The ultrastructure of the spermatozoon of the oligochaetoid polychaete Questa sp. (Questidae, Annelida) and its phylogenetic significance

JOURNALOF ULTRASTRUCTURERESEARCH 84, 238-251 (1983)

The Ultrastructure of the Spermatozoon of the Oligochaetoid Polychaete Questa sp. (Questidae, Annelida) and Its Phylogenetic Significance B. G. M. JAMIESON Department of Zoology, University of Queensland, Brisbane 4067, Australia Received June 13, 1983 Questids are clitellate annelids with oligochaetoid features but spermatozoal ultrastructure confirms that they are not oligochaetes. The very elongate acrosome vesicle and contained tubular perforatorium, somewhat exceeding 5 #m, are followed by a highly condensed elongate nucleus. Between acrosome and nucleus a subacrosomal and a narrow nuclear plate intervene. Behind the nucleus a complex centriolar apparatus with evidence of two centrioles, of which the distal has triplets and accessory microtubules, gives rise to a long glycogen-containing flagellum. At least 12 ~m of the axoneme, constituting the midpiece, is invested by two mitochondrial derivatives, each with a narrow crescentic transverse profile, posteriorly limited by an annulus. The axoneme is of the 9 + 2 type but in the mitochondiral region as many as 27 accessory peripheral microtubules, in 9 triads, encircle it. Elongation of the primary vesicle and of the mitochondrial derivatives, and presence of an extensive axial rod, are seen in sperm of protodrilid archiannelids with which, as with dinophilids, questids have somatic similarities indicative of a relationship closer than with other annelids. The accessory flagellar microtubules, unique in the Annelida, may prove a distinctive autapomorphy of the Questidae. Questids are retained in the Polychaeta pending clarification of polychaete-archiannelid relationships and the new name Euclitellata is proposed for the former class Clitellata.

Questids are small, littoral and sublittoral interstitial worms, maximally a few millimeters long, provisionally referred to the Polychaeta in the absence of a clear apomorphy for that class. The distribution of the five known species (Pacific and Atlantic coasts of North America, Galapagos Islands, Bermuda and, in the present account, the Great Barrier Reef of Australia) suggests that the group will be found to have a world wide distribution as has been shown for mai n e oligochaetes, though first indications are that species diversity will prove to be much lower than that for marine oligochaetes. The family Questidae, and the genus Questa, was first defined and named by Hartman (1966) for a single species, Q. caudicirra, collected in marine benthos from 6 fathoms at Santa Catalina Island and from 68 fathoms at Lasuen Sea Mount, southern California. This species was later reported

from British Columbia, at a depth of 7 m, by Hobson (1976). A further species collected from depths of 7-19 metres in Cape Cod Bay was referred to a new genus and species, Novaquesta trifurcata, by Hobson (1970) and a third species, Q. media, from the sublittoral of Santa Cruz, Galapagos Islands, has recently been added by Westheide (1981). In an interesting and seminal paper, Giere and Riser (1981) have added to our knowledge of the anatomy of N. trifurcata and Q. caudicirra from types and from additional material from the intertidal of northern Maine and New Brunswick, correcting and extending the descriptions of both species. They allude to the existence of a fourth, undescribed, species from Bermuda. Hartman (1966) at first considered Questa to have affinities with oligochaetes but rejected this in favor of an alliance with the paraonid polychaetes. Fauchald (1977) has 238

0022-5320/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

SPERMATOZOON OF Questa since revived the idea that they m a y be oligochaetes. Giere and Riser (1981), recognizing the phylogenetically enigmatic position ofquestids, have coined for t h e m the epithet "oligoehaetoid polychaetes." They have shown that questids, though dioecious, resemble oligochaetes in having a clitellum and producing a cocoon, in storing sperm in spermathecae (presumably received by mutual exchange o f sperm in copulation) and in the limited n u m b e r o f gonadal segments. Hair and bifid setae are further similarities. They have demonstrated, nevertheless, in a p e n e t r a t i n g analysis, t h a t questids do not fit into the concept o f true Clitellata. Their observations "tenuously allow the Questidae to be retained in the class Polychaeta" though they rightly refer to the heterogeneous and possibly polyphyletic nature o f polychaetes, including archiannelids. The present investigation is directed to an ultrastructural description o f the sperm o f a fifth species o f questid, from Lizard Island on the Great Barrier Reef, as an aid to determination o f the affinities o f the Questidae. This species has the posterior gills which at present distinguish Questa from Novaquesta and will be described taxonomically in a separate work. The s p e r m a t o z o a o f the Oligochaeta show a high structural consistency and, with those o f other true clitellates which have been investigated (leeches and branchiobdellids), are unique in possessing an acrosomal tube which supports and usually contains the acr o s o m e vesicle (see Jamieson, 1981, 1983) excepting the apparently convergent acquisition o f an acrosome tube in the n e m a t o m o r p h Gordius (Lora L a m i a D o n i n and Cotelli, 1977). Oligochaete sperm resemble only the O n y c h o p h o r a in interpolation o f the m i t o c h o n d r i a o f the midpieee between the nucleus and the basal b o d y o f the flagellum. Where advanced sperm occur in polychaetes and arehiannelids they are more diverse than in oligochaetes and lack an acrosome tube and interpolated m i t o c h o n dria. Presence or absence o f an acrosome

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tube will therefore be a key character in determining the relationships o f the Questidae, MATERIAL AND METHODS Specimens of the questid which is the subject of this paper were kindly collected by Dr. C. Ersrus, at the author's request, from sublittoral coral sands at Lizard Island in November 1982. All specimens were fixed whole in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 2 hr at 4°C and transferred to the buffer for transportation to Brisbane. Some days later they were postfixed in similarly buffered 1% osmium tetroxide solution for 80 min, rinsed in the buffer, dehydrated in a graded ethanol series (at 4°C from 20 through 70%, thereafter at 20°C), and embedded in Spurr's low-viscosityepoxy resin. Thin sections of regions containing testes and seminal vesicles or spermathecae were cut with glass knives on an LKB 2128 UM IV ultrotome and mounted on Formvar-coated H200 copper grids. Sections were stained with 5% aqueous uranyl acetate for 40 rain and Reynold's lead citrate for 20 min. Micrographswere taken on a Philips 400 and an Hitachi 300 electron microscope at 60-80 kV. RESULTS

General Morphology of the Spermatozoon N o differences were observed in mature sperm from the testes or seminal vesicles. S p e r m a t h e c a l s p e r m a p p e a r e d to be undergoing histolysis; in view o f the presence o f developing stages, and the low frequency o f patently a b n o r m a l sperm, in the testes this dissolution would not seem to indicate parthenogenesis and is possibly a seasonal p h e n o m e n o n in the female or the result o f adverse conditions in the few hours that they were maintained in the laboratory after sorting.

The Acrosome The acrosome (Figs. 1, 29, 34) is a cylinder gradually tapering to the d o m e d tip, 0.47 # m wide at its greatest, basal diameter and 0.23 # m wide at its narrow anterior end at the base o f the apical dome. Its length, from tip to the base o f the subacrosomal plate, is 5.2 and 5.6 # m in two complete profiles obtained. It, and the remainder o f the sperm, is covered by by a well-defined plasma m e m b r a n e which consists o f a very

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dense layer and, external to this, a slightly wider m o d e r a t e l y lucent glycocalyx. The d o m e d tip (Figs. 1, 30), beneath the p l a s m a m e m b r a n e , consists in longitudinal section o f an inverted U - s h a p e d profile, 0.18 ~tm long, o f the p r o x i m a l region o f the p r i m a r y a c r o s o m e vesicle which surrounds an electron-pale axial (and, strictly, subacrosomal) cavity into which the perforatorium, to be described, protrudes. T h e a c r o s o m e Vesicle is i m m e n s e l y elongate, extending posteriorly almost to the nuclear plate and forming, with the overlying p l a s m a m e m b r a n e , the wall o f the acrosome. The r o u n d e d posterior r i m o f this elongate vesicle usually displays clearly the investing m e m b r a n e o f the vesicle. T h e r i m is succeeded by, a n d fits into, an electron-dense saucer-shaped pad, concavity posterior, the s u b a c r o s o m a l plate (Figs. 1, 31), which bears a short c o l u m n like anterior extension, giving it an inverted T - s h a p e d longitudinal profile. The nuclear plate is a separate density behind this and is f o r m e d by thickening o f the nuclear envelope o v e r the slightly convex or centrally depressed tip o f the nucleus. In longitudinal section (Figs. 1, 30) an inner, axial, layer o f the a c r o s o m e vesicle is usually delimited as an electron-dense zone f r o m a paler outer zone o f similar appearance to the apical d o m e d region o f the vesicle. Although it lies within the m e m b r a n e o f the p r i m a r y vesicle, the dense zone c o m mences abruptly at the base o f the d o m e shaped apical region so that a definite " s t e p " occurs where the d o m e d paler part o f the vesicle acquires the zone adaxially (Figs. 1, 30). Further posteriorly the two zones m a y continue to be recognizable or (Fig. 31) the a c r o s o m e contents between outer and inner a c r o s o m e m e m b r a n e s m a y b e c o m e unif o r m l y electron dense. Internal to the adaxial layer, the core o f the a c r o s o m e is filled by an anteriorly tapering, thick-walled dense tube, the putative perforatorium (Figs. 1, 29-31). The peglike anterior end o f the p e r f o r a t o r i u m (Figs. 1, 30) fills the length o f the terminal d o m e -

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FIGS. 1--8. Questasp., spermatozoon. Acrosome and proximal nucleus, slightly diagrammatized from micrographs. All × 55 000. FIG. 1. Longitudinal section showing the very elongate primary acrosome vesicle (of which only 0.35 of the total length is shown here) enclosing, in the subacrosomal space, the long tubular perforatorium. FIGS. 2-8. Transverse sections corresponding with regions of the longtudinal section with which they are linked by lines; the contents of the primary acrosome vesicle are shown uniformly dense though in longitudinal section, as shown, they may be paler peripherally.

shaped cavity and its r o u n d e d apical region is reminiscent o f the capitulum o f the acr o s o m a l rod o f oligochaete sperm. At the base o f the anterior a c r o s o m a l d o m e the p e r f o r a t o r i u m is 0.033 g m wide a n d has no or a very restricted lumen; the l u m e n be-

SPERMATOZOON OF Questa comes progressively wider posteriad, though irregular and in places slit-like and basally, near the nucleus, is 0.15 ~m wide with a wide lumen. The wide basal lumen is sometimes seen to contain an ill-defined core of granular material which in the younger acrosome is continuous with the column-like axial extension of the subacrosomal plate from which it subsequently detaches. In most cases the extreme terminal region of the anterior dome of the vesicle is paler than the remainder and thus has a micropyle-like appearance (Figs. 1, 30) although the vesicular membranes are intact. T r a n s v e r s e sections d e m o n s t r a t e the above-mentioned changes in the width and shape o f the lumen of the perforatorium (Figs. 2-7, 22-27). They also reveal that, although the external outline of the acrosome vesicle is circular basally (Figs. 7, 27), it has a six-rayed symmetry for most of its length. This stellate appearance is due to development of six equally spaced peripheral protrusions of the vesicle. The external membrane of the acrosome vesicle where it covers the protrusions or rays is joined to a corresponding local plaque-like thickening of the overlying plasma membrane by a variable amount, greatest anteriorly, of paler interstitial material. The rays vary from circumferentially very restricted abrupt projections posteriorly (Figs. 5, 6, 25, 26) to petal-like lobes with progressively smaller intervening subplasmalemmal spaces anteriorly (Figs. 2-4, 22, 23). The contents of the vesicle, including the rays, usually appear moderately to strongly electron dense in transverse section and differentiation of the axial dense layer is less apparent than in longitudinal section or is negligible. The nuclear plate (Figs. 1, 31) which lies directly posterior to, and often contiguous with, the subacrosomal plate is a thickening o f the nuclear envelope over the tip of the nucleus. The envelope continues laterally, unthickened, to contact and internally line the plasma membrane around the elongate nucleus.

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The Nucleus Limitations of material have not permitted examination of whole sperm by light microscopy and no full length profiles of the nucleus have been seen by electron microscopy. The greatest length of a nucleus recorded, 8 izm, may approach entirety, however (Fig. 35). Basal and apical widths are 0.31 and 0.23 lzm, respectively. The nucleus is circular in transverse section (Figs. 8, 28); it is uniformly electron opaque and does not exhibit the occasional circular vacuities often seen in oligochaete sperm nuclei. At its base the nucleus is concave in longitudinal section with a short but conspicuous peg-like extension into the core of the axoneme (Figs. 9, 32).

The Centriolar Complex The basal body of the flagellum (Figs. 10, 36) has the typical structure of a longitudinally orientated centriole, with nine triplets each consisting of an A and B microtubule (corresponding with those of the axonemal doublets but with A internal to and on the same radius as B) and an additional C microtubule. An apparent difference from typical invertebrate centrioles is, however, the persistence of two central singlets (as in rat sperm, Wooley and Fawcett, 1973) as no profile, either transverse or longitudinal, has been obtained in which they are absent. A further difference from typical centrioles is the presence of two additional microtubules, here termed D and E associated with each triplet (Figs. 10, 36). The D tubule lies approximately in the same circle as, and anticlockwise to, C. The E tubule is peripheral to C and D, forming the apex of a triangle made up of the triad C, D, and E. D and E are consistently smaller in diameter than C which equals A and B in diameter. Tubule C, though conjoined to B, is displaced in a clockwise direction so that C and D, and therefore the triad, fills the space between the "doublets" A and B. All microtubules of the centriole are embedded

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FIGS. 9-21. Questasp., spermatozoon. Hagellum and distal nucleus, slightly diagrammatized from micrographs. All × 55 000. FIG. 9. Longitudinal section in the plane of the central singlets. Axonemal microtubules, though hollow, are shown in solid black for clarity. F~os. 10-21. Transverse sections of the flagellum corresponding with regions of the longitudinal section with which they are linked by lines. In Fig. 10, extension of the mitochondria (stippled crescents) around the centriole is enigmatic and presumably reflects variation in the anterior extent of these, a, annulus; amt, accessory microtubules; cc, centriolar complex; dc, dense cylinders; gc, glycocalyx; gl, glycogen; m, mitochondrion; n, nucleus.

in a common moderately electron-dense m a t r i x (Fig. 36). T h e n i n e t r i a d s c o n t i n u e as accessory m i c r o t u b u l e s a l o n g the axon e m e (see below).

I n l o n g i t u d i n a l s e c t i o n (Figs. 9, 32), the c e n t r i o l a r c o m p l e x , w i t h its m a t r i x m a t e rial, is a p p a r e n t as m o d e r a t e l y e l e c t r o n d e n s e m a t e r i a l w h i c h fans o u t f r o m the a n -

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terior ends of the peripheral doublets, and of the accessory microtubules, adjacent to the nucleus and is interrupted axially by the anterior extension, to the nucleus, of the central singlets. This centriolar ring is seen in some longitudinal profiles to send extensions laterally to the plasma membrane and in others (Figs. 9, 32) to have a unilateral adjunct which is interpreted as a persistence o f the proximal centriole demonstrated for the spermatid (Jamieson, in preparation). Behind the centriolar complex, immediately internal to the ring of doublets are two thick hollow cylinders or sheaths of electron-dense material in single file with a short hiatus between them. Each cylinder is approximately 0.13 #m long.

The Midpiece The mitochondrial portion of the axoneme, comprising the midpiece, is immensely long compared with that of most annelid spermatozoa. The length, from centriolar complex to what appears to be a

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glancing section of the annulus, is 12.3 ~m in the longest longitudinal profile obtained. The several rounded mitochondria of the spermatid have transformed, presumably by fusion and possibly also elimination, into the two elongate mitochondrial derivatives which in transverse section form two crescents enclosing the axoneme like parentheses (Figs. 11, 15, 16, 36-40). If the axonemal doublet number 1 (that at a right angle to a line joining the two central singlets to each other) be arbitrarily regarded as dorsal, the mitochondrial derivatives are seen to be almost dorsal and ventral. They are, however, consistently rotated 20 ° anticlockwise relative to the diameter which passes through doublet 1. The center of one mitochondrial crescent thus coincides with the interval between adjacent doublets 1 and 9 and the center of the other with doublet 5 (or less frequently 5-6, 6, or 7). The gap between the two mitochondrial crescents on each side generally corresponds with a sector of 40 °, commonly demarcated by radii passing

FI~S. 22-35. Questa sp., spermatozoon. FIGS. 22-28. Successive transverse sections of the acrosome and, Fig. 28, nucleus in anterior-posterior sequence, corresponding with Figs. 2-8. All × 90 000. FIG. 29. Longitudinal section of the entire length of an acrosome. X 37 200. For explanation see Fig. 1. Fits, 30-33. Successive longitudinal sections of the spermatozoon through, Fig. 30, acrosome tip; Fig. 31, acrosome base; Fig. 32, base of nucleus, the centriolar complex and proximal axoneme; and Fig. 33, posterior end of mitoehondrial derivatives and annulus. All X 90 000. For explanation see Figs. 1 and 9. FIGS. 34 AND 35. Longitudinal sections showing the organization of the entire spermatozoon excepting the greater part of the postcentriolar flagellum. Figure 34, the acrosome and proximal nucleus; Fig. 35, montage of the greatest length of the nucleus observed and the proximal region of the flagellum. Both × 12 000. Fx6s, 36-43. Questasp., spermatozoon. Successive transverse sections in anterior-posterior sequence through the flagellum including the mitochondrial midpiece region. All X 120 000. FI~. 36. Centriole with accessory microtubules. The outer microtubule of each centriolar triplet is an anterior extension of accessory tubule C of the axoneme. The other two accessory tubules, D and E, make up a triad, with C, which extends into the axoneme. Persistence of the crescentic mitochondrial derivatives into the centriolar region is apparently variable. FIGS. 37-40. Midpiece. Figure 37, central to the crescentic mitochondrial derivatives, the 9 + 2 axoneme is surrounded by nine triads of accessory microtubules of which one basal member, D, and the apical member, E, are consistently smaller than the other, C. Peripherally directed radial arms separate the triads; Fig. 38, the apical member of some of the triads has discontinued, leaving six complete triads; Fig. 39, only four complete triads remain; Fig. 40, only the larger, C, tubule of triads persists though in one the smaller, D, basal tubule is still present. FIGS. 41-43. Axoneme posterior to annulus. Accessory mierotubules are sparse or, here, absent. Terminally, Fig. 43, the 9 + 2 pattern has disrupted.

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through adjacent doublets 2 and 3, or, on the other side, the intervals on each side o f doublet 7, with some variation. Cristae, so clearly visible in spermatids, are not generally observable in the narrow mitochondrial derivatives though short lengths o f longitudinal cristae are occasionally seen anteriorly; the mitochondrial lumina (matrices) appear granular and moderately electron dense and each is b o u n d by a single membrane. This m e m b r a n e is separated by a wide, clear gap from a m e m brane which is adherent to the inner face o f the plasma m e m b r a n e and continuous with a m e m b r a n e closely circumscribing the axoneme. This continuous m e m b r a n e is here considered to be the outer mitochondrial membrane; the clear space is presumably the cisterna o f the mitochondrial wall. Immediately behind the m e m b r a n e bounding the posterior limit o f the mitochondria, the annulus (Fig. 17) is seen as a well-defined, circular, dense ring, 0.04 ~m wide on each side, the total width o f the annulus being 0.36 #m. Although several longitudinal profiles o f the annulus have been obtained, only one transverse profile has been seen the quality o f which leaves some doubt that the ring is as continuous as represented diagrammatically in Fig. 17. In transverse section o f the spermatid it consists o f two concentric circles each of nine distinct but closely adjacent elements. The portion o f the axoneme enclosed by the mitochondria has a structure unique for the Annelida and, as far as is known, for spermatozoa o f all groups, for while its core has the classical 9 + 2 configuration, it is s u r r o u n d e d by a c c e s s o r y m i c r o t u b u l e s which, although present also in Onychophora and higher insects, have a peculiar arrangement. At each o f the nine intervals between adjacent doublets is a triad o f three microtubules (Figs. 15, 37) which are clearly continuous with the centriolar triads. Each triad differs, however, in that the C microtubule, which in the centriole is joined to the B microtubule o f the AB doublet-equivalent, is free from the adjacent doublet. A

cross link is often seen connecting the more anticlockwise o f the two basal tubules (D) with the A microtubule o f the anticlockwise axonemal doublet, junction occurring where the dynein arms arise from the A tubule. A link is often seen between members o f the same triad also. D and E are without exception slightly smaller than C which tends to be nearest the radius bisecting the space between adjacent doublets. The larger size and the position o f C identifies it as the outer, C, microtubule o f each centriolar triplet while the small size and the position o f D and E identify them with D and E in the centriole. More posteriorly in the mitochondrial region the n u m b e r o f accessory microtubules is reduced. First the apical m e m b e r o f the triad is lost, often with an intermediate condition (Figs. 11, 38, 39) in which only some (to as few as four) triads are retained. Subsequently all apical microtubules o f the triads are lost (Figs. 16, 40). Ultimately (Fig. 40), as at the annulus (Fig. 17), only nine accessory microtubules typically remain; each lies in the inter-doublet radius and is a persistence o f the larger microtubule. Each doublet o f the 9 + 2 a x o n e m e in the mitochondrial region (Figs. 11, 15, 16, 3 7 40) has two dynein arms on the A tubule, o f which the outer is the more conspicuous, and a radial arm which extends peripherally from the B tubule at its junction with the A tubule and as such partitions the accessory triads or singlets from each other. This radial arm can sometimes be seen to fan out immediately peripherally to the inner ring o f accessory microtubules. Although it has not been traced to the outer plasma m e m brane, it is presumably the homologue o f a Y-link (see Baccetti and Afzelius, 1976). A departure from the classical situation is that while the B microtubule is, as normal, well developed, the A microtubule though clearly visible in some doublets is small and observable with difficulty in others. Behind the annulus accessory microtubules are absent (Figs. 12, 41) or occasionally one to several m a y occur. Posteriorly the flagellum nar-

SPERMATOZOON OF Questa rows, accessory microtubules are absent (Figs. 13, 42), the doublets become singlets (Figs. 14, 18, 19, 43), and all microtubules become disarrayed (Figs. 14, 18, 19, 43) and, in the narrowest, terminal region (Figs. 20, 21) cease. Disruption of the 9 + 2 pattern varies in longitudinal location and the pattern may persist almost to the caudal limit. Glycogen persists for some distance behind the annulus.

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The intrasegmental, virtually equatorial position and ciliated nature of the female ducts contrasts with oligochaetes; in the latter the female duct is unciliated and opens at or behind the posterior septum of the ovarian segment. The dorsal and unpaired spermathecal pores are, as Giere and Riser (1981) note, unusual but not unknown in oligochaetes. In addition to the tubficid Aktedrilus monospermathecus and the enchytraeid Grania monospermatheca cited by those authors, spermathecae are unpaired DISCUSSION and dorsal in some Alluroididae (see BrinkBefore evaluating the evidence which hurst and Jamieson, 1971). It is clear that spermatozoal ultrastructure presents for the a dorsal location (seen also for the single affinities ofquestids, we may note that Giere spermatheca of Questa sp.) has developed and Riser (1981) have already cited char- by parallelism within oligochaetes and as a acters of questids which, despite the re- convergence between these, questids and markably "oligochaetoid" features of the some polychaetes. The dorsal location of family (see Introduction) distinguish them the male pores seen in questids is unknown from oligochaetes. Among the distinguish- in oligochaetes. The a u t h o r (Jamieson, ing features shown by them for Novaquesta 1983b) has already drawn attention to the are: the presence in the vascular system of fact that the male ducts of oligochaetes are an antecerebral loop unrecorded for oligo- not homologues of nephridia contrary to a chaetes and polychaetes; the shape of the statement of Giere and Riser (1981). The intraprostomial brain; and the exact form same appears to be true of the ducts ofquesof the male reproductive apparatus. In this tids, which are probably coelomoducts as apparatus a pair of testes on the posterior presumed for oligochaetes. face of septum 11/12 discharges into paired Dioecism is a further distinction between "sperm sacs" each of which tapers to form questids and the consistently hermaphroa coiled duct near the border of 12/13. Each dite oligochaetes. An exceptional questid duct swells to form a "seminal vesicle" specimen with anlages of testes and vestiges which in turn opens dorsolaterally in 14 of ovaries was interpreted, probably rightly, (from their diagram, not 12 as stated in the by Giere and Riser (1981) as an abnormality text) at the paired male pore in the vicinity in a gonochorist and not as evidence of conof the glandular "dorsal folds." Location of secutive hermaphroditism. The presence of testes in segment 12 is not seen in oligo- a ventral pharyngeal bulb or pad (confirmed chaetes. The sperm-sac should, more cor- for Questa sp.) similar to that of the arrectly, be termed a testis-sac and has a re- chiannelids Dinophilus and Trilobodrilus markable similarity to the testis-sac of (see also Rieger and Rieger, 1975) is a very moniligastrid oligochaetes (Jamieson, 1977). significant difference from oligochaetes. The seminal vesicle is not the equivalent of Demonstration by Giere and Riser (1981) the oligochaete seminal vesicle which is of a clitellum producing a cocoon in quessimply a saclike evagination of the septa of tids (confirmed for Questa sp.) supports the the testis-segment and, although in com- observation of Chapman (1965) that epiparison with some groups (e.g., trematodes) dermal specializations for the production of the term is correct, the term "sperm reser- egg capsules have developed several times voir" is here substituted to distinguish it in polychaetes. It also deprives the Clitellata from the oligochaete seminal vesicle. of one of their two traditionally recognized

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autapomorphies, the clitellum (the other is 1.* The tubular form of the very elongate hermaphroditism). However, these workers perforatorium. This apomorphy is shared rightly do not question the validity of the only with Nereis japonica (Takashima and class. Their recognition that, because the Takashima, 1963) and N. diversicolor (Berquestid clitellum is anterior to the female tout, 1976) but there the perforatorium is pores the cocoon cannot be filled with eggs much shorter. by backward movement of the worm, as 2. The very elongate primary acrosome occurs in oligochaetes, provides a basis for vesicle, immediately beneath the plasma distinguishing the true Clitellata. To distin- membrane. Its length, mean 5.4 urn, far exguish such clitellates (Oligochaeta, Hirudin- ceeds that in most polychaetes with adca, and Branchiobdellida) with preclitellar vanced sperm. In the archiannelids Protoor intraclitellar female pores, and monoe- drilus rubropharyngeus and Dinophilus sp., cism, the name Euclitellata is here intro- however, the acrosomes (measured from line duced for the old class Clitellata. drawings) are approximately 9 and 3 ~m Spermatozoal ultrastructure assumes a long, respectively (Franzen, 1974, 1977b). special significance in providing additional The length of the vesicle varies in oligoapomorphies for euclitellates (see Jamieson, chaetes from less than 1 ~m in nonphreo1983a,b). First, the acrosome tube, lying di- drilid microdriles to more than 14 ~m in rectly under the plasma membrane and car- the l u m b r i c i d Allolobophora chlorotica rying apically, or also enclosing, the primary (Jamieson et al., 1983) but there it is inacrosome vesicle and, secondly, in oligo- vested by the acrosome tube. A vesicle of chaetes, the secondary tube dependent from somewhat similar appearance to that in the posterior rim of the primary vesicle are Questa occurs in some gastropod sperm, e.g., confirmed as autapomorphies of euclitel- Pyrazus ebeninus (see Healy, 1982). lates, notwithstanding convergent acquisi3.* The six-rayed symmetry of the acrotion of an acrosome tube in Gordius referred some vesicle is unique. to in the introduction, as these are entirely 4. A nuclear plate is constant for oligoabsent from the sperm of Questa sp. It is, chaete sperm but an additional subacrohowever, likely that the perforatorium has, somal plate, seen in Questa, has been rein addition to its presumed role in fertiliza- ported elsewhere in annelids only in the tion, an important supporting function for syllid Grubea clavata (Franzen, 1974). 5. The elongate nucleus, with a length of the long acrosome vesicle as the acrosome tube has in euclitellates though in Questa at least 8 ~m, is in this respect the most internal to rather than external to the ves- apomorph for the Polychaeta. This length icle. It must be noted, nevertheless, that the i s , however, approached or exceeded in primary function of the euclitellate acro- sperm of protodrilid and dinophilid arsome tube is effectively adjustment of the chiannelids (Franzen, 1974, 1977b) and in length of the acrosome in relation to the oligochaete sperm (references in Jamieson, thickness of the zona pellucida of the oocyte 1981). Its chromatin is highly condensed in and that it acquires the role of investing the all annelid classes. 6. The axoneme shows remarkable unique vesicle secondarily in a progression toward apomorphies for the Annelida. the higher oligochaetes and the leeches a. Mitochondrial derivatives ensheathing (Jamieson, 1983a,b; Jamieson et al., 1983). Apomorph features of spermatozoal ul- the proximal axoneme are seen in some trastructure of Questa sp. include the fol- polychaetes, e.g., Chitinopoma, and in Dilowing. Those which are not known for oth- nophilus and parallel the axoneme in Proer annelids and which may prove to be todrilus. In Questa they are of a greater length additional autapomorphies for the Questi- (> 12 urn) than is known in any polychaete dae as a whole when the sperm of further but a length for the midpiece of approxispecies are examined are asterisked. mately 70 um in Protodrilus rubropharyn-

SPERMATOZOON OF Questa

geus is the longest known for the Annelida. The longest known for the Oligochaeta is 2.3 ~m in Phreodrilus (Jamieson, 1983a). Rotation of the mitochondrial derivatives 20 ° from the dorsoventral axis in Questa, may be unique. A remarkably similar arrangement of mitochondria, though four in number, similarly limited posteriorly by an annulus occurs in some gastropod sperm, e.g., Pyrazus ebeninus (Healy, 1982). An annulus has not been reported but, because observations are only preliminary, may well be present in archiannelids. An annulus occurs in the mature sperm of the spionid Polydora ciliata where it is known to consist of nine separate elements (Franzen, 1974). An annulus posteriorly delimiting the mitochondria also exists in the sperm of the serpulid Chitinopoma serrula (=Miroserpula inflata) (Franzen, 1982) in which, however, it is separated from the mitochondria by a slight invagination of the plasma membrane. b.* Presence of two postcentriolar dense cylinders has not been reported in other annelids. c.* The presence in mature sperm of accessory microtubules peripheral to the nine normal doublets is unknown in other annelids. Peripheral accessory tubules are seen in Onychophora and in insects from the campodeid Diplura upward (Baccetti, 1979). The arrangement in nine triads in Questa is, nevertheless, unique. Cortical microtubules, seen in onychophoran sperm, are absent. Persistence ofcentrioles and centriolar triplets are plesiomorph features apparently absent from mature euclitellate sperm. d. Glycogen in the tail is apomorph relative to primitive sperm but is widespread in and probably plesiomorph for advanced sperm. Thus, for a modified flagellate sperm (i.e., type 2 spermatozoon sensu Franzen, 1977a) that of Questa is highly advanced, especially in great elongation of the acrosome vesicle and mitochondrial derivatives, presence of a peculiar tubular perforatorium, and presence of accessory axonemal microtubules. The apomorphies enumerated above, and

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particularly the putative autapomorphies, have utility in defining the Questidae from other groups and may be added to other questid autapomorphies, sometimes less satisfactory in being shared or approached by other annelids, cited by Giere and Riser (1981). The ventral Dinophilus- or Trilobodrilus-like buccal organ relates the quesrids to archiannelids and is seen in some polychaetes (e.g., ctenodrilids, amphinomids, and cirratulids). It cannot be regarded as a pleisomorphy as there is no reason to envisage that the first annelids possessed the buccal organ (for a discussion of its origins see Rieger and Rieger, 1975). Presence in archiannelids and some polychaetes may merely be a synapomorphy between two classes of undoubtedly related organisms but it must at least be considered as a possible subgroup autapomorphy, with others, in future attempts to analyze the archiannelidpolychaete assemblage in terms of Hennigian phylogenetic systematics. Whether the Polychaeta which, as at present constituted, lack a distinctive autapomorphy (Giere and Riser, 1981) will survive such an analysis is questionable. If it be considered unrealistic to expect the Polychaeta to be totally monothetic, the possession ofacicular parapodia might define the class polythetically. The significance of the absence of true parapodia in questids and species attributed to the Archiannelida is therefore debatable. Crediting questids with parapodia (Hartman, 1966; Hobson, 1970) has no foundation. Questids thus resemble protodrilid and dinophilid archiannelids not only in some apomorph somatic features such as the buccal organ but also in derived features of their spermatozoa, including elongation of the acrosome and nucleus and development of peri- or para-axonemal mitochondrial derivatives. These specializations of the sperm, also approached in some polychates, are expressions of modified fertilization biology, involving internal fertilization, and contrast with the primitive, externally fertilizing sperm which occur in p o l y g o r d i d archiannelids (Franzen, 1977b) and which

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appear to be basic to the Polychaeta. Sperm structure and related reproductive system and fertilization biology thus support the view of Hermans (1969) that archiannelids are not primitive but are modified annelids primarily adapted for interstitial life but it is debatable that, with or without inclusion of questids, they can be considered as a monophyletic group placeable in the Polychaeta as Hermans proposes. The validity of recognizing them as a monophyletic group is strongly contested by Orrhage (1974). Despite shared spermatozoal apomorphies, questid, protodrilid, and dinophilid sperm do not show the homogeneity which characterizes the Oligochaeta (Jamieson, 1981, 1983a); on the other hand greater diversity occurs within subgroups of some other phyla (for instance the variation in scorpion sperm within the phylum Chelicerata). It is here considered worthy o f consideration that similar trends, if not identical products, in q u e s t i d - p r o t o d r i l i d dinophilid sperm are evidence, bearing in mind pharyngeal similarity, of parallelism in related forms. If this relationship were accepted, it would be necessary if archiannelids were to be regarded as a coherent and monophyletic group to accept that all members, including the polygordids with their simple sperm, and nerillids and saccocirrids the sperm of which await ultrastructural description, are the sole descendants of a common ancestry. Data are as yet sufficient for an attack on this problem using cladistic methods. Furthermore, as Franzen (1977b) has stated, the different types of spermatozoa among archiannelids cannot yet tell us about the relations of the archiannelid genera to specific polychaete families, such relationships representing the antithetic view of archiannelid relationships. The problem of questid and archiannelid affinities will not be settled by summarily relegating them to the Polychaeta without profound analysis, especially in view of the tenuous nature of the latter class. On the other hand, .those families attributed to the Archiannelida should not occupy a special

place merely because of the historical existence of the name. For the present, it is proposed that the Questidae be retained in the Polychaeta while recognizing that their closest affinities appear to lie with non-polygordid archiannelid families. What is made certain by the spermatozoal study, in confirmation of the careful work of Giere and Riser, is that questids are not oligochaetes. It remains to be said that similarities of questids to oligochaetes cannot unequivocally be dismissed as convergences. They may conceivably be the product of parallelism by virtue of mutual descent from an early annelid stock which was in the process of diverging into polychaetes and oligochaetes and which may have been highly variable in fertilization biology. Clark (1964) has argued for an oligochaete-like protoannelid (though a clitellate reproductive mode would not have been envisaged). The author will present spermatozoal evidence elsewhere for a special relationship between euclitellates and onychophorans which suggests that early annelids (possibly before divergence of polychaetes) may have been oligochaete-like not only in somatic morphology but also in development including, conceivably, absence o f a t r o c h o p h o r e . Questids, though not oligochaetes, may be a relict, albeit specialized, of the "experimentation" in morphology and fertilization biology which occurred in this early stock. The author is deeplyindebtedto Dr. Christer Ers6us for collectingmaterial of Questa and thanks Mrs. Lina Daddow and Mr. Richard Webb for technical assistance. The work was made possible by Australian Research Grants Scheme funding which is gratefullyacknowledged. All drawings are by the author.

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SPERMATOZOON OF Questa The Aquatic Oligochaeta of the World. Oliver and Boyd, Edinburgh. CHAPMAN, G. (1965) Biol. Bull. (Woods Hole Mass.) 128, 189-197. CLAaK, R. B. (1964) Dynamics in Metazoan Evolution. Clarendon, Oxford. FAUCHALD,K. (1977) Nat. Hist. Mus. Los Angeles Cty Sci. Set. 28, 1-190. FRANZ~N,A. (1974) in AFZELIUS,B. A. (Ed.), The Functional Anatomy of the Spermatozoon, pp. 267-277, Pergamon, Oxford. FRANZI~N, A. (1977a) Verh. Dtsch. Zool. Ges. 1977, 123-138. FRANZI~N,A. (1977b) Zoon 5, 97-105. FRANZI~N,A. (1982) Int. J. Invertebr. Reprod. 5, 185200. GIERE, O. W., AND RISER, N. W. (1981) Zool. Scr. 10, 95-103. HARTMAN, O. (1966) Allan Hancock Pac. Exped. 19, 187-456. HEALY, J. M. (1982) Zoomorphology 100, 157-175. HERMANS, C. O. (1969) Syst. Zool. 18, 85-102.

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HOBSON, K. IS).(1970) Proc. Biol. Soc. Wash. 83, 191194. HOBSON, K. D. (1976) Canad. J. Zool. 54, 591-596. JAMIESON, B. G. M. (1977) Evol. Theory 2, 95-114. JAMIESON, B. G. M. (1981) The Ultrastructure of the Oligochaeta, Academic Press, London/New York. JAMIESON, B. G. M. (1983a) Zool. Scr. 12, 107-114. JAMIESON,B. G. M. (1983b) Hydrobiologia, in press. JAMIESON,B. G. M., RICHARDS,K. S., FLEMING,T. P., AND ERS~US, C. (1983) Gamete Res., in press. LORA LAMIA DONIN, C., AND COTELLI, F. (1977) J. Ultrastruct. Res. 81, 322-332. ORRHAGE, L. (1974) Z. Morphol. Tiere 79, 1-45. RIEGER, R. M., AND RIEGER,G. E. (1975) Tissue & Cell 7, 267-279. TAKASHIMA,R., AND TAKASHIMA,Y. (1963) Tokushima J. Exp. Med. 10, 117-123. WESTHEIDE, W. (1981) Abh. Math.-Naturwiss. Kl., Akad. Wiss. Lit., Mainz. Mikrofauna Meeresbodens 82, 1-24. WOOLEY, D. M., AND FAWCETT, D. W. (1973) Anat. Rec. 177, 289-301.