INTEG.
AND
COMP. BIOL., 42:641–651 (2002)
An Introduction to Loricifera, Cycliophora, and Micrognathozoa1
REINHARDT MøBJERG KRISTENSEN2
Zoological Museum, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark
SYNOPSIS. Loriciferans, cycliophorans and micrognathozoans are amongst the latest groups of animals to
be discovered. Other than all being microscopic, they have very different body plans and are not closely
related. Loriciferans were originally assigned to the Aschelminthes. However, both new molecular and ultrastructural researches have shown that Aschelminthes consist of two unrelated groups, Cycloneuralia and
Gnathifera. Cycloneuralia may be included in the Ecdysozoa, including all molting invertebrates, and Gnathifera are more closely related to Platyhelminthes. The phylum Loricifera shares many apomorphic characters (e.g., scalids on the introvert) with both Priapulida and Kinorhyncha, and can be included in the
taxon Scalidophora, a subgroup of Cycloneuralia. Cycliophora was originally allied to the Entoprocta and
Ectoprocta (Bryozoa) based on ultrastructual research. Subsequent molecular data show they may be related
to Rotifera and Acanthocephala, within the taxon Gnathifera. The phylogenetic position of Cycliophora is
therefore not settled, and more ultrastructural and molecular data are needed. Micrognathozoa is the most
recent major group of animals to be described. They show strong affinities with both Rotifera and Gnathostomulida (within the taxon Gnathifera), especially in the fine structure of the pharyngeal apparatus,
where the jaw elements have cuticular rods with osmiophilic cores. Furthermore the micrognathozoans have
two rows of multiciliated cells that form a locomotory organ, similar to that seen in some gastrotrichs and
interstitial annelids. This character is never seen in Rotifera or in the monociliated Gnathostomulida. Rotifera and Acanthocephala always have a syncytial epidermis (Syndermata). Micrognathozoa lack this characteristic feature. Therefore, they are postulated to be placed basally in the Gnathifera, either as a sistergroup to Gnathostomulida or as a sister-group to Rotifera 1 Acanthocephala.
INTRODUCTION
Since the discovery of the Loricifera (Kristensen,
1983) 11 species have been described (Higgins and
Kristensen, 1986; Kristensen and Shirayama, 1988;
Todaro and Kristensen, 1998), and more than 80 recorded species await description (Kristensen, 1991a,
b; Gad, 2000). The loriciferans seem to be one of the
most abundant groups of meiofauna in the deep-sea,
but also occur in mud on shallower water (Soetaert et
al., 1984), and they may be one of the dominant meiofauna groups. Recently, Warwick (2000) has postulated that loriciferans are simply paedomorphic priapulids. This was based on the fact that the larvae of
some loriciferans (Higgins and Kristensen, 1986) look
superficially similar to the larvae of some priapulids
(van der Land, 1970; Higgins et al., 1993). No comments were made concerning the unique adult features
of loriciferans, including the cuticular buccal tube,
myoepithelial pharyngeal apparatus with placoids
(Kristensen, 1991a, b) and the very complex life cycle
of the different species of loriciferans (Kristensen,
1999). Below I will discuss the different types of life
cycles in loriciferans, totally different from all other
known meiobenthic animals.
Cycliophora is the most recently described phylum,
consisting of marine acoelomate epibionts on setae of
mouthlimbs of lobsters (Funch and Kristensen, 1995).
Today three species are known, all in the genus Sym-
bion. The only described species, Symbion pandora,
has a complicated life history with several attached
and several free-living stages. The life cycle varies between asexual and sexual reproduction (Funch and
Kristensen, 1997). The asexual and the sexual parts of
the life cycle each have a unique larva. The life cycle
seems to be more complex than we originally thought
(Funch and Kristensen, 1995), and accordingly the life
cycle has been redrawn in this paper. Their phylogenetic position is still uncertain. Cycliophorans have
been considered to be a sister-group to Entoprocta and
Ectoprocta (Funch and Kristensen, 1995; Funch,
1996), a view mainly based on ultrastructural investigation, or alternatively as a sister-group to the clade
Rotifera-Acanthocephala (Winnepenninckx et al.,
1998), a conclusion arrived at on the basis of molecular evidence.
Micrognathozoa was described by Kristensen and
Funch (2000) as a new class within the Gnathifera (see
Ahlrichs, 1995, 1997). This clade now contains Gnathostomulida, Micrognathozoa and Syndermata. Syndermata consists of all Rotifera (Eurotatoria and Seisonida) and the parasitic Acanthocephala. The sistergroup relationship between Gnathostomulida and Rotifera 1 Acanthocephala was first mentioned by Rieger
and Tyler (1995). The discovery of Micrognathozoa,
in a cold spring from Greenland, strongly supports the
above mentioned relationship (Ahlrichs, 1997; Herlyn
and Ehlers, 1997). Only one species, Limnognathia
maerski, has been described.
1 From the Symposium Lesser-Known Protostome Taxa: Evolution, Development, and Ecology presented at the Annual Meeting of
the Society for Integrative and Comparative Biology, 3–7 January
2001, at Chicago, Illinois.
2 E-mail: [email protected]
CURRENT STATE OF KNOWLEDGE
Loricifera
Adult loriciferans are bilaterally symmetrical marine
metazoans 108–485 mm long (Higgins and Kristensen,
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1986). The body is divided into five regions: mouth
cone, head (introvert), neck, thorax and abdomen. The
introvert of the adult has nine rings of scalids, which
may have both sensory and locomotory functions. Two
families, Nanaloricidae and Pliciloricidae, have been
described, and two new families are under description.
Nanaloricidae with one genus (Nanaloricus), is acoelomate both as larva and adult. The abdomen of the
adult may be covered with a thick cuticular lorica consisting of 6 plates. Pliciloricidae with the two genera
(Pliciloricus and Rugiloricus) may be acoelomate as
larva, and pseudocoelomate as adult (Kristensen,
1995). The abdomen is usually covered with a thin
lorica with many folds (plicae, in Higgins and Kristensen, 1986). The lorica of pliciloricids seems always
to lack thick cuticular plates. All investigated loriciferans have chitin in the procuticle (Neuhaus et al.,
1997).
The first loriciferans had already been collected in
the seventies, but the first species, Nanaloricus mysticus, was described by me in 1983, from shell gravel
off the coast of Brittany, France (Kristensen, 1983).
The reason why it took so long from discovery to description was that these microscopic animals have a
very complex life cycle, with a larva (Higgins-larva)
which looks totally different from the adults (Kristensen, 1991a). Both the Higgins larvae and the adults
may adhere strongly to sand grains or gravel by adhesive glands, so a new technique, freshwater-shocking on large samples of sand to release the animals
from the bottom sediments had to be developed. Eleven species have been described, but about 80 other
undescribed species have been collected, and many of
the new species are found in deep-sea sediments (Gad,
2000). All known species are marine, microscopic, and
live within the bottom sediments as adults. The Higgins-larvae can have very large swimming-toes (flippers) and have been taken free in the plankton. Alternatively, the larvae may be benthic and have tubeshaped toes with adhesive glands. Usually there exists
a sexual secondary dimorphism in the first ring (clavoscalids) and last ring (trichoscalids) of scalids.
In the family Nanaloricidae the male has the middorsal, dorsolateral and ventrolateral pairs of clavoscalids divided into three branches but the two midventral scalids are undivided, so the clavoscalids consist of 20 chemoreceptors; the female always has only
eight primary clavoscalids (Kristensen, 1991a). Sexual
dimorphism may also be apparent in the trichoscalids
(Kristensen, 1991b). In the male of Nanaloricus the
two lateroventral secondary elements are modified as
claw-shaped organs, called claspers. The claspers are
assumed to function in copulation. Recently sexual dimorphism has also been recognised in the trichoscalids
of Rugiloricus, where the female has seven of the 15
trichoscalids reduced in length (Kristensen, unpublished).
In several species of Rugiloricus the larva develops
a mature ovary (neoteny), and this produces between
4 to 12 larvae inside the old larval exuvium (Kristensen and Brooke, 2001). These neotenous larvae look
exactly like the larva emerging from the free-laid egg.
The Rugiloricus-larvae may be common for most of
the year, and outnumber the adults by 100:1. Postlarvae, when they are present (seemingly absent in Pliciloricus), lack toes, setae and locomotory spines on
the abdomen. Often the postlarva appears very similar
to the adults, but it always has fewer scalids and a
thinner lorical cuticle than the males and females, and
it lacks the gonads. The life cycle of Rugiloricus is
surprisingly variable (Fig. 1). It varies from one species to another and, more confusingly, may vary depending on the feeding condition of the larva in the
same species.
Three types of life cycles have been discovered in
Rugiloricus. In R. carolinensis the Higgins larva
moults direct to a mature male or female (Fig. 1A, B,
C and H1, H2). In R. cauliculus the Higgins larva
moults into a postlarva without toes and gonads. The
postlarva moults into the adult (Fig. 1A, B, C, G and
H1, H2). In a new species of Rugiloricus from Faroe
Bank (North Atlantic Sea) the Higgins larva (Fig. 1D)
grows bigger than the adult and develops a large single
ovary. This larva forms a cyst-like stage (Fig. 1E).
Inside the cyst moulting occurs and, after forming several eggs from the ovary, the Higgins larva totally disintegrates. The eggs mature to neotenous larvae inside
the cyst walls. The larvae escape later from the cyst
(Fig. 1F). The neotenous larva moults several times
and grows to a new very large larva, which again develops an ovary. This part of the life cycle, called the
parthenogenic life cycle (Fig. 1D, E, and F), recur and
so explains why the Higgins larvae can outnumber the
adults by a factor of 100. Under certain unspecified
conditions the Higgins larva moults to a postlarva,
which again moults into the adult. It is unclear if other
species of Rugiloricus also have this parthenogenic life
cycle, but there is some evidence that both R. carolinensis and R. cauliculus may also form neotenous larvae (Higgins and Kristensen, 1986; Kristensen, 1999;
→
FIG. 1. The proposed life cycles in the genus Rugiloricus (Loricifera). All stages are seen in a new species from Faroe Bank. The larvae and
the female are shown ventrally, the male from the dorsal side. A. The free-laid egg. B. Embryo inside the egg. C. First instar larva (Higgins
larva). D. Last instar larva with a single ovary developed by parthenogenesis and neoteny. E. The cyst-like larval formation with the old
larval exuvium with toes outside and a new thin cuticle inside. Between the two cuticles the eggs and embryos in different stages are seen.
F. The neotenous larva escapes from the old larval exuvium. G. Postlarva without toes. H1. Female with ovaries. H2. Male with testes. In R.
carolinensis the Higgins larva can develop directly into the adult (C to H). In R. cauliculus the postlarva is always involved in the life cycle
(C, G to H), but new evidence indicates that neoteny may be present in all species of Rugiloricus under certain circumstances (Modified after
Kristensen and Brooke, 2001).
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Kristensen and Brooke, 2001). Recently Gad (2000)
has found neotenous larvae in several species of deepsea pliciloricid loriciferans.
The fine structure of the mouth cone, the introvert
with scalids, and the circumenteric brain indicate that
this meiofaunal phylum is monophyletic, related to Kinorhyncha and Priapulida. The three groups are referred to as the Scalidophora (see Lemburg, 1995;
Schmidt-Rhaesa et al., 1998). Autapomorphies for
Loricifera may include the unique location in both
males and females of the protonephridia inside the gonads, the complexity of the spinoscalids with muscles,
and the special toe glands. The hexagonal arrangement
in both adults and larvae of the internal armature in
the buccal apparatus may be plesiomorphic, a shared
character with Nematoda and Nematomorpha (Nematoida, subgroup of Cycloneuralia). Annulation of the
flexible buccal tube, telescoping of the mouth cone and
three rows of placoids in the triradiated pharynx bulb
are found only in Tardigrada and Loricifera, but these
similarities between tardigrades and loriciferans may
be either convergent or plesiomorphic to all Cycloneuralia and Tardigrada (Kristensen, 2002). The thick
lorica consisting of plates seems to be developed only
in the family Nanaloricidae, for which it is a convincing autapomorphic character. The plesiomorphic condition in both adults and larvae of the lorica seems to
be a thin lorica with many plicae.
Unfortunately, no molecular data are yet available
for loriciferans.
Cycliophora
The marine Cycliophora is the most recently described phylum in the animal kingdom. Only one microscopical species, Symbion pandora, has been described (Funch and Kristensen, 1995). The sessile
stage had been known since the late sixties, but was
never described (Funch and Kristensen, 1997). It is
approximately 350 mm in length and 100 mm wide,
and is attached with an acellular stalk and adhesive
disc to crustacean mouth parts, which can be encrusted
with thousands of individuals of the sessile stage. Investigations of mouth appendages of many decapod
crustaceans world-wide have resulted in further discoveries of this new type of epibiont, with a very complex life cycle, living mainly on the setae of lobsters’
mouthparts.
The sessile stage is the feeding stage, and has a
circular mouth surrounded by a ring of compound cilia
used for filtering small food particles, such as bacteria
or food particles from the host. Cycliophorans are
acoelomate metazoans with bilateral symmetry and a
well-differentiated cuticle, and the epidermis is cellular
rather than syncytial. They cannot, therefore, be assigned to the Syndermata (Rotifera and Acanthocephala). The internal anatomy is relatively simple, with
a U-shaped gut and the anus located outside the ciliated feeding apparatus, a similar arrangement to that
found in Ectoprocta (anus outside the tentacular
crown, Nielsen, 2001) and some sessile rotifers (anus
outside the corona, Winnepenninckx et al., 1998).
It is the life cycle consisting of an asexual and a
sexual generation (metagenesis) of the cycliophorans
that makes the phylum unique (Fig. 2). The asexual
part of the life cycle consists of two types of feeding
animals. The first type houses the so-called Pandora
larva, the second type houses the primary male and
female inside a brooding chamber. Furthermore, the
sessile animal shows internal budding, whereby it loses the head (buccal funnel) and entire digestive system,
replacing them with a new bud arising from embryonic
cells in the posterior part of the trunk. This process of
internal budding is repeated several times in the life
of the sessile animal and is coupled to the asexual
formation of the Pandora larva. The Pandora larva
forms a new feeding-stage, also by internal budding.
It settles very close to the maternal individual, and in
this way the epibiont can multiply very quickly and
completely cover the mouth limbs of its host. Later,
the feeding stages may switch to produce a primary
male or a sexually mature female instead of the Pandora larva. This marks the beginning of the sexual part
of the life cycle. The primary male is released from
the brooding chamber without a penis or a true testis.
It settles on a feeding animal. Only if there is a young
female inside this feeding animal can the attached primary male develop one or two new secondary males
inside by internal asexual budding. Each secondary
male has a tubular cuticular penis and a single compartment with spermatozoa. The female is about double the size of the primary male, and she looks similar
to the Pandora larva except for lacking internal budding; rather anteriorly she has a single large oocyte.
Fertilisation of the egg occurs inside the female, perhaps just before or during the release of the female
from the brooding chamber. She settles very close to
her maternal feeding animal, and broods inside herself
a new type of larva, the chordoid larva, so named after
a mesodermal chordoid structure of approximate 50
stacked muscle cells (see Funch, 1996). The growing
larva engulfs all her tissue, leaving only the cuticle.
The chordoid larva hatches from the female exuvium,
head first. It is a good swimmer and represents the
dispersal stage. This larva settles on a new host and
by internal budding forms a new feeding animal, so a
new life cycle can begin. The complex life cycle of
Symbion pandora as illustrated in Figure 2 differs from
the original description only by the recognition of the
free swimming secondary male (Fig. 2.17), where it
can be shown that it develops by budding inside the
primary male (Kristensen and Funch, 2001). We had
not originally discovered the free-swimming secondary male because of its tiny size, only 37 mm long, so
easily passing through our 63 mm net.
The type species was found on the Norway lobster,
Nephrops norvegicus from Scandinavian waters. More
recently two other and still undescribed species of the
cycliophorans have been found on the mouth appendages of other lobsters such as Homarus gammarus
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FIG. 2. The modified life cycle of Symbion pandora indicating that the primary male develops two dwarf secondary males inside by budding.
It is the secondary male (17) that fertilizes the female (18). The asexual part of the life cycle includes the sessile feeding stage, which forms
the Pandora larva (3–11). The sexual part of the life cycle includes two types of sessile feeding stages, each forms a primary male and female,
the secondary male and the chordoid larva (12-2). The chordoid larva is the dispersal stage (Modified from Funch and Kristensen, 1995,
1997).
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(Europe) and H. americanus (North America). So far
the cycliophorans have only been found on marine
decapods from the Northern hemisphere. Records of
cycliophorans from other crustaceans and nematodes
all seem to be false alarms. All these records seem to
represent chonotrich ciliates. Only by transmission
electron microscopy it can be resolved that the ciliate
only consists of one cell with several nuclei. Even so,
the convergences between cycliophorans, and especially the chonotrich, Isochonopsis kergueleni, are surprising. In particular, this ciliate has a cryptogemmic
budding like the cycliophorans (for detail, see Batisse
and Crumeyrolle, 1988). Therefore the chonotrich ciliates often are recorded as cycliophorans on copepods
or nematodes.
In the original description we stated that the Cycliophora are related to Entoprocta/Ectoprocta (Funch
and Kristensen, 1995). The overall similarities with
sessile rotifers (Collothecacea), and the presence of a
dwarf male with the penial structure in both groups
were considered to be superficial resemblances. This
reluctance to ally cycliophorans and rotifers was based
mainly on the ultrastructure of the epidermis covered
with a cuticle and the lack of the mastax. All known
rotifers have a mastax and a syncytial epidermis
(Sørensen et al., 2000). These authors placed cycliophorans close to entoprocts on the basis of a careful
cladistic approach. Unfortunately the phylogenetic relationships were only examined in the light of morphological evidence. The molecular data (study of 18S
rRNA/DNA sequences) indicate that cycliophorans are
a sister-group to the Syndermata which includes Rotifera and Acanthocephala (Winnepenninckx et al.,
1998). Most recently Giribet et al. (2000) placed the
cycliophorans within the Gnathifera. Their investigation was a combined approach based on both 18S
rDNA sequences and morphology across the range of
triploblasts, but their placement of Cycliophora was
entirely dependent on our molecular data (1,542 base
pairs of the 18S rRNA gene sequences) from alcoholpreserved material of Symbion pandora (see Winnepenninckx et al., 1998). The phylogenetic position of
Cycliophora is, therefore, very uncertain, and new ultrastructural and molecular studies are urgently needed.
Micrognathozoa
A new microscopic animal, Limnognathia maerski,
with complicated jaws, was found in a cold spring at
Disko Island, Greenland in 1994, but first described by
Kristensen and Funch (2000), where it was assigned
to a new class, Micrognathozoa, in the Gnathifera
(Ahlrichs, 1997). The clade Gnathifera now includes
Gnathostomulida, Micrognathozoa and Syndermata
(Eurotataria, Acanthocephala and Seisonida), see Figure 3.
Limnognathia maerski has dorsal plates formed by
an intracellular matrix, as in Rotifera and Acanthocephala (Storch and Welsch, 1969), but ventrally the
epidermis is ‘‘naked’’ only with a thin glycocalyx. The
gross anatomy of the body consists of a two-part head,
accordion-like thorax and ovoid abdomen with a small
tail. The total length of the holotype was only 142 mm,
and the maximum width of the abdomen was 55 mm.
As in many marine interstitial animals, e.g., gnathostomulids, gastrotrichs and polychaetes, special forms
of tactile bristles or sensoria are found on the body.
The locomotory organ consists of two rows of cells
with cilia. These cells are similar to the ciliophores in
annelids (Eibye-Jacobsen and Kristensen, 1994; Kristensen and Niilonen, 1982). An adhesive ciliary pad is
located ventro-posteriorly. It consists of ten ciliated
cells, which form a very sticky glue. The cells do not
contain the two types of granulae which are characteristic in the duo-gland system (Fig. 16, Kristensen
and Niilonen, 1982). Instead the ten cells are dominated by the cross-striated ciliary root passing through
the entire cell. The cilia are ordered in register like
those of the trunk ciliophores. The ciliary pad is very
different from the adhesive toes of rotifers, gastrotrichs
and annelids, and the structure may be a good autapomorphy for the micrognathozoans.
Because the new taxon lacks syncytia, a key character for the Rotifera and Acanthocephala (Syndermata, Ahlrichs, 1997), the Micrognathozoa cannot be
included in Syndermata. Two pairs of protonephridia
are found in the trunk of Limnognathia maerski, the
nephridiopore of which seems to open laterally on the
abdomen. The terminal cells are monociliated, a plesiomorphic character Micrognathozoa shares with
Gnathostomulida (Ax, 1996). Only the female reproductive system has been observed, suggesting that
Limnognathia maerski reproduces by parthenogenesis,
as do many other freshwater meiofaunal animals, such
as gastrotrichs, rotifers and heterotardigrades. The reproductive system is anatomically simple, and it seems
that the two ovaries derive nutrition directly from the
midgut, a feature also seen in freshwater chaetonotoid
gastrotrichs. There is a possibility that the female lays
two types of eggs, as in limnic gastrotrichs and rotifers: a smooth egg may be quick-developing summer
→
FIG. 3. Phylogeny of the clade Gnathifera, to show the position of Micrognathozoa. The cladogram is slightly modified from Kristensen and
Funch (2000). The dotted and broken lines with question mark are alternative suggestions (see Ahlrichs, 1997; Kristensen and Funch, 2000).
The black squares 1–11 mark synapomorphies used in the description of Micrognathozoa (see text). A–F: ventral views of the animals and
their jaws. A. Haplognathia rosea (Filospermoidea, Gnathostomulida). B. Gnathostomula microstyla (Bursovaginoidea, Gnathostomulida). C.
Limnognathia maerski (Micrognathozoa). D. Synchaeta vorax (Monogononta, Eurotatoria, Rotifera). E. Echinohynchus gadi (Palaeacanthocephala, Acanthocephala), note that jaws are lacking. F. Seison annulatus (Seisonida, Digononta, Rotifera). Note that clade Syndermata is the
same as the clade Rotifera, if Acanthocephala is an ingroup in Rotifera (Modified from Kristensen and Funch, 2000).
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egg, and a sculptured equivalent may be the resting
winter stage (Funch and Kristensen, 2002).
Most of the digestive system in micrognathozoans
is anatomically very simple, except for the pharyngeal
apparatus with large array of true cuticular elements,
the sclerites. In fact, Limnognathia maerski has one of
the most complicated jaw systems in microscopical animals, and it is still not well understood. Until now
only the main parts have been described by SEM investigations (Kristensen and Funch, 2000). They consist of two ventral basal plates, each plates associated
with large teeth (manus), two dorsal oral lamellae and
two lateral pharyngeal lamellae, two fibularia with
small teeth anteriorly, and three sets of jaws, where
the main jaws (mid jaws) are fused and consist of a
dentarium (basket formed), and an articularium
(Sørensen, 2002). The posterior part of the main jaws
has a paired cauda, as in some species of gnathostomulids (Riedl and Rieger, 1972). Furthermore, most of
the lumen of the pharynx is covered with a thin cuticle,
and several accessory sclerites attaching the ventral
and dorsal jaws are still not fully described (Sørensen,
2001). All sclerites seem to be built of microtubules,
consisting of lucent, osmiophobic material surrounding
a dense osmiophilic core. This structure was first described by Rieger and Tyler (1995) in the jaws of Gnathostomulida and Rotifera. The rest of the digestive
system is surprisingly simple. A short dorsal esophagus leads into the large midgut, which occupies most
of the thoracic region and the anterior part of the abdomen. The midgut lacks cilia completely, and has
only a brush border of short microvilli. A true cuticlelined hindgut is lacking. The anus is located dorsally
and opens only periodically, as seen in Gnathostomulida and some Gastrostricha that also lack a permanent anus (Knauss, 1979).
The ultrastructure of the micrognathozoan jaws is
similar to jaws of advanced scleroperalian gnathostomulids (Rieger and Tyler, 1995; Herlyn and Ehlers,
1997). However, the jaw apparatus might also possess
some of the characteristics of the rotiferan mastax (Koehler and Hayes, 1969). Jaw-like structures are found
in other protostome taxa as well, for instance in the
proboscises of kalyptorhynch turbellarians, in dorvilleid polychaetes and aplacophoran molluscs, but studies of their ultrastructure (e.g., polychaetes, Purschke,
1987) show that none of these jaws is homologous
with the jaws in Gnathostomulida, Rotifera, and Micrognathozoa (Sørensen, 2000; Sørensen et al., 2000).
Our proposed phylogeny of the clade Gnathifera is
shown in Figure 3. The solid line is the suggestion of
Kristensen and Funch (2000). The dotted line with a
question mark denotes the suggestion of Ahlrichs
(1997) that Acanthocephala and Seisonida are sister
groups. The broken line with a question mark is our
alternative suggestion (Kristensen and Funch, 1995)
that Gnathostomulida and Micrognathozoa are sister
groups. This preliminarily proposed idea was later
confirmed by molecular data (Garey, personal communication). The black squares with numbers 1–11
mark synapomorphies: 1. Gnathifera. Cuticular jaws
with tubes composed of lucent material surrounding
an electron dense core. 2. Gnathostomulida. Monociliated epidermis. Hermaphrodites with complex reproductive system. 3. Micrognathozoa 1 Syndermata.
Epidermis with apical intracellular matrix and dorsal
and lateral trunk regions without motile cilia. 4. Filospermoidea. Elongation of the body with a long and
slender rostrum. Compact jaws with 8 to 12 longitudinally arranged rods in lamellae symphyses. 5. Bursovaginoidea. Fibularization of the jaw apophyses.
Complex female organs with a bursa and often a vagina. Male opening with an injectory penis often consisting of 8 to 12 intracellular rod-like structure. Spermatozoon strongly modified with lack of flagellar or
centriolar structures. 6. Micrognathozoa. Ventral locomotory organ consists of two rows of multiciliated
cells (trunk ciliophores). Ventral epidermis covered
only with a thin glycocalyx. Cuticular outer oral plate
and dorsal and lateral intracellulary plates present. The
solid parts of the jaws very complex with paired basal
plates, each associated with denticulate manus, paired
striated oral and pharyngeal lamellae, paired fibularia
and three sets of paired jaws. 7. Syndermata. No motile trunk ciliation. Eutelic, syncytial epidermis with
apical crypts. Spermatozoon with anterior inserted flagellum. 8. Eurotatoria 1 Acanthocephala. Spermatozoon without acrosome. 9. Seisonida. Epizoic. The
head ciliation consists of few tufts of stiff bristles. The
trophi forms an anterior cuticular sheath. Presence of
spermatophore. 10. Eurotatoria. Corona, unpaired retrocerebral glands, female reproductive organs with vitellarium. 11. Acanthocephala. Endoparasitic with a
two-host life cycle. Epidermal lacunar system. Proboscis with intraepidermal hooks. Females with an
uterine bell.
Molecular data are still not available for Micrognathozoa, but a study of 18S rRNA/DNA sequences
is underway to be published (Garey, personal communication).
CONCLUSIONS
Loriciferans were originally assigned to the paraphyletic or polyphyletic group called Aschelminthes
(Kristensen, 1983). Subsequently, both molecular
(Winnepenninckx et al., 1995) and ultrastructural (Ehlers et al., 1996; Ahlrichs, 1997) studies have shown
that Aschelminthes consist of two unrelated groups,
Cycloneuralia and Gnathifera. Cycloneuralia may belong within the Ecdysozoa, including all molting invertebrates (Aguinaldo et al., 1997; Garey and
Schmidt-Rhaesa, 1998; Schmidt-Rhaesa et al., 1998),
while the Gnathifera are more closely related to either
Platyhelminthes or Annelida.
The phylum Loricifera clearly shares many apomorphic characters (e.g., scalids on the introvert) with
both Priapulida and Kinorhyncha, and can be included
in the taxon Scalidophora (Ehlers et al., 1996). Furthermore they have several plesiomorphic characters,
such as a myoepitheal pharyngeal bulb, comparable to
LORICIFERA, CYCLIOPHORA,
the other Cycloneuralia, especially the Nematoda and
Gastrotricha. The parasitic Nematomorpha, whose
adults lack the pharyngeal bulb, has a larva with an
introvert similar to Scalidophora. The loriciferans
have, therefore, also been assigned to the Cephalorhyncha that includes Priapulida, Kinorhyncha and
Nematomorpha (Malakhov, 1980; Adrianov et al.,
1990). The phylogenetic position remains controversial, but the close relationship of loriciferans to kinorhynchs and priapulids can hardly be doubted (Ehlers
et al., 1996; Neuhaus et al., 1996; Nielsen, 2001).
The idea that loriciferans are no more than ‘‘neotenous’’ or progenetic priapulids (Warwick, 2000) is
based on misunderstandings of the supposed resemblances between adult loriciferans and the loricate larvae of priapulids. There are very few similarities, and
it is incorrect that they are ‘‘virtually identical in morphology, except for the absence of gonads’’ (Warwick,
2000). It is correct that there are some similarities between the larvae of priapulids and loriciferans, but
nearly all these similarities are plesiomorphic characters seen in all Cephalorhyncha (Nematoda, Nematomorpha, Priapulida, Kinorhyncha and Loricifera, see
Nielsen, 2001). Furthermore if loriciferans are evolved
from priapulids by progenesis, the larvae in loriciferans should not be present. This is the case in many
progenetic polychaetes, e.g., Neotenotrocha sterreri
(see Eibye-Jacobsen and Kristensen, 1994; Westheide,
1987), which secondarily are adapted to the interstitial
way of life. Loriciferans do not lack larvae. In fact,
they have several types of larva; ironically one type
produces a mature ovary (i.e., neoteny). Therefore, I
reject the hypothesis that the loriciferans are simply
progenetic priapulids. Loricifera is a monophyletic
group, and is the sister group to the Priapulida 1 Kinorhyncha clade, within the Scalidophora (Nielsen,
2001).
In our original description (Funch and Kristensen,
1995) the Cycliophora was related to the Entoprocta
and Ectoprocta (Bryozoa) on the basis of ultrastructual
research. Cladistic morphological analyses (Zrzavý et
al., 1998; Sørensen et al., 2000) place the Cycliophora
as sister-group to Entoprocta. On the other hand, Cavalier-Smith (1998) considered them to be members of
the phylum Kamptozoa (5Entoprocta). This latter hypothesis should be rejected because it necessitates a
total redefinition of the Entoprocta. As the name implies, entoprocts always have the anus inside the tentacular crown (Nielsen, 2001), whereas the cycliophorans have the anus outside the ciliated mouth ring
(Funch and Kristensen, 1997). However, our molecular
data (Winnepenninckx et al., 1998) show they may be
related to Rotifera 1 Acanthocephala inside the taxon
Gnathifera (Giribet et al., 2000). The phylogenetic position of Cycliophora is, therefore, not settled, and
more ultrastructural and molecular research is needed.
Micrognathozoa is the latest described group of the
new animals. They show strong affinities with both
Rotifera and Gnathostomulida within the taxon Gnathifera, especially in the fine structure of the pharyn-
AND
MICROGNATHOZOA
649
geal apparatus. Only in these three groups do the jaw
elements have cuticular rods with osmiophilic cores.
Such microtubular structures are found in the fulcrum
of all Rotifera and in several cuticular sclerites of both
Gnathostomulida and Micrognathozoa (Sørensen,
2000). Furthermore, the micrognathozoans have two
rows of multiciliated cells that forms a locomotory organ, similar to that in some gastrotrichs and interstitial
annelids. This character is never seen in Rotifera, nor
in the monociliated Gnathostomulida. Rotifera and
Acanthocephala always have syncytia in the epidermis
(Syndermata). Micrognathozoa lack this characteristic
feature and, therefore, are postulated to be more basal
in the Gnathifera, as a sister-group either to Gnathostomulida or Rotifera/Acanthocephala.
The jaws of Gnathostomulida, Rotifera, and Micrognathozoa, with their microtubes, are considered to
be homologous structures. Limnognathia maerski is
probably related to both Rotifera and Gnathostomulida. Syndermata 1 Micrognathozoa might be sistergroups, while Gnathostomulida could be the sistergroup to this assemblage (Kristensen and Funch,
2000). Gnathostomulida was originally described as an
order of the Platyhelminthes (Ax, 1956). More recently Ax (1996) postulated a sister-group relationship of
Gnathostomulida with Platyhelminthes (‘‘Plathelminthomorpha hypothesis’’). This hypothesis is rejected
(Kristensen and Funch, 2000), and the finding of Limnognathia maerski supports the monophyly of Gnathifera (see Ahlrichs, 1997). The ultrastructural similarities of the new animal to certain chaetonotoid gastrotrichs and interstitial polychaetes may be either convergent or plesiomorphic features.
The discovery of Micrognathozoa has clearly
thrown new light on the polyphyletic origin of the ‘‘socalled’’ Pseudocoelomata or Aschelminthes. Gnathifera are more closely related to other platyzoan acoelomates (Gastrotricha and Platyhelminthes) than to introvertan pseudocoelomates. Cycliophora may be included in Platyzoa, too (Giribet et al., 2000). Introverta
(Cycloneuralia without Gastrotricha) form part of the
clade Ecdysozoa (Aguinaldo et al., 1997). Already in
1991—before the molecular evidence for Ecdysozoa—
I had stated (Kristensen, 1991a) that some aschelminth
groups (later named Scalidophora) and arthropods may
be related. Therefore, similarities (i.e., chitin in procuticle) between Panarthropoda and Scalidophora
could be true homologies. Proarthropoda (Tardigrada
and Onychophora) could have developed from Scalidophora-like animals similar to Opabinia and Anomalocaris of the Cambrian Burgess Shale Fauna, and
not from annelid-like animals without a moulting cuticle. The evidence for a clade Ecdysozoa comprising
all moulting invertebrates, is strongly supported by the
discovery of Loricifera, especially the life cycles of
the new pliciloricid loriciferans that indicate a close
relationship between Panarthropoda and Scalidophora.
ACKNOWLEDGMENTS
I gratefully acknowledge the collaboration with my
teacher Dr. Robert P. Higgins for the 20 yr of work
650
REINHARDT MøBJERG KRISTENSEN
together on meiobenthos, and to Dr. James Garey, who
invited me to SICB 2001 in Chicago and for the interesting discussions on a number of matters relevant
to this article. Especially I thank Dr. Andreas SchmidtRhaesa, Dr. Simon Conway Morris and an anonymous
reviewer who carefully read the manuscript and added
helpful comments and linguistic corrections.
I am indebted to Mrs. Stine B. Elle and Mrs. Birgitte
Rubæk who made the final drawings of loriciferans,
cycliophorans and micrognathozoans. Danish financial
assistance came from the Carlsberg Foundation
(970345/30-488) and the Danish Natural Science Research Council (9701589).
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