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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: micro-
anatomy 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 col-
lected 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. ba-
varica 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 ar-
rangement 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 Mün-
chen, Germany; and Department Biology II, BioZentrum, Ludwig-Maximilians-
Universität, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany; e-mails:
gastropoda@gmx.de, bastian.brenzinger@arcor.de, michael.schroedl@zsm.mwn.de
Introduction
The vast majority of gastropods is grouped into the
sister taxa Caenogastropoda and Heterobranchia,
together forming the taxon Apogastropoda (Salvini-
Plawen & 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, includ-
ing 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, valvati- to 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 autapo-
morphies 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 microana-
tomical 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 spe-
cies assigned to Sadleriana currently (Szarowska &
Falniows ki 2013) and their taxonomy is still unclear.
The genus itself was originally described as a subge-
nus of Lithoglyphus C. Pfeiffer, 1828 by Clessin (1890;
see Szarowska & Wilke 2004) and considered as a
distinct genus by Giusti & Pezzoli (1980). One spe-
cies, 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 re-
garded to be an isolated remnant of glacial deposits
stemming from the Riß period, and is thus older
than comparable habitats in the southern proxim-
ity, 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 Slo-
venia. 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 his-
tological 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 resolu-
tion, 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 Hetero-
branchia (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 us-
ing 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. ba-
varica for two reasons: 1) to evaluate and supple-
ment 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 Brunn-
bach, its type locality and single known habitat. Speci-
mens were taken close to the Brunnbach spring (Her-
zogpark 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 Ru-
thensteiner (2008) (8W9, 9W0, 9W1: section thickness 1.5
µm, 9W2: 2.0 µm). The ribbons were collected on micro-
scope 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-re-
construction. For photography a ProgResC3 ccd camera
(Jenoptik, Jena, Germany), mounted on a Leica DMB-
RBE microscope (Leica Microsystems, Wetzlar, Germa-
ny), 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 pho-
tographed. The female specimen (9W0) was photo-
graphed 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 fe-
male 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 co-
pulatrix 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 Muse-
um of Natural History, Frankfurt (Figs 1A-C). – Mate-
rial examined: 5 specimens (ZSM Mol 20131107-
2013111), 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. 1 F-I). A corneous,
reddish-brown operculum is attached to the foot-
end’s upper side measuring 1.1 to 1.7 mm (Figs 3 B,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 differ-
entiated 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 recon-
structed from the male specimen (9W2). The mantle
cavity fills nearly half of the first whorl and then nar-
rows 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 2 B,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, am-
pulla; 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 cav-
ity’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 or-
gans of the circulatory and excretory systems and
is located at the posterior left of the mantle cav-
ity, 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 con-
tact 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 two-
chambered 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 diges-
tive 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), tube-
like 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. 4 A,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 abun-
dant 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 defer-
ens 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. 5 A-C); the distalmost vas def-
erens 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 sub- and suprae-
sophageal 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 con-
nective 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 6 A,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 local-
ity (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 Cles-
sin (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 perma-
nent 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. ba-
varica, 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, reproduc-
tive 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 microanatomi-
cal examination of at least a few representatives is de-
manding 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 delimita-
tion, 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. Fron-
tal 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, au-
ricle; 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, pericar-
dium; pe, penis; ph, pharynx; pr, prostate; rm, retractor muscle; sg, salivary gland; sn, snout; st, stomach; tn, ten-
tacle; 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, intesti-
ne; jw, jaw; oe, oesophagus; og, oral gland; ot, oral tube; ph, pharynx; rc, radula cartilage; rd, radula; sg, saliva-
ry gland; ss, style sac; st, stomach.
10
Comparative morphology
This is the first detailed 3D-microanatomical ap-
proach on a typical, i. e. snail-like caenogastropod.
As shown for Heterobranchia, this approach based
on serial semithin sections is powerful for revealing
full-scale histological and anatomical data on tissues
and organs even from small specimens, and is also
suitable for minute caenogastropods such as hydro-
biids. However, also macroscopic caenogastropods
or other animals, or complex parts thereof, e. g.
central nervous systems, can be promising targets
of comparative morphological studies using that
technique. In the following we use the detailed his-
tological and microanatomical data from Sadleriana
bavarica as a representative of Caenogastropoda for
comparisons with their heterobranch sister group,
which has already been much better explored from
a microanatomical point of view.
External morphology
The head of Sadleriana bavarica bears long and narrow
cephalic tentacles, with eyes on swellings at their
outer bases, as usual for caenogastropods (Ponder et
al. 2007), but similarly present in lower heterobranchs
and certain “archaeopulmonate” panpulmonates
(‘Basommatophora’, see e. g. Nordsieck 1993). The
wedge-like broadened propodium includes a well-
developed propodial gland (Ponder & Lindberg
1997). It was suggested that the propodium, with
a distinct anterior pedal gland opening beneath it
into a groove, is a synapomorphy of all gastropods
other than adult patellogastropods (Ponder &
Lindberg 1997). This character has become lost or
modified in some ‘higher’ heterobranchs (Ponder
& Lindberg 1997).
Mantle cavity and pallial organs
The mantle cavity of Sadleriana bavarica contains a
single monopectinate ctenidium on its left side, as
usual in caenogastropods (Ponder & Lindberg 1997,
Ponder et al. 2007). A central rachis is not present,
and the gill leaflets are attached directly to the roof
of the mantle cavity. The ctenidia of the herein inves-
tigated specimens have 8 lamellas with apical ciliary
bands. This contradicts Boeter’s (1989) description of
15 lamellas in specimens with the same size, which
might be a matter of intraspecific variability.
Behind the ctenidium of Sadleriana bavarica,
towards the right side, a well-developed gland
is located. This ‘mantle gland’ or ‘hypobranchial
gland’ is usually assumed to be a homologous
structure throughout the gastropods (Ponder &
Lindberg 1997). Because of the reduction of the
mantle cavity organs, in caenogastropods, there
is only a single hypobranchial gland left (Ponder
2007). In Heterobranchia, there is higher complex-
ity with several described types of ‘mantle cavity
glands’ that are histologically different (Wägele et
al. 2006, Brenzinger et al. 2014). While the largest
structure is commonly called hypobranchial gland
in the literature, it is not clear whether smaller, ad-
ditional glandular structures and cell types found
in some taxa are derived from the same stock of
‘hypobranchial’ gland cells.
The anus of Sadleriana bavarica opens into the right
part of the mantle cavity, as it does in many shelled
heterobranchs. According to Ponder and Lindberg
(1997), at least in caenogastropods this results from
the extension of the rectum along the right side of
the mantle cavity, correlated with the loss of the
right ctenidium.
The osphradium of Sadleriana is relatively large
(compared to similarly-sized heterobranchs) but
otherwise morphologically similar to that of other
caenogastropods (e. g. Hydrobia ulvae as examined
by Haszprunar 1985a).
Pericardial complex
On the left side of the mantle cavity, Sadleriana
bavarica possesses a voluminous ‘kidney’ (Ponder
& Lindberg 1997, Ponder 2007). The organ partly
extends into the left side of the mantle cavity roof, and
is histologically characterized by its typical uniform
and vacuolated appearance of the excretory tissue
as known for other caenogastropods (Strong 2003).
The kidney is topped by a gland-like structure,
with unclear homology and function. Fretter & Gra-
ham (1962) described a nephridial ‘gland’ of caeno-
gastropods as a mass of connective tissue and muscle
fibers between kidney and pericardium, lined with
ciliated cells and penetrated by haemocoelic spaces.
According to Strong (2003), the histology of the
nephridial gland is remarkably uniform across the
caenogastropods. Ponder & Lindberg (1997) suggest
that the caenogastropod ‘nephridial’ or ‘renal’ gland
is probably not homologous to the nephridial glands
of other groups, such as vetigastropod Trochoidea,
for there are different regions in which these sup-
posed nephridial glands are located. Similar organs
have been reported for some ‘lower’ heterobranch
clades (e. g. Haszprunar 1985c, Hawe et al. 2014).
Furthermore, organs of similar histology, comparable
position (associated with the circulatory or excretory
systems) and thus perhaps comparable function are
found in some more derived heterobranchs, e. g. as
the so called ‘blood gland’ of nudibranchs (Schrödl &
Wägele 2001, Wägele 2004). The latter was shown to
be a loose aggregation of rhogocytes and connective
tissue by Fahrner & Haszprunar (2002). Interestingly,
11
none of these so-called ‘glands’ seems to be a real
gland composed of a secretory, glandular cell type.
In caenogastropods, tissue on top of the kidney forms
a distinct, superficially gland-like structure, which
sometimes consists of a simple mass or a single series
of lamellae (Ponder & Lindberg 1997). This surface
enlargement may be the same as seen in Sadleriana
bavarica, where the kidney epithelium forms crypts
extending into the nephridial gland. Whether these
organs have homologues throughout Gastropoda
remains to be investigated.
The number of auricles varies within the mol-
luscs from one to four and is usually correlated
with the number of gills (Ponder & Lindberg 1997).
In caenogastropods, there is thus a single auricle
combined with a single left ctenidium (Ponder 2007)
as observed in Sadleriana bavarica. This condition can
be regarded as a shared character of Apogastropoda.
Variability exists in the orientation of the heart,
especially in partially detorted heterobanchs. Blood
vessels such as an aorta could not be reconstructed
from the present material.
Digestive system
In most gastropods (see Warén & Bouchet 1990),
there is a single pair of fused, dorso-lateral jaws,
in the buccal cavity. Histologically, minute rod-like
structures can be detected at the jaws of Sadleriana
bavarica. This agrees with the hypothesis of Ponder
& Lindberg (1997) based on Starmühlner (1969),
that the jaws of caenogastropods in general and in
particular those of the sorbeoconchans (Strong 2003)
are at least partially composed of rods, which is also
the case in opisthobranchs and lower heterobranchs
(Mikkelsen 1996, Wägele & Willan 2000).
The radula of Sadleriana bavarica shows a ple-
siomorphically taenioglossate condition; it bends
longitudinally and enables the marginal teeth to
sweep inwards. This ‘flexoglossate’ radula was re-
garded as a synapomorphy of gastropods (Golikov
& Starobogatov 1975, Ponder & Lindberg 1997),
with patellogastropods establishing a stereoglossate
condition independently (see also Stöger et al. 2013).
In the case of Sadleriana bavarica, the radula lies on
top of two cartilaginous cushions, and is topped by
a smaller third cartilage. All cartilages of Sadleriana
show the typical large cells embedded in extracellular
matrix, forming homogeneous units (see Hall 2005,
Schmidt-Rhaesa 2007). The unpaired dorsal cartilage
is unusual, as most caenogastropods possess only a
single pair of buccal cartilages (Strong 2003, Ponder
2007). Structural diversity of cartilages was found
to be not directly related to feeding ecology, and
therefore regarded as a potentially useful character
for phylogenetic study (Golding et al. 2009).
Usually, the salivary glands of gastropods open
dorsally into the buccal cavity near the connection
to the oesophagus (Ponder & Lindberg 1997). In
Sadleriana bavarica, they open into the pharynx much
more anteriorly, nearby the mouth opening, instead.
The stomach of S. bavarica is separated into a
smaller (= style sac) and a bigger part (= gastric cham-
ber) that has an angular gastric shield. According to
Strong (2003) and Salvini-Plawen (1988), a gastric
shield might be the antagonist of a protostyle within
the style sac. In S. bavarica, there was no protostyle
found, which could be a matter of fixation. The
stomach of heterobranchs is much simpler (gastric
shields and a style sac have been lost except for in
the lower heterobranch Valvatoidea; e. g. Hawe et al.
2014), but larger forms have sometimes re-evolved a
more complex stomach with at least epithelial folds
(e. g. the sea hare Aplysia). As usual in gastropods,
oesophagus and digestive gland ducts open into the
gastric chamber and the intestine leaves the stomach
anteriorly (Ponder & Lindberg 1997, Strong 2003),
a situation which could be observed in Sadleriana
bavarica as well.
Reproductive system
As other caenogastropods, Sadleriana bavarica is a
dioecious species. The condition found in the inves-
tigated specimens generally agrees with the usual
situation in hydrobiids (Radoman 1983). Both sexes
possess a voluminous gonad, which is located at
the upper whorls of the shell, and a gonoduct lead-
ing to the genital opening at the right hand side of
the mantle cavity. Male snails have a rather simple
prostate and penis, as described from histological
reconstruction herein, while females, according to
Boeters (1989), develop a bursa copulatrix and two
receptaculi. Histological and functional details of
the female reproductive system could not be inves-
tigated yet. The number, structure and distribution
of allosperm receiving organs within heterobranchs
is highly heterogeneous, and their homology is
controversely discussed (e. g. Wägele & Willan
2000 versus Valdés et al. 2010). The homology of
allosperm receptacles across major apogastropod
groups remains to be evaluated based on micro-
anatomical detail.
Central nervous system
The central nervous system, e. g. the arrangement
of the circum-oesophageal ganglia, has always been
considered important for gastropod classification
(e. g. Ponder & Lindberg 1997). The nervous system
of Sadleriana bavarica is concentrated, with well-
defined pairs of cerebropleural and pedal ganglia,
as typical in caenogastropods (Ponder et al. 2007).
12
Fig. 5. Three-dimensional reconstruction and schematic overview of the male reproductive system of Sadleriana
bavarica. A. Dorsal view. B. Ventral view. C. Schematic overview. D. Penis of Sadleriana fluminensis modified after
Radoman (1983). E. Penis of Sadleriana bavarica modified after Boeters (1989). Abbreviations: am, ampulla; gd, gono-
duct; gn, gonad; pe, penis; pr, prostate; vd, vas deferens; vdo, vas deferens opening.
13
Fig. 6. Three-dimensional reconstruction of Sadleriana bavarica central nervous system. A. Frontal view. B. Posteri-
or view. C. Lateral view. Abbreviations: bcm, buccal commissure; bg, buccal ganglion; ccm, cerebral commissu-
re; cpc, cerebro-pedal connective; cpg, cerebro-pedal ganglion; le, lens; osg, osphradial ganglion; osn, osphradial
nerve; pan, pallial nerve; pg, pedal ganglion; pna, anterior pedal nerve; pnl, lateral pedal nerve; subg, suboeso-
phageal ganglion; supg, supraoesophageal ganglion; sc, statocyst; vg, visceral ganglion; vn, visceral nerve.
14
We identified the presence of cerebropleural ganglia
and the location of the pleural portion of the latter
according to the presence of two close-by connec-
tives to the pedal ganglia, a condition also found
in numerous Heterobranchia (e. g. Brenzinger et al.
2013a,b). Regarding the pleural ganglia as not exter-
nally demarcated, the three ganglia on the visceral
nerve loop are the following (from left to right): a
suboesophageal ganglion (lying rather ventral), a
visceral ganglion (with thick nerve running posteri-
orly), and a supraesoophageal ganglion (lying more
dorsally). The first and last are anteriorly more or
less completely fused with the pleural ganglia, and
the supraesophageal ganglion additionally carries
an osphradial ganglion, which is shifted to the left
and is connected to a further, peripheral ganglion.
Therefore, two ganglia are associated with the os-
phradium: one just below the sensory epithelium
(osg1 herein), and the aforementioned one connected
to the supraesophageal ganglion (osg2).
Save the additional ganglion found underneath
the osphradial epithelium, this condition is morpho-
logically essentially identical to what was reported
for hydrobiids (e. g. Davis 1967: pl. 20, Hershler &
Davis 1980). The ganglion connected to the visceral
loop but shifted to the side (left side in caenogastro-
pods, right side in many heterobranchs) is generally
referred to as the osphradial ganglion in the literature
(e. g. Haszprunar 1985a). Our nomenclature of the
visceral loop ganglia is in conflict with descriptions
in the literature: Davis (1967) called the single left
ganglion (osg2 herein) the supraesophageal gan-
glion, and the one on the visceral loop ‘right pleural
ganglion’ (supraesophageal ganglion herein). The
same nomenclature could be used for Sadleriana,
where the ‘sole’ osphradial ganglion would then
be the subepithelial one (osg1), but this would be
inconsistent with our detection of the right pleural
ganglion as part of a fused cerebropleural ganglion.
Further research is necessary to homologize hetero-
branch and caenogastropod ganglia and to correlate
terminology.
Interpretational difficulties also pertain to the
nerves found herein. From the right side, a single
long nerve arises between the supraesophageal and
visceral ganglion, which leads into the mantle cav-
ity. The visceral ganglion bears a single nerve that
runs into the visceral sac, which is therefore the
visceral nerve as in other gastropods; no nerves in
the subesophageal ganglion were detected. Anterior
accessory ganglia are reported for other hydrobiids
(“propodial” ganglia by Hershler & Davis 1980). Ac-
cessory ganglia are present on the cerebral nerves of
many heterobranchs, but pedal accessory ganglia are
rare and were reported only for some lower hetero-
branch Rhodopemorpha (see Brenzinger et al. 2013b).
Cerebral nerves
In S. bavarica, three pairs of cerebral nerves were
found. Staubach (2008) and Klussmann-Kolb et al.
(2013) comparatively investigated the cerebral nerves
of several heterobranchs and the caenogastropod Lit-
torina using stain backfilling techniques. We follow
the authors’ nomenclature of cerebral nerves here,
as the nerves in Sadleriana fit in size and position
with those described for Littorina littorea (Linnaeus
1758) and other Apogastropoda: an ‘oral’ one (N1),
which innervates the lip and anterior head region, a
very thick tentacle nerve (a fused N2+N3 according
to Staubach 2008) with basal thickening (not a gan-
glion), and the N4 (as ‘Nclc’) innervating the anterior
head region (snout in Sadleriana). The thick N2+N3
of S. bavarica innervates the head tentacle, i. e. is the
“tentacular” or “tentacle” nerve, soon branching and
innervating the eye as well and dividing in three
further branches. In a phylogenetic context, a bifur-
cate tentacular nerve as found in S. bavarica has been
thought to be typical for Apogastropoda (Haszpru-
nar 1985b, 1988a,b, Salvini-Plawen & Haszprunar
1987, Salvini-Plawen 1988, Ponder & Lindberg 1997).
However, an analysis by Strong (2003) revealed
that there is an unappreciated diversity of branch-
ing patterns in Caenogastropoda, including trifid,
quadrifid and multifid tentacle nerves in addition
to the commonly recognized single and bifid pat-
terns. In a previous study, Huber (1993) examined
the nervous systems of many Heterobranchia and
some caenogastropods, including the littorinoidean
Skeneopsis planorbis (Fabricius, O., 1780), and found a
somewhat different situation. Huber (1993) described
a thick tentacle nerve (‘te’ therein), but additionally
an optic nerve (op) that arises directly beneath from
the cerebral ganglion, instead of branching from the
tentacle nerve as in Sadleriana bavarica and Littorina
littorea. Furthermore in Skeneopsis planorbis, there are
four ‘labial’ nerves (li, ls, le, lm), instead of only two
(i. e. N1, N4). The homology of these nerves cannot
be clarified definitely here, but there might be some
evidence due to the position of origin at the cerebral
ganglion described by Huber: Referring to its anterior
origin, N1 might refer to Huber’s nerve ‘le’, while N4
might be the homologue of ‘ls’. The differences in the
setting of cerebral nerves might have observational
or systematic reasons. In traditional systematics all
three mentioned species belong to the superfamily
Littorinoidea, molecular phylogenetic studies are
necessary to clarify their actual relationships within
Caenogastropoda.
Homology and evolution of the tentacle nerve
There is an important question behind evaluating
the homology and distribution of cerebral nerves
15
in apogastropods. Huber (1993) considered that a
nervus tentacularis, i.e. a tentacle nerve as in Sadle-
riana, is present in Caenogastropoda, lower hetero-
branchs, Rissoellidae, Pyramidellidae, Pulmonata
and Thecosomata, and that this nerve is homologous
to the Nclc (N4 herein) of some headshield-bearing
opisthobranchs. In contrast, Staubach (2008) proved
unambiguously by tracing nervous features by
immunocytochemistry, that the caenogastropod
tentacle nerve contains all neurons and axons of the
separate nerves N2 (= labiotentacular nerve) and N3
(= rhinophoral nerve) of euthyneurans and acteo-
noids (Staubach 2008: figs 33 and 45), but no parts
of the N4 (Nclc). This clearly contradicts Huber’s
assumption of direct homology of the tentacle nerve
with the Nclc, and there is no indication for even just
partial homology yet. Since caenogastropods and
lower heterobranchs possess a thick tentacle nerve
(N2/N3), its presence is likely symplesiomorphic
for heterobranchs and caenogastropods. In stark
contrast to paradigms conveyed by Huber (1993),
this means that ancestral apogastropods already had
a rhinophoral nerve, which was basally combined
with the labial tentacle nerve. We suggest that head
tentacles innervated by both the N2 and N3, such
as in S. bavarica, serve the special sensory functions
of both of these nerves, i.e. short and long distance
chemosensory perception.
In the light of modern hypotheses on euthyneu-
ran relationships (e. g. Jörger et al. 2010, Schrödl et
al. 2011a,b, Brenzinger et al. 2013b, Wägele et al.
2014), we hypothesize that the ancestrally combined
apogastropod N2/N3 became fully separated in an
ancestral euthyneuran, or in the common ancestor
of euthyneurans and acteonoideans. In groups with
2 pairs of head tentacles (or separated anterior and
posterior sensory organs sensu Klussmann-Kolb et
al. 2013), e. g. most nudibranchs, the N2 innervates
the anterior labial tentacles, while the N3 innervates
proper rhinophores, organs specialized to short
and long distance olfactory functions, respectively.
Several instances of secondary fusion of nerves and/
or tentacles may have occurred, e. g. thecosoma-
tous pteropods show a potentially paedomorphic
tentacle nerve (Kubilius et al. in press). Within
panpulmonates, in most acochlidians the separate
N2 and N3 innervate different tentacles, while in
several sacoglossans they jointly innervate a single
pair of head tentacles (Huber 1993, Jensen et al. in
press) or a flattened head without tentacles in Gas-
coignella (Kohnert et al. 2013). Ancestrally separate
nerves apparently have fused to form a bifurcating,
pseudoancestral tentacular nerve (N2/N3) again in
(non-monophyletic) “archaeopulmonate” groups.
Conclusion and outlook
This study presents a full anatomical 3D model
of Sadleriana bavarica, thus providing an (almost)
complete anatomical description of a member of
Hydrobiidae, which are so far mainly described on
the basis of shells, and the first comprehensive his-
tological and microanatomical 3D data of a typical
snail-like caenogastropod.
In order to further elucidate the species status
and specific characters of Sadleriana bavarica, in
particular mature female specimens remain to be
analysed anatomically, showing the arrangement,
shape and variation of allosperm receptacles. In ad-
dition to macropreparations, histological 3D models
need to be reconstructed, to evaluate functions and
homologies. SEM studies exploring the details and
variability of hard structures, such as shell and
radula, are warranted as well. In addition, molecular
investigations have to be considered. Currently, Sad-
leriana bavarica is regarded as an endemic taxon with
divergent, extremely restricted relictual habitat. COI
barcodes from S. bavarica and Slovenian and Croatian
S. fluminensis show minimum uncorrected pairwise
distances of 2 percent (source: BOLD Barcode of Life
Data Systems, and GenBank; Swarowska & Wilke
2004, Swarowska & Falniowski 2013). This currently
neither supports nor rejects the morphology-based
idea that Sadleriana bavarica is specifically distinct
rather than an isolated population of Sadleriana
fluminensis.
Furthermore, the detailed 3D-microanatomical
data on S. bavarica is a first set of data for minute
Caenogastropoda which can be used for comparison
to other taxa including their histologically more com-
prehensively explored sister, the Heterobranchia.
The potential of detailed comparative work is
exemplified here briefly discussing the homology
of nephridial glands or visceral loop ganglia. It is
important to note that gross-anatomical dissectings
or paraffin-based histology, which were standard
methods for examining macroscopic caenogastro-
pods, may not be sufficient to reveal all delicate
features. Also, as shown from comparative studies
of pulmonates and opisthobranchs (e. g. Jörger et al.
2010, Schrödl et al. 2011a) to discover overlooked
aspects and to understand functions, homology and
evolution of structures, it is crucial to compare them
across conventional taxon borders.
Considering modern tree hypotheses and com-
parative research on cerebral nerves (Staubach 2008)
clearly challenges Huber’s (1993) paradigms on the
evolution of apogastropod cerebral nerves and ten-
tacles. Of particular interest to us is the homology of
caenogastropod and heterobranch tentacular nerves
with the labiotententacular plus rhinophoral nerves
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 com-
missure; 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, stato-
cyst; 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, inner-
vation of the tentacles by the N3 (plus N2), similar
association with the eye (nerve), and continuous
distribution across the apogastropod tree, we sug-
gest that caenogastropod and lower heterobranch
head tentacles are at least partly homologous to
opisthobranch rhinophores, to pyramidellid poste-
rior 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 Jans-
sen and Sigrid Hof (both Senckenberg Museum Frank-
furt, SMF) provided photographs of the holotype.
Frauke Lücke (Landesbund für Vogelschutz in Bayern
(LBV), Kreisgruppe München) is thanked for providing
literature on the Brunnbach. Two referees provided
constructive comments. The GeoBioCenter of the LMU
and the DFG (Project SCHR667/13 to MS) provided
17
Amira licenses and covered lab costs. The Barcoding
Fauna Bavarica (BFB) and German Barcode Of Life
(GBOL) projects supported us with computer equip-
ment and sequences.
References
Altnöder, A., Bohn, J. M., Rückert, I. & Schwabe, E.
2007. The presumed shelled juvenile of the para-
sitic gastropod Entocolax schiemenzii Voigt, 1901
and its holothurian host Chiridota pisanii Ludwig,
1886 (Gastropoda, Entoconchidae – Holothuroidea,
Chiridotidae). Spixiana 30: 187-199.
Boeters, H. D. 1989. Unbekannte westeuropäische Pro-
sobranchia 8. Heldia 1: 169-170.
Brenzinger, B., Padula, V. & Schrödl, M. 2013a. In-
semination by a kiss? Interactive 3D microanato-
my, biology and systematics of the mesopsammic
cephalaspidean sea slug Pluscula cuica Marcus,
1953 from Brazil (Gastropoda: Euopisthobranchia:
Philinoglossidae). Organisms, Diversity & Evolu-
tion 13: 33-54.
– – , Haszprunar, G. & Schrödl, M. 2013b. At the lim-
its of a successful body plan – 3D microanatomy,
histology and evolution of Helminthope (Mollusca:
Heterobranchia: Rhodopemorpha), the most worm-
like gastropod. Frontiers in Zoology 10: 37.
– – , Wilson, N. G. & Schrödl, M. 2014. Microanatomy of
shelled Koloonella sp. (Gastropoda: ‘Lower’ Hetero-
branchia: Murchisonellidae) does not contradict
a sistergroup relationship with enigmatic Rho-
dopemorpha slugs. Journal of Molluscan Studies
doi:10.1093/mollus/eyu036.
Clessin, S. 1890. Die Molluskenfauna Österreich-Un-
garns und der Schweiz. 858 pp., Nürnberg (Bauer
& Raspe).
Criscione, F. & Ponder, W. F. 2013. A phylogenetic
analysis of rissooidean and cingulopsoidean fami-
lies (Gastropoda: Caenogastropoda). Molecular
Phylogenetics and Evolution 66: 1075-1082.
DaCosta, S., Cunha, C. M., Simone, L. R. & Schrödl, M.
2007. Computer-based 3-dimensional reconstruc-
tion of major organ systems of a new aeolid nudi-
branch subspecies, Flabellina engeli lucianae, from
Brazil (Gastropoda: Opisthobranchia). Journal of
Molluscan Studies 73: 339-353.
Davis, G. M. 1967. The systematic relationship of Po-
matiopsis lapidaria and Oncomelania hupensis for-
mosana (Prosobranchia: Hydrobiidae). Malacologia
6: 1-143.
Fahrner, A. & Haszprunar, G. 2002. Anatomy, ultra-
structure, and systematic significance of the excre-
tory system and mantle cavity of an acochlidian
gastropod (Opisthobranchia). Journal of Molluscan
Studies 68: 87-94.
Fretter, V. & Graham, A. 1962. British prosobranch
molluscs; their functional anatomy and ecology.
London (Ray Society).
Giusti, F. & Pezzoli, E. 1980. Guide per il riconoscimento
delle specie animali delle acque interne italiane, 8:
Gasteropodi, 2. (Gastropoda: Prosobranchia: Hy-
drobioidea, Pyrguloidea). 67 pp., Roma (Consiglio
Nazionale delle Richerche, AQ/1/47)
Glöer, P. 2002. Die Tierwelt Deutschlands – Die Süßwas-
sergastropoden Nord- und Mitteleuropas: Bestim-
mungsschlüssel, Lebensweise, Verbreitung. 327 pp.,
Hackenheim (ConchBooks).
Golding, R. E., Ponder, W. F. & Byrne, M. 2009. Three-
dimensional reconstruction of the odontophoral
cartilages of Caenogastropoda (Mollusca: Gastropo-
da) using micro-CT: Morphology and phylogenetic
significance. Journal of Morphology 270: 558-587.
Golikov, A. N. & Starobogatov, Y. I. 1975. Systematics of
prosobranch gastropods. Malacologia 15: 185-232.
Hall, B. K. 2005. Bones and cartilage: developmental
and evolutionary skeletal biology. 788 pp., London,
(Elsevier/Academic Press).
Haszprunar, G. 1985a. The fine morphology of the
osphradial sense organs of the Mollusca. I. Gastro-
poda, Prosobranchia. Philosophical Transaction of
the Royal Society of London B 307: 457-496.
– – 1985b. The Heterobranchia – a new concept of the
phylogeny of the higher Gastropoda. Zeitschrift für
Zoologische Systematik und Evolutionsforschung
23: 15-37.
– – 1985c. Zur Anatomie und systematischen Stellung
der Architectonicidae (Mollusca, Allogastropoda).
Zoologica Scripta 14: 25-43.
– – 1988a. On the origin and evolution of major gastro-
pod groups, with special reference to the Strepto-
neura (Mollusca). Journal of Molluscan Studies 54:
367-441.
– – 1988b. A preliminary phylogenetic analysis of the
streptoneurous gastropods. Malacologial Review
Supplement 4: 7-16.
– – & Koller, K. 2011. Barcoding Fauna Bavarica – eine
Chance für die deutsche Malakologie. Mitteilungen
der Deutschen Malakologischen Gesellschaft 84:
25-27.
– – , Speimann, E., Hawe, A. & Heß, M. 2011. Interactive
3D-anatomy and affinities of the Hyalogyrinidae,
basal Heterobranchia (Gastropoda) with a rhipi-
doglossate radula. Organisms, Diversity & Evolu-
tion 11: 201-236.
Hawe, A., Heß, M. & Haszprunar, G. 2013. 3D-recon-
struction of the anatomy of the ovoviviparous (?)
freshwater gastropod Borysthenia naticina (Menke,
1845) (Ectobranchia: Valvatidae). Journal of Mol-
luscan Studies 79: 191-204.
– – , Paroll, C. & Haszprunar, G. 2014. Interactive
3D-anatomical reconstruction and affinities of the
hot-vent gastropod Xylodiscula analoga Warén &
Bouchet, 2001 (Ectobranchia). Journal of Molluscan
Studies. doi:10.1093/mollus/eyu017
Hershler, R. & Davis, G. M. 1980. The morphology
of Hydrobia truncata (Gastropoda: Hydrobiidae):
relevance to systematics of Hydrobia. The Biological
Bulletin 158: 195-219.
18
– – & Ponder, W. F. 1998. A review of morphological
characters of hydrobioid snails. Smithsonian Con-
tributions to Zoology 600: 1-55.
Huber, G. 1993. On the cerebral nervous system of
marine Heterobranchia (Gastropoda). Journal of
Molluscan Studies 59: 381-420.
Jensen, K. R., Kohnert, P., Bendell, B. & Schrödl, M.
in press. A miniature sacoglossan (Gastropoda:
Heterobranchia: “Opisthobranchia”) feeding on the
seagrass Halophila ovalis in Thailand and Australia.
Journal of Molluscan Studies.
Jörger, K. M. & Schrödl, M. 2013. How to describe a
cryptic species? Practical challenges of molecular
taxonomy. Frontiers in Zoology 10: 59.
– – , Stöger, I., Kano, Y., Fukuda, H., Knebelsberger,
T. & Schrödl, M. 2010. On the origin of Acochlidia
and other enigmatic euthyneuran gastropods, with
implications for the systematics of Heterobranchia.
BMC Evolutionary Biology 10: 323.
– – , Wilson, N. G., Norenburg, J. L. & Schrödl, M. 2012.
Barcoding against a paradox? Combined molecular
species delineation reveals multiple cryptic lineages
in elusive meiofaunal sea slugs. BMC Evolutionary
Biology 12: 245.
Klussmann-Kolb, A., Croll, R. P. & Staubach, S. 2013.
Use of axonal projection patterns for the homologi-
sation of cerebral nerves in Opisthobranchia, Mol-
lusca and Gastropoda. Frontiers in Zoology 10: 20.
Kohnert, P., Brenzinger, B., Jensen, K. R. & Schrödl, M.
2013. 3D-microanatomy of the semiterrestrial slug
Gascoignella aprica Jensen, 1985 – a basal plakobran-
chacean sacoglossan (Gastropoda, Panpulmonata).
Organisms Diversity & Evolution 13: 583-603.
Kubilius, R. A., Kohnert, P., Brenzinger, B. & Schrödl, M.
in press. 3D-microanatomy of the straight-shelled
pteropod Creseis clava (Gastropoda, Heterobranchia,
Euthecosomata). Journal of Molluscan Studies.
Küster, H. C. 1853. Die Gattungen Paludina, Hydrocaena
und Valvata. Systematisches Conchylien-Cabinet
2: 1-96.
– – 1862. Die Gattungen Limnaeus, Ampipeblea, Chilina,
Isidora und Physopsis. Systematisches Conchylien-
Cabinet 1 (17b): 1-48.
LBV (Landesbund für Vogelschutz in Bayern e.V., Kreis-
gruppe München) 2007. Quellschutz in München.
44 pp.
Martynov, A., Brenzinger, B., Hooker, Y. & Schrödl,
M. 2011. 3D-anatomy of a new tropical Peruvian
nudibranch gastropod species, Corambe mancorensis,
and novel hypotheses on dorid gill ontogeny and
evolution. Journal of Molluscan Studies 77: 129-141.
Mikkelsen, P. M. 1996. The evolutionary relationships of
Cephalaspidea s.l. (Gastropoda: Opisthobranchia):
a phylogenetic analysis. Malacologia 37: 375-442.
Neusser, T. P., Heß, M., Haszprunar, G. & Schrödl, M.
2006. Computer-based three-dimensional recon-
struction of the anatomy of Microhedyle remanei
(Marcus, 1953), an interstitial acochlidian gastro-
pod from Bermuda. Journal of Morphology 267:
231-247.
– – , Martynov, A. V. & Schrödl, M. 2009. Heartless and
primitive? 3D reconstruction of the polar acochlid-
ian gastropod Asperspina murmanica. Acta Zoologica
90: 228-245.
Nordsieck, H. 1993. Phylogeny and system of the Pul-
monata (Gastropoda). Archiv für Molluskenkunde
121: 31-52.
Ponder, W. F. & Clark, G. A. 1990. A radiation of hydro-
biid snails in threatened artesian springs in western
Queensland. Records of the Australian Museum
42: 301-363.
– – & Lindberg, D. R. 1997. Towards a phylogeny of
gastropod molluscs: an analysis using morpho-
logical characters. Zoological Journal of the Linnean
Society 119: 83-265.
– – , Colgan, J. M., Healy, J. M., Nützel, A., Simone, L.
R. L. & Strong, E. E. 2007. Caenogastropoda. Pp.
331-383 in Ponder, W. F. & Lindberg, D. L. (eds).
Phylogeny and evolution of the Mollusca. Berke-
ley, Los Angeles, London (University of California
Press).
Radoman, P. 1983. Hydrobioidea a superfamily of
prosobranchia (Gastropoda) I Sistematics. Serbian
Academy of Sciences and Art, Monographs 547:
1-256.
Richardson, K. C., Jarett, L. & Finke, E. H. 1960. Em-
bedding in epoxy resins for ultrathin sectioning in
electron microscopy. Stain Technology 35: 313-323.
Ruthensteiner, B. 2008. Soft part 3D visualization by
serial sectioning and computer reconstruction. Zo-
osymposia 1: 63-100.
Salvini-Plawen, L. v. 1988. The structure and function
of molluscan digestive systems. Pp. 301-379 in
Trueman E. R. & Clarke, M. R. (eds). The Mollusca
11: molluscan form and function. New York (Aca-
demic Press).
– – & Haszprunar, G. 1987. The Vetigastropoda and
the systematics of streptoneurous Gastropoda (Mol-
lusca). Journal of Zoology 211: 747-770.
Schmidt-Rhaesa, A. 2007. The evolution of organ sys-
tems. USA (Oxford University Press).
Schrödl, M. & Wägele, H. 2001. Anatomy and histol-
ogy of Corambe lucea Marcus, 1959 (Gastropoda:
Nudibranchia), with discussion of the systematic
position of Corambidae. Organisms, Diversity &
Evolution 1: 3-16.
– – , Jörger, K. M., Klussmann-Kolb, A. & Wilson, N.
G. 2011a. Bye bye “Opisthobranchia”! A review
on the contribution of mesopsammic sea slugs to
euthyneuran systematics. Thalassas 27: 101-112.
– – , Jörger, K. M. & Wilson, N. G. 2011b. A reply to
Medina et al. 2011: Crawling through time: Transi-
tion of snails to slugs dating back to the Paleozoic
based on mitochondrial phylogenomics. Marine
Genomics 4: 301-303.
Sedlmeier, H. & Schwab, U. 2006. Die Biotope des
Münchner Stadtaußenbereichs. Zustand – Konflikte
– Maßnahmeempfehlungen. In: LBV (Landesbund
für Vogelschutz in Bayern e.V., Kreisgruppe Mün-
chen, ed.). Managementpläne für Münchner Bioto-
pe Teil 3, 36: 1-5.
19
Seidl, F. jun. & Colling, M. 1986. Ein Vorkommen von
Sadleriana fluminensis (Küster) in der Bundesrepu-
blik Deutschland. Mitteilungen der Zoologischen
Gesellschaft, Braunau 4: 345-354.
Starmühlner, F. 1969. Die Gastropoden der Madagas-
sischen Binnengewässer. Malacologia 8: 1-434.
Staubach, S. 2008. The evolution of the cephalic sen-
sory organs within the Opisthobranchia. 155 pp.,
Dissertation Johann Wolfgang Goethe Universität,
Frankfurt am Main.
Stöger, I., Sigwart, J., Kano, Y., Knebelsberger, T.,
Marshall, B., Schwabe, E. & Schrödl, M. 2013. An
integrative approach supports a new perspective
on early molluscan evolution. BioMed Research
International: 407072.
Strong, E. E. 2003. Refining molluscan characters: mor-
phology, character coding and phylogeny of the
Caenogastropoda. Zoological Journal of the Lin-
nean Society 137: 447-554.
– – , Gargominy, O., Ponder, W. F. & Bouchet, P. 2008.
Global diversity of gastropods (Gastropoda; Mol-
lusca) in freshwater. Freshwater Animal Diversity
Assessment. Hydrobiologia 595: 149-166.
Szarowska, M. & Falniowski, A. 2013. Species distinct-
ness of Sadleriana robici (Clessin, 1890) (Gastropoda:
Rissooidea). Folia Malacologica 21: 127-133.
– – & Wilke, T. 2004. Sadleriana pannonica (Frauenfeld,
1865): a lithoglyphid, hydrobiid or amnicolid taxon?
Journal of Molluscan Studies 70: 49-57.
Valdés, Á., Gosliner, T. M., Ghiselin, M. T. 2010. Chap-
ter 8: Opisthobranchs. Pp. 148-172 in: Leonard,
J. L. & Córdoba-Aguilar, A. (eds). The evolution
of primary sexual characters in animals. Oxford
University Press.
Wägele, H. 2004. Potential key characters in Opistho-
branchia (Gastropoda, Mollusca) enhancing adap-
tive radiation. Organisms, Diversity & Evolution
4: 175-188.
– – & Willan, R. C. 2000. Phylogeny of the Nudi-
branchia. Zoological Journal of the Linnean Society
130: 83-181.
– – , Ballesteros, M. & Avila, C. 2006. Defensive glan-
dular structures in opisthobranch molluscs – from
histology to ecology. Oceanography and Marine
Biology Annual Review 44: 197-276.
– – , Klussmann-Kolb, A., Verbeek, E. & Schrödl, M.
2014. Flashback and foreshadowing – a review of
the taxon Opisthobranchia. Organisms, Diversity &
Evolution 14 (1): 133-149. doi:10.1007/s13127-013-
0151-5.
Warén, A. & Bouchet, P. 1990. Laubierinidae and Pisani-
anurinae (Ranellidae), two new deep-sea taxa of
the Tonnoidea (Gastropoda: Prosobranchia). The
Veliger 33: 56-102.
Wilke, T., Haase, M., Hershler, R. Liu, H. P., Misof, B.
& Ponder, W. 2013. Pushing short DNA fragments
to the limit: phylogenetic relationships of ‘hydro-
bioid’ gastropods (Caenogastropoda: Rissooidea).
Molecular Phylogenetics and Evolution 66: 715-736.
