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ue8ma Trans. iepid, Soc. Japan 59 (4): 305-311,September 2008

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

Kei HAsH[MoTo and Osamu YATA

Biosystematics Laboratory, Faculty of Social and Cultural Studies, Kyushu University,

Repponmatsu, Fukuoka, 81e-8560 japaii

Abstract The genus Tirumala (Nympha]idae, Lepidoptera) is a group belonging to Danainae and consists of 9 species, 2 from Africa and 7 from the Indo-Australian region (Ackery and Vane- Wright. 1984). This is one of the most difiicul{ genera because its rnember species resemble each other not only in general appearance but also in morphology of the male gcnitalia. A comparison was made of the microstructure of pheromone transfer particles (PTPs) in the alur poueh for all spccies in the genus. Here, we report that some definite diffbrences were detected in the microstruc- ture of PTPs between species. The cvaluation of PTPs suggests that. in an area where several species fiy togcther, (he morphology of PTPs di[TZ)rs from species to species.

Key words 7:ir"mala, Danainae, PI]Ps, microstructure, SEM, courtship behavior, sexual isolation, taxonomie charaeter.

Introduction

The genus nr"inala belongs to the Danainae (Nymphalidae, Lepidoptera) and comprises 9 species, 2 (T. formosa, T. petiverana) firom Africa and 7 (T, gautama, T. choaspes, T. Iimni- ace, T. septentrionis, T, ishmoides, T. hamata, T. etoptoeomorpha) from the Indo-Australian Region (Ackery and Vtane-Wright, 1984). As in many other Danainae butterfiies, the larvae feed on Asclepiadaceae and Apocynaceae which contain noxious ingredients.

7irttmata is one of the most diMcult genera due to the resemblance of member species not only in general appearance but also in morpholegy of the male genitalia, There are only a few studies which examined in detail the morphology of the male and female genitalia of 7Vrumata (Shir6zu, 1960; Kawazo6 and Wakabayashi, 1977, etc.). As fOr grouping of species, Ackery and Vane-Wright (1984) only dichotomized the genus, with Afrotropical T. `the ,forntosa treated as the sister-group of the other eight species, called limniace species- complex',

A unique morphological character of 7"iruntala is the presence of a sex patch, called the alar pouch, on the hindwing cell Cu,, of males (Fig. 2A-B), Inside the alar pouch, there are scale sockets (Fig. 2A, 1) and disk-shaped sockets (Fig. 2A, 2) on the surface and, from these disk-shaped sockets, characteristic long and narrow scales (called cushion scales) grow. A large number of pheromone transfer particles (PTPs) are produced by fragmenta- tion of the cushion scales after a certain period of time (Beppr6 and Vane-Wright, 1989), It is be]ieved that PTPs migrate to the tip of the abdominal hairpencils and enable females to recognize and accept their partner by scattering pheromones or by directly sticking to their antennae during courtship behavior. If the chemical constituents of the pheromone and their ratio differ from species to species, it is possible that the morphology of PTPs is also species-specific.

Boppr6 and Viine-Wright (1989) examined the microstructure of PTPs by SEM, but for Tirumala petiverana only, We conducted comparative morphological studies focusing on

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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 (venation, markings and sex patch, etc.) and male and female genitalia, and found definite differences in the microstructure of PTPs among seven species (Hashimoto and Yata, 2007),

In the present study, the microstructure of PTPs was examined in detail in alt nine species including two additienal species. The morphological differences detected ear]ier in PTPs were confirmed by the results.

Materials and methods

Forty sjx individuals were examined representing all the 9 species [and 16 subspeciesl of the genus nrumala according to Ackery and Vane-Wright (1984). Males of each species were dissected and exemnined, Materials used in this study were all dried specimens ([Elabie 1). Specimens of T. gautama were borrowed from the Research Institute of Evolutionary Biology, Tokyo, those of T, fonnosa from Kitakyushu Museum of Natural History & Human History and T. euploeomorpha from the Natural History Museum, London and the Museum of Nature aRd Human Activities, Hyogo; and for the other species, dried speci- rnens in the collection of the Graduate School of Social and Cultural Studies, Kyushu University were used.

For observation of PTP microstructure, a 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-coated three times for four minutes each using a JEOL JSM-35 ion sputtering system; the inside of 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 BtttonVietnamHainanN.August 2004

T. septentrionis septentrionis June 1997

DittoDittoT. March 2004

Thai1and July 2005 ishmoides ishmoides ButonIrian ?February

T. hamata leucctptera Jaya 2003

T. h, orientalis Philippines December 1969, August 1998

Z h. paryadres MoaVanuatuSantaAnaJanuary 2003 T. h. tnoderata October 1982

T. eaploeomotpha August 1965 DittoT. San Cristbal September 1 954

Iimniace limniace ThailandN. August 2001 DittoDitto71 IndiaTaiwanLuzonS.June 1992

August 1952

l. orestilla December 1969

T.I.makassara Su]awesi August 2003

T. I. hentenga SalayarKenyaCameroonJanuary 2007 T. petiverana November 20e4 DittoT. March 2004 fo rmosa mereedonia UgandaZaireJuly 1980 Ditto February 2007

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307 Comparative Morphological Study in 71rumala: Sexual Isolation and PTPs

Results

When examined by SEM, though slight]y diffbrent, the shapc of PTPs of T. gautama, T, choaspes, T. ishmoides, T. septentrionis, T. hamata and T. ettptoeomorpha was similar and T. almost spherical (Fig, 1 , A-F). Characteristic wrinkles are seen on the surface in ga"tama (Fig. 1, A) and T. choaspes (Fig. 1, B). They are peculiarly polyhedral and closely similar in T. Iimniace (Fig, 1, G) and T, petiveftzna (Fig. 1, H). They are globulai; and covered with many projectionsin T, formosa (Fig. 1, I),

Particles measure 1.2-1,5 ,ttm in diameter in T. gautama and T. choaspes and 1.5-2.0 pam in T. ishmoides, T. septentrionis, T. hamata and T. euptoeomorpha. They are a Iittle smaller in T. Iimniace and T. petiverana, measuring 1.0-1.5 ptm in diameter. They are larger in 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 (multiple specimens from different ]ocalities for widely distributed subspecies) are examined,

Fig. 1. Microstructure of 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. Iimnictce. H. 71 petiverana, I. T..fbrmosa (× 10,OOO),

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308 Kei H?xsHiMoTo and Osamu YATA

pt ma/

'F'} ,...･ .t, /rm; , .. ma/ ,ma,t

t/ '

, Jt '''' tw,

tv.'

Fig, 2.A-C. Microstructure of IrlrPs of 71rumata. A. Interior of pouch of Tirttmata timniace show- ing cushion (]) and pouch (2) scales (× 2,500). B. Interior or pouch of T. .fbrmosa. C. Cushion scales of T. petiverana. D. Cushion scales of T. fbrmosa. c-d: Model of cushion scalcs.

Discussion

The morphology and function of PTPs

[I]he results showed that species with similar PTPs are allopatric, In other words, the evalu- ation of PTPs suggests that, in areas where several species fiy together, the morphology of PTPs differs from species to species (Fig. 3). For 7Irumala species resembling each other in general appcarance, some kind of mechanism for sexual isolation is required. In the genus Pieris it is said that the incense material differs among species in ingredients or their ratio (Yata et al, 1986). It is probable therefore that the chemical composition of the

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309 Comparative Morphological Study in Tirumala：Sexual Isolation and PTPs

ゴ 薗 で 一 xgautama 醜 二 が」 ． ヘ 等 1砲・’寮ヒ 「” 匚＝ ＝ ：＝］ τ choaspes 〔 》 〆 a 「 ／ ヤ書 塊 丶ぐ ｛齢 惣 舳 “ 評 簡 拶 丶． 魅 潯 “ 気／ 一 ． ・： k A ．「．／1 N こ二 丶

一 − Fig．3． Distrlbution maps of some 7 ’厂ufnala specics ． A ． T ． gttutama and 7 ‘／hoaspes． B ， T． seρten 〃 め 加 3 ，7 hamata， T． ishmoides and T． euptoeom （冫rpha ． C ： T． petiverana， T ． formosa and T・ li〃 〜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. (Although Ackery and Vane-Wright (1984) reported PA (pyrrolizidine alkaloids) plants of 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) Characteristic wrjnkles, paTticles measuring 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 (Tl septentrionis, T, ishmoides, T. hamata, T. eupleeomorpha) (Fig. 1, C-F). (3) Peculiarly polyhedral, 1.0-1.5 pam in diameter (T. Zimniace, T. petiveiuna) (Fig. 1, G-H). (4) Globular and covered with many projections, more than 2 "m in diameter (T. formosa) (Fig, 1, I).

The noteworthy point is the resemblance of PTP morphology between T, limniace from tropical Asia and T. petiverana firom Africa in spite of geographical isolation, (Fig. 3, C) suggesting a close relationship between the two (probably vicariant species) as evidenced by the fact that they had been treated as different subspecies of the same species from [falbot (1943) to D'Abrera (1980). Moreover, the morphology of PTPs is also quite similar among fOur species (T, sqptentrionis, T. ishmoides, T, hamata and T. euploeomoT;pha). The same applies to the morphology of the male genitalia in these fOur species (Ackery and 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. formosa from Africa are globular and covered with many projections, which is quite ditferent from the other species, Moreover, they are two or more times as 1arge as those of other species. This size is associated with the method of division of the cushion scale (Fig. 2, C-D, c-d,). The cushion scales are divided gradually with time, generating a 1arge number of PTPs (Boppr6 and Vane-Wright, 1989). The cushion scale is constricted like a rosary in T, fbrmosa, while it is divided minutely into a tassel to- wards the tip in the remaining 8 species, T, fonnosa is thus peculiar not only in the appear- ance and morphology of the male genitalia but also in its PTPs,

As mentioned above, the present study showed the species specificity of the PTP morphology and its usefulness as a taxonomic character. The classification of the genus 7irumata has remained diMcult due to the scantiness of ditferences among species. The microstructure of the PTPs will ensure the identification of species and evaluation of characters diMcult so

`migrant far, In Japan, four 7riru.mata species have been recorded as butterflies' (Shir6zu, 2005). So the microstructure of PTPs seems to be useful for the identjfication of close species, especially Z limniace and Z hamata,

These results are interesting when considering the speciation process of the genus. Further taxonomic study of the whoie genus based on thc morphological findings obtained this time and taking into consideration the biogeographic infbrmation would clarify the phylogenetic relationships among species,

Acknowledgements

Hashimoto especially thanks Prof. Y Abe, Assoc. Prof. K. Araya and Prof. Emeritus H. Shima of Biosystematics Laboratory, Kyushu University (BLKU) for their critical review of the manuscript and continuous guidance. Her corclial thanks are due to Dr K. Ueda (Kitakyushu Museum of Natural history and Human history) fbr constant guidance and en- couragement. Moreover, we wish to express our sincere thanks to Mr R. I. Vane-Wright

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311 Comparative Morphelogical Study in Tirumata ：Sexual工solation and PTPs

（NHM ）， Dr Alexander L ． Monastyrskii （Russia Vietnam Tropical Research Center）， Dr M Yago （T（）kyo University）， Mr Y． Nishiyama （Tokyo）and the curators of Museum of Nature materials or infor皿 ation ． Finally and Human Activities，Hyogo fbrprovidingvaluable ， Hashimoto appreciates the kind friendship of the members of BLKU for supporting and en −

couragement of her study ．

References − Ackery ， P ． and R ． Vane Wright，1984． Milkweed Butte，flies， their Ctadisties and 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）： functional morphology ， − ofAmauris ， Danaus ， Tirumala and Eupl〔）ea ． Zool．」、 LiFui．∫oc ．97 ： 101 133． ’ ’ D Abrera B 1980 Danaidae Butterflies of the Afrotr（〜 ’Region xx ． ， ，， ． ． pica ， ，593 pp，Melboume Hashimoto ， K ． and O ． Yata，2007 ． Microscopic gtructure of pheromone transfer particles （PTPs ）in the genus Tinim．ala ． Trans． tepid．∫oc ． japan 58 ： 177−182． ’ Howarth ， T ． G ．， Kawazo6， A ． and A ． Sibtttani，1976． A new mimetic species of Danaus （1irumalの from − Solo皿 on Islands（Lepidoptera：Danaidac）． Ty6 G α 27： 131 137． Kawazo6 ， A ． and M ． W 漁 bayashi，1976． Coloured IUustratioi↓s Of the Buttenj7ies of Japan ［rev ． Edn 」． viii ， 422pp ．，72 pls． Hoikusha ， Osaka． ’ T ． i Buttetflies Fornzosa in Coioul．481pp ．76 ． Hoikusha Osaka ．ln Japan巳se ． Shir6zu， ，960． （ゾ ， pls ， （ ） − − ，2005 ．Migrated Butteijiiesげ ノapan I Family Danaidae、 family Satyrldae． viii ，730 pp． Chouken Publishing Co ，， 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． Relationship between androconial scale and the lnale wi 皿 g Qdour in − Pieris metete M6nEtrj．es（Lepidoptera， Pieridae）．518わol 跏 5 ω ： 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 Tirttni，ala 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 Lepidopterological Society efJapan ，

−20 Motoyokoyama 2 Hachi（’ Tokyo 192−0063 Japan 5 ， ， ji， ，

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