Comparative morphological study in the genus Tirumala (Lepidoptera, Nymphalidae, Danainae) : sexual isolation and PTPs

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TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
ue8ma Trans.iepid,Soc.Japan 59 (4)3:05-311,September 2008

Comparative morphological study in the genus Zrumain (Lepidoptera,
Nymphalidae, Danainae): sexual isolatio nand PTPs
Kei HAsH[MoTo and Osamu YATA
Biosystematic sLaboratory, Facult yof Socia alnd Cultura lStudies ,Kyushu University,
Repponmatsu,Fukuoka, 81e-8560japaii

Abstrac tThe genus Tirumala (Nympha]id aLeep,idoptera) is a group belongin gto Danainae and
consists of 9 species, 2 from Afric aand 7 from the Indo-Australia nregion (Acker yand Vane-
Wright. 1984). This is one of the most difiicu lg{enera because it srnember species resemble each
other not only in general appearance but also in morphology of the male gcnitali a.A comparison
was made of the microstructure of pheromone transfer particl e(sPTPs )in the alur poueh for all
spccies in the genus. Here, we report that some definit deiffbrence swere detect eidn the microstruc-
ture of PTPs between species. The cvaluation of PTPs suggests that. in an area where several
species fi ytogcther, (he morphology of PTPs di[TZ) rfsrom species to species.
Key words 7:ir"mala ,Danainae, PI]Ps, microstructure, SEM, courtship behavio rs,exual isolation,
taxonomie charaeter.
Introduction
The genus nr"inala belongs to the Danainae (Nymphalid Laeepi,doptera) and comprises 9
species, 2 (T .formos aT., petiverana f)iro mAfric aand 7 (T ,gautama, T. choaspes, T. Iimni-
ace, T. septentrionis, T, ishmoides T,. hamata, T. etoptoeomorpha) from the Indo-Australian
Region (Acke ranyd Vtane-Wright, 1984). As in many other Danainae butterfii tehse, larvae
feed on Asclepiadacea aend Apocynaceae which contain noxious ingredients.
7irttmata is one of the most diMcult genera due to the resemblance of member species not
only in genera lappearance but also in morpholegy of the male genitali aT,here are only a
few studies which examined in detai lthe morphology of the male and femal egenital iofa
7Vrumata (Shir6 z1u9,60; Kawazo6 and Wakabayashi, 1977, etc.). As fOr grouping of
species, Ackery and Vane-Wright (1984 )only dichotomized the genus, with Afrotropic aTl.
,forntosa treated as the sister-group of the other eight species, called `the limniac especies-
complex',
A unique morphological character of 7"irunta lias the presence of a sex patch ,called the alar
pouch, on the hindwing cell Cu,, of males (Fig .2A-B), Insid ethe alar pouch, there are
scale sockets (Fi g2.A, 1) and disk-shaped sockets (Fi g2.A, 2) on the surface and, from
these disk-shaped sockets, characteristic long and narrow scales (call ecdushion scales)
grow. A larg enumber of pheromone transfe rparticl e(sPTPs a)re produced by fragmenta-
tion of the cushion scales after a certain period of time (Beppr 6and Vane-Wright ,1989) ,It
is be]ieve dthat PTPs migrate to the tip of the abdominal hairpenci lansd enable female sto
recognize and accept their partne rby scattering pheromones or by directl ysticking to their
antennae during courtship behavior. If the chemical constituents of the pheromone and
thei rratio diffe frrom species to species, i tis possibl ethat the morphology of PTPs is also
species-specific.
Boppr6 and Viine-Wright (1989 e)xamined the microstructure of PTPs by SEM, but for
Tirumala petiverana only, We conducted comparative morphological studies focusin gon
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306 Kei HAsHiMoTo and Osamu YATA
the microstructure of PTPs in the alar pouch as weli as on the external morphology includ-
ing appearance (venati moarnki,ngs and sex patch ,etc.) and male and female genitali aan,d
found definit edifferenc eisn the microstructu rofe PTPs among seven species (Hashimoto
and Yata ,2007),
In the presen tstudy, the microstructu rofe PTPs was examined in detai lin alt nine species
includin gtwo additienal species. The morphological differenc edsetecte dear]ier in PTPs
were confirmed by the results.
Materials
and methods
Forty sjx individua lwesre examined representing all the 9 species [an d16 subspeciesl of
the genus nrumala according to Ackery and Vane-Wright (1984 )M.ales of each species
were dissecte adnd exemnined, Material ussed in this study were all dried specimens ([Elabie
1) . Specimens of T. gautama were borrowed from the Research Institut eof Evolutionary
Biology, Tokyo, those of T, fonnos afrom Kitakyush uMuseum of Natural History &
Human Histor yand T. euploeomorpha from the Natura lHistor yMuseum, London and the
Museum of Nature aRd Human Activitie Hsy,ogo; and for the other species, dried speci-
rnens in the collection of the Graduate School of Socia land Cultura lStudies ,Kyushu
University
were used.
For observation of PTP microstructu rae ,part of the pocket-shaped alar organ was taken
from the male hindwing ,opened so that the inside could be seen; the dissected organs were
glued onto a metal stub and gold-coate dthree times for four minutes each using a JEOL
JSM-35 ion sputtering system; the insid eof the alar pouch was then examined by using a
SEM JEOL JSM-5600LV at 151(V voltage and at magnifications of 800 × 1,OOO× 2,500 ×
1O,OOO × and 15,OOO × , , ,
.
Table 1,Used materials for observation of PTPs microstructure of ma]e 7irumata,
speciesfsubspecies locality dateofcapture number
T, gat{tama gautama HainanS. December 1980 251122144111]1214113151
T. ehoaspes choa,sT?e,s Sulawesi August 2002-March 2004
T. e. kroeseni BtttonVietnaAmuHgauisnt a2n0N0.4
T. septentrionis septentrionis June 1997
DittoDittoT. March2004

Thai1and July2005

ishmoidesishmoides ButonIrian ?February

T.hamata leucctptera Jaya 2003

T. h, orientalis Philippines December 1969, August 1998
Z h. paryadres MoaVanuatuSanJtaanAunaary 2003
T. h. tnoderata October 1982
T. eaploeomotpha August 1965
DittoT. San Cristbal September1954

Iimniacelimniace ThailandN. August 2001

DittoDitto71 IndiaTaiwanJLuunzeon1S9.92

August 1952

l .orestilla December 1969
T. I .makassara Su]awesi August 2003
T.I.hentenga SalayarKenyaJCaanmuearroy2o0n07

T. petiverana November 20e4
DittoT. March2004

fo rmosa mereedonia UgandaZaireJuly 1980
Ditto February2007

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Comparativ eMorphologica lStudy in 71rumala: Sexual Isolation and PTPs 307
Results
When examined by SEM, though slight]y diffbren tth,e shapc of PTPs of T. gautama, T,
choaspes, T. ishmoides, T. septentrionis, T. hamata and T. ettptoeomorpha was similar and
almost spherical (Fig 1, , A-F). Characteristi wcrinkles are seen on the surface in T. ga"tama
(Fig .1, A) and T. choaspes (Fig 1., B). They are peculiar lpyolyhedral and closely similar
in T. Iimniace (Fi g1,, G) and T, petiveftz n(aFig 1., H). They are globula ian;d covered with
many projections iTn, formos (aFi g1., I),
Particle smeasure 1.2-1,5 ,ttm in diamete rin T. gautama and T. choaspes and 1.5-2. 0pam in
T. ishmoides T,. septentrionis, T. hamata and T. euptoeomorpha. They are a Iitt lsmealler in
T. Iimniace and T. petiverana, measuring 1.0-1. 5ptm in diameter .They are large rin T. for-
mosa than in other species of the genus ,measuring more than 2 pam in diameter.
Morphology and size were almost stable within each species when several subspecies were
available (multi pspleceimens from differe n]tocalit ifeosr widely distribut seudbspecies) are
examined,
Fig. 1. Microstructur eof PTPs of male Ttrumala. (× 15,OOO ),A. T, gautama. B. T. choaspe.s. C.
T. sqptentrionis. D. T. ishmoides ,E: T. hamata, F. T, euploeomorpha. G. T. Iimnict cHe..
71 petiverana ,I. T..fbrmosa (× 10,OOO),
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308 Kei H?xsHiMoT aond Osamu YATA

pt ma/
'F'} ,...･
/r m, ;. . ,m.at,,t
ma/
t/
'
, Jt tw,
''''
tv.'
Fig ,2.A-C. Microstruct uorf eIrlrP sof 71rumata. A. Interi oofr pouch of Tirttmat atimniace show-
ing cushion (] )and pouch (2) scales (×2,500) .B. Interio ror pouch of T. .fbrmosa .C.
Cushion scales of T. petiverana .D. Cushion scales of T. fbrmos a.c-d: Model of cushion
scalcs.
Discussion
The morphology and functio nof PTPs
[I]h eresults showed that species with similar PTPs are allopatric, In other words, the evalu-
ation of PTPs suggests that ,in areas where several species fi ytogether, the morphology of
PTPs diffe rfsrom species to species (Fig .3). For 7Irumala species resembling each other
in general appcarance, some kind of mechanism for sexual isolati oins required. In the
genus Pieri sit is said that the incens ematerial diffe rasmong species in ingredien tors their
ratio (Yat aet al, 1986) . It is probabl etherefore that the chemical composition of the
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Comparative 　Morphological　Study　in　Tirumala：Sexual　Isolati　oannd　PTPs 309
1
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Fig．3．　Distrlbutio　mnaps 　of　some 　7一’厂ufnala 　specics．　A．　T．　gttutama　and 　7 ‘／hoaspes．　B，　T．　seρten−
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　　　 l〃i〜niace ，
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310 Kei HAsHiMoTo and Osamu YATA
pheromone is species-specific and that this specificity is reflected in the morphology of
PTPs. (Althou gAchkery and Vane-Wright (1984 r)eported PA (pyrrolizi dalikanleoids)
plant sof eac.h species, they did not show thcir chemical components.)
Four types based on microstructure of PTPs
According to SEM observation, 9 species can be divided into fbur types based on the mor-
pho]ogy and size of PTPs. (1 )Characteris twirjcnkles, paTticl emseasuring 1,2-1. 5,um in
diameter (T .gautama, T. choaspe,y) (Fig .1, A-B). (2 )Almost spherical, 1.5-2, O"m in di-
ameter (T lseptentrionis, T, ishmoides, T. hamata, T. eupleeomorpha) (Fi g1., C-F). (3)
Peculiar lpyolyhedral, 1.0-1. 5pam in diamete r(T .Zimniace, T. petiveiuna) (Fig .1, G-H).
(4 )Globula rand covered with many projection msor,e than 2 "m in diamete r(T .formosa)
(Fig1,,I).

The noteworthy point is the resemblance of PTP morphology between T, limniac efrom
tropical Asia and T. petiveran afiro mAfrica in spite of geographica ilsolatio n(,Fig .3, C)
suggesting a close relationship between the two (proba bvlicyariant species) as evidenced
by the fact that they had been treated as differe nsutbspecies of the same species from [falbot
(1943 t)o D'Abrera (1980 )M.oreover, the morphology of PTPs is also quit esimilar among
fOur species (T ,sqptentrionis, T. ishmoides, T, hamata and T. euploeomoT;pha). The same
applies to the morphology of the male genital iian these fOur species (Acker aynd Vhne-
Wright, 1984; Howarth et al. 1976, etc,) These resemblances in thcse morphological char-
acters which tend to be specics specific suggest a close relationship among them.
On the other hand, the PTPs of T. formos farom Africa are globula rand covered with many
projections ,which is quit editferen tfrom the other species, Moreover, they are two or more
times as 1arg eas those of other species. This size is associated with the method of division
of the cushion scale (Fi g2., C-D, c-d,). The cushion scales are divide dgraduall ywith
time, generatin ga 1arg enumber of PTPs (Boppr 6and Vane-Wright ,1989) .The cushion
scale is constricted lik ea rosary in T, fbrmos awh,ile it is divide dminutely into a tassel to-
wards the tip in the remaining 8 species, T, fonnos ias thus peculia nrot only in the appear-
ance and morphology of the male genital ibaut also in it sPTPs,
As mentioned above, the presen tstudy showed the species specificity of the PTP morphology
and it susefulness as a taxonomic character. The classification of the genus 7irumata has
remained diMcult due to the scantiness of ditference samong species. The microstructure of
the PTPs will ensure the identificat iofo nspecies and evaluation of characters diMcult so
far ,In Japan ,four 7riru.ma stpaecies have been recorded as `migrant butterflie s('Shir6zu,
2005) . So the microstructure of PTPs seems to be useful for the identjficat iofo nclose
species, especially Z limniac eand Z hamata,
These results are interesti wnhgen considering the speciation process of the genus. Further
taxonomic study of the whoie genus based on thc morphological finding osbtained this time
and taking into consideration the biogeographi icnfbrmatio wnould clarify the phylogenetic
relationships among species,
Acknowledgements
Hashimoto especially thanks Prof .Y Abe, Assoc. Prof .K. Araya and Prof .Emeritu sH.
Shima of Biosystemati cLsaboratory K,yushu Universit (yBLKU) for their critical review of
the manuscript and continuous guidance .Her corclial thanks are due to Dr K. Ueda
(Kitakyu sMuhsueum of Natural histor yand Human histor yf)br constant guidance and en-
couragement. Moreover, we wish to express our sincere thanks to Mr R. I. Vane-Wright
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Comparative 　Morphelogical　Study　in　Tirumata：Sexual工solation　and 　PTPs 311
（NHM ），　Dr　Alexander　L．　Monastyrskii（Russia　Vietnam　Tropica　lResearch　Center），　Dr　M
Yago （T（）kyo　Universit），y　Mr　Y．　Nishiyama　（Tokyo）and　the　curators 　of　Museum 　of　Nature
and 　Human 　Activiti，e　sHyogo　fbr　providin　gvaluable　materials 　or　infor皿 ation ．　 Finally
Hashimoto 　appreciates　the　kind　friendshi　opf　the　members 　of　BLKU 　for　supporting 　and 　en −，
couragement 　of　her　study ．
References
Ackery，　P．　and 　R．　Vane−Wright，1984．　Milkweed　Butte，fli，　ethseir　Ctadistie　sand 　Biolog．v，425　pp．　British
　　Museum （Na巳ural　History），　Hong　Kong．
Boppr6　M ，and 　R．　Valle−Wright，且989、　Androconial　systems 　in　Danainae（Lepidoptera）：functiona　lmorphology
　D ’　AbroefAr，maa，u　Bris，，19，　8D0a．n　aDuasna，　iTdiareu．m　aBluat　taendrf　lEiu　eposfl〔　）tehae　．　AfZroootl’r．（」〜pi、　LciaF’uRi．e∫goicon．9，　7x：x1，0519−31　p3p3．，　Melboume．
Hashimoto，　K．　and　O．　Yata，2007．　Microscopic　gtructure 　of　pheromone　transfer　particl　e（PsTPs）in　the　genus
　　Tinim．ala ．　Trans．　tepid．∫oc．　japan　58：177−182． ’
Howarth，　T．　G．，　Kawazo6，　A．　and　A．　Sibttta，n1i976．　 A　new 　mimetic 　species 　of　Danaus （1irumalの from
　　Solo皿on　Islands（Lepidoptera：Danaidac）．　Ty6　Gα27：131−137．
Kawazo6 ，　A．　and　M ．　W 漁 bayash，i1976．　Coloured　IUustratio↓si　Of　the　Buttenj7i　eosf　Japan ［rev ．　Edn」．　viii，
　　Sh　　ir　462　z2，　upT，p．2．，　，0i7　09256　p．0l．　sM．B　iuHgtortiaekttuefsdlhi　aBe（u，sゾ　OtFtsoeariknjazi．oiseasげ　in　ノCapoainou　lI’．−481Fpapmil．，y7　6D　apnlas．i　dHaoei、k　ufsahmai，　lO　ysSaakaty．r（llnd　−aJaep．　avnii巳is，e）7．30　pp．
　　Chouken　Publishi　nCgo，，　Osaka，
Taユbot，　G．，1943．　RevisiQml　notes 　on　the　genus　Z）anaus 　Kluk （Lep．　Rhop，　Danaidae）．　Trans，　R．　ent．＆，己
　　 Lond．93：115−148．
Yata，0．，　Abe，　M ．　and 　M ．　Nakai，1986．　Relationshi　pbetween　androconial 　scale　and 　the　lnale 　wi 皿g　Qdour 　in
　　Pieri　msetete　M6nEtr．ejs（Lepidopter，　aPieridae）．518わol跏 5ω：131−136，
摘 要
コ モ ンマ ダラ属 Tirumalaにおける比較形態学的研究：生殖隔離とフェ ロ モ ン 運搬粒子PTPs
・
（橋本　恵 矢田　脩）
タテハ チ ョ ウ科マ ダラチ ョ ウ亜科に属するコ モ ンマ ダラtt　Tirttn，alia 全9種を対象に して，雄の性標内
で生成されるフ ェ ロ モ ン運搬粒子PTPs の微細構造の 比較形態学的研究を行なっ た．本属の種は，外
観だけでなく交尾器の形状も相互に酷似するため，種間の識別が難 しい とされてい たが，本研究におい
て，　PTPs の 形態に明確な相違が見出され，本属内の PTPs の微細構造は少な くとも4つ の タイ プに分
けられることが明らか になっ た．さ らに同所的に分布する種の PTPs の形態は異なるとい うこ とが示
唆された．揮発性物質を運ぶPTPsは，同種の雌に交尾相手を正確に認識させる機能をもつ ものである
．
と考えられる．つ まり，　Tirumataは種同士の外観が酷似するため，特に他の種との生殖隔離機構が重要
となる．モ ンシロ チ ョウnt　Pierisにおいて，雄の発香物質の化学的成分やその成分比が種によっ て異な
るこ とがすで にわかっ てい るこ とか ら，おそらく本属に もこの ような種特異性があり，それとともにフ
ェ ロ モ ン を運ぶPTPs の形態に も反映してい る可能性は十分ありうる．
（Accepted　Apri124，2008）
Published　by　the　Lepidopterologica　Slociety　efJapan ，
5−20，Motoyokoyama 2，Hachi ji，Tokyo 192−0063 Japan
　 　 （’ 　 ， 　
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