegroupsofhilsashad,ana- Thefivesamplesweregroupedintotwoclustersonthe
dromous, potamodromous and marine, as reported by basisofgeneticdistanceamongpopulation-pairs.Neverthe-
PillayandRosaJr(1963).Now,thequestioniswhetherthe less,thetwoclusterswerefinallydividedintothreegroups
T.ilishafeedingintheChandpurareamigratefurtherup- correspondingtothethreedifferentenvironments,riverine,
streamtospawninthePadmaandtheJamunariversornot. estuarineandmarine(sea).Theslightdistancebetweenthe
NosignificantdifferenceinF valuebetweenthesamples BalashiandChandpurpopulationindicatestheirbelonging
ST
from Balashi (the Jamuna) and Chandpur (the Meghna) tothesamestock.However,thesharingoftwohaplotypes
suggestasinglepanmicticriverinepopulationofT.ilishain byallthepopulationshasledustopostulatethepresenceofa
Bangladesh.However,thisconclusionmaybecontradicted certaindegreeofgeneflowamongthosestudied.
through the presence of some private haplotypes in the Hilsa shad (T. ilisha) is the national fish of Bangla-
Balashi and Chandpur populations. These results also run desh. The delicious taste and contribution of this fish has
counter to those of Shifat et al. (2003), who reported that leadtoitspositionasoneofthemosteconomicallyimpor-
thetworiverpopulationsofT.ilisha,theMeghnaandthe tantfishinthiscountry.Furthermore,hilsafisheryconsti-
Padmaweredifferentstocks,thoughtheyobservedcertain tutes the largest single species fishery in the riverine,
allelessharedbysomeindividualsofthesetworiversam- estuarineandmarineecosystemsofthecountry.Itisessen-
ples. The presence of private haplotypes indicates the ex- tialtorecognizethatgeographicallyandgeneticallydiffer-
tentofmixingbetweenpopulations.Duetotheirlocations ent populations as different stocks should be managed
(Figure 1) and the mode of migration of the species, the separately (Carvalho and Hauser, 1994). Our studies on
possibility of mixing in the Chandpur (the mid Meghna mtDNA PCR-RFLP in T. ilisha indicate that population
river), Kuakata and Sundarbans populations is higher, re- sub-divisiondoesindeedexistinthisspecies.Onthebasis
196 Geneticvariabilityinhilsashad
oftheAMOVA,wecanconcludethattherearethreestocks IslamMSandAlamMS(2004)Randomlyamplifiedpolymorphic
ofhilsawithasubstantiallevelofinter-populationgenetic DNAanalysisoffourdifferentpopulationsoftheIndianma-
divergenceamongriver,estuarineandmarinepopulations. jorcarpLabeorohita(Hamilton).JApplIchthyol20:407-
412.
The high level of haplotype variability found in only five
MartinAP,HumphreysRandPalumbiSR(1992)Populationge-
populations underlines the usefulness of RFLPs of the
netic structure of the armorhead, Pseudopentaceros
D-loopregionasmolecularmarkerstoinvestigatethegeo-
wheeler, in the North Pacific Ocean: Application of the
graphicstructureofT.ilisha.Weanalyzedonly18individ-
polymerasechainreactiontofisheriesproblems.CanJFish
ualsfromeachpopulation,andtheelectrophoreticanalysis AquatSci49:2386-2391.
ofrestrictionfragmentsisalsonotsoperfectassequencing MiltonDAandChenerySR(2001)Canotolithchemistrydetect
thePCRproduct.Therefore,toreachamoredefinitecon- thepopulationstructureoftheshadhilsaTenualosailisha?
clusion, larger samples from throughout the distribution Comparisonwiththeresultsofgeneticandmorphological
range of the species in the country should be analyzed by studies.MarEcolProgSer222:239-251.
NurulAminSM,RahmanMA,HaldarGC,MazidMA,Milton
sequencing the mtDNA D-loop region and/or with faster
DAandBlabberSJM(2004)Stockassessmentandmanage-
evolvingmolecularmarkers,suchasmicro-satelliteloci.
ment of Tenulosa ilisha in Bangladesh. Asian Fish Sci
17:50-59.
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