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Phylogenetic branching of trichoptera and lepidoptera: An ultrastructural analysis on comparative spermatology
J O U R N A L OF U L T R A S T R U C T U R E RESEARCH
83, 141-147 (1983)
Phylogenetic Branching of Trichoptera and Lepidoptera: An Ultrastructural Analysis on Comparative Spermatology MICHAEL FRIEDL)~NDER
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel Received November 23, 1982 The divergence during the evolution of the sister orders of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from their common ancestors is still subject to disagreement. This is partially due to lack of clarity concerning the phylogenetic position of Zeugloptera which is considered to be either a very primitive suborder of Lepidoptera or an order of its own, more archaic than both Trichoptera and Lepidoptera. The ultrastructural analysis on comparative spermatology reported here shows that dichotomous spermatogenesis producing concomitant, normal, anucleate (apyrene) and nucleate (eupyrene) spermatozoa: (1) is generalized in Lepidoptera as it occurs, in addition to higher Lepidoptera, also in the primitive Hepialoidea, (2) is present in Zeugloptera, and (3) is absent from Trichoptera which produce only nucleate spermatozoa. It is concluded that dichotomous spermatogenesis is an evolutionary novelty of Lepidoptera (including Zeugloptera) that was established at the origin of the order, after the divergence of Trichoptera and Lepidoptera.
Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) are sister orders of insects. Their close relationship is sustained by abundant morphological data, mainly (Ross, 1967; Common, 1975), but also by studies on comparative embryology (Ando and Tanaka, 1980; Miyakawa, 1975; Kobayashi et al., 1981) and by their sharing of unusual cytogenetic features such as female heterogamety, achiasmatic oogenesis, and holocentric chromosomes (Kiuta, 1971; Suomalainen, 1969). Although the close phylogenetic affinity of the two orders appears to be well established, their divergence from their common ancestors is still a controversial subject, partially due to the lack of clarity concerning the phylogenetic position of the Zeugloptera. Many taxonomists consider the Zeugloptera to be a primitive suborder of Lepidoptera which has retained functional mandibles in the adult (Common, 1975), while others recognize Zeugloptera as an order of its own (Hinton, 1958) which is more archaic than either Trichoptera or Lepidoptera (Hinton, 1946). In addition to discrepancies in the evaluation of'the morphologic data, disagreements on comparative karyological analyses (Kiuta,
1968), and the relative paucity of fossils of Trichoptera and Lepidoptera, make the solution of the question of their divergence still more difficult. Comparative spermatology, which has been used as a phylogenetic index in problems of insect taxonomy and evolution (Baccetti, 1979; Dallai, 1979), has not yet been applied to the question of the divergence of Trichoptera and Lepidoptera. Spermatogenesis of"higher" Lepidoptera is dichotomous and produces concomitant nucleate (eupyrene) and anucleate (apyrene) spermatozoa (Meves, 1903; Friedl~inder and Gitay, 1972). Data on spermatozoa of systematic groups regarded as primitive among Lepidoptera, such as Eriocraniidae or Hepialidae, could not be found in the literature. The situation in Trichoptera is not clear; early light-microscope studies (Klingstedt, 1931 a,b) purport to show that dichotomous spermatogenesis is found in this order too, but a more recent ultrastructural analysis demonstrates that only nucleate spermatozoa are produced in hydropsychid caddisflies (Friedl~inder and Morse, 1982). No data on spermatozoa of Zeugloptera could be found in the literature.
141 0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
142
MICHAEL FRIEDLANDER
To elucidate whether the origin o f the Zeugloptera could have indeed preceded the divergence o f Trichoptera and Lepidoptera, we studied the presence o f apyrene sperm a t o z o a in the two suborders o f Trichoptera, in the " p r i m i t i v e " lepidopteran Hepialus sequoiolus and in the zeuglopteran species Epimartyria pardella, and used it as a phylogenetic index. MATERIAL AND METHODS The following species were studied: Higher Lepidoptera: Bombyx mori (Bombycidae), Ephestia cautella (Pyralidae), Ectornyelois ceratoniae (Pyralidae), Hyalophora cecropia (Saturnidae); lower Lepidoptera: Hepialus sequoiolus (Hepialidae); Trichoptera: Agrypinia vestita (Phryganeidae, Integripalpia), Pychnosyche sonso (Limnephilidae, Integripalpia), Glossosoma sp. (Glossosomatidae, Integripalpia), Rhyacophila minora (Rhyacophilidae, Integripalpia), Psilotreta frontalis (Odontoceridae, Integripalpia), Polycentropus sp. (Polycentropodidae,Annulipalpia), Chimarraflorida (Philopotamidae, Annulipalpia); Zeugloptera: Epimartyria pardella (Micropterigidae). Testes, ejaculatory ducts, and female genital ducts were dissected out and fixed in 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. Subsequently, the tissues were postfixed in 1% OsO4, dehydrated, and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and lead citrate and studied under the electron microscope. RESULTS
Higher Lepidoptera. Testes, ejaculatory ducts, and genital ducts o f inseminated females contain eupyrene (nucleate) and apyrene (anucleate) s p e r m a t o z o a which differ profoundly at the ultrastructural level. Only outstanding characteristics which are relevant for distinguishing between the two types o f s p e r m a t o z o a and for their c o m p a r i s o n with s p e r m a t o z o a o f the other systematic groups, are reported here. For detailed descriptions, see Phillips (1971), R i e m a n n and T h o r s o n (1971), and Friedl~inder and Gitay
(1972). The intratesticular eupyrene sperm a t o z o o n has an elongate nucleus and two apposed mitochondrial derivatives that fuse into one structure having a crescent shape in transverse sections. F r o m the cell surface protrude the lacinate appendages which appear in transverse sections as rays o f alternating electron-opaque, electron-lucid bars (Fig. 1). The intratesticular apyrene sperm a t o z o o n has an electron-opaque truncate cone replacing the nucleus at the anterior tip o f the cell. Transverse sections o f the flagellum show two separate m i t o c h o n d r i a l derivatives forming a " V " configuration. The surface o f the apyrene s p e r m a t o z o o n bears no protruding appendages (Fig. 2) similar to those found in the eupyrene sperm a t o z o o n . In the ejaculatory duct, the eup y r e n e s p e r m a t o z o a r e m a i n in b u n d l e s which are surrounded by scattered apyrene spermatozoa. The extratesticular eupyrene s p e r m a t o z o o n lacks the appendages f o u n d in the intratesticular one but is wrapped by a cylindrical sleeve and contains an elect r o n - o p a q u e rod having a c o m m a - s h a p e d transverse section (Fig. 3). The extratesticular apyrene s p e r m a t o z o o n (Fig. 3) is similar to the intratesticular one. Lower Lepidoptera. Testes o f H. sequoiolus contain both eupyrene and apyrene sperm a t o z o a (Fig. 4). However, in contrast to the eupyrene s p e r m a t o z o o n o f the higher Lepidoptera, that o f Hepialus displays (a) a clear separation between the two m i t o c h o n drial derivatives which show well-defined electron-lucid cristae and (b) a lack o f appendages or other cell surface differentiation (Fig. 5). The apyrene s p e r m a t o z o a resemble those o f the higher Lepidoptera (Fig. 6). Trichoptera. The testes and the genital ducts o f the species studied here contain only one kind o f nucleate s p e r m a t o z o a and
F~s. 1-3. HigherLepidoptera. Fla. 1. Intratesticular eupyrene spermatozoa of Ephestia cautella. Transverse sections through the nuclei (N) or the mitochondrial derivatives (M) which are apposed and form a crescent-shaped structure. The lacinate appendages protrude from the cell surface and appear as rays made of alternating electron-opaque, electronlucid bars. × 25 000. FI~. 2. Intratesticular apyrene spermatozoa of E. cautella. Transverse sections showing the separate mito-
TRICHOPTERA AND LEPIDOPTERA BRANCHING
143
chondrial derivatives in a " V " configuration a n d the lack o f appendages protruding f r o m the cell surface. C o m p a r e with Fig. 1. × 25 000. FIG. 3. S p e r m a t o z o a in the ejaculatory duct o f Hyalophora cecropia. T h e eupyrene s p e r m a t o z o a r e m a i n in b u n d l e s w h i c h are s u r r o u n d e d by scattered apyrene spermatozoa. T h e eupyrene s p e r m a t o z o o n (E) lacks the lacinate appendages, w h i c h were present while in the testes, a n d is s u r r o u n d e d by a c o m p l e x sleeve. T h e apyrene s p e r m a t o z o o n (A) r e m a i n s u n c h a n g e d b u t is also s u r r o u n d e d by a sleeve. × 15 000.
144
MICHAEL FRIEDLANDER
FIGS. 4-6. Lower Lepidoptera. Hepialus sequoiolus. Fla. 4. Transverse section through two adjacent testicular cysts containing either eupyrene (E) or apyrene (A) spermatozoa. × 8 000. F~a. 5. Transverse sections of intratesticular eupyrene spermatozoa. The two mitochondrial derivatives are separated and the lacinate appendages are absent, thus differing from the eupyrene spermatozoa of the higher Lepidoptera. Compare with Fig. 1. × 30 000. Fla. 6. Transverse sections of intratesticular apyrene spermatozoa which resemble those of the higher Lepidoptera. Compare with Fig. 2. X 30 000.
lack a p y r e n e s p e r m a t o z o a (Figs. 7 - 1 0 ) . T h e five species s t u d i e d t h a t b e l o n g to the s u b o r d e r I n t e g r i p a l p i a d i s p l a y the g e n e r a l i z e d model of insect spermatozoa containing an
a x o n e m e c o m p o s e d b a s i c a l l y o f the 9 + 2 g r o u p s o f m i c r o t u b u l e s (Figs. 7 a n d 8). T h e s p e r m a t o z o a o f the t w o species o f the s u b o r d e r A n n u l i p a l p i a s t u d i e d here s h o w de-
TRICHOPTERA AND LEPIDOPTERA BRANCHING
145
FIGS. 7-10. Trichoptera. FIGS. 7 AND 8. Integripalpia. Transverse sections through intratesticular spermatozoa of Agrypinia vestita (Phryganeidae) (Fig. 7) and Psychnosyche sonso (Limnephilidae) (Fig. 8), showing the typical 9 + 2 axoneme. × 26 000. FIGS. 9 AND 10. Annulipalpia. Transverse sections through intratesticular spermatozoa showing departure from the generalized model of insect spermatozoa. In Polycentropus sp. (Polycentropodidae) (Fig. 9) the axoneme contains three centrally located microtubules. In Chimarraflorida (Phillopotamidae) (Fig. t0), no typical axoneme is present but the "flagellar" portion of the cell shows two concentric electron-opaque circles separated by a lesser electron-opaque zone. Fig. 9, × 27 000; Fig. 10, × 36 000.
vmtions from the generalized scheme of the i n s e c t s p e r m a t o z o a . I n Polycentropus sp., the axoneme contains three centrally locat-
e d m i c r o t u b u l e s (Fig. 9) i n s t e a d o f t h e t w o f o u n d i n t h a t o f t h e t y p i c a l a x o n e m e . I n C. florida, n o t y p i c a l a x o n e m e c o u l d b e f o u n d .
146
MICHAEL FRIEDLANDER
FI~s. 11 AND 12. Zeugloptera. Epirnartyria pardella (Micropterygidae). FIo. 11. Intratesticular eupyrene spermatozoa lacking lacinate appendages and resembling extratesticular eupyrene spermatozoa of higher Lepidoptera. Compare with Figs. 1 and 3. × 40 000. FIG. 12. Intratesticular apyrene spermatozoa resembling those of the higher and lower Lepidoptera. Compare with Figs. 2 and 6. × 40 000. T h e portion o f the cell corresponding to the flagellum contains an electron-opaque tubular structure with an electron-lucid lumen. In transverse sections this tubular structure shows two concentric electronopaque circles separated by a lesser elect r o n - o p a q u e zone (Fig. 10). Zeugloptera. Testes o f Epimartyria pardella contain b o t h eupyrene a n d apyrene s p e r m a t o z o a which are very similar to the corresponding s p e r m a t o z o a o f the higher L e p i d o p t e r a (Figs. 11 and 12). T h e intratesticular eupyrene s p e r m a t o z o o n o f Epimartyria, however, resembles the extratesticular eupyrene s p e r m a t o z o o n rather than the i n t r a t e s t i c u l a r s p e r m a t o z o o n o f the higher Lepidoptera: it is w r a p p e d by an elaborate cylindrical sleeve and contains, in addition to the m i t o c h o n d r i a l derivatives, an opaque structure showing a c o m m a shaped profile in transverse sections (Fig. 11, c o m p a r e with Fig. 3).
DISCUSSION We a s s u m e that the d i c h o t o m o u s eupyrene-apyrene spermatogenesis is an evolutionary novelty o f L e p i d o p t e r a a n d that the solely nucleate spermatogenesis found in T r i c h o p t e r a is a s y n a p o m o r p h i c character o f the c o m m o n ancestors o f these two orders. T h e following data support this assumption: (1) T h e e u p y r e n e - a p y r e n e spermatogenesis appears to be generalized within Lepidoptera; in addition to their presence in a d v a n c e d Lepidoptera, apyrene sperm a t o z o a are reported here in Hepialidae, considered to be an isolated lepidopteran family conserving m a n y p r i m i t i v e features o f external and internal a n a t o m y . (2) A p y rene spermatogenesis is absent f r o m m o s t o f the systematic groups o f animals, including b o t h apterygote and pterygote insects. (3) It is absent particulary f r o m Trichoptera, as has been reported here for several f a m -
TRICHOPTERA AND LEPIDOPTERA BRANCHING ilies o f t h e t w o s u b o r d e r s o f T r i c h o p t e r a . Earlier light microscope reports (Klingsted, 1931a; 1931b) o n t h e p r e s e n c e o f a p y r e n e s p e r m a t o z o a in l i m n e p h i l i d s m a y b e e r r o n e o u s since we c o u l d n o t find t h e s e s p e r m a t o z o a in t h i s f a m i l y , e i t h e r in m a l e s o r in i n s e m i n a t e d f e m a l e s . T h e s e e a r l y r e p o r t s o n t h e p r e s e n c e o f a p y r e n e s p e r m a t o z o a in limnephilids might stem from the sporadic a p p e a r a n c e in s o m e i n d i v i d u a l s o f a b n o r mal or degenerating nucleate spermatozoa, which could have been confused with normal apyrene spermatozoa. Indeed, degeneration of typical nucleate spermatozoa due to f l u c t u a t i o n s o f t h e e x t e r n a l c o n d i t i o n s h a s b e e n r e p o r t e d for L i m n e p h i l i d a e (Le L a n n ic, 1975, 1976). (4) A p y r e n e s p e r m a t o z o a h a v e n o t b e e n r e p o r t e d in s t u d i e s o n s p e r matogenesis of the closely related Mecopt e r a ( B a c c e t t i et al., 1969; B a c c e t t i , 1979) which have many characters that are more a r c h a i c t h a n t h e h o m o l o g o u s o n e s f o u n d in either Trichoptera or Lepidoptera. The presence of the dichotomous eupyr e n e - a p y r e n e s p e r m a t o g e n e s i s in Z e u g l o p tera strongly supports the idea that they are t y p i c a l l e p i d o p t e r a n s a n d is i n c o n g r u e n t w i t h t h e s u g g e s t i o n t h a t t h e y c o u l d b e m o r e archaic than Trichoptera lacking apyrene s p e r m a t o z o a . F o r t h e l a t t e r to b e true, e u pyrene-apyrene spermatogenesis would e i t h e r h a v e to b e p r e s e n t in t h e c o m m o n a n c e s t o r s o f t h e t w o o r d e r s , o r to d e v e l o p in L e p i d o p t e r a b e f o r e T r i c h o p t e r a b r a n c h e d from their common ancestors, neither of w h i c h is s u p p o r t e d b y a n y d a t a . I thank Professor John C. Morse for identifying the caddisflies and David Wagner for identifying and sup-
147
plying the hepialids and zeuglopterans. This work was funded in part by a Clemson University Alumni Visiting Professorship to M.F. REFERENCES ANDO, H., ANDTANAKA,M. (1980) Int. J. Insect Morphol. Embryol. 9, 67-77. BACCETTI,B. (1979) in GUPTA,A. P. (Ed.), Arthropod Phylogeny, pp. 609-644, Van Nostrand-Reinhold, New York. BACCETT1, B., DALLAI,R., AND ROSATI, F. (1969) J. Microsc. (Paris) 8, 233-248. COMMON, I. F. (1975) Annu. Rev. Entomol. 20, 183203. DALLAI,R. (1979) in FAWCETT,D. W., AND BEDFORD, J. M. (Eds.), The Spermatozoon, pp. 253-265, Urban and Schwarzenberg, Baltimore/Munich. FRIEDL~NDER,M., AND GITAY, H. (1972) J. Morphol. 138, 121-130. FRIEDE~NDER, M., AND MORSE, J. C. (1982) J. Ultrastruct. Res. 78, 84-94. HINTON, H. E. (1946) Trans. R. Entomol. Soc. London 97, 1-37. HINTON, H. E. (1958) Annu. Rev. Entomol. 3, 181206. KrUTA, B. (1968) Genen Phaenen 12, 110-113. KrUTA, B. (1971) Ergeb. Wiss. Unters. Schweis. Nat. Park 9, 174-185. KLINOSrEDT, H. (1931a) Acta Zool. Fenn. 10, 1-69. KI.INOSTEDT,H. (1931b) Not. Entomol. 11, 132-134. KOBAYASHI,Y., TANAKA,M., AND ANDO, H. (1981) Kontyu (Tokyo) 49, 641-652. LE LANNIC, J. (1975) Bull. Soc. Zool. Fr. 100, 539550. LE LAt,~IC,J. (1976) Th~se 36 cycle, Universit6 Rennes. MEVES, F. (1903) Arch. Mikrosk. Anat. Entwicklungsmech. 61, 1-84. MIYAKAWA,K. (1975) Kontyu (Tokyo) 43, 55-74. PHILLIPS, D. M. (1971) J. Ultrastruct. Res. 34, 567585. RIEMANN, J. G., AND TnORSON, B. J. (1971) Int. J. Insect Morphol. Embryol. 1, 11-19. Ross, H. H. (1967)Annu. Rev. Entomol. 12, 169-206. SUOMALAINEN,E. (1969) Chromosomes Today 2, 132138.
83, 141-147 (1983)
Phylogenetic Branching of Trichoptera and Lepidoptera: An Ultrastructural Analysis on Comparative Spermatology MICHAEL FRIEDL)~NDER
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel Received November 23, 1982 The divergence during the evolution of the sister orders of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from their common ancestors is still subject to disagreement. This is partially due to lack of clarity concerning the phylogenetic position of Zeugloptera which is considered to be either a very primitive suborder of Lepidoptera or an order of its own, more archaic than both Trichoptera and Lepidoptera. The ultrastructural analysis on comparative spermatology reported here shows that dichotomous spermatogenesis producing concomitant, normal, anucleate (apyrene) and nucleate (eupyrene) spermatozoa: (1) is generalized in Lepidoptera as it occurs, in addition to higher Lepidoptera, also in the primitive Hepialoidea, (2) is present in Zeugloptera, and (3) is absent from Trichoptera which produce only nucleate spermatozoa. It is concluded that dichotomous spermatogenesis is an evolutionary novelty of Lepidoptera (including Zeugloptera) that was established at the origin of the order, after the divergence of Trichoptera and Lepidoptera.
Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) are sister orders of insects. Their close relationship is sustained by abundant morphological data, mainly (Ross, 1967; Common, 1975), but also by studies on comparative embryology (Ando and Tanaka, 1980; Miyakawa, 1975; Kobayashi et al., 1981) and by their sharing of unusual cytogenetic features such as female heterogamety, achiasmatic oogenesis, and holocentric chromosomes (Kiuta, 1971; Suomalainen, 1969). Although the close phylogenetic affinity of the two orders appears to be well established, their divergence from their common ancestors is still a controversial subject, partially due to the lack of clarity concerning the phylogenetic position of the Zeugloptera. Many taxonomists consider the Zeugloptera to be a primitive suborder of Lepidoptera which has retained functional mandibles in the adult (Common, 1975), while others recognize Zeugloptera as an order of its own (Hinton, 1958) which is more archaic than either Trichoptera or Lepidoptera (Hinton, 1946). In addition to discrepancies in the evaluation of'the morphologic data, disagreements on comparative karyological analyses (Kiuta,
1968), and the relative paucity of fossils of Trichoptera and Lepidoptera, make the solution of the question of their divergence still more difficult. Comparative spermatology, which has been used as a phylogenetic index in problems of insect taxonomy and evolution (Baccetti, 1979; Dallai, 1979), has not yet been applied to the question of the divergence of Trichoptera and Lepidoptera. Spermatogenesis of"higher" Lepidoptera is dichotomous and produces concomitant nucleate (eupyrene) and anucleate (apyrene) spermatozoa (Meves, 1903; Friedl~inder and Gitay, 1972). Data on spermatozoa of systematic groups regarded as primitive among Lepidoptera, such as Eriocraniidae or Hepialidae, could not be found in the literature. The situation in Trichoptera is not clear; early light-microscope studies (Klingstedt, 1931 a,b) purport to show that dichotomous spermatogenesis is found in this order too, but a more recent ultrastructural analysis demonstrates that only nucleate spermatozoa are produced in hydropsychid caddisflies (Friedl~inder and Morse, 1982). No data on spermatozoa of Zeugloptera could be found in the literature.
141 0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
142
MICHAEL FRIEDLANDER
To elucidate whether the origin o f the Zeugloptera could have indeed preceded the divergence o f Trichoptera and Lepidoptera, we studied the presence o f apyrene sperm a t o z o a in the two suborders o f Trichoptera, in the " p r i m i t i v e " lepidopteran Hepialus sequoiolus and in the zeuglopteran species Epimartyria pardella, and used it as a phylogenetic index. MATERIAL AND METHODS The following species were studied: Higher Lepidoptera: Bombyx mori (Bombycidae), Ephestia cautella (Pyralidae), Ectornyelois ceratoniae (Pyralidae), Hyalophora cecropia (Saturnidae); lower Lepidoptera: Hepialus sequoiolus (Hepialidae); Trichoptera: Agrypinia vestita (Phryganeidae, Integripalpia), Pychnosyche sonso (Limnephilidae, Integripalpia), Glossosoma sp. (Glossosomatidae, Integripalpia), Rhyacophila minora (Rhyacophilidae, Integripalpia), Psilotreta frontalis (Odontoceridae, Integripalpia), Polycentropus sp. (Polycentropodidae,Annulipalpia), Chimarraflorida (Philopotamidae, Annulipalpia); Zeugloptera: Epimartyria pardella (Micropterigidae). Testes, ejaculatory ducts, and female genital ducts were dissected out and fixed in 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. Subsequently, the tissues were postfixed in 1% OsO4, dehydrated, and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and lead citrate and studied under the electron microscope. RESULTS
Higher Lepidoptera. Testes, ejaculatory ducts, and genital ducts o f inseminated females contain eupyrene (nucleate) and apyrene (anucleate) s p e r m a t o z o a which differ profoundly at the ultrastructural level. Only outstanding characteristics which are relevant for distinguishing between the two types o f s p e r m a t o z o a and for their c o m p a r i s o n with s p e r m a t o z o a o f the other systematic groups, are reported here. For detailed descriptions, see Phillips (1971), R i e m a n n and T h o r s o n (1971), and Friedl~inder and Gitay
(1972). The intratesticular eupyrene sperm a t o z o o n has an elongate nucleus and two apposed mitochondrial derivatives that fuse into one structure having a crescent shape in transverse sections. F r o m the cell surface protrude the lacinate appendages which appear in transverse sections as rays o f alternating electron-opaque, electron-lucid bars (Fig. 1). The intratesticular apyrene sperm a t o z o o n has an electron-opaque truncate cone replacing the nucleus at the anterior tip o f the cell. Transverse sections o f the flagellum show two separate m i t o c h o n d r i a l derivatives forming a " V " configuration. The surface o f the apyrene s p e r m a t o z o o n bears no protruding appendages (Fig. 2) similar to those found in the eupyrene sperm a t o z o o n . In the ejaculatory duct, the eup y r e n e s p e r m a t o z o a r e m a i n in b u n d l e s which are surrounded by scattered apyrene spermatozoa. The extratesticular eupyrene s p e r m a t o z o o n lacks the appendages f o u n d in the intratesticular one but is wrapped by a cylindrical sleeve and contains an elect r o n - o p a q u e rod having a c o m m a - s h a p e d transverse section (Fig. 3). The extratesticular apyrene s p e r m a t o z o o n (Fig. 3) is similar to the intratesticular one. Lower Lepidoptera. Testes o f H. sequoiolus contain both eupyrene and apyrene sperm a t o z o a (Fig. 4). However, in contrast to the eupyrene s p e r m a t o z o o n o f the higher Lepidoptera, that o f Hepialus displays (a) a clear separation between the two m i t o c h o n drial derivatives which show well-defined electron-lucid cristae and (b) a lack o f appendages or other cell surface differentiation (Fig. 5). The apyrene s p e r m a t o z o a resemble those o f the higher Lepidoptera (Fig. 6). Trichoptera. The testes and the genital ducts o f the species studied here contain only one kind o f nucleate s p e r m a t o z o a and
F~s. 1-3. HigherLepidoptera. Fla. 1. Intratesticular eupyrene spermatozoa of Ephestia cautella. Transverse sections through the nuclei (N) or the mitochondrial derivatives (M) which are apposed and form a crescent-shaped structure. The lacinate appendages protrude from the cell surface and appear as rays made of alternating electron-opaque, electronlucid bars. × 25 000. FI~. 2. Intratesticular apyrene spermatozoa of E. cautella. Transverse sections showing the separate mito-
TRICHOPTERA AND LEPIDOPTERA BRANCHING
143
chondrial derivatives in a " V " configuration a n d the lack o f appendages protruding f r o m the cell surface. C o m p a r e with Fig. 1. × 25 000. FIG. 3. S p e r m a t o z o a in the ejaculatory duct o f Hyalophora cecropia. T h e eupyrene s p e r m a t o z o a r e m a i n in b u n d l e s w h i c h are s u r r o u n d e d by scattered apyrene spermatozoa. T h e eupyrene s p e r m a t o z o o n (E) lacks the lacinate appendages, w h i c h were present while in the testes, a n d is s u r r o u n d e d by a c o m p l e x sleeve. T h e apyrene s p e r m a t o z o o n (A) r e m a i n s u n c h a n g e d b u t is also s u r r o u n d e d by a sleeve. × 15 000.
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MICHAEL FRIEDLANDER
FIGS. 4-6. Lower Lepidoptera. Hepialus sequoiolus. Fla. 4. Transverse section through two adjacent testicular cysts containing either eupyrene (E) or apyrene (A) spermatozoa. × 8 000. F~a. 5. Transverse sections of intratesticular eupyrene spermatozoa. The two mitochondrial derivatives are separated and the lacinate appendages are absent, thus differing from the eupyrene spermatozoa of the higher Lepidoptera. Compare with Fig. 1. × 30 000. Fla. 6. Transverse sections of intratesticular apyrene spermatozoa which resemble those of the higher Lepidoptera. Compare with Fig. 2. X 30 000.
lack a p y r e n e s p e r m a t o z o a (Figs. 7 - 1 0 ) . T h e five species s t u d i e d t h a t b e l o n g to the s u b o r d e r I n t e g r i p a l p i a d i s p l a y the g e n e r a l i z e d model of insect spermatozoa containing an
a x o n e m e c o m p o s e d b a s i c a l l y o f the 9 + 2 g r o u p s o f m i c r o t u b u l e s (Figs. 7 a n d 8). T h e s p e r m a t o z o a o f the t w o species o f the s u b o r d e r A n n u l i p a l p i a s t u d i e d here s h o w de-
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FIGS. 7-10. Trichoptera. FIGS. 7 AND 8. Integripalpia. Transverse sections through intratesticular spermatozoa of Agrypinia vestita (Phryganeidae) (Fig. 7) and Psychnosyche sonso (Limnephilidae) (Fig. 8), showing the typical 9 + 2 axoneme. × 26 000. FIGS. 9 AND 10. Annulipalpia. Transverse sections through intratesticular spermatozoa showing departure from the generalized model of insect spermatozoa. In Polycentropus sp. (Polycentropodidae) (Fig. 9) the axoneme contains three centrally located microtubules. In Chimarraflorida (Phillopotamidae) (Fig. t0), no typical axoneme is present but the "flagellar" portion of the cell shows two concentric electron-opaque circles separated by a lesser electron-opaque zone. Fig. 9, × 27 000; Fig. 10, × 36 000.
vmtions from the generalized scheme of the i n s e c t s p e r m a t o z o a . I n Polycentropus sp., the axoneme contains three centrally locat-
e d m i c r o t u b u l e s (Fig. 9) i n s t e a d o f t h e t w o f o u n d i n t h a t o f t h e t y p i c a l a x o n e m e . I n C. florida, n o t y p i c a l a x o n e m e c o u l d b e f o u n d .
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MICHAEL FRIEDLANDER
FI~s. 11 AND 12. Zeugloptera. Epirnartyria pardella (Micropterygidae). FIo. 11. Intratesticular eupyrene spermatozoa lacking lacinate appendages and resembling extratesticular eupyrene spermatozoa of higher Lepidoptera. Compare with Figs. 1 and 3. × 40 000. FIG. 12. Intratesticular apyrene spermatozoa resembling those of the higher and lower Lepidoptera. Compare with Figs. 2 and 6. × 40 000. T h e portion o f the cell corresponding to the flagellum contains an electron-opaque tubular structure with an electron-lucid lumen. In transverse sections this tubular structure shows two concentric electronopaque circles separated by a lesser elect r o n - o p a q u e zone (Fig. 10). Zeugloptera. Testes o f Epimartyria pardella contain b o t h eupyrene a n d apyrene s p e r m a t o z o a which are very similar to the corresponding s p e r m a t o z o a o f the higher L e p i d o p t e r a (Figs. 11 and 12). T h e intratesticular eupyrene s p e r m a t o z o o n o f Epimartyria, however, resembles the extratesticular eupyrene s p e r m a t o z o o n rather than the i n t r a t e s t i c u l a r s p e r m a t o z o o n o f the higher Lepidoptera: it is w r a p p e d by an elaborate cylindrical sleeve and contains, in addition to the m i t o c h o n d r i a l derivatives, an opaque structure showing a c o m m a shaped profile in transverse sections (Fig. 11, c o m p a r e with Fig. 3).
DISCUSSION We a s s u m e that the d i c h o t o m o u s eupyrene-apyrene spermatogenesis is an evolutionary novelty o f L e p i d o p t e r a a n d that the solely nucleate spermatogenesis found in T r i c h o p t e r a is a s y n a p o m o r p h i c character o f the c o m m o n ancestors o f these two orders. T h e following data support this assumption: (1) T h e e u p y r e n e - a p y r e n e spermatogenesis appears to be generalized within Lepidoptera; in addition to their presence in a d v a n c e d Lepidoptera, apyrene sperm a t o z o a are reported here in Hepialidae, considered to be an isolated lepidopteran family conserving m a n y p r i m i t i v e features o f external and internal a n a t o m y . (2) A p y rene spermatogenesis is absent f r o m m o s t o f the systematic groups o f animals, including b o t h apterygote and pterygote insects. (3) It is absent particulary f r o m Trichoptera, as has been reported here for several f a m -
TRICHOPTERA AND LEPIDOPTERA BRANCHING ilies o f t h e t w o s u b o r d e r s o f T r i c h o p t e r a . Earlier light microscope reports (Klingsted, 1931a; 1931b) o n t h e p r e s e n c e o f a p y r e n e s p e r m a t o z o a in l i m n e p h i l i d s m a y b e e r r o n e o u s since we c o u l d n o t find t h e s e s p e r m a t o z o a in t h i s f a m i l y , e i t h e r in m a l e s o r in i n s e m i n a t e d f e m a l e s . T h e s e e a r l y r e p o r t s o n t h e p r e s e n c e o f a p y r e n e s p e r m a t o z o a in limnephilids might stem from the sporadic a p p e a r a n c e in s o m e i n d i v i d u a l s o f a b n o r mal or degenerating nucleate spermatozoa, which could have been confused with normal apyrene spermatozoa. Indeed, degeneration of typical nucleate spermatozoa due to f l u c t u a t i o n s o f t h e e x t e r n a l c o n d i t i o n s h a s b e e n r e p o r t e d for L i m n e p h i l i d a e (Le L a n n ic, 1975, 1976). (4) A p y r e n e s p e r m a t o z o a h a v e n o t b e e n r e p o r t e d in s t u d i e s o n s p e r matogenesis of the closely related Mecopt e r a ( B a c c e t t i et al., 1969; B a c c e t t i , 1979) which have many characters that are more a r c h a i c t h a n t h e h o m o l o g o u s o n e s f o u n d in either Trichoptera or Lepidoptera. The presence of the dichotomous eupyr e n e - a p y r e n e s p e r m a t o g e n e s i s in Z e u g l o p tera strongly supports the idea that they are t y p i c a l l e p i d o p t e r a n s a n d is i n c o n g r u e n t w i t h t h e s u g g e s t i o n t h a t t h e y c o u l d b e m o r e archaic than Trichoptera lacking apyrene s p e r m a t o z o a . F o r t h e l a t t e r to b e true, e u pyrene-apyrene spermatogenesis would e i t h e r h a v e to b e p r e s e n t in t h e c o m m o n a n c e s t o r s o f t h e t w o o r d e r s , o r to d e v e l o p in L e p i d o p t e r a b e f o r e T r i c h o p t e r a b r a n c h e d from their common ancestors, neither of w h i c h is s u p p o r t e d b y a n y d a t a . I thank Professor John C. Morse for identifying the caddisflies and David Wagner for identifying and sup-
147
plying the hepialids and zeuglopterans. This work was funded in part by a Clemson University Alumni Visiting Professorship to M.F. REFERENCES ANDO, H., ANDTANAKA,M. (1980) Int. J. Insect Morphol. Embryol. 9, 67-77. BACCETTI,B. (1979) in GUPTA,A. P. (Ed.), Arthropod Phylogeny, pp. 609-644, Van Nostrand-Reinhold, New York. BACCETT1, B., DALLAI,R., AND ROSATI, F. (1969) J. Microsc. (Paris) 8, 233-248. COMMON, I. F. (1975) Annu. Rev. Entomol. 20, 183203. DALLAI,R. (1979) in FAWCETT,D. W., AND BEDFORD, J. M. (Eds.), The Spermatozoon, pp. 253-265, Urban and Schwarzenberg, Baltimore/Munich. FRIEDL~NDER,M., AND GITAY, H. (1972) J. Morphol. 138, 121-130. FRIEDE~NDER, M., AND MORSE, J. C. (1982) J. Ultrastruct. Res. 78, 84-94. HINTON, H. E. (1946) Trans. R. Entomol. Soc. London 97, 1-37. HINTON, H. E. (1958) Annu. Rev. Entomol. 3, 181206. KrUTA, B. (1968) Genen Phaenen 12, 110-113. KrUTA, B. (1971) Ergeb. Wiss. Unters. Schweis. Nat. Park 9, 174-185. KLINOSrEDT, H. (1931a) Acta Zool. Fenn. 10, 1-69. KI.INOSTEDT,H. (1931b) Not. Entomol. 11, 132-134. KOBAYASHI,Y., TANAKA,M., AND ANDO, H. (1981) Kontyu (Tokyo) 49, 641-652. LE LANNIC, J. (1975) Bull. Soc. Zool. Fr. 100, 539550. LE LAt,~IC,J. (1976) Th~se 36 cycle, Universit6 Rennes. MEVES, F. (1903) Arch. Mikrosk. Anat. Entwicklungsmech. 61, 1-84. MIYAKAWA,K. (1975) Kontyu (Tokyo) 43, 55-74. PHILLIPS, D. M. (1971) J. Ultrastruct. Res. 34, 567585. RIEMANN, J. G., AND TnORSON, B. J. (1971) Int. J. Insect Morphol. Embryol. 1, 11-19. Ross, H. H. (1967)Annu. Rev. Entomol. 12, 169-206. SUOMALAINEN,E. (1969) Chromosomes Today 2, 132138.