Table Of ContentOdonatologica33(4):399-412 December 1,2004
Macro-andmicroscaledistribution patterns
of twoclosely related Japanese Mnaisspecies
inferredfromnuclear ribosomalDNA,
its sequences andmorphology
(Zygoptera: Calopterygidae)
F.Hayashi¹S,.Dobata²andR.Futahash³i
‘DepartmentofBiology,TokyoMetropolitanUniversity,Minamiosawa 1-1,Hachioji,Tokyo192-0397, Japan
e-mail;[email protected]
“DepartmentofSystemsSciences(Biology),UniversityofTokyo,Komaba3-8-1,Meguro,
Tokyo 153-8902,Japan
“DepartmentofIntegratedBiosciences,GraduateSchool ofFrontierSciences,UniversityofTokyo,
Bioscience Bldg.501,Kashiwanoha5-1-5,Kashiwa,Chiba277-8562,Japan
Received October16,2003/Revised andAcceptedApril28, 2004
Much variation occursin morphologyand colouration amongindividuals ofJapanese
Mnaisspp.Ithasbeennotedthat2groupsofMnaisoftencohabitastreaminwesternJapan.
Thereis,however,noclearmorphologicaldifferencein6 appendagesandpenisbetween
the2groups,andthismakesitdifficultto determinetheirtaxonomic status.Inthisstudy,to
clarifytherelationshipsbetweenthe sympatricspeciesonboth small(alongastream)and
large(acrossJapan)geographicscales, sequences oftheinternaltranscribed spacers 1and
2(ITS1and2)ofnuclearribosoraalDNAarecompared.Basesubstitutionsoccurred at4
sitesof223bpsoftheITS1region,andbytheircombinations,thefour sequence typescould
bedistinguishedamongatotalof800individuals. Inthe ITS2region(total411bps includ-
ing5.8SrRNAregion),allexaminedindividualshadthesamesequence.Thegeographical
distributionofeachITS1sequencetypeandmorphologicaldataofwingsandapterostigma
suggestthatJapaneseMnaisincludes 2distinctspp.,M.strigataSelys,1853andM.costalis
Selys, 1869.TheirdistributionrangesoverlapwidelyinwesternJapan,whereM.strigatais
usuallyfoundatsmallerandupperstreamsthanM.costalis.
INTRODUCTION
In Japan,the damselfliesofthegenusMnais(Odonata: Calopterygidae) are widely
distributed,excluding the Ryukyu Islandsand severalsmallislands (SUGIMURA et
al., 1999).The Japanese Mnaisgroupisalsorecordedfromthe KurileIslands, Russia
(HARITONOV&MALIKOVA, 1998),anuncertainrecordofM. pruinosais known
fromKorea(cf.LEE,2001),butnospecies hasever beenfoundincontinentalRussia.
400 F. Hayashi,S.Dobata&R. Futahashi
Thus,Japanese Mnaisis anisolatedgroupinthe distributionrangeofMnais(Fig. 1).
Mnaisdamselfliesare stream-dwellerswithlow dispersal ability(TAGUCHI& WA-
TANABE, 1992).This genushasbeenintensively studiedfromtheviewpoint ofbe-
haviouralecology. Ithasbeenshown,copulating malesremove rivalspermstoredin
thefemale(SIVA-JOTHY&TSUBAKI, 1989a)andtwo wing-colour formsof males
coexistwithin a population usually corresponding to their mating tactics, territorial
malesandnon-territorial,sneaking males(HIGASHI. 1976, 1981;UBUKATA, 1979;
SUZUKIetal., 1980:NOMAKUCHIetal„ 1984;NOMAKUCH1& HIGASHI,1985;
NOMAKUCHI, 1988, 1992;SIVA-JOTHY & TSUBAKI, 1989b;WATANABE&
TAGUCHI, 1990, 1997;TSUBAKI et al., 1997;HOOPER etal., 1999;PLAISTOW
& TSUBAKI.2000;HIGASHI& NOMAKUCHI.2002;TSUBAKI,2003).It seems
interesting tosearchforanevolutionarypattemof matingbehaviourofMnaisusing a
phylogenetically comparative method.
However,agreatvariabilityinbody size, wing colouration,andothermarking pat-
ternsofindividualsmake itdifficultto distinguish themtaxonomically. A consensus
view has not yet beenobtainedto distinguish inter- or intraspecific populations. At
present, fourtaxonomictreatments coexist; (1) onespecies including threesubspecif-
ic groups(e.g., ASAHINA, 1975b,1976;HAMADA& INOUE, 1985),(2) two spe-
cies, oneofwhichseparating intoseveral subgroups ortwo subspecies (e.g., HIURA,
1972;ISH1DA etal„ 1988;SUG1MURA etal„ 1999),(3) threespecies thatrecognize
thethreegroupsin(1)and(2)as alldifferentspecies (e.g.,SUZUKI, 1980, 1981),and
(4) fourspecies by adding another undescribedspecies to(3)(e.g., SUZUKI, 1984b,
1985).Despite thedifferenceintheirtaxonomictreatments,allagreedwith aphenom-
enon thattwo groupsofMnaisoftencohabita stream inwestern Japan, segregating
theirmicrohabitats,oneprefers theupperreachesof streamsandtheotherlives inthe
lowerreaches (YAMAMOTO. 1955, 1956;H1ROSE& ROKUYAMA, 1966; 1SH-
IDA, 1969;SUZUKI&EGUCHI, 1979),and probably being isolatedreproductively
becausefield-caught mating pairs werealways intra-group members(SUZUKIetal.,
1980).Difficulty intaxonomicconclusionsmayresultnot only from a greatmorpho-
logical variationacrossallJapanesepopulations butalsoduetonoclearmorphological
differenceinmaleterminaliaandpenis (ASAHINA, 1975b).
DNA sequencing techniques andtheircomparison contributetounderstandspatial
andtemporal patternsofspeciation ofanimals, inparticular theclosely relatedspecies
(reviewed by CATERING et al., 2000). The nuclearribosomal internaltranscribed
spacers 1and2 (ITS1 and2) are noncoding, rapidlyevolving DNAregions andhave
proved usefulforcomparingcloselyrelatedinsectspecies, subspecies, or populations
(ALVAREZ& HOY,2002).WEEKERSetal.(2001),SAMRAOU1etal.(2002,2003)
and PILGRIMetal.(2002) sequenced ITSregions forphylogenetic analysis of some
damselfliesanddragonflies. Inthisstudy, weanalyze populations ofJapanese Mnais
damselfliesby sequencing ITS1and2andbycomparing somemorphological characters
ofspecimens taken(1) throughout theJapanese Archipelago (macro-scaledistribution)
and(2) taken alongasingle orneighboringriver systems(micro-scale distribution).
Distribution patternsinJapaneseMnaisbasedonDNAanalyses 401
MATERIALANDMETHODS
Atotalof800Mnaisspecimens, 556adultmales, 140 adultfemalesand 104larvae,wereusedforDNA
analysis.Mostadultswerepreservedinpure ethanol,butaboutaquarterweredried.Larvae wereallpre-
servedinethanol.Specimenswerecollected tocovertheirentiredistribution rangein Japan.Onthe other
hand,toexaminethemicro-scalegeneticstructure, intensivecollectionsweremadeinthefourriversystems;
theYodo-gawaandKumozu-gawaRivers(ShigaandMieprefectures),theWaidani-gawaRiver(Wake-gun,
Okayamaprefecture),the Tsukitani-gawaRiver (Soja-shi,Okayamaprefecture),and the Chikugo-gawa
andYamakuni-gawaRivers(Oitaprefecture)(Fig.1).IntheYodo-gawaandKumozu-gawaRivers, which
flow inoppositedirection fromacommonwatershed,Mnaiswerecollectedatfoursamplingsites(Sites 1-
4;Oishiodawara inthebranch Uji-gawa,andMaruyama,Kozu and Nishiaoyamainthebranch Kizu-gawa)
and attwositesinthe latter (Sites5-6;HigashiaoyamaandIsegi).Theminimum distancebetweenSites 1
and6is55km. Samplingwasmadeat 1kraintervalsintheWaidani-gawaRiver(fivesites) and200-400m
intervalsintheTsukitani-gawaRivers(fivesites).IntheChikugo-gawaandYamakuni-gawaRivers,each
flowingin anoppositedirectionfrom the samemountain,Mnaiswerecollected atUchikawano (Site 1)in
theChikugo-gawa,andatNakama,KanayoshiandNishiyakata(Sites2-4) inthe Yamakuni-gawa.Site 4
locates35kmnortheastfromSite1.
Musculartissuewasisolatedfrom thethorax inadultspecimens andfrombasal segmentsoflegsin the
larvae.Thetissuewashomogenizedinasterile1.5-mlcentrifugetubecontaining500pioflysis buffer(100
mMTris-HCI,5mMEDTA,0.2% SDS,200mMNaCl),50pg ofProteinaseK,and 10pg ofRNase.The
mixturewasincubated 3hat55°C.Followingaseriesofphenol-chloroformandchloroform-isoamylalcohol
Fig. 1.Thedistribution rangesofJapaneseand theothergroups ofMnaisdamseiflies andthefourriver sys-
tems(a-d) wheresmallscalegeneticstructureswereexaminedbasedon ITS1 sequences(seeFig. 4).
402 F.Hayashi,S.Dobata&R.Futahashi
extractions, DNAwasprecipitatedwith anequalvolume ofpure ethanol.Thepellet wasrinsed with70%
ethanol,dried,anddissolved inTEbuffer(10mMTris-HCl, ImMEDTA).
Ribosomalspacers(ITS1andITS2)andtheribosomal5.8SrDNAwereamplifiedbyuseofthepolymer-
asechainreaction(PCR).Theprimers5'-TAGAGGAAGTAAAAGTCG-3’and5'-GCTTAAATTCAGCGG-
3'(WEEKERSetal.,2001)wereusedtoamplifytheentireITS1,5.8S,andITS2regions.Thereverseinter-
nalprimerintheconservedregionsofthe5.8SrDNAwasusedforconfirmation(5-CCGAGTGATCCAC-
CGl 11AGGAT-3').Thereaction mixwasatotalvolumeof30piina500-pltube,composedof19.7piof
distilled water,3.0pioflOxPCRbuffer,2.4pi of25mMdNTPmix, 1.5pi ofeachprimer(10pM),0.3pi
Fig.2.Geographicaldistributions ofITS1sequencetypesofMnaisdamselfliesinJapan.Dottedlinesindicate
prefecturalboundaries. Twogroups,TypeAl/2(upper)andtheothers(lower),arerevisedasM.costalisand
M.strigata,respectively. ArrowsindicatetwohybridFI individuals ofTypesAl/CandTypesC/Dl.
DistributionpatternsinJapaneseMnaisbasedonDNAanalyses 403
ofTaqpolymerase(5U/pl;Takara,Japan),and 1.5pioftemplateDNA.Thereactionmixwaslayeredwitha
dropofmineral oil(Sigma)andthetubeswereplacedinathermalcycler(PTC-100TM;MJResearch,Inc.).
Cyclingconditions were94°Cfor 1min,50°Cfor1min,and72°Cfor1.5minfor30 cycles.PCRproducts,
afterpurifiedbyacentrifugalfilterdevice(MicroconYM-100;MilliporeCorporation),wereusedfordirect
sequencingwiththeBigDyetechnology,theprotocoloftheABIPrismBigDyeterminatorcyclesequencing
readyreactionkit, andthereafteranalyzedonanABIPrism377 DNAsequencer(PEAppliedBiosystems).
Thewidthofheads(betweentheoutermarginsofeyes)andthetotallengthoftheleftforewing(rightoneif
theleftwasbroken)weremeasuredwithaslide calipertothenearest0,05mm.Apterostigmaoftheforewing
wasalsomeasuredforitslengthalongthecostaandwidthatthewidestpart(includingvein'swidth),undera
binocularmicroscopewithanocularmicrometer, tothe nearest0.01mm(IOxmagnification).
RESULTS
DISTRIBUTIONPATTERNS
Thereweresix sequencetypesof the223-bp ITS1region (accession numbersinthe
GenBank database,AB125623-AB125628)amongthe 800individualsstudied.Base
replacements occurredat77th, 116th, 132nd,and 156thsites(5'-3')andrepresented as
AGTA(Type Al, N=270),GGTA(TypeA2, N=53),AGTG(Type B.N=35),AGCA
Fig. 3.Distributions ofITS1sequencetypesofMnaisdamselfliesinKantoDistrict andadjacentareas,cen-
tralJapan(forlocation,seeFig. 1).Dotted linesindicateprefecturalboundaries. Individuals ofTypeAare
revisedasM.costalisandthoseofTypesBandCareM.strigata.
404 F.Hayashi,S.Dobata&R.Futahashi
Fig.4. Genetic structuresofMnaisdamselfliescollected atfourtosixsitesalongasinglestreamorbetween
twoadjacentstreamswithacommonwatershedatthefourriver systems:(a)theYodo-gawaandKumozu-
-gawaRivers; —(b)theWaidani-gawaRiver—(c)theTsukitani-gawaRiver;and —(d)theChikugo-gawa
andYamakuni-gawaRivers. For locationofthestudiedriver systems,seeFig. 1.Each squarerepresents a
singledamselflywithinformationoftheforewinglength(FL),sexes, andITS1sequencetypes.Arrows in-
dicatethedirectionofstreamflows fromapeak(p).Twogroups.TypeA1andtheothers,arerevisedasM.
costalisandM.strigata,respectively.
Distribution patternsin JapaneseMnais basedonDNAanalyses 405
(Type C,N=328), AGCG(Type D1,N=104),andACCG (TypeD2, N=8).Onefemale
andonemaleadultshadhybrid DNAofTypes A1 andC, AG(T/C)A,andofTypes C
andDl, AGC(A/G),respectively. Thebasereplacement ofAtoG atthe77th bp oc-
curredonly inTypeA2,andthereplacement ofGtoCatthe116thbp occurredonly in
Type D2.Therefore,TypeA2was closelyrelatedto TypeA1 (so thatType A group),
andType D2was closeto Type Dl (Type Dgroup).
Thesequenceincluding the5.8SrDNAandITS2region(411bps, accessionnumber,
AB125622)was readforatotalof45individuals,butdidnotdifferamongthesix types
ofITS1; TypesA1 (N=9), A2(N=4), B (N=5), C(N=21), Dl (N=4),andD2(N=2).
Thegeographical distributionofType A1 was widest,whileA2appeared in anar-
row area, Ngano, Niigata, and YamagataPrefectures (Fig. 2upper). Theothertypes
werefoundmoreor less allopatrically inwestern Japan (Fig. 2lower). The distribu-
tionrangeofType Cwas separated by therangeofTypesDl andD2,andTypeB was
foundinasmallrangeincentralJapanandalsoinoneplaceofwestern Japanthatwas
occupied by Type Dl.Figure 3shows more detaileddistributionpatternsofthe ITS1
typesinandaroundKantoDistrict, centralJapan, wherethereis theeast limitofthe
rangeoftheTypes B-D group.The populations ofType C intheMiuraPeninsulaand
thesouthernpart oftheBoso Peninsulawereisolatedfromthe identicaloneby Type
Apopulation.
Along thesingle riverandbetween the neighboring rivers, individualsof Type A
(all subtype Al) andthoseofTypes Cor D(subtypes D1/D2)coexisted(Fig. 4).Type
Aindividualshadusually larger wingsanddominatedinalowerreachthanthe other
ITS1type(s).
MORPHOLOGICALCHARACTERS
Of556adultmaleswhoseITS1typesweredetermined,adatasetofforewinglength/
headwidth and pterostigma length/width was obtainedfor548individuals(8 males
lackedhead/wings).Therelationshipsbetweentheheadwidthandforewing lengthwere
similaramongthe malesofTypes B, C and D,butmalesofType Ahadarelatively
longerforewingthanthosemales(Fig.5).Theregression equationwasY=7.810X-9.144
(N=213, r=0.907,P<0.001)forthe malesof Type AandY=6.334X-2.467(N=335,
r=0.781,P<0.001)forthoseofTypes B, CandD.Theslopesoftheseregression lines
differedsignificantly (ANCOVA;F, P<0.0001).Themeanratioofforewing
length/head widthwas 6.35 (N=213i SD=0.18) inType A,6.00(N=18,SD=0.23)in
Type B, 5.94(N=229,SD=0.21)inTypeC, and5.92(N=88,SD=0.20) inType D.
Therelationships betweenthepterostigmalength andwidthweresimilaramongthe
malesofTypes B, CandD(Fig.6 upper).Flowever, thepterostigma wasmoreelon-
gated inType A malesthanothertypesofmales(Fig.6 lower). Theregression equa-
tionwas Y=0.121X+0.550(N=213, r=0.524,PcO.OOl) forthe malesofType Aand
Y=0.130X+0.634(N=335,n=0.379,P<0.001)forthoseofTypes B,CandD. Theslopes
didnotdifferbetweentheseregression lines(ANCOVA; Fj =0.15,P=0.70),buttheir
544
406 F. Hayashi,S.Dobata&R. Futahashi
Fig.5.TherelationshipsbetweentheheadwidthandforewinglengthamongthreetypesofITS1 sequences
(upper),and between thetwoITS1 groups(lower)ofMnaisdamselflies. Thetwogroups.TypeAandthe
others,arerevisedasM.costalisand M.strigata,respectively.
Distributionpatternsin JapaneseMnaisbasedonDNAanalyses 407
interceptsdifferedsignificantly (Ft =122.8,PcO.OOOl). Themeanlength/width ratio
545
ofthepterostigma was 2.56 (N=213, SD=0.36)inType A, 1.83(N=18,SD=0.26)in
TypeB, 1.76(N=229, SD=0.28)inType C, and 1.82(N=88,SD=0.28)inType D.
DISCUSSION
Sequencing ofITS1 revealedthatTypeA1 iswidelydistributedinJapanwithitssub-
type,TypeA2.Theothertypes(TypesB, C,andD1 withsubtype D2) occupywestern
Japan,replacingeachother.Inwestern Japan wherethepopulations ofTypeAandthe
othertypes usuallycoexistalongasingle stream,theformerwasfoundmorefrequently
inlowerreachesandthelatterinupperreaches.Severalresearchers pointedoutthatthe
thoraxsizeandthepterostigmashape ofmalesarekey characterstodistinguish these
two groupsinwestern Japan (e.g.,YAMAMOTO, 1956;ASAHINA, 1975b;ISHIDA
etal., 1988).Thoracicsize maybecorrelatedwithwingsizebecauseflightmusclesare
containedwithinthethorax.Inourmeasurements,therelativelength offorewingsand
theshapeofpterostigma differedbetweenmalesofTypeAandTypesB, CandD.These
results suggestthat Japanese Mnaisare dividedintotwogroups.Type Aandtheother
types.Only asingle femaleof800individualsexaminedwas confirmeda hybrid FI
betweenthetwo(Type A/C) despiteexaminationofseveralapparenthybrids basedon
morphology.Thisfact suggests thatthetwo species arewellisolatedreproductively.
Ourconclusiondiffersfrompreviousspecific/subspecific groupings.Thegeographical
boundariesobservedamongeachITS1typedonotcoincidewithanyboundariesofpre-
viously proposed groups. Amongthepreviously knownthreegroupscorresponding to
subspecific/specific names,pruinosa(including Mnaissp. insenseofSUZUKI,1984b,
1985),costalisandnawai,YAMAMOTO(1956)andASAHINA(1975b) pointedout
theirmorphological similaritiesbetween costalisandnawai,butHIURA (1972),SU-
ZUKI (1984a), ISHIDA et.al.(1988), SUGIMURA et. al.(1999)andotherresearch-
ersregarded costalisandpruinosaas morerelatedtaxathannawai.Inthisrespect, our
conclusionisthatcostalisandnawaiarenotheterogenous butasingle group,andpru-
inosadiffersfromit.Thetwo populations ofType AandTypes B-D arerenamedM.
costalisandM.strigata, respectively, withoutsubspecific division.
MNAISCOSTALISSELYS, 1869
MnaiscostalisSELYS, 1869:651.Lectotype(6)designatedby ASAHINA(1975a).
Mnaispruinosaf.costalis;SELYS, 1883: 128-129.
Mnaispruinosacostalis;ASAHINA, 1975b:31-40.
MnaisstrigatanawaiYAMAMOTO, 1956: 19-22with2pis.Holotype(6)byoriginal
designation.Newsynonym.
Mnaispruinosanawai; ASAHINA, 1975b: 34-36.
BothtypespecimensofM.costalisandM.strigatanawaihaveamuchnarrowerpter-
ostigma in forewings (YAMAMOTO, 1956;ASAHINA, 1975a); therefore,Type A
408 F,Hayashi,S.Dobata&R.Futahashi
Fig.6.Therelationshipsbetween thepterostigmalengthandwidthamong threetypes ofITS1 sequences
(upper),andbetween thetwoITSI groups (lower)ofMnaisdamselflies. Thetwo groups,TypeAandthe
others,arerevisedasM.costalisandM.strigata,respectively.
