The Rise of Eversion Techniques in Lepidopteran Taxonomy (Insecta: Lepidoptera) SHILAP Revista De Lepidopterología, Vol

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Mikkola, K. The rise of eversion techniques in lepidopteran taxonomy (Insecta: Lepidoptera) SHILAP Revista de Lepidopterología, vol. 35, núm. 139, septiembre, 2007, pp. 335-345 Sociedad Hispano-Luso-Americana de Lepidopterología Madrid, España

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SHILAP Revta. lepid., 35 (139), septiembre 2007: 335-345 CODEN: SRLPEF ISSN:0300-5267

The rise of eversion techniques in lepidopteran taxonomy (Insecta: Lepidoptera) K. Mikkola

Abstract

During the last decades of the 20th century, a new step to the traditional way of preparing lepidopteran microscope slides was widely adopted, the technique of inflating and fixing the soft parts of internal genitalia. The taxonomic resolution in revising problematic species groups, particularly allopatric relationships, improved considerably. At the same time, the so-called lock-and-key hypothesis has been revived, since usually the sexes show corresponding structural details in their internal genitalia. On the basis of intercontinental studies on the Noctuidae, it is considered that the divergence of internal genitalia in pairs of sister species is based on genetic drift. The history of the eversion technique is revised. KEY WORDS: Insecta, Lepidoptera, internal genitalia, male vesica, female bursa, lock-and-key, microscopic slides.

El aumento de las técnicas de eversión en la taxonomía de los lepidópteros (Insecta: Lepidoptera)

Resumen

Durante las últimas décadas del siglo veinte, un nuevo paso para la manera tradicional de realizar las prepara- ciones microscópicas de los lepidópteros fue adoptada extensamente, la técnica de inflar y adaptar las zonas blandas de las genitalias internas. Mejora considerablemente, la revisión de la solución taxonómica en los grupos de especies problemáticas, particularmente las relaciones alopátricas. Al mismo tiempo, se ha revisado la conocida hipótesis llave-cerradura, ya que generalmente los sexos muestran los detalles estructurales correspondientes en sus genitalias. Sobre la base de los estudios intercontinentales de los Noctuidae, se considera que la divergencia de sus genitalias en especies gemelas, está basada en la tendencia genética. Se revisa la historia de la técnica de la eversión. PALABRAS CLAVE: Insecta, Lepidoptera, genitalia interna, vesica del macho, bursa de la hembra, llave-cerradura, preparación microscópica.

Introduction It does not happen frequently that the basic technique in research is improved by decisively new methods. Towards the end of the 20th century, such a change took place in the treatment of lepidopteran genitalia, particularly those of the Noctuidae, when most taxonomists started everting and expan- ding the soft internal parts of both sexes. A similar step forward in the 21st century may be DNA sequencing, particularly the so-called barcoding. However, like the earlier electrophoretic analysis of isozymes, sequencing is not a technique for every lepidopterist. In the 1980s and onward, the male sclerotized aedeagus (= phallus or penis) was no longer left as such on microscope slides but the membranous vesica (= endophallus) was inflated from the inside of the aedeagus using a hypodermic syringe (Fig. 1). Soon it became evident that by the same technique the female bursa copulatrix should be expanded, so as to have the sexes comparable. Both organs were

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stained and fixed on slides. Slides are more practical than preservation in microtubes or on pins, as, for purposes of comparison under the microscope, slides are quicker to handle and can be placed on top of each other, and they last forever. This technique opened a new world and endless row of anatomical secrets have been detected that no-one had previously seen. Actually, the technique is imitating what is happening in vivo: ac- cording to CALLAHAN & CHAPIN (1960), other than muscular action, air (or fluid?) pressure is the main force everting the vesica, since it is equipped with special unbranched tracheae (air tubes). The differences to nature are: that the (probable) air is replaced by alcohol; the pressure is applied into the folded vesica, where the spermatophore is expected to proceed, and not along the walls of it; and the cut ductus ejaculatorius is blown out of the vesica, forming an artificial simple extension of the latter (during copulation, eversion ends when the vesica and ductus inside it are connected; the primary gonopore). As a reasonable new addition, the skins of the moths have also been preserved, since particularly the male hair brushes, or absence of them, may provide important species characters (MIKKOLA & LAFONTAINE, 1986). The happy side of this development has been that it has not made the earlier achievements use- less. Thus, in the late 1980s I was excited to soak a slide of a type specimen of the genus Xestia (Noc- tuidae) from the Naturhistoriska Riksmuseet (Stockholm), fixed in the early 1940s by famous Charles Boursin, but could evert the vesica as is routine today. The new technique is reversible and is just one step further to the traditional way of preparing genitalia slides. This development led inevitably to the observation that in the male vesica we can see parts that correspond exactly to structures in the female bursa of the same species. Thus, in closely related species the vesicae are usually different and in the bursas we can see accordant distinguishing features. Dr Jeremy Holloway (oral. comm.) confirmed that in the tropics where the sexes of unknown species often look confusingly different, it has frequently been possible to “make marriages”, i. e. to find, on the basis of internal genitalia, the correct companions for opposite sexes. Thus, they fit each other like lock-and-key. There has been much confusion in evolutionary discussion about lock-and- key theories. The lock-and-key hypothesis, that the genitalia structures constitute an isolation mechanism between related species, originally arose from the observations of the complicated external genitalia of insects, particularly the valvae. Even if the idea about such a function of the external genitalia has been abandoned, some kind of general resistance toward the hypothesis seems to be irresistibly con- tinuing. Such an idea is considered dilettantish and the “real evolutionarists” speak mainly about the different effects of sexual selection on genitalia, as opposed to the lock-and-key hypothesis. The development has taken place despite the pioneering studies of CALLAHAN & CHAPIN (1960) that clearly unravelled the background anatomy of lepidopteran internal genitalia. Already these authors stated that “definite ‘lock-and-key’ barrier…prevents crossing” of species. In Holarctic zoogeography we see the order of divergence of characters during speciation: the internal genitalia go ahead in the process (MIKKOLA, unpubl.). Continental speciation is not a conse- quence of adaptation, because populations were already living in their ecological niches and long-las- ting species guilds, and no external selection affected the internal genitalia. Sexual selection does not, either, play a role. Most female noctuids re-mate only in their older age for the nutritive substances in the male spermatophore. The absence of female choice is best documented in Agrotis segetum, where the calling female always accepts the first male courting (SVENSSON et al., 1998). In addition, the locking structure of genitalia and the mechanism to deposit the spermatophore in its highly specific and springlike position, mean that for the female it is too late to abandon the male when internal coupling has started. Therefore it was considered that, in general, genetic drift drives taxa apart, originally because of hybrid inferiority. Schematically, the rate of divergence in pairs of sister species can be used as a rough measure of divergence in a given time of any (lepidopteran) taxon from its ancestral taxon. The problems in comparing of different insect classes are (1) that eversible intromittent organs do not occur in all insect groups, and (2) that the eversion technique is difficult and needs much training

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and the researchers in groups other than Lepidoptera hardly master it. Thus, in spite of some attempts (LAFONTAINE & MIKKOLA, 1987; MIKKOLA, 1992), the miraculous world of the mechanisms of internal genitalia has largely remained a secret of lepidopterists. From the texts of researchers of other insect groups it is often hard to know whether they speak about external or internal genitalia. In Lepidoptera, the internal genitalia constitute the following: in males, locking and spermatophore-transmitting organs, the aedeagus and vesica, with the juxta often functionally connected to the lamella antevaginalis, and in females, the bursa copulatrix, from the ostium bursae to the opening of the ductus seminalis, with the periostial posterior margins of the lamella ante- and postvagi- nalis included. With new preparation methods, evidence of the lock-and-key mechanisms (LKMs) in the internal genitalia of insects has been rapidly accumulating. They seem to be a rule in the ditrysian Lepidoptera but, for example, they also appear in the Carabidae (Coleoptera; for literature, see MIKKOLA, unpubl.). In the late 1980s and 1990s illustrations of lepidopteran internal genitalia invaded, except many revisions (eig. VARGA, 1998, NIKKOLA, 1998), the main handbooks (LAFONTAINE, 1987; RONKAY & RONKAY, 1994), along with the techniques (LAFONTAINE, 1987, FIBIGER & GOATER in FIBIGER, 1997) and the morphological nomenclature (LAFONTAINE 1987; GOATER et al., 2003: 34–35). Characters of vesica have already been provided for applied purposes (POGUE, 2004). The aim of this article is to find out how lepidopterists ended up at this line of research, who started to evert vesicas, who inflated them by syringe and how the technique slowly came into the general use. It is also briefly noted what the role of internal genitalia is in the mechanics of copulation and what kind of taxonomical significance they have.

The history of the lock-and-key ideas in Lepidoptera

Examination of external genitalia characters, and mainly the male valvae, have a long tradition in lepidopteran taxonomy. Since DUFOUR (1844) worked on Diptera, claims of so-called lock-and-key mechanisms as tools for species isolation have repeatedly occurred. However, external genitalia rarely show counterparts in the opposite sex. JORDAN (1905), working on Papilionidae, was seemingly the first to refute the lock-and-key hypothesis. The subject of later criticism against the lock-and-key hypothesis is somewhat obscure because no clear distinction was made between external and internal genitalia (for instance SHAPIRO & PORTER, 1989). The mechanism of internal coupling in Lepidoptera was first fully described by CALLAHAN & CHAPIN (1960), using techniques of serial morphology. Already PETERSEN (1904) had described several details correctly and was acknowledged by CALLAHAN & CHAPIN (1960). The controversy is that many recent authors do not refer to the work by CALLAHAN & CHAPIN, except for EBER- HARD (1985) where he cites the number of spermatophores only. Similarly, ARNOLD & FISCHER (1977), publishing in the same journal as CALLAHAN & CHAPIN (1960), did not mention their work, nor did ARNQVIST & NILSSON (2000) include them in their review. In the handbook on Le- pidoptera by SCOBLE (1995), the LKMs are not mentioned. KRISTENSEN (2003) presents illustrations from CALLAHAN & CHAPIN (1960), without mentioning their conclusions regarding LKMs. HOSKEN & STOCKLEY (2004) have recently reviewed the role of sexual selection in genitalic evolution. Their statement that the lock-and-key hypothesis is not consistent with “evidence based on the morphology of the female reproductive tract” and “genital associations during copula” is surpri- singly erroneous. In otherwise interesting analyses, MUTANEN et al., (2006) and MUTANEN & KAITALA (2006) have unfortunately misunderstood the function of the sclerotized aedeagus. The latter authors exclude the aedeagus from the internal genitalia by claiming CALLAHAN & CHAPIN (1960), saying that it “is positioned on the female ostium bursae but is not placed in the female ductus bursae”. Ac- cording to them, the internal genitalia should be soft. However, CALLAHAN & CHAPIN (1960: 778) state that in both Peridroma saucia (Hübner) [as margaritosa (Haworth)] and Pseudaletia unipuncta

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(Haworth) the aedeagus is short and therefore “fits only a short distance up the female ductus bursae”. In P. saucia “the keellike carina of the aedeagus hooks behind the anterior inner edge of the lamella antevaginalis” [i.e. inside the ductus bursae] (1960: 778), and in P. unipuncta the aedeagus inserts “in the ductus bursae up to the aedeagal caecum” [as deep as possible] (1960: 777). For Heliothis zea (Boddie), CALLAHAN (1958b: fig. 6) illustrates how the aedeagus has passed the ostium bursae and is inside the ductus bursae. It is evident that the aedeagus has to be rigid (sclerotized) because it is the most important intromittent organ, and because it also protects the membranous vesica. In copulation the aedeagus mostly penetrates the entire ductus bursae, and thereafter does the vesica get space to expand and to produce the spermatophore.

The mechanics of lepidopteran copulation

The following two phases of lepidopteran copulation may be distinguished (MIKKOLA, unpubl.): (1) External coupling: the male uses external genitalia. This is mostly a transient fixing phase be- fore internal coupling but may have extended sensory functions. There is no direct bearing upon reproduction. (2) Internal coupling: performed with internal genitalia. The internal genitalia first lock the sexes together for copulation proper (cf. CALLAHAN, 1958b: fig. 1). Thereafter follows the transfer of the spermatophore, which may or may not result in sperm transfer. The external genitalia usually have no morphological counterparts in the female anatomy (for ex- ceptions, see MIKKOLA, unpubl.). Their main function is to keep the female abdomen in a position allowing the insertion of the aedeagus; the male may rub the female abdomen with valves during internal coupling (Pieridae; LORKOVIC, 1952) and this also occurs in Aglaope infausta (L.) (Zy- gaenidae; H. Fänger pers. comm.). Many photographs in the literature prove that only the male aedeagus (a sclerotized, partially transparent tube) connects a pair in copula, and this is also illustrated as a drawing by CALLAHAN (1958a: fig. 1). The external genitalia have no locking function at this stage. The internal genitalia of Lepidoptera form a postcopulatory mechanism in that the success of sperm transfer comes primarily from the ability of the male spermatophore to fit into the female genitalia. The frenum end of the spermatophore (with aperture) is placed against the opening of the female ductus seminalis (CALLAHAN & CHAPIN, 1960; PROSHOLD et al., 1975). The couple is then locked together. In a few lepidopteran species, mechanical isolation is precopulatory in that specific external structures do not permit heterospecific couplings. LAFONTAINE & MIKKOLA (1987) and MIKKOLA (1992) listed the anatomical correspondences when male and female noctuids couple: (1) The diameter, length and shape of the male aedeagus fit into the female ostium bursae and ductus bursae (2) The apical part of the aedeagus, often with the carina, fits into the distal part of the ductus bursae, which has a receiving fold for carina (3) The basal part of vesica protruding first out from the aedeagus (often with sclerotized, spiny and muscular ridges guiding the spermatophore), fits into the distal part of the ductus bursae and the most proximal part of the bursa copulatrix, with sclerotizations receiving the ridges (4) The body of the vesica, with its diverticula and cornuti, fits into the pockets and sclerotizations of the proximal part of the corpus bursae and the appendix bursae (= cervix b.) (5) The shape of the spermatophore fits into the bursa from the most distal (anterior) part of the corpus bursae all the way to the appendix bursae with the opening of the ductus seminalis. In addition, (6) the male juxta (of the fultura inferior) often shows specific structures corresponding to the caudal margin of the female lamina antevaginalis. Similarly, in the Zygaenidae the main internal correspondences are found between the male vesica and the female ductus bursae (FÄNGER & NAUMANN, 1997).

The history of preparation techniques of internal genitalia

The preparation of lepidopteran internal genitalia includes techniques that have been in general

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use for two-three decades only. However, already JANSE (1932) in South Africa pioneered in everting the male vesicas of Noctuidae, blowing them out through the anterior end of abdomen (then as emptied and cleaned skin with genitalia in the posterior end). During the 1930s and 1940s, J. McDun- nough, working in Canada, removed the vesica from the aedeagus with the aid of a small hook (J. D. Lafontaine pers. comm.). Already in the 1960s and 1970s, in Finland and Sweden, P. Grotenfelt and S. Överby, respectively, everted and expanded vesicas by injection, but the method did not become a general praxis in taxonomy. To inflate the internal male genitalia using syringe and to also expand the female bursa in moths, particularly in Noctuidae, has been mainly a North American tradition (e.g. HARDWICK, 1950, 1965; FRANCLEMONT, 1952, 1963; DONAHUE & NEWMAN, 1966; LAFONTAINE, 1974, 1981; LAFONTAINE et al., 1983). In Europe, DUFAY (1978, 1981) in France was an early user of vesica characters of Noctuidae, and in Japan KISHIDA & YOSHIMOTO (1977, 1979), SUGI (1979) and YOSHIMOTO (1982) started to evert vesicas at the same time. In the British Museum (presently The Natural History Museum), S. Fletcher pulled out vesicas with forceps in the 1950s and 1960s. A. Watson was the first to evert them with syringe in the late 1950s (WATSON, 1957, 1961); G. Robinson and J. Holloway continued this work in the 1970s (ROBINSON, 1971; HOLLOWAY, 1977). By the late 1980s this procedure had become commonplace in Europe in that the most well-known noctuid taxonomists were using it (e. g. FIBIGER et al., 1984, 1985; RONKAY & HACKER, 1985; RONKAY, 1986; RONKAY & RONKAY, 1986; RONKAY & VARGA, 1986). After 1986, the number of papers reporting vesica evertions in- creased abruptly. The method has been reviewed by, e. g., HARDWICK (1950), LAFONTAINE & MIKKOLA (1987) and FIBIGER & GOATER (1997). In addition to Noctuoidea, the eversion technique has been applied to or analysed in relatively few groups of Lepidoptera, e.g. in Sphingidae (McCABE, 1984), and Tortricidae (DANG, 1993). In Geometroidea, the vesicas have been used in the genera Cleora (ROBINSON, 1971), Eupithecia (BOLTE, 1990, MIKKOLA ,1993), Operophtera (TROUBRIDGE & FITZPATRICK, 1993), Rho- dostrophia (KAILA et al., 1996) and Entephria (TROUBRIDGE, 1997), in the tribe Cidarini (CHOI, 1997), in a species pair of the genus Selidosema (SIHVONEN & MIKKOLA, 2002) and in the drepanids (WATSON, 1957, 1961). BOLTE (1990) and DANG (1993) improved eversion technique in small specimens by placing aedeagus in the lumen of the injection needle. The same technique is necessary even in such large but- terflies as Colias with extremely thin aedeagus (MIKKOLA, unpubl.).

The taxonomic significance of internal genitalia characters

MIKKOLA et al., (1991) studied Holarctic pairs of sister species in the Noctuidae and noted that “if the divergence concerns the inner organs which are crucial for the sperm transfer, and if the corresponding differentiation is observed in both sexes, the conclusion that the taxa are not conspecific has a sound and functional basis”. As mentioned, internal genitalia have proved crucial in the research of relationships among the Holarctic taxa. In current taxonomic investigations of certain lepidopteran groups, such as the Noctuidae, the internal genitalia are routinely everted. The characters are by and large qualitative, such as “vesica with a subapical diverticulum” instead of two lateral ones or “with two coils” instead of one. Correspon- ding anatomy is found in the females, although the walls of many structures are folded and flexible and they may therefore look smaller in size than the reciprocal parts of the male. We have not utilized any characters such as “vesica longer” or “bursa more massive” but such characters may occur along with qualitative ones. Recently MUTANEN (2005) has demonstrated how deceptive relative characters can be. There is a very limited amount of intrapopulation or geographical variation in internal genitalia. For instance, no transcontinental clines were found. This is different to the external genitalia. Thus, the shape of the valva of the noctuid Celaena leucostigma (Hübner) shows a cline through the

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Palaearctic region (MIKKOLA, unpubl.). Unexpectedly, MUTANEN et al., (2006) found less variation in the external genitalia in the genus Euxoa than in the internal genitalia, but this may be connected with the facts that in this genus the external genitalia show unusually low interspecific variation and that only vesica was included in the internal genitalia. The general shape of the male vesica and, of course, the female bursa, is often a synapomorphy of all species in a genus (for instance in noctuids such as Euxoa, Xylomoia, possibly Xestia) or of all taxa in a species group (e.g. Apamea). The specific level is denoted by more detailed characteristics, which may be synapomorphic inside infrageneric groups or autapomorphic in single species. More general characters may be particularly useful when constructing family-level cladograms, while detailed characters are best suited to classification of taxa from specific to generic level.

Conclusions

The eversion technique has produced much credibility and stability in lepidopteran taxonomy, and, correspondingly, decreased the proportion of subjective identifications. It has also shed light on the evolution of the diversity of insect genitalia in general. Unfortunately, the characters of internal genitalia do not provide much help in the problems of suprageneric taxonomy, particularly in such a derived group as Noctuoidea. It is hoped that in the near future the characters of internal genitalia be used more thoroughly in many other groups of Lepidoptera (such as Papilionoidea) creating compa- risons with the Noctuidae as well as in other insect groups where eversible intromittent male organs exist.

Acknowledgements

I am indebted to Drs. J. D. Holloway (London) and J. D. Lafontaine (Ottawa) for notes on the history of eversion technique, to Mr. Michael Fibiger (Sorø) for notes on literature, and to Dr. Ph. Gould (Rothamsted) for the thorough linguistic revision.

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K. M. Finnish Museum of Natural History University of Helsinki P. O. Box 17 FIN-00014 Helsinki FINLANDIA / FINLAND E-mail: [email protected]

(Recibido para publicación / Received for publication 12-III-2007) (Revisado y aceptado / Revised and accepted 16-IV-2007)

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a. KOH H2O + 40 % alk.

c. abs. alk.

d. abs. alk.

Fig. 1.– The technique of everting a vesica (after LAFONTAINE & MIKKOLA, 1987).

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Figs. 2.-3.– 2. The grand old man of eversion technique of lepidopteran genitalia, D. F. Hardwick (†; right), and his successful successor, J. D. Lafontaine (middle); the author on the left. Ottawa, Canada, 25th August, 1992. Photo: KM. 3. Social study of lepidopteran genitalia at the VII Congress of Societas Europaea Lepidopterologica at Lunz am See, Austria, September 1990. Videomicroscope was for the first time used. Yela has provided Fibiger with a Cucullia slide, Skou, Sauter and Malicky (the organiser), watch attentively but Ronkay prefers to check the specimens. Photo: K. M.

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