A caenogastropod in 3D: microanatomy of the Munich endemic springsnail Sadleriana baoarica Boeters, 1989

1 SPIXIANA 37 11-19 München, August 2014 ISSN 0341-8391 A caenogastropod in 3D: microanatomy of the Munich endemic springsnail Sadleriana bavarica Boeters, 1989 (Caenogastropoda, Hydrobiidae) Katrin Koller, Bastian Brenzinger & Michael Schrödl Koller, K., Brenzinger, B. & Schrödl, M. 2014. A caenogastropod in 3D: microanatomy of the Munich endemic springsnail Sadleriana bavarica Boeters, 1989 (Caenogastropoda, Hydrobiidae). Spixiana 37 (1): 1-19. Comparative 3D-microanatomy reconstructed from histological sections has become a powerful method for investigating anatomical details of small animals, and has been particularly applied to heterobranch gastropods. Here we present first comprehensive 3D data on a member of the Caenogastropoda, the putative sister clade of Heterobranchia. The limnic hydrobiid snail Sadleriana bavarica was collected from its type locality, the Brunnbach, a creek within the city limits of Munich, Germany. External features are described and compared with the holotype; S. bavarica is supported as a valid species based on morphological evidence, but further morphoanatomical examination of allosperm receptacles and molecular analyses are required. Five specimens were embedded in epoxy resin, sectioned serially, and described histologically. With the software Amira 3D models of all major organ systems were generated. Anatomy is compared to other caenogastropods and heterobranchs; special reference is given to the central nervous system. The arrangement and homology of cerebral nerves are discussed, contrasting earlier opinions on the origin and evolution of tentacular nerves. Katrin Koller (corresponding author), Bastian Brenzinger & Michael Schrödl, SNSB – Zoologische Staatssammlung München, Münchhausenstraße 21, 81247 München, Germany; and Department Biology II, BioZentrum, Ludwig-MaximiliansUniversität, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany; e-mails: [email protected], [email protected], [email protected] Introduction The vast majority of gastropods is grouped into the sister taxa Caenogastropoda and Heterobranchia, together forming the taxon Apogastropoda (SalviniPlawen & Haszprunar 1987); these relationships were supported by multi-locus sequence analyses (Stöger et al. 2013). Caenogastropods include over 120 families, and about 60 % of all living gastropod species (Ponder et al. 2007, Strong 2003). Although mainly marine, caenogastropods have undergone several successful radiations into freshwater, including the species-rich family Hydrobiidae Troschel, 1857 (Strong 2003, Ponder et al. 2007, Strong et al. 2008). Hydrobiidae is considered to be one of the largest gastropod families with more than 400 genera and over 1250 species assigned (Strong et al. 2008, Wilke et al. 2013). Because most hydrobiids are small animals, living in small and isolated freshwater bodies such as artesian springs, caves, or crevices in groundwater, their collection – especially of live specimens – is often difficult (Glöer 2002, Ponder & Clark 1990). Most hydrobiids show a dextrally coiled, smooth, valvatito slightly turriform shell and a head with a distinct snout and a pair of long and thin tentacles (Glöer 2002), as do other closely 2 related taxa. Currently, only few anatomical autapomorphies are described to distinguish Hydrobiidae, e. g. the presence of a closed ventral wall of the female capsule gland (Wilke et al. 2013). Gross-anatomical information is available from dissections of several hydrobiids (e. g. Hershler & Davis 1980, Hershler & Ponder 1998), but detailed histological or microanatomical information on tiny organs and structures is largely lacking. Shell features have been used as taxonomic characters for central European snails for a long time. However, taken alone they can be misleading and are often not reliable to distinguish on a species level (Haszprunar & Koller 2011). Individuals of a single species, for example, may develop different shells, size and shapes in dependency of their habitats (ecomorphs) (Glöer 2002). Particularly in hydrobiids, the taxonomy is primarily shell-based, and recent molecular studies suggest that their traditional taxonomy is widely deficient (Criscione & Ponder 2013, Wilke et al. 2013). Hydrobiids of the genus Sadleriana Clessin, 1890 are small freshwater snails characterized by a round shell, open umbilicus and reddish-brown operculum (Clessin 1890, Glöer 2002). There are only 8 species assigned to Sadleriana currently (Szarowska & Falniows ki 2013) and their taxonomy is still unclear. The genus itself was originally described as a subgenus of Lithoglyphus C. Pfeiffer, 1828 by Clessin (1890; see Szarowska & Wilke 2004) and considered as a distinct genus by Giusti & Pezzoli (1980). One species, Sadleriana pannonica (Frauenfeld, 1865) from the eastern parts of Slovakia and Hungary, recently was transferred to the genus Bythinella Moquin-Tandon, 1856 (Szarowska & Wilke 2004, Wilke et al. 2013). The distribution of the genus Sadleriana is largely restricted to limestone areas of southern Europe, with species in e. g. Italy, Slovenia and Croatia (Szarowska & Wilke 2004, Szarowska & Falniowski 2013). That is why the affiliation of S. bavarica Boeters, 1989, a single species occurring north of the Alps, to this group was in doubt. Sadleriana bavarica is a highly endemic species that occurs only within the city limits of Munich, Bavaria, where it is found only in a short (approx. 3 kilometer long) cool stream leading from a small groundwater spring into the Isar river (Seidl & Colling 1986, Boeters 1989, Glöer 2002, Szarowska & Wilke 2004). This habitat (the “Brunnbach”) is regarded to be an isolated remnant of glacial deposits stemming from the Riß period, and is thus older than comparable habitats in the southern proximity, which were remodelled by moraines during the subsequent Würm glaciation (Sedlmeier & Schwab 2006, LBV 2007). Seidl and Colling (1986) originally determined their specimens to belong to an isolated population of Sadleriana fluminensis (Küster, 1853), a species otherwise found south of the Alps in Slovenia. Further analyses by Boeters (1989) revealed morphological differences between S. bavarica and S. fluminensis, namely conchological aspects and characters of the male and female genital systems. 3D-microanatomy reconstructed from serial histological sections, e. g. using the software Amira, has shown its power for the detailed investigation and visualization of smaller gastropods (Neusser et al. 2006, DaCosta et al. 2007). Generating anatomical 3D models of minute structures from labelled semi-thin histological slices this method shows higher resolution, accuracy and reproducibility compared to the traditional examination via dissection, which is not always efficient and usually destructive to small specimens. To date, 3D-microanatomical studies of apogastropods are mainly restricted to Heterobranchia (e. g. Neusser et al. 2009, Haszprunar et al. 2011, Martynov et al. 2011, Brenzinger et al. 2013a,b, Hawe et al. 2013, Kohnert et al. 2013). Altnöder et al. (2007) examined a juvenile eulimid parasite using Amira software, but, to our knowledge, adult representatives of typical snail-like caenogastropods are still unexplored. In this paper, we undertook a histological and 3D-microanatomical study of the hydrobiid S. bavarica for two reasons: 1) to evaluate and supplement the original description, especially soft-body characteristics, and 2) to investigate the anatomy of a typical caenogastropod as basis for comparison to microanatomically better-known heterobranch taxa. Of special interest is obtaining reliable information on the central nervous system and on cerebral nerves, in order to evaluate contradictory hypotheses by Huber (1993) and Staubach (2008) on the homology and evolution of apogastropod nervous systems. Materials and methods Specimens of Sadleriana bavarica were collected by hand from stones and submerged driftwood from the Brunnbach, its type locality and single known habitat. Specimens were taken close to the Brunnbach spring (Herzogpark area, 48°9'21" N, 11°36'45" E) and towards St. Emmeram (16.08.2013, 01.03.2014; end of stream: 48°10' 45" N, 11°37'34" E) near the river Isar in Munich (31.05. 2012, 16.08.2013 and 01.03.2014). Collection permits were obtained as part of the project Barcoding Fauna Bavarica (BFB). Live specimens and the habitat were documented with digital cameras. For histology, specimens were relaxed with menthol and preserved in formalin (2.5 %). For 3D reconstruction five specimens were embedded in epoxy-blocks (Epon, block-numbers: 8W8, 8W9, 9W0, 9W1, 9W2; collection numbers ZSM Mol 20131107- 3 20131111). All but the first block were trimmed and sectioned serially with a microtome using a HistoJumbo diamond knife (Diatome, Biel, Switzerland), following the method described by Neusser et al. (2006) and Ruthensteiner (2008) (8W9, 9W0, 9W1: section thickness 1.5 µm, 9W2: 2.0 µm). The ribbons were collected on microscope slides, stretched by heat, stained with aqueous methylene blue/azure-II (after Richardson et al. 1960), and sealed with araldite resin. The slides of a male specimen (9W2, cross section) and a female specimen (9W0, longitudinal sections) were chosen for 3D-reconstruction. For photography a ProgResC3 ccd camera (Jenoptik, Jena, Germany), mounted on a Leica DMBRBE microscope (Leica Microsystems, Wetzlar, Germany), was used. In case of the male specimen (9W2) the complete animal (5 ×) and, its anterior body containing the nervous system and buccal organs (20 ×) were photographed. The female specimen (9W0) was photographed under a 20 × lens. Using Adobe Photoshop (Adobe Systems, Mountain View, CA), the photographs were stack processed (resized, changed to greyscale, unsharp-masked) and imported into Amira 5.2 software (Visage Imaging, Berlin, Germany). For the male (9W2) a resolution of 1200 × 890 pixels (complete animal), and 2080 × 1542 pixels (nervous system) were used. The female slides (9W0) were imported with a resolution of 2080 × 1542 pixels. Photographs were aligned and for each image-stack, the organs were labelled manually onto the sections, using different colours. Rendered 3D models of the organ systems were created for both specimens. In specimen 9W2 the complete 3D model is based on 507 photographs, with every second section being used. Details of the nervous system were analysed in a separate aligned stack (307 photos, every section used). The model of 9W0 based on longitudinal sections consists of 373 slides, with every second section used. For labelling, specimens were chosen according to their state of fixation and clarity of organ structures: habitus, digestive system and mantle cavity are reconstructed from the male specimen (9W2). Pericardial complex and central nervous system were reconstructed based on the female specimen (9W0). The reproductive system was reconstructed from a male and an immature female specimen, for that reason, structures like the bursa copulatrix and the receptacula could not be investigated. Systematics Caenogastropoda Cox, 1960 Sorbeoconcha Ponder & Lindberg, 1997 Truncatelloidea Gray, 1840 Family Hydrobiidae Stimpson, 1865 Subfamily Belgrandiinae de Stefani, 1877 Genus Sadleriana Clessin, 1890 Type species: Sadleriana fluminensis (Küster, 1853) (as Paludina), by original designation. Sadleriana bavarica Boeters, 1989 Holotype: HYD1008, stored at the Senckenberg Museum of Natural History, Frankfurt (Figs 1A-C). – Material examined: 5 specimens (ZSM Mol 201311072013111), collected at the type locality (Brunnbach, Munich). Embedded in epoxy resin, 4 of them sectioned. Several further specimens (two lots: ZSM Mol 20131112, 20131113). Natural habitat In its natural habitat (Fig. 1D), S. bavarica is found abundantly in shallow water (10-40 cm), dwelling actively at day on rocks and driftwood that are covered by a biofilm of green algae and diatoms (Fig. 1E). Male and female specimens of Sadleriana bavarica occur mixed with individuals of another hydrobiid species (here tentatively identified as Bythiospeum sp.). Specimens of S. bavarica are most abundant close to the Brunnbach spring. External morphology Sadleriana bavarica has a thick and brown shell between 3 to 4 mm in height and 3 to 3.5 mm in width, with 3.5 or 4 rapidly increasing whorls. The umbilicus is open and slotted. In most specimens the shell is covered in green algae (Fig. 1F-I). A corneous, reddish-brown operculum is attached to the footend’s upper side measuring 1.1 to 1.7 mm (Figs 3B,G; op). The soft body is black, except for the foot sole and the tentacle bases. The body is subdivided into a dextrally coiled visceral sac, which lies inside the shell curling up to the apex, and a broad muscular head-foot. The foot is broadened at the front and contains a well-developed foot gland (propodial gland), which is 0.6 mm width and 0.5 mm height (Figs 2F, 3A,C,G; fg, fgo). The head is clearly differentiated from the foot and possesses a pair of thin tentacles (0.6 mm long), which cannot be retracted. Eyes are located at the outer side of each tentacle base (Figs 2G, 3A,C,D; ey, tn). Here there is also a cushion-like batch of vacuolated cells. The snout is long, flexible, and protrusible (Figs 3C,D; sn). Mantle cavity and pallial organs The mantle cavity and pallial organs were reconstructed from the male specimen (9W2). The mantle cavity fills nearly half of the first whorl and then narrows to its rear parts increasingly (Figs 2B, 3D; mc). On its left side, the ctenidium is formed by 8 leaflets each of about 0.2 mm length (Figs 2A,B, 3A-C; ct, ctf). Behind the ctenidium, there is a voluminous semicircular mantle gland (length: 0.75 mm, height: 0.2 mm) (Figs 2B,D, 3A,C,D; mg). Histologically, the gland consists of a strip of particularly tall epidermal 4 A B D E C F G H I A B D E C F G H I Fig. 1. Sadleriana bavarica. External morphology and habitat. A-C. Holotype of Sadleriana bavarica, Senckenberg Museum Frankfurt (HYD1008). Photos by Sigrid Hof, section malacology, SMF. D-E. Natural habitat of Sadleriana bavarica, Brunnbach, Munich. F-I. Living specimens. Shell diameter approximately 2 mm. 5 Fig. 2. Sadleriana bavarica. Histology, cross sections; overview and details. A. Ctenidium. B. Anterior part, overview. C. Osphradium and connected ganglion. D. Mantle gland. E. Buccal gland. F. Foot gland. G. Eye. H. Radula cartilage. I. Radula. J. Gonad. K. Stomach. L. Posterior overview. M. Nephridial gland. Abbreviations: am, ampulla; an, anus; bg, buccal gland; bm, buccal mass; cdf, ctenidium filament; cl, ciliata; ct, ctenidium; dg, digestive gland; dgl, digestive gland lumen; fg, foot gland; fgo, foot gland opening; fr, food remains; gc, gastric chamber; gn, gonad; gs, gastric shield; in, intestine; kd, kidney; le, lens; mc, mantle cavity; mg, mantle gland; nc, nucleus; nch, nephridial channel; ng, nephridial gland; osp, osphradium; osg, osphradial ganglion; pe, penis; pg, pigments; rcc, radula cartilage cell; rdt, radula tooth; sg, salivary gland; ss, style sac; vc, visual cells. 6 cells that are stained in a very dark blue (Fig. 2B,D). Two distinct body openings lead into the mantle cavity’s right part: the anus opens into the right corner of the mantle cavity (Figs 2B, 3A; an); in males, the genital opening is located on the right side of the head-foot. The osphradium is a crescent-shaped, ciliated groove with tall yet narrow epithelial cells situated on the anterior left side of the mantle cavity roof, anterior to the gill; there is an oval ganglion just below the epithelium (Fig. 2C; osp and osg). The pericardial complex is located posteriorly, dorsally and to the left. Circulatory and excretory systems The pericardial complex comprises the main organs of the circulatory and excretory systems and is located at the posterior left of the mantle cavity, near the mantle gland (Fig. 3E,F). The kidney measures 1.8 mm in length and 0.43 mm in width. It is characterized by a vacuolated and unstained epithelium (Fig. 2L). Superior to the kidney there is a nephridial ‘gland’, a mass that contains loosely organized, irregular and unstained cells with darker blue nuclei (Fig. 2M); the part of the kidney in contact with this structure is thin and not vacuolated, forming short, apparently blind-ending ducts that project into the nephridial gland (Fig. 2M; nch and ng). On its posterior side, the kidney is attached to a thin-walled pericardium, which surrounds a twochambered heart: a thicker-walled ventricle and an auricle (Fig. 3E,F). Digestive system The digestive system consists of a short pharynx, followed by the short esophagus leading into the stomach, which is connected to a voluminous digestive gland. The intestine runs from the stomach to the mantle cavity’s right side. The mouth opening lies medially on the tip of the snout and leads into a wide pharynx. The pharynx contains a pair of small chitinous jaws, which are fused dorsally and located just behind the mouth opening, and two pads of epithelial single-celled glands in the posterior lower part. The radula (Figs 2I, 4B) is quite long (about 1 mm) and shaped like a question mark. It extends through much of the snail’s headfoot and is equipped with roughly 60 rows of teeth which are stained in a dark blue. The radula is bedded on two lower cartilaginous pillows and is topped by a smaller upper cartilage, all characterized by voluminous unstained cells with big and well-apparent nuclei and minute darker granules (Figs 2H, 4B). Two long (ca. 0.8 mm), tubelike salivary glands lie on the pharynx and open nearby the mouth opening. Histologically, they are glandular with numerous small vesicles and stained blue. Centrally, the salivary glands have a narrow lumen (Fig. 4A,C,E). The pharynx narrows to a ciliated oesophagus leading into the stomach. The stomach wall is muscular and thick (66 µm). The stomach is separated into a smaller, ciliated, upper part (style sac) and a bigger lower bag (gastric chamber) (Fig. 4A,C-E). The latter is equipped with a light blue-stained, angular and cuticular shield that carries a strongly elevated ridge (Figs 2L, 4D; gs). The stomach’s interior is voluminous and shows some amorphous remains of food, including abundant shells of diatoms (Fig. 2K,L). Attached to the stomach, the big digestive gland extends as a spiral to the apex. The digestive gland cells are stained bright with large vacuoles; the gland itself has thick walls and a big lumen which distinguish it from the finer structured gonad, with which it is interlaced (Figs 2J, 5A-C). The ciliated intestine leaves the stomach centrally and features a single loop that is about 2.5 mm long and quite thick (0.2 mm). The loop is thick-walled and muscular, and at some parts bulging, with irregular surface, due to food pellets inside. Narrowing slightly, it opens into the right hand side of the mantle cavity (anus) (Figs 3A,B, 5A-C). Reproductive system The male reproductive system comprises a gonad, a prostate and a penis (Fig. 5A-C). The gonad is slightly coiled, spacious, overlaying the digestive gland. It is histologically characterized by a finely dotted appearance, and moderately stained (Fig. 2J). Emerging from the gonad, a thin-walled proximal male gonoduct first forms an undulated ampulla (84 µm wide) and then a rather straight vas deferens portion, which leads into the posterior end of prostate. The prostate is kidney-shaped, measuring 0.49 mm in length and 0.23 mm in width (Fig. 5A-C). Its lumen is slightly stained, surrounded by a thick wall of blue stained cells. The distal vas deferens is a long, thin, and darkly staining tube (45 µm thick) opening at the tip of the penis (Fig. 5B). The penis is about 1 mm long, flat and tapering towards its tip. The outer surface is rough and covered by concentric and regular folds (Fig. 5A-C); the distalmost vas deferens is not demarcated externally (see Discussion). The female reproductive system is not described here, as examined individuals were immature, with indistinctly developed reproductive organs. Central nervous system The central nervous system consists of paired pedal and cerebropleural ganglia, and a smaller pair of buccal ganglia. The nerve-ring is circumoesopha- 7 geal and epiathroid. The visceral loop is short, with three ganglia that are close together (Figs 6A, 7): on the left and right, the respective suband supraesophageal ganglia are closely annexed anteriorly to the cerebropleural ganglia. The middle, visceral ganglion is situated slightly to the right. From the supraesophageal ganglion emerges a long connective that runs to the left, where it carries the osphradial ganglion (Figs 6A, 7; but see Discussion). The osphradial ganglion carries two nerves, one of which runs to the osphradium, where it carries another, distal ganglion just below the osphradial epithelium (Fig. 2C). Histologically, the ganglia are characterized by distinct neurons in the periphery and lighter-stained nerve fibers in the centre. The pedal ganglia are biggest (length: 0.44 mm, width: 0.23 mm) and interconnected by a single, short and thick (80 µm) commissure (Figs 6A-C, 7). Each pedal ganglion bears three nerves, the two thick, anterior ones carry a small ganglion each (ca. 60 µm in diameter; Fig. 7). Attached to the upper, posterior surface of the pedal ganglia are the two statocysts (0.13 mm diameter; Fig. 6B), with a single spherical statolith. The static nerve was not detected. The paired cerebropleural ganglia (length: 0.43 mm, width: 0.16 mm) are connected to each other via a long commissure (connective width: 60 µm), and to the pedal ganglia by two connectives per side, the cerebropedal and pleuropedal connectives (Figs 6, 7). Three nerves emerge from each cerebral ganglion (Figs 6A,C, 7): nerve 1 (N1, 30-35 µm thick) emerges anteroventrally and innervates the sides of the snout, nerve 2 (65 µm thick; a fused N2+N3, see Discussion) innervates the tentacle and the eye after splitting into three branches. The third nerve (N4, 30 µm thick) emerges at the base of N2 and also innervates the snout. Paired buccal ganglia (0.12 mm) are located in front of the cerebral ganglia and are connected to each cerebral ganglion by a single, short connective (Figs 6A,C, 7); each buccal ganglion carries a nerve that runs to the sides of the pharynx (Figs 6A, 7). Discussion Remarks on taxonomy Our specimens are identified as Sadleriana bavarica Boeters, 1989, for they were found at the type locality (Brunnbach) in the same habitat, and they agree in external characters. According to the original description, the shell of Sadleriana bavarica is pressed conical with 3.5 to 4 whorls and a size from 3.5 to 4 mm in width, with an umbilicus that opens into a crescent-bordered channel, while the operculum is reddish brown. This description agrees with the studied holotype (HYD1008; shell height = 3.4 mm, shell width = 2.8 mm). All those features coincide with the specimen studied herein, which also fit the shell-based diagnosis of the genus Sadleriana by Clessin (1890). Boeters’ description of Sadleriana bavarica also includes some reproductive characters. The smooth, flat and distally rounded appearance of the penis described from dissected specimens by Boeters (see Fig. 5E) differs from our 3D-reconstruction, which shows a tapering penis covered by regular folds (Fig. 5A). These folds could be explained as a contraction artefact during fixation of not optimally relaxed specimens, or may alternatively be a permanent feature. Having regular folds, the reconstructed penis of Sadleriana bavarica thus comes closer to the drawing of a S. fluminensis penis by Küster (1862) (see Fig. 5D), but still is distinct. In the case of female specimens, Boeters (1989) differentiated between Sadleriana bavarica and its congeners using the shape and length of the bursa copulatrix and the receptaculi. Boeters emphasized the unequal length of the two receptaculi of S. bavarica, in contrast to other species where the length of the receptaculi is approximately similar. Receptaculi could not be found in the single immature female specimen that was investigated histologically, so we cannot evaluate the presence and potential variation of such characters in Sadleriana bavarica. Both the somewhat variable penis shape and the difficulties investigating females, however, point to the general taxonomic problem that reproductive characters depend on ontogenetic stages, reproductive condition and preparation or relaxation of the specimens available for study. In order to study the range of morphological variation of putative hydrobiid species, careful micro-dissectings such as performed by Boeters (1989) need be efficiently applied to an adequate number of specimens and stages. Additional histological and 3D microanatomical examination of at least a few representatives is demanding but useful even within a merely taxonomic framework, providing accurate and permanent structural information, and supplementing often ephemeral gross-morphological observations. Once comparative molecular data is available on relevant species, barcoding approaches may be more efficient to identify ontogenetic stages of hydrobiids. For purposes of species discovery, i. e. species delimitation, we think integrative approaches will become more useful (e. g. Jörger et al. 2012), and recently, methods have been developed to use sequences as characters in descriptions of morphologically more or less cryptic species (Jörger & Schrödl 2013) that should be applied to elusive hydrobiids also. 8 Fig. 3. Sadleriana bavarica. Three-dimensional reconstruction of general anatomy and pericardial complex. A. Frontal overview. B. Dorsal overview. C. Mantle cavity; frontal view. D. Mantle cavity; dorsal view. E. Pericardial complex; lateral view. F. Pericardial complex; dorsal view. G. Habitus; ventral view. Abbreviations: an, anus; au, auricle; ct, ctenidium; dg, digestive gland; ey, eye; fg, foot gland; gd, gonoduct; gn, gonad; in, intestine; jw, jaw; kd, kidney; mg, mantle gland; ng, nephridial gland; oe, oesophagus; og, oral gland; op, operculum; pc, pericardium; pe, penis; ph, pharynx; pr, prostate; rm, retractor muscle; sg, salivary gland; sn, snout; st, stomach; tn, tentacle; ve, ventricle. 9 Fig. 4. Three-dimensional reconstruction and schematic overview of Sadleriana bavarica. digestive system. A. Dorsal overview. B. Lateral view of buccal organs in the pharynx. C. Ventral overview. D. Stomach. E. Digestive system schematic view. Abbreviations: an, anus; dg, digestive gland; gc, gastric chamber; gs, gastric shield; in, intestine; jw, jaw; oe, oesophagus; og, oral gland; ot, oral tube; ph, pharynx; rc, radula cartilage; rd, radula; sg, salivary gland; ss, style sac; st, stomach. 16 Fig. 7. Sadleriana bavarica. Schematic overview of central nervous system. Dorsal view, anterior at top. Lower-lying structures with darker shading. Abbreviations: bcm, buccal commissure; bg, buccal ganglion; ccm, cerebral commissure; cpc, cerebro-pedal connective; cpg, cerebro-pedal ganglion; le, lense; osg1, distal osphradial ganglion; osg2, proximal osphradial ganglion; osn, osphradial nerve; pan, pallial nerve; pg, pedal ganglion; pna, anterior pedal nerve; pnl, lateral pedal nerve; subg, suboesophageal ganglion; supg, supraoesophageal ganglion; sc, statocyst; vg, visceral ganglion; vn, visceral nerve. of acteonoideans and many euthyneurans (Staubach 2008), implying separation(s) and multiple secondary fusions during heterobranch evolution. Because of their similar posterior position on the head, innervation of the tentacles by the N3 (plus N2), similar association with the eye (nerve), and continuous distribution across the apogastropod tree, we suggest that caenogastropod and lower heterobranch head tentacles are at least partly homologous to opisthobranch rhinophores, to pyramidellid posterior tentacles, to basommatophoran-style pulmonate tentacles, and also to systellommatophoran and stylommatophoran “eye-stalks”. The evolution of caenogastropod and heterobranch cerebral nerves and head tentacles appears to be excitingly different from conventional views, and remains to be studied in more anatomical and comparative detail. Acknowledgements We want to thank Eva Lodde-Bensch (ZSM) who helped with histological preparations. Hans Boeters kindly shared information on Sadleriana bavarica. Ronald Janssen and Sigrid Hof (both Senckenberg Museum Frankfurt, SMF) provided photographs of the holotype. 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