complex epidermal organs of phascolion (sipuncula): insights into the...

Complex epidermal organs of Phascolion (Sipuncula):

insights into the evolution of bimodal secretory cells in

annelids

Carsten H.G.M€uller,1,2 Jørgen Hylleberg3 and Peter Michalik1

1Zoologisches Institut undMuseum, Ernst-

Moritz-Arndt-Universit€at Greifswald, Jo-

hann-Sebastian-Bach-Str. 11/12, D-17487,

Greifswald, Germany; 2Department of

Neuroscience, University of Arizona, 1040

E 4th Street, PO Box 210077, Tucson,

AZ, 85721, USA; 3Department of Marine

Ecology, Institute of Biology, Aarhus Uni-

versity, Bld. 1135, Ole Worms alle 1,

8000, Aarhus C, Denmark

Keywords:

ultrastructure, evolutionary morphology,

phylogeny, polychaetes, multicellular

glands, secretory cells, receptor cells, cilia

Accepted for publication:

09 April 2014

Abstract

M€uller, C.H.G., Hylleberg, J. and Michalik, P. 2014. Complex epidermal organs of

Phascolion (Sipuncula): Insights into the evolution of bimodal secretory cells in anne-

lids.—Acta Zoologica (Stockholm) 00: 000–000.

The epidermal organs of an undescribed Phascolion species from the Balearic

Islands were investigated using SEM, TEM, LM, CLSM and lCT methods.

We found axial receptor cells confirming the previously assumed sensory func-

tion of epidermal organs. Our analyses also revealed six types of secretory cells.

Some secretory cells types are capable of secreting filamentous and amorphous

secretion in two different ways simultaneously (bimodal secretion). The high

diversity of cell types, the complex pattern of acinar units, and the absence of a

common gland pore make epidermal organs of Phascolion unique amongst

sipunculans (Phascolion type). Our reconstruction of the evolution of the epider-

mal organs of Sipuncula revealed that Phascolion-type epidermal organs may

have derived from either Golfingia-, Sipunculus- or Phascolosoma-type epidermal

organs. The oldest known sipunculans were Golfingia-like and had epidermal

organs, which might resemble the architecture of the Golfingia-type epidermal

organs in extant taxa. Thus, it can be hypothesized that bimodal secretory cells

(e.g. basophilic secretory cells) were part of the sipunculan ground pattern.

Moreover, bimodal secretory cells of Phascolion look strikingly similar to those

found in various annelid glands and thus might even be part of the ground pat-

tern of stem species of Sipuncula + Pleistoannelida.

Carsten H.G. M€uller, Department of Neuroscience, University of Arizona,

1040 E 4th Street, PO Box 210077, Tucson, AZ 85721, USA.

E-mail: [email protected]

Introduction

Sipunculans (peanut worms) are tough and tentaculate mar-

ine filter and deposit feeders comprising about 150 species.

This clade of unsegmented coelomate worms has always

puzzled zoosystematicists who assigned them to different

protostomian groups. Most of these investigators, however,

linked them to Echinodermata, Mollusca or Annelida (e.g.

Siewing 1976; Green and Bergquist 1982; Cutler 1994;

Nielsen 2001, Ruppert et al. 2004; see also historical sum-

mary by Dordel et al. 2010). Cutler (1994) stated that

Sipuncula, Mollusca and Annelida most likely shared a com-

mon ancestor, but suggested Mollusca and Sipuncula to be

most closely related. A sister group relationship of Sipuncula

and Annelida, named Pulvinifera, was proposed by Ax

(1999). Following morphological characters were interpreted

as synapomorphies supporting the Pulvinifera hypothesis: (i)

the presence and particular texture of the collagenous cuticle;

(ii) the coelom used as hydrostatic organ; and (iii) the spe-

cific organisation of the coelothelium (e.g. Moritz and Storch

1970; Goffinet et al. 1978; Bartolomaeus 1994; Ax 1999).

Recent molecular phylogenetic analyses place Sipuncula

either as sister group (Boore and Staton 2002; Mwinyi et al.

2009; Paps et al. 2009; Sperling et al. 2009) or as an ingroup

We dedicate this article to the late Dr. Bertil �Akesson (1927–2013).He was the skilful and inspiring pioneer of comparative studies of epi-

dermal organs in sipunculans.

© 2014 The Royal Swedish Academy of Sciences 1

Acta Zoologica (Stockholm) doi: 10.1111/azo.12082

of Annelida (e.g. Bleidorn et al. 2006; Colgan et al. 2006;

Rousset et al. 2007; Struck et al. 2007, 2011; Shen et al.

2009; Zrzav�y et al. 2009; Bleidorn 2009; Dordel et al. 2010;

Kvist and Siddall 2013; Weigert et al. 2014 in press). Struck

(2011) introduced the taxon Pleistoannelida. This hypothesis

allies ‘mobile’, often actively hunting Errantia with the pre-

dominantly (hemi-) sessile, often tube-dwelling Sedentaria.

The obligatory terrestrial Clitellata are considered derived

Sedentaria. In such analyses, Sipuncula often shape up as

sister group of Pleistoannelida. However, morphological

characters linking Sipuncula to annelids are scarce (e.g. spe-

cific collagen cuticle: Moritz and Storch 1970). One possi-

ble, so far underrated character complex is the exocrine

glandular apparatus and, specifically, the complex multicellu-

lar glands that are associated with parapodia (e.g. Meißner

et al. 2012).

Multicellular glands of tube-dwelling annelids producing

fibrillous secretions have recently drawn some attention.

For example, the pyriform or tubiparous glands of vestim-

entiferan and frenulate Siboglinidae (formerly called Pogon-

ophora) and the parapodial glands of representatives of the

spionid genus Spiophanes seem to be linked by secretory

cells forming cup-shaped microvilli along their entire apical

membrane (Shillito et al. 1993, 1995; Southward et al.

2005; Meißner et al. 2012). In both taxa, microfilaments

emerge from the centre of these microvillar cups and are

assembled to thicker microfibrils. In Siboglinidae, these mi-

crofibrils were proven to contain b-chitin and to become

part of the tube (Shillito et al. 1995). The production of b-chitin fibrils was assumed to be secreted by parapodial

glands of Spionidae as well (Meißner et al. 2012). Consid-

ering the recently proposed inter-relationships within

Annelida (e.g. Struck et al. 2011) multicellular units of epi-

dermal glands of tube-dwelling sipunculans might be

homologous to those of Spionidae and/or Siboglinidae.

The sipunculan integument bears numerous multicellular

glands, called epidermal organs, which are externally visible as

distinctive knobs or papillae. In both larval and adult sipuncu-

lans, epidermal organs are externally encapsulated by a cuticle

and internally delimited by regular epidermal cells. If pock-

eted deeply into the body ball, epidermal organs are also

delimited by subepidermal musculature. Since Keferstein and

Ehlers’ (1861) first account on the integument and epidermal

bodies (‘Hautk€orper’) of Sipunculus nudus, epidermal organs

of Sipuncula have been the subject of many comparative mor-

phological studies based on histological examinations (Kefer-

stein 1865; Th�eel 1875; Andreae 1882; Selenka et al. 1883;

Andrews 1890; Shipley 1890; Jourdan 1891; Ward 1891;

Cu�enot 1900; Metalnikoff 1900; Nickerson 1901; Shitamori

1936; Gerould 1939; Stehle 1953; �Akesson 1958). Although

being highly variable in some sipunculan taxa, it is possible to

distinguish certain types of epidermal organs. According to

their location on the sipunculan body (distal, medial and

proximal region of the introvert and trunk), unique structures

may be encountered, such as the holdfast papillae. These

organs are exclusively found in the median and proximal part

of the trunk of Phascolionidae. Amongst them, holdfast papil-

lae are particularly distinct in the gastropod shell-inhabiting

Phascolion species (e.g. �Akesson 1958; Rice 1993; Hylleberg

1995). Besides secretory cells, most sipunculan epidermal

organs contain ciliated receptor cells (�Akesson 1958). If cen-

tred, these receptor cells are termed ‘axial cells’ (Hylleberg

1995). Albeit some variation on the species level (depending

on size and shape of the epidermal organ), the diversity and

organization of secretory cell types seem to be more deter-

mined on higher taxonomic level (�Akesson 1958; Hylleberg

1995). The highest diversity of secretory cell types was hith-

erto found in the smooth and holdfast epidermal organs of

Phascolion species (�Akesson 1958; Hylleberg 1995).

Very little is known about the fine structure and the pre-

sumably complex function of epidermal organs in Sipuncula.

Few TEM micrographs of epidermal organs have been

printed in literature. Rice (1993) presented three sections

through epidermal organs located at the posterior trunk region

of an unidentified interstitial Phascolion species (Fig. 57, p.

257) and Sipunculus nudus (‘epidermal glandular organ’, Figs.

60–61, p. 258). Hylleberg (1995) described six types of epi-

dermal organs in Phascolion strombus, including some insights

from the fine structure of secretory cells (Figs 7, 9, 10, pp.

22–25). Micrographs in Rice’s and Hylleberg’s contributions

reveal fibrillous secretion stored in a huge gland reservoir.

However, it was impossible to verify the existence of special-

ized microvilli at the apices of contributing secretory cells and

to evaluate whether they were similar to those described by

Shillito et al. (1993, 1995) or Meißner et al. (2012) for sibo-

glinid and spionid annelids.

To analyse the ultrastructure of sipunculan epidermal

organs and its connections to the nervous system in detail,

we investigated a putatively new, tube-dwelling species of

Phascolion from the Balearic Island Ibiza using both invasive

(histology, immunohistochemistry and transmission electron

microscopy) and non-invasive (scanning electron micros-

copy, micro-computed tomography) methods. Based on

their location on anterior half of the trunk and potential

involvement in tube formation, we focused on smooth and

holdfast epidermal organs. Furthermore, we investigated the

qualitative and quantitative variation in cells constituting the

smooth epidermal organs. We develop a hypothesis suggest-

ing a tentative scenario of the evolution of multicellular

glands specialized in bimodal secretion in Sipuncula and

Annelida.

Materials andMethods

Specimens of Phascolion sp. (n = 17) were collected from

sandy sediment at the margins of Posidonia oceanica seagrass

meadows in the upper infralittoral (6–10 metres of depth) of

the beach Cala Llenya, located at the north-eastern coast of

the Balearic Island Ibiza (Spain). Collecting permits were

granted by local authorities: Govern de le Illes Balears,

© 2014 The Royal Swedish Academy of Sciences2

Epidermal organs of Phascolion sp. � M€uller et al. Acta Zoologica (Stockholm) 0: 1–32 (May 2014)

Conselleria d’Agricultura, Medi Ambient I Territori, Direcc�ıo

General de Medi Rural I Mari; http://vd.caib.es/13480628

74200-8316584-1321477938270970481). The specimens

were found in small-sized, marine gastropods, such as Bittium

ssp., Gibberula miliaria (Linnaeus, 1758), Nassarius cuvieri

(Payraudeau, 1826), Cyclope pellucida Risso, 1826, and Rissoa

auriscalpium (Linnaeus, 1758), crammed into the first 2–3whorls of the shell. Shells inhabited by Phascolion sp. were

easily identified by the presence of clay tubes incrusted by

sand particles (Fig. 1A,B). Specimens were cautiously

removed from the gastropod shells using a bench vice. For

identification, external and internal (e.g. apparatus of external

muscles) characters were evaluated based on Hendrix (1975);

Sa�ız-Salinas (1986, 1993); Cutler (1994); Ferrero-Vicente

et al. (2012) (see Appendix S1).

Dissection and fixing of the material

Adult specimens of Phascolion sp. were fridge-anaesthetized

for 30 min and then fixed in the following primary fixative

solutions: (i) Karnovsky’s (1965) solution for electron micros-

copy; (ii) paraformaldehyde for immunohistochemistry; (iii)

Bouin’s solution for differential histological staining; and (iv)

80% ethanol for species identification. Vouchers are deposited

in the Zoological Museum of the University of Greifswald

(ZIMG).

As in our study, we focused on a detailed examination of

the smooth and holdfast epidermal organs, the trunks of pri-

marily fixed animals were cross-cut in three pieces by a fresh

razor blade: the anterior, medial and proximal third of the

trunk, including various sections of the invaginated and con-

tracted introvert oozing out the respective trunk parts. Ante-

rior parts of the trunk, carrying papillated, smooth and some

holdfast epidermal organs, were processed with priority.

Transmission electron microscopy (TEM)

Anterior trunk pieces of six individuals of Phascolion sp.

were directly fixed in a fresh fixative solution modified

after Karnovsky (1965) containing 2.5% glutaraldehyde,

2.5% paraformaldehyde, 1.5% NaOH and 5% D-glucose,

buffered with 0.1 M sodium phosphate buffer adjusted at

pH 7.4. After rinsing the trunk pieces three times for each

5 min in the same buffer solution, postfixation in 1%

OsO4 solution (same buffer) was conducted at room tem-

perature for 4 h followed by dehydration in graded etha-

nols and embedding in Araldite (FLUKA) or Spurr media

(SIGMA-ALDRICH). Ultrathin sections (55–70 nm) were

Fig. 1—A–C. Phenotype of Phascolion sp. as viewed through a dissection microscope.A. Anterior trunk region of a living specimen withdrawn

into the tube projecting from the occupied gastropod shell. The cream-whitish coloured animal is observed from above by zooming into the entry

of the tube. Some papillated epidermal organs are clearly visible, the introvert is retracted.B. Example of an occupied gastropod shell: Bittium re-

ticulatum (da Costa, 1778). Note the projecting sediment tube incrusted with anorganic sand particles.C. Ethanol-conserved specimen cautiously

removed from its shell. Some parts of the body are not visible due to retraction of the introvert and winding of the trunk. Note the existence of

commensalistic Kamptozoa of the genus Loxosomella attached to the medioanterior region of the trunk. atr anterior trunk region, hepo holdfast epi-

dermal organs, Lox Loxosomella sp.,mtrmedian trunk region, pepo papillated epidermal organs, ptr posterior trunk region, sepo epidermal organs of

the smooth region, tpro projection of the sediment tube.


Acta Zoologica (Stockholm) 0: 1–32 (May 2014) M€uller et al. � Epidermal organs of Phascolion sp.

made using a Leica UCT ultramicrotome. Serial ultrathin

sections were mounted on Formvar-coated slotgrids

(PLANO: model G2500C), stained with uranyl acetate

and lead citrate for 4 min each, and then examined under

a ZEISS 902A (located at Electron Microscopic Centre,

University of Rostock) and a JEOL JEM-1011 (located at

Dept. General and Systematic Zoology, University of

Greifswald) transmission electron microscopes operated at

80 kV. In some specimens, up to 30 digital micrographs

showing sections of the generally voluminous epidermal

organs had to be stitched, using the software iTEM, to

provide an overview of an entire section plane.

Scanning electron microscopy (SEM)

Two specimens of Phascolion sp. were fixed in toto in modified

Karnovsky’s (1965) fixative solution. After washing in 0.1 M

sodium phosphate buffer adjusted at pH 7.4, the animals were

critical-point-dried, mounted on standard conductive adhe-

sive tubs, sputter-coated with gold, and examined at an accel-

erating voltage of 15–30 kV under a ZEISS EVO LS10

(Imaging Center, Biology unit, University of Greifswald).

Histology

Semithin sections (approximately 0.5 lm in thickness) were

made through the anterior trunk region of Phascolion sp.

(n = 4). Semithin sections were stained with 1% toluidine

blue in a solution of 1% sodium tetraborate (borax), modified

after Richardson et al. (1960).

Moreover, for Azan histology, entire specimens of Phaso-

lion sp. were fixed in Bouin’s solution (5 mL 40% formalin,

1 mL glacial acetic acid and 15 mL saturated picric acid solu-

tion; Mulisch and Welsch 2010) and paraffine-embedded.

Serial thick transverse sections at thickness of 5–7 lm were

stained with Azan as established by Heidenhain (1892). Light

micrographs were taken with the aid of an NIKON Eclipse

90i microscope (Nikon Instruments Inc., Melville, NY, USA)

equipped with an NIKON D-2MBWc camera driven by an

NIKONNis-Elements Ar 3.10 software.

Immunohistochemistry and fluorescence microscopy/confocal laser-

scanning microscopy

Two entire specimens of Phascolion sp., prefixed in 4% para-

formaldehyde for several days, were exclusively used for

immunohistochemistry. Following fixation, the trunks of

three individuals were cut transversally into discs of varying

shape and thickness (approximately 80–140 lm) using a ZE-

ISS Hyrax V-50 vibratome. Trunk pieces were washed in

several changes of sodium phosphate buffer (PBS) for at

least 4 h. All trunk parts were then pre-incubated in PBS-

TX (containing 1% normal goat serum, 0.3% Triton X-100

and 0.05 Na-azide) for 1 h and then incubated over night in

antityrosinated a-tubulin primary antibody from mouse

(1:2000; SIGMA) diluted in PBS-TX at room temperature.

Trunk parts were then rinsed for at least 2 h in several

changes of PBS and subsequently incubated in secondary

antibody against mouse proteins conjugated to Cy3 (1 : 500;

JACKSON Immuno research) for another 4 h. Finally, the

tissues were washed for at least 2 h in several changes of

PBS and then mounted in MOWIOL (Calbiochem). We

waived control experiments to evaluate unspecific binding of

the secondary antiserum, as anti-tyrosinated tubulin antibod-

ies revealed their significance as highly reliable markers of a-tubulin aligned in neurotubuli of axonal processes (e.g. Ken-

ning et al. 2013; Mayer et al. 2013). However, tubulin

occurs in several isoforms in living cells (Marotta et al.

1978). As it is likely that the antibody binds to any peptide

ending with the corresponding amino acid sequence, we will

refer to the labelled structures as ‘tyronisated tubulin-like

immunoreactivity (TUBir)’ throughout our manuscript. A

detailed statement on antibody specifity is issued in

Appendix S2.

Anti-tyrosinated a-tubulin immunoreactivity of the axial

cells was visualized 3-dimensionally using a LEICA SP5

(Leica Microsystems CMS GmbH, Mannheim, Germany)

confocal laser-scanning microscope (CLSM). Images were

volume-rendered using AMIRA 5.4. software and processed

in Adobe Photoshop CS4.

X-ray micro-computed tomography (micro-CT)

For micro-CT analysis, three specimens, prefixed in Karnov-

sky’s (1965) fixative for several days, were incubated in 1%

OsO4 (dissolved in 0.1 M sodium phosphate buffer) for 6 h,

with the addition of several crystals of red prussiate of potash

adjusted at pH 7.4. After being washed several times (same

buffer), specimens were incubated in potassium iodide for

another 8 h, before they were dehydrated in a graded series of

ethanol. The three dehydrated specimens were then critical-

point-dried, glued to pinheads and scanned for 9.5 h with an

Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss X-

ray Microscopy Inc., Pleasanton, USA) at 20 kV and 4W

using a 4.0 scintillator-objective lens unit.

Evolutionary analyses

To assess the evolutionary history of epidermal organs in Si-

puncula, we allocated the observed diversity of epidermal

organs based on �Akesson (1958) and the findings of the pres-

ent study into one phylogenetic character:

Type of epidermal organ

Type of epidermal organ. 0 – Sipunculus type; 1 – Golfingia

type; 2 – Phascolosoma type; 3 – Phascolion type.All characters are conceptualized unordered. The evolu-

tion of characters was reconstructed under parsimony using



Fig. 2—A–H.Diversity and histological anatomy of epidermal organs across body of Phascolion sp. A. Volume rendering of l-computed tomogra-

phy. B–H. Azan histology of various types of epidermal organs.A. Virtual parasagittal section through body. Various organs of trunk region and

posterior part of introvert are visible. The anterior part of introvert is retracted into the trunk and only ‘cut’ tangentially. Arrow heads with capital

letters mark the body region from which the histological sections were obtained.B. Anterior epidermal organs of anterior tip of introvert sitting

between introvert hooks which are rounded at their tip (transition zone to perioral tentacle crown). Longitudinal section.C. Papillated epidermal

organ frommost posterior region of introvert with axial cell cluster. Cross section.D. Papillated epidermal organs from anterior trunk region.E.

Smooth epidermal organs, anterior part of smooth trunk region with more voluminous, sac-like organs. Note massive amount of secretory cells

inside organs. Cross section. F. Smooth epidermal organs, posterior part of smooth trunk region with more flattened organs. Cross section.G.

Holdfast epidermal organ frommedian trunk region. The cuticle lining anterior tip of the organ is strongly sclerotized. Cross section.H. Posterior

epidermal organs from posterior trunk tip containing few secretory cells and an axial cell cluster. Longitudinal section. acc axial cell cluster, aepo

epidermal organ of anterior introvert region, an anus, axb axon bundles, bgc basal group of cells (associated with pepo), bsc basophilic secretory

cells, c cilia, cm circular muscles, csc coarse granular secretory cells, cte tentacles of crown region, cu cuticle, epc epidermal cells, fm fixing muscle,

fsc fine granular secretory cells, hepo holdfast epidermal organs, hsc heteromorphic secretory cells, in intestine (coiled), inv introvert, inv* with-

drawn anterior part of introvert, invh introvert hook, lm longitudinal muscles,mucmucous cells, oes oesophagus, pepo papillated epidermal organs,

rec rectum, res gland reservoir, rm retracting muscle, sc secretory cells, sct sclerotized anterior tip of holdfast epidermal organ, sepo epidermal organs

of smooth region, tr trunk, vnc ventral nerve cord.



the modular software package Mesquite version 2.75 (build

564) (Maddison andMaddison 2009).

Results

Typology and general organisation of epidermal organs

Individuals of Phascolion sp. from Ibiza (Figs. 1C, 2A)

carry five different types of epidermal organs that are typi-

cal for particular region of the trunk’s and/or introvert’s

epidermis. However, transition zones from one type of epi-

dermal organ into another do occur and are especially

obvious on the trunk. We illustrate the morphology of any

type of epidermal organ according to their sequential

appearance from anterior (introvert) to posterior (trunk).

Histological anatomy is shown for all types (Figs. 2 and

4). In this work, we focus on the ultrastructure of smooth

(Figs. 6–11) and holdfast (Figs. 12–13) epidermal organs.

The ultrastructural organization of papillated epidermal

organs is only briefly described, as the cellular architecture

of these organs is extensively treated in another work

(M€uller et al. accepted).

Anterior epidermal organs. The first kind of epidermal organs,

termed here as anterior epidermal organs, is found posterior

of the perioral tentacle crown, scattered between the introvert

hooks (Fig. 2B). These organs contain only a few secretory

cells. Furthermore, these organs neither show an obvious api-

cal opening nor a distinctive axial pile of receptor cells.

Papillated epidermal organs. At the posterior end of the intro-

vert as well as at the anterior tip of the trunk, there are numer-

ous, densely packed papillae with a conspicuous apical bulb,

the sensory cap, which carries the sensory pore at its tip

Fig. 3—A–D. A–C.Types of epidermal organs situated on the anterior trunk region of Phascolion sp. observed with scanning electron micros-

copy:A. Papillated epidermal organs from anterior tip of the trunk,B. Epidermal organs of the smooth anterior trunk region (slightly posterior to

A),C.Holdfast epidermal organ frommedioanterior trunk region, acute anterior tip of the organ is well visible.D. Scanning electron micrograph

showing Loxosomella kamptozoans firmly attached to the cuticle of median trunk region.



Fig. 4—A–E.Histology of smooth epidermal organs from anterior trunk region in Phascolion sp. Toluidine blue staining of 500 lm sections pro-

vides differentiation of various types of secretory cells within organs. A–D.: Longitudinal or oblique sections relative to trunk and organs; E.: Cross

section.A. Parasagittal view of a particularly voluminous smooth epidermal organ with large content of mucous cells. A basophilic secretory cell is

cut in full.B. Periphery of a longitudinal obliquely cut smooth epidermal organ showing coarse granular secretory cells.C. Same organ as in B

(with same section orientation) but a little closer to sagittal plane. Note circle of basophilic secretory cells occupying centre of the organ.D.Two

apposing smooth epidermal organs showing almost all types of secretory cell types typically observed therein. Heteromorphic granular secretory

cells are always highlighted in violet staining, probably due to polychromasic nature of toluidine blue.E. Several smooth epidermal organs of vari-

ous sizes and growth stages. The number of centrally aggregated basophilic secretory cells varies strongly with size of the organ. Diversity of secre-

tory cell types likewise becomes higher with increasing size: note the voluminous organ in the centre of the section. acc axial cell cluster (receptor

cells and glial-like sheath cells), bsc basophilic secretory cells, cc canal cells, cm circular muscles, csc coarse granular secretory cells, cu cuticle, ecm

extracellular matrix, epc epidermal cells, fsc fine granular secretory cells, hsc heteromorphic granular secretory cells, lm longitudinal muscles,msc

mosaic granular secretory cells,muc1 type 1 mucous cells,muc2 type 2 mucous cells, res gland reservoir.



(Fig. 3A). These papillated epidermal organs measure

approximately 100 lm in diameter and may appear either

prominent (Figs. 2D and 3A) or bulged inward the thickened

cuticle (Fig. 2C). They are also characterized by an axial clus-

ter including 5–12 multiciliated receptor cells the somata of

which are often located at the base of the sensory cap. Proxi-

mally, each set of receptor cells project an axon bundle, which

are connected with the subepidermal nerve plexus (Fig. 2D).

The axial cell cluster also contains several sheath cells

equipped with disc-like granules and two types of immature

secretory cells (see Figs. 2C,D and 9A). Sheath cells and pre-

sumably non-functional secretory cells envelop the central pile

of receptor cells. One type of secretory cells contains coarse,

acidophilic and electron-dense granules. The other one

houses basophilic, moderately osmiophilic granules that form

particularly dense aggregations around the apex. Bimodal

secretion activity is observed in basophilic secretory cells. Fila-

mentous secretion emits from tips of elongated microvilli and

aggregates in the centre of a spacious reservoir not yet con-

nected to a pore, whereas amorphous secretion is released at

the interface of microvillar bases. The axial cell cluster is sepa-

rated from peripheral epidermal cells by a spacious extracellu-

lar cavity, widely free of fibrillous contents. This cavity is

continuous with the strongly filamentous dermis below the

papillae and around the axons of the receptor cell wedged into

minute proximal canal. In the transition zone to smooth epi-

dermal organs, the glandular portion within the axial cell clus-

ter continuosly becomes more voluminous, whilst in return

the dermis becomes thinner.

Smooth epidermal organs. Posterior-to-anterior tip of the trunk

and in part intermingling with papillated epidermal organs,

we encountered dense populations of epidermal organs with

clearly dominating secretory structures. These organs are

voluminous, prominent and dome-shaped (lacking a sensory

cap!) in the transition zone to papillated epidermal organs.

More posteriorly, they occur as externally flattened excava-

tions in the integument. Hence, these organs are characterized

as smooth epidermal organs or, synonymously, as organs of

the smooth region of the trunk (sensuHylleberg 1995). In lon-

gitudinal histological sections, these smooth epidermal organs

exhibit an elongated sac-like (Fig. 2A,E) or flattened dome-

shaped (Fig. 2F and 3B) profile. They measure up to 150 lmin diameter. The size of these organs varies strongly, indepen-

dent of their particular position on the trunk. Smaller organs

are situated between bigger ones (e.g. Fig. 4E), but a clear

topographic pattern is not discernible. More details of the his-

tological and ultrastructural organisation are given further

below.

Holdfast epidermal organs. Towards the median region of the

trunk, the belt of smooth epidermal organs becomes more

and more loosened and nested between large epidermal

organs displaying a conspicuous, heavily sclerotized and

rounded cuticular scale located at the anterior margin

(Figs. 2A,G and 3C). These organs, approximately 100–200 lm in diameter, have been named organs of the holdfast

region (Hylleberg 1995), holdfast papillae (e.g. Rice 1993;

Cutler 1994) or just holdfasts (�Akesson 1958). We call them

holdfast epidermal organs. Here, the dominating cell type is

the mucous-producing secretory cells (Figs. 2G and 12A,B)

to which occasionally fine and coarse granular secretory cells

may be added.

Posterior epidermal organs. The holdfast region almost extends

to the posterior end of the trunk. At the posterior tip of

the trunk, holdfast epidermal organs are replaced by a dense

palisade of small and elongated posterior epidermal organs

that contain axial receptor cells and few secretory cells

(Fig. 2A,H).

Epibionts

Occasionally, the trunk of Phascolion sp. is infested with indi-

viduals of commensalistic Kamptozoa of the genus Loxosom-

ella (Figs. 1C and 3D). In fixed condition, individuals of

Loxosomella are barely recognizable as animals, because the

tentacular crown is retracted into the calyx (Fig. 3D). As the

Fig. 5—A–D. Semischematic reconstructions showing selected functional components of a fully developed smooth epidermal organ (trunk

region) in Phascolion sp. with emphasis on degree of organisational complexity.A. Sensory component of the epidermal organ comprising an axial

cell cluster: several receptor cells surrounded by two kinds of sheath cells. The monociliated central receptor cell (collar receptor) is highlighted by

an extensively dotted cytoplasm, whereas dotting is less intense in surrounding multiciliated receptor cells. For technical reasons, only two cilia

could be drawn per peripheral receptor cell. Note that proximal portion of receptor cells and their connection to the CNS is not illustrated. The

frayed mass plugging the sensory pore is only weakly indicated by punctuation to clearly highlight the cilia. B–D. Glandular components of the

epidermal organ comprising unicellular units (B: basophilic secretory cell producing amorphous and filamentous secretion) and multicellular

units (C: acinar formation of coarse granular secretory cells with each secretory cell having its own reservoir;D: compound-acinar formation of

fine granular secretory cells discharging their secretion into a common reservoir). In all cases, secretion is guided through a cuticle-lined conduct-

ing canal formed by one or several canal cells. Note that secretory cells are widely wrapped by processes of sheath cells containing flattened, disc-

like granules. bsc basophilic secretory cell, c receptor cilia, cc canal cell, con cuticularized conducting canal, csc coarse granular secretory cell, cu

cuticle, epc epidermal cell(s), exc extracellular cavity, fsc fine granular secretory cell, gp gland pore, rcmulticiliated receptor cell, rc*monociliated

receptor cell (collar receptor), res gland reservoir, rpr radial processes of sheath cells with disc-like granules, shc glial-like sheath cell (surrounding

receptor cells), shc* circumglandular sheath cell(s) with disc-like granules.



stalk passes into the cuticle of Phascolion sp. without display-

ing any kinds of grooves or similar marks on the cuticle

(Fig. 3D), these kamptozoans, if observed under the SEM,

may be easily confused with more exposed types of epider-

mal organs.

Interaction between epidermal organs and tube

In living specimens, papillated and smooth epidermal organs

were observed to come in close range to or even permanently

touch the clay tube projecting from the shell’s aperture. In

‘inactive’ condition (introvert retracted), the anterior tip of

the animal is well visible from above. Being half hidden in the

clay tube, the papillated epidermal organs are pointed

upwards and face the observer (Fig. 1A). Only few of them

are in contact with the inner line of the clay tube. The epider-

mal organs of the smooth region (abbreviated here as ‘smooth

epidermal organs’), however, are pointed laterally and come

in close contact with the clay tube, along with the holdfast epi-

dermal organs. Phascolion sp. moves its trunk towards the

opening of the clay tube and extracts the introvert, which then

measures up to three to four times the length of the trunk. If

so, at least the posterior population of the papillated epider-

mal organs are in contact with the inner margin of the clay

tube. We observed that any time the introvert is retracted the

epidermal organs of the posterior region of the introvert may

be as well in contact with the tube. As their glandular and sen-

sory architecture is somehow similar, the fine structure of

papillated and holdfast epidermal organs has been only briefly

described in the chapter above and only with regard to cellular

and submicroscopic patterns differing from those of the

smooth epidermal organs.

Histochemistry and fine structure of smooth epidermal organs

In Phascolion sp. (Fig. 1C), smooth epidermal organs com-

prise a central pile of receptor cells (= axial cells). Receptor

cells are wrapped by mostly granulated, glial-like sheath cells

(Figs. 5A, 6 and 7), and, predominantly, by secretory compo-

nents (e.g. Figs. 4, 5B–D and 10, 11). According to histo-

chemical stainings of related epidermal organs in Phascolion

strombus (�Akesson 1958; Hylleberg 1995), cellular arrange-

ment and fine structural appearance of secretory granules, we

are able to distinguish the following cell types: (i) basophilic

secretory cells; (ii) secretory cells with coarse granules; (iii)

secretory cells with fine granules; (iv) secretory cells with het-

eromorphic granules; (v) secretory cells with mosaic-like gran-

ules; (vi) sheath cells with disc-like granules; and (vii) two

kinds of mucous cells. Except for the sheath cells with disc-

like granules, all secretory cells described in this work extrude

their amorphous or filamentous secretion through a conduct-

ing canal the distal part of which is formed by one or several

canal cell(s). The duct portion formed by the canal cells is

lined by a distinct cuticle. Secretory cells may remain solitary

(e.g. basophilic secretory cells, e.g. Fig. 5B) or are grouped

together in circular glandular units reminding of an acinar or

acino-tubular formation. These acini may remain solitary (e.g.

Figs. 5C, 10F) or come together, then forming compound

and branched glandular units (e.g. Figs. 5D, 10D,E). A

monolayered epithelium of interdigitating epidermal cells

secretes the cuticle overlaying the smooth epidermal organs at

most parts (e.g. Figs. 5, 6A,D, 10G and 11E). At the top of

the organ, canal cells contribute to the cuticle (Fig. 6A). The

epidermal cells often contain Golgi stacks, a well-developed

ER, numerous free ribosomes and conspicuous aggregations

of electron-dense granules and late lysosomal bodies (Figs.

10B and 11A). The cuticle is traversed by thin and elongated

microvilli of the epidermal cells (e.g. Fig. 8A). It exhibits a

dense meshwork of stratified fibrils (presumably collagen).

Fibrils of apposed layers are aligned perpendicular to each

other (Figs. 6D and 10D,G). To the lateral margins of the

epidermal organ, the overlaying epidermal cells also secrete a

conspicuous, fibrillous extracellular matrix (ECM) that sepa-

rates the basal part of the organ from the circular musculature

(Figs. 4C,E and 6A). At the bottom of the epidermal organ,

this ECM is mainly secreted by the circumglandular sheath

cells containing disc-like granules (Figs. 6A and 9D; see fur-

ther below for details), as this cell type predominantly lines

the ECM and regular epidermal cells are absent in the basal

region. There, the epidermal organs directly nest in the subja-

cent trunk musculature, only separated by the ECM (e.g.

Figs. 4, 5 and 9D).

Axial cell cluster: receptor cells and glial-like sheath cells

Axial cells are restricted to the axial plane of the smooth epi-

dermal organ of Phascolion sp. This pile of cells consists of a

population of 4–8 mono- and multiciliated receptor cells that

are supported by 2–4 strongly ramified sheath cells resem-

bling glial cells (Fig. 5A). The spindle-shaped somata of the

receptor cells are aggregated in the axial region of the

smooth epidermal organ (Figs. 4C, 5, 6, 7E and 8C). The

cytoplasm in the somatic region is almost completely occu-

pied by polymorphic nuclei (Fig. 7E). The somata are

crammed into the median interspace of the basophilic secre-

tory cells (Figs. 6A,C, 7E and 8C). TEM and immunolabel-

ing against tyrosinated tubulin indicates that the receptor

cells are bipolar (e.g. Fig. 6B). No synapses were found

along the soma and receptor neurites within the epidermal

organ. To its distal end, each soma tapers and extends into

a dendritic process. Each dendrite displays a moderately

electron-dense cytoplasm with small, elongated mitochondria

of the tubular-type, polymorphic, electron-lucent vacuoles,

ribosomes, cisternae of both rough and smooth endoplasmic

reticulum, and numerous microtubules (Fig. 7B–D). Close

to the sensory pore, each dendrite forms at least one (collar

receptor cell with most central position within the cluster) or

3–4 (all other receptor cells of the cluster surrounding the

central cell) short cilia (Fig. 5A). The cilia are protruded

into an extracellular cavity, which is traversed by numerous



microvilli and separate from the outer environment

(Figs. 6D and 7B,C). The single cilium formed by the cen-

tral collar receptor cell is surrounded by thicker microvilli,

the coronal configuration of which resembles that of stereov-

illi (Figs. 5A and 7B,C). Those cilia located more peripher-

ally in the extracellular cavity seem to be surrounded by a

corona of thinner microvilli, as well. However, these coronal

patterns are less obvious. The cilia project through the extra-

cellular cavity to the sensory pore (Figs. 5A, 6C,D and 7A).

The cilia reach the outer environment but barely exceed the

Fig. 6—A–D. Smooth epidermal organs on anterior trunk region in Phascolion sp. as observed by TEM (A,C,D) and confocal laser-scanning

microscopy (B): sensory portion comprising receptor cells and supporting glial-like sheath cells.A.Overview of nearly all types of secretory, recep-

tor and sheath cells. Sheath cells with disc-like granules are clearly visible forming a sleeve around receptor cells as well as basophilic secretory cells

and heteromorphic granular secretory cells.B. Immunolabelling of tyrosinated tubulin, receptor cells are present, with their somata aggregated in

the centre from which strongly labelled neurites project to the distal tip and bottom of the organ (arrows). Note weaker immunofluorescence signal

in the centre due to presence of nuclei: arrowheads. Mostly diffuse staining of tyrosinated tubulin is also observed throughout the periphery of the

epidermal organ (double arrow).C–D. Apical terminations of mono- and multiciliated receptor cells surrounded by glial-like cells in overview (C)

and high-power magnification (D). D shows detail of cilia projected through sensory pore in longitudinal section, apposed epidermal cells inter-

digitate along lateral parts of the cell membrane (arrowhead). Voluminous glandular reservoirs of two basophilic secretory cells are illustrated inC.

acc axial cell cluster (predominantly receptor cells), bsc basophilic secretory cell, c receptor cilia, cc canal cells, cm circular muscles, csc coarse gran-

ular secretory cells, cu cuticle, ecm extracellular matrix, epc epidermal cells, exc extracellular cavity, fsc fine granular secretory cells, gp gland pore,

micmicrovilli,mscmosaic granular secretory cells,muc1 type 1 mucus cells, rc receptor cell(s), res gland reservoir, shc glial-like sheath cells (sur-

rounding the bsc), shc* circumglandular sheath cells with disc-like granules, sp sensory pore.



Fig. 7—A–F. Smooth epidermal organs on anterior trunk region in Phascolion sp. as observed by TEM: sequence of cross sections of receptor

cells and supporting glial-like sheath cells from apical (A) to basal (F) region.A. Tangential section through tip of epidermal organ. Several cilia

of receptor cells project through the cuticle, next to pore of a basophilic secretory cell. B. Lower part of extracellular cavity housing the apical tips

of dendrites of four multiciliated receptor cells. Several basal bodies can be assigned to each receptor cell highlighting its multiciliated nature. Cilia

are emitted into extracellular cavity which is also traversed by numerous microvilli formed by receptor cells and surrounding glial-like sheath cells.

The single cilium of the central (collar) receptor cell is encircled by distinctive stereovilli.C.Close-up of upper middle section of B highlighting

apex of a receptor cell forming three (basal bodies) cilia.D. Section slightly more proximal to C showing extensive granulation of glial-like sheath

cells. One of four visible receptor cells is cut on the level of basal bodies of two cilia. E. Region of receptor cell somata located in the centre of the

epidermal organ enclosed by several basophilic secretory cells. F.Most proximal axial region of two smooth epidermal organs each showing an

axon bundle encompassed by processes of one or two glial-like sheath cells. ax axons (of receptor cells), bb basal bodies (of receptor cilia), bsc

basophilic secretory cell, c receptor cilia, cu cuticle, ecm extracellular matrix, exc extracellular cavity, dicGolgi stack, g electron-dense granule, gp

gland pore,mimitochondrion, micmicrovillus/microvilli, mitmicrotubules, nu nucleus, rc receptor cell, sej septate junctions, sER smooth endo-

plasmic reticulum, shc glial-like sheath cell(s), shc* sheath cell(s) with disc-like granules, sic single cilium of the central collar receptor, stv stereov-

illus, za belt desmosome (Zonula adhaerens).



Fig. 8—A–I. Smooth epidermal organs from anterior trunk region in Phascolion sp. as observed by TEM: basophilic secretory cells cut at vari-

ous section levels and orientations. A. Longitudinal section of the apices of basophilic secretory cell and associated canal cells as well as of

gland pore. The gland reservoir is also endowed with thicker microvilli of uncommon origin (black arrowheads); slender, elongated microvilli of

the epidermal cells traverse the cuticle (white arrowhead). B.Details of canal cell surrounding the conducting canal which is lined by a distinct

cuticle. Cross section.C. Cross section of smooth epidermal organ containing three basophilic secretory cells that enclose the axial cell cluster

(asterisk) and are wrapped intensively by sheath cells.D. Close-up of reservoir of basophilic secretory cell in cross section. Reservoir space is

traversed by long, slender microvilli and a filamentous mass in its centre; simultaneously secreted amorphous substances are noticeable overly-

ing the apical membrane of secretory cell. E–F.Detail of apical part of microvilli formed by basophilic secretory cell (longitudinal section).

Note the electron-dense plaque covering the tip of microvilli (arrowhead) from which a brush of microfilaments emanates.G.Detail of tangen-

tial section through periphery of gland reservoir showing microvilli in cross section. Note occasional appearance of ‘scatter plots’ indicative of

aggregated microfilaments.H. Close-up of periphery of basophilic secretory cell densely filled with locally fusing secretory granules. Note

radial ramifications of sheath cells tightly enclosing the secretory cell (arrowheads). I. Aspects of cytoplasmic composition of basophilic secre-

tory cell close to the nucleus: note presence of biosynthesis-active organelles, densely packed secretory granules filled with an amorphous sub-

stance. Longitudinal section. bsc basophilic secretory cell, cc canal cell, cu cuticle, dicGolgi stack, epc epidermal cell(s), gp gland pore, mfl

microfilaments, mimitochondrion, micmicrovilli, nu nucleus, res gland reservoir, se amorphous secretion (discharged), sg secretory granules

(containing amorphous secretion), shc circumglandular sheath cells (with disc-like granules).



Fig. 9—A–D. Smooth and papillated epidermal organs from anterior trunk region in Phascolion sp. as observed by TEM: Sheath cell apparatus.

A.Cross section of papillated epidermal organ situated on the anterior tip of the trunk. At least three axial cells are clearly identifiable enveloped

by several sheath cells containing numerous disc-like or spherical granules. This central aggregation of receptor and sheath cells is separated from

peripheral epidermal cells by a voluminous dermis.B–C.Circumglandular sheath cells within smooth epidermal organ in transverse (B) and lon-

gitudinal (C) section, cut in part at nuclear level. Disc-like granules are sectioned in various orientations.D. Proximolateral margin of smooth epi-

dermal organ comprising basophilic and coarse granular secretory cells surrounded by ramified sheath cells with disc-like granules. Longitudinal

section. acc axial cell cluster (receptor cells and sheath cells), bsc basophilic secretory cell, cm circular muscles, cog coarse secretory granules, csc

coarse granular secretory cells, cu cuticle, de dermis, dlg disc-like granule(s), ecm extracellular matrix, epc epidermal cells, nu nucleus, shc glial-like

sheath cells (with spherical granules). shc* circumglandular sheath cells with disc-like granules.



cuticular surface of the epidermal organ. To its basal end,

each receptor cell soma tapers into an axonic process

(Fig. 5A), likewise endowed with numerous microtubules.

The axons remain closely grouped together and in axial posi-

tion, heading straight to the bottom of the epidermal organ

(Fig. 6B). At the bottom of the epidermal organ, we

observed distinct neurite bundles that are surrounded by

proximal processes of glial-like sheath cells located in the

epidermal organ (Fig. 7F). The neurite bundle pierces the

extracellular matrix. Given the continuity of tubulin-

immunolabelled strands (Fig. 6B), these neurites visible in

TEM most likely are the axons heading towards subepider-

mal parts of the central nervous system.

The axial receptor cells are tightly wrapped by special-

ized, glial-like sheath cells (Figs. 5A, 6A,C,D and 7). These

sheath cells accompany the axial receptor cells along their

entire length. Sheath cells tend to form radial extensions

particularly around the soma region of the receptor cells,

where they intertwine with similarly thin radial processes of

circumglandular sheath cells equipped with disc-like gran-

ules (e.g. Figs. 5A and 7E). The spatial interconnection of

both types of sheath cells results in a complex glial multi-

layer. Apically, these sheath cells form and surround the

extracellular cavity containing the receptor cilia. The moder-

ately osmiophilic cytoplasm of the sheath cells contains

small, ovoid or spherical, electron-dense granules (0.2–0.4 lm in diameter), some tubular mitochondria (some-

times branched) and locally aggregated microtubules. The

high abundance of microtubules and the 3-dimensional

spreading of radial processes fit the diffuse reticular pattern

of tyrosinated tubulin immunostaining throughout the epi-

dermal organ (Fig. 6B).

Secretory units comprising one or two secretory cells

Uni- and bicellular secretory units are more or less

restricted to the centre of the epidermal organ. Basophilic

secretory cells can be distinguished from heteromorphic

granular secretory cells by (i) affinity towards certain

alkaline histochemical components present in toluidine

blue and Azan (also in comparison to P. strombus: see�Akesson (1958); Hylleberg (1995) (ii) ultrastructure of

their granular content; and (iii) position of the gland

pores:

Basophilic secretory cells belong to the only type of gland

cells present in each smooth epidermal organ, irrespective of

its size and position on the trunk. Secretory material in these

cells remains widely unstained by Azan (Fig. 2E). However,

strong violet metachromasic staining in toluidine blue

indicates that basophilic biochemicals are predominantly pro-

duced by them (Fig. 4). The number of basophilic secretory

cells gathered within a given epidermal organ varies almost

linearly with size (Fig. 4E). In smaller organs (diameter

<50 lm), we counted 1–4 basophilic secretory cells (Figs. 4E

and 8C), whereas 5–7 basophilic secretory cells were observed

in larger ones (diameter >50 lm; see Fig. 4E for example).

To the centre of the epidermal organ, the basophilic secretory

cells enclose the apparatus of axial receptor cells and associ-

ated sheath cells (e.g. Figs. 5 and 8C), whilst to their periph-

ery they make contact to multicellular glandular units (acini)

made up by various types of secretory cells (see Figs. 4B–Dand 6A and description below). Each basophilic secretory cell

displays an elongated, bottle-like shape (Figs. 4A, 5B and

6A). The cell corpus may be divided into three different com-

partments: (i) the proximal biosynthetic zone, where the

secretion is produced and enclosed into numerous secretory

granules; (ii) the maturation zone dominated by secretory

granules in different successive stages; and (iii) the apical

zone, where amorphous and fibrillous secretion masses are

released into the gland reservoir (e.g. Fig. 5B). In addition,

the periphery of each secretory cell is further compartmental-

ized by radial, finger-like processes of those sheath cells

endowed with disc-like granules (Figs. 7E and 8H). The

proximal biosynthetic zone roughly occupies 50% of the entire

cell body and is characterized by a high diversity of cytoplas-

mic organelles. Besides a small, often rounded nucleus which

is rich in euchromatin, we found numerous, locally aggregated

mitochondria of the tubular-type, free ribosomes, rough and

smooth ER cisternae (often restricted to the cell’s periphery),

and large Golgi stacks (Fig. 8I). Most conspicuous organelles

are the polymorphic, densely packed and locally fusing weakly

osmiophilic secretory granules (e.g. Figs. 8H and 9B). These

granules are usually filled with an amorphous substance, but

occasionally, may also exhibit a fine granular or flocculent

matrix. In the proximal biosynthetic zone, aggregations of

secretory granules are nested within a meshwork of cytoplas-

mic inclusions housing all other kinds of organelles (Fig. 8H,

I). In the maturation zone, abundance of the secretory gran-

ules is even higher resulting in the restriction of remaining

cytoplasm to thinned interstitial meshwork (Figs. 6A,C, 9B,C

and 10A). Here, only mitochondria are found in considerable

abundances. At the deeply invaginated apex, the electron-

lucent and widely amorphous secretion is released into a tubu-

lar reservoir (=‘receptacle’ sensuHylleberg 1995) by exocytosis

(e.g. Fig. 8E). In cross sections, the reservoir looks ovoid or

spherical, filled in its centre by a mass of locally highly ordered

microfilaments (Fig. 8C,D). Numerous microfilaments are

formed at the tip of elongated, slender (<100 nm in diameter)

and unbranched microvilli diversifying the apex of the baso-

philic secretory cell (Fig. 8D–G). The tip of each microvillus

is covered by a highly osmiophilic plaque (‘sholder’ structure)

from which a brush of microfilaments is emitted (Fig. 8F,G).

The reservoir passes into a microvilli-surrounded, cuticular-

ized, and mostly triangular duct established by one or two

canal cells. Here, shorter microvilli are embedded into an elec-

tron-dense subcuticle (Figs. 5B, 6C and 8A,B). The centre of

the canal contains a filamentous mass (Fig. 8B). The volu-

men of the cuticularized duct becomes smaller and looks

spherical or slit-like if approaching or traversing the cuticle

(Figs. 6C, 7A and 8A), before it re-widens at the pore region



(e.g. Fig. 8A). The pores of the basophilic secretory cells have

paraxial positions relative to the nearly coaxial sensory pore

(compare Figs. 6B,C and 7A).

One or two units of heteromorphic granular secretory cells

may be present in a smooth epidermal organ. These secretory

cells are generally similar to the basophilic secretory cells with

regard to their location in the centre of the smooth epidermal

organ. Some of them, however, are displaced to a more

mediolateral position (e.g. Fig. 2E). The pores associated

with the heteromorphic granular secretory cells usually have

paraxial positions, meaning a lateral position in relation to the

sensory pore. The secretory component within one glandular

unit is also made up mainly by a single secretory cell.

Secretory granules are also very frequent, often aggregated (or

fusing), equally sized and highly polymorphic (Fig. 10A).

There is a high exocytotic activity at the apical cell membrane.

Simultaneously, there is also filamentous material produced

and originated from the tips of plaque-covered elongated and

slender microvilli (Fig. 10B,C). However, there are differ-

ences in comparison with basophilic secretory cells as well,

such as the high electron-optic heterogeneity of the cyto-

plasm in general and of the matrix in secretory granules in

particular. Second, there is a much stronger affinity towards

toluidine blue causing a conspicuous, dark-violet staining

(Fig. 4D). Third, the electron-dense subcuticle lining the

duct reaches further proximally down into the gland reservoir

that displays lower concentration of filamentous secretion

(Fig. 10B,C).

Glandular units comprising more than two secretory cells

Multicellular secretory units normally occur at the periphery

of the smooth epidermal organs (Fig. 5C,D). However, these

units may extend to the centre as well, especially if the organ

is larger and contains not more than four basophilic secretory

cells. According to their (i) histochemical affinities (also in

comparison to P. strombus: see �Akesson 1958 and Hylleberg

1995); (ii) architectural complexity of the acini; (iii) ultra-

structure and dimensions of their secretory granules (includ-

ing ultrastructure of the secretion discharged into the

reservoir); (iv) specific pattern of canal cells (including ultra-

structure of the conducting canal); and (v) position of the

gland pores, we distinguish coarse granular, fine granular and

mosaic granular secretory cells as well as type-1 and type-2

mucous cells:

Because of the enormous, strongly varying size and the

highly variable osmiophyly of their spherical secretory gran-

ules (1–5 lm in diameter), coarse granular secretory cells are

easily noticeable, even on the histological level (e.g. Fig. 4).

Incubations of thick sections in Azan distinctly highlight these

granules. However, it is impossible to differentiate physiologi-

cal stages. The granules are contrasted in red against a pale

cytoplasm (Fig. 2E). Toluidine blue stainings contrast the

secretory granules much better, ranging from light-bluish to

deep blue (Fig. 4B–D). Considerable differences in size and

biochemical composition of the coarse secretory granules are

also yielded by TEM, as we found both moderately and highly

electron-dense contents. The matrix of the secretory granules

appears always homogenous; electron-density does not corre-

spond to diameters (Figs. 6A, 9D and 10D,G). 3–6 coarse

granular secretory cells constitute one acinar glandular unit

(Fig. 5C). The secretory granules of each acinus are embed-

ded into a moderately osmiophilic cytoplasm, which is widely

poor in other organelles; only some mitochondria, ER cister-

nae (at the cell’s periphery), free ribosomes, and few small

Golgi stacks were observed. The elongated nucleus is rich in

heterochromatin and housed at the basal pole of the cell

(Figs. 5C and 9D). The secretory granules head towards the

cell apex. Its contents are discharged into a separate tubular

gland reservoir diversified by short microvilli (Figs. 5C and

10G). The relatively short reservoirs pass their portion of

secretion into a spacious and cuticularized conducting canal

(Figs. 5C and 10G). The canal cells that build and impreg-

nate the conducting canal form proximal processes that reach

between the apices of the secretory cells giving them a collar-

like appearance (Figs. 5C and 10G). The gland pores are sit-

uated mediolaterally or laterally on the organ’s surface.

Fine granular secretory cells are generally restricted to the

distal half of the smooth epidermal organ (e.g. Fig. 6A). They

are more frequent in the posterior, flattened organs (Fig. 2F).

The comparatively small and slim cell bodies are typically

found in acinar formation (Fig. 5D). Each acinus comprises

4–8 secretory cells (cf. Fig. 10E,F). In these acini, every secre-

tory cell contributes to lining a particular part of the common

gland reservoir penetrated by numerous microvilli. The reser-

voir does not contain a distinctive, filamentous secretion; the

microvillar tips are devoid of plaque structures and outgrows

of microfibrillar brushes (Fig. 10E). The reservoir contains an

amorphous substance (Fig. 10F) that is discharged via mero-

crine secretion. Acini may stay alone or cooperate with a sec-

ond acinus, thus forming a compound glandular unit

(Fig. 5D). Then, the number of secretory cells contributing

to a glandular unit may be doubled or even tripled. In this

case, the canal cells act as collar structure between apposed

and collaborating acini. Thus, a conducting canal is formed,

which is traversed by numerous microvilli and lined by a dis-

tinctive subcuticle (Figs. 5D and 10E). The secretory gran-

ules are numerous, mostly spherical, homogenously electron-

dense, toluidine blue affine and comparatively small barely

exceeding diameters of 1 lm (Figs. 4A,D,E and 10D–F).Further, frequently found organelles are tubular mitochon-

dria, rough and (especially) smooth ER cisternae, dictyo-

somes, glycogen rosettes and multivesicular bodies.

Glandular units constituted by mosaic granular secretory

cells resemble those of fine granular secretory cells in many

aspects, such as their mediolateral position within the epider-

mal organ and the tendency to build up compound glandular

units consisting of up to three acini. A generally enhanced

number of secretory cells, usually more than 6 (Figs. 6A and

11A), contribute to a given acinus. The most striking feature



Fig. 10—A–G.Diversity of secretory cells in smooth epidermal organs on anterior trunk region in Phascolion sp. as observed by TEM: heteromor-

phic granular, coarse granular and fine granular secretory cells.A.Oblique section of heteromorphic granular secretory cell crammed between

basophilic and coarse granular secretory cells. The granules are polymorphic and exhibit contents of varying electron-density.B. Apex of hetero-

morphic granular secretory cell in cross section. The gland reservoir is traversed by numerous microvilli that are stuck together by electron-dense

cuticle. The secretory cell is wrapped around itself, marked by a mesaxonal membrane (arrowhead).C.Close-up of microvillar border formed by

heteromorphic granular secretory cell. A brush of microfilaments is emitted from an electron-dense plaque on each microvillar tip (arrowhead).D.

Cross section through one half of the median region of smooth epidermal organ comprising glandular units of aggregated coarse or fine granular

secretory cells.E–F. Apical region of glandular unit (acinus) formed by several fine granular secretory cells. Cross section inE. reveals collar-like

arrangement of canal cells which build a conducting canal collecting secretion from two acini. Note that only canal cell(s) line the conducting

canal with a cuticle. F shows acinus and gland pore associated with this glandular unit in longitudinal section.G. Longitudinal section of acinus

composed of coarse granular secretory cells. Note the collar-like nature of canal cell caused by many cytoplasmic processes reaching between the

apices of secretory cells (arrowheads). bsc basophilic secretory cell, cc canal cell, cog coarse secretory granules, con conducting canal (collector), csc

coarse granular secretory cells, cu cuticle, epc epidermal cells, fg small and rounded (fine) secretory granules, fsc fine granular secretory cell, gp

gland pore, heg heteromorphic secretory granules, hsc secretory cell with heteromorphic granules,mflmicrofilaments,micmicrovilli,msc secretory

cells with mosaic-like granules, nu nucleus, res gland reservoir, shc circumglandular sheath cells with disc-like granules.



of this cell type surely is the secretory granules that have a

strictly spherical profile, measure 0.5–1.5 lm in diameter

(e.g. Fig. 11B), and are strongly stained with toluidine blue

(Fig. 4B,D). The inner matrix of the secretory granules

exhibits a distinctive mosaic-like arrangement of sausage- or

kidney-shaped, osmiophilic bodies contrasting with extremely

osmiophilic inclusions (Fig. 11A–D). The narrowed inter-

granular cytoplasm is particularly rich in glycogen rosettes

(Fig. 11D). Mitochondria, the elongated nucleus and aggre-

gated Golgi stacks are found at the base of the cells. The

number of cells forming one acinus may easily exceed 10,

thereby representing the biggest multicellular unions encoun-

tered in all smooth epidermal organs investigated (Fig. 11B).

The spacious reservoir may appear crescent in cross sections

and is bordered by numerous microvilli (Fig. 11A–C). The

tips of the microvilli are plaque covered and are the growth

zone of brushes of microfilaments (Fig. 11C). The reservoir

contains dense accumulations of microfilaments. It also

includes an amorphous electron-dense substance that fits the

sausage-shaped bodies in the mosaic-like secretory granules

that are found aligned to the apical cell membrane (Fig. 11A–C). Both kinds of secretions are guided outside via a cuticu-

larized conducting canal built up by several canal cells that

opens to the side of the organ’s surface (Fig. 11A). The

mosaic granular secretory cells are long and bottle shaped.

Some of them may reach between the basophilic secretory

cells (Fig. 6A).

In large-sized smooth epidermal organs, we detected volu-

minous, sac-like type-1 and type-2 mucous cells (e.g.

Fig. 4E). These cells are characterized by large (up to 6 lm in

diameter), polygonal, homogenous and tightly adjoined secre-

tory granules (Fig. 11F). Dense packing of secretory granules

causes displacement of other cytoplasmic organelles to the

utmost periphery of the mucous cell, except for the apex

where granular formation does not appear to be particularly

dense (Fig. 11E). According to different affinities towards

toluidine blue and osmiumtetroxide, we distinguish type-1

mucous cells with granules of varying electron density

(Figs. 4A,D,E, 6A and 11E,F) from type-2 mucous cells with

equally weakly osmiophilic granules (Fig. 4D). At the bottom,

each mucous cell houses the ovoid nucleus. The apex is dee-

ply invaginated and surrounds a tubular gland reservoir nar-

rowed by a dense border of microvilli not affiliated to

microfilaments (Fig. 11E). Up to four mucous cells have been

counted to form a glandular unit. The configuration resem-

bles that described for coarse granular secretory cells (com-

pare Fig. 5C). However, given the enormous size and spatial

extension of these cells, it is sometimes difficult to recognize

the acinar configuration in oblique sections not including the

gland reservoirs. Each mucous cell may have its own reservoir

or contribute to a compound reservoir (Fig. 11E). Either way,

amorphous secretion, primarily released into the reservoir by

merocrine pathway, fills up completely the cuticularized con-

ducting canal that opens to the side of the organ’s surface

(Fig. 11E).

Circumglandular sheath cells with disc-like granules

By the aid of many radial processes, circumglandular sheath

cells are the only ones that stay in contact with all types of

secretory cells within a smooth epidermal organ (e.g. Fig. 5).

This is accomplished by numerous ramifying, glial-like pro-

cesses protruding from the more voluminous, nucleus-

containing part of the cell (Figs. 5 and 9D). The entity of

these sheath cells forms a meshwork across the entire organ

and separates glandular units from each other. Wrapping is

especially dense around centrally located secretory cells, such

as the basophilic secretory cells (e.g. Figs. 6A, 8C,H and 9B–D), heteromorphic granular secretory cells (Fig. 10A) and, if

present, the mucous cells. Then, a glial multilayer may be

formed. Numerous disc-like granules are present in a highly

electron-dense cytoplasm (Figs. 6A and 9B–D). These discs

may be cut tangentially, then appearing nearly spherical

(around 1 lm in diameter), or longitudinally, then looking

ovoid or elliptical (e.g. Figs. 5 and 9B). Besides these gran-

ules, the cytoplasm is poorly supplied with other organelles

(mitochondria, Golgi stacks).

Fine structure of holdfast epidermal organs

Despite their location on the trunk, their greater size and

characteristic strengthening of anterior portion of the cuticle,

holdfast epidermal organs resemble smooth epidermal organs

in many aspects. Holdfast epidermal organs share with the

latter (i) the side-by-side occurrence of organs of various dif-

ferential stages (Fig. 12A); (ii) an axial sensory pore through

which several short cilia project outside (Fig. 12C); (iii) an

apparatus of axial receptor cells comprising a central mono-

ciliated (collar) cell with 10 surrounding stereovilli, 5–7 sur-

rounding multiciliated cells, and three peripheral glial-like

sheath cells containing electron-dense granules (Fig. 12D–G); (iv) two kinds of mucous cells with separate or common

pores (Figs. 12A,B and 13C,F); (v) acini of fine granular

secretory cells producing both merocrine and filamentous

secretion simultaneously and guided outside the body via

separate and cuticularized conducting canals (formed by spe-

cialized canal cells) and gland pores (Fig. 13A,B,F,G); and

(vi) sheath cells with disc-like granules filling the interspace

of glandular and sensory compartments (Figs. 12D–F and

13A,C–G). As with smooth epidermal organs, axial receptor

cells are connected to the orthogonally arranged central ner-

vous system via long axons that become bundled below the

glandular apparatus (Fig. 12G). At this subepidermal inter-

face to circular musculature, sheath cells with small, ovoid as

well as with disc-like granules are found wrapping the axon

bundles (Fig. 12G).

Holdfast epidermal organs differ from smooth ones by the

more spacious dermis especially to lateral margins of the

organ. Hemidesmosomes serve for stable connection of

the dermis to the outer epidermal cell sheath. The dermis is

rich in filamentous material and occasionally interspersed by



Fig. 11—A–F.Diversity of secretory cells in smooth epidermal organs from anterior trunk region in Phascolion sp. as observed by TEM: mosaic

granular and mucus-producing secretory cells.A. Longitudinal section of acinus built up by several mosaic granular secretory cells. The gland res-

ervoir is filled with a filamentous and an amorphous mass; reservoir passes into cuticularized duct leading to slit-like gland pore.B.Cross section

of apical border of acinus of several mosaic granular secretory cells.C.Detail of gland reservoir in cross section, acinus of mosaic granular secre-

tory cells as indicated in B. Fibrillar portion is secreted at the tips of inward-pointing microvilli (arrowhead).D.Cytoplasmic details of mosaic

granular secretory cell. Note large amount of free ribosomes and glycogen rosettes spotted between mosaic-like secretory granules. Cross section.

E. Apical region of acinus showing several type-1 mucous cells in longitudinal section. Mucous secretion is discharged into gland reservoir (cutic-

ularized in its distal part) and finally released at slit-like pore. F.Cytoplasmic detail of a type 1 mucous cell filled with numerous large and tightly

packed secretory granules. Cross section. cc canal cell, con conducting canal (collector), cu cuticle, ecm extracellular matrix, epc epidermal cells, gly

glycogen rosettes, gp gland pore, lys late lysosomes,mflmicrofilaments,micmicrovilli,mogmosaic-like secretory granule,msc secretory cell(s) with

mosaic-like granules,muc1 type 1 mucous cell(s),mugmucous-containing secretory granules, nu nucleus, res gland reservoir, se amorphous secre-

tion (discharged), za belt desmosome (Zonula adhaerens).



Fig. 12—A–G.Holdfast epidermal organs on midposterior trunk region in Phascolion sp. as observed by LM (A–B) and TEM (C–G).A–B.

Toluidine blue-stained semithin sections of several immature (arrowheads) and fully differentiated holdfast epidermal organs in cross sections (A)

and longitudinal section (B). Note the spine-like cuticular protuberance of the cuticle pointing towards anterior site and rich in non-layered, dark-

stained inclusions.C–G. Sequence of cross sections of axial receptor cells and supporting glial-like sheath cells from apical (A) to basal (G) region.

C.Tangential section through tip of the organ showing sensory pore traversed by several receptor cilia.D. Lower part of extracellular cavity sur-

rounded by apical tips of dendrites of five multiciliated receptor cells and three granulated sheath cells. Several cilia and numerous microvilli are

located to periphery of the cavity. Centre of cavity is occupied by cilium of central (collar) receptor cell; collar is built by ten stereovilli. E. Section

level approximately 2 lm farer proximal to D. Apices of altogether eight receptor cells are visible, amongst them one monociliated collar receptor

cell; note the presence of several basal bodies in some receptor cells. F. Section level approximately 10 lm farer proximal to E. Glial-like sheath

cells are cut at their nuclear level, wrapping dendritic processes of receptor cells.G.Most proximal region of a holdfast epidermal organ showing

an axial bundle surrounded by granulated, glial-like sheath cells. ax axons (of receptor cells), bb basal bodies (of receptor cilia), c receptor cilium,

ccu condensed, non-layered cuticular inclusions, cm circular muscles, crc central (collar) receptor cell, cu cuticle, ecm extracellular matrix, exc extra-

cellular cavity, ecm extracellular matrix, epc epidermal cell(s), g electron-dense granule, lm longitudinal muscles,mlbmultilamellar bodies,mimito-

chondrion,micmicrovillus/microvilli,mitmicrotubules,muc1 type-1 mucous cell,muc2 type-2 mucous cell, nu nucleus, pm frayed plug mass, rc

receptor cell, sej septate junctions, sER smooth endoplasmic reticulum, shc glial-like sheath cell, shc* sheath cell with disc-like granules, sic single

cilium of collar receptor, stv stereovilli, za belt desmosome (Zonula adhaerens).



Fig. 13—A–G.Glandular portion in holdfast epidermal organs frommidposterior trunk region in Phascolion sp. as observed by TEM: fine granu-

lar (A–B, F–G) and mucous-producing secretory cells (C–G).A–B.Cross section of small glandular unit of fine granular secretory cells; two

secretory cells are visible including oblique sight of conducting canal cuticularized if formed by canal cells. B shows sector of the conducting canal

seen in A; note projection of microfilaments frommicrovillar tips (arrowhead) indicating bimodal secretion activity.C.Oblique cross section of

holdfast epidermal organ housing several voluminous mucous cells.D.Close-up of part of specialized anterior cuticle enforced by numerous,

polymorphic inclusions showing no layered collagen texture.E. Lateral aspect of holdfast epidermal organ comprising spacious dermis containing

granulocytes and separating mucous cells of peripheral epidermal cell layer. F.Cross section through centre showing several locally expanded fine

granular secretory cells. Note presence of interstitial sheath cells with disc-like granules.G. Longitudinal section of immature holdfast epidermal

organ accommodating type-1 mucous cells with primary stages of secretory granules. Mucous cells, sheath cells and fine granular secretory cells

are well distinguishable by specific ultrastructure of their granules. cc canal cell, ccu condensed, non-layered cuticular inclusions, cm circular mus-

cles, con conducting canal (collector), cu cuticle, de dermis, ecm extracellular matrix, epc epidermal cell(s), fbm fibrillous material, fg small and

rounded (fine) secretory granule, gc granulocyte,mflmicrofilaments, hds hemidesmosomes,micmicrovilli,muc1 type 1 mucous cell,muc1 type-1

mucous cell,muc2 type-2 mucous cell,mugmucous-containing secretory granule, nu nucleus, rc receptor cells (somata), shc glial-like sheath cell,

shc* sheath cell with disc-like granules, za belt desmosome (Zonula adhaerens).



granulocytes (Fig. 13E). The anterior-thickened portion of

the cuticle is rich in polymorphic inclusions of amorphous,

highly electron-dense material produced by acute, anterior-

wards projections of particularly large epidermal cells

(Figs. 12B and 13C,D).

Discussion

Interspecific diversity of epidermal organs in Phascolion

Our data confirm previous findings of �Akesson (1958) and

Hylleberg (1995) on Phascolion strombus according to which

the papillated, smooth and holdfast epidermal organs com-

bine components of sensory and secretory functions. As secre-

tory cells appear to be not abundant and, moreover, non-

functional in papillated epidermal organs (see also M€uller

et al. accepted), it seems likely that the tube material is mainly

secreted and maintained by the various secretory cells of the

smooth epidermal organs. Holdfast epidermal organs may

contribute as well, but are located too far posteriorly on the

trunk to establish at least those parts of the sand-incrusted

tube projecting out from the shell’s aperture. According to

our observations, living individuals of Phascolion sp. avoid to

expose their trunks. At the utmost, trunks are moved back

and forth within the tube projection, whereas the introvert is

extended out from the tube. Furthermore, holdfast epidermal

organs predominantly contain mucous secretory cells (also

described from P. strombus by Hylleberg 1995), bimodal (fine

granular) secretory cells are much rarer. Basophilic secretory

cells appear to be present in P. strombus (see Fig. 12 in Hylle-

berg 1995), but are absent in investigated Phascolion species.

If at all relevant for tube building, mucous cells may provide a

sticky matrix which later on accumulates filamentous material

produced by various bimodal secretory cells. This assumption

implies the mucous secretion to be transported anteriorwards,

perhaps enabled by peristaltic movements of the trunk with

the shell. However, Phascolion species seem to mainly utilize

holdfast epidermal organs to prevent from being pulled out of

the shell. Behavioural observations revealed that P. strombus

uses these organs to scrape off deposits that got stuck to the

inner walls of the tube (Hylleberg 1975).

Basophilic secretory cells in smooth epidermal organs of

P. strombus and Phascolion sp. are most likely homologous.

They may produce similar sorts of muco-polysaccharides

via merocrine pathway. However, our results do suggest

that basophilic secretory cells also produce microfilaments

via microvilli rather than releasing amorphous secretion

alone as depicted by Hylleberg (1995). In our opinion,

Hylleberg’s (1995) fixation of epidermal tissue of P. strom-

bus may have suffered from treatment with 4% formalin

prior to TEM-prefixing in 4% glutaraldehyde solution. For-

malin penetrates the target tissues fast, but also has the

potential to extract proteins from target tissues and causes

swellings or distortions of cytoplasmic organelles (Hayat

1981). This massive extraction may also explain incongru-

encies between the present description and that of Hylle-

berg (1995) and, in particular, the absence of

microfilaments in the reservoir of basophilic secretory cells

of P. strombus. Variations in number of basophilic secretory

cells present in smooth epidermal organs of P. strombus

(n = 5–9: �Akesson 1958 and Hylleberg 1995) and Phasco-

lion sp. (n = 1–7: this study) may be correlated with body

size. Smaller body sizes, as encountered in Phascolion sp.,

usually result in epidermal organs that are comparatively

smaller and thus comprise fewer cells. Smooth epidermal

organs of P. strombus and Phascolion sp. also share fine gran-

ular and coarse granular secretory cells. The electron-optic

appearance of coarse granules in P. strombus, as described

by Hylleberg (1995), may again be attributed to formalin-

induced fixation artefacts (see above). The unidentified

interstitial Phascolion species examined by Rice (1993) at

least includes coarse granular secretory cells (‘GC2’ on Fig.

57, p. 257). In addition, Rice (1993) identified type-1

secretory cells in the same Phascolion species (‘GC1’ on Fig.

57, p. 257) that appear to be homologous with type-1

mucous cells in our Phascolion species from Ibiza and,

moreover, with mucous cells in holdfast epidermal organs

of P. strombus (�Akesson 1958; Rice 1993; Hylleberg 1995).

Although previous descriptions dealing with epidermal

organs were mostly confirmed, our analysis revealed new

insights from the organization of smooth epidermal organs of

Phascolion spp.:

1. Three hitherto undescribed types of secretory cells: het-

eromorphic granular secretory cells, mosaic granular

secretory cells and a second type of mucous cells (type-2

mucous cells),

2. Bimodal secretion mechanisms working simultaneously in

certain types of secretory cells (basophilic secretory cells,

heteromorphic granular secretory cells, mosaic granular

secretory cells and fine granular secretory cells); extrusion

of amorphous secretion via exocytosis and brushes of mi-

crofilaments at the tip of microvilli; bimodal secretion is

not linked to a certain degree of organisation (unicellular/

multicellular gland units),

3. Presence of multicellular gland units exhibiting two differ-

ent formations: solitary or compound acini with each

secretory cell contributing to a common gland reservoir

(fine granular secretory cells, mosaic granular secretory

cells) or having its own reservoir compartment by dint of a

deeply invaginated apex (coarse granular secretory cells,

type-1 and type-2 mucous cells),

4. Presence of canal cells forming cuticularized ducts in uni-

cellular and multicellular/compound gland units,

5. Presence of sheath cells with disc-like granules separating

the gland units from each other and building a ‘connective

tissue’ within the epidermal organs (illustrated but not

noticed by Rice (1993) in an interstitial Phascolion sp., see

cells located at the bottom of the organ in her Fig. 57, p.

257), are also found in papillated and holdfast epidermal

organs of this study species (partim M€uller et al.



accepted); this kind of sheath cells looks closely similar to

granulated cells described from ‘regeneration string’ of the

ventral nerve cord and subepidermal reservoirs in

P. strombus (Storch and Moritz 1970) and may therefore

be the cytological source of postembryonic growth, func-

tional differentiation and local replacement of lost epider-

mal organs (see Discussion in M€uller et al. accepted and

Appendix S3),

6. Presence of uni- and multiciliated receptor cell(s) within

the axial cluster which is surrounded by granulated and

ramified glial-like sheath cells, the uniciliated cell is a

regular collar receptor cell that is also present in papil-

lated and holdfast epidermal organs (partim M€uller et al.

accepted); collar receptors are widespread amongst

aquatic, worm-shaped invertebrates (e.g. Plathelminthes:

Sopott-Ehlers 1984; Priapulida: Moritz and Storch

1971; ‘Polychaeta’: Purschke 2005; see summary in

Tab. 7.1 of Schmidt-Rhaesa 2007) and known to be

specialized mechanoreceptors (see Thurm et al. 1983,

1998 for mechanisms triggering stimulus transduction);

this finding apears to be the first record of collar recep-

tors in adult sipunculans,

7. Tendency to gradual increase in cellular and functional

complexity with growth: the larger the smooth epidermal

organ, the higher the typological diversity of secretory

cells.

The total number of six secretory cell types in smooth epi-

dermal organs of Phascolion sp. by far exceeds previous

accounts for other Phascolion species (compare Appendix S4 –Table). Based on histological sections and histochemical affin-

ities, �Akesson (1958) identified two types of secretory cells

(‘basophilic glandular cells’, ‘acidophilic glandular cells’) in

P. strombus. Hylleberg (1995) found four types of secretory

cells instead of two in the same species. Amongst portion of

‘acidophilic granular cells’, he further distinguished cells con-

taining ‘fine granular cellular material’ from those housing

‘coarse granular cellular material’. A forth secretory cell type

with ‘amorphous contents’ was observed at the margin of the

smooth epidermal organ, but was left without in-depth ultra-

structural description (see aspect 11 in Fig. 5 in Hylleberg

1995, p. 22). Our data suggest that the secretory cells in the

anterior trunk region may be mainly responsible for the pro-

duction of the protruding tube. P. strombus does not form

tubes; its epidermal glands are less complex on cellular level

(e.g. Hylleberg 1975; Cutler 1994). Hence, the newly defined

heteromorphic granular secretory cells, mosaic granular secre-

tory cells and mucous cells may be considered candidates to

support tube building. This process does not only imply

secreting a (muco-) protein and/or (muco-) polysaccharid

matrix but also maintaining its integrity (hardening and

impregnation). The latter function has to be performed occa-

sionally or continuously by smooth epidermal organs present

on anterior part of the trunk. Instead, papillated epidermal

organs seem to fulfil sensory functions, as this type is widely

devoid of operable secretory cells (M€uller et al. accepted). It

seems plausible that heteromorphic granular and mosaic gra-

nular secretory cells form a large amount of filamentous mate-

rial in Phascolion sp. This filamentous mass may be useful for

stabilizing the multilayered tube wall as it is for instance

known from tubes of the siboglinids Riftia pachyptila and Ne-

reilinum punctatum (see Gaill and Hunt 1986; Southward et al.

2005). Admittedly, we do not know yet the internal structure

of the tube projection of Phascolion sp. In addition, even

though we consider our cell typology consistent and well

founded, we cannot entirely exclude that some of the secre-

tory cells types described herein may transform into another.

This functional turnover of secretory cells in epidermal organs

has been assumed by some authors (e.g. Nickerson 1901).

Apparently, such turnover may happen in those types of secre-

tory cells that differ only little in their apical structure, secre-

tion mode (uni- or bimodal) or configuration (multicellular

gland units). An example might be the heteromorphic granu-

lar secretory cells that appear closely similar to basophilic

secretory cells. Albeit our study is based on a sufficiently

broad scale of sections and individuals, it is possible that baso-

philic secretory cells in Phascolion sp. transform into hetero-

morphic granular ones with age or produce different secretion

due to environmental/ecological influences. Secretory cells of

annelids not only display a wide structural diversity of secre-

tion (filamentous, fine granular, strongly condensed amor-

phous, flocculent or fibrillous material), but also produce

many different substances ranging from organic (e.g. muco-

polysaccharids, proteins, enzymes, phenols) to inorganic com-

pounds (e.g. Dorsett and Hyde 1970a,b; Defretin 1971; Kryvi

1971, 1972; Storch and Welsch 1972; Moermans 1974; Anc-

til 1979; Vovelle 1979; Hausmann 1982; Welsch et al. 1984;

Hilbig 1986a,b; Vovelle and Gaill 1986; Storch 1988; Gardin-

er 1992; Vovelle et al. 1994; Hausen 2005; Mastrodonato

et al. 2005; Meißner et al. 2012). Various secretory cells in

the annelid epidermis were proven to produce different carbo-

hydrates at the same time (Welsch and Storch 1986). More-

over, the structural and functional entity of secretory cells

during differentiation has not been investigated so far in anne-

lids. Neither has there been any comparative study that clearly

links (histo-) chemistry to specific ultrastructure of secretory

granules found in the cytoplasm (Hausen 2005).

Diversity of epidermal organs in Sipuncula

The diversity of the secretory cells in smooth epidermal organs

of investigated Phascolion sp. is astonishingly high and, to our

knowledge, has no equivalent amongst other Sipuncula (com-

pare Th�eel 1875; Andreae 1882; Shipley 1890; Andrews

1890; Jourdan 1891; Ward 1891; Metalnikoff 1900; Cu�enot

1900; Shitamori 1936; Gerould 1939; Stehle 1953; �Akesson

1958; Rice 1993; Hylleberg 1995 and Appendix S4 – Table).

Admittedly, depth of description depends on microscopic

methods applied, and in fact, some of the granulated secretory

and sheath cell types may remain hidden in previous accounts



based on standard light microscopy, especially that of�Akesson

(1958). But, our present work shows specific correlation of

secretory cell typology and pattern of organisation (acini, sep-

arate pores, etc.) at least in species of Phascolion. These excep-

tional characters legitimate special typological treatment of

epidermal organs of Phascolion (see below). Our analyses

moreover indicate that the papillated, smooth, holdfast and

posterior epidermal organs of Phascolion sp. share several axial

receptor cells, basophilic secretory cells and granulated sheath

cells. In contrast, epidermal organs at the anterior region of

the introvert appear to contain secretory cells only. This pre-

sumed absence is, however, not in accordance with observa-

tions of �Akesson (1958) who identified three cell types

(receptor cells, basophilic glandular cells, acidophilic (granu-

lar) glandular cells: p. 187) in the anterior epidermal organs of

Phascolion strombus. �Akesson (1958) classified sipunculan epi-

dermal organs into Golfingia-, Sipunculus-, and Phascolosoma-

type organs (see Appendix S5 for histological details and dis-

tribution record). Except for Sipunculus spp. (‘bicellular glan-

dular organs’) and Siphonosoma cumanense (‘vesicular

organs’), all sipunculan epidermal organs examined so far

share a multicellular arrangement and integrate receptor and

secretory cells. Basophilic secretory cells and their voluminous

reservoir (‘receptacle’), well visible in light microscopic sec-

tions by their spacious, deep-racing reservoir, are also wide-

spread in Sipuncula. Based on�Akesson’s (1958) observations,

basophilic secretory cells are only completely absent in epider-

mal organs of the Phascolosoma type. In Sipunculus- and Gol-

fingia-type organs, they do occur frequently. The basophilic

secretory cells seem to be merely arranged in multicellular,

acinar formations. This is in contrast to the pattern of scat-

tered basophilic secretory cells described herein and by �Akes-

son (1958) and Hylleberg (1995) for Phascolion species.

Future studies should address whether simultaneous, bimodal

secretion is performed by at least some of these granulated

secretory cells and, subsequently, whether there is a hidden

diversity of cell types in other sipunculans. To date, the poor

quality of the few light micrographs and the simplifying nature

of �Akesson’s (1958) graphic reconstructions make it extre-

mely difficult to decide which one of the granulated secretory

cells of our Phascolion species is actually equivalent to those of

other Sipuncula. If taking into account the extension of the

secretory cells within the epidermal organ, their arrangement

and the relative dimensions of the secretory granules, as repro-

duced by �Akesson (1958) and preceding anatomists, the

coarse granular or mosaic granular secretory cells in the

smooth epidermal organs of Phascolion sp. could be potentially

homologous to them. Moreover, we interpret the mucous-

producing cells with densely packed granules (‘cellules

diff�erentes’: Cu�enot 1900) in the epidermal organs on the

trunk of Golfingia vulgaris vulgaris to be homologous to

mucous type-1 and/or type-2 secretory cells in Phascolion sp.

Beyond Phascolionidae, the potential existence of glial-like

and circumglandular sheath cells, however, inevitably needs

to be reinvestigated in higher detail using TEM methods. We

expect that sheath cells of this kind will be found in the epider-

mal organs of other Sipuncula, too.

In conclusion, the unique organization of smooth and

holdfast epidermal organs of Phascolion spp. justifies adding a

new type of epidermal organs to �Akesson’s (1958) classifica-

tion. We therefore introduce the term Phascolion-type epider-

mal organs. This new type is generally similar to the Golfingia

type but differs from the latter in the presence of (i) strictly

axially located receptor cells; (ii) more than two types of secre-

tory cells; and (iii) more than two distinct gland pores. At least

three, but mostly over ten gland pores are found (�Akesson

1958: cf. Fig. 82 and description; Rice 1993: Figs. 56–57; Hy-

lleberg 1995: Figs. 5,6). We refer here to Phascolion sensu

Th�eel 1875 including all Phascolion species with holdfast epi-

dermal organs (=holdfast papillae) and occupying gastropod

or scaphopod shells.

Zones on the trunk and introvert where the various types

of epidermal organs merge into another have raised suspicion

that at least those epidermal organs encountered in some si-

punculan species may be considered developmental stages

rather than distinct types of independent developmental origin

(e.g. �Akesson’s 1958). In Phascolion sp., continuous increase

in cell number and diversity in papillated (see M€uller et al.

accepted) and smooth epidermal organs from anterior to pos-

terior may indicate the existence of such transition zones.

However, to date arguments in favour of validity of epidermal

organ types are more convincing (M€uller et al. accepted,

Appendix S3).

Evolution and phylogenetic implications of epidermal organs in

Sipuncula

It is premature to conduct a phylogenetic analysis including

epidermal organ characters, because further TEM studies

have to unravel at first the true diversity of secretory cells in

epidermal organs of golfingiid, sipunculid, aspidosiphonid

and phascolosomatid Sipuncula. Second, potential intraspe-

cific variation in cellular architecture of epidermal organs of

those to this day disregarded taxa have to be re-evaluated with

the same set of methods as applied for this work. To hypothe-

size at least a preliminary scenario of the evolution of the epi-

dermal glands in Sipuncula, we traced the epidermal organ

types described above on the morphology-based tree of Cutler

and Gibbs (1985) as well as the latest, probably most substan-

tial molecular topology provided by Kawauchi et al. (2012)

(Fig. 14). In both topologies, the ancestral condition is

ambiguous, and thus, it is unclear what type of epidermal

organ is present in the ground pattern of Sipuncula. In the

light of Cutler and Gibbs’ (1985) hypothesis, it would be

equally parsimonious to assume that the stem species of the

Sipuncula either evolved quite compact and structurally

diverse epidermal organs of theGolfingia type or, alternatively,

started with compact epidermal organs without basophilic

secretory cells, corresponding to the Phascolosoma type

(Fig. 14). The latter scenario of evolution of epidermal organs



evolved in Sipuncula was preferred by Cutler (1994), starting

from simply constructed organs that became more complex in

higher derived taxa.

If using the hypothesis of Kawauchi et al. (2012), again

the last common ancestor of the Sipuncula may have been

equipped with epidermal organs of the Golfingia type or the

Fig. 14—Reconstruction of the evolution of epidermal organs within Sipuncula based on two partly conflicting phylogenetic hypotheses of Sipun-

cula. Four morphotypes of epidermal organs defined by�Akesson (1958) and in the present study were traced on both topologies to reveal the evo-

lutionary transformation.



Phascolosoma type. However, sipunculan stem species could

also have had disintegrated epidermal organs of the Sipunculus

type comprising combined ciliary sense organs, glandular

organs and combined glandular and sensory epidermal organs

(Fig. 14). Assuming epidermal organs of the Phascolosoma

type to reflect ancestral condition would be least compatible

with our hypothesis that bimodal secretory cells in sipunculan,

spionid and siboglinid Annelida are homologous (see chapter

below). An independent evolution of bimodal, simultaneous

secretion strategies can certainly not be excluded, but the

combination of specific ultrastructures is very complex, espe-

cially if considering the microvillar apparatus with its structur-

ally and functionally coherent, membrane-bound building

domains of microfilaments (see Discussion in chapter below).

In the light of tree topology presented by Kawauchi et al.

(2012), the polytomy of Golfingiidae is problematic, and in

particular, the suggested polyphyly of Phascolion with some

remote species (e.g. the interstitial Phascolion psammophilus

Rice 1993) and Onchnesoma steenstrupii Koren and Daniels-

sen, 1875, nesting within the main branch of Phascolion spe-

cies (see topologies in their Figs. 2 and 3). According to�Akesson (1958), epidermal organs of O. steenstrupii are uni-

form, tiny (measuring only 15–20 lm in diameter) and

merely contain 12 cells, amongst them only 3–4 are secretory

cells. To date, factors causing the miniaturization of and

reduction of basophilic secretory cells in epidermal organs of

Onchnesoma spp. are unknown. In conclusion, we consider

these organs to be strongly modified and unique amongst

Sipuncula (�Akesson 1958).�Akesson (1958) suggested that the plesiomorphic organi-

zation of epidermal organs resemble the Golfingia type. Inter-

estingly, the oldest known sipunculan fossils (e.g.

Cambrosipunculus spp., Archaeogolfingia caudata: See Huang

et al. 2004) date back to Lower CambrianMaotianshan Shale

(from southwest China) and show a golfingiid appearance.

These golfingiid-like fossil taxa were endowed with ‘fine papil-

lae’ on the trunk (Huang et al. 2004) which might resemble

the prominent trunk papillae of extant golfingiid taxa (�Akes-

son 1958). Thus, it can be hypothesized that bimodal secre-

tory cells (e.g. basophilic secretory cells) were part of the

sipunculan ground pattern. However, as the detailed ultra-

structure of epidermal organs cannot be revealed from fossil

taxa, it would be necessary to study more extant taxa within

and outside Sipuncula to test this scenario.

In an evolutionary scenario with the Golfingia type as

ancestral condition, epidermal organs became transformed

several times independently (Fig. 14). The disintegration of

epidermal organs and isolation of basophilic secretory cells

likely occurred only once (Sipunculus type), irrespective of tree

topology. The reduction of basophilic secretory cells, coincid-

ing with the loss of bimodal secretion ability (Phascolosoma

type), happened either two (based on Kawauchi et al. 2012)

or three times independently (based on Cutler and Gibbs

1985). The Phascolion type of epidermal organs might be

derived from ‘Golfingian’ ground pattern by augmentation

and diversification of glandular units and pores associated

with them. Alternatively, it might have derived from ancestors

equipped with Phascolosoma-type epidermal organs. Taking

into account previous studies of�Akesson (1958), Rice (1993),

and Hylleberg (1995), we hypothesize that the ground pattern

of epidermal organs of the last common ancestor of Phascolion

included receptor (axial) cells, basophilic secretory cells and at

least two types of granulated secretory cells. If assuming a

‘Golfingian’ ground pattern, Golfingia-type epidermal organs

got transformed by two parallel or sequential steps of evolu-

tion. First, the diversity of secretory cells was increased. Sec-

ond, the formerly clearly arranged glandular epithelium the

secretory cells of which discharge their secretion through a

common, axially located gland pore (retained in majority of

sipunculans investigated so far), disintegrated into a more

complex system of uni- and multicellular glandular units

(Fig. 5B–D) guiding different kinds of secretion through a

variety of pores in the cuticle. The increase in secretory cell

diversity could be linked to a spatial and perhaps also typologi-

cal diversification of sheath cells. Variations in cellular com-

plexity in a given species, as for instance found in papillated,

smooth and holdfast epidermal organs of Phascolion sp.

(M€uller et al. accepted and this work), may be explained by

growth gradients or, more likely, by functional specializations

of a given body region (see Discussion in M€uller et al.

accepted and Appendix S3).

Phylogenetic implications of the ultrastructure of multicellular glands

Across Annelida, combined glandular and ciliary sense

organs, similar to epidermal organs of Sipuncula and aggre-

gated in a papilla-like protrusion of the epidermis, are found

occasionally in various sedentarian annelids, such as represen-

tatives of the Flabelligeridae (compare Schlieper 1927; Boro-

din 1930), Sternasipdae (Cirratuliformia, compare Dahl

1955), Opheliidae (Vodopyanov et al. 2014) and Capitellidae

(compare Michel 1972). However, many older data on poly-

chaete glands seem to be ambiguous as well, as they are solely

based on light microscopy. Best available basis for comparison

with papillated and smooth epidermal organs of Phascolion

spp. is given byMichel (1972), who analysed the proboscidian

papillae in the capitellidNotomastus latericeus Sars, 1851, using

TEM. Here, prominent epidermal papillae are stiffened by

regular epidermal cells that surround a ‘central sensorial bud

made up with intermixed supporting cells and ciliated sensory

cells’ and ‘glandular cells’ (p. 482). These receptor cells pro-

ject axons that connect to the subepidermal nerve plexus.

Thus, this configuration generally matches the set-up of si-

punculan epidermal organs. However, glands described by

Michel (1972) are unicellular and clearly lack a bimodal secre-

tory mechanism as outlined above. Perhaps, functional aggre-

gations of unicellular glands and ciliated cells might be more

common than reported so far from the annelid epidermis (see

reviews of Richards 1978; Welsch et al. 1984; Storch 1988;

Gardiner 1992; Hausen 2005). The key to evaluate the phylo-



Fig. 15—A–D. Putative evolution of bimodal secretory cells in multicellular glands of Annelida. Annelida in general share a collagen cuticle char-

acterized by a unique system of crossed fibres. Microfilamentous material, considered sort of glycocalyx, is secreted by regular epidermal (support-

ing) cells from tip of microvilli, especially well-noticeable in polychaete and oligochaete annelids with an extraordinarily thick cuticle (A: microvilli

are over-dimensioned for didactic clarity). Adapted from several authors (e.g. Gupta and Little 1970; Storch andWelsch 1970; Burke 1974; Rie-

ger and Rieger 1976; Welsch et al. 1984; Hilbig 1986a; Hausen 2005). Ordinary unicellular glands are widespread and locally condensed in the

annelid’s integument (B); each secretory cell is primarily merocrine, producing secretory granules and releasing them into a self-built duct (here

devoid of microvilli) by exocytosis. Adapted from several authors (e.g. Gupta and Little 1970; Storch andWelsch 1970; Welsch et al. 1984;

Storch 1988; Hausen 2005).C–D. Bimodal secretory cells are present in multicellular glandular organs of various polychaetes, display structural

characteristics of regular epidermal cells and unicellular glands (intermediate stage) and, therefore, may be considered an synapomorphy of either

Pulvinifera (Sipuncula + Annelida sensu Ax 1999; Sperling et al. 2009; Eibye-Jacobsen and Vinther 2012) or Sipuncula + Pleistoannelida as large

ingroup of Annelida (sensu Struck et al. 2011; Struck 2011):C. In pyriform or tubiparous glands of frenulate and vestimentiferan Siboglinidae as

well as in parapodial glandular organs (type-5 secretory cells) of Spiophanes pisinnus, amorphous secretion is produced in a merocrine manner.

Simultaneously, microfilaments are released and assembled to microfibrils by cup-shaped microvilli diversifying the entire apical membrane of the

secretory cells (only a small section of the glandular epithelium of the huge, sac-like organs is shown in C, drawing modified after micrographs pre-

sented by Southward 1984; Shillito et al. 1993; Southward et al. 2005 andMeißner et al. 2012).D. Bimodal secretory cells aggregated in multi-

cellular glandular units in smooth and papillated epidermal organs from anterior trunk region of Phascolion sp. as unfolded in the present work

and inM€uller et al. (accepted). Simultaneous to merocrine secretion activity, brushes of microfilaments are released from tips of simple,

unbranched microvilli reaching into tubiform reservoirs of each glandular unit. cc canal cell, con conducting canal (collector), csm cup-shaped

microvillus, cu cuticle, ecm extracellular matrix, epc epidermal cell, gp gland pore,mfbmicrofibril,mfl brushes of microfilaments,micmicrovillus,

res gland reservoir, sc secretory cell, sg secretory granule(s).



genetic value of epidermal glands in Annelida (including epi-

dermal organs of Sipuncula) may be to target multicellular

glands. It is likely that early in sipunculan evolution such mul-

ticellular glandular units with bimodal secretory cells were

assembled with receptor cells to form epidermal organs.

Some types of secretory cells/units we found in smooth

and holdfast epidermal organs of Phascolion sp. show ultra-

structures that if further evaluated and conceptualized, may

have features supporting the recently proposed superclade of

Annelida (including Siboglinidae, Myzostomida and Sipuncu-

la). Furthermore, these specific gland characters may contrib-

ute to reconstruct certain inter-relationships within Annelida.

The most promising new character seems to be the bimodal

secreting mechanism. Secretion may be produced via mero-

crine pathway which implies biosynthesis of a secretion at the

basal pole of the cell, collection in regular secretory granules,

their transport to the cell apex using motor domains in the

cytoskeleton (including a successive maturing of the secretion

combined with subsequent fusion of secretory granules), and

subsequent discharge of secretion into a, however, structured

duct system. This consecutive secretion appears to work in all

kinds of secretory types of cells described herein. At the apex

of basophilic, heteromorphic granular, fine granular and

mosaic granular secretory cells in the smooth epidermal

organs of Phascolion sp., however, brushes of microfilaments

emanate from the tips of microvilli. In Annelida, this mecha-

nism is known from many ordinary epidermal cells. In longi-

tudinal sections, annelid epidermal cells reveal elongated

microvilli with tips covered by an electron-dense plaque struc-

ture and associated with an apparatus of filamentous material

‘outgrowing’ the plaque and establishing a glycocalyx

(Fig. 15A). In supporting cells, these specific apical microvil-

lar differentiations are called ‘shoulders’ (Richards 1978;

Hausen 2005; Meißner et al. 2012). Obviously, supporting

cells use this mechanism of making up fibrillous secretion to

establish the multilayered collagen cuticle (Fig. 15A). How-

ever, we have no reasons yet to assume that epidermal organs

of Phascolion spp. produce collagens at all. Nevertheless, the

various types of bimodal secretory cells in smooth epidermal

organs of Phascolion sp. may produce simultaneously amor-

phous secretion via the merocrine granule system and micro-

filaments via microvillar apparatus (Fig. 15D). Therefore,

these cell types represent an intermediate stage by performing

functions typical for both epidermal cells and merocrine secre-

tory cells. The latter ones are widespread components of glan-

dular epithelia in the annelid integument. Glandular epithelia

may be either more or less dense aggregation of solitary secre-

tory cells (Fig. 15B) or complexes of compound glands (see

reviews of Richards 1978; Welsch et al. 1984; Storch 1988;

Hausen 2005). Bimodal secretory cells have been docu-

mented for some Spionidae (type-4 and type-5 secretory cells

in parapodial glands of Spiophanes pisinnus Meißner and

Hutchings, 2003: see ultrastructural description of Meißner

et al. 2012) and Siboglinidae (secretory cells of pyriform

glands in Riftia pachyptila Jones, 1981: see description of

Shillito et al. 1993) (Fig. 15C). Admittedly, the architecture

of bimodal secretory cells in Sipuncula, Spionidae and Si-

boglinidae is not totally congruent. In S. pisinnus and

R. pachyptila, microfibrils are formed on the top of cup-

shaped microvilli instead of thinner microfilaments. These

microfibrils are known (Siboglinidae: Gaill et al. 1992a,b;

Shillito et al. 1995) or assumed (Spionidae: Meißner et al.

2012) to contain b-chitin. In contrast, those microvilli-pro-

jecting microfilaments produced in some secretory cells of

smooth epidermal organs in Phascolion sp. are simple,

unbranched projections. In addition, chitin seems to be

almost completely absent in Sipuncula (Nielsen 2012). Ear-

lier records of chitinous hooks and holdfast epidermal

organs are most likely the result of inaccurate analyses, only

the finding of chitin in the hooks of Cloeosiphon spp. is

trustable (Cutler 1994). Moreover, the possession of bimo-

dal secretory cells in multicellular glands does not seem to

be linked to a certain tube consistency and (chemical) com-

position noted for the mentioned annelids (e.g. Gaill and

Hunt 1986; Meißner et al. 2012; this work). Instead, the

evolution of bimodal secretory cells may be correlated with

tube dwelling enabling sedentarian annelids to add fibrillous

material to a primarily mucous-hardened tube. However, it

is inevitable to link ultrastructural diversity of epidermal

glands to tube composition. So far, the pattern of type-

equivalent tubes present in species-rich sedentarian groups

such for instance Spionida or Sabellidae is so diverse that it

seems virtually impossible to reveal a phylogenetic signifi-

cance of this character on a higher systematic level (see

summary of Defretin 1971). We also have to admit that

palaeoecology of ancestral sipunculans is still unclear,

namely their affinity to tube dwelling. U-shaped recurved

guts, as found in fossil sipunculans, may be interpreted

mandatory preadaptations to live in sediment burrows (Hu-

ang et al. 2004). It is not known yet whether endopsammic

or endolithic lifestyle was linked to tube formation from the

very beginning of sipunculan evolution.

It is reasonable to assume a common origin of bimodal

secretion in multicellular glands across Annelida, even at this

preliminary state of knowledge. Proper microfibril synthesis is

anything but trivial. It requires a high degree of

cytophysiological regulation starting from controlled transport

of microfibril precursors to the top of the target microvilli, fol-

lowed by the enzyme- and cytoskeleton-supported synthesis

and subsequent release of polysaccharides (cellulose, chitin)

at the terminal complexes along the apical membrane of the

microvilli, and resulting in the formation of microfibrils within

the gland reservoir (chain assembly). Shillito et al. (1993)

described this mechanism to work in the cup-shaped microvil-

li of R. pachyptila (Fig. 15C). The same mechanism seems to

be realized in the smooth and holdfast epidermal cells of Phas-

colion sp., as well as in some papillated epidermal organs of

the same species, however, in an immature state (M€uller et al.

accepted). The electron-dense plaque structure at the tip of

the microvilli may correspond to biosynthesizing terminal,



membrane-associated complexes in cup-shaped microvilli of

R. pachyptila and Spiophanes spp. (Shillito et al. 1993;

Meißner et al. 2012). However, as cup-shaped microvilli are

not developed in epidermal organs of Phascolion sp., there is

no cup cavity or compartmentalization of the extracellular

space. Hence, the efficiency of the microfibril formation is

reduced (Shillito et al. 1993). The structural and functional

complexity of bimodal secretion makes a random, convergent

evolution of bimodal secretory cells less likely and leads us to

assume a common origin of this cell type across hitherto

examined Sipuncula, Spionidae, Siboglinidae, and perhaps

also Oweniidae (see Fig. 30 in Gardiner 1992).

As simultaneous bimodal secretion seems to be exclusive

for annelids (cf. Shillito et al. 1993), the evolution of bimodal

secretory cells in multicellular glands may be interpreted

according the two main competing scenarios regarding the

annelid phylogeny. This character may be added to the

ground pattern of a taxon unifying Sipuncula and Annelida as

sister groups (sensu Sperling et al. 2009; Eibye-Jacobsen and

Vinther 2012), formerly postulated as Pulvinifera in the light

of the Articulata concept (Ax 1999). However, topology test-

ing of Dordel et al. (2010) significantly rejected sister group

relationship of Sipuncula and Annelida, but unambigiously

indicated that sipunculans are nested within annelids. Based

on this work and later contributions of the same working

group (e.g. Struck et al. 2011), it seems more plausible to sug-

gest bimodal secretory cells in multicellular glands as a syna-

pomorphy of Sipuncula + Pleistoannelida or of a grouping

Pleistoannelida + (Sipuncula + Amphinomidae) (Weigert

et al. 2014 in press). Moreover, this scenario regards the fact

that so far neither in so-called “Archiannelida” (compare Rie-

ger and Rieger 1976) nor in Chaetopteridae (compare Anctil

1979) multicellular glands or bimodal secretory cells have

been described. However, both taxa are supposed to nest

basally within Annelida (e.g. Struck et al. 2011; Struck 2011;

partim Struck et al. 2007; Zrzav�y et al. 2009). Besides molecu-

lar sequencing data, annelid relationship of Sipuncula is indi-

cated by the common possession of multicellular eyes (e.g.

‘ocellar tubes’) made of everse photoreceptor cells, accessory

pigment cells and canal-like, inward-pointing protrusion of

the cuticle (compare Rice 1993 with Purschke 2005, 2011

and Purschke et al. 2006).

We hypothesize that the evolution of cup-shaped microvilli

was due to a functional preadaptation towards forming micro-

fibrils, perhaps in connection with the secretion and process-

ing of b-chitin-containing microfilaments. This might have

been a further step in the evolution of the gland system of

sedentarian annelids to construct fibrillar tubes of higher

strength.

Acknowledgements

Our electron microscopic studies were supported by Prof.

Gabriele Uhl from University of Greifswald, PD Dr. Markus

Franck and his technician team from the Electron Micro-

scopic Centre of the University of Rostock, and by Gabriele

Ladwig working at the Electron Microscopic Unit of the Uni-

versity Hospital Essen. Matthes Kenning (University of Gre-

ifswald) vibratomized material of Phascolion sp. and provided

technical assistance getting proper CLSM stacks of immuno-

labelled trunk pieces. Furthermore, Dr. Andy Sombke and

Elisabeth Lipke (University of Greifswald) helped us to carry

out lCT scans of an undissected specimen of Phascolion sp.

and provided us with highly informative volume-rendered

images. Three anonymous reviewers made a fantastic job by

delivering highly detailed and useful comments that helped us

to improve this work.

Supporting Information

Additional supporting information may be found in the online

version of this article:

Appendix S1. Taxonomic and biogeographic remarks on

Mediterranean Phascolion species.

Appendix S2. Antibody specifity of antityrosinated

a-tubulin immunolabeling used in the present study.

Appendix S3. Variability and developmental origin of

epidermal organs in Phascolion sp.

Appendix S4. Table with a compilation of morphological

data on epidermal organs covering 150 years of research on

Sipuncula.

Appendix S5. Review of anatomical and distributional

details of three morphotypes of epidermal organs as defined

by�Akesson (1958).

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