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metxx 1)ans, iepial Soc Jopan 53 (4): 234-240, Seprember 2002

Cytogenetic studies of seyen species of Charaxinae (Nymphalidae, Lepidoptera) from Brazil and Peru

Azuma ABEi> and Yoshinori KuMAGAi2)

i)Sakaimachi 4-12-2, Hirosaki-city, Aornori Prefi, 036-8336 Japan 2)Kashiwa Junior-High-School, Matsubaranishi 2-6-I4,

Hirosaki-city, Aomori Prefi, 036-8142 Japan

Abstract The meiotic chromosomes of seven charaxine species belonging to the genera Archaecrpmpena, RTepona and Agrias from Brazil and Peru were investigated with primary and secondary spennatocytes. The haploid numbcrs of chromosomes were tbund to be n=I6 for

Arehaeopepona demophon and An dlemophoon, n=15 for An ticomedes, n]9 for Ar, an7- phimachus and An meander, n= 12 fbr Pmpona phendamas and Agrias claudina AII these Neotropical butterflies contained one to ten large-sized chromosomes in their metaphase plates. In view ofthe correlation between choromosome size and modal number ofchromosornes (n= 31) in Nymphalidae, the largesized chromosomes noticed here might have been fbrmed through chromosome fusion. which resu!ted in decrease of chromosome number.

Key words Charaxinae, ArchaeopTepena, Jlrepona, Agrias, meiotic chromosomes.

Introduction

To date the chromosomes ofnymphalid butterfiies have been studied in more than 280 species (Stevens, 1906; Kerne"'itz, 1915; Beliajeff; 1930; Federlay, 1938; Lorcovi6, 1941, 1958; Lesse, 1952, !953, 1960, 1967a, b, 1970a, b; Maeki, 1953a, b; Maeki & Makino, 1953; Maeki &

Remington, 1961; Maeki et aL, 1965; Saitoh & Abe, 1969, 1981; Brown et aL, 1992; Abe,

1997). Almost halfof these species have the haploid number of n=31 which is regarded as a modal number of chromosomes in this butter[fly group. The lowest and highest haploid numbers are n= 12 in Bnenthts claphne iwatensis (Saitoh et aL, 1985) and n=37 in Atgynnis anadyomene (Maeki, 1953a, b; Maeki & Makino, 1953), respectively, in Heliconiinae, Nymphalinae, Limenitidinae, Apaturinae and Charaxinae,

Recently Lesse (1967a, b, 1970a, b) presented the chromosome numbers ofabout I60 species of S. American butterflies, in which Archaeopmpona demophon (as Plrepona demophon) was reported as n= 16 in the haploid number. This is markedly lower than the modal number

normally counted in Nymphalinae, Heliconiinae, Limenitidinae, Apaturinae and Charax- inae, but no description of either the karyotypes or photographs was given.

From 1987 to 1996 the authors collected seven species of Charaxinae belonging to the genera Archaeopmpona, Rrqpona and Aghas for chromosome study in the Neotropical region, Brazil and Peru. These genera are systematically closely related to one another, fbrming a part of the tribe Preponini', and Archaeopnepana is known from about 9 species, Pmpona from about 2I species and Agn"as from about 7 species (D'Abrera, 1987). In five species ofArchaeopre- pona, one species of Prepona and one species of Agn'as, which the authors examined, all were confirrned as having the haploid numbers less than n =J 16, including n == 9, as shown by Lesse (1970a) for Archaeopnepona dlemophon. The haploid number n::=9 is a new finding being the lowest so far reported, In this report, the authors discuss a plausible mechanism of

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Cytogenetie Studies ot' SevenSpecies of Charaxinae from Brazil ttndPeru 235

decrease in choromosome number in seme charaxine species, paying attention to the existence of large-sized chromosomes which have not been fbund in the nymphalid butterflies carrying n=3L

Materials and methods

Seven charaxine species, in total fifteen male spocimens, were collected at four localities of Brazjl and Peru from l987 to 1996 (Table 1). The testes taken from liye butterfiy specimens were fixed in PFA3 fixative, Chromosome preparations were made according to the paraffin sectioning method (10 #m) and stained with Heidenhein iron-haematoxylin. In the authors' preparations, spermatogonial metaphases were not suitable for chromosome observation due to the cohesion of chromosomes, while many well-spread metaphases could be obtained in the primary (MI) and secondary (MII) spemiatocytes. Such metaphase plates were basically composed of dot-like small chromosomes and rather targe round chromosomes. So, the

number and size of chromosornes were the main subject of this study.

In the paratiin sectioning method, chromosome size occasienally depends on the sectioning angle and sectioning phase for chromosemes, So, the frent and rear sections of a given metaphase plate were always checked. In case ofdiMculty for determination ofchromosome "3 size, the number of chromosomes of given size was expressed with a certain range like to 4",

Results

The results of choromosome analysis in MI and MII spermatocytes fi'om the seven charaxine species are summarized in Table 2. No heteroploidy was fbund in the present study. The haploid numbers of these buttefly species ranged from n=9 to n:=16. In each species,

chromosomes were morphologically classified into four types, namely markedly large-sized (LL), large to medium (L-M), and srnal1-sized (S) and rnarkedly srnal1 (SS) chromosomes, though this classification was occasionally not clear-cut. In general, the chromoseme size of each class was almost two times larger in MI than in MII, apparently reflecting meiotic

division, The chromosome findings in each species are described as fbllows,

Archaeopmpona amphimachus (Fabricius)

The haploid number of this species is n=9, as determined with only one specimen. This

Table 1, Species of Neotropical charaxine butterflies examined in this study.

no. of specles date collecting loculity speclmenscollecting

Archaeoprepona amphimachus 1,ln2cl26]2dii1cli129. Sept. 1992 Nova orinda (Brazil) Ar. `iemophon 28. Sept. 1992 Nova orinda (Brazil) 29. Sept. I992 Nova orinda (Brazil) 1. Jan. 1993 Kanuma (Brazil) 2. Jan. 1993 Kanuma (Brazil)

,fny1cim1 3. Jan. 1996 Katumba (Brazi]) Ar, dlemophoon li. Jan. I987 Tingomaria (Peru) Ar. meancler ciny2di1"16i 7. Jan. 1987 Tingomaria (Peru) Ar, lieomedus 28. Sept. 1992 Nova orinda (Brazil) Ptepona pheridomas 29. Sept. I992 Nova orinda (Bruzil) Ag7'ias clauLfina 28. Sept. 1992 Nova orinda (Brazil)

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236 Azuma ABE and Yoshino- KuM/4GAI

tuadi"////'/'"' ,,,',/.,/,"rm",de.{/,s'#'.'."",/,, "- /tt t. .w't/ "as ' ,,/t,//t,t////,/ tww//ti /t/ , "x$" st ""l estspts *asew tl ew"b /tt/･.1.IIi,'111I:ee,mu.,,,,,,i'rt'"':' "; #" lilE.iillt;･:,:, ". - , , ,:' "/##1/lil"'/'' /'i,y//la't',v"es//1",' / ,/, ,,,'

la lb 2a 2b 3a 3b

4a 4b 5a 5b 6a 6b

10ptm

7a 7b

Figs 1-7. Chromosornes ofseven Preponini species from Brazil and Peru. 1. Archaeoprepona amphimachus (a: MI, b: MII, Brazil). 2. A. dlemophon (a: MI, b: MII, Brazil; c: MI, Peru). 3. A. demqphoon (a: M], bi MI], Brazil). 4. A. meandler (a: MI, b: MII, Brazil). 5, A. Iibomedes (a; MI, b: MII, Brazil). 6. Prepona pheridamas (a: MI, b: MII, Brazil). 7. Agrias clattdina (a: MI, b: MII, Brazjl).

species has4LL, 4L-M, and 1S chromosomes (Figs la and lb). Both the haploid number and chromosome constitution are quite stable, so far as 10MI and 32M" spermatocytes observed are concerned.

Archaeopmpona ciemophon (Linnaeus)

All of the eight specimens examined have n= 16 as already reported by Lesse (1970b), But the diflerentiation of chromosome size is rather indistinct as compared with An anzphima- chus (Figs 2a and 2b), and thus the numbers of L-M and S chromosomes could not be deteimined as fixed numbers (Table 2).

Archaeopiepona cien2ophoon mtdsen (Cramer)

This species also has n=: 16, resembling Az demophon in its chromosorne constitution. But An a muson is apparently different from An ciemophon in which one ofthe S chromosomes is markedly smal1, as confirmed by observing consecutive parathn sections (Figs 3a and 3b). "SS" This unusually small chromosome was termed as here (Table 2).

Archaeoptepona meander (Cramer)

This species has the haploid number n=9 characterized by 4-5 LL, 3-4 L-M and 1 S, closely resembling An amphimachtAs in chromosome constitution as well as in haploid number of

chromosomes, though An meander seemed to have one more LL chromosome than An amphimachus (Figs 4a and 4b).

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Cytogenetic Studies of Seven Species of Charaxinae from Brazil and Peru 237

Archaeoprepona Ebomedbs (Cramer)

The haploid number of this species is n= 15. Diflerentiation of chromosome size seemed to be of low degree in this species, as shown in Figs 5a and 5b. But most ofthe spermatocytes examined contained 12-14L-M and 1-3 S chromosomes.

Prepona pheriddmas (Cramer)

The haploid number is n=12, as determined with only one specimen, In most of the spermatocytes examined, all 12 chromosornes were of M-L size without S chromosomes, independent of defiection of the section angle (Fig. 6a), The MII plate of Fig 6b contains mostly M chremosomes due to subtle deflection of the section angle,

Agrias claudina sardnnapakts (Bates)

This species has n=12 equal to R pkeridamas in haploid number, carrying 9-11 L-M and 1-3 S chromosomes (Figs 7a and 7b).

Discussion

Literature reported to date proved that the modal haplo{d number of chromosomes in Nymphalinae, Heliconiinae, Limenitidinae, Apaturinae and Chai;axinae is n=31. The haploid numbers found in the present Neotropica] species are any of n= 16, n= 15, n= 12 or n=9, being apparently far lower than the modal number n=31. Assuming that the ancestral group of Nymphalidae had the modal number n=31, the present Neotropical species would be derivative. In other words, these Neotropical species might have evolved in the direction of diminishing chromosomes by means of chromosome fusion or chromo- some loss. As an example of the forrner case, Maeki (1961) reported the chromosomal relationship among three apaturine species, Apatura metts (as illa) (n = 3 l ), Hestina 1'aponica (n=30) and Sasakia charoncia (n=29). The first species contains only small chromosomes,

Table 2. Summary ofchromosome analysis in seven nymphaline spocies from Brazil and Peru.

no. haPLOidofnUMber of speorrbnsg:eecdytes specles iocality remarks chromosomes J II

Archaeqpieponaanu)himachus Brazil n, 9n, 10 321423t5145815251242472812114LL, 4L-M, IS An demophon Brazil 16n, 7 I3-14LmM. 2-3S

An dlemophon Brazil t6n, 20 IL, 2-3S

Ar. demqphon Brazil I6n, 39 4L, 2-3S

AL demcrphon Brazil 16n, 27 4L, 2-3S

An dlemophon Brazil 16n, 542627 1-3L, 2-3S

An demophon Brazil 16n, 1-3L, 2-3S

An clemophon Peru 16n, 1-3L, 3S

Ai: ttemophon Peru 16n, 14 13-14L-M, 2-3S

Ait dbmophoon Peru 16n, 12110 ll-12L-M, 3-4S, ISS meander An Peru 9n, 4-5LL, 3-4L-M, IS

An tibomedes Brazil 15n, 7 12-14L-M, 1-3S

i4n ticomedes BrEizil l5n, 1247 12-14L-M

Rrepona pheridamas Brazil l2n, 12L-M, OS

Agn'as elaudina Brazil I2 7 9-11L-M. 1-3S

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238 Azuma ABE and Yoshinori KuMAGAi

and the latter two species contain one and two large-sized chromosomes, respectively, Taking these findings into consideration, he speculated that, in the course of differentiation of Il japonica and S charoncla, fusion of two pairs ofchromosomes might have taken pait in the formation of one pair of !arge chromosomes, resulting in reduction of one bivalent in MI. Maeki (1958) also proposed, for polymorphic occurrence of Pileiis rapae (n=25, n-- 26), chromosornal reduction by means of chromosome loss through elimination inte cyto- plasm. In case of fus{on, the resulting fusion-mediated chromosome may inevitably become larger in size. In the present charaxine species, the spermatocytes of each species contain markedly 1arge, large-to-medium, small and markedly smal1 classes ofchromosomes (LL, L- M, S and SS) which are morphologically discernible to each other. The LL chromosomes were fbund in two Arehaeopmpona species (n=9), and an SS was fbund only in An demophoon. Of special interest js the general tendency that the L-M chromosomes of these seven species appear to decrease in number and become larger as the haploid number decreases, These findings may suggest participation of chromosome fusion as in the cases reported by Maeki (196l), especially involvement of more than two bivalents in chromosome fusion in two ArchaeopTepona species (n=9), Apart from the propriety of this notion, An amphimachus and An meancier are karyosystematically clese to each other,

Of seven species examined, An ctemophon and An ciemophoon have the largest haploid

number n= 16. In both species, diflerentiation of chromoseme size seemed to be not so

remarkable as compared with the other species with lower haploid number, and reflecting

such ambiguity of chromoseme size, L-M and S chromosomes varied in number from cell to cell as well as from indjvidual te individual (Table 2). But the latter species has one markedly small chromosome (1SS) which is the only chromosomal marker distinguishing An dlemophon from An demqphoon (Fig.3a), It is still uncertain whether the SS can be rega:;ded as a marker chromosome specific to this species and stable within species, since only one specimen was examined in this study. Many more specimens should be examined to

certify this finding. It may be reasonable lo consider, based on the close similarity of

chromosomal features, that AE clemophon is systematically closer to An cleniophoon than to

the remaining three congeners,

Both Rrepana and Agn'as, examined in only one specimen each, have an intermediate haploid number (n= I2), so far as the seven species exarnined are concerned, showing similar chromosome constitution to each other (Table2). So, the Neotropical chai:axine species, including ArehaeopnElpona species, may be characterized, at least as to the present species, by a low number ef chromosomes, The present findings suggest chromosomal evolution of Neotropical charaxine species to a direction of diminution in chromosome number. In order to discover whether this pattern is more general, many other species of Nymphalidae, especially species belonging to Prepona and Agrias, both of which have well adapted to the tropical jungle zone and differentiated to a large number of species, should be examined fbcussing on the haploid number and chromosome constitution. This kind ofbasic chromo- some analysis may give a clue to inquiry into speciation process ef Neotoropical charaxine butterflies.

Acknowledgements

The authors are gratefu1 to the late Dr Kazuo Saitoh and Dr Yoshitaka Obara of Hirosaki University for their valuable advice and suggestions.

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Cytogenetjc Studies of Seven Species of Charaxinae from Brazil and Peru 239

References

Abe, A. 1997. Chromosorne study ofbutterfiies in Primorye region. A'attire inseets 32 (1): 15-18 (in Japanese). BeliajefT; N.K., 1930. Die chromosomen Komplexe und: ihre Bezichung zur Phylogenie bei den Schmatterlingen. Z indukt. Abstammu. PlererbLehre 54: 319-339. Brown, K. Jr, Emrnel, T. C,, Eliazer, P. J. & E. Suomalainen, 1992. Evolutionary patterns in chromosome numbers in neotropica] Lepidoptera, 1. Chromosomes of the He]iconiini (Fam. Nymphalidae: Subfam. Nymphalinae). Hlareditas 117i I09-I25. D'Abrera, B., 1987. Buttet:f2ies of the iVeetropical Ragion. Part4. Nyrnphalidae (Partim). xxx pp. Hill House.

FedeTley, H,, 1938. Chromosomenzahlen finnlljndischer Lepidopteren. 1. Rhopalocera. Hereditas 24: 397-464.

Kernewitz, B., 19I5. Spermiogenese bei Lepidopteren mit besonderer BerUcksichtigung der Chromosomen. Anch, Ntiturgesch. (A)81: 1-34. Lesse, H., de, I9S2. Note sur les genres Prects Hb. et. Junonia Hb. (Lep. Nymphalidae). Buza Soc. ent F>: 57: 74-77, chromosomiques , 1953. Formu]es de Boloria aquilonaris Stichel., B. pales D. et. Schiff., B, ncu)cea HofTfr]. et. auta:es L6pidopteres Rhopaloceres. Revue Lopidbpt. queLques .fr, 14:24-26. et variation , 1960. Speciation chromosomes chez. Ies LEpidopteres. Annts Sei nat. (Zool. BioL anim.) (12) 2: 1-223. 1967a. Les nombres de chromosomes les , chez Lepidopteres neotropicaux. Annls Soc ent ". (N. S.) 3: 6.

1967b. Les nombres chromosomes chez Lepidopteres neotropicaux. Annls ent , de les Soa F>･. (N. S.) 3: 67-136. Les , 1970a, nombres de chrornosemes chez les Lepidopteres Rhopaloceres en Amerique eentrale et Colombie. Annls Soc, ent F>'. (N. S.) 6: 347-358, figs2, 1970b. Formules chromosomiques . de quelques Lepidopteres Rhopaloceres de Guyane. Annls Soc. enit ft. (N. S.) 6: 849-855. Lorkoyid, Z., 1941. Die chromosomenzahlen in der Spermatogenese der Taglalter. Chromesoma 2: i55-191.

Merkmale der unvo]lstandigen spezLationsstufe , 1958. Die und die Frage der Einfuhrung der Semispezies in die Systematik. Cippsala Uhiv. Dratsskm 1958: 159-168. Maeki, K., 1953a. Chromosome numbers of some butterflies (Lepidoptera: Rhopalocera). Jdp. X Genet. 28: 6-7.

1953b. Cbromosome numbers of some Rhopalocera). , butterfiies(Lepidoptera: A. Stual Kwansei Gakuin Uhiv. 1] 67-70.

, 1958. The chromosomes of Pierts rapae, Jap. X GeneL 33: 398-404, The survey ofchromosomes ofJapanese ,1961. Nymphalidae. Jap. J Genea 36: 137-146. Maeki, K. & S, Makino, 1953. Chromosome numbers ofJapanese Rhopalocera, Lepid IVbws 7: 36-38. Maeki, K., Ogata, M., & T, Shir6zu, 1965. A study of the chromosomes in twenty-five species of Formosan Rhopalocera. SPec, Buza iepid Soc. Japan (1): 1-10. Maeki, K. & C, L. Remington, 1961. Studies of the chromosomes of North American Rhopalocera. 4. Nymphalinae, Charaxidinae, Libytheinae, X LE7Jid Sbc, 14: 179-201. Saitoh, K. & A.Abe, 1969, Chromosome numbers of some Himalayan butterfiies (Lepidoptera: Danaidue, Nymphalidae and Lycaenidae), 1

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240 Azuma ABE and Yoshinori KuMAGAI

） − sex determillation． 1 め な Camaegie 肋 跏 （Zool ．）（36）； 33 74．

摘 要 ー ・ ブ ラ ジ ル お よ びペ ル 産 フ タオ チ ョ ウ亜 科プ レ ポ ナ 族 7種 の 染色体 （阿部 東 熊 谷義則 ）

ー ブ ラ ジ ル と ペ ル 産 の タ テ ハ チ ョ ウ 科 フ タ オ チ ョ ウ亜 科 Preponiniに 属 す る 7 種 に つ い て 第 1 精母 細 − 胞 （MI ）及 び第 2 精母 細胞 （MII ） の 分裂 に お け る 半数 の 染 色体 を 調 査 し た ． そ の 結果， A rchaeopre pona amph imaeh us ， n ，9； A ． demophon ， n ，16； A ． demophoon ， n ，16； A ． meander ， n ，9； A ． licomedes， n ，

15； Prepona pheridamas ， n ，12； Agrias claudina ， n ，12 で あ っ た ． こ れ ら は ． タ テ ハ チ ョ ウ 科 に お け る 最頻 染色 体 数 （modal ・ chromosome ・ number ）， n ，31 に 較 べ 少 な い 染色 体 数 で あ る ． こ れ ら各種 の 染色 体 一 の 中 に は大 型 又 は大 中型 の 染色 体 を含 み ， こ の 大 き な染色 体 は，染色 体 の 融合 に よ っ て 生 じた と考 こ こ Preponinj え られ た ． の と か ら， 族 で は融 合 に よ っ て 染 色 体数 を減 少 さ せ ， 進 化 し て 来 た も の と 考 え られ る．

（Accepted February 3，2002）

Published by the Lepidopterological Society of 亅apan ， − − 5 20，Motoyokoyama 2， Hachioji ， Tokyo ， L920063 Japan

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