全 文 ：应用与环境生物学报　 2007, 13(6):753～ 758 　 　
ChinJApplEnvironBiol=ISSN1006-687X 　 2007-12-25
　
Received:2007-01-09　　Accepted:2007-04-17
＊SupportedbytheOutstandingYoungScientistProgramoftheNational
NaturalScienceFoundationofChina(No.30525036)andtheChina
NationalKeyProgramoftheInternationalCooperationforScienceand
Technology(No.2005DFA30620)
＊＊Correspondingauthor(E-mail:licy@cib.ac.cn)
GeneticVariationofHippophaerhamnoidesPopulationsatDiferentAltitudes
intheWolongNatureReserveBasedonRAPDs＊
ZHAOChunfang1, 2 , CHENGuojuan1, 2 , WANGYuhua1
KORPELAINENHelena3＆ LIChunyang1＊＊
(1ChengduInstituteofBiology, ChineseAcademyofSciences, Chengdu610041, China)
(2GraduateUniversityofChineseAcademyofSciences, Beijing100039, China)
(3DepartmentofAppliedBiology, UniversityofHelsinki, HelsinkiFI-00014, Finland)
Abstract　ToestimategeneticvariationandgeneticstructureofHippophaerhamnoidesssp.sinensisatdiferentaltitudes, five
naturalpopulationsintheWolongNatureReserveataltitudesrangingfrom1 800 to3 400 mabovesealevel(a.s.l.)were
surveyedusingrandomamplifiedpolymorphicDNAmarkers(RAPDs).Basedon11decamerprimers, atotalof151reproduc-
ibleDNAlociwereyielded, and143 ofthemwerepolymorphic, accountingfor94.7%.Amongthefivepopulationsinvestiga-
ted, thetotalgenediversity(HT)andgenediversitywithinpopulationBequaled0.289 and0.315, respectively, whichare
modestforasubspeciesofH.rhamnoidesthatisanoutcrossedandlong-livedwoodyplant.Theamountofgeneticvariationa-
mongpopulationsvariedfrom0.098withinpopulationE(3 400 ma.s.l.)to0.315 withinpopulationB(2 200ma.s.l.).
Thecoefficientofgenedifferentiation(GST)amongpopulationsequaled0.406, andrevealedthat40.6% ofthegeneticvari-
anceexistedamongpopulationsand59.4% withinpopulations.ThepopulationA(1 800 ma.s.l.)differedgreatlyfromthe
otherfourpopulations, whichcontributestohighgeneticdiferentiation.AUPGMAclusteranalysisandprincipalcoordinate
analysesbasedonNeisgeneticdistancesfurthercorroboratedtherelationshipsamongthefivepopulationsandallthesampling
individuals, respectively.Manteltestsdetectedasignificantcorelationbetweengeneticdistancesandaltitudinalgradients(r
=0.646, P=0.011).Fig3, Tab4, Ref42
Keywords　altitudinalgradient;geneticdiferentiation;geneticvariability;Hippophaerhamnoides;RAPD
CLC　S793.602(271):Q75
利用 RAPD标记分析卧龙自然保护区不同海拔沙棘种群的遗传变异＊
赵春芳 1, 2　陈国娟 1, 2　王芋华 1　KORPELAINENHelena3　李春阳1＊＊
(1中国科学院成都生物研究所　成都　610041;2中国科学院研究生院　北京　100039)
(3赫尔辛基大学应用生物学系　芬兰赫尔辛基　FI-00014)
摘　要　从 1 800m到 3 400 m五个海拔连续取样 ,用 RAPD分子标记研究了卧龙自然保护区中国沙棘种群的遗传结
构和遗传变异.用 11条寡核苷酸引物 ,扩增得到 151个重复性好的位点 , 其中 143个多态位点 ,多态率达 94.7%.在 5
个沙棘种群中 ,总遗传多样性值(HT)为 0.289, B种群内的遗传多样性值为 0.315, 这完全符合沙棘这种多年生 、远交
的木本植物高遗传变异的特性.5个种群内遗传多样性随海拔升高呈低—高—低变异趋势 , 在2 200 m海拔处的 B种
群遗传多样性达最大值 0.315, 3 400m海拔处的 E种群则表现最小 , 仅 0.098.5个种群间的遗传分化值 GST=0.406,
也即是说有 40.6%的遗传变异存在于种群间 , 59.4%存在种群内.1 800m海拔处的 A种群与其他种群的明显分离是
造成种群间遗传分化大的原因.UPGMA聚类图和 PCoA散点图分别进一步确证了 5个种群间关系和所有个体间的关
系.最后 , 经过 Mantel检测 , 遗传距离与海拔表现了明显的相关性(r=0.646, P=0.011).图 3表 4参 42
关键词　海拔梯度;遗传分化;遗传变异;沙棘;RAPD
CLC　S793.602(271):Q75
　　ThespeciesofthegenusHippophaeL.(Elaeagnaceae)are
perennialdeciduousshrubs, smaltreesortrees, whicharedioe-
cious, wind-polinatedpioneerplants.Theyreproducesexualyby
bird-dispersingseedsorasexualybyrootproliferation.Thenatural
genushasawidedistributionextendingfrom NorthernEurope
throughCentralEuropeandCentralAsiatoChina, rangingfrom
27°Nto69°Nandfrom7°Wto122°E[ 1] .Theplantscangrowon
seashores, riverdeltas, riverterraces, valleyslopesandsometimes
inmeadows, atsupremealtitudeupto5 000 ma.s.l.[ 1～ 3] .
Accordingtothelatesttaxonomy[ 4] , thegenusHippophaeis
dividedintosixspeciesand12 subspecies.ThesubspeciesH.
rhamnoidesssp.sinensisoccursonlyinChinaanditisrecognized
astheprimordialgenusofHippophae[ 5] .Itsdistributionextends
fromtheeasternpartoftheQinghai-TibetanPlateauthroughthe
QinlingMountainstotheTaihangMountains.Itsoccurrenceina
largerangeofhabitatsacrossdifferentaltitudesfrom 550 mto
3 700 ma.s.l.[ 6] , althoughmainlyrestrictedtosunny, south-fa-
cingslopes, implieswideadaptationtodifferentenvironments[ 7 , 8] .
Asthepresentstudyarea, theWolongNatureReserveinSichuan
Province, China(Fig.1-a), possessestheclimaticconditionsof
theQinghai-TibetanPlateau, thisareaischaracterizedbyonly
littlevariationinannualtemperature, butdistinctdryandwetsea-
sons, heavyrainsandsignificantverticalclimatevariation.Be-
causeoftheblockingeffectofhighmountainsontheairflowcoming
fromthewestandeast, wetairformsabundantrainsinthearea.
Theannualrainfalequals1 500 ～ 1 800 mm[ 9] .Thedistribution
ofvegetationvariesconsiderablyaccordingtothealtitude(Fig.1-
b).TheareaofoccurrenceforH.rhamnoidesintheWolongNa-
tureReserveisatthealtitudesvaryingfrom1 800to3 400 ma.s.
l.[ 10] .
Fig.1　a.LocationoftheWolongNatureReserveinSichuanProvince, China
　　　　　　　　　　b.DistributionofvegetationalonganaltitudinaltransectintheWolongNatureReserve
　 　 Itis wellknown thataltitudinalgradients impose
heterogeneousenvironmentalconditions, includingruggedtopogra-
phy, acomplexpatternofvegetationandadelayinflowering, and
theyarelikelytomarkedlyafectthegeneticvariationpaternof
plants.Numerousstudiesonthemorphological, phenological,
physiologicalandmolecularresponsesofmanyplantspeciesoccup-
yingsitesatvaryingelevationshavebeenconducted[ 11 ～ 14] , inclu-
dingafewreportsrelatedtothemorphological, physiologicaland
biochemicalresponsesofH.rhamnoidesalongaltitudinalgradi-
ents[ 15 , 16] .However, nostudiesonthegeneticstructureofH.rh-
amnoidesinrelationtothealtitudehavebeenconductedbefore.
AccordingtoourpreviousfieldinvestigationsintheWolongNature
Reserve, thegrowth, spatialpaternandpopulationmorphological
structureofH.rhamnoidesarecloselyrelatedtoaltitudinalgradi-
ents, althoughthechangeinthesecharacteristicshappensnon-lin-
earlyalongincreasingaltitudes.
Thispresentstudyaimstoinvestigatethelevelofgeneticvari-
ationandthepaternofgeneticstructureinfivenaturalpopulations
ofH.rhamnoidesoccurringintheWolongNatureReserveatalti-
tudesrangingfrom1 800 to3 400 ma.s.l.
1　Material＆Methods
1.1　Plantmaterial
Duringthe2004 growingseason, freshleaveswerecolected
from124 adultindividualsofH.rhamnoidesssp.sinensisatfive
differentaltitudesintheWolongNatureReserveinsouthwestern
China(102°25′～ 103°24′E, 30°45′～ 31°25′N).Fivesampling
locations(A, B, C, D, andE)wereselectedalongavertical
transectthatspannedapproximately1 600moveralineardistance
ofabout50kmat400 mintervalsfrom1 800 m(populationA)to
3 400 m(populationE)elevation.Ineachpopulation, twenty-
threetotwenty-sixadultplantswererandomlysampled, andthe
754 　　　　　　　　应 用与 环 境生 物学 报　　ChinJApplEnvironBiol　　　　　　　　　　　　　　 　　　13卷
sampledindividualswereseparatedbyadistanceofatleast50 m
upwards.Formolecularanalyses, thesefreshleaveswerefrozen
quicklyinliquidnitrogenandstoredat-86 ℃ untilDNAextrac-
tion.
1.2　DNAextractionandPCRamplification
DNAwasisolatedfrom 0.5 gfrozenleavesandpurifiedfol-
lowingtheprocedureofIsabel, etal.[ 17] .Thequalityandconcen-
trationofDNAwascheckedbyaSmartSpecTM 3000 DNA-Protein
instrument(Bio-Rad).Amongthescreened140 RAPDprimers
(OperonTechnologies), elevenwerechosenforthepresentstudy
(Table1)basedontheclarityandreproducibilityofthebandpat-
terns.Theamplificationreactionswereperformedinthefinal
volumeof25 μLcontaining2.5 μLofthe10 ×reactionbuffer
(TaKaRa, Dalian), 250μmol/LdNTP(Promega), 0.25 μmol/
Lprimer, 1.0 UTaqpolymerase(TaKaRa, Dalian)and40 ～ 50
nggenomicDNA.Alamplificationreactionswereperformedina
GeneAmp PCRSystem 9700 (PerkinElmerCorp., Norwalk,
CT, USA)usingthefolowingprogram:Aninitialstepof4 minat
94 ℃, folowedby40cyclesof1minuteat94℃, 1 minuteat39
℃ and2 minutesat72℃, andanincubationof10minat72 ℃.
ThePCR productswereseparatedon1.8% agarosegelsand
stainedwith0.1%ethidiumbromide.Molecularweightswereesti-
matedusingaGeneRulerTM 100bpDNALadderPlus(Fermen-
tas).ThegelimageswererecordedunderaGelDoc2000 (Bio-
Rad).ThereproducibilityoftheRAPDreactionswastestedbyre-
peatingtheamplificationprocessatleasttwiceforeachprimer.
Table1　ThesequencesofelevenRAPDprimersandnumberof
lociperprimerscoredinH.rhamnoides
Primer RAPDproductsperprimer
Polymorphic
RAPDproducts
Primersequence
(5-3)
OPA-07
OPA-13
OPA-18
OPB-10
OPB-12
OPB-18
OPD-05
OPD-15
OPD-20
OPE-03
OPH-20
Total
Polymorphism
16
17
7
11
15
16
10
8
21
11
19
151
16
17
7
9
13
15
10
7
20
10
19
143
94.7%
1.3　Dataanalysis
Onlyclearandreproduciblelociwereincludedinthefurther
analysis.TheRAPDfragmentswerescoredforeachindividualas
present(1)orabsent(0)onthebasisofsizecomparisonwithex-
ternalstandards(GeneRulerTM 100bpDNALadderPlus).Thefol-
lowingparametersweregeneratedusingtheprogramPOPGENE
1.32 todescribegeneticvariation:thepercentageofpolymorphic
loci(P, 5% criterion), Neisgenediversity(h), Shannonsin-
formationindex(i), theobservednumberofalleles(Na)andthe
effectivenumberofaleles(Ne)[ 18, 19] .Thegeneticstructurewas
investigatedusingNeisgenediversitystatistics, includingthetotal
geneticdiversity(HT), geneticdiversitywithinpopulations(HS),
andtherelativemagnitudeofgeneticdifferentiationamongpopula-
tions〔GST=(HT-HS)/HT〕[ 18] .Anestimateofgeneflow(Nm)
wascomputedusingtheformulaofNm=(1-GST)/2GST[ 20] .All
theseparameterswerecalculatedusingPOPGENEprogramversion
1.32[ 19] .ByemployingtheUPGMA(Unweightedpairgroupmean
average)method, adendrogramwasconstructedbasedonNei′s
(1973)unbiasedgeneticdistancestoanalyzegeneticrelationships
amongpopulations[ 18] .Thegeneticrelationshipsamongindividuals
andamongpopulationswerefurtheranalyzedbytheprincipalcoor-
dinateanalysis(PCoA)ofageneticdistancematrixaccordingto
theextractedEigenvectorsinGenAlExversion6.Thepossiblere-
lationshipbetweengeneticdistancesandaltitudinalgradientswas
assessedbytheManteltest[ 21] inGenAlExversion6[ 22] .
2　Results
2.1　RAPDanalysis
TheRAPDpatternsobtainedusingthe11 selectedprimers
werefoundtobehighlyreproducible.Thenumberoflocivaried
from7 to21, withanaverageof13.7 perprimer(Table1).Ato-
talof151 reproduciblelociwereproduced, withfragmentsizes
rangingfrom 400 bpto1 500bp, and143 lociofthemwerefound
polymorphic, accountingfor94.7% ofthetotal.
2.2　Geneticvariabilitywithinpopulations
Inthepopulations, thepercentageofpolymorphicloci(P)
rangedfrom27.8% to90.1% withameanof48.3%.Neisgene
diversity(h)variedfrom 0.098 to0.315 withanaverageof
0.170, andShannonsindex(i)rangedfrom0.146 to0.471 with
anaverageof0.253.Themeanobservednumberofaleles(Na)
rangedfrom1.278 to1.901, whilethemeanefectivenumberof
alleles(Ne)variedfrom1.168 to1.543(Table2).PopulationB
wasobservedwiththehighestgeneticdiversity, whilepopulationE
withthelowest.Whencalculatedacrosspopulations, thehandi
valuesequaled0.289and0.438, respectively, andtheNaandNe
valueswere1.947and1.489, respectively.
Table2　GeneticvariabilityparametersofH.rhamnoides
basedonRAPDdata
Population Samplesize P/% h i Ne Na
A
B
C
D
E
Mean
Total
26
25
25
25
23
124
35.8
90.1
54.3
33.8
27.8
48.3
94.7
0.131
0.315
0.186
0.118
0.098
0.170
0.289
0.194
0.471
0.278
0.176
0.146
0.253
0.438
1.229
1.543
1.320
1.206
1.168
1.293
1.489
1.358
1.901
1.543
1.338
1.278
1.483
1.947
P, Percentageofpolymorphicloci;h, Neisgenediversity;i, Shannonsin-
formationindex;Na, Observednumberofalleles;Ne, Effectivenumberofaleles
2.3　Geneticdiferentiationamongpopulations
Theanalysisontheapportionmentofgeneticvariationshowed
thatthemeangenediversitywithinpopulations(HS)andthetotal
755　 6期 ZHAOChunfang, etal.:GeneticVariationofHippophaerhamnoidesPopulationsatDiferentAltitudesintheWolongNatureReserveBasedonRAPDs 　　
genediversity(HT)equaled0.170and0.289, respectively.Con-
sequently, variationoccurringamongpopulations(GST)equaled
0.406 (Table3), indicatingthat40.6% ofgeneticdifferentiation
existedamongthefivepopulations.However, theGST valuee-
qualed0.193 whenpopulationAwasexcludedfromtheanalysis.
Theobservedstructureofgeneticvariabilityshowedthattherewas
ahighdegreeofpopulationdifferentiationamongthepopulationso-
vershortgeographicdistances.Theoverallrateofgeneflow(Nm)
amongthepopulationswaslow, thatis, 0.732.
Table3　Apportionmentofgeneticstructureofthefivenatural
populationsofH.rhamnoides
Population HT HS GST Nm
5 0.2858 0.1698 0.4060 0.7315
2.4　Geneticrelationships
Geneticdistanceswerecalculatedforeachpairofpopulations
toestimatetheextentofthedivergenceamongthefivepopulations
ofH.rhamnoides(Table4).Thedistancesrangedfrom 0.016
betweenpopulationsDandEto0.476 betweenpopulationsAand
E, withameanof0.197.
Table4　Neisgeneticdistancesandgeneticidentitiesamong
populationsofH.rhamnoides
Population A B C D E
A
B
C
D
E
＊＊＊＊＊
0.2014
0.4307
0.4578
0.4762
0.8176
＊＊＊＊＊
0.0802
0.1107
0.1093
0.6501
0.9230
＊＊＊＊＊
0.0416
0.0418
0.6327
0.8952
0.9592
＊＊＊＊＊
0.0157
0.6211
0.8965
0.9590
0.9849
＊＊＊＊＊
Abovediagonal:Neis(1973)geneticidentities;Belowdiagonal:genetic
distances
TheUPGMAclusteranalysisbasedonNeisunbiasedgenetic
distanceswasperformedtoshowthegeneticrelationshipsamong
thepopulations(Fig.2).Thefivenaturalpopulationswere
groupedintotwomainclusters.PopulationsB, C, DandEformed
acluster, whilepopulationAwasdistinctfromtheothers.Within
thecluster, populationsDandEweremostcloselyrelated.The
Manteltestshowedthattherewasasignificantcorrelationbetween
geneticandaltitudedistancesamongthefivenaturalpopulations(r
=0.646, P=0.011).
Fig.2　UPGMAdendrogramofthefivenaturalpopulationsof
H.rhamnoidesbasedonNeis(1973)geneticdistances
2.5　Principalcoordinateanalysis
Basedonthespatialrepresentationoftherelativegeneticdis-
tancesacrossallthe151 lociamongindividuals, thegeneticrela-
tionshipsamongpopulationswerefurtherillustratedbythePCoA
plotofcoordinates1 and2.Theprincipalcoordinates1 and2 ob-
tainedintheanalysisaccountedfor74.0% and8.2% ofthetotal
variation, respectively(cumulativevalue=82.2%).Innon-
agreementwiththeclusteranalysis, eachindividualformedasepa-
rateplotandsomeindividualsfromonepopulationcouldnotbe
clearlydistinguishedfromindividualsoriginatingfromotherpopula-
tions(Fig.3).The124 samplesweredividedintotwogroups.A-
mongalthesamples, 30 individualsofpopulationAand9 indi-
vidualsofpopulationBownedonegroup, whichexpressedlarge
divergencetotheothersamples, whilepopulationBwasconstitu-
tedoflooseindividualsoftwoclustersandexpressedhigh
differencesofindividualgeneticstructure.
Fig.3　Principalcoordinatesanalysis(PCoA)basedon
RAPDprofilesof124samplesofH.rhamnoides
Population:A(◆);B(◇);C(★);D(×);E(■)
3　Discussion
BasedonthepresentRAPDinvestigation, thefiveexamined
naturalpopulationsofH.rhamnoidesssp.sinensis, locatedalonga
heterogeneousaltitudinalgradientintheWolongNatureReservein
China, appearedtopossessahighlevelofgeneticdiversity.The
totalgenediversityequaled0.289 andgenediversitywithinpopu-
lationBequaled0.315.Theobserveddiversitiesweresimilaror
higherwhencomparedtothevaluespreviouslyreportedforthe
populationsofH.rhamnoides[ 23, 24].Ingeneral, thelevelofgenet-
icdiversityofoutcrossedperennialwoodyspecieswithwidelydis-
persingseedsandwithbothsexualandasexualreproductionlargely
relatestothewideregionaldistribution, largegeographicrangeand
differentlife-historycharacteristics[ 25 , 26] .Besidessuchfeaturesas
relativelywidedistribution, dominantlyoutcrossedmatingsystem
andefectiveseeddispersalbysmalanimalsandbirds[ 26] , itis
sometimesarguedthathardclimaticconditionsandheterogeneous
habitatsmayalsocontributetohighlevelofgeneticdiversity[ 27] .
OurinvestigationdemonstratesthatthegeneticdiversityofH.
rhamnoidesvarieswithchangingelevationandshowsatrendthat
populationsBandC(2 200 mand2 600m)aregeneticallymore
diversethanpopulationA(1 800 m)andpopulationsDandE
756 　　　　　　　　应 用与 环 境生 物学 报　　ChinJApplEnvironBiol　　　　　　　　　　　　　　 　　　13卷
(3 000 mand3 400 m).Thedetectedpaternofwithin-popula-
tiongeneticdiversityislikelyatributedtovariableenvironmental
factors, e.g., temperatureandrainfall, atcorrespondingalti-
tudes.AccordingtoLi, etal.[ 16] , H.rhamnoidesisstressedby
droughtandhightemperatureatlowelevations, andbycoldtem-
peratureathighelevations.Unfavourableenvironmentsatbothlow
andhighaltitudinalzonesmayleadtoanincreaseinvegetativere-
productionanddecreaseinresource-demandingsexuality, poten-
tialyresultinginalossofgeneticvariation.Ingeneral, every100
mofelevationrisingreducestemperatureby0.56 ℃[ 28] .Thus,
thetemperaturediferencebetweenthesitesofeachinvestigated
populationpairofH.rhamnoideswouldbe2.24 ℃, andthe
differencebetweenthelowestaltitudeofpopulationAandthehigh-
estofpopulationEwouldbe9 ℃.Thisvariationintemperature
maypartlyaccountforthetrenddetectedingeneticvariation.The
lowlevelofgeneticvariabilitypresentinpopulationAmaybeat-
tributedtotherestrainedgrowthandstrongcompetitionwithother
treesunderdryandhotenvironmentalconditions.Sincealthe
studiedpopulationsaresampledfromtheWolongNatureReverse,
humaninterferencemaycontributelittletothelowlevelofgenetic
variability, thoughseverehumaninterferencemaycauseincrease
inasexualreproductionandreductioningeneticvariability[ 29] .
PopulationEislocatedrightontheboundarybetweenaH.rham-
noidesshrubandamountainmeadow, wheretheclimateconditions
aredryandcold.Thepresenceoflowergeneticdiversityinpopu-
lationsDandEislikelycausedbyadecreaseinsexualreproduc-
tionimposedbypollinationlimitationandfloweringasynchronydue
tothediferenceintemperaturebetweenpopulations[ 30] .Onthe
otherhand, populationsBandCarethoughttobemostlyfreefrom
bothdroughtandsub-optimaltemperaturestresses.Previously, Li,
etal.havedocumentedtheevidencethatthereexistsanoptimum
zoneforH.rhamnoidesintheWolongNatureReserve[ 16] .The
beneficialclimateconditionsofthesesitesforthegrowthandsexual
reproductionofH.rhamnoidescouldcontributetothehighergenet-
icdiversitypresentinpopulationsBandC.
Thepartitioningofthegeneticdiversityrevealedthat40.6%
ofthevariationwasmaintainedamongthepopulations.Although
thisresultisconsistentwithapreviousstudyonH.rhamnoidesin
ChinawiththeGSTvalueof0.418obtainedforthepopulationsfrom
awidegeographicalareaandwiththeGST valuesof0.377 and
0.357 detectedintwomorelimitedgeographicregions[ 23 , 31] , the
levelofdifferentiationamongthepopulationsappearstobeclearly
higherthanthevaluesobservedfromotherstudiesonH.rham-
noides[ 24] , andissimilartothelevelofdiferentiationinanendan-
geredplant[ 32] .TheUPGMAdendrogrambasedongeneticdis-
tancesandthePCoAanalysisbasedonpair-wiseindividualgenetic
distancesshowedthatpopulationAdiferedconsiderablyfromthe
otherpopulationsandwasclusteredaloneasagroup.Infact, the
GSTvalueequaled0.193 whenpopulationAwasexcludedfromthe
analysis.Thissituationislikelytoresultfromrestrictedgeneflow
betweenpopulationAandtheotherfourpopulations.SinceH.
rhamnoidesisgenerallywind-pollinated, inbreedingandassociative
matingareunlikelythefactorscontributingtothegeneticstructure
ofH.rhamnoides.Theestimatesofgeneflowamongalthesam-
pledpopulationsandamongthefourpopulationsexcludingpopula-
tionA, Nm =0.732 andNm =2.089 (numberofmigrantsex-
changedbetweenpopulationspergeneration)[ 33] , respectively, in-
dicatealowrateofgeneexchangebetweenpopulationAandthe
otherpopulations.Theverticaldistributionofvegetationvariessig-
nificantlyintheWolongNatureReserve.Anevergreenbroad-
leavedtreebeltoccursbetweenpopulationAandpopulationB,
anditmaybethemainbarrierforgeneexchange.Moreover, itis
unknownhowthewindpolinationfunctionsbetweenvertically
differentsites.Geneflowmaymainlydependonseedspreadingby
birdsratherthanpollinationbywind.Bellusci, etal.[ 12] havesug-
gestedthatgeneflowviapolinationislesseficientthanthatvia
seeddispersionbetweenpopulationsoccupyingsitesatdifferentel-
evations.
Mountain environmental conditions are typically
heterogeneous, sincesomeimportantecologicalfactors, suchas
temperature, vegetationpaternandfloweringtimechangerapidly
withelevation, whicharelikelytomarkedlyafectthegeneticvari-
ationpaternofplants.Afasttransitioninthegeneticstructurea-
longaltitudinalgradientshasbeenobservedinseveralplantspecies
duetothechangingenvironmentalconditions[ 34 ～ 38] .However,
therearealsoinvestigationsindicatingoppositeresults[ 14, 27 , 39 , 40]
orshowingthatgeneticvariationanddiferentiationdonotcorrelate
withaltitude[ 41 , 42] .Inthisstudy, thepaternofgeneticvariation
detectedfromthepopulationsofH.rhamnoideshasaclearrela-
tionshipbetweengeneticandaltitudedistances, asrevealedbythe
Manteltest.Phenologicalvariationandmorphologicalstructureof
H.rhamnoidesarecloselyrelatedtoaltitudinalgradientsfromour
fieldobservations, andthephysiologicalandbiochemicalofH.rh-
amnoidesalongaltitudinalgradientsareevident[ 16] , althoughthese
characteristicschangenon-linearly.Thus, thesecharacteristic
changesarelikelycausedbygeneticvariationofH.rhamnoides
withaltitudes.Theobservedrelationshipbetweenaltitudeandge-
neticdistancesandtheresultoftheclusteranalysisclassifying
populationAintoadiscretegroupshowthataltitudinalvariationis
importantforinter-populationgeneticdivergenceofH.rhamnoides
evenoverasmalgeographicscale.
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