Population genetic differentiation in three sympatric damselfly species in a highly fragmented urban landscape (Zygoptera: Coenagrionidae)

2008·1.2 MB·English

by  Sato, M

Odonatologica37(2):131-144 June I,2008
Populationgenetic differentiationin three
sympatric damselfly species ina highly
fragmentedurbanlandscape
(Zygoptera: Coenagrionidae)
M.Sato¹*Y.Kohmatsu²,M.Yuma³ and Y.Tsubaki¹
1CenterforEcologicalResearch,Kyoto University,509-3,Hirano 2,Otsu,Shiga,520-2113,Japan
2Research InstituteforHumanityand Nature,457-4,Motoyama,Kamigao,Kitaku,
Kyoto,603-8047,Japan
3DepartmentofEnvironmental Solution Technology,FacultyofScienceandTechnology,
Ryukoku University,Seta-Oe,Otsu,Shiga,520-2194,Japan
ReceivedJuly24,2007/RevisedandAcceptedNovember 12,2007
TheAFLP (AmplifiedFragment LengthPolymorphism) techniquewasused to
comparethelevelsofgeneticdiversityanddifferentiation amongParacercion calam-
orum, Ischnura senegalensisand I. asiaticaand tocompare thegeneticstructureof
populationsfound in highlyfragmentedurban habitatstopopulationsin relatively
continuousruralhabitats. Forall3spp., highgeneticdiversitywasfoundinbothareas.
However,populationgeneticdifferentiation amongurban populationswasapproxi-
matelytwice that ofrural populations,indicatingthat movementsbetween habitat
patchesaremorerestrictedin urbanareas,probablyduetohuman disturbancesthat
mayfunctionasbarriers. Inter-specificdifferencesregardinggeneticdiversityanddif-
ferentiation arefurtherdiscussed intermsofhabitat specificity.
INTRODUCTION
Habitatdestructionandfragmentation due to humanactivity are a common
phenomenon occurring throughout theworldandisarguably thegreatestthreat
to biodiversity (PRIMACK, 1998;WILCOVE et al., 1998). Thusroads, build-
ings and towns often provide barriers that interferewith anorganisms’ move-
ment betweentheremaining habitatpatches, causing population fragmentation
(PRIMACK, 1998).
Fragmented populations directlyimpactgeneflowwithinaspecies, leading toa
*Correspondingauthor:MayumiSato,[email protected]
132 M.Sato,Y.Kohmatsu,M.Yuma& Y.Tsubaki
lossofgenetic diversitywithineachpopulation andanincreasein genetic differ-
entiationbetween populations (FRANKHAM, 2006). Thisgenetic force, along
withdemographicandenvironmentalforces, coulddrivefragmented populations
toextinction(GILPIN &SOULE,1986;CROOKS&SANJAYAN,2006).How-
ever, despite a growing concern over theinfluenceof habitatfragmentation on
genetic structure, fewpopulation genetic studieshave been conducted in urban
landscapes, wherehabitats are expected to bemorefragmented andindividual
movements between fragmented patches may bemore restricted than thosein
rural landscapes.
Ideally, population genetic structure presumably resulting fromhabitatfrag-
mentationshouldbecompared to thatofacontrol site(i.e., unfragmentedhabi-
tat)(VAN DONGEN etah, 1998) withthesamespatial scale attheintra-specific
level.However,theuseofsuch acontrolsiteisoftenignored (WILLIAMSetah,
2003; SOFIAetah,2006). Furthermore,bothsitesshouldbelocatedinrelatively
closeproximityinordertoeliminateany possible geographiceffectson dispersal
such as climaticconditionsandaltitude.Moreover, inorderto detectlowgenetic
differentiationsresulting fromasmallspatialscale,molecularmarkers withhigh-
resolutionpowershould beemployed (OUBORGet ah, 1999). Therefore,find-
ing aspecies inhabiting bothfragmentedandunfragmented habitatsoverasmall
geographical rangeandusingamolecularmarkerwithhigh-resolutionpowerare
important issues forexamining theeffectsof habitatfragmentation onpopula-
tiongenetic structure.
AmplifiedFragmentLength Polymorphism (AFLP) (VOSetah, 1995) isamo-
lecular markerwhichhasbeenusedinthe fieldof molecularecology andevolu-
tion since 1995. Although themethodhasbeenwidely employed inthestudy of
plants, fungi, and bacteria, theadvantages of the method(short start-up time,
nopriorsequenceknowledge andgood repeatability) shouldmakeita valuable
tool to investigate thepopulation genetics ofwild animalspecies (BENSCH &
AkESSON, 2005). Amajorconcernaboutthetechnique isthatmarkersaredom-
inant, and it is impossible to distinguish homozygous (1/1) from heterozygous
(1/0) genotypes(BENSCH& AKESSON,2005). Thisrequires previous know-
ledgeoftheinbreeding coefficient(HOLSINGERetah,2002)and,therefore,the
Hardy-Weinberg equilibrium is sometimesassumed to estimate allele frequen-
cies. However, thelarge numberoflocicounterbalancesthis drawbackof domi-
nant markers (PARSONS& SHAW,2001)as ifcanreveal subtlebutsignificant
population structures (BENSCH& AKESSON,2005).
We usedthethreeZygoptera species, Paracercioncalamorum, Ischnurasenega-
lensis, and I. asiatica to compare levelsof genetic diversityand differentiation
amongpopulations foundinhighlyurbanizedfragmented habitatrelativetopop-
ulationsin continuousrural habitatandthereby determineany effectofhabitat
fragmentation ontheirgenetic structure.Adultodonatesareknowntobesensi-
tiveto thedestructionand modificationoftheiraquatic habitats(e.g. CARLE,
Populationgeneticdifferentiationin sympatricZygoplera 133
1979;BROWN. 1991;CLARK & SAMWAYS, 1996;SUHLING etal„ 2006;
SATO & RIDDIFORD, 2008), and this, along withtheirease ofcapture and
wide-spread distributionmakethesethreespecies idealforsuch astudy.Allthree
damselfly species occursympatrically intheKantoregion in Japananduselentic
habitats, mainly occurring in agricultural pondsoftenconnectedby ditchesand
rice paddies in rural areas,andin distantly spaced city park ponds in urbanar-
eas. However, habitatspecificity seemsto differamong them.P. calamorumhas
anarrowerselectionofhabitatwithrespect totherelativestructureandarrange-
mentofvegetation atthewatersidecompared to theothertwo species, andofthe
two congeneric species, I. senegalensis selects amore confinedrange ofhabitat
typescompared to I. asiatica. Thesampling designallowed usto conduct both
intra-specificcomparison betweentwocontrasting areas andinter-specific com-
parisonamongthethreespecies since differencesinhabitatspecificitycouldhave
profound influencesonthegenetic structure.
MATERIALAND METHODS
STUDYSITESANDSAMPLECOLLECTION — Wecollected adultP. calamorum,I. senega-
lensis,and I. asiaticafromatotal of23different sites (11,10,and 13sitesforeach,respectively) in
twocontrastingareasontheKantoPlain in Japan(Fig. I).Sincethe mainaim ofourstudy wasto
examine theeffectsofhabitat fragmentationondamselflydispersal,wechose the most intensively
urbanized areain Japan. 11 ofthe study sites werepondsincity parks in theTokyometropolitan
area,andexceptforsite U11,theywerewithin the23wardsofTokyo,occupying muchofthecenter
oftheTokyometropolitanarea.This areaisvery built-upwith business,commercial,and high-rise
apartment buildings,which arepotentialphysical barriers for dispersaland hence resultin habitat
fragmentation.The other 12siteswereselected inthe surroundingcountrysideasacontrol against
the urban data; 10 ofthese werelocated in Ibarakiprefecture, the other twoontheborder ofIba-
rakiandChibaprefectures.These studysiteswereeitherponds(natural,agriculturalorpark ponds)
orrice paddiesand werewithin an areaapproximately 50to60km NE ofTokyo andmainlysur-
rounded byrelativelycontinuousagriculturalland. Althoughthereissomeurbanization inthisarea,
much oftheareahas still been used forrice paddies,lotus-cultivation,andarable lands due tothe
closeproximityofLake Kasumigaura,which isthesecond largestlakein Japan.Irrigatedricepad-
dies,withirrigation ditchesand shallow holdingponds,sustain manyspecies ofaquaticorganisms
andareconsidered tobe artificialshallow wetlandsinthisrespect(NAKAMURA&SHORT,2001).
Landcovertypeswithinapproximately 1500km2ofeachrural andurbanareacoveringall studysites
aresummarized inTableI.Thespatialscales ofthetwostudyareasarecomparable.Theminimum
and maximumlinear geographicaldistances betweenstudysiteswere4.5kmand 25.7kmintheur-
ban areacomparedto4.6km and 27.4kmin therural area,respectively,for P.calamorum,8.6km
and 27.0kminthe urbanareacomparedto7.0kmand25.1 km intherural area,respectively,forI.
senegalensisand5.3kmand 38.9km in theurbanareacomparedto4.5km and38.2kmintherural
area, respectively,forI.asiatica. Samplingwasconducted in JulyandAugust2004. Adult damsel-
flies werecollected with netsand immediatelystored in99%ethanol inthefield. Anaverage of 15
individuals foreachsiteforeachspecieswerecaughtand usedfortheanalyses.
DNA EXTRACTIONAND AFLPPROCEDURE — The head and thorax weregroundin
liquidnitrogenasa basematerial fromwhich toextract totalDNA followingtheslightly modified
cetyltrimethylammoniumbromide (CTAB)method suggestedspecificallyforAFLPanalysisforin-
sect material(REINEKEetal., 1998).DNA wasextracted withchloroforrmisoamylalcohol(24:1),
134 M.Sato,Y.Kohmatsu,M.Yuma&Y.Tsubaki
and theDNApelletswereethanol-precipitatedandthensuspendedinTEbuffer. DNAextractswere
furtherpurifiedusingaQIAquick PCR Purification Kit (QIAGEN K.K.,Tokyo)accordingtothe
manufacturer’s protocols.
AFLPreactions wereconducted usingtheAFLPPlant MappingKit(AppliedBiosystems,Foster
City, CA)foraregulargenomefollowingtheprocedures outlined inthemanufacturer’sinstructions
with minormodifications. Tento 150ngofinitial DNAper individual weredigestedwith restriction
enzymes (Mseland EcoRl;New England Biolabs,Beverly,MA)before ligationofrestriction site-
Fig. 1.Location ofthe samplingsitesin urban (Tokyo: Ul-UII) and rural (Ibaraki and Chiba:
R1-R12)areas.
Populationgeneticdifferentiation insympatric Zygoptera 135
specificadaptors.Thepre-selective and selectiveamplifications werecarried outin aPerkin Elmer
GeneAmpPCRSystem9600thermalcycler(AppliedBiosystems).Modifications included reduction
ofthetotalvolume ofthesolution toahalfofthevolume suggestedin themanufacturer’sinstruc-
tions,which was5.5pLforrestriction and adaptor-ligationand lOpLforpre-selective and selective
amplifications.Inordertoremovetheinhibitorysubstancesforamplifications, 1.6pL ofArapdirect
(ShimadzuCorporation,Kyoto, Japan)was added tothepre-selectiveamplificationreaction mix-
ture.Twoprimercombinations (EcoRI-ACA/Msel-CTG,EcoRI-ACT/Msel-CAC)thatamplified
areasonable number ofclearbandswereused.
The fluorescence-labeled selectiveamplifiedproductswereresolved with aGene-Scan 500 ROX
internal sizestandard(AppliedBiosystems),usinganABI3100automatedsequencer (AppliedBio-
systems). Theelectrophoresisimageswereobtainedfrom each run,and peakswereassignedbase-
pair sizes usingtheGENESCAN programversion 3.1 (AppliedBiosystems). OnlyDNA fragments
ranginginsizefrom 50to500base pairswerediscerned andmanuallyscoredas 1(presence)or0(ab-
sence)toassemble abinarydata matrix consistingofatotalof189,215,and203 bands foreach of
theP.calamorum,I.senegalensis,and I.asiaticaindividuals sampled,respectively.TenDNAsamples
from each specieswereamplifiedatleast twicetoverifythereproducibility ofthe AFLPtechnique,
and onlyintense and unambiguousbands werescored.
DATAANALYSIS —We assumed theHardy-Weinbergequilibriumtoestimategeneticdiversity
and differentiation asnopriorinformation onthematingsystem ofthethree damselflieswasavail-
able.Allele frequencieswereestimatedaccordingtoaBayesianmethod withnon-uniform priordis-
tribution ofallelefrequencies(ZHIVOTOVSKY, 1999),usingthecomputerprogram AFLP-SURV
1.0(VEKEMANS,2002).Basedontheestimatesofallele frequencies,wedetermined geneticdiver-
sityasNei’s unbiased expectedheterozygosity(NEI, 1973).We estimatedthegeneticdifferentiation
amongpopulationswithin eachareausingtwo different methods. Wefirstcalculated Wright’sFsp
thefixationindex,which isaparameterindicatinghowpopulationsaregeneticallydifferentiated ac-
cordingtotheLynch andMilliganmethod(LYNCH&MILLIGAN,1994),usingAFLP-SURV 1.0
(VEKEMANS,2002).Themethodwasspecificallydevelopedforcalculatingdifferentiation andsuit-
ableforusewithdominantdatatypes.ThesignificanceofFSTwastestedbycomparingtheobserved
F with thedistribution ofthesetofF generatedby 1000randompermutationsfromindividuals.
sr ST
We alsoused the method thatdoesnotassumethe Hardy-Weinbergequilibrium,but instead,uses
the Euclidean distance toestimatethe componentsof variance attributable to differencesamong
populationsand among individuals within populations(EXCOFFIER etal., 1992).TheAnalysis
ofMOlecular VAriance(AMOVA)(EXCOFFIER etal., 1992)wasperformedtocalculate 4> (F
ST ST
Table I
Percentageofmajorlandcover typeswithin about 1500knr foreachurban (Tokyo)and rural (Iba-
raki andChiba)area.Vegetationand landusedata derivedfrom 15 gridcells,each ofwhich isap-
proximately10x10km oftheNational SurveyontheNatural Environment(BiodiversityCenterof
Japan,1999),werere-categorizedintofourlandcover types.Urbanlandscapesincluderesidential ar-
eas,factories,and buildings.AG-testshowed thatthecompositionoflandcover typesinthe urban
areaissignificantlydifferentfromthat oftherural area(G=921.5,/?<0.001)
Urban area Ruralarea
Landcovertype Area(km2) %oftotal Area(km2) %oftotal
Paddyfield/Wetlandvegetation 50.5 3.4 433.2 32.1
Arableland/Meadow 217.1 14.6 413.9 30.6
Urban landscapes 1002.0 67.3 233.8 17.3
Others 218.9 14.7 267.4 19.8
Total 1488.6 100 1348.4 100
136 M.Sato,Y.Kohmatsu,M.Yuma &Y.Tsubaki
TableII
The number ofrural and urban populationssampled(n), estimatesofgeneticdiversity (Flw), and
geneticdifferentiation(Fsr<1>ST) in P.calamorumI. senegalensis,and I.asiaticaamongpopulations
ineachurban and rural area.—indicate significantdifferencesfrom0atthep < 0.05levelbased on
1000 and 1023randompermutationsforF and rl> respectively
ST s[,
UUrrbbaann aarreeaa RRuurraall aarreeaa
nn HHww FFs„t <<1I>>sStI nn HHww FfSsTt <<1t>>SS|T
PP..ccaallaammoorruumm 66 00..227744 00..007799** 00..114455** 55 00..330000 00..003366** 00..007766**
II..sseenneeggaalleennssiiss 55 00..332244 00,,003311** 00..006655** 55 00..330088 00..001188** 00..003366**
II..aassiiaattiiccaa 77 00..330044 00..000066** 00..003311** 66 00..331133 00..000033** 00..001155
analogue),usingtheprogram Arlequin3.1 (EXCOFFIERetal., 2005).This <t>STissimilar toWeir
andCockerham’s 6(WEIR&COCKERHAM, 1984),which iscalculated within ananalysisofvari-
anceframeworkandsupposedtobeabetterestimatewhensamplesizesarefewer than30individuals
(LOWEetal.,2004).Arlequinalsocalculated pairwised> valuesforallpairsofpopulationswithin
ST
eacharea.The significanceof<1>S1 andpairwise<I>STweretested usingnon-parametricpermutation
ofthedata setwith 1023permutations.
Forallpairsofpopulationsineach area,geneticdistanceswerecalculated (NEI,1972)usingAFLP-
SURV 1.0(VEKEMANS,2002),andgeographicaldistanceswereobtainedassimplelinear distances,
usingKashmir 3D (SUGIMOTO,2002).Wethen testedwhetherthere wasany correlation between
thegeneticand geographicaldistancefor each species.Since theindependenceofpairwiseelements
ofdistancematricesviolatesthebasicassumptionsforusingasimplecorrelation coefficient,weused
the Manteltest (e.g.MANTEL, 1967;MANLY,1986;SOKAL & ROHLF, 1995).
RESULTS
Nostatisticaldifferenceswerefoundinthegeneticdiversity,asmeasuredby the
meanexpected heterozygosity, betweenurbanand ruralareas forP.calamorum,
I. senegalensis and I. asiatica(Unpaired t-test; p = 0.130,/? =0.120,/? = 0.201,
respectively) (Tab. II).However,incomparisons betweenspecies, asignificant dif-
ference was foundin the totalmean expected heterozygosity (combined urban
and ruralpopulations) (ANOVA; F = 6.781,/? =0.003). Apost hoc test revealed
that P. calamorumhad significantly less genetic diversity thanthatof I.senega-
lensisandI. asiatica(LSD test;p=0.001 andp =0.009, respectively), butno dif-
ferencewas foundbetween I. senegalensis andI. asiatica(LSD test;p=0.357).
Significant population differentiationswithan F and <t> were foundforall
SJ s[
three species except a3> valueofI. asiaticaamongruralpopulations thatwas
ST
not significantly differentfromzero(Tab. II). Therewas a consistent trend for
all species withFsxand<I>STvaluesabouttwiceas high in urbancompared toru-
ralpopulations (Tab. II), demonstrating thatpopulations intheurbanareawere
more differentiatedthan intheruralones.Thepairwise4> valuesrevealedthat
ST
significant geneticdifferentiationwas morecommonamongurbancompared to
ruralpopulations and thepattern was consistentfor allthreespecies (Tab. III).
For P. calamorum, of the 15 pairwise comparisons withinthe urban area, all
Populationgeneticdifferentiation insympatricZygoptera 137
pairwise 4> values(100 %)were significant,compared to seven(70 %) outof 10
ST
pairwise comparisons withintherural area. For I. senegalensis, 10 (100 %) out
of 10 pairwise <h valueswithinthe urbanareawere significant, andseven (70
ST
%) outof 10withinthe ruralarea. For I. asiatica, ofthe21 pairwise <hSTvalues
TableIII
Pairwise estimates of<1> in(a)P. calamorum,(b)I. senegalensis,and (c) I. asiatica forurban and
S1
rural populations.- indicate significantdifferences from0atthep<0.05 level
((aa))PP..ccaallaammoorruumm
UU11 UU22 UU33 UU44 UU55 RRll RR77 RR88 RR1IO0
UU22 00..006600**
UU33 00..118899** 00..220000**
UU44 00..008888** 00..009966** 00..225500**
UU55 00..009966** 00..006699** 00..119922** 00..009966**
UU77 00..110011** 00..117755** 00..332200** 00..114477** 00..117711**
RR77 00,,009944**
RR88 00..008888** 00..007722**
RR1IO0 00..006600** 00..008800** 00,,006655**
RR1122 00..111177** 00..005533 00..007700 00..005500
((bb))II..sseenneeggaalleennssiiss
UU11 UU33 UU77 UU99 RR33 RR44 RR55 RR66
UU33 00..113377**
UU77 00..004433** 00..111133**
UU99 00..005522** 00..008822** 00..002266**
UU1100 00..005511** 00..110044** 00..003322** 00..002211**
RR44 00..007788**
RR55 00..004411** 00..004411**
RR66 00..004444** 00..004433** 00..001111
RR88 00..002288** 00..004455** 00..001177 00..001100
((cc))II..aassiiaatliiccaa
UU22 UU66 UU77 UU88 UU99 UU1lO0 RR22 RR33 RR44 RR88 RR99
UU66 00..001133
UU77 00..008833** 00..005533**
UU88 00.,002299 00..110077** 00..004400
UU99 00..000000 00..000066 00..002277 00..004444
UU1lO0 00..000000 00..001133 00..008833** 00..008855 00..000000
UUllll 00..001100 00,,002222 00..005500** 00..006600** 00..000077 00..000000
RR33 00..000000
RR44 00..000000 00..000000
RR88 00..001177 00..000000 00..005522**
RR99 00..000066 00..000000 00..002266 00..002255
RRlllI 00..002244 00,.001144 00..005599** 00,.008855** 00..003399
138 M.Sato, Y.Kohmatsu, M.Yuma &Y. Tsubaki
withinthe urban area, six (29 %) were significant, andof 15 pairwise <I> val-
ST
ues withinthe ruralarea, three(20 %)were significant. There was also a trend
that,in bothareas, P. calamorumshowedthehighest F and 4> valuesamong
ST S|
thethree species, andthe F and(1> valuesofI. senegalensis were higher than
ST ST
thoseofI. asiatica(Tab. II). TheManteltestshowedno significant relationship
betweengenetic distanceand lineargeographical distance ineach areaforeach
ofthethreespecies (p >0.1).
DISCUSSION
Inour study, wefoundrelatively high genetic diversity, butthepatternsof in-
creasedgeneticstructuring (as estimatedby F /fU ),amongpopulations from
ST ST
urbanhabitatscompared to thepopulations fromruralhabitats,were consistent
withthe predicted effects of habitatfragmentation on genetic structure forall
threespecies (Tab. II).
DITCHINGS & BEEBEE (1997), CAIZERGUES et al. (2003), and WIL-
LIAMS(2003) all founddecreasedgenetic diversityandincreasedgeneticdiffer-
entiationin either naturally or anthropogenically fragmented landscapes when
compared to thoseincontrol sites. Interestingly, however, no decreasedgenetic
diversity was detectedin our study andthoseby GIBBS (1998), KNUTSEN et
al. (2000) and ARNOUD et al. (2003) although increased genetic differentia-
tion was found. We propose the following two non-exclusive explanations for
thehigh genetic diversity foundin thepresentstudy. Habitatfragmentationmay
result inareducedgenetic diversitythrough randomgenetic drift(BAHL etal.,
1996;ARNAUD et al., 2003). As randomgenetic driftis knownto depend on
effectivepopulation size(BEEBEE &ROWE, 2004;LOWEetal., 2004),thatof
eachpopulation intheurbanareaislargeenough to maintainhighgeneticdiver-
sity. Analternativeexplanation is thatthereis sufficientgeneflowamong urban
populations to maintainhighgenetic diversity.
We didnotfindanycorrelationbetweenthegeneticdistanceandthelineargeo-
graphical distanceeitherintheurbanorin theruralareas. Wheneversuch acor-
relationis found, thefactor limiting dispersal amongpopulations is considered
tobegeographical distance, andthis isthe basic premiseof thetheory of ‘isola-
tion-by-distance’ (WRIGHT, 1946). Theabsence of such an associationin the
presentstudymaysimply reflectinsufficienttimeforanequilibrium betweengene
flowanddrifttobereachedandhencetheexpected geneticpatternsofisolation-
-by-distance to build up(HUTCHISON & TEMPLETON, 1999;KNUTSEN
et al.,2000). However,pairwise <h valuesbetween siteU3 andothersites were
s|
consistently higher forboth P. calamorum(range0.189-0.320) andI. senegalensis
(range0.104-0.137) compared tothepairwise<1> valuesbetweensitesisolatedby
ST
equalor greaterdistances.Considering thatsiteU3islocatedinthecentreofthe
Tokyo metropolitanarea, wesuspect thathumandisturbancessuch as buildings
Populationgeneticdifferentiation insympatricZygoptera 139
androadscouldbelimiting geneflowandhavethus createdtheobserved genetic
differentiationwhichcannot beexplained by geographical distance.
Although all three species showed the samepattern,with urbanpopulations
being moregenetically differentiatedthanruralpopulations, the F /<I> values
SJ S|
of P. calamorumwere twiceas high as thoseofI. senegalensis, whichwere also
morethan twiceas high as those ofI. asiatica. Inaddition,thegeneticdiversity
of P. calamorumwas significantly lowerthanthatofI. senegalensis and/. asiat-
ica. To ourknowledge, thisis one ofonly afewstudies thathavedemonstrated
howtheeffects ofhabitatfragmentation onpopulation genetics differedamong
closely-related, sympatricspecies. Habitatspecificity couldbeoneexplanation for
the observed differencesingenetic diversity anddifferentiationamongthe three
species.
P. calamorumhasspecifichabitatrequirements, generallyinhabiting pondswith
submerged orfloating vegetation butwith relatively littleemergent aquatic veg-
etation(UEDA, 1985). They alsorequire trees incloseproximityto thewater as
maleroosting sites(UEDA, 1976).On theotherhand, bothI. senegalensis and
I. asiaticauseawiderangeofwater typesincluding ponds,smallchannels, sew-
agedrainsestuarieswithemergentaquatic vegetation andrice paddies (INOUE
& TANI, 1999;WATANABE& MATSU'URA,2006). Dueto the higher habi-
tatspecificityof P calamorumcompared tothatofI.senegalensis and I.asiatica,
P. calamorumutilizedasmallerrangeof habitats,whichmayresultin restricted
gene flow betweenavailable patches, especially in the highly fragmented urban
landscape.
Whencomparing thetwo Ischnuraspecies, thewiderrangeofhabitatrequire-
ments forI. asiatica mightalso explain the observed differencesin F /<I> In
ST sT
additionto thehabitattypesusedby both species described above, /. asiaticais
also seenaroundponds withgrassy typesofvegetation (e.g. Poasceae, Cyperace-
ae) thatgrowinwet areaseventhough thepond itselfdoesnot haveany aquatic
vegetation (pers. obs.). Theseeurytopiccharacteristics ofI. asiaticamayenable
themtofindavailablepatches ofhabitatandcolonizesuccessfully inotherpatch-
es, eveninfragmented areas.
Although the three damselfly species investigated in this study are neither
threatenednor endangered, the factthatthe population genetic differentiation
differedamongclosely-related,sympatricspecies hasimportantmanagementim-
plications. Habitatspecificity is awell-knownecologicalattributeofspecies that
increases vulnerability toextinction(WALDRON et al.,2000). Thus, if habitat
fragmentation hasamoresevere impacton habitatspecialists, as this study indi-
cates,thismaybeone ofthe mechanismsexplaining why habitatspecialists are
more vulnerableto extinction.
It is worth noting that I. senegalensis has a larger body size than I. asiatica
(adult body length (mm): I. senegalens■=23-25; I. asiatica = 20-25, SUGIMU-
RA etal.,2001). Contraryto our intuitionthatlarger species arebetterdispers-
140 M.Sato,Y.Kohmatsu,M.Yuma &Y.Tsubaki
ers, the smallerbody size of I. asiatica showed a lowergenetic differentiation.
CORBET(1999)consideredthesetwo Ischnuraspeciesas migrantsbasedonthe
reports thatadultsofI. asiaticawere caught more thanoccasionally over water
far fromland(ASAHINA & TURUOKA, 1970),and larvaeof I.senegalensis
inhabitedtemporary pools in seasonal rainfallareas (WEIR, 1968; WATSON,
1980as citedby CORBET, 1999;DUMONT, 1981as citedby CORBET 1999).
If being small is aprerequisite for migrant Zygoptera to movea long distance
by exposing themselves toaircurrents as CORBET (1999) inferred, I. asiatica,
which hasa smaller body than I. senegalensis, should havean advantage indis-
persing.
An F valueof 1 indicatesperfect genetic differentiationand 0 indicatesno
sx
differentiation, withmoderate differentiationat values between 0.05 and 0.15
(WRIGHT, 1978). Themagnitude ofthe F / d> valuesfoundforboth urban
ST ST
and rural populations suggests arelatively high dispersal capability ofallthree
damselfly species. Thisfinding is not inaccordance withthegeneralization that
damselfliesare weak fliersthatcan disperse only betweenwater bodiesless than
I kmapart(CONRAD etal.,2002). Since moderategenetic differentiationwas
detectedfor P. calamorumin theurbanarea, theresolutionpowerofthegenetic
markerusedin thisstudy was consideredtobe high enough to detectdifference
in dispersal ability. Oneshould bearinmindthat dispersal ability measuredby
direct observations (i.e. Mark-Release-Recapture) and indirect measurements
(i.e.F /4> values) havesubstantially differentmeanings. Theformerisasnap-
st st
shot ofa one-timedispersal event. Not all the marked individualsare located
andlong-distance andrare movements tendto beparticularly difficultto detect
because of the spatio-temporal constraints of the method(e.g. RIECKEN &
RATHS, 1996;NATHAN,2001;OSBORNE et al.. 2002). On theotherhand,
dispersalmeasuredas F /<1> valuesis acumulativeeffect ofgeneflow.Individ-
ST S|
uals mightsuccessfully disperse to new habitatpatches, eitherby flying directly
or in astepping-stone way, or evenby being passively dispersed by aircurrents.
Thiscolonizationeventmighthappenwithinageneration insomecasesandover
several generations inothercases,andmayresult ingeneflow.Therefore, whilst
the resultsof this study suggest thaturbanpopulations are experiencing theef-
fectsofhabitatfragmentation, relatively highdispersal events areexpected tobe
occurring among the populations. Caution should betaken as this cumulative
effectofgeneflowexpressed as F /ffi Imaynotbe reflecting thecurrent status
ST S
of dispersal where habitatdestructionandmodificationare stilllikely tobeoc-
curring.
Inconclusion, humandisturbancesinurbanareasare likely torestrictthedis-
persal ofindividuals, andtheextentofhabitatfragmentationeffectsonthepopu-
lationgeneticdifferentiationdifferedamongthethreedamselflyspecies probably
becausetheyhavedifferenthabitatspecificity. However,thisstudyis onlythefirst
step towardsdetermininghowweshouldconserve Odonataspecies andmaintain