Journal of Natural History
Vol. 44, Nos. 37–40, October 2010, 2255–2286
Comparative morphology and evolution of the nephridia in Nemertea
0022-2933
WTNAH
Journal
1464-5262
of Natural History
History, Vol. 1, No. 1, Jul 2010: pp. 0–0
Thomas Bartolomaeus* and Jörn von Döhren
Journal
T.
Bartolomaeus
of Natural
and
History
J. von Döhren
Institute for Evolutionary Biology and Animal Ecology, An der Immenburg 1, 53121 Bonn,
Germany
(Received 27 November 2009; final version received 27 May 2010)
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In various discussions on the phylogenetic position of the Nemertea, nephridial
morphology seems to support current hypotheses for a close relationship to
lophotrochozoan subtaxa. These arguments are based on isolated findings and
suffer from the lack of a phylogeny-based inference. In order to fill in this gap, the
structure of the nemertean nephridia is reviewed. Based on the structural data
presently available for nemertean nephridia, 30 characters are described. Their
ancestral states are inferred from cladograms derived from recent molecular analyses.
According to these, paired protonephridia restricted to the foregut region and
associated with the blood vascular system represent the primary state in nemerteans.
The terminal cells are covered by the blood vessel endothelium and have no open
connection to the lumen of the blood vascular system. Serially arranged protonephridia that could support the hypothesis of an annelid–nemertean–relationship
are either apomorphic for Cephalothricidae, a nemertean subgroup, or a cephalothricid ingroup.
Keywords: phylogeny; excretory organs; ribbon worms; ultrastructure
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Introduction
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The first mention of excretory organs in nemerteans is generally credited to Schultze
(1851 [for Cyanophthalma obscura]). These organs represent protonephridia and are
meanwhile known from all nemertean species, except pelagic polystiliferans. Presumably
transitory nephridia have recently also been found in the planuliform larvae of two
palaeonemertean species (own unpublished data). Generally consisting of a single
pair of protonephridia, these organs are often enlarged and multiplied in freshwater
and terrestrial nemertean species. Some cephalothricid species possess series of
nephridia that superficially resemble metanephridia (Wijnhoff 1912; Coe 1930, 1943).
Since the 1980s, electron microscopy contributed detailed insight into the organization of the protonephridia in hoplonemertean species (Bartolomaeus 1985, 1988; Jespersen 1987a, b; Jespersen and Lützen 1988, 1989). There is, however, still limited
knowledge on their ultrastructure in palaeonemertean species (Jespersen and Lützen
1987).
Protonephridia belong to a class of organs called filtration nephridia, which basically consist of a terminal unit serving in ultrafiltration and an excretory duct along
which fluid, soluble wastes, and water are transferred out of the body (Ruppert and
Smith 1988). Within these organs body fluid is ultrafiltered by special filtration cells
when passing through extracellular material covering them. Since ultrafiltration is
*Corresponding author. Email: tbartolomaeus@evolution.uni-bonn.de
ISSN 0022-2933 print/ISSN 1464-5262 online
© 2010 Taylor & Francis
DOI: 10.1080/00222933.2010.503941
http://www.informaworld.com
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largely a non-specific process, the ultrafiltrate also contains essential substances for
the animal’s metabolism which are reabsorbed by the duct cells. Thus, the ultrafiltrate
becomes modified prior to its release into the environment (Webster 1972; Wilson
and Webster 1974; Sato et al. 2002; Kusel et al. 2009). Since these two processes –
ultrafiltration and subsequent modification of the ultrafiltrate – are characteristic for
filtration nephridia, they are defined by their functional operation rather than by
their structure (Ruppert and Smith 1988, Bartolomaeus and Quast 2005).
Ultrafiltration depends on pressure differences between two different body
compartments, so that the filtration barrier has to cope with mechanical forces.
The filtration barrier or diaphragm generally consists of a thin layer of dense extracellular material. To withstand the mechanical forces, the diaphragm must be
spanned by cellular components for support. The entire filtration element of each
filtration nephridium, thus, consists of a porous or perforated cellular supporting
structure, and of extracellular diaphragms that bridge small clefts and pores of the
cellular supporting structure. In protonephridia terminal cells mediate ultrafiltration, while the duct serves in modifying the ultrafiltrate; both are connected by cellular junctions and form a structural unit (Figure 1A). This structural integrity is a
major difference to metanephridial systems, the second kind of filtration nephridia
in Bilateria (Figure 1B; Ruppert and Smith 1988; Bartolomaeus and Ax 1991). In
the latter systems the site of ultrafiltration (diaphragms bridging clefts between
podocytes) and the site of modification (metanephridia) are always spatially separated (Bartolomaeus and Quast 2005).
A protonephridium consists of at least one terminal cell, one duct cell and one
nephridiopore cell (Figure 1A). The terminal cell bears a cytoplasmic hollow cylinder which surrounds cilia and microvilli of the terminal cell. The hollow cylinder
contains numerous pores and clefts, which are bridged by diaphragms representing
the actual site of ultrafiltration. Functionally, the hollow cylinder represents the
suspending structure for the diaphragms which prevents them from being destroyed
by the mechanical forces of filtration (Figure 1D). Such a hollow cylinder has been
termed “filter” (Lammert 1985; Bartolomaeus 1989; Bartolomaeus and Ax 1991). If
two or more terminal cells form the suspending structure of the filtration barrier
(Figure 1C), it is called “compound filter” (Neuhaus 1988). It has been shown for
several bilaterian taxa that supporting structures may display a large variation of
forms (Kieneke et al. 2008). This variation might be informative for phylogenetic
inferences.
The structure and topological position of protonephridia has been used to argue
on a close relationship between nemerteans and annelids. In nemerteans the protonephridia are generally spatially related to the lateral vessels of the blood vascular system
(circulatory system sensu Ruppert and Carle [1983]). This observation led Friedrich
(1935b) and Ruppert and Carle (1983) to suggest that the blood vessels are homologous
to the annelid coelomic cavities. This hypothesis seemed to find support in Oudemans’
(1885), Nawitzki’s (1931) and Friedrich’s (1935a, b) observations of a direct connection
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filtration barrier: (C) compound filter is formed by several terminal cells; (D) filter formed by
a single terminal cell; (E) transcellular duct. Notes: ECM, extracellular matrix; DC, duct cell;
NPC, nephridiopore cell; PC, podocyte; TC, terminal cell; (A, B) modified from Bartolomaeus
and Ax (1991); (C–E) modified from Kieneke et al. (2008).
Journal of Natural History
2257
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Figure 1. Functional organization of protonephridia and metanephridial systems in Bilateria. (A) Protonephridia; (B) metanephridial system; arrows indicate the direction of filtration as well as secretory and resorption processes in the duct; (C, D) supporting structure for the
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T. Bartolomaeus and J. von Döhren
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between the blood fluid and nephridial lumen in certain palaeonemertean species.
Turbeville’s (1986) study of the hoplonemertean Prosorhochmus americanus revealed
a schizocoelous mode of blood vessel formation, similar to coelomogenesis in annelids,
a finding that implies a close annelid–nemertean relationship (Turbeville 2002; Struck
and Fisse 2008). The idea of a nemertean–annelid relationship, however, involved
another, contradictory string of argumentation. Within the framework of the gonocoel theory Goodrich (1895, 1945) considered the annelid coelomic cavities as homologous to the serially arranged gonads in nemerteans. He accordingly assumed that in
nemerteans the protonephridia also are primarily serially arranged – a state that can
be found in Recent cephalothricid species (Coe 1930). Coe (1930, 1943), and later
Friedrich (1935: p. 350), subsequently interpreted the cephalothricid nephridia as
metanephridia. This interpretation seemingly provided evidence for a close
nemertean–annelid relationship, assuming that cephalothricids maintain the ancestral state of nephridial arrangement in nemerteans.
In this review, the morphology and evolution of the nemertean nephridia is evaluated
comparatively in the light of recent molecular-based phylogenetic studies (Sundberg
et al. 2001; Thollesson and Norenburg 2003; Sundberg and Strand 2007; Sundberg et al.
2009), (Figure 2) to address the following questions: (1) What is the ancestral design
of nemertean protonephridia? (2) What is the ancestral relationship between the
blood vessels and the nephridia in nemerteans? (3) Do cephalothricid nephridia represent modified metanephridia? Special emphasis is placed on the palaeonemertean
taxa Tubulanidae, Carinomidae, Cephalothricidae, and Hubrechtidae, since palaeonemerteans appear to be paraphyletic (Thollesson and Norenburg 2003; Sundberg and
Strand 2007). Because they form a basal grade within the Nemertea, similarity in
morphology between the representatives of these four palaeonemertean groups likely
represents the ancestral condition for the nemerteans.
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Carinomidae
Cephalothricidae
Carinomidae
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Tubulanidae
Tubulanidae (part.)
palaeonemerteans
Tubulanidae (part.)
Hubrechtidae
Hubrechtidae
Heteronemertea
Heteronemertea
PILIDIOPHORA
Cephalothricidae
Hoplonemertea
A
NEONEMERTEA
Hoplonemertea
B
Figure 2. Competing hypotheses on the interrelationships among the high-ranking subgroups
of Nemertea based on molecular data. (A) Inferred from the combined analysis of 28S rRNA,
H3, 16S rRNA, and COI (Thollesson and Norenburg 2003); (B) majority rule consensus tree
inferred from 18S rDNA (Sundberg et al. 2009).
Journal of Natural History
2259
Materials and methods
Findings
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Procephalothrix filiformis (Johnston, 1828) was collected in the Odde Watt (List/Sylt,
Germany) in February 2005; Amphiporus imparispinosus Griffin, 1898 between rocks
at Cattle Point on San Juan Island (Washington, USA); and Callinera monensis Rogers,
Gibson and Thorpe, 1992 on a sand flat in the bay of Poldouhan close to Concarneau
(Brittany, France) in May 2008. For analyzing the nephridial histology the species
were fixed in a Bouin–Dubosc–Brasil solution, dehydrated in a graded alcohol series
and embedded in paraplast via methylbenzoate and butanol. Series of 5-µm sections
were cut on a steel knife (grade C) in a Reichert microtome, mounted on glass slides
and Azan stained (Döhren and Bartolomaeus 2006). The sections were analyzed
using an Olympus BX 51 light microscope with a ColorView digital camera for documentation. For electron microscopy, P. filiformis was fixed in 1.25% glutaraldehyde
buffered in 0.05 M phosphate buffer with 0.3 M NaCl (PBS) for 12 hours (pH 7.2, 4° C),
washed in the same buffer and postfixed in 1% OsO4 buffered in PBS. The animals
were dehydrated in an acetone series and embedded in Araldite. Series of silver interference sections (70 nm in thickness) were cut on a diamond knife using a LEICA
UC6, kept on formvar-covered single slot grids, automatically stained in a Nanofilm
TEM Auto Stainer and analyzed under a PHILIPS Biotwinn CM 120. Micrographs
were taken on DITABIS imaging plates and processed using the AnalySIS software.
All metadata and additional micrographs are deposited in the MorphDBase data base.
Voucher specimens from the populations of the collecting sites are deposited in the
Collection of the Institute of Evolutionary Biology and Animal Ecology (University of
Bonn). Various literature sources (see references) were screened for details on
nephridia.
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Protonephridia in palaeonemertean taxa
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Light microscopical data on the single pair of nephridia in the palaeonemertean
Callinera monensis (Tubulanidae) revealed that in fertile animals each nephridium
measures between 750 and 900 µm in length and, thus, constitutes about 1/100 of the
entire body length. The terminal section, which bulges into the lateral blood vessel,
measures 270–290 µm (Figure 3A, B). This section is composed of several terminal
units that serve in filtration and are overlain by the endothelial cells of the blood vessel. These organs have a ciliary flame and are connected to small and short capillaries
that branch from a 500–550 µm-long duct located dorso-laterally to the blood vessel.
This duct is the main duct of the protonephridium (Figure 3C). It has a wide lumen
and is intensely ciliated. Posteriorly, its diameter is reduced to one quarter of its original cross section; here, the duct bends to run almost perpendicular to the longitudinal body axis and passes through the muscle layer above the lateral nerve cord
(Figure 3D). This small, last section of the protonephridial duct is the efferent duct; it
opens to the exterior via a single dorso-lateral, intraepidermal nephridiopore (Figure 3E).
Basically, the same structure and topological position has been reported for the
paired nephridia in Callinera buergeri (Bergendahl 1900), Callinera bergendahli (Gibson
and Sundberg 1999), C. nishikawai (Kajihara 2006) and C. zhirmunskyi (Chernyshev
2002). The terminology applied here for Callinera monensis is consistently used in the
following sections (for different terminologies see Table 1).
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Figure 3. Callinera monensis (Tubulanidae, Nemertea) protonephridium: histological
sections, Azan staining (for method, see Döhren and Bartolomaeus [2006]). (A) Terminal
units (arrow) bulging into blood vessel (BV); (B) capillaries (arrows) in the terminal region;
(C) main duct (MD) running dorsal to blood vessel; (D) efferent duct (arrow); (E) dorsolateral nephridiopore (arrow). Note: LN, lateral nerve.
Journal of Natural History
2261
In nearly all palaeonemertean species the distal portion of the protonephridium
bulges deeply into the lumen of the lateral blood vessel (Figure 4A, B, D; Bürger
1895a, b). This anterior part of the protonephridium had been termed nephridial
gland in tubulanids and carinomids, because of its compact structure (Oudemans
1885). An open connection between blood fluid and nephridial lumen, which has
been suggested by some authors (Oudemans 1885; Hubrecht 1885 [for Tubulanus species and Carinoma armandi]; Nawitzki 1931; Friedrich 1935a, b [for Carinina spp.]),
has not been confirmed by others (Hubrecht 1887; Bürger 1895a). In almost all palaeonemertean species the nephridia are very short and measure less than 1/30 of the
entire body length (Bürger 1897–1907).
Tubulanidae
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The nephridial organization briefly described here for the tubulanid Callinera monensis
is largely representative of all tubulanid species, except Tubulanus lutescens in which,
according to Cantell (2001), protonephridia could not be found. All tubulanid species
possess a single pair of protonephridia that is very short and restricted to a part of the
foregut region (Figure 3; Oudemans 1885; Bürger 1895a, 1897–1907; Coe 1901, 1943;
Friedrich 1935b, Gerner 1969; Jespersen and Lützen 1987).
In Carinina species the single pair of protonephridia is located in the posterior
foregut region (Bürger 1895a; Friedrich 1935a; Hylbom 1957; Gibson and Sundberg
1999; Kajihara 2006). Each protonephridium consists of a long main duct extending
parallel to the lateral blood vessel. Only the anterior section of this duct closely
approaches the blood vessel, as this part of the duct forms several small capillaries
that extend into the vessel, interdigitate, and finally end up in a terminal unit (Figure 4A
for Carinina grata). Capillaries and terminal units are covered by endothelial cells;
there is no direct contact between blood fluid and the lumen of the protonephridia
(Bürger 1895a). This interpretation differs from Nawitzki (1931) and Friedrich
(1935a: p. 71, 1935b), who described an open connection between blood fluid and
nephridial lumen for Carinina remanei, C. buddenbrocki and C. poseidoni. In these
species numerous capillaries branch off the main duct (Friedrich 1935a: Figure 34).
The posterior section of the main duct is dislocated from the blood vessel (Bürger
1895a; Nawitzki 1931; Friedrich 1935b). In Carinina grata the main duct bears several caeca that lie between the longitudinal and the circular muscles of the body wall
(Bürger 1895a). Posterior to these caeca the duct bends almost perpendicularly and
passes the body wall muscles dorsal of the lateral nerve cord to open to the exterior
(Figure 4A). Except for the distal caeca of the main duct the protonephridia correspond in structure and composition to those of C. arenaria; C. coei; C. johnstoni; C. plecta
and C. sinensis (Hylbom 1957; Gibson and Sundberg 1999; Senz 2000; Kajihara 2006).
Earlier studies showed that protonephridia in Tubulanus species are largely identical
to those described above for Carinina species, especially in terms of position of the protonephridia (posterior region of the esophagus), course and position of the efferent duct,
and position of the nephridiopore (Bürger 1895a; Coe 1901, 1940; Friedrich 1936; Gibson
and Sundberg 1999; Cantell 2001; Ritger and Norenburg 2006). The main difference lies
in the formation of the so-called collecting tubules that serially branch off the main duct
(Figure 4B for Tubulanus superbus) (Table 2). These ramify into small capillaries and enter
the blood vessel to terminate in terminal units, called “flame bulbs” by Oudemans (1885)
and Bürger (1895a [for T. linearis, T. polymorphus and T. superbus] or “cyrtocytes” by
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Table 1. Synopsis of terms applied to nephridia and their histological and ultrastructural characteristics.
Coe (1930)
Cephalothrix
spp.
Nephrostome
Collecting
tubules
Not termed
Flame cell
Endkölbchen,
Wimperkolben
Bartolomaeus
(1985, 1988)
Terminal cell
Jespersen
(1987a,b);
Jespersen and
Lützen (1987, 1988,
1989)
Terminal organ,
terminal
element,
terminal cell,
flame bulb
Terminal
chamber,
end canal
Canälchen, feine
Canäle, Aestchen,
feine
Nephridial-canäle,
Exkretionskanäle
Proximal
Protonephridial
efferent duct
capillaries
Convoluted
tubule
Convoluted
tubule
Distal efferent
duct
Collecting tubules,
protonephridial
tubules
Efferent duct
Efferent duct
Sprossen,
Nebengefäße,
Zweigkanäle,
Zweige,
Längsgefäß,
Hauptcanal,
Nephridialcanal,
Hauptgefäße,
Exkretionsgefäßstämme,
Hauptstämme,
Längscanal
Ausführgang,
Ausführductus
Distal efferent
duct
Efferent duct
Nephridiopore
Nephridiopore Porus
Nephridiopore
Nephridiopore
p
y
c
o
Main
longitudinal
duct
Thin-walled, leading Small lumen, short, cilia, Efferent duct
microvilli
to pore,
rectangular branch
of main duct
Nephridiopore Intraepidermal pore Intraepidermal pore
Nephridiopore
Efferent duct
Bürger (1895a)
u
t
Terminal organs
Coe (1929)
terrestrial
species
'r s
Main duct
Ultrafilter, terminal
cell(s) with
supporting
structure for
filtration
barrier
Thin-walled,
Small diameter, few
branched
cilia, or few
microvilli, smooth
adluminal surface,
transcellular or
intercellular between
two cells
Thick-walled, ciliated Larger diameter,
intercellular between
more than two cells,
lumen numerous
cilia, adluminal
infolding
Coe (1943);
Gibson (1972)
h
o
Capillary
Terminal ciliary
flame
Ultrastructure
a
Terminal
unit
Histology
T. Bartolomaeus and J. von Döhren
Present study
Journal of Natural History
2263
Carinomidae
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Ritger and Norenburg (2006 [for T. riceae]). Jespersen and Lützen (1987) and Turbeville
1991 [for T. cf. pellucida] confirmed Bürger’s (1890, 1895a) statement that, in contrast to
Oudemans’ observation (1885), the nephridium does not open into the blood vessel.
Details on the protonephridial ultrastructure in Tubulanidae were published by
Jespersen and Lützen (1987) for Tubulanus annulatus and Turbeville (1991) for Tubulanus
cf. pellucidus. They clearly provided evidence that the anterior terminal units may be
bathed directly in the blood fluid, but are nevertheless separated from the blood by at
least an extracellular matrix (ECM), (Bürger 1895a: p. 305; Jespersen and Lützen
1987; Turbeville 1991: Figure 17). According to Jespersen and Lützen (1987) several
terminal cells comprise the terminal units (flame bulbs) and form a compound filter.
Each terminal cell bears a distal cytoplasmic lobe that interdigitates with those of
adjacent terminal cells. The meandering cleft between the adjacent cells is bridged by
extracellular material (diaphragm). Terminal cells and filtration area are overlain by
extracellular matrix. If covered by the blood vessel endothelium, its cells are fenestrated to allow ultrafitration of the blood fluid. In Tubulanus annulatus, there are several of these terminal units. The ultrafiltrate produced in each of them passes small,
sometimes branching capillaries, which are weakly ciliated and have a transcellular
lumen and a smooth adluminal surface. They are connected to collecting tubules that
branch from the large main duct (Figure 4 B for T. superbus). Ultrastructurally, collecting
tubules and main duct are similar in being heavily ciliated and having deeply invaginated
adluminal cell surfaces. It is likely that the entire genus has similar ultrastructure of
the protonephridia, as described by Jespersen and Lützen (1987) and briefly
reviewed here. The sole difference between Tubulanus species concerns the number
of collecting tubules (Bürger 1895a; Coe 1901, 1940; Friedrich 1936; Ritger and
Norenburg 2006).
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The nephridia of carinomids were studied in Carinoma armandi (Oudemans 1885;
Bürger 1895a), Carinoma mutabilis and Carinomella lactea (Coe 1901, 1943). The single pair of protonephridia lies in the foregut region; their anterior section is rather
long and bulges deeply into the blood vessel (Figure 4C, D for C. mutabilis). In contrast to Oudemans (1885), who described an open connection between blood fluid
and nephridial lumen, Bürger (1895a) described terminal units (“Wimperkolben”)
that are covered by the endothelium of the blood vessels. Small capillaries connect
the terminal units to the terminally branched main duct, which runs parallel to the
blood vessel. The number and details of morphology of the capillaries and of the
main duct branches are difficult to infer from Bürger’s (1895a) description, but can
be derived from Coe’s (1901, 1943) descriptions and illustrations (Figure 4D). Terminal units and capillaries have not yet been studied ultrastructurally in any Carinoma
species. The narrow lumen of the main duct gradually expands from anterior to posterior. Posteriorly, the main duct loses its intimate contact with the blood vessel, makes
a U-turn to run parallel to its posterior section and, finally, bends towards the epidermis, passing through the body wall muscles above the lateral nerve cord, and opening
to the exterior via a single dorso-lateral pore. The overall nephridial structure is quite
uniform in all carinomid species; interspecific differences occur in the structure and size of
the anterior region of the protonephridum, and the U-shaped part of the main duct
(Friedrich 1956), which is absent in Carinomella lactea.
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T. Bartolomaeus and J. von Döhren
frontal
TU
TU
TU
BV
BV
Ca
caudal
MD
BV
MD
C
BV
au
ED
MD
NP
MD
th
Ce
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or
ED
ED
A
NP
D
co
NP
B
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NP
BV
TU
ED
E
MD
Ca
TU
Figure 4. Nephridia of palaeonemertean species. Fronto-caudal axis applies to A, B and D. (A)
Carinina grata (Tubulanidae); (B) Tubulanus superbus (Tubulanidae); (C, D) Carinoma mutabilis
(Carinomidae): (C) cross-section showing contact area between blood vessel and terminal region
at the level indicated in D; (D) entire nephridium; (E) Procephalothrix major (Cephalothricidae).
Notes: BV, blood vessel; Ca, capillaries; Ce, caecae; ED, efferent duct; MD, main duct; NP,
nephridiopore; TU, terminal organ; figures modified from different sources: (A) Bürger (1895a:
Figure 11.1); (B) Bürger (1895a: Figure 28.2); (C, D) Coe (1943: Figure 15); (E) Coe (1930: Figure 9).
Journal of Natural History
2265
Cephalothricidae
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Most cephalothricid species possess serially arranged protonephridia. Up to 300 of
these organs have been described for Procephalothrix major (Coe 1930) and other
large Cephalothrix and Procephalothrix species (Wijnhoff 1912; Coe 1940, 1943;
Friedrich 1935b). Each protonephridium has a mushroom-shaped terminal unit with
a large terminal compartment (Figure 4E for Procephalothrix major; Figure 5 for
Procephalothrix filiformis). Most of these organs extend into the blood vessel (Figure 5C),
but like in other nemertean species, there is no direct connection between blood fluid
and nephridial duct. At the light microscopical level, a small cytoplasmic layer separates the large terminal compartment from the blood vessel. Opposite to this layer,
large ciliated cells send their cilia into the thin-walled capillary which leads to the
convoluted thick-walled main duct, which continues into a long thin-walled efferent
duct, opening to the exterior via a small, dorso-lateral nephridiopore (Figure 4E). Cephalotricid terminal units superficially resemble ciliated funnels (“nephrostomes”) of
annelid metanephridia. However, not all representatives of Procephalothrix and Cephalothrix possess this peculiar nephrostome-like protonephridial terminal unit (Gerner
1969; Gibson et al. 1990). In Procephalothrix spiralis only females have them, while
the protonephridia in males show several flame cells instead (Coe 1930). The meiofaunal species Cephalothrix atlantica, C. pacifica, and C. germanica, have a single pair
of protonephrida located anterior to the mouth and equipped with one to four terminal cells (Gerner 1969). No mushroom-shaped terminal structure, as present in the
protonephridia of larger Cephalothrix and Procephalothrix species, is seen in these
miniaturized species. In C. atlantica two to four additional rudimentary nephridia
without terminal cells were described by Gerner (1969).
Ultrastructural data of Procephalothrix filiformis provide strong evidence that
these peculiar metanephridium-like organs are actually protonephridia. The entire
mushroom-shaped terminal unit is formed by several terminal cells (Figure 5A). Each
terminal cell bears a cytoplasmic lobe that interdigitates with lobes of adjacent terminal cells (Figure 5B). The meandering cleft between the two neighbouring lobes is
bridged by extracellular matrix. Adhaerens junctions connect the lobes and guarantee structural integrity of the entire construction. Each terminal cell is multiciliated,
and each cilium has a 9 × 2 + 2 axoneme emanating from a basal body anchored by
ciliary rootlets. As in Tubulanus annulatus the supporting structure of the filtration
barrier is formed by several terminal cells, thus forming a compound filter.
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Hubrechtidae
Among hubrechtids, protonephridia are only known from Hubrechtia desiderata.
Bürger (1895a) described them as being most similar to those of heteronemerteans.
H. desiderata has a single pair of protonephridia located in the foregut region. In this
region the foregut vessel bears several lacuna-like extensions. Each protonephridium
lies lateral to these blood vessels, in such close proximity that it appears to be connected to the inner wall of the vessel. However, it is always separated from the blood
fluid by the vessel endothelium. The main duct ramifies into branches of similar
width which terminate in flame-bulb-like terminal units. Bürger’s (1895a) description
does not allow to discriminate between the main duct, its branches, or the capillaries,
or to infer whether the terminal units consist of single or multiple terminal cells. Half
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Figure 5. Procephalothrix filiformis, ultrastructure (A, B) and histology (C) of the terminal
unit (TU) of the protonephridium. (A) Sagittal section of the terminal region showing several terminal cells (TC) and their cilia; (B) filtration barrier is sustained by slashed cytoplasmic lobes (asteriscs) of the terminal cell and represents a compound filter (filtration slits are
Journal of Natural History
2267
way from anterior to posterior a small efferent duct branches off from the main duct,
pierces the body wall muscles dorsal to the lateral nerve cord, and opens to the exterior via a single dorso-lateral nephridiopore. The interpretation of a group of cells in
the foregut region of Hubrechtella atypica as protonephridium (Senz 1992) requires
confirmation, since no such structure has been found in other Hubrechtella species
(Chernyshev 2003; Kajihara 2006).
Gibson and Sundberg (1999) described nephridia in Parahubrechtia jillae, a species they classified as a hubrechtid nemertean. According to their description, the
nephridia resemble those of tubulanid species in terms of position, composition and
relation to the blood vascular system. This corroborates Chernyshev’s (2003) statement that Parahubrechtia jillae should not be classified as a hubrechtid nemertean.
Protonephridia in Heteronemertea
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Heteronemertea are a monophyletic group within the Nemertea, characterized by a
unique subepidermal layer called dermis (Hyman 1951) or cutis (Bürger 1895a; Coe
1943). The nephridia of the Heteronemertea are quite well understood at the light
microscopical level (Bürger 1895a, 1897–1907); however, ultrastructural data are
available only for Lineus viridis (Anadon 1976; Bartolomaeus 1985).
In general, heteronemerteans have a single pair of protonephridia located in the
foregut region. In Baseodiscus delineatus, as in in Hubrechtia desiderata, they are
associated with the foregut blood lacunae (Bürger 1895a). The main duct is ramified
and laterally opposed to the wall of the blood vessel; its lumen is much smaller than
in carinomid and tubulanid species. Branches of the main duct are opposed to the
blood lacunae and terminate with terminal units (“flame bulbs”), both being covered
by the vessel endothelium. It is not clear from Bürger’s (1895a) description whether
the capillaries are present, but small branching ducts (“Canälchen”) leading to terminal units might represent them and will be coded as such. Posteriorly, the main
duct continues into the efferent duct, which bends toward the lateral body wall,
pierces the body wall muscles, and opens via a single midlateral nephridiopore dorsal
to the lateral nerve cord (Figure 6B for B. delineatus). Oudemans (1885) described
several nephridiopores for each protonephridum of Baseodiscus curtus. Each pore is
connected to its own efferent duct branching off from the main duct. The nephridiopores are sequentially aligned along a midlateral line in the epidermis. Because of
this difference, Gibson’s (1979: p. 145–146) synonimization of B. delineatus and
B. curtus seems unjustified.
Several efferent ducts and nephridiopores have also been described for Valencinia
longirostris (Bürger 1895a) and Valencinura bahusiensis (Bergendahl 1902). In these
species the nephridiopores are arranged irregularly along the midlateral line; the
efferent ducts run dorsal to the lateral nerve cord (Figure 6A for V. longirostris).
Each species has a pair of protonephridia extending between middle and posterior
foregut region, but being more expanded than in Hubrechtia desiderata and Baseodiscus
species (compare Figure 6A and B). The general protonephridial organization, the
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marked by white arrows); (C) cross-section of the terminal organ, Azan stained histological
section. Notes: BV, blood vessel; CM, circular muscles; cr, ciliary rootlets; ECM, extracellular
matrix; IC, intestinal cells; LM, longitudinal muscles.
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T. Bartolomaeus and J. von Döhren
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structure of the terminal units, the organization of the duct, and the association with
the blood vascular system are similar in Valencinia longirostris, Valencinura bahusiensis,
Baseodiscus species and Hubrechtia desiderata, although the number of nephridiopores varies.
Species of Lineidae have a single pair of protonephridia mostly restricted to the
foregut region. Their general organization is similar to that in the heteronemertean
species mentioned above. The protonephridia in lineids are closely associated with
the blood vascular system, but exhibit some topological differences owing to the
course of the blood vessels in the foregut region. The blood vessels are located on
either side of the rhynchodeum anteriorly, and surround the rhynchocoel almost
completely posteriorly. This position is maintained in the anterior foregut region, but
toward the posterior, the blood vessels gradually descend to a ventro-lateral position,
extending ventrally almost side-by-side in the midgut region (Bürger 1895a; personal
observation). In several lineids the protonephrida are confined to the posterior
foregut region. Since the blood vessels in this region are located ventro-laterally, the
main duct and its branches are also ventro-lateral (Bürger 1895a). In species in which
the nephridia are shorter and located further anteriorly, e.g. Cerebratulus marginatus,
Lineus gilvus, L. parvulus, L. ruber, and L. viridis, their association with the blood
vessels results in a dorso-lateral position of both, the nephridia and the nephridiopores (Bürger 1895a; personal observation). In Ramphogordius lacteus a portion of
the main duct is anterior to the mouth and dorsal; the nephridiopores are also dorsal.
In those lineids in which the nephridia are located more posteriorly, the main duct
extends almost ventrally and parallel to the blood vessels. The nephridiopores are
located along the midlateral line of the animal; the efferent ducts, however, extend
dorsal to the lateral nerve cord.
Anadon (1976) described the ultrastructure of the duct in adults of Lineus viridis,
and Bartolomaeus (1985) has described details of the entire protonephridia in different developmental stages of this species. No association with the blood vessel is found
in the hatchlings. Terminal cells are multiciliated and possess a filter connected to the
adjacent duct cell by adhaerens junctions. Terminal cells are initially monociliated;
multiplication of centrioles during development gives rise to additional cilia (Bartolomaeus 1985). Since the protonephridia in hatchlings are quite short and possess only
a few terminal cells, it is likely that they grow during development. In hatchlings
the main duct ramifies proximally; the lumen is intercellular. All duct cells are primarily monociliated and acquire additional cilia later in development (Bartolomaeus
1985). The duct cell situated next to the terminal cell has a transcellular lumen.
It remains to be shown whether these cells represent precursors to capillaries. So far
no study of heteronemertean protonephridia clearly demonstrated the presence of
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Figure 6. Protonephridia of neonemertean species. Fronto-caudal axis applies to A, B, C and
E. (A) Valencinia longirostris (Heteronemertea); (B) Baseodiscus delineatus (Heteronemertea);
(C) Prostoma graecense (Hoplonemertea); (D) Pantinonemertes agricola (Willemoes-Suhm,
1874) (Hoplonemertea); (E) Malacobdella grossa (Bdellonemertea). Notes: BV, blood vessel;
CA, capillaries; cf, ciliary flame; ED, efferent duct; fb, flame bulb; MD, main duct; NP,
nephridiopore; TU, terminal unit; figures modified from different sources: (A) Bürger (1895a:
Figure 28.15); (B) Bürger (1895a: Figure 28.27); (C) Gibson (1972: Figure 15E); (D) Coe
(1930: Figure 16); (E) Bürger (1895a: Figure 28.39).
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T. Bartolomaeus and J. von Döhren
capillaries in adults. Despite this fact, capillaries are coded as present according to
the descriptions given in the older literature (see Tables 1 and 2).
Protonephridia in Hoplonemertea
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The general organization of hoplonemertean protonephridia is well known from
older literature (Bürger 1895a, b; Coe 1930) and the ultrastructure has been described
by several authors (Jespersen 1987a, b; Bartolomaeus 1988; Jespersen and Lützen
1988, 1989).
Bürger (1895a) described one pair of protonephridia in Emplectonema gracile. In
this species, several collecting ducts branch off from the main duct. They further ramify into capillaries, which interdigitate to form a net-like structure, and end in a
flame-bulb-like terminal unit. The exact number of terminal cells is unknown. Each
main duct gives rise to a single efferent duct at the level of the stomach. This duct
extends dorsal to the lateral nerve cord, penetrates the body wall muscles, and opens
via a single nephridiopore. This pattern is also found in other marine hoplonemerteans,
like Tetrastemma, Prostomatella, Drepanoporus and some Amphiporus species
(Bürger 1895a). In E. gracile and Nemertopsis bivittata (Bürger 1895a) protonephridia extend from the cerebral region to the midgut region, and are located
between the anteriormost gonads. The protonephridia in species of Amphiporus and
Tetrastemma, as well as in Prostomatella arenicola, Malacobdella grossa and Paradrepanophorus crassus, are confined to the stomach region (Bürger 1895a; Mock 1981; own
unpublished data), (Figure 6E). Except for Prostomatella arenicola, the protonephridia of
these species are shorter, but more voluminous than in Emplectonema and Nemertopsis
species. The protonephridia of several species possess more than one nephridiopore
(Figure 7 A, B; Table 2). Friedrich (1935b) reported that the protonephridia of
Cyanophthalma obscura are associated with the lateral blood vessels, but their terminal
cells never bulge into their lumen (Friedrich 1935b; Norenburg 1986).
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MD
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Ca
TU
LN
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caudal
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Figure 7. Protonephridia in Amphiporus species (Hoplonemertea). Fronto-caudal axis applies
to A only. (A) Amphiporus lactifloreus (redrawn from Bürger [1895a: Figure 28.9]); (B)
Amphiporus imparispinosus, Azan stained histological section of the protonephridium showing
an efferent duct (ED), capillaries (Ca), and the topological position of the organ. Notes: BV,
blood vessel; LN, lateral nerve; MD, main duct; P, proboscis, TU, terminal organ.
Journal of Natural History
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Neither the terminal region nor the ducts extend into the blood vascular system in
hoplonemerteans. According to Bürger (1895a) and Friedrich (1935b) this correlates
with the reduced number and narrow lumen of the blood vessels. Malacobdella species, however, which have large, branched and anteriorly located nephridia and a
well-defined blood vascular system, also lack an intimate connection between
nephridia and blood vascular system (Bürger 1895a). In M. grossa the main duct
ramifies and each ramus gives rise to branching capillaries that end in a terminal unit.
The efferent duct leads to a single ventral nephridiopore (Figure 6E).
Terrestrial and freshwater hoplonemerteans possess an extremely large excretory
system, which extends the entire body length and consists of more than one pair of
protonephridia (Coe 1929; Pantin 1969), (Figure 6C). Juveniles of Prostoma graecense
possess a single pair of protonephridia, which is reported to divide into several pairs
found in adults (Böhmig 1898). Ultrastructure of the nephridia in these non-marine
hoplonemerteans is well known (Jespersen 1987a, b; Jespersen and Lützen 1988,
1989). Among the marine hoplonemerteans, protonephridia have been studied only
in the intertidal Prostomatella arenicola (Bartolomaeus 1988).
The terrestrial and semi-terrestrial species of Geonemertes, Prosadenoporus and
Pantinonemertes have a large number of protonephridia (Coe 1929). Their terminal
units are large, binucleate, densely staining structures with a strong ciliary flame (Coe
1929, 1943; Moore and Gibson 1981, 1985; Gibson et al. 1982; Maslakova and Norenburg 2008; Sundberg et al. 2009) (Figure 6D). Electron microscopical studies by Jespersen and Lützen (1988, 1989) demonstrated that in Pantinonemertes californiensis
and Geonemertes pelaensis each terminal unit consists of two interdigitating terminal
cells, each with a single nucleus. They showed that the close proximity of the terminal
cells to each other causes the false impression that the two nuclei belong to the same
cell (Sundberg et al. 2009). Each terminal cell bears a strong distally oriented cytoplasmic column with a series of circular bars; the bars of both terminal cells interdigitate.
Since every bar possesses several short cytoplasmic protrusions extending into the
terminal compartment and alternating with those of the other terminal cell, an irregular system of clefts is formed between the terminal cells. The clefts are bridged by
diaphragms which represent the site of ultrafiltration. Thus, both Pantinomertes californiensis and Geonemertes pelaensis possess a compound filter. Both terminal cells
form a single strong ciliary flame. Identical orientation of central microtubule pair in
the ciliary axonemata indicates that all cilia beat in the same direction. Microvilli
arise from the wall of the compound filter. This organization can be assumed for all
species of both genera, since light microscopical studies clearly indicate that their terminal units are very similar (Moore and Gibson 1985). In the species of Pantinonemertes, Geonemertes and Prosadenophorus terminal units are distributed throughout
the body, each being connected to a small capillary that joins other capillaries and
continues into a convoluted ciliated main duct with an intercellular lumen and adluminal invaginations. The main duct continues into a thin-walled efferent duct which
opens to the exterior via a single intraepidermal nephridiopore (Figure 6D).
Compared to Pantinonemertes and Geonemertes species, the protonephridia of
the terrestrial Acteonemertes bathamae, the limnetic Prostoma graecense and the
marine Prostomatella arenicola differ in their supporting structure (Jespersen 1987a, b;
Bartolomaeus 1988). In Acteonemertes bathamae and Prostomatella arenicola a
distally oriented cytoplasmic hollow cylinder formed by a single terminal cell is perforated by numerous clefts bridged by diaphragms, thus, forming a simple filter. In
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T. Bartolomaeus and J. von Döhren
both species cilia and microvilli arise from the perikaryon; additional microvilli and
cilia may arise from the inner wall of the filter. In Prostoma graecense the perikaryon
of the terminal cell lies at the base of the supporting structure which forms a domelike extension. Several cilia emanate from the wall of the filter and extend deeply into
the adjacent duct. While the inner wall of the filter bears several irregular and partly
branched cytoplasmic protrusions, long and slender microvilli arise only from the
perikaryon and distal margin of the filter. They extend deeply into the duct surrounding the cilia of the terminal cell. The proximal duct cells adjacent to the terminal cells, i.e.
the capillaries, have a very few (Acteonemertes bathamae) or no microvilli (Prostomatella
arenicola, Prostoma graecense). The distal portion of the duct, i.e. the main duct and
the collecting duct, contains numerous microvilli and cilia. Only in Acteonemertes
bathamae the distal portion of the main duct lacks microvilli. This species is also
unique in lacking an efferent duct (Jespersen 1987a); the main duct apparently opens
directly to the exterior via an intra-epidermal nephridiopore.
Patterns in the construction of nemertean protonephridia
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The comparison of nephridial structures across nemerteans revealed that in all
marine species nephridia are restricted to the anterior end. Here, they show a
strong topological relation to the blood vessels, probably for functional reasons
(see later). There is no evidence for an open connection between blood and nephridial lumen; terminal units are always at the inner (distal) end of the duct and are covered by the blood vessel endothelium. These terminal units represent the site of
ultrafiltration and consist of one or more terminal cells. While electron microscopy
is needed to determine the structure of the ultrafilter, the relative position of the
terminal units can be inferred from histological sections. The different terms
applied to parts of the excretory apparatus in classical studies are summarized in
Table 1.
Classical studies have applied a vast number of different terms for components
of the nephridial duct (Bürger 1895a; Coe 1943; Table 1). Three different sections
of the duct can always be distinguished (Table 1): the efferent duct, the main duct
and the capillaries. The efferent duct leads to the nephridiopore and is characterized primarily by its position. As the efferent duct, the capillaries are very small in
diameter, less ciliated and thin-walled than the main duct. The main duct and its
branches are intensely ciliated, have an intercellular lumen and the main duct cells
are higher than those of the capillaries and the efferent duct. Ultrastructural studies, especially those by Jespersen (1987a, b) and Jespersen and Lützen (1987, 1988,
1989), also analyzed protonephridia in species previously studied histologically
(Bürger 1895a; Böhmig 1898; Coe 1929; Pantin 1969). They confirmed that the differences revealed at the level of light microscopy are also observed at the ultrastructural level. All regions of the protonephridial duct, i.e. the efferent duct, the main
duct, and the capillaries, are unambiguously described in all nemertean species,
except in heteronemerteans and Hubrechtia desiderata, for which additional studies
are needed.
Presently, the following phylogenetic characters and character states can be
inferred from the available data (Table 2). Some of them are presently parsimony
uninformative, but have been included for the sake of completeness.
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2.
3.
Number and arrangement of protonephridia: (0) a single pair; (1) multiple pairs in
irregular arrangement; (2) multiple pairs in serial arrangement.
Most marine species have a single pair of protonephridia. Multiple irregularly
arranged nephridia are found in freshwater, semi-terrestrial and terrestrial species
(Figure 6C). Multiple pairs are serially arranged in most studied cephalothricid species.
Extension of protonephridia: (0) restricted to the foregut region; (1) from the
foregut region to the anterior gonad region; (2) preoral to the anterior gonad
region; (3) throughout the entire body, (4) restricted to the preoral region.
In general protonephridia are restricted to the foregut region, or as in certain
lineids, extend to the anterior gonad region. In some species they are found
immediately behind the foregut region, while in freshwater, semi-terrestrial and
terrestrial species they are found throughout the entire body. In the marine species Cyanophthalmus obscura a single pair of protonephridia exends throughout
the entire body (Norenburg 1986). In some interstitial cephalothricid species, the
nephridia are located in front of the mouth opening (Gerner 1969).
Polarity of protonephridia in adults: (0) polarized along the antero-postrior axis;
(1) polarity absent.
The nephridia of the palaeonemertean species (except Hubrechtidae) are polarized, so that the terminal region is at the anterior end, and the efferent duct and
pore are at the posterior end. Hubrechtia desiderata, heteronemerteans and
hoplonemerteans do not appear to exhibit such polarity, instead, the efferent duct
and the pore are situated half way between the the anterior and posterior ends of
the branched main duct. The terminal cells, positioned at the inner (distal) end of
each branch, are distributed along the entire nephridial region. The protonephridia
of L. viridis juveniles exhibit the same polarity as those of tubulanid and carinomid
species (Bartolomaeus 1985: Figure 1), but the polarity is lost in later developmental stages, most likely because new terminal cells and duct branches are added.
Contact of protonephridia with blood vessel: (0) all terminal cells bulge into the
blood vessel wall (Figure 4C); (1) some terminal cells bulge into the blood vessel
wall, others are found within the matrix among muscle or parenchymal cells
(Coe 1943: p. 172); (2) the terminal cells never bulge into the blood vessels
(Norenburg 1986; Bartolomaeus 1988: Figure 2A).
Position of the terminal unit: (0) confined to the proximal section of the protonephridium; (1) spread over the entire length of the protonephridium.
In tubulanid, carinomid and cephalothricid species as well as in some terrestrial nemerteans, terminal units are restricted to the terminal section of the protonephridium, while in the remaining species they are distributed along the
entire length of the protonephridium.
Clustering of terminal units: (0) a single cluster of few terminal units; (1) several
clusters of terminal units forming a nephridial gland (Figure 4A, B; Figure 6D);
(2) absent (a single terminal unit per protonephridium [Figure 4E]).
This character is only applicable to species with terminal units confined to
the proximal section of the protonephridium (character 5, state 0).
Aggregation of terminal units: (0) all terminal units are isolated from each other
(Figure 6B, E); (1) in addition to isolated terminal units, some terminal units
form clusters (Figure 6D).
This character is only applicable to species possessing terminal units spread
along the entire length of the protonephridium (character 5, state 1).
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Composition of terminal unit: (0) a single terminal cell (Figure 1A); (1) a pair of terminal cells (Jespersen and Lützen 1988, 1989); (2) several terminal cells (Jespersen
and Lützen 1987); (3) terminal cell and adjacent duct cell (Jespersen 1987a).
Site of ultrafiltration is: (0) a filter formed by a single terminal cell (Figure 3D);
(1) a compound filter formed by two or more terminal cells (Figure 3C).
This character is not identical to character 8, since a pair of terminal cells
does not necessarily from a compound filter, nor do terminal and duct cell necessarily form a weir (e.g., Acteonemertes bathamae [Jespersen 1987a]). A weir is a
supporting structure formed by the terminal cell and the adjacent duct cell (Bartolomaeus and Ax 1991). Here, cytoplasmic rods of both cells interdigitate to
span the filtration matrix. No such structure has been described in nemerteans
thus far, but it is known from some plathelminth and annelid species (Ehlers
1985; Wessing and Polenz 1974; Bartolomaeus and Quast 2005).
Transverse bars of terminal cells: (0) absent (or indistinguishable at the light
microscopical level); (1) appears on histological sections as densely staining horizontal lines (Figure 6D; Coe 1930).
Reinforcement of transverse bars of terminal cells: (0) by fibrils (Jespersen and
Lützen 1989: Figures 2, 3); (1) by microtubules (Jespersen and Lützen 1988:
Figures 2, 3).
Although information on the substructure of the bars could be summarized
by character 10, it is coded separately because state 1 of character 10 is visible in
histological sections, while electron microscopy is needed to determine the composition of the bars.
Longitudinal reinforcement of terminal cells: (0) absent, (1) by fibrils; (2) by
microtubules.
Ciliation of terminal cells: (0) multiple cilia per terminal cell (Figure 5A, B); (1)
single cilium per terminal cell (Bartolomaeus 1985: Figure 8).
Capillaries: (0) bulging into the blood vessel lumen (Figure 3B); (1) bulging
partly into blood vessel lumen, and partly into parenchyma; (2) never bulge into
the blood vessel lumen (Figure 7B).
Capillary lumen: (0) exclusively transcellular (Figure 1E); (1) exclusively intercellular; (2) both transcellular and intercellular.
If intercellular, only two cells per cross-section have been described thus far
(Jespersen 1987a, b; Jespersen and Lützen 1987, 1988, 1989).
Capillary branching: (0) absent (unbranched); (1) present.
Capillary course: (0) straight; (1) curved; (2) convoluted.
This character can be applied to species showing branched as well as
unbranched capillaries.
Topological relation of capillaries and main duct: (0) main duct or main duct
branches give rise to a single capillary which may ramify terminally (Figure 4E,
6D); (1) several capillaries branch irregularly from the main duct and/or main
duct branches (Bürger 1895a: Figure 27.1).
Topological relation of main duct and blood vessel: (0) anterior section of main
duct bulges into lateral blood vessel; (1) main duct is adjacent to lateral blood
vessel; (2) main duct is not immediately adjacent, but parallel to lateral blood
vessel; (3) main duct is completely independent of the lateral blood vessel.
Main duct branching: (0) absent (unbranched); (1) main duct ramifies terminally; (2) main duct ramifies regularly forming a series of ladder-like branches;
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20.
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(3) main duct ramifies irregularly forming several irregularly arranged
branches; (4) main duct with bulbs and star-like radiating capillaries.
Series of ladder-like branches of the main duct have only been described in Tubulanus species (Figure 4B; Bürger 1895a; Jespersen and Lützen 1987). Capillaries that
radiate in star-like manner from bulbs of main duct branches are described for the
hoplonemertan Nemertopsis bivittata (Bürger 1895a: Figure 9.16).
Main duct course: (0) straight; (1) U-shaped; (2) convoluted.
This character is only applicable to species showing either unbranched main
ducts (character 20, state 0), or main ducts that ramify terminally (character 20,
state 1).
Lumen of main duct: (0) uniform throughout its length; (1) distal section of main
duct widens.
Main duct extensions: (0) absent (no extensions); (1) main duct forms caeca.
Caeca are described to branch from the distal portion of the main duct in
Carinina grata (Bürger 1895a).
Efferent duct: (0) absent; (1) present.
In nearly all studied nemertean species a narrow efferent duct branches from
the main duct almost rectangularly to open to the exterior. A distinct duct of this
kind is definitively lacking in Acteonemertes bathamae (Jespersen 1987a).
Number of efferent ducts per protonephridium: (0) single efferent duct; (1) several
efferent ducts (Figures 6A, 7A).
Efferent duct branching: (0) absent (unbranched); (1) present. The efferent duct
of nemertean protonephridia is usually unbranched, but in some species the
efferent duct ramifies and opens to the exterior via several nephridiopores (e.g.,
Prostomatella arenicola, personal observation).
Fusion of efferent ducts: (0) absent; (1) present.
This character is only applicable to species showing more than one efferent
duct per protonephridium. Here, two neighbouring ducts may fuse to a single
one leading to a nephridiopore, e.g., the hoplonemertean Amphiporus lactifloreus (Figure 7A)
Relative position of efferent duct to main duct: (0) efferent duct is a distal extension of the main duct; (1) efferent duct branches off from the main duct.
This character is only applicable to species showing a single efferent duct per
protonephridium (character 25, state 0).
Opening of protonephridium: (0) intra-epidermal (via nephridiopore); (1) intraepidermal and intra-intestinal; (2) exclusively intra-intestinal.
Nemertean protonephridia usually open to the exterior via epidermal
nephridiopores (Figure 3D). No such pores are described for the heteronemertean species Apatronemertes albimaculosa (Coe 1906; Wilfert 1974). In this
species, the excretory system apparently opens into the gut. In the heteronemertean Baseodiscus cingulatus Coe (1906) described such a connection to the
gut in addition to the intra-epidermal pores.
Position of nephridiopore(s): (0) dorsal to lateral midline; (1) ventral to lateral
midline.
The midline is defined by the position of the lateral nerve cords in the
nephridial region. Nemerteans distinctly differ in whether the nephridiopores lie
dorsal or ventral to this midline.
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Comparative and evolutionary evaluation
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The evolution of nemertean protonephridia can be inferred from the molecular phylogenies only because the interrelationships of the major nemertean clades have not
been analyzed cladistically with morphological characters, Morphological character
matrices exist only for several subgroups: Palaeonemertea (Sundberg and Hylbom
1994; Sundberg et al. 2003), Eureptantia (Härlin and Sundberg 1995; Härlin and
Härlin 2001), Enopla (Sundberg 1990), Pelagica (Maslakova and Norenburg 2001),
Heteronemertea (Schwartz and Norenburg 2001), Monostilifera (partim) (Sundberg
1989a, b; Crandall 2001). Most molecular studies of nemertean phylogeny focused on
18S and/or cytochrome oxidase I (COI) DNA sequences (Sundberg et al. 2001; Sundberg
and Strand 2007; Sundberg et al. 2009), while a single study applied a multi-gene
approach (28S rRNA, histone H3, 16S rRNA, and COI; Thollesson and Norenburg
2003). The results of these analyses consistently favour the non-monophyly of the Palaeonemertea, but they differ in placement of individual palaeonemertean subgroups. For
example, Cephalothricidae is resolved either as the sister group of the Tubulanidae
(Thollesson and Norenburg 2003) (Figure 2A), or as the sister group of the Hoplonemertea (Sundberg et al. 2001; Sundberg and Strand 2007; Sundberg et al. 2009)
(Figure 2B).
The most comprehensive taxon sampling has been applied by Thollesson and
Norenburg (2003). The main results of their study are (Figure 2A): (1) Heteronemertea and Hubrechtidae are sister taxa – because these taxa share a pilidium larva,
the clade has been named the Pilidiophora; (2) Hoplonemertea is the sister group of
the Pilidiophora – together they form the clade Neonemertea; (3) Carinomidae is the
sister group of the Neonemertea; (4) Carinomidae + Neonemertea form the sister
group to the Cephalothricidae + Tubulanidae.
Here we use these molecular phylogenies to (1) reconstruct the ancestral design of
nephridia in nemerteans; (2) summarize major evolutionary changes of the nemertean
nephridia; and (3) evaluate the status of serial protonephridia in Cephalothricidae.
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Ancestral design of nemertean protonephridia
Molecular phylogenies presently favour the following reconstruction of ancestral
character states.
(1) In the Nemertea, the number of protonephridia is restricted to a single pair
located in the foregut region. This state is maintained in most marine species,
but transformed in Cephalothricidae (discussed later) as well as in freshwater,
semi-terrestrial and terrestrial nemerteans.
(2) The terminal units of the protonephridia are composed of several terminal
cells, which bulge into the wall of the blood vessel. This condition is maintained in the palaeonemertean and heteronemertean taxa, while in
hoplonemerteans merely the anteriormost terminal units are apposed to
the vessel wall. In general, a layer of endothelial cells separates the terminal cells from the blood fluid; the few reports of lack of endothelial cells
in this region require confirmation (Bürger 1895a; Jespersen and Lützen
1987). An open connection between protonephridial and vascular systems
(fide Oudemans 1885; Nawitzky 1931; Friedrich 1935a) has not been
Journal of Natural History
2277
confirmed by any ultrastructural study. Available data favour a retro-vascular position (abluminal side of the blood vessels) of terminal cells as
ancestral in nemerteans. Presently, the ancestral composition of the ultrafilter cannot be evaluated, since the number of studies of its ultrastructure
in palaeonemerteans is limited.
(3) The ancestral protonephridia are in line with the antero-posterior axis of the
animal. The nephridial duct system consists of a main duct which terminally
branches into collecting tubules and proximally leads into a single efferent
duct. The efferent duct is rectangular to the main duct; it passes through the
body wall muscles dorsal to the nerve cord, and opens to the exterior via a
single, dorso-lateral nephridiopore. These states are found in all carinomid
and tubulanid species, as well as in most neonemerteans, and are accordingly
favoured as ancestral for the phylum.
Functional and evolutionary changes in nephridia
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In most marine species the nephridia are extremely short relative to the body
length. Provided that these organs are used to eliminate nitric wastes from the
body and to osmoregulate (Lechenault 1965; Ferraris 1984, 1985a, b; Ferraris
and Schmidt-Nielsen 1982; Ferraris and Norenburg 1988), nemerteans have to
solve a transport problem caused by the position of the nephridia in the foregut
region. This might explain both the presence of a blood vascular system, and the
close contact between nephridia and blood vessels. The blood vascular system
connects all parts of the body to the nephridia, allowing rapid fluid transfer, and
a close contact guarantees effective filtration of the blood fluid. Protonephridia
are formed in the foregut region during development. Thus, the topology of the
protonephridia in marine species can be explained not only by functional, but
also by developmental reasons.
There is some evidence that species that have several protonephridia as adults
had a single pair as juveniles (Böhmig 1898 [for Prostoma graecense]). Freshwater
and humid terrestrial habitats cause an increased need for osmoregulation. In
nemerteans living in such habitats, fluid transport by the blood vessels alone does not
seem to be sufficient and their nephridia extend throughout the entire body (Moore
1985; Moore and Gibson 1985; Pantin 1947). They are multiplied and have up to several thousands of pores, but they are associated with the blood vessels only in the
very anterior end of the animal. A strict topographical correlation between lateral
blood vessels and nephridia, as observed in palaeonemertean species, is not shared by
non-palaeonemertean species. The retro-vascular position of the terminal units is
maintained only in the anterior-most section of the nephridia (Coe 1943: Figure 21
[for Geonemertes pelaensis]). Everywhere else in the animal, the terminal units of
additional nephridia are embedded in the connective tissue beneath the body wall
muscles. Filtration occurs nearly everywhere in the body, which doubtless increases
the osmoregulatory capacity.
Since nemerteans are primarily marine animals, the enlargement and multiplication of the nephridia shown by freshwater and terrestrial species can readily be
explained as adaptive transformations (Moore and Gibson 1985). A functional
explanation, however, cannot be given for the multiplied protonephridia in the
marine cephalothricids. Presently, there is also no obvious selective advantage for
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2278
Taxa
Characters
--00?00111
--?0?00?11
-?00?1?110
--?0?00210
--?0?00200
--?0?00200
--00?11112
--00?1?112
--00?1?112
--00?1?112
???0?1010?
-0?0?0?11?
-0??????11
-000?00000
-0?1??0010
-002?01030
-002?01030
-002001030
-002?01030
?00????11?
-001?1?111
-001?1?111
-001??0010
a
000001-??0
000001-??0
000001-??0
000000-0?0
000000-0?0
000000-0?0
00101-1210
00001-1??0
00001-1??0
00001-1??0
000001-???
000001-??0
000001-??0
040000-0?0
040000-1?0
231002-2?0
231002-2?0
231002-210
231002-2?0
00101-0???
00101-0??0
00101-0??0
000001-000
2222222223
1234567890
h
o
'r s
c
o
000100-000
000100-000
001100-000
000100-000
000100-000
000100-000
-00100-100
-00100-000
-00100-000
-00100-000
110100-000
110100-000
010100-0??
000100-000
000100-000
200100-000
200100-000
200100-000
200100-000
?00100-100
000100-100
0001100-00
000100-000
u
t
1111111112
1234567890
p
y
Callinera monensis Rogers, Gibson and Thorpe, 1992
Callinera buergeri Bergendahl, 1900
Carinina grata Hubrecht, 1887
Carinina poseidoni Friedrich, 1935
Carinina buddenbrocki Friedrich, 1935
Carinina remanei (Nawitzky, 1931)
Tubulanus annulatus (Montagu, 1804)
Tubulanus polymorphus Renier, 1804
Tubulanus superbus (Kölliker, 1845)
Tubulanus linearis (Mclntosh, 1873-1874)
Carinoma armandi McIntosh, 1875
Carinoma mutablilis Griffin, 1898
Carinomella lactea Coe, 1905
Cephalothrix germanica Gerner, 1969
Cephalothrix pacifica Gerner, 1969
Procephalothrix major (Coe, 1930)
Procephalothrix spiralis (Coe, 1930), female
Procephalothrix filiformis (Johnston, 1828)
Procephalothrix kiliensis Friedrich, 1935
Hubrechtia desiderata (Kennel, 1891)
Baseodiscus delineatus (Delle Chiaje, 1825)
Baseodiscus curtus (Hubrecht, 1879)
Riserius pugetensis Norenburg, 1993
0000000001
1234567890
Reference
Present study
Bergendahl (1900)
Bürger (1895a)
Friedrich (1935a)
Friedrich (1935b)
Nawitzki (1931); Friedrich (1935b)
Jespersen and Lützen (1987)
Bürger (1895a)
Bürger (1895a)
Bürger (1895a)
Bürger (1995a)
Coe (1901, 1943)
Coe (1943)
Gerner (1969)
Gerner (1969)
Coe (1930, 1943)
Coe (1930)
Present study
Friedrich (1935b)
Bürger (1895a)
Bürger (1895a)
Oudemans (1885); Bürger (1895a)
Norenburg (1993)
T. Bartolomaeus and J. von Döhren
Table 2. Character matrix for nephridial structures derived from various literature sources.
a
0001100-00
0001100-00
000?00-101
-00100-100
-00100-100
-00100-100
-001101-01
000??1-?00
200100-000
200100-10?
2000----0?
-00??0-?0?
000100-100
-00100-001
200100-11?
u
t
?0?1?1?11?
-001011111
-001?11110
-002?12123
-002?12123
-002?12224
-002??2123
-002012?20
??02?11030
0102011030
-202102?30
-002102133
?002?01120
???2?11123
1202011030
h
o
00101-????
00101-0000
00101-0000
00101-???0
01111-0??0
01111-0??0
00101-???0
00120-0000
13?100-1?1
131100-111
131100-300
13111-0000
03121-0??1
00111-0???
131100-111
'r s
Valencinia longirostris Quatrefages, 1846
Lineus viridis (Müller, 1774)
Ramphogordius lacteus Rathke, 1843
Paradrepanophorus crassus (Quatrefages, 1846)
Emplectonema gracile (Johnston, 1837)
Nemertopsis bivittata (Delle Chiaje, 1841)
Amphiporus lactifloreus (Johnston, 1828)
Prostomatella arenicola Friedrich 1935
Pantinonemertes agricola (Willemoes-Suhm, 1874)
Geonemertes pelaensis Semper, 1863
Acteonemertes bathamae Pantin, 1961
Prostoma graecense (Bohmig, 1892)
Cyanophthalma obscura (Schultze, 1851)
Malacobdella grossa (Müller, 1776)
Pantinonemertes californiensis Gibson, Moore and
Crandall, 1982
Bürger (1895a)
Anadon (1976); personal observation
Bürger (1895a); personal observation
Bürger (1895a, 1987–1097)
Bürger (1895a, 1987–1097)
Bürger (1895a, 1987–1097)
Bürger (1895a); personal observation
Mock (1981); Bartolomaeus (1988)
Coe (1929, 1930)
Jespersen and Lützen (1989)
Jespersen (1987a)
Jespersen (1987b)
Norenburg (1986)
Bürger (1895a)
Jespersen and Lützen (1988)
Journal of Natural History
p
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c
o
Note: Only those species are coded for which a sufficient number of details were described. Among the species mentioned in this review, the following
species have not yet been included: Amphiporus imparispinosus Griffin, 1898; Apatronemertes albimaculosa Wilfert and Gibson, 1974; Baseodiscus cingulus
(Coe, 1906); Callinera bergendahli Gibson and Sundberg, 1999; Callinera nishikawai Kajihara, 2006; Callinera zhirmunskyi Chernyshev, 2002; Carinina
arenaria Hylbom, 1957; Carinina coei Hylbom,1957; Carinia johnstoni Senz, 2000; Carinina plecta Kajihara, 2006; Carinina sinensis Gibson and Sundberg,
1999; Cephalothrix atlantica Gerner, 1969; Cerebratulus marginatus Renier, 1804; Hubrechtella atypica Senz, 1992; Lineus gilvus Bürger, 1892; Lineus
parvulus Bürger, 1892; Lineus ruber Müller, 1774; Parahubrechtia jillae Gibson and Sundberg 1999; Prosorhochmus americanus Gibson, Moore,
Ruppert and Turbeville, 1986; Tubulanus lutescens Cantell, 2001; Tubulanus pellucida (Coe, 1895); and Valencinura bahusiensis Bergendahl, 1902. The
validity of species names is according to Gibson (1995).
2279
2280
T. Bartolomaeus and J. von Döhren
an increased number of nephridiopores per protonephridium, which occurs in
many lineages.
Serial protonephridia in Cephalothricidae
In the context of nemertean origins, two aspects of cephalothricid nephridia are
particularly relevant. (1) Are these organs modified metanephridia as suggested by
Coe (1930, 1943) and Friedrich (1935b)?; (2) Is a serial arrangement of nephridia
ancestral for Cephalothricidae?
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(1) There is no evidence that other types of filtration nephridia than protonephridia were ever present in nemerteans. Ultrastructural data demonstrate that the peculiar mushroom-shaped nephridia in cephalothricids
are in fact protonephridia with a compound filter that bulges into the
blood vessel, but is always separated from the blood fluid by the vascular
endothelium. The compound filter forms the advascular limit of the large
terminal compartment of the protonephridium. Since the perikarya of the
terminal cells are connected to duct cells, they seem to form a funnel-like
structure at the distal end of the duct (Figure 4D, 5c). These funnel-like,
large terminal compartments made Friedrich (1935b) believe that this
structure is the remnant of a coelomic cavity, while Coe (1930, 1943)
interpreted the perikarya of the terminal cells as a metanephridial funnel.
Both authors agreed in regarding the nemerteans as annelid relatives, due
to similarity in nephridial structure. Friedrich’s (1935b) interpretation
was also inspired by Oudemans’ (1885) observation of an open connection
between the blood fluid and the nephridial lumen in Tubulanus linearis,
which Friedrich (1935a, b) also observed in Carinina species. According to
his view, certain Carinina species represented the ancestral nemertean
organization, and he regarded the cephalothricid nephridium as a step
toward a reduction of the coelom. These interpretations are not corroborated by the available ultrastructural data.
(2) Larger cephalothricid species possess up to 300 serially arranged pairs of
nephridia, while the smaller interstitial species possess a single pair which
lies anterior to the mouth. Two interpretations might be provided for this
observation: (a) the meiofaunal species exhibit the ancestral condition,
and serial nephridia evolved secondarily within cephalothricids; or (b)
progenetic evolution of the interstitial species could explain the paired
protonephridia in adults as retained larval head kidneys, because larvae
of Procephalothrix filiformis and Procephalothrix ostrymnicus possess a
single pair of head kidneys (own unpublished data), while the adults possess multiple pairs. This latter interpretation implies that in large cephalothricid species during ontogenesis protonephridia are serially added to
the head kidneys, as is indicated by the state in Cephalothrix atlantica,
which shows two more posteriorly located, incompletely developed
nephridia in addition to the preoral protonephridia (Gerner 1969). As
long as the placement of the Cephalothricidae on the nemertean phylogeny remains unresolved, the second interpretation seems more likely,
because progenetic evolution is a common trait of interstitial animals
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Journal of Natural History
2281
(Westheide 1987). This means that series of multiple protonephridia are
likely to be ancestral for the Cephalothricidae.
Conclusions
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The available data on nemertean nephridia and the current view of the phylogeny
suggest that the palaeonemertean taxa retained much of the ancestral design of the
protonephridia, with the exception of the Cephalothricidae. In the latter taxon, protonephridia resemble modified metanephridia only superficially, and their serial
arrangement is unambiguously a derived state. Previous conclusions of a close relationship between Nemertea and Annelida, mainly drawn from characters of cephalothricid nephridia, are accordingly no longer compelling. The strong topographical
and functional correlation between the nephridia and the blood vessel has been
emphasized by Ruppert and Carle (1983), when they proposed the term “circulatory
system” for the blood vascular system in nemerteans. They assumed that not only
functional, but also historical constraints underlie this correlation, since the lateral
position of the two main blood vessels, and the endothelial lining of the blood vessels
are reminiscent of coelomic cavities in annelids. Similarities in the mode of blood vessel formation in the nemertean Prosorhochmus americanus and the coelomogenesis in
the annelid Magelona sp. (Turbeville 1986) seem to support this view. The occurrence
of a group of cleavage-arrested cells during early cleavage of Carinoma tremaphoros
that can be homologized with the trochoblast in trochophore larvae (Maslakova
et al. 2004) supports nemertean affinities with other trochozoans, but not necessarily
with annelids. Several recent molecular phylogenies based on expressed sequence tags
(EST) data suggested a clade of Nemertea, Brachiopoda, and Phoronida (Dunn et al.
2008; Bourlat et al. 2008; Hejnol et al. 2009), which prompts us to extend the comparative survey of nephridial structures to other lophotrochozoan taxa. Presently, this
clade does not seem to receive any support from characters of the excretory system.
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Acknowledgements
Our thanks a due to Markus Koch, who gave important and valuable remarks on earlier versions of the manuscripts and discussed details on the character coding. We also would like to
thank three anonymous reviewers for their efforts and am grateful to Svetlana Maslakova who
perfectly edited this manuscript. This study was supported by the German Research council
(BA 1520/11-1).
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