Abstract The reproductive systems and especially thenidamental glands of 20 species of Opisthobranchia be-longing to the “Cephalaspidea s. l.”, Anaspidea, Saco-glossa, Tylodinoidea and Pleurobranchoidea, have beeninvestigated histologically and ultrastructurally. Thenidamental glandular system is responsible for the for-mation of the egg masses. In all investigated species it isdivided into three distinct parts. The most proximal partcan be an albumen gland (some “Cephalaspidea s. l.”,Anaspidea and Sacoglossa) or can exhibit a capsulegland (some “Cephalaspidea s. l.”, Tylodinoidea andPleurobranchoidea). All species additionally possess amembrane gland and a distally lying mucous gland. Insome species the most distal part of the oviduct was alsofound to be glandular. The structure of the nidamentalglands is described and compared within the Opistho-branchia. Albumen and capsule glands are found to behomologous glandular parts of the system. It can be con-cluded that the albumen gland has undergone a structuraland functional change within the evolution of theOpisthobranchia.

A. Introduction

The Opisthobranchia are hermaphroditic animals whichpossess structurally and functionally complex reproduc-tive systems. Many functional problems occur when thesame individual has to perform both sex roles (such asexpulsion of gametes, reception of allosperm and forma-tion of the egg masses). Thus, within the evolution, theOpisthobranchia have evolved a diverse array of her-maphroditic structures to meet these needs. The nida-mental glands are the part of the female system held responsible for building the gelatinous egg masses.

Our current knowledge about the reproductive systems of the Opisthobranchia is mainly based on ana-

tomical studies (see, for example, Guiart 1901; Pruvot-Fol 1960; Schmekel 1970; Sanders-Esser 1984). Infor-mation about the histology and ultrastructure of thenidamental glands in the various opisthobranch taxa arescattered throughout the literature (Cephalaspidea: Fretter and Graham 1954; Rudman 1971, 1972a, b, c,1974; Kress and Schmekel 1992; Anaspidea: Mazzarelli1891; Eales 1921; Thompson and Bebbington 1969; Beeman 1970; Coggeshall 1972; Thomas 1975; Saco-glossa: Sanders-Esser 1984; Pleurobranchoidea: Wägeleand Hain 1991), but extensive comparative studies with-in the major opisthobranch taxa are lacking to date. Inthe Euthyneura a three-part nidamental glandular systemwith a proximally lying albumen gland, a membrane anda distally located mucous gland is found. This situationhas been described for the Gymnomorpha (Fretter1943), the Pulmonata (De Jong Brink 1969; Plesch et al.1971) and the Pyramidellidae (Fretter and Graham1949), and also for the Opisthobranchia (see, for exam-ple, Ghiselin 1965). A three-part nidamental glandularsystem, as proposed for the hypothetical ancestor of theOpisthobranchia by Ghiselin, thus represents the plesio-morphic state. Although Ghiselin himself investigatedonly very few Nudibranchia, he still drew his overallconclusions for all Opisthobranchia. In Ghiselin’s opin-ion a three-part nidamental glandular system is alsofound in all recent opisthobranch taxa. Schmekel(1985), in contrast, believed that the Pleurobranchoideaand the Nudibranchia have lost the albumen gland. Onthe other hand, Wägele (1989) and Klussmann-Kolb(2001), described three different glandular structureswithin the nidamental glands of nudibranchs. This in-consistency demonstrates the need for clarification ofthis matter. Moreover a hypothesis about the homo-logies of the different glandular structures in the nida-mental glands within the Opisthobranchia based on ex-tensive comparative studies is lacking to date. The ter-minology of the different glandular parts is very incon-sistent throughout the literature which makes it difficultto homologise the various parts within the Opisthobran-chia. Thus a uniform terminology, as presented here, is

A. Klussmann-Kolb (✉ )Lehrstuhl für Spezielle Zoologie, Gebäude ND05/755, Ruhr-Universität Bochum, 44780 Bochum/GermanyTel.: +49-234-3224585, Fax: +49-234-32-14114

Zoomorphology (2001) 120:215–235 © Springer-Verlag 2001

O R I G I N A L A RT I C L E

Annette Klussmann-Kolb

Comparative investigation of the genital systems in the Opisthobranchia (Mollusca, Gastropoda) with special emphasison the nidamental glandular system

Accepted: 26 December 2000

necessary for future comparative studies of this organsystem. This paper is part of an investigation of the ho-mology and functional morphology of the nidamentalglands of the Opisthobranchia. Histology and fine struc-ture of the nidamental glands of representatives of theOpisthobranchia except for the Nudibranchia are in thefocus of the present study. Histochemical investigationsserve to further characterise the substances which areproduced in the different glandular parts. The results arecompared to previous comparative studies on the nida-mental glands (Klussmann-Kolb 2001) and the eggmasses of Opisthobranchia (Klussmann-Kolb and Wägele2001). Based on this comparison various parts of thenidamental glands in the Opisthobranchia species stud-ied are homologised in order to find possible traits ofnidamental gland evolution within the Opisthobranchia.

B. Materials and methods

Twenty species of Opisthobranchia have been investigated. Referto Table 1 for species names, collection sites and methods applied.The animals were collected in the intertidal or subtidal areas andmost were kept in aerated aquaria overnight in the hope of egglaying.

For histological and histochemical investigations the sampleswere preserved in 4–6% formalin buffered in seawater. After about4 weeks of fixation the samples were transferred to 70% ethanol.Shelled animals were treated for a short time (one to a few hours)with a solution of 10% formalin and 5% formic acid in seawater todecalcify the shell. After dehydration in a graded ethanol series of increasing concentration, animals or dissected reproductive systems were embedded in methacrylate resin (Technovit 7100;Kulzer). Serial sections (2.5 µm thick) were made for reconstruc-tion of the organ systems and structures of interest. The sectionswere stained with toluidine blue and additional sections were alsostained with standard histochemical stains such as periodic acid-Schiff (PAS; indication of neutral mucopolysaccharides), alcianblue (indication of acidic, sulphated mucopolysaccharides), bromo-phenol blue (indication of proteins) and azan (differentiation of secretory products). For the azan reaction samples had to be em-bedded in paraffin. For various staining reactions refer to Table 2.

Small samples were also preserved in glutaraldehyde bufferedin seawater or cacodylate buffer. Afterwards the samples werepostfixed in OsO4 also buffered in seawater or cacodylate buffer,respectively. Ultrathin sections (70–90 nm) were stained with uranyl acetate and lead citrate and studied using a Hitachi H 500TEM and a Zeiss EM 109.

C. Results

I. Terms and general structure of the nidamental glands

As can be seen from the literature on Opisthobranchia,the terminology of the different parts of the nidamentalglandular system has been very inconsistent in the past(Table 3). This strengthens the need for a uniform termi-nology in future studies dealing with this organ system.In this paper the terms of Ghiselin (1965) and others(Beeman 1970; Schmekel 1970, 1971, 1985; Thompson1976; Hadfield and Switzer-Dunlap 1984) are adopted,because, as will be shown in this study, these terms bestmatch the actual situation found in the various parts in-

vestigated. During the discussion it will be shown thatby using these terms presumed homologous structureswill be termed in the same manner.

In the Opisthobranchia, gametes, produced in the hermaphroditic gonad, are transported via the hermaphro-ditic duct through the ampulla to the postampullary duct.This duct can lead to an undivided pallial gonoduct (monaulic) or can split into two (diaulic) or three (triaulic)separate channels (vas deferens, vagina, oviduct). Oftenassociated with the vas deferens are a glandular prostateand a muscular penis. Two different kinds of pouches canbe attached to the vaginal duct, a proximal receptaculumseminis and a distal bursa copulatrix. The oviduct is themost complex structure of the system. It can be dividedinto the ciliated non-glandular proximal oviduct and theglandular distal oviduct, which comprises the nidamentalglands. In the nidamental glands of various Opisthobran-chia, three parts can be distinguished on their histologicalbase (from proximally to distally): an albumen or alterna-tively a capsule gland, a membrane and a mucous gland.The most distal part of the oviduct, which leads into thevestibule or just into the female genital aperture is normal-ly non-glandular and ciliated. However, in some speciesthe duct can be lined by glandular tissue which is termedadhesive region here (after Schmekel 1971; Wägele1989). In the Anaspidea, almost the complete length of theoviducal channel of the spermoviduct is lined by glandularepithelium. It is here called oviducal gland in accordancewith Beeman (1977). The male part of the spermoviductin Anaspidea is also lined by a glandular epithelium,which is termed spermoviduct gland (Beeman 1977). Insome species small glands are attached to the vestibulumor distal oviduct, termed accessory glands. An atrial glandis attached to the vestibule of the Anaspidea.

The epithelia of all glandular parts in the nidamentalglands have the typical arrangement of alternating glan-dular cells and supporting cells. The glandular cells aremostly columnar to highly columnar and contain secreto-ry products of various shapes, textures and histochemicalstaining properties, whereas the supporting cells areslender, wedge-shaped and bear cilia of various lengths.The supporting cells generally also contain an apicallylying nucleus and ultrastructurally numerous mitochon-dria are visible.

The albumen gland can have the shape of a sac (Fig. 4B) or a tube (Fig. 2C, D). The secretory productsare mostly packed in the form of large round or elliptically shaped vesicles and have a homogeneous(Figs. 7A, 8A, B, C) or amorphous structure (Figs. 4A,5A, B, D). In addition to the secretory products, theglandular cells contain active nuclei and large areas ofendoplasmic reticulum and Golgi complexes in the basalpart of the cell, even if the cell is not actively secreting.The supporting cells bear short cilia.

The capsule gland is always tubular (Fig. 2A) andmostly narrowly coiled. The secretory products have theshape of distinct round to irregularly shaped granules(Fig. 7E) and their size is generally smaller than the sizeof the albumen vesicles. The granules can be of homoge-

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neous texture or they can contain distinct substructures.As has been described for the albumen gland, the capsulegland also contains large active nuclei, prominent endo-plasmic reticulum and Golgi complexes.

The membrane gland is also tubular and often as nar-rowly coiled as the capsule gland (Figs. 4C, 7F). In con-trast to the latter it comprises a smaller glandular area.The mostly heterogeneous secretory contents of the pris-

matic to columnar glandular cells are typically packedinto small mucous vesicles (Fig. 6A) and either form adense meshwork of mucous filaments or compact mu-cous coagulations. Usually endoplasmic reticulum can-not be found in the glandular cells, and the nuclei oftenhave a pycnotic appearance. It is noticeable that in con-trast to the other glandular parts the supporting cells ofthe membrane gland always bear very long cilia.

Table 1 Species, collection sites and methods applied. (fix Fixed length, M methacrylate histology, P paraffin histology, TEM transmis-sion electron microscopy)

Species Speci- Collection site Length Histology Histo- TEMmens chemistry

“Cephalaspidea s. l.”Acteocina atrata 2 Indian River Lagoon, USA 4 mm (fix) M – –Mikkelsen and Mikkelsen, 1984Acteon tornatilis 2 Irish Sea 7 mm (fix) M, P + –(Linné, 1758)Pupa sulcata 1 Western Australia 8 mm (fix) M – –(Gmelin, 1791)Philine alata 1 Antarctica 14 mm (fix) M + –Thiele, 1912Scaphander nobilis 1 21°36.2’ N; 18°40.,6’ W; 33 mm (fix) M + –Verrill, 1884 depth 2710 mRuncina adriatica 2 Corse, France 1.5 mm (fix) M – –Thompson, 1980Haminoea cymbalum 2 Dingo Beach, Australia 9 mm (alive) M + –(Quoy and Gaimard, 1835)Chelidonura inornata 2 Keeper Reef, Australia 21, 23 mm (alive) M, P + –Baba, 1949Philinopsis gardineri 2 Dingo Beach, Australia 20, 63 mm (alive) M – +(Eliot, 1903)

AnaspideaAplysia punctata 3 Helgoland, Germany; 18, 35, 63 mm (fix) M + +(Cuvier, 1803) Villefranche, FranceBursatella leachii 2 Townsville and 78, 82 mm (alive) M, P + –(de Blainville, 1817) Dingo Beach, AustraliaPhyllaplysia taylori 1 Victoria, B.C., Canada 18 mm (fix) M + –Dall, 1900Petalifera petalifera 1 Dingo Beach, Australia 6 mm (alive) M – –(Rang, 1828)

SacoglossaOxynoe viridis 1 Cottesloe Reef, Australia 15 mm (fix) M + –(Pease, 1861)Elysia ornata 2 Dingo Beach, Australia 30, 35 mm (alive) M, P + –(Swainson, 1840)Elysia viridis 1 Helgoland, Germany 25 mm (alive) M + –(Montagu, 1810)Thuridilla hopei 3 Roses, Spain 6, 8, 9 mm (fix) M + +(Verany, 1853)

TylodinoideaTylodina perversa 1 Chalkidike, Greece 18 mm (fix) M + –(Gmelin, 1790)

PleurobranchoideaBerthella stellata 1 Townsville, Australia 8 mm (fix) M + –(Risso, 1826)Euselenops luniceps 1 Dingo Beach, Australia 110 mm (alive) M + –(Cuvier, 1817)

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Table 2 Histochemical staining reactions. – Not stained, + weakly stained, ++ moderately stained, +++ brightly stained, blank space noresults. (BPB Bromophenol blue, PAS periodic acid-Schiff)

Species Alcian Alcian Azan BPB PAS ToluidinepH 2.5 pH 1.0 blue

“Cephalaspidea s. l.”A. tornatilis Albumen gland Blue – + Blue-pink

Membrane gland – – VioletMucous gland + – – ++ Red-violet

S. nobilis Albumen gland – – + BlueMembrane gland VioletMucous gland + – ++ Red-violet

H. cymbalum Capsule gland – Dark blueMembrane gland – RedMucous gland + – Violet

C. inornata Capsule gland Blue + Dark blueMembrane gland – – VioletMucous gland – – Violet

P. gardineri Capsule gland Dark blueMembrane gland PinkMucous gland + – Violet

AnaspideaA. punctata Albumen gland – – – ++ Blue

Membrane gland + ++ – + Violet-pinkMucous gland + ++ ++ Red-violetOviducal gland – +++ Dark purpleSpermoviduct gland – – Blue-green

B. leachii Albumen gland – – Blue + + BlueMembrane gland + + Light blue – – RedMucous gland ++ + – – ++ VioletOviducal gland – – Blue – +++ Dark blueSpermoviduct gland – – Pink + – Blue

P. taylori Albumen gland – – + – BlueMembrane gland – + – Light violetMucous gland + ++ – ++ VioletOviducal gland – – – +++ PurpleSpermoviduct gland – – – – Blue

SacoglossaO. viridis Albumen gland + + Blue

Membrane gland + PinkMucous gland + – +++ VioletAdhesive region +++ – +++ Dark purple

E. ornata Albumen gland Blue Dark blueMembrane gland VioletMucous gland Pink

E. viridis Albumen gland – + +++ Dark blueMembrane gland + – – + Violet-pinkMucous gland +++ +++ – +++ Violet

T. hopei Albumen gland – +++ BlueMembrane gland ++ Dark violetMucous gland – + Pink

TylodinoideaT. perversa Capsule gland – – – – Blue

Membrane gland – – – – VioletMucous gland + + – + Purple

PleurobranchoideaB. stellata Capsule gland – – – Blue

Membrane gland + ++ VioletMucous gland ++ – +++ Red-violet

E. luniceps Capsule gland ++ – +++ BlueMembrane gland + + PinkMucous gland + – ++ Violet

Table 3 Terminology of the different parts of the nidamental glands in the Opisthobranchia taken from the literature in comparison tothe terminology applied in the present study

Taxon Albumen Capsule Membrane Mucous Adhesive region/ Referencegland gland gland gland oviducal gland

Opisthobranchia in Albumen gland Membrane Mucous Ghiselin (1965)general [based on (note that gland glandinvestigation of DendrodorisDendrodoris does not posses analbopunctata albumen gland!)(Cooper, 1863)]

“Cephalaspidea s. l.” Albumen Capsule gland Posterior Rudman (1971)(Haminoea) gland mucus gland

“Cephalaspidea s. l.” Capsule gland Rudman (1972a)(Melanochlamys)

“Cephalaspidea s. l.” Albumen gland Membrane Mucous Oviducal Robles (1975)(Bulla) gland gland gland

Anaspidea (Aplysia) Albumen gland Convoluted Mucous Glandular Eales (1921)(winding) portion gland oviductof mucous gland

Anaspidea (Aplysia) Albumen gland Winding gland Mucous gland Female channel Thompson andof wide distal Bebbingtonhermaphroditic (1969)duct

Anaspidea Albumen gland Membrane gland Mucous gland Oviducal gland Beeman (1970)(Phyllaplysia)

Anaspidea Winding gland Mucous gland Oviducal channel Fretter and Hian(Dolabella) (1984)

Sacoglossa Albumen gland Shell gland Glandular Fourth region Gascoigne (Acteonia, oviduct of glandular (1956)Limapontia) oviduct

Sacoglossa Eiweißdrüse Faltiger Ovidukt; Sander-Essererster drüsiger (1984)Oviduktabschnitt

Pleurobranchoidea Capsule Proximal Distal mucous Wägele and Hain(Tomthompsonia) gland mucous gland gland (1991)

Nudibranchia Capsule Membrane gland Mucous gland Adhesive region Klussmann-Kolbgland (2001)

investigated “Cephalaspidea s. l.” species show monaulicanterior reproductive systems with separated seminalgrooves and penes (not shown here).

The nidamental glandular mass is divided into threedistinct parts in all cephalaspid species examined. InActeocina atrata, A. tornatilis, P. sulcata, Philine alata,Runcina adriatica and Scaphander nobilis the mostproximal part can be referred to as an albumen gland.Haminoea cymbalum, Chelidonura inornata and Phili-nopsis gardineri show a capsule gland there. Histologi-cal differences are explained below. All species have amembrane gland and a mucous gland. P. sulcata, P. alataand S. nobilis show specially differentiated glandularcells along the most distal part of the oviduct (adhesiveregion; Fig. 1C–E). In some species the hermaphroditicduct leads distally into a muscular vestibule, which itselfdischarges into the genital opening. No distinct vestibulecould be found in A. atrata, P. alata, R. adriatica and C.inornata. In P. gardineri a small accessory gland is at-

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The mucous gland always comprises the largest part inthe glandular system. It has the shape of a large tube withmostly wide coils (Figs. 2C–F, 4D) in contrast to the nar-rowly coiled membrane gland. The secretions in the mu-cous gland can also take various textures, from homoge-neous masses to heterogeneous filaments or irregulardroplets. The glandular cells do not contain endoplasmicreticulum and often have small or even pycnotic nuclei.As in the membrane gland it seems that the cells stop se-cretion once they have reached full maturity. Usually dif-ferent functional stages of the glandular cells are presentin the same species or even individual. The supportingcells generally bear short cilia in the mucous gland.

II. “Cephalaspidea s. l.”

Except for Acteon tornatilis (Fig. 1B) and Pupa sulcata(Fig. 1C), which have a diaulic genital system, all other

tached to the vestibulum (Fig. 1I). For schematic out-lines of the anterior reproductive systems of the investi-gated “Cephalaspidea s. l.” refer to Fig. 1A–I.

1. Albumen gland

The albumen gland mostly exhibits a pouch-like or bulbous organ. The albumen glands of A. tornatilis(Fig. 2C), S. nobilis (Fig. 2D) and R. adriatica are locat-ed in between the folds of the mucous glands. The secre-tory products contain neutral mucopolysaccharides andproteins (Table 2). Sometimes prominent nucleoli arevisible in the nuclei of the glandular cells. The albumen

gland and membrane gland are not separated by a dis-tinct duct in A. atrata, A. tornatilis and P. sulcata(Fig. 1C). In P. alata the two glands are connected by asmall non-glandular duct (Fig. 1D), whereas the albumengland of S. nobilis discharges into the spermoviduct (Fig. 1E). R. adriatica presents a different situation: the

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Fig. 1A–I Schematic outlinesof the anterior reproductivesystems of several “Cephalas-pidea s. l.”. A Acteocina atrata.B Acteon tornatilis. C Pupasulcata. D Philine alata.E Scaphander nobilis. F Runc-ina adriatica. G Haminoeacymbalum. H Chelidonurainornata. I Philinopsis gardin-eri. acgl Accessory gland,adr adhesive region, agl albu-men gland, amp ampulla,bc bursa copulatrix, cgl capsulegland, fc fertilisation chamber,megl membrane gland,mugl mucous gland, rs recep-taculum seminis

Fig. 2A–F Histological sections through the nidamental glands ofseveral “Cephalaspidea s. l.”. A C. inornata; capsule and mem-brane gland. B P. gardineri; capsule gland. C A. tornatilis; albu-men and mucous gland. D S. nobilis; albumen and mucous gland.E H. cymbalum; mucous gland. F R. adriatica; mucous gland.agl Albumen gland, cgl capsule gland, dct duct, megl membranegland, mugl mucous gland, vest vestibulum

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albumen gland discharges into a dilation of the oviduct,which is considered to be a fertilisation chamber.

2. Capsule gland

In H. cymbalum the capsule gland is coiled a few times,whereas in the other two species the pseudo two-layeredepithelium forms pouch-like structures branching offfrom the central duct of the gland (Fig. 2A, B). The secretory granules are round and ultrastructurally appearhomogeneous in P. gardineri. In this species the granulesalmost completely fill the upper two-thirds of the cell.Basally the number of mature granules decreases and, in-stead, a few immature granules can be found. These havea slightly different structure to the mature ones: theyhave an electron-dense centre which is surrounded bycoarse material forming a lighter rim. The granules arecomposed of neutral mucopolysaccharides and proteins(Table 2). In P. gardineri rough endoplasmic reticulumcan be found especially around or near the basal nucleus.Further away from the nucleus only vesicular endoplas-mic reticulum is found. Few Golgi complexes are pres-ent in the basal half of the cell. Spread throughout theglandular cells are elongated mitochondria showing dis-tinct cristae. The apical surface of the cell bears micro-villi. The supporting cells bear short cilia having a typi-cal 9+2 pattern in the shaft in all glands described. InH. cymbalum the capsule gland discharges into thespermoviduct (Fig. 1G) whereas in P. gardineri bothglands are connected by a small duct (Fig. 1I). C. inor-nata is missing a connecting duct. In this species bothglands form a continuous transition (Figs. 1H, 2A).

3. Membrane gland

The mucus in the membrane gland can take the form ofsmall droplets or be of heterogeneous texture. Acidic,sulphated mucopolysaccharides are produced in the glan-dular cells of the membrane gland (Table 2). Small nu-clei are always located basally in the glandular cells. Thenuclei contain dense chromatin in P. gardineri.

4. Mucous gland

In P. sulcata the proximal part of the mucous gland is a small, narrow tube, which dilates further distally toform the major part of the gland. In A. tornatilis andH. cymbalum the proximal part of the mucous gland isnarrowly coiled (Fig. 2C), whereas the distal part is alsowidely coiled as in the other species (Fig. 2E, F). Themucus can either be homogeneous or filamentous. Some-times it has the form of small droplets or the mucus ispacked into larger vesicles which sometimes dissolve,thus filling the cells with homogeneous patches of mu-cus. It was observed in P. gardineri that the mucous con-tents in the vesicles sometimes coagulate and form com-

pact droplets. The secretory products generally containneutral mucopolysaccharides and acidic, sulphated mu-copolysaccharides (Table 2). In most species the mucousgland discharges into the distal spermoviduct or oviduct,but in H. cymbalum it connects distally to a vestibulum(Fig. 1G).

5. Adhesive region

The secretory products of the adhesive region have dif-ferent structures. The mucous secretions can either havethe form of droplets of different sizes or they can be fila-mentous or the droplets or mucous coagulations dissolveand form homogeneous patches. The nuclei found in theglandular cells are small and located near the base of thecell. Alternating supporting cells bear short cilia andcontain apically lying nuclei in S. nobilis. In the othertwo species the nuclei are not visible in the supportingcells.

III. Anaspidea

The Anaspidea studied here show monaulic reproductivesystems (Fig. 3A–D). The nidamental glands are at-tached to the pallial spermoviduct. This latter duct is in-ternally folded and bears three incompletely separatedchannels, namely the oviducal channel, the internal au-tospermal groove (see Thompson and Bebbington 1969;Thomas 1975) and the vaginal channel. The oviducalchannel is always lined by glandular tissue (oviducalgland). In Aplysia punctata this glandular part of the ovi-ducal channel is very large and forms a pouch (Fig. 3A).In Bursatella leachii it is also prominent (Fig. 3B),whereas in the other two investigated species (Fig. 3C, D)this structure is confined to a smaller area. All speciesalso show another type of glandular tissue related to theautospermal groove (spermoviduct gland). The postam-pullary duct forms a dilation in its distal region, here referred to as a fertilisation chamber. This area is thecentral part of the system and all parts join here. Thenidamental glands originate in the fertilisation chamber,as do the spermoviduct and the oviducal glands.The nidamental glandular complex can be divided intothree parts. Most proximally a large sac-like albumengland is found. Adjacent to this is the membrane glandfollowed further distally by the large mucous gland. Thislatter gland always shows a continuous connection to theoviducal gland. All species that were investigated havean atrial gland attached to the atrium.

1. Albumen gland

The albumen gland internally builds secondary folds,which are very obvious in A. punctata, B. leachiiand Phyllaplysia taylori (Fig. 4A) but less distinct inPetalifera petalifera (Fig. 4B). The number and size of

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the secretory vesicles increases towards the apical part ofthe cell. In A. punctata the vesicles show contents of different structures: large areas within the vesicles arecomposed of electron-dense, homogeneous substanceswhereas other areas are of a loose, filamentous structure(Fig. 5B, D). Within the electron-dense substance morecompact, filamentous substructures are visible at highmagnifications (Fig. 5B arrow). The secretory contentsof the vesicles in P. taylori are of heterogeneous, fila-mentous structure. B. leachii shows two types of vesiclesin the albumen gland: apically few round to ellipticallyshaped vesicles with heterogeneous contents can befound, whereas basally vesicles with less condensed, homogeneous contents are present. The secretory vesi-

cles of P. petalifera have amorphous contents. When thevesicles are ejected, the amorphous content of the vesi-cles is poured into the lumen (Fig. 5C arrow). Histo-chemical staining (Table 2) indicates the production ofneutral mucopolysaccharides and proteins. In A. puncta-ta additionally to a basal nucleus, prominent Golgi com-

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Fig. 3A–I Schematic outlinesof the anterior reproductivesystems of several Anaspidea,Sacoglossa, Tylodinoidea andPleurobranchoidea. A Aplysiapunctata. B Bursatella leachii.C Phyllaplysia taylori. D Peta-lifera petalifera. E Oxynoe viri-dis. F Thuridilla hopei. G Tylo-dina perversa. H Berthella stel-lata. I Euselenops luniceps.acgl Accessory gland, adr ad-hesive region, agl albumengland, amp ampulla, atgl atrialgland, bc bursa copulatrix,cgl capsule gland, fc fertilisa-tion chamber, megl membranegland, mugl mucous gland,ovgl oviducal gland, pe penis,pegl penis gland, pr prostate,rs receptaculum seminis,spgl spermoviduct gland

Fig. 4A–F Histological sections through the nidamental glandsand accessory glands at the oviduct of several Anaspidea. A P.taylori; albumen gland. B P. petalifera; albumen and membranegland. C–F A. punctata. C Membrane gland. D Mucous gland.E Oviducal and spermoviduct gland. F Atrial gland. agl Albumengland, atgl atrial gland, megl membrane gland, mugl mucousgland, ov oviduct, ocgl oviducal gland, spgl spermoviduct gland,vch vaginal channel

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Fig. 4A–F Legend see page 223

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Fig. 5A–D Legend see page 226

plexes and large areas of endoplasmic reticulum and mitochondria are spread throughout the glandular cells,their number decreasing towards the apical part. The apices of the glandular cells bear small microvilli, as dothe alternating ciliated supporting cells.

2. Membrane gland

In A. punctata the secretions of the membrane glandhave the shape of small vesicles, all containing a mesh-work of heterogeneous mucous fibres (Fig. 6A). In someareas these vesicles seem to fuse into one large vesicle,and the mucous fibres seem to coagulate to form elec-tron-dense mucous lumps. The number and size of thevesicles decrease towards the basal part of the cell,where only small vesicles are found, mostly with condensed contents. In P. petalifera and P. taylori themucous secretions take the shape of small droplets. Themucus is composed of neutral and acidic, sulphated mu-copolysaccharides (Table 2). In A. punctata basally largeGolgi complexes can be found close to the nucleus. Fewmitochondria and vesicular endoplasmic reticulum arepresent.

3. Mucous gland

The mucous gland is similarly folded internally (Fig. 4D) as is the albumen gland. The mucous secre-tions can be of homogeneous structure or in the form ofvesicles with homogeneous or heterogeneous contents.The mucus contains neutral and acidic, sulphated muco-polysaccharides (Table 2). The small nuclei lie basally inthe glandular cells and are seemingly pycnotic inA. punctata and P. petalifera. In B. leachii the nuclei arelarge and contain nucleoli.

4. Oviducal gland

In A. punctata the oviducal gland forms secondary widefolds (Fig. 4E). The glandular cells are stout, prismaticor highly columnar. They contain numerous small gran-ules of irregular shape. The granules cluster in the apicalhalves of the cells. In A. punctata they are membranecoated and have a very characteristic structure: within ahomogeneous substance that is completely filling thegranule, electron-dense lamellae-like substructures arepresent giving the granule a mitochondrion-like pattern(Fig. 6B inset). When ejected the granule matrix seemsto dissolve (Fig. 6B). Nascent granules with a still very

amorphous shape are found in the centre of large Golgiareas in the basal part of the cell (Fig. 6D). The granulesare composed of neutral mucopolysaccharides (Table 2).The nuclei contain visible nucleoli in P. petalifera. InA. punctata endoplasmic reticulum is found in closeproximity to the nucleus. The alternating supportingcells have apically a triangular shape. In A. punctata andB. leachii the supporting cells bear long cilia, whereas inP. taylori the cilia are of moderate length, and inP. petalifera short cilia are found. In between the ciliamicrovilli are found in A. punctata. Numerous mitochon-dria are found close to the ciliary rootlets (Fig. 6B). Apically lying nuclei are visible in the supporting cellsof all species except for P. petalifera.

5. Spermoviduct gland

The glandular tissue is composed of alternating glandu-lar and supporting cells. The glandular cells are colum-nar to highly columnar. The secretory vesicles containproteins in B. leachii (Table 2). In P. taylori the vesiclesseem to have alveolar substructures when examined withhigh magnification. Generally large nuclei are found in abasal position. The supporting cells are very conspicuousand bear short to moderately long cilia.

6. Atrial gland

This gland has the shape of a narrowly coiled tube (Fig. 4F). The glandular cells are prismatic or columnar.They contain secretory vesicles which have a character-istic structure in A. punctata: electron-dense centres aresurrounded by less electron-dense material (Fig. 6C).The number of vesicles decreases towards the base of thecells, where nuclei with sometimes prominent nucleoliare found. Few Golgi complexes, rough endoplasmic reticulum and mitochondria are also located basally inthe glandular cells. The supporting cells, which alternatewith the glandular cells in all species investigated, con-tain an apically lying nucleus. The cells bear short tomoderately long cilia. Microvilli were found in betweenthe cilia in A. punctata.

IV. Sacoglossa

The Sacoglossa studied here have an androdiaulic systemwith the vas deferens and the accessory prostate glandseparated (Fig. 3E, F). In Oxynoe viridis (Fig. 3E) thenidamental glands comprise a compact glandular mass

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Fig. 5A–D Ultrastructure of the albumen gland of A. punctata.A Schematic outline of a glandular and a supporting cell. B, D Al-bumen vesicles; arrow in B indicates electron-dense substructures.C Amorphous albumen material in lumen of the gland (see ar-row). avs Albumen vesicle, bl basal lamina, ci cilia, er endoplas-mic reticulum, gc Golgi complex, mi mitochondrion, nuc nucleus

Fig. 6A–D Ultrastructure of the membrane, oviducal and atrialgland of A. punctata. A Membrane gland, B, D Oviducal gland;arrow in B indicates active secretion. C Atrial gland. ci Cilia,gc Golgi complex, mi mitochondrion, mvs mucous vesicle, nuc nu-cleus, sc supporting cell, vs vesicle

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located in the central body cavity, whereas the albumen glands in Elysia viridis, Elysia ornata andThuridilla hopei (Fig. 3F) are tubular and branched within the lateral parapodia. In the Elysia species andT. hopei only the mucous glands of the nidamental glan-dular systems are compact and located in the central body cavity. Small ducts connect the follicles of the albumen gland with the oviduct. All studied sacoglossanspecies show three separate glandular parts in the nida-mental gland systems as also described for the “Cephal-aspidea s. l.” and Anaspidea. The albumen gland is locat-ed most proximally. In O. viridis the membrane gland isset apart clearly from the mucous gland whereas in theElysia species and T. hopei membrane and mucous glandsare closely connected. In O. viridis the distal oviduct islined by glandular tissue (adhesive region; Fig. 3E). Sincethe anatomy of the reproductive systems of the Elysiaspecies and T. hopei is very similar, only one representa-tive schematic drawing is shown in Fig. 3F.

1. Albumen gland

In O. viridis the albumen gland is a compact organ lyingin the central body cavity and is composed of small tubules (Fig. 7A). The broad, columnar glandular cells inthis species contain vesicles of round to elliptic shapeand different size. A large round nucleus is lying basally.At the distal end of the albumen gland the efferent ductleading to the following membrane gland is lined byglandular epithelium slightly different from the albumengland cells: the glandular cells are more slender and con-tain numerous small dark blue staining round granules.The basally lying nuclei do not contain visible nucleoli.The alternating supporting cells are more prominent herethan in the proximal albumen gland. In the Elysia spe-cies and T. hopei the club-shaped glandular cells sur-round a small central duct into which they discharge(Fig. 7B). The ducts are connected to a larger efferentduct lined by columnar, non-glandular, ciliated cells. Theglandular cells contain numerous large round to ellipti-cally shaped secretory vesicles. In T. hopei they arefound to be composed of two distinct substances, oneelectron dense substance showing a mottled pattern and aless electron dense substance of homogeneous structure(Fig. 8A–C). The relative amount of the more electrondense substance seems to be higher in the vesicles thanthe less electron dense substance. In small areas withinthe electron dense substance the mottled pattern is notobvious but homogeneous patches are apparent. In im-mature vesicles the electron-dense substance seems to be

less dense than in mature vesicles. Neither dischargingof the vesicles nor of the substances composing themcould be observed. But a dissolution of the less electrondense substance is visible in vesicles close to the apicalcell membrane (Fig. 8B arrow). The secretory productsof the albumen glands generally contain neutral mucopo-lysaccharides and proteins (Table 2). Microvilli emanatefrom the apical surface of the glandular cells into the lumen of the ductules into which the glandular cells discharge. The glandular cells alternate with small columnar cells lining the central ductule.

2. Membrane gland

In O. viridis the membrane gland directly joins to theglandular epithelium of the efferent duct of the albumengland described above (Fig. 3E). The membrane gland isnarrowly coiled in this species, whereas it is lining theefferent duct of the mucous gland in its most proximalpart in E. viridis and T. hopei. In O. viridis the glandularcells contain homogeneous mucus in the apical two-thirds of the cell and irregularly formed mucous dropletsbasally. In E. viridis the mucus has the form of irregulardroplets, whereas in T. hopei heterogeneous mucous fibres are present. The mucus is composed of neutral andacidic, sulphated mucopolysaccharides (Table 2).

3. Mucous gland

The mucous gland forms a large, widely coiled tube inO. viridis and comprises a two-folded tube in E. viridis(Fig. 7C). In T. hopei it is separated into two distinctparts (Fig. 3F). Proximally the columnar glandular cellsare arranged around a small central duct. This duct leadsto the second, more distal part which is tubular. InO. viridis the columnar glandular cells contain numeroussmall, irregularly formed mucous vesicles. In E. viridisthe glandular cells are not distinguishable as single cells.But mucous droplets can be observed in the cells. Theglandular cells in T. hopei contain mucous vesicleswhich are larger than those in the membrane gland andcontain less electron dense and less coagulated mucousfibres. Neutral mucopolysaccharides as well as acidic,sulphated mucopolysaccharides are present in the glan-dular cells of all species (Table 2). Nuclei are located basally and in some cells of O. viridis they can also be found medially. Golgi complexes are found in theglandular cells of T. hopei.

4. Adhesive region

In O. viridis this part is very prominent (Figs. 3E, 7D).The glandular epithelium lines a large tubular and con-voluted organ. The glandular cells are broad basally andnarrow slightly towards the apex. They contain numer-ous small irregularly shaped granules. Large elliptically

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Fig. 7A–F Histological sections through the nidamental glands ofseveral Sacoglossa, Tylodinoidea and Pleurobranchoidea. A O.viridis; albumen gland. B Elysia viridis; albumen gland. C E. viri-dis; mucous gland. D O. viridis; adhesive region. E T. perversa;capsule gland. F E. luniceps; membrane gland. agl Albumengland, cgl capsule gland, dct duct, megl membrane gland,mugl mucous gland, ov oviduct

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shaped nuclei are located in various positions. Nucleoliare always visible. The supporting cells are small andwedge-shaped and bear short to moderately long cilia.They contain an apically lying nucleus.

V. Tylodinoidea

Tylodina perversa possesses a monaulic genital system(Fig. 3G). The distal male part of this system has notbeen investigated in further detail. The female part con-sists of a very short proximal oviduct, a large nidamentalgland mass and an accessory gland at the vestibulum.The nidamental glands are composed of three distinctparts, namely a capsule gland, a membrane and a mucous gland.

1. Capsule gland

The capsule gland is a large organ with narrow coils(Fig. 7E). The glandular cells contain round granules. At high magnification the granules seem to have finesubstructures. The supporting cells are very small andhardly visible.

2. Membrane gland

Capsule gland and membrane gland are connected viathe distal oviduct. The glandular cells are prismatic andfilled with heterogeneous mucous masses. The mucuspartly forms droplets. The supporting cells have a trian-gular apex which partly overlaps the glandular cells api-cally.

3. Mucous gland

This large organ both originates and discharges into thedistal spermoviduct (Fig. 3G). No direct connection tothe previous membrane gland has been observed. Themucous vesicles in the glandular cells seem to dissolvein parts to form homogeneous mucous masses. The mucus is composed of neutral and acidic, sulphated mucopolysaccharides (Table 2). The alternating support-ing cells are not as prominent as in the membrane gland.

VI. Pleurobranchoidea

Berthella stellata and Euselenops luniceps both possessandrodiaulic reproductive systems (Fig. 3H, I). In B.

stellata the proximal oviduct is short and soon dis-charges into the capsule gland. E. luniceps presents a slightly different picture: the proximal oviduct is very long and joins distally to the vestibulum, to which the nidamental glands are also connected. Both species show three distinct parts in the nidamentalglandular system: a capsule, a membrane and a mucousgland.

1. Capsule gland

The capsule gland is tubular in both species, but inE. luniceps it is narrowly coiled, whereas in B. stellatait comprises only few, slightly wider coils. The glandularcells contain numerous round granules in both species,which are smaller in B. stellata than in E. luniceps. Inthe latter species the average size of the granules andtheir number are smaller in the basal part of the cell thanin the apical part. The texture of the granules is homo-geneous in both species. The granules contain neutralmucopolysaccharides (Table 2). In E. luniceps black pig-ments are visible at the apical rim of the supporting cells.The capsule gland shows a continuous transition to themembrane gland in B. stellata (Fig. 3H), whereas inE. luniceps a small ciliated duct connects both glands(Fig. 3I).

2. Membrane gland

In B. stellata the mucus in the membrane gland has theshape of heterogeneous fibres, partly coagulating to formdroplets. In E. luniceps the mucus is heterogeneous inthe proximal part of the gland and forms distinct dropletsof different sizes further distally. The secretory productscontain acidic mucopolysaccharides (Table 2). The sup-porting cells are very prominent in E. luniceps, their triangular apices partly overlapping the apices of the adjacent glandular cells.

3. Mucous gland

In B. stellata the mucus has the shape of distinct mucousvesicles of different sizes and shapes. But they can alsodissolve or fuse, thus filling the cells with a homogene-ous mass of mucus. Neutral as well as acidic, sulphatedmucopolysaccharides can be found in the cells (Table 2).Relatively large nuclei are located medially to basally inthe glandular cells. Nuclei are not visible in the cells. InE. luniceps the glandular cells in the proximal part of thegland contain heterogeneous mucous masses often form-ing compact droplets. The alternating supporting cellsare prominent, have a triangular apex and overlap theglandular cells. Further distally the glandular cells aremore slender, more elongate and the mucus forms smallvesicles. The mucous vesicles sometimes fuse to build ahomogeneous mass of mucus.

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Fig. 8A–C Ultrastructure of the albumen gland of T. hopei.A Whole glandular cell. B, C Albumen vesicles. B Dissolving secretory substances (see arrow) at luminal tip of glandular cell.C Membrane-bound albumen vesicle. avs Albumen vesicle, ci cil-ia, er endoplasmic reticulum, mi mitochondrion, mv microvilli,nuc nucleus, sc supporting cell

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D. Discussion

Although the hermaphroditic reproductive systems of theOpisthobranchia are very complex organs, the nidamen-tal glands seem to be rather uniformly structured. For allinvestigated species three structurally different glandularparts within the nidamental glandular system have beendescribed. These findings are in contrast to previousstudies which stated that the Pleurobranchoidea and the Nudibranchia possess only two glandular parts(Schmekel 1970, 1985; Hadfield and Switzer-Dunlap1984). In contrast, Wägele (1989) and Gosliner (1994)stated that aeolid Nudibranchia possess three distinctglandular regions within the nidamental glands.

The most proximal part (with respect to the gonad)described in the present study has two different struc-tures in the investigated taxa: it comprises either an albumen gland, as in most “Cephalaspidea s. l.”, all Anaspidea and all Sacoglossa, or it can be arranged as a capsule gland as in some “Cephalaspidea s. l.”, all Tylodinoidea and Pleurobranchoidea. The structure ofthe capsule gland found here in some Opisthobranchia issimilar to the one described for Nudibranchia in an earli-er investigation (Klussmann-Kolb 2001). Additionallyall investigated Opisthobranchia have a membrane glandand further distally a mucous gland. These two glandsare uniformly structured in all investigated Opisthobran-chia. Moreover, in some species additional glandular tis-sue lines the distal oviduct (adhesive region) or an acces-sory gland is attached to the most distal region of the oviduct. The terminology used here and in the following is based on the detailed comparative studieswhich have been described before.

The glandular parts of the nidamental glandularsystem can be divided into two types of glands, based ontheir histochemical staining properties and on their modeof secretion: on the one hand the basophilic albumen andcapsule glands can be found which contain neutral poly-saccharides as well as proteins and which are merocrine(i.e. they keep the capability of active secretion through-out the span of reproductive activity of the animal), andon the other hand the acidophilic membrane and mucousglands are present, which produce neutral polysaccha-rides and acidic, sulphated mucopolysaccharides. Theseglands stop their production of secretory material oncethey have reached full maturity. The structures of the se-cretory products in the two glandular types differ consid-erably. The secretions of the albumen and capsule glandsare packed into distinct vesicles or granules, whereas thesecretions of the membrane and mucous glands are eithera homogeneous mass, heterogeneous fibres or irregulardroplets.

I. Comparison within the Opisthobranchia

Although a detailed comparative investigation of the his-tology and fine structure of the nidamental glands of theOpisthobranchia has never been undertaken before, some

data are available from the literature to which the presentfindings can be compared.

For the “Cephalaspidea s. l.” some papers on the his-tology of the reproductive system have been publishedby Rudman (1971, 1972a, b, c, 1974), but his descrip-tions are not detailed enough to allow a comparison withother Opisthobranchia and lack data on the fine structureof the glandular products. Rudman described the mostproximal part of the nidamental glands in species ofHaminoea, Chelidonura and Philinopsis as an albumengland, not noting the difference to the “real” albumengland in other “Cephalaspidea s. l.” and erroneouslytermed the gland following the most proximal part a cap-sule gland (for terminology of the different parts in theliterature also refer to Table 3). In the present study themost proximal part in these taxa was found to be a cap-sule gland, followed by a membrane gland. The only ul-trastructural investigation of the nidamental glands in acephalaspidean species has been undertaken by Kressand Schmekel (1992). The authors also found three histologically and ultrastructurally distinct parts in thegland mass of Runcina coronata (Quatrefages, 1844) andRuncina ferruginea Kress, 1977. The authors describedan albumen gland followed by an egg-capsule glandforming a transitional duct between the albumen glandand the mucous gland. From their descriptions I assumethat their capsule gland is identical to the membranegland described in the present study for R. adriatica.

A more detailed investigation of the nidamentalglands of three different species of Anaspidea has beenundertaken by Thompson and Bebbington (1969). Theauthors also found three different parts, which theytermed albumen, winding and mucous gland. The wind-ing gland accords with what has been described as themembrane gland in the present study. Thomas (1975)also described a winding gland for Bursatella leachiiplei (Rang, 1828), but in his opinion the secretory cellsof the winding gland are interspersed among the support-ing cells, which stands in contrast to the findings of thepresent study. Thompson and Bebbington (1969), more-over, described a prostate to line the spermoviduct ofAplysia fasciata Poiret, 1789, A. depilans Gmelin, 1791and A. punctata. According to their description of posi-tion and structural properties of this glandular tissue it is very probable that the prostate of Thompson andBebbington accords with the spermoviduct gland studiedhere. A glandular oviducal channel was also describedby Eales (1921) and Thompson and Bebbington (1969)for A. punctata, by Beeman (1970) for P. taylori and byThomas (1975) for B. leachii plei. This gland is alwaysshown to have a direct connection to the mucous glandas also presented for the four Anaspidea studied here.Coggeshall (1972) presented a detailed description of theultrastructure of the nidamental glands in Aplysia cali-fornica Cooper, 1863. He distinguished four types of secretory cells in the albumen, winding and mucousgland. His descriptions of the fine structure of the glan-dular cells are mostly congruent with those described forA. punctata here. Much attention has been paid to the

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structure of the atrial gland of different species ofAplysia. This organ is supposed to produce a hormoneresponsible for egg laying (Kelner et al. 1984). Beard et al. (1982) and van Heumen et al. (1995) presented ul-trastructural studies of the atrial gland of A. californica.Their findings regarding the fine structure of the vesiclesin the secretory cells accord with the descriptions pre-sented here for A. punctata.

One detailed analysis of the histology and fine struc-ture of the nidamental glands in the Sacoglossa has beenpresented by Sanders-Esser (1984). Her findings on thenidamental glands are very similar to the ones describedin the present study. Still, some differences are worthdiscussing. Sanders-Esser described the supporting cellsin the albumen gland of E. viridis to be very broad attheir apex, thus overlapping the apices of the glandularcells. This could neither be observed with low power microscopy in this species nor with TEM in T. hopei.Sanders-Esser did not distinguish between two differentparts of mucous glands as presented for all Sacoglossaspecies here (membrane and mucous gland), but she dis-cussed the possibility that the first part of the glandularoviduct accords with the membrane gland of the Anaspi-dea. This structure corresponds to the membrane gland described here (see above). In his classification of thefamilies of the Sacoglossa, Gascoigne (1985) distin-guished three different glandular parts in the female reproductive system, namely an albumen, a capsule anda large oviducal gland.

No histological data on the reproductive systems ofTylodinoidea species are available from the literatureand only few descriptions can be found for Pleurobran-choidea. Jensen (1994) described the anatomy of the an-terior reproductive system of E. luniceps. In a drawingof the reproductive system of this species she distin-guished between an albumen, a capsule and a mucousgland in the nidamental glandular system, but she didnot describe these parts any further. She also did notmention the capsule gland in the describing text. Thus itis difficult to compare her findings to the present data.As far as I know, only one histological investigation of apleurobranchoid reproductive system has been undertak-en so far (Wägele and Hain 1991). The authors also de-scribed three glandular parts in the female glands ofTomthompsonia antarctica (described as T. spiroconcha-lis Wägele and Hain, 1991), namely the capsule gland,the proximal and the distal mucous gland. From the position of the proximal mucous gland in this species it seems obvious that it corresponds to the membranegland described in this study. The distal mucous glandof T. antarctica accords with the mucous gland observedhere.

II. Homology of the nidamental glands

As mentioned in the section on terminology this compar-ative survey allows to hypothesise that within theOpisthobranchia those structures which were termed in

the same manner are actually homologous. This homolo-gy hypothesis concerns the albumen gland, capsulegland, the membrane and the mucous gland and is sup-ported by the following arguments:

1. Identical position of the glandular parts within a (hy-pothesised) homologous glandular system; the albu-men gland and the capsule gland always occur at the most proximal end of the oviduct or spermovi-duct, respectively. The next gland further distally isthe membrane gland and the mucous gland occursmost distally.

2. Similar histology and ultrastructure of the glandularepithelium; the vesicles in the albumen gland are ofamorphous structure and composed of substances ofdifferent electron densities. The texture of the gran-ules in the capsule glands is either homogeneouslyelectron dense or the granules contain more electrondense substructures. The texture of the mucous vesi-cles in the membrane glands are also similarthroughout the taxa (dense meshwork or compactcoagulations) and differ from the mucous vesicles inthe mucous gland (loose meshwork). The supportingcells in the membrane glands always bear extremelylong cilia in contrast to the other glandular parts. Allglands show the same pattern of alternating glandu-lar and supporting cells.

3. The similar mode of secretion; the albumen glandsare merocrine, so are the capsule glands, whereasmembrane and mucous glands stop secreting whenthe cells have reached full maturity.

4. The similar histochemical staining properties; albu-men glands and capsule glands are basophilic andcontain neutral polysaccharides and proteins, whereasmembrane and mucous glands are acidophilic andcontain neutral polysaccharides and acidic mucopoly-saccharides.

Due to the identical position and the similar histochemis-try and ultrastructure it is further concluded that the al-bumen glands and the capsule glands are homologousglandular parts of the system. Gosliner (1994) has alsoalready hypothesised that the most proximal part of thenidamental glandular system in aeolid Nudibranchia ishomologous to the albumen gland of other Opisthobran-chia. His hypothesis is thus confirmed here and can evenbe extended to the rest of the Nudibranchia as well as the Tylodinoidea, Pleurobranchoidea and part of the“Cephalaspidea s. l.”.

The conclusion presented here finally dismisses the speculations about possible homologies of the “membrane” or “winding glands” in Anaspidea and Sacoglossa with the “capsule glands” in the Nudibranchia, as have been put forward by Schmekel(1985), Sanders-Esser (1984) and Hadfield and Switzer-Dunlap (1984). Whether the adhesive regionin the “Cephalaspidea s. l.” and the Sacoglossa and the oviducal gland in the Anaspidea are homologouscannot be conclusively clarified at this point in time.

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III. Evolutionary implications

Since only one organ system has been investigated in thepresent study, thus providing a small data set, it is notaimed at using the present results to resolve phylogenyof the Opisthobranchia. Nevertheless, based on the datapresented here, especially considering the likely homo-logies of the various glandular parts within the nidamen-tal glands, it is possible to reconstruct the ground patternof the nidamental glands in the Opisthobranchia. More-over a possible scenario for evolutionary change of thisorgan system within the Opisthobranchia can be pro-posed. This leads to certain phylogenetic implicationswhich will be discussed and compared to existing phylo-genies.

The division of the nidamental glands into a proxi-mally lying albumen gland, followed by a membranegland and a distally lying mucous gland has been observed in Acteon, a basal taxon within the “Cephalasp-idea s. l.” (compare Mikkelsen 1996), as well as in thePulmonata and Gymnomorpha (Fretter 1943; De JongBrink 1969; Plesch et al. 1971; Haszprunar 1985) repre-senting sister groups to the Opisthobranchia. Similar his-tological properties of the nidamental glands were foundin the latter two taxa compared to the Opisthobranchia(personal observations) and thus this configuration of thenidamental glands is proposed for the ground pattern ofthe Opisthobranchia. This accords with the hypotheses ofGhiselin (1965) and Gosliner (1981). Since in the out-groups (Pulmonata, Gymnomorpha, Pyramidelloidea)and in the “Cephalaspidea s. l.” tubular albumen glandsare present it is suggested that the tubular arrangement isancestral, which complies with Ghiselin (1965), and sac-cular albumen glands have evolved within the Opistho-branchia. The membrane and mucous glands have mostprobably also been tubular in the ground pattern of theOpisthobranchia. An arrangement of alternating glandu-lar and supporting cells, which has been found in allglandular parts in all taxa studied, represents the basichistological pattern.

The plesiomorphic condition of a proximal albumen,a membrane and a distal mucous gland can also be proposed for the ground pattern of the “Cephalaspidea s. l.”, since it is found in the most basal investigated taxon of this group (Acteon) as well as in the out-groups (Anaspidea and Sacoglossa). In the investigatedAnaspidea the albumen gland is sac-like whereas in thesacoglossan species of Elysia and in T. hopei it is tube-like and highly branched within the parapodia. The sac-like albumen gland in the Anaspidea obviously rep-resents an apomorphic condition, whereas the compacttube-like arrangement in the Sacoglossa (Oxynoe as abasal representative) is plesiomorphic. This tubularstructure secondarily got branched in the stem lineageof this group (compare Elysia and Thuridilla). A majorevolutionary change with regards to structure and func-tion of the nidamental glands has taken place within the Opisthobranchia. The cephalaspid taxa Haminoea,Chelidonura and Philinopsis as well as the Tylodino-

idea and Pleurobranchoidea and also the Nudibranchia(see Klussmann-Kolb 2001) do not possess an albumengland at the proximal end of the nidamental glands buta capsule gland instead. The function of the proximalpart of the nidamental glands (i.e. the albumen gland)has changed in so far that within the egg masses ofthese taxa the albumen fluid is replaced by a compact“capsule-like” layer. In contrast, albumen fluid is pres-ent in those species which possess an albumen gland.This has been shown by Klussmann-Kolb and Wägele(2001) for various Opisthobranchia. Because of the cor-responding complexity of the albumen-capsule glandsin all taxa, as shown before, I assume that this changehas taken place only once within the Opisthobranchia.Two hypotheses are possible to explain the evolution ofthe albumen-capsule glands:

1. The albumen gland has been reduced in the ancestorof the above named taxa and a “new” capsule glandhas been developed.

2. The glandular cells in the albumen gland have under-gone a structural change, resulting in the capsulegland, which now serves a different function. Thiswould demand that the albumen and capsule glandsare homologous structures, which has been proposedbefore. This second explanation seems to be moreprobable, because of the structural similarity of thetwo glands and because less evolutionary steps needto be assumed to lead to this functional change; hencethe evolutionary scenario would be more conceivableand more parsimonious.

These considerations also imply preliminary conclu-sions about the phylogeny of the Opisthobranchia. Ifthe change from an albumen gland into a capsule glandis considered to have evolved only once within theOpisthobranchia, this would consequently mean thatthe “Cephalaspidea s. l.” are a paraphyletic taxon. Theparaphyletic status of the “Cephalaspidea s. l.” has already been assumed for a long time and has beenshown by Mikkelsen (1996) in her detailed cladisticanalysis. Furthermore, the cephalaspid taxa Haminoea,Chelidonura and Philinopsis, the Tylodinoidea, Pleu-robranchoidea and the Nudibranchia would share thesynapomorphy of possession of a capsule gland andcould be joined in one monophylum. A capsule glanddoes not occur in any other taxon outside these groupsand it has been found in all representatives of thesetaxa. A unification of these taxa into one monophylumhas not been proposed before. But it has to be kept inmind that only one organ system has been investigatedin the present study, and that more studies on other or-gan systems are necessary before a sound phylogeneticanalysis can be performed. Nevertheless, I consider the present findings as the basis for further comparativeanalyses, which promise to give new exciting insightsinto the evolution and phylogeny of these remarkableanimals.

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Acknowledgements I am very grateful to Heike Wägele for hervaluable ideas and many helpful discussions about the phylogenyof opisthobranchs. Helpful comments on the manuscript were pro-vided by Paula Mikkelsen. Also, two unknown referees have madevery constructive comments that helped to improve the manuscriptconsiderably. One essential part of this study was conducted at theDepartment of Marine Biology of James Cook University,Townsville, Australia. This would not have been possible withoutthe help of Gilianne Brodie, whom I wish to thank for her constantsupport and encouragement. Material was kindly provided by Gilianne and Jon Brodie, Clay Bryce, Paula Mikkelsen, SandraMillen, Heike Wägele and the Zoologische Staatssammlung München. This study was supported by the Deutsche Forschungs-gemeinschaft (Wa 618/3) and the Deutscher Akademischer Aus-tauschdienst.

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