Acta Zoologica (Stockholm) 89: 137–148 ( April 2008) doi: 10.1111/j.1463-6395.2007.00301.x On the fine structure of the creeping larva of Loxosomella murmanica: additional evidence for a clade of Kamptozoa (Entoprocta) and Mollusca Blackwell Publishing Ltd Gerhard Haszprunar1 and Andreas Wanninger2 Abstract 1 Zoologische Staatssammlung München, Münchhausenstrasse 21, D-81247 München, Germany; 2Department of Cell Biology and Comparative Zoology, Institute of Biology, University of Copenhagen, DK-2100 Copenhagen, Denmark Keywords: Entoprocta, Kamptozoa, larva, Mollusca, phylogeny, ultrastructure Accepted for publication: 26 June 2007 Haszprunar, G. and Wanninger, A. 2008. On the fine structure of the creeping larva of Loxosomella murmanica: additional evidence for a clade of Kamptozoa (Entoprocta) and Mollusca. – Acta Zoologica (Stockholm) 89: 137–148 The creeping larva of the kamptozoan (entoproct) Loxosomella murmanica was investigated using transmission electron microscopy. The late larva exhibits a prominent apical organ connected to the ‘cerebral’ commissure of large cerebral ganglia, which supply the paired frontal organ. From the cerebral ganglia two paired nerve cords project backwards, closely resembling the tetraneuralian pattern of basal molluscs. In addition, a neural ring supplying the prototroch is present. The epidermis is composed of myoepithelial cells. Dorsally its cuticle is covered by granules of unknown composition. The prototroch consists of two ciliary rings; a downstream collecting system is not present. Although there is a one-way gut with a lumen throughout, the larva obviously does not feed. A single pair of protonephridia is present. The foot sole shares distinct similarities with basic molluscs, particularly with those of the aplacophoran Solenogastres:The anterior part shows a huge, subepidermal pedal gland and several bundles of cirri consisting of compound cilia. The posterior part is ciliated with intraepithelial mucous cells interspersed. The dorsoventral muscle fibres show the mollusc-like ventral intercrossing. The present results and previous findings, in particular the chitinous, non-moulted cuticle, the sinus circulatory system, and a number of neural features shared by Kamptozoa and Mollusca, provide substantial evidence for a direct sistergroup relationship between these phyla. In addition, the basal position of the Solenogastres (Neomeniomorpha) within the Mollusca is corroborated. Gerhard Haszprunar, Zoologische Staatssammlung München, Münchhausenstrasse 21, D-81247 München, Germany. E-mail: haszi@zsm.mwn.de Introduction Whereas the systematic position of Kamptozoa (= Entoprocta) is fairly settled among the spiralian Lophotrochozoa by phenotypic and genotypic characters, clear affinities of this phylum are still a matter of debate (see the recent review by Nielsen 2002a). Contrary to the statement of Nielsen (2002a: p. 687) that ‘no entoproct character indicates a closer relationship with any specific spiralian phylum’, Bartolomaeus (1993a,b) and Haszprunar (1996) have both argued in © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences favour of a direct sister-group relationship with the Mollusca. These assumptions were based on several similarities between Kamptozoa and basal Mollusca: (1) a distinct division of the body into a dorsal area covered by a cuticle and a ventral area that is specialized as a ciliated creeping sole with anterior cirri is shared by basal Mollusca (particularly the aplacophoran Solenogastres) and creeping entoproct larvae; (2) an epidermal, chitinous cuticle, which is not moulted, in contrast to the ecdysozoan Nemathelminthes and Arthropoda; (3) a sinus circulatory system, where the  Fine structure of Loxosomella larva (Entoprocta) • Haszprunar and Wanninger blood flows in distinct spaces lacking any epithelial border, combined with a pumping organ (Emschermann 1969). Based on a cladistic analysis of morphological characters of the spiralian phyla, Haszprunar (1996: p. 22) concluded that ‘the assumption of the proposed sister-group relationship [of Mollusca and Kamptozoa] is now more probable than previously thought,’ but ‘that data on ... the kamptozoan larva are still very poor’. Indeed, the aberrant morphology and biology of the sessile, filter-feeding kamptozoan postlarval stages makes it improbable that distinct morphological similarities with any other spiralian phylum will be found. It is much more likely that the trochophore-like larvae of the Kamptozoa do exhibit such similarities. However, up to now only two studies dealt with the fine-structure of kamptozoan larvae: Woollacott and Eakin (1973) described the fine structure of the cerebral eyes of the creeping larva of Loxosomella cf. harmeri, and Sensenbaugh (1986, 1987) investigated the holoplanktonic larva of Loxosoma pectinaricola using transmission electron microscopy (TEM). In addition, Nielsen provided scanning electron micrographs (SEM) of the larval Loxosoma pectinaricola and of an unidentified creeping loxosomatid larva from the Bahamian plankton (Nielsen 2002a,b). Based on widespread and thorough studies on the kamptozoan life cycle, Nielsen (1971) concluded that the creeping type with direct metamorphosis is probably the most primitive larval type among the Kamptozoa. Therefore, we concentrated our studies on this larval type to look for peculiarities with phylogenetic significance. Most recently, Wanninger et al. (2007) pointed out the characteristic nervous system of this larval type revealed by immunocytochemistry and confocal laser scanning microscopy (CLSM). Here we describe the fine structure of the kamptozoan creeping larva and discuss the molluscan affinities of the Kamptozoa. Materials and Methods Animals Adult Loxosomella murmanica (Nilus 1909) live on the body wall of the sipunculan Phascolion strombus, which inhabits empty shells of the gastropod Turritella (cf. Nielsen 1971). Turritella shells inhabited by Phascolion were dredged from 30 m depth off the Kristineberg Marine Research Station, Sweden, transported to the laboratory, and maintained in large plastic containers in natural seawater. Water was changed twice per day; the seawater was filtered through a 70-µm mesh and checked using a stereomicroscope for larvae that had been released by the mother animal. Fixation and TEM All individuals were relaxed in a solution of 3.5% MgCl2 before fixation, which was carried out using a solution of 4% glutaraldehyde in 0.2  sodium cacodylate buffer (SCB) for  Acta Zoologica (Stockholm) 89: 137–148 (April 2008) 2 h at room temperature. Subsequently, the samples were washed three times for 15 min in SCB and stored at 4 °C. Alternatively, larvae that were fixed in 4% paraformaldehyde with 0.05% glutaraldehyde and stored in phosphate-buffered saline with 0.1% NaN3 were used with the same results. In the following, the specimens were post-fixed in buffered 1% OsO4 for 1 h at room temperature, washed in deionized water (three times for 15 min), dehydrated in an ethanol series and embedded in low-viscosity resin (Spurr 1969). Ultrathin sections with gold–silver interference colour (about 70 nm thick) were cut with a diamond knife and stained for 30 min with saturated uranyl acetate and for 5 min with lead citrate after Reynolds (1963). The sections were examined and photographed with a Philips CM 10 TEM. Results External morphology The larvae investigated closely resemble the general descriptions given by Jägersten (1964) for Loxosomella, and by Franzén (1970) and Nielsen (1967, 1971) for the same species. Peculiarities of this creeping larva are a prominent foot sole, the lack of pigmented eye spots, the prominent pair of ciliary frontal organs, and the covering of the dorsal side by granules of unknown composition (Fig. 1). Compared with other trochophore larvae, the axes in Loxosomella are somewhat different in that the pretrochal episphere is extended and covers the whole dorsal body of the larva. The larva is of the ciliary-gliding type with a prominent foot sole that bears some cirri at the frontal end. Most anteriorly there are the paired, frontal organs (with extended cilia in live animals, but contracted in the preserved specimens), each of which has a prominent ciliary tuft, but they lack the pigmented eye spots found in the larvae of many other loxosomatid species. The dorsal area (episphere) is covered by a thin cuticle and shows fine granules. The prototroch forms a ring around the whole ventral side of the body and can be contracted so that the foot is enclosed in a cavity. Episphere The epidermal cells of the episphere lack cilia and are covered by a thin microvillous cuticle (Rieger 1984). Over a distance of about 200 nm the glycocalyces of the microvilli form a dense layer, the microvilli themselves are slightly longer than this distance (Fig. 2A). This type of cuticle also covers the prototroch region, where the cilia penetrate the cuticle (Fig. 2B). At the episphere there are additional granulelike elements outside the cuticle, which strongly react with the electron beam in the TEM showing dynamic interference patterns (Fig. 2C). We could not detect any evidence that these particles are produced by the animal. Most epidermal cells of the episphere are myoepithelial cells, where the © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 89: 137–148 ( April 2008) Haszprunar and Wanninger • Fine structure of Loxosomella larva (Entoprocta) Nervous system The anatomy of the serotonergic portion of the nervous system has been recently described in detail by Wanninger et al. (2007). The TEM sections confirm and supplement the theory that the general pattern of the nervous system matches the results provided by antiserotonin staining: we found a paired cerebral ganglion supplying the frontal organs, and we confirm the subepidermal, tetraneuran condition of the longitudinal neural cords (Fig. 3). One pair of prominent nerves (Fig. 4A) runs above the foot sole, a second pair, which is less prominent (Fig. 4B), is situated laterally at half the level of the episphere. Pericarya of nerve cells are rarely found, although they are quite large. In addition, there is a very large apical ganglion with – contrary to the apical organ itself – almost no serotonergic elements, and the likewise subepidermal, circumprototrochal nerve ring is prominently developed (Fig. 3). There are no further main nerves or ganglia after those revealed by the antiserotonin staining. Apical organ The apical organ consists of many, very narrow, polyciliary cells without any cuticle formation, but with a dense microvillous border and few supporting cells interspersed. The cilia of the apical organ show the regular microtubular pattern and have short roots (Fig. 4C). The basal neural processes of the ciliary cells run directly downwards into the underlying, very large apical ganglion. Frontal organ Fig. 1—The creeping larva of Loxosomella murmanica. Scale bars represent 50 µm. —A. SEM image in ventral aspect showing the ciliated gliding sole of the foot (ft), the ciliated area of the paired frontal organ (f1, f2), as well as the prototroch (pt). —B. Drawing of a live specimen in lateral left view. Note the cirri (cr) situated at the anterior region of the foot and the apical organ (ao), which is located opposite the foot and so is shifted by 90° relative to the anterior–posterior axis of the larva. After Nielsen (1971), reproduced with kind permission of the author. The paired frontal organ lies anteriorly between the apical organ and the mouth opening and is extended in the living larva (Fig. 1). In the preserved specimens it consists of two deep invaginations that are filled by densely arranged cilia from several polyciliary cells (Fig. 4D). The cilia show the regular pattern of microtubuli and long roots. The cerebral ganglia are situated immediately behind the cavities of the frontal organs. The short cerebral commissure fuses mid-dorsally with the ventral part of the apical ganglion (Fig. 5A). Prototroch myofilaments are obliquely striated and are mainly situated in the distal part of the cells interconnecting the zonula adherens of the belt desmosomes (Fig. 2D). Large, solitary mucous cells occur occasionally in the epidermis of the episphere (Fig. 2E). The nucleus rests on the base and is often highly homogeneous, the cytoplasm shows several large vacuoles; a distinct opening was never detected. © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences The circular prototroch surrounds the whole body of the larva and forms a distinct fold. The prototroch itself consists of two cell rows separated from each other by a cell row of obliquely striated myoepithelial cells (Fig. 5B), whereas a ciliated food rim is lacking. The dorsal ciliary ring (i.e. the ‘outer prototroch’ of Jägersten 1964) is formed by cells with very many (up to 40 have been counted), long, accurately arranged cilia with very long roots reaching to the basis of these cells (Fig. 5B,C). The myoepithelial  Fine structure of Loxosomella larva (Entoprocta) • Haszprunar and Wanninger Acta Zoologica (Stockholm) 89: 137–148 (April 2008) Fig. 2—Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards, scale bars represent 1 µm. —A. Cross-section of the ‘pallial region’ between prototroch and foot showing the microvillous border (mv) with distinct glycocalyx layer (arrows). —B. Cross-section of the outer prototroch showing distally situated mitochondria (asterisks), accurately arranged cilia with long roots (arrowheads), microvillous border (mv), and glycocalyx layer (arrow). —C. Epidermis of the episphere showing microvillous border (mv) with glycocalyx (obliquely sectioned) and the enigmatic granules (gr). Below the epidermis, muscle fibres (mu) are visible. —D. Tangential section of the epidermal myoepithelial cells in the prototroch region with cilia (ci). Note that the myofilaments (mf ) are situated distally of the cell. —E. Solitary mucous cells of the episphere. Note the nucleus (nu) with homogeneous chromatin, the large, bright vacuoles (va), and the sparse regular cytoplasm (cy). cells form a ring, which probably corresponds with the ‘prototroch constrictor’ described by Nielsen (1971: Fig. 31) for Loxosomella pectinaria, although the trochus conditions differ in that species. The ventral ring (‘middle metatroch’ of Jägersten 1964) is much smaller than the outer ring, the respective cells bear fewer and much shorter cilia. According to the orientation of the ciliary microtubules, the beating direction of the cilia (always perpendicular to the line of the central tubuli towards the cleft between tubuli pairs 4 and 5) of both rings is from dorsal to ventral.  Foot The cilia of the most anterior portion of the foot typically form cirri (Mariscal 1965; Nielsen 1967, 1971; Franzén 1970). The TEM images show, however, that a special connection between the cilia does not exist. Between the polyciliary cells of this region there are the openings of the cells of the pedal glands (Fig. 5D). These symmetrically arranged glands reach deep into the head region of the larva and occupy a large volume dorsal to the gut (Fig. 6A). The cytoplasm of these subepidermal gland cells show numerous, © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 89: 137–148 ( April 2008) Haszprunar and Wanninger • Fine structure of Loxosomella larva (Entoprocta) because of the positional shift of the prototroch by 90° relative to other trochozoan larvae, the axis of the gut corresponds to the longitudinal axis of the prototroch and does not cross the latter as in the typical trochophore-type larva. The mouth opening is placed behind the anterior fold of the prototroch. The most anterior part of the gut makes a short loop forwards and upwards and then runs straight backwards. The hindgut opens dorsally near the posterior end of the foot (Fig. 6D). The lateral cells of the oral opening and of the most anterior part of the gut have a microvillous border and cilia. The anterior portion of the gut opens quite abruptly into the midgut (Fig. 7A). The epithelium of the midgut shows a dense microvillous border and small yolk vesicles but no cilia, the lumen is extremely narrow. The cilia of the anterior part reach backwards for some distance, later only microvilli are visible in cross-sections. The cells of the posterior alimentary channel and the anal opening again have cilia. There was no trace of food or nourishment throughout the whole gut, the cells of which still show some small yolk vesicles. Protonephridia Fig. 3—Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards. —A. Cross-section of the larval body at half length showing the general organization (cuticularized episphere, ciliated prototroch, pallial rim lateral to the ciliated foot sole), in particular the central gut and the position of the lateral and pedal nerve cords (see inset figure): two asterisks indicate artificial cleft caused by fixation above the gut, one asterisk indicate a hole in the section caused by a sticky stone. Scale bar = 25 µm. —B. Same photograph with marked gut (dark grey, central position), lateral and pedal nerve cords (white; the right lateral cord is swollen because of the presence of a nerve cell body with nucleus, see Fig. 4B), and the subepidermal prototroch nerve (grey, lateral). small, homogeneous vesicles (Fig. 6B), while their necks are quite narrow. More posteriorly, the foot sole becomes regularly ciliated, mucous cells are rare in this region and always truly epithelially positioned. The microvillous border of this area again shows distally a very delicate, but distinct glycocalyx layer. In this area there are dorsoventral muscles, which are obliquely striated and run from the dorsolateral region to the foot. These muscles are obliquely striated and intercross just above the foot sole’s epithelium (Fig. 6C). Alimentary tract The free larval stage investigated has a gut, the lumen of which runs throughout the body. It is worthwhile noting that, © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences A single pair of laterally situated protonephridia could be detected in the TEM sections. There is a single, multiciliated terminal cell building up the ultrafiltration weir, its nucleus is semicircular in shape and is thus visible twice in the sections (Fig. 7B). The channel is formed by a string of single cells, its lumen is extremely narrow and is entirely filled with cilia (Fig. 7C). Close to the excretory opening the channel bends towards the ventral side, and after this the lumen is widened and the cells bear microvilli (Fig. 7D). The excretory opening is provided with a sphincter muscle and is placed in the lateral ‘pallial’ rim between the prototroch and the foot. Discussion The kamptozoan gliding larva as a trochophore – molluscan mosaic The present results provide novel insights into the organization of the kamptozoan creeping larva. According to Nielsen (1971), this larval type is more primitive for the phylum than both the holopelagic type with a reduced foot sole and the intermediate forms that have been described (Nielsen 1971). The larva of L. murmanica exhibits a mosaic of (1) autapomorphic characters, which may additionally define the phylum but need confirmation by studies on larvae of further species, (2) typical features of a trochophore larva being defined in the broad sense of Rouse (1999) as the synapomorphic, lecithotrophic larva of Trochozoa, i.e. Kamptozoa, Mollusca, Sipuncula and Annelida (possibly also Nemertinea; cf. Maslakova et al. 2004), (3) characters that are shared with the adult Mollusca, particularly with  Fine structure of Loxosomella larva (Entoprocta) • Haszprunar and Wanninger Acta Zoologica (Stockholm) 89: 137–148 (April 2008) Fig. 4 —Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards, scale bars represent 1 µm in A–C and 10 µm in D. —A. Detail of Fig. 3. Cross-section of the pedal nerve above the ciliated epithelium of the foot sole (cf ) lateral to the dorsoventral muscle fibre (mf ). —B. Detail of Fig. 3. Cross-section of the lateral nerve below the cuticularized epidermis (ep) of the episphere showing a (rare) neural nucleus (nu) with two portions of neuropile (ne) adjacent to various muscle fibres (mf ). —C. Cross-section of the apical region showing many narrow sensory cells (asterisks), bearing long cilia (ci) with short roots (arrowheads). In living larvae the cilia form a ciliary tuft. —D. Cross-section of the most anterior part of the larva showing the retracted frontal organs (fo) with dense ciliation (ci). In the living larva the frontal organs are extended (see Fig. 1). Fig. 5—Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards, scale bars represent 10 µm in A and 1 µm in B–D. —A. Cross-section of the larva just in front of the apical organ (ao) showing the large, centrally placed apical ganglion (ag) and more ventrally the posterior portion of the paired cerebral ganglion (cg). Dorsally right several obliquely striated muscle fibres of the dorsoventral muscles (dvm) are visible. —B. Cross-section of the prototroch showing the dorsally placed main trochus, the cilia (ci) with very long roots marked by arrowheads, the myoepithelial cell row (me), and the much weaker ventral trochus (vt). —C. Oblique section of the outer prototroch showing accurately arranged cilia (ci) with long roots (dark dots in the cytoplasm) between many distally situated mitochondria (asterisks). —D. Crosssection of the anterior end of the foot sole showing the releasing necks and openings of the pedal gland cells (pg) between the polyciliary cells bearing the cirri (cr).  © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 89: 137–148 ( April 2008) Haszprunar and Wanninger • Fine structure of Loxosomella larva (Entoprocta) Fig. 6—Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards, scale bars represent 10 µm in A, 1 µm in B and C, and 2 µm in D. —A. Cross-section posterior to the cerebral ganglia. In the centre lies the foregut (fg), the lumen of which is filled by cilia. Dorsally placed are the pedal glands (pg), the cleft is an artefact. Ventral side with ciliary foot sole (fs), flanked by the releasing channels (rc) of the pedal glands. Most laterally are the polyciliary cells of the prototroch (pt). —B. Cells of the pedal glands showing nuclei (nu), adjacent dictyosomes (di), smooth endoplasmic reticulum, and numerous vesicles. —C. Cross-section of the central foot sole with dense ciliation (ci). Note the dorsoventral muscles (dvm), which intercross ventrally (square). —D. Cross-section of the posterior end of the larva showing the prototroch (pt) and the anal opening (a) slightly dorsal to the rear of the foot sole (fs). Fig. 7—Loxosomella murmanica, TEM. Orientation is with the dorsal side facing upwards, scale bars represent 1 µm. —A. Cross-section of the point where the foregut (fg) with many cilia enters the midgut (mg) with much denser microvilli and few cilia. —B. Oblique section through the terminal end of the protonephridium showing the cilium (ci) of the flame cell with the ultrafiltration weir (arrows) with many microvilli and (because of bending) a crosssection of the most terminal part of the releasing channel (rc) filled with cilia. The various sections all belong to the single nucleus (nu) of the terminal cell. Dorsoventral and longitudinal muscle fibres (mf ) are situated nearby. —C. Cross-section of the releasing channel (rc) of the protonephridium filled with cilia adjacent to the lateral nerve (ne). —D. Cross-section of the protonephridial releasing channel (rc) close to the excretory opening. Note the cilia and microvilli in the lumen and the adjacent sphincter muscle (m). © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences  Fine structure of Loxosomella larva (Entoprocta) • Haszprunar and Wanninger the Solenogastres (Neomeniomorpha), and (4) neuroarchitectural apomorphies shared with larval polyplacophorans (Friedrich et al. 2002; Voronezhskaya et al. 2002; the larval nervous system of Solenogastres and Caudofoveata is still insufficiently known). All character sets coexist beside each other in this larva, which as a whole may be interpreted as a trochophore–ancestor mosaic with some peculiarities. The general morphology of the creeping larva is twofold: on the one hand it is a typical trochophore larva in the sense of Rouse (1999), showing a ciliated apical sensory organ, a prominent prototroch arising from the 1d-lineage (Marcus 1939; recently reviewed by Nielsen 2005), and a pair of protonephridia. On the other hand, it exhibits several characters that are typical for adult Mollusca: the division of the body into a cuticularized episphere and a microvillous– ciliary hyposphere, the ventral foot sole with its anteriorly placed cirri, and the anal opening, which is situated slightly dorsally to the rear of the animal. Moreover, the larval serotonergic nervous system shows a mosaic of larval and adult molluscan characters including a complicated larval apical organ comprising a set of central flask cells and several peripheral cells, a preoral nerve loop, an oral nerve ring, and true mollusc-like tetraneury (Wanninger et al. 2007). The episphere again shows the mosaic character of the larva. Certainly the clearest apomorphic character of the creeping larva of L. murmanica is found in the myoepithelial cells of the episphere and prototroch (see also Sensenbaugh 1987). Such a cell-type is not known from any other trochophore type, but from adult Kamptozoa (Emschermann 1982). Eeckhaut and Jangoux (1993) have described similar conditions in the epidermis of adult Myzostomida (the finestructure of their trochophore larvae is unknown), a phylum probably closely related to the Annelida. It is thus unlikely that this character represents a synapomorphy between the two taxa. Again apomorphic, and possibly restricted to this species, are the enigmatic granules on the epispheral cuticle, the function and origin of which are unknown. The general type of microvillous cuticle with a distally placed glycocalyx is found in many protostomian phyla and is considered a primitive stage of cuticle (Rieger 1984). The large, solitary mucous cells of the episphere resemble similar cells (many large vacuoles, again lacking a clear opening) in the notum of aplacophoran molluscs, Solenogastres and Caudofoveata (e.g. Hoffman 1949). It needs to be shown whether the larval cuticle already includes chitin, like the cuticle of the adult Kamptozoa and aculiferan Mollusca (Salvini-Plawen and Nopp 1974; Jeuniaux 1982; Unger and Bartolomaeus 2004). The significance of the various aspects of the nervous system concerning kamptozoan–molluscan affinities has been outlined recently by Wanninger et al. (2007). Here, we note again that the concurrence of an apical ganglion and true cerebral ganglia, as well as the presence of a trochal ring in addition to the lateral and ventral cords in the same stages, reflects the larval–adult mosaic nature of the kamptozoan  Acta Zoologica (Stockholm) 89: 137–148 (April 2008) creeping larva. Our TEM sections clearly confirm the tetraneuran condition of the whole (not just serotonergic) nervous system, which so far has been considered as a diagnostic character for the Mollusca. Whereas a ciliated apical organ is found in the overwhelming majority of (non-ecdysozoan) invertebrate larvae (for details see Wanninger et al. 2007), the frontal organs have in the past been considered as a kamptozoan apomorphy (Mariscal 1965; Nielsen 1971, 2005). The frontal organs are paired basally and fused in more derived forms. Photoreceptive structures, which may be interpreted as cerebral eyes, have been found in many kamptozoan species within the frontal organs. Cerebral eyes in Mollusca are restricted to Gastropoda and Cephalopoda and are unlikely to be a feature of the molluscan ground pattern (Salvini-Plawen 1981; Haszprunar 2000). In addition, the kamptozoan photoreceptors are of the ciliary type (Woollacott and Eakin 1973), which in Mollusca is restricted to few, long-planktonic larvae (e.g. Blumer 1995, 1998). Whereas photoreceptor cells are probably plesiomorphic for Bilateria or even Metazoa, the homology of cerebral eyes as organs between phyla remains doubtful (see recent discussion in Arendt 2003; Gehring 2004; Plachetzki et al. 2005; or Fernald 2006). Interestingly, the bryozoan (ectoproct) larvae likewise exhibit ciliary photoreceptors (e.g. Woollacott and Zimmer 1972; Hughes and Wollacott 1978, 1980; Reed 1988; Reed et al. 1988). The presence of a paired (respectively symmetrical), invaginated and contractile, preoral sensory system is shared with the Solenogastres, where such a structure is known as ‘atrial sensory organ’. The fine structure of the latter is still poorly known (Haszprunar 1986; reproduced in Scheltema et al. 1994) and suggests chemoreception as in the case of the kamptozoan frontal organ. Differences, however, occur in that the kamptozoan frontal organ is clearly pretrochally situated, whereas the atrial sense organ of Solenogastres appears to be a post-trochal structure (Baba 1938; Okuso 2002). The latter is also true for the so-called ‘pedal shield’ (in fact cerebrally innervated) of the Caudofoveata (Chaetodermomorpha), which is a sensory structure rather than a burrowing organ (Tscherkassky 1989; Scheltema et al. 1994; Shigeno et al. 2007). Again, direct homology is doubtful. At first glance, and based on external morphology alone, the prototroch of the larva of L. murmanica closely resembles a trochophore with a so-called downstream system, which is usually correlated with a planktotrophic mode of development (Nielsen 1987, 2001, 2004, 2005). However, no less than three features do not correspond with this hypothesis: (1) the gut does not contain any trace of food; (2) the cilia of the ‘metatroch’ beat downwards as do the cilia of the ‘prototroch’, whereas metatroch cilia beat in the opposite direction; (3) there is no ciliary band towards the mouth opening to transport filtered particles. Accordingly, this larva – although provided with a double ciliary band consisting of compound cilia – neither has a downstream system nor is planktotrophic. This result raises doubts on many other cases © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 89: 137–148 ( April 2008) Haszprunar and Wanninger • Fine structure of Loxosomella larva (Entoprocta) throughout the spiralian phyla, where SEM data alone have been used to infer the presence of a downstream system. The exact mode of feeding in kamptozoan larvae, as reported by Jägersten (1964), needs to be investigated to confirm a downstream mode of feeding. The condition of the foot of the kamptozoan gliding larva provides the most significant similarities with basal Mollusca: Nearly identical to the conditions of the Solenogastres, the anterior part of the foot is provided with cirri consisting of from several to many regularly structured cilia. Somewhat similar organs have been described for several larvae of Bryozoa as ‘vibratile plume’ (e.g. Reed et al. 1988; Zimmer and Woollacott 1989, 1993), but these larvae are planktonic rather than of the benthic creeping-gliding type. The larvae of Loxosomella and Solenogastres share a so-called pedal gland, which occupies a considerable volume of the head region and is continued more posteriorly by differently structured mucous cells (sole glands). The most exciting finding, however, is the presence of the intercrossing dorsoventral muscle fibres – up to now a diagnostic character of the Mollusca. In summary, the foot of the kamptozoan larva shares all major characteristics with the foot of basal molluscs, particularly of the Solenogastres, because the Caudofoveata (Chaetodermomorpha) entirely lack a pedal sole and the Polyplacophora show a pedal gland only in the larval stage (Hammarsten and Runnström 1926). As outlined above, the gut does not show any trace of larval feeding. It is interesting to note that the position of the anal opening exactly corresponds to that of the Polyplacophora and Monoplacophora, i.e. slightly dorsal to the rear of the foot. The presence of a single pair of laterally situated protonephridia is typical for spiralian larvae. Since we could not follow the larvae through metamorphosis, it remains open, whether the adult protonephridia, the fine-structure of which somewhat differs from those of the larva (Franke 1993), are the retained larval ones or of secondary origin. A clade of Mollusca and Kamptozoa The present investigation provided significant similarities between Mollusca and Kamptozoa. Whereas molecular data up to now have only occasionally contributed significantly to resolving the interrelationships of the phyla within the Spiralia or Lophotrochozoa (e.g. Boore and Staton 2002), phylogenetic considerations and cladistic analyses of morphological characters revealed a clade ‘Lacunifera’, consisting of both phyla, more than 10 years ago (Bartolomaeus 1993a,b; Haszprunar 1996, 2000; Ax 1999). At present, and with the new data provided herein, an impressive list of potential synapomorphies uniting Mollusca and Kamptozoa can be presented: 1 The division of the body into a cuticularized episphere and a ciliary–microvillous pedal part. 2 A chitinous cuticle that is not moulted (Unger and Bartolomaeus 2004). © 2007 The Authors Journal compilation © 2007 The Royal Swedish Academy of Sciences 3 A pedal gliding sole with (3a) anteriorly placed cirri, (3b) a symmetrical/paired pedal gland, which reaches deeply into the head region of the body, and (3c) a pedal sole with epidermal mucous cells. 4 Dorsoventral muscles that intercross above the pedal sole (so far diagnostic for Mollusca). 5 A sinus (tubes that lack endothelia) circulatory system (Bartolomaeus 1993a,b). 6 An anal opening that opens slightly dorsal of the rear of the pedal sole. 7 A tetraneurous nervous system (so far diagnostic for Mollusca) with (7a) pedal commissures, (7b) a buccal nerve ring, and (7c) an anterior nerve loop (Wanninger et al. 2007). 8 A complex apical organ consisting of several serotonergic cells (contrary to most other trochozoans, which usually only express very few serotonergic cells in the apical organ) and of two cell types (serotonergic ciliary flask cells and additional peripheral cells) (Voronezhskaya et al. 2002; Wanninger et al. 2007). 9 Possibly also the solitary mucous cells in the episphere, which may correspond to similar structures found in the notum of Solenogastres. To summarize, we think that Lacunifera, the clade consisting of Kamptozoa and Mollusca, is currently among the best documented interrelationships of two metazoan phyla. Whereas we agree that phylogenetics is always a matter of probabilities and can never be proven in a strict sense, we believe it will be hard to overcome this list of distinct similarities with an alternative hypothesis. Implications for molluscan origin and the position of the Solenogastres Accepting the similarities listed above as synapomorphies of Mollusca and Kamptozoa, and thus accepting Lacunifera as a clade, also has implications for the hypothetical ancestor of the Mollusca. In addition, an accepted sister-group relationship between Kamptozoa and Mollusca also roots character polarity concerning the phylogenetic position of molluscan subgroups. A clade Lacunifera is a direct counter-hypothesis against the proposed sister-group relationship of the Mollusca to the Sipuncula (Scheltema 1993, 1996). The latter hypothesis has already been weakened by cladistic analysis of morphological characters (Haszprunar 1996, 2000), developmental data (Wanninger et al. 2005), and by molecular analyses (Boore and Staton 2002). We therefore regard this hypothesis as no longer valid. Maybe the most important point is the deduced hypothesis that a molluscan-like pedal sole with pedal gland was already a feature of the premolluscan (i.e. sinusoidean) ancestor and not an evolutionary novelty within the Mollusca. Accordingly, the hypothesis of Salvini-Plawen (1972, 1981, 1985, 1991, 2003) of a plesiomorphic turbellaria-like (cerebrally  Fine structure of Loxosomella larva (Entoprocta) • Haszprunar and Wanninger and pedally innervated) gliding sole and the basic division of the Mollusca into Scutopoda (with a retained cerebral part) and Adenopoda (with a retained pedal part) should no longer be upheld (see also Salvini-Plawen 2006). A direct sister-group relationship of the acoelomate Kamptozoa and the coelomate (but not eucoelomate; cf. Salvini-Plawen and Bartolomaeus 1995; Haszprunar 1996; for discussion) Mollusca renders homology of the molluscan coelomic cavities (i.e. the gonopericardial system) and the coelom of Sipuncula or Annelida unlikely. The present results also shed additional light on the assumed basic position of the Solenogastres (Salvini-Plawen and Steiner 1996; Haszprunar 2000) within the Mollusca: only this molluscan group (still) exhibits a pedal sole with anterior cirri, only this taxon shows the distinct solitary mucous cells in the notum. Outlook The present findings shed light on several anatomical features of the kamptozoan creeping larva. However, several questions, and in particular the variation of most characters within the Kamptozoa, remain to be studied. It is time to pay more attention to this neglected group. Acknowledgements We thank the Marine Biological Station in Kristineberg for hospitality. Eva Lodde (ZSM) and the late Alenka Kerin (Department Biology I of LMU Munich) provided technical support during preparation, Prof. Dr M. Starck (Department Biology II of LMU Munich) and his team were responsible for the TEM facilities. Marianne Müller (ZSM) helped with the photographic and digital work. We also thank two anonymous referees for detailed and very valuable comments on the draft of the typescript. This work was supported by funds from the Danish Research Agency (FNU; grant 21-04-0356 and 272-05-0174 to A.W.) and the European Commission (ARI-programme, 5th framework, to A.W.). References Arendt, D. 2003. Evolution of eyes and photoreceptor cell types. – International Journal of Developmental Biology 47: 563–571. Ax, P. 1999. Das System der Metazoa. II. Stuttgart 383 pp, Gustav Fischer. Baba, K. 1938. 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