Larval development and morphogenesis of the sea spider Pycnogonum litorale (Ström, 1762) and the tagmosis of the body of Pantopoda.

Abstract

Aspects of pantopod ontogeny have been known for a long time, but specific information is available for only a few species. Our account of the postembryonic development of Pycnogonum litorale is based on laboratory-reared individuals and SEM studies. We documented particularly all early developmental stages, with emphasis on morphogenetic changes of head structures and appendages. In P. litorale the protonymphal limbs, the chelicerae and two more uniramous legs, degenerate already during the larval phase; only the third one, the ovigers, reappears in male juveniles. Other Pantopoda vary in this aspect from retention of all three protonymphal appendages to their complete reduction, as in P. litorale. Accordingly, the two post-cheliceral larval appendages are separate legs in front of the walking legs in the adults, the 'parapalps' and the 'ovigers', but they do not occur in all pantopods. The scarcity of studies of the ontogeny of Pantopoda prevents us from a more conclusive picture, but our data are promising to state that additional such studies will increase the usability of ontogenetic data for a phylogenetic analysis of Pantopoda, the crown group of the Pycnogonida. We also discuss the phylogenetic implications of our data in the light of new information from Hox genes and developmental-biological data on body segmentation and tagmosis of the Chelicerata. These suggest the homology of chelicerae and antenn(ul)ae of other euarthropods. Accepting this, we conclude that the adult pycnogonid/pantopod head, the cephalosoma, corresponds to the euarthropod head and that the protonymph with three appendage-bearing segments may represent an even shorter, possibly phylogenetically older larval type than the euarthropod 'head larva' bearing four pairs of appendages. In further consequence, the fourth walking legs of Pycnogonida/Pantopoda should correspond to the first opisthosomal appendages, the chilaria, of euchelicerates. This implies that within Pycnogonida the post-prosomal region became compacted during evolution to a single leg-bearing segment plus a tubular end piece. Accordingly, neither the anterior nor the posterior functional boundaries of the walking-leg region correspond to the original tagma borders.

Full text

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Larval development and morphogenesis of the sea spider Pycnogonum
litorale (Stro ¨m, 1762) and the tagmosis of the body of Pantopoda
Kathia Vilpouxa, Dieter Waloszekb,*
aDepartment of Neurobiology, University of Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
bSection for Biosystematic Documentation, University of Ulm, Helmholtzstrasse 20, D-89081 Ulm, Germany
Received 12 May 2003; accepted 11 September 2003
Abstract
Aspects of pantopod ontogenyhave been known for a longtime, but specific information is available for only a few species. Our account of
the postembryonic development of Pycnogonum litorale is based on laboratory-reared individuals and SEM studies. We documented
particularly all early developmental stages, with emphasis on morphogenetic changes of head structures and appendages. In P. litorale the
protonymphal limbs, the chelicerae and two more uniramous legs, degenerate already during the larval phase; only the third one, the ovigers,
reappears in male juveniles. Other Pantopoda vary in this aspect from retention of all three protonymphal appendages to their complete
reduction, as in P. litorale. Accordingly, the two post-cheliceral larval appendages are separate legs in front of the walking legs in the adults,
the ‘parapalps’ and the ‘ovigers’, but they do not occur in all pantopods. The scarcity of studies of the ontogeny of Pantopoda prevents us
from a more conclusive picture, but our data are promising to state that additional such studies will increase the usability of ontogenetic data
for a phylogenetic analysis of Pantopoda, the crown group of the Pycnogonida. We also discuss the phylogenetic implications of our data in
the light of new information from Hox genes and developmental-biological data on body segmentation and tagmosis of the Chelicerata.
These suggest the homology of chelicerae and antenn(ul)ae of other euarthropods. Accepting this, we conclude that the adult
pycnogonid/pantopod head, the cephalosoma, corresponds to the euarthropod head and that the protonymph with three appendage-bearing
segments may represent an even shorter, possibly phylogenetically older larval type than the euarthropod ‘head larva’ bearing four pairs of
appendages. In further consequence, the fourth walking legs of Pycnogonida/Pantopoda should correspond to the first opisthosomal
appendages, the chilaria, of euchelicerates. This implies that within Pycnogonida the post-prosomal region became compacted during
evolution toa single leg-bearing segment plus a tubular end piece. Accordingly,neither the anterior nor the posterior functional boundaries of
the walking-leg region correspond to the original tagma borders.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Ontogeny; Appendages; Segmentation; Pycnogonida/Pantopoda; Phylogeny; SEM study
1. Introduction
Ontogeny has been documented for only a few species
of extant Pantopoda as the crown group of the taxon
Pycnogonida, which embrace also its fossil stem taxa
(Waloszek and Dunlop, 2002). Isolated stages were
recorded from the field (Dohrn, 1881; Hoek, 1881; Morgan,
1891; Meisenheimer, 1902; Dogiel, 1913; Korschelt and
Heider, 1936), but descriptions of the developmental cycle
are very rare and often incomplete (Nakamura, 1981 for
Propallene longiceps [Bo ¨hm, 1879], Behrens, 1984 for
Pycnogonum litorale [Stro ¨m, 1762]). In various cases, the
earliest stages were missed due to their small size and
differing life habits (e.g. Merton, 1906; Sawaya, 1941).
From what is known, the Pantopoda demonstrate a large
variety of ontogenetic patterns and exhibit in part strong
morphological differences between early larval and adult
stages. This is not least due to significant differences in the
life strategies of the successive semaphoronts. This
ontogenetic and morphological plasticity makes the Panto-
poda an interesting group for related studies.
On the basis of field studies of pantopod larval
development (Dogiel, 1913), the Department of General
Zoology, University of Ulm, has successfully bred the
littoral P. litorale since the 1970s. The focus was to monitor
the development of this species under controlled conditions
1467-8039/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.asd.2003.09.004
Arthropod Structure & Development 32 (2003) 349–383
www.elsevier.com/locate/asd
* Corresponding author. Tel.: þ49-731-503-1000; fax: þ49-5731-503-
1009.
E-mail address: dieter.waloszek@biologie.uni-ulm.de (D. Waloszek).

Page 2

(Lotz and Bu ¨ckmann, 1968; Schmidt and Bu ¨ckmann, 1971;
Tomaschko et al., 1997), and Behrens (1984), using light
and scanning electron microscopy (SEM), was the first to
document its general postembryonic development.
The goal of our investigation is to fully document the
ontogeny of P. litorale from both a comparative-morpho-
logical and a systematic-phylogenetic point of view. Hence,
we have attempted to study all of the major aspects of its
external morphology, especially the development of the
head region and the appendages. Our paper also aims to
establish enhanced standards for the descriptive and
illustrative documentation of the larval cycle of Pantopoda,
for better comparability between taxa in the future. This
may also facilitate more detailed phylogenetic analyses,
which are still scarce (Arango, 2002).
2. Material and methods
All of the investigated specimens of P. litorale have been
raised by one of us (KV) in the laboratory of the Department
of General Zoology at the University of Ulm. Rearing of the
larvae followed basically the methods of Behrens (1984).
Individuals were kept in tanks together with their host, the
actinian Metridium senile (Linne ´, 1767), using artificial
salt water (Tropic Marine, Dreieich) of a salinity having
27–30%. The aquarium system was a closed circuit, with
an attached biofilter and continuous aeration at a room
temperature of 15 8C. Egg batches were removed from
males and transferred to a tank with seawater from the North
Sea. Water was taken from the Jadebusen near Wilhelm-
shaven, Germany, the locality of the original egg-carrying
adults and that of the first animals to start the culture in Ulm
(collected by E. Wilhelm). Freshly hatched protonymphs
(stage I larvae) were placed manually on the stolons of the
host, the hydrozoan polyp Clava multicornis Forsskal, 1775,
to which the early larval stages attach and start sucking
(Dogiel, 1913). At stage VI, the larvae were removed from
the stolons and placed onto single M. senile specimens.
Subsequent juvenile stages were raised, but since develop-
ment from the egg to the adult takes more than one and a
half years (Tomaschko et al., 1997) and each specimen
would have to be kept isolated, these stages were collected
only randomly.
Nevertheless, we were able to document the first six
successive stages, several undetermined juveniles, and
females and males, including egg-carrying specimens. All
of the specimens were fixed with 2.5% glutaraldehyde and
dehydrated in 30, 50, 70, 80, 90, 96 and 100% acetone. The
dehydrated specimens were critical point dried with liquid
carbon dioxide. For better attachment of the animals to the
SEM stubs, we used special oil for thin metals (Mixtion
Lefranc by Eckart-Werke). Gold-platinum sputtered speci-
mens were studied using a scanning electron microscope
(Zeiss DSM 962) at the Zentrale Einrichtung Elektronen-
mikroskopie of the University of Ulm and images were
captured digitally. Measurements for the generalized views
of the different stages were obtained from laser printouts of
the SEM images. The lengths given for the youngest five
stages are body length; all subsequent data refer to the
length from the tip of the proboscis to the tip of the trunk
end. If not stated otherwise, size proportions refer to body
length (see Table 2).
We also documented each stage of development by
drawings in ventral and dorsal aspect. The appendages were
drawn as if held laterally and in plane with the visible body
surface. This procedure aimed to improve comparability
between the stages and to emphasize position and insertions
of the different body parts and changes between them along
the ontogenetic sequence. In addition, posterior views of the
legs were drawn. Terminology follows, in general, that of
Helfer and Schlottke (1935) and Gruner (1993). Histori-
cally, investigators of Pantopoda adopted a number of terms
introduced for other arthropod taxa, but which have caused
difficulties due to the uncertain homology of the structures
in question. In order to improve comparability, we have
substituted some critical terms by more neutral ones
(Table 1). For example, in the ground-pattern of Euarthro-
poda there was only a single stem portion, basipod, which
carries the two rami endopod and exopod (the basipod
should not be mismatched or even termed a ‘coxa’ in
Chelicerata, because this is a specific structure developed
from a precursor structure, a median enditic lobe, proximal
to the basipod in the stem lineage of Crustacea (Walossek
and Mu ¨ller, 1990; Walossek, 1999, 2003a,b; Siveter et al.,
2001; Maas et al., 2003). Chelicerata apparently retained the
original, plesiomorphic condition having a basipod and two
rami, as can be deduced from the Silurian probable
euchelicerate Offacolus kingi Orr, Siveter, Briggs, Siveter
and Sutton, 2000) (Sutton et al., 2002) on most prosomal
and opisthosomal legs or from the Recent horseshoe ‘crabs’
on their last prosomal and all post-chilarial opisthosomal
legs (e.g. Walossek and Mu ¨ller, 1998; Walossek, 1999,
2003a; see also Fig. 13C). Because there is no distinct
proximal portion on pantopod walking legs that could be
referred to the basipod, it remains unclear if the leg portions
correspond to podomeres or articles of endopods only or if
they include the original basipod. Therefore, we termed all
limb subdivisions ‘limb segments’ and number them
consecutivelyfrom one onwards, the last beingthe ‘terminal
segment’. More detailed and large-scale comparative-
morphological investigations of chelicerate and euarthropod
legs, as for example attempted by Wolf and Harzsch (2002a,
b), may clarify this point further.
3. Results
P. litorale passes through six larval stages followed by a
series of juvenile stages. Juveniles are, as stated above,
difficult to culture; therefore, we can present only a gross
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Table 1
Abbreviations used in figures and text
ABD
agr
an
ant
aoc
aov
app
a2
b
ce
ch
chi
CLS
dscp
f
fi fix
fi mov
fu
gr
go
h
hib
hu
ic
jm
lab
lah
lbud
lis
ll1, 2
m
md
Abdomen (limb-bearing in insects, therefore in brackets there)
Abaxial groove
Anus
First appendage or first appendage-bearing segment of euarthropods, ¼ a1, antennula or antennular segment of crustaceans
Anlage of ocular tubercle
Anlage of oviger
Appendage
Second appendage or second appendage-bearing segment, ¼ antennal segment of crustaceans or intercalary segment of insects
Body
Cerci of insects
Chelicera; cheliceral segment of chelicerates
Chilaria-bearing segment of euchelicerates ( ¼ last prosomal segment)
Cephalosoma of pycnogonids/pantopods
Dorso-caudal spine of eucrustaceans
Field
Fix fixed finger of chela
Movable finger of chela
Furca of crustaceans
Groove
Genital opening
Head region of insects and crustaceans
Hind body
Hump
Intercalary segment of insects (corresponding to a2 or antennal segment of crustaceans and ‘pedipalp’ segment of euchelicerates)
Joint membrane
Labial segment of insects (corresp. to mx2 ¼ maxillary segment of crustaceans)
Larval head
Leg bud
Lip structure
Limbs of larval (protonymphal) head
Mouth
Mandibular segment of insects and crustaceans (corresponding to oviger segment of pycnogonids ¼ 3rd appendage-bearing segment of
euarthropods)
Middle region
Maxillary segment of insects (corresp. to mx1 ¼ maxillularly segment of crustaceans)
Maxillularly and maxillary segments of crustaceans
Ocular tubercle
Opisthosoma of chelicerates
Opercular segment (genital) of euchelicerates
Oviger
Oviger bud
Pre-antennal segment (status uncertain)
Pivot joint
Pore
Pedipalp segment of euchelicerates ¼ parapalps of pycnogonids
Proboscis
Process
Prosoma of chelicerates
Segment (e.g. of early larval appendages)
Spinning-gland process
Socket (e.g. of early larval appendages)
Spine
Trunk bud
Trunk end
Telson portion (uncertain or missing in insects, therefore with question mark there)
Thorax of insects and crustaceans (not the same region; original number of segments unclear for crustaceans, also if and how many true
limb-less abdominal segments were present)
Terminal segment
Walking legs of pycnogonids
mir
mx
mx1, 2
oc
OP
opl
ov
ovb
pas
pi
po
pp
pr
pro
PS
s
sgp
so
sp
tbud
te
tel
TH
ts
wal1-4
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scheme of development during that phase, with Table 1
including all major morphometric measurements.
3.1. Stage I (protonymph, hatching stage) (Figs. 1, 2A and
B, and 3; Tables 2–4)
Body. Almost circular, unsegmented, bearing three
pairs of appendages (Figs. 1 and 3A). Dorsal body surface
convex, slightly overhanging the flat ventral side (Fig. 3A
and B). Shallow abaxial groove located dorsally approxi-
mately between the insertions of the second and third
appendages (Fig. 3B and D). Cuticle of the fixed specimens
finely wrinkled except for the appendages, which are
smooth (unclear if this is an artifact, due to fixation, or
reflects true differences in cuticular softness). Eye structures
not detectable externally in this stage (measurements see
Table 2).
Proboscis. Only an anlagen, with the shape of a
tetrahedron arising behind the insertions of the chelicerae
and extending between these. Tip of proboscis anlage
bearing a small, oval opening, the mouth.
Appendages. Anteriormost limbs, the chelicerae, insert-
ing antero-ventrally and pointing almost ventrally. Articu-
lation with body oval and almost half as wide as the width of
the body and in long axis one-third of the length of the body
(Table 2). Proximal cheliceral segment is the largest,
making up 50% of the entire appendage, being barrel-
shaped in posterior view but antero-posteriorly compressed.
Segment carrying a soft, whip-like and curled outgrowth
latero-distally, which is longer than the body and becomes
flattened terminally. Numerous fine hairs except in the
proximal and distal areas cover this thread-like outgrowth;
its smooth tip bears the small opening of the ‘spinning-
gland’ ( ¼ spinning-gland process; Figs. 2A and B, and 3A
and D). Second segment less wide than the proximal one,
arising from the medio-distal edge of the latter. Its inner
edge is drawn out into a long, outwardly curving hook-
shaped process, the immovable or fixed finger. Third
segment acting as its movable counterpart, being similarly
shaped but less robust, arising from a well-developed joint
on the obliquely slanting outer distal edge of the second
limb segment. Movable finger bent inwards, even sharper
distally, and exceeding the length of the immovable finger
(Figs. 2A and B, and 3A and C). The two distal segments act
as a claw or chela (some use this term for the whole leg, but
we prefer the term chelicera and restrict the term chela to the
claw itself). Chela about as long as the proximal cheliceral
segment. Immovable finger with a small spine distally, at its
sharpoutwardcurvature, and outer edge, facing the movable
finger, carrying a row of short stout spines (Fig. 3A).
Movable finger may bear fine, short, individually different
spines along its concave inner edge (Fig. 3A). Each fingertip
with a pore of a gland of unknown function distally.
Proximal cheliceral segment oriented straight ventrally.
The two pivots, one medially and one dorsally (but slightly
anteriorly shifted), limit the movements of the whole
appendage in an antero-posterior direction. Joint between
first and second segment also with dorsal and median pivots
to permit bending in anterior–posterior direction, but due to
the location of the whole joint and an anterior bordering
hump at the first segment, the chela swings more backwards
than towards the anterior. Pivot joints between second and
third segment located antero-dorsally and postero-medially
permitting inward-outward movements of the movable
finger against the immovable counterpart (Table 3, top
row). Second and third appendages similar to each other,
with a wide ventral sternal area spanning the sockets of
these (particularly Fig. 3A). Second limb positioned
immediately behind the chelicera but more laterally, arising
from an almost circular articulation area. Third limb inserts
behind the latter, both arising from short conical sockets
provided by the body proper, pointing ventro-laterally to
laterally. Proximal limb segment tubular to conical being,
only a little longer than wide. Outer distal edge bearing a
thread-like outgrowth. Outgrowth similar to that of the
chelicera, also being covered with fine hair and carrying a
pore terminally, but much shorter and process-like,
narrower and almost straight. Second limb segment also
conical but twice as long as the proximal segment. Terminal
Fig. 1. First larval stage, protonymph, of Pycnogonum litorale (Stro ¨m,
1762).ForabbreviationsinthisandallfurtherfiguresandtablesseeTable1.
K. Vilpoux, D. Waloszek / Arthropod Structure & Development 32 (2003) 349–383352

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segment similar in length to the second segment, but
narrower and thorn-like tapering toward its slightly curved,
pointed tip. Surface of the terminal segment is adorned with
fine hairs similar to the process of the proximal segment,
except for short smooth areas proximally and distally.
Terminal segment also bearing an opening distally. Pivot
joints between the limb segments being rather indistinct
except for the ones between second and third ones. Due to
the anterior and posterior position of these pivots the distal
part of the limb can be flexed inward. A medio-distal
widening of the joint area of the second segment facilitates
this bending, while an outward curvature of the terminal
segment is limited by a small hump on the second segment
(Table 3, second row). The second and third limbs differ in
their orientation with respect to the main body axis: the
second one points slightly antero-medially, while the third is
oriented slightly backwards. Orientation of these limbs and
the flexure of their terminal segments enable the proto-
nymph to attach to the substrate (¼Klammerbeine in
German).
Hind body. No distinct trunk end; larval body ends
immediately behind the third limbs in a rounded rear,
lacking any signs of an anus or additional appendages.
Besides various pores all over the body, there are two small
surface protuberances between the limb sockets each bear a
pore distally (Figs. 1 and 3A and E; ¼ Buckelporen after
Behrens, 1984; function unclear).
3.2. Stage II (Figs. 2C and D and 4; Tables 2–4)
Body. Undivided, as in the protonymph, but more ovoid
due to appearance of a short, rounded to sub-triangular hind
body behind the last pair of limbs. Cuticle wrinkled except
for the limbs (see Stage I). Dorsal side slightly domed
(Fig. 4D), ventral side straight (Fig. 4A). No marked head-
shield borderline detectable (Fig. 4G; but see Behrens,
1984) and no eyes visible (Fig. 4D and F). Abaxial dorsal
furrow now almost indistinct but surface with six smooth,
clearly demarcated fields instead (Fig. 4D and F): one
located antero-medially immediately behind the chelicerae,
followed by a pair of fields more laterally, another single
one medially behind the pair and a pair posterior to it and
positioned less far laterally than the preceding pair (Figs. 2D
and 4F). Ventrally, a distinctive sub-oval groove of
unknown origin or function is located in the center of the
inter-limb area (Fig. 4A).
Proboscis. Anlage larger than in Stage I, made of a
proximal pyramid-shaped socket, as in the Stage I,
continuing in a trumpet-like ventrally pointing extension
(Figs. 2C and 4B). Mouth opening deeply recessed into
this trumpet and surrounded by three initial lip structures
(Fig. 4C).
Appendages. Unchanged in number and gross shape
(Figs. 2C and D, and 4A). Chelicera with a spinning-gland
outgrowth shorter relative to that of protonymph and more
like a process or spine (Table 2). Process only as long as the
limb, pointed and its surface free of hair, ending with the
opening of the spinning-gland (Fig. 4A and G). Articulation,
axes of swing of the limb parts, and orientation of the
chelicera as in Stage I (Fig. 4A; Table 4, two top rows).
Proximal cheliceral segment slightly smaller and stouter,
while the second segment is more voluminous and rounded
than in Stage I. Both fingers of the chela broader and
straighter than in Stage I, the outer processes being greatly
reduced relative to those of Stage I, but both finger tips still
carrying openings of glands. Size, proportions and articula-
tions of the second limbs not significantly different from
Stage I, except their enlarged body sockets (Table 2;
Table 2
Measurements of major body parts in the different developmental stages (Roman letters in row 1) and the adult (ad) of Pycnogonum litorale (Stro ¨m, 1762).
Body equals the head in stage I; p no length given, because of missing boundary at this stage, otherwise in mm. Data for adults refer to males, if not stated
otherwise. Dash: structure not present
Measured structure (mm)I II IIIIVV VI Adult
Body, length
Body, width
Larval head, length
Larval head, width
Chelicera, length
2. Larval appendage, length
3. Larval app. ¼ oviger in adults, length
4. App. (1st walking leg), length
5. App. (2nd walking leg), length
6. App. (3rd walking leg), length
7. App. (4th. Walking leg), length
Spinning gland protrusion, length
Spinning hair, length
Grasping-leg processes, length
Proboscis, length
Mouth opening diameter
Egg, diameter
120
120
120
120
110
140
140
170
140
160
140
110
130
130
260
170
–p
170
130
130
130
490
270
230
240
130
130
130
430
580
350
230
290
140
130
130
700
530
1.100
450
230
340
–
–
–
1.020
1.020
930
–
–
–
–
390
8.000–10.000
3.000
600
1.700
–
–
2.270
7.500
7.700
7.500
6.600
–
–
–
3.200
330
100
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
260
13
50
50
–
20
–
14
50
–
14
–
14
50
–
14
–
20
60
–
–
–
25
–
2 27
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Fig. 4E). Proximal limb segment broader than high and,
relative to that of the protonymph, with a shorter, narrower
process lacking hair; tip still with a pore. Second segment
unmodified, terminal segment shorter but broader than in
the previous stage, bearing a short spine anteriorly. Another,
longer spine is located on its inner side guided by several
small areas with short spines. Unlike the first stage, the
terminal segment lacks hairs, while the tip also bears a small
opening. Articulation between body and proximal limb
segments not clearly determinable, whereas, the anterior
and posterior pivots of the subsequent joints segments
permit an inward swing of the two distal segments, as in
stage I (Fig. 4E). Third limb similar to second one in all
aspects (Fig. 4A). Absolute size of all appendages only
slightly changed relative to Stage I, while the most obvious
change is the increase in body length (Table 2); this results
in a slightly larger distance between the bases of the second
and third limbs and in a relative size reduction of all
appendages relative to the body.
Hind body. A humped region with no signs of anus or
additional legs (Fig. 4A, F and G).
3.3. Stage III (Figs. 2E and F and 5; Tables 2–4)
Body. More flattened in dorso-ventral aspect and more
rectangular in dorsal or ventral view than in Stage II; also
dorsal surface more domed, but cross-wise groove very
weakly defined. Six smooth fields present as in Stage II, but
larger proportionally and their cuticle is no longer smooth
but porous (Figs. 2F and 5D). Ventral central groove distinct
(Fig. 5A). Cuticle offixed specimens finely wrinkled, that of
the limbs is much smoother (Fig. 5A). No eye structures
visible.
Proboscis. Still an anlage similar to that of Stage II but
enlarged relative to the latter (Table 2). Pyramid-shaped
base broadened and constricted more distally, appearing to
be squeezed in between the chelicerae (Fig. 5A and B).
Tube longer than in Stage II; mouth unchanged (Fig. 2E
and 5A).
Appendages. Chelicera more rounded and its segments
more bulged than in Stage II. First segment with a ventrally
pointing spinning-gland process, which is as long as the
limb. Few spines on the fingers compared to previous stage,
fingertips now at the same height, but fixed finger turned
backwards and movable finger tilted anteriorly distally
(Fig. 5A). Pores on fingers and spinning-gland, process as in
Stage II (Fig. 5A and B). Second and third limbs arising
from well-developed ventro-laterally, extending sockets
(Fig. 5A and E). In reverse to the further elongation of the
posterior body relative to the preceding stage, the head
(protonymphal) portion is shortened. The effects of this are
the location of the protonymphal limbs in the anterior half of
the body, the relatively smaller size of all limbs and the
relatively shorter distance between their sockets (Table 2;
Fig. 5A and C). Again, the proximal segments of second and
third limbs are shorter relative to those in Stage II and
almost ring-shaped. Subsequent limb segments unchanged,
as are their short processes (Fig. 5E), the openings of the
terminal segments, and the articulations (pivot joint
encircled in Fig. 5E; Table 4, upper two rows).
Hind body. Body extended into a short, rounded trunk
bud, which is flanked by the buds of the fourth appendages,
i.e. the first pair of the walking legs (characterizing this
stage; Fig. 5A, C and D). Limb buds circular in diameter,
first tubular and distally tapering to a slightly ventrally
curved, pointed tip (shaped like a bullet). Buds first curving
slightly outwards and extending straight posteriorly there-
after. Each bud with one spine dorso-laterallyclose to the tip
(Fig. 5D). Segment boundaries absent (Fig. 5A, C and D).
No anus.
3.4. Stage IV (Figs. 2G and H, and 6; Tables 2–4)
Body. Nearly twice as long as broad (Table 2), still
undivided (no segment boundaries), but three regions can be
distinguished now: the protonymphal head, now termed
‘larval head’, a middle region comprising the segment of the
first pair of walking legs, and the hind body made of one
segment carrying the pair of bullet-shaped buds of the
second walking legs, which flank the similarly bullet-shaped
trunk bud. Cuticle finely wrinkled except on the appen-
dages, their sockets, the proboscis, the dorsal humps and the
trunk bud (Fig. 6A and B).
Larval head ¼ protonymphal body region: domed dor-
sally and ventrally slightly vaulted. Margins of dorsal area
form a kind of collar around the anterior three pairs of
appendages and the anlage of the proboscis. Six fields on
dorsal surface, as in Stage III (Figs. 2F and 6B). Frontal field
correspondingtothefutureeyehump(Fig.6BandC),butno
eye structures visible. Ventral groove still present (Fig. 6G).
Proboscis. Anlage unchanged (Figs. 2G and 6G).
Appendages. Three-segmented chelicera and the two
pairs of grasping limbs insert ventro-laterally on the larval
head, as in Stage III. Chelicera unchanged in general shape,
position and absolute size (Fig. 6E; Table 2), but the chela is
very small compared to the basal segment of the limb
(Fig. 6G). Spinning-gland process as in Stage III (Fig. 6D
and G). Second and third larval legs similar to those in Stage
III (Fig. 6G) and also unchanged in size, therefore
proportionally smaller than in the preceding stage (Table 2).
First walking leg elongate, distinctly divided into seven
segments. Leg length nearly matching the body length
(prominent structure of this stage). Leg attaching laterally
in the middle of its body segment, arising from a broad,
Fig. 2. Drawings of developmental stages I–IV of Pycnogonum litorale (Stro ¨m, 1762). On the left: ventral views of whole specimens, on the right: dorsal
views. A, B Stage I; C, D Stage II; E, F Stage III; G, H Stage IV.
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rounded socket formed by the body (annulations may
indicative the original body-leg joint). According to its
outwards orientation, the lateral side of the leg, when fully
stretched, points dorsally and the median side ventrally.
Joints are developed betweenthe leg segments, but are fairly
indistinct. The differentiated shape of the limbs segments
andorientationoftheirjoints(locationofthepivots;Fig.6A,
D and F) result in a characteristic S-curvature of the walking
leg and a limited but different swing of each of the segments
to move the limbs in various ways and directions (Table 4).
Proximal segment broader than all distal segments, pos-
sibly twisted 908 around its socket, as is derived from a
posteriorly pointing protuberance. This results in a medio-
lateral location of the pivots causing a more anterior–
posterior swing of the distal part of the leg. Second segment
annular but medially and posteriorly bulged and with
anterior and posterior pivots, which permit the larva to
raise the remaining leg part (outward flexure of the leg).
Subsequent third segment bulged medially and also
swinging inward outward (dorsally–ventrally), so that the
elongate fourth segment is even further raised dorsally. Its
enlarged and convex outer side results in a shift of the
insertion of the subsequent short fifth segment almost to the
inner side and a sharp inward curvature of the distal leg part.
Fifth segment also longer laterally than medially, enhancing
this curvature. Sixth segment elongate and conical carrying
the inwards curved, thorn-like seventh segment slightly
inwards of its apex. Accordingly, the thorn can act similar
to a subchela, rotating against the penultimate segment
(Fig. 6A, D and F). Fourth, fifth and sixth segments with a
long spine laterally on their distal edge, which can be
accompanied by patches of smaller spines (Fig. 6F).
Middle region. Tube-shaped, wider than long. The
dominating structure is the pair of sockets for the fourth
appendages stemming from the mid-level of the region and
extending laterally, narrower proximally than distally
( ¼ inverted conical). Ventrally there is only a small gap
between the bases of the sockets (Fig. 6A). Dorsal surface
of the middle part with a median hump with a spine on top,
but indistinctly set off from neighboring segments (Fig. 6B
and D).
Hind body. Comprising the segment of the bullet-shaped
buds of the second pair of walking legs flanking the non-
separated, similarly bullet-shaped trunk bud. Shape of this
region virtually as those in Stage III, except that this is one
segment further back. No anus developed (Fig. 6A and B).
3.5. Stage V (Figs. 7A and B, and 8A–I; Tables 2–4)
Body.As in Stage IV (Figs. 7A and B and 8A and D), also
cuticle of fixed specimens finely wrinkled except for the
appendages, their sockets, the proboscis, the dorsal humps
and the trunk bud (Fig. 8A, B and D).
Larval head. Outline almost circular in ventral view,
slightly wider than long. Margin around the proboscis and
anterior three limbs collar-like and finely wrinkled and
collar-like, as in Stage IV, as is the dorsal cuticle except for
the fields (Fig. 8A, C, D and G). Early larval head distinctly
set offfrom the rest of the body by a lateral constriction and
shallow abaxial groove ventrally, less so dorsally. Dorsal
fields as in Stage IV. No eye structures visible on the anlage
of the eye hump (Fig. 8D and E). Ventral side without
median groove (Fig. 8B).
Proboscis. Anlage located ventrally in the center of the
larval head; shape as in Stage IV (Fig. 7A). Lip structures
distinct, causing a Y-shaped mouth opening (Fig. 8F).
Appendages. Early larval appendages unchanged in
shape, size and articulations from Stage IV (Figs. 7A and
8A–C; Tables 3 and 4), but due to the enlargement of the
head, appearing considerably smaller relative to those in
Stage IV (Table 2); their insertion areas appear to be located
more inwards of the margins (Fig. 8A and B). Two pairs of
developed walking legs in the middle region and one pair of
buds of walking legs three at the rear of the hind body.
Compared to Stage IV, the first walking leg is longer and the
number of segment has increased eight (Tables 2 and 3).
Joint membranes are more distinctive than in preceding
stage, particularly between the more proximal segments.
Otherwise shape and proportions similar, also with the two
major bends between the second segment and the rest and
the fourth and the rest, causing the S-curvature of the
walking legs (Fig. 8A, D and G–I). Fifth limb segment
apparently representing an additional element compared to
Stage IV (marked in Table 4). Fourth to sixth segments
carrying several strong spines latero-distally, segment 7
bears one spine laterally. Cuticle of the terminal segment
covered with small pits of unknown function. Second
walking leg seven-segmented and resembling the fourth
appendage of Stage IV both in shape and number of
segments (Fig. 8G and I).
Middle region. Made of two tubular segments, both
wider than long and broadening into leg sockets laterally.
Diameter of each of the prominent and inverted conical-
shaped sockets, are almost as wide as length of the whole
middle region. Segments separated by deep constrictions,
and each segment bearing a domed hump with wart-like
protuberances and a spine dorsally surrounded by wrinkled
cuticle (Fig. 8G).
Hind body. Similar to that of Stage IV, but now
comprising the segment with the initial third pair of walking
legs and the rear end (Fig. 8A and B). Leg buds slightly
postero-laterally oriented and bullet-shaped, flanking and
Fig.3. Developmental StageI (protonymph) ofPycnogonum litorale(Stro ¨m,1762).A.Ventral viewofthe whole specimenwithextendedlegs.B. Dorsal view.
C. View of the cranial part of a specimen with its chelae widely gaping. D. Lateral view, legs extended. E. Posterior end of the body with two pores on small
protuberances.
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the vaguely separated trunk bud, which, in contrast to
previous stages, is more rod-shaped, becoming broader first
and slims again caudally towards its rounded end. Trunk
bud less wide but also more elongate than that in Stage IV.
Dorsal side of leg buds with a spine or setae; trunk end with
a vertical slit, the anlage of the anus.
3.6. Stage VI (Figs. 7C and D, and 9; Tables 2 and 3)
Body. Divided into larval head region, region of the
walking legs, and hind body, but segment of first walking
leg indistinctly demarcated off from larval head, initiating
the formation of the adult cephalosoma (Figs. 7D, 9A and
B). Cuticle wrinkled in fixed specimen on the ventral side of
the head and between the leg sockets just to the area
between the leg buds (Fig. 9A).
Larval head. Dorsal side domed, ventral side slightly
vaulted. Frontal margin of dorsal side bearing the elevated
abaxially wider than long oval eye hump, which is followed
by a smaller and a larger, caudally vaulted hump, both
originating from the anterior fields of the six smooth dorsal
fields of previous stages. First two lateral fields still present
Table 3
Important characters and their modification during different stages of development in the ontogeny of Pycnogonum litorale (Stro ¨m, 1762). The term head is
placed in apostrophes because of its changing segmental status during ontogeny and uncertainty in evolutionary terms
Stage IStage II Stage IIIStage IVStage V Stage VI JuvenilesAdult
Feature
Body Round,
undivided
Oval,
undivided;
head þ trunk
bud
Elongatedly
oval,
undivided;
head þ hind
body
Body to border
of hind body
–
Buds of 1st
walking leg
þtrunk bud
3-Segmented
3-Segmented
grasping limb
3-Segmented
grasping limb
Twice as long
as wide,
undivided but
visible 3
portions
Anterior body
part
1-Segmented
Buds of 2nd
walking leg þ
trunk bud
3-Segmented
3-Segmented
grasping limb
3-Segmented
grasping leg
limb
7-Segmented
walking leg
Limb bud with
dorsal seta
–
Twice as long
as wide,
undivided but
visible 3
portions
Anterior body
part
2-Segmented
Bud of 3rd
walking leg
þtrunk bud
3-Segmented
3-Segmented
grasping limb
3-Segmented
grasping limb
Longer than
wide; head set
off from 2-
partite trunk
Longer than
wide; head set
off from 2-
partite trunk
Longer than
wide; head set
off from 2-
partite trunk
“Head”Whole bodyBody without
trunk bud
–
Central caudal
hump
Anterior body
part
3-Segmented
Bud of 4th
walking leg þ
trunk end
–
–
Anterior body
part
4-Segmented
Rod-shaped
hind body
Anterior body
part
4-Segmented
Rod-shaped
hind body
Middle region
Hind body
–
–
App1 ¼ ch
App2
3-Segmented
3-Segmented
grasping limb
3-Segmented
grasping limb
3-Segmented
3-Segmented
grasping limb
3-Segmented
grasping limb
–
–
–
–
App3f-/m shallow
hump with
distal seta
9-Segmented
walking leg
9-Segmented
walking leg
9-Segmented
walking leg
Limb bud with
dorsal seta
Anlage of eye
hump
Develops from
bud to a 10-
segm. oviger
9-Segmented
walking leg
9-Segmented
walking leg
9-Segmented
walking leg
9-Segmented
walking leg
Hump bearing
4 lens eyes
10-Segm.
oviger
4. app. wal1––Limb bud with
dorsal seta
–
8-Segmented
walking leg
7-Segmented
walking leg
Limb bud with
dorsal seta
–
9-Segmented
walking leg
9-Segmented
walking leg
9-Segmented
walking leg
9-Segmented
walking leg
Hump bearing
4 lens eyes
5. app. wal2––
6. app. wal3–––
7. app. wal4 ––––
Eye hump– Bordered
depression
Bordered
depression
with porous
cuticle
Short, no
threads
Pyramidal
anlage with
trumpet-like
tube, mouth
inside funnel
Lengthwise
oriented hump
Anlage of eye
hump
Outgrowth
on app 1–3
Proboscis
Long, with fine
threads
Pyramidal
anlage with
elongatedly
pear-shaped
mouth
Short, no
threads
Pyramidal
anlage with
trumpet-like
tube, mouth
inside funnel
Short, no
threads
Pyramidal
anlage with
trumpet-like
tube, mouth
inside funnel
Short, no
threads
Pyramidal
anlage with
trumpet-like
tube, mouth
inside funnel
with 3 lips
Slit-shaped
anlage
–––
Cylinder-
shaped, dist.
rounded, 3 lips
closing mouth,
with 1 tooth
each
Slit-shaped
Bottle-shaped,
distally
rounded, 3 lips
closing mouth,
with 1 tooth
each
On papilla,
slit-shaped
Bottle-shaped,
distally
rounded, 3 lips
closing mouth,
with 1 tooth
each
On papilla,
slit-shaped
Anus––––
Fig. 4. Developmental Stage II of Pycnogonum litorale (Stro ¨m, 1762). A. Ventral view of a whole specimen. B. Anterior view of proboscis with its funnel-
shaped tip. C. View into the tip of the proboscis. D. Antero-dorsal view showing the proximal segments of the chelicerae and the proboscis. E. Lateral view of
grasping leg (app 2). F. Dorsal view of a whole specimen. G. Lateral view of a specimen, terminal segments folded inwards.
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but smaller and vaulted relative to Stage V, the last couple
of fields reduced to small spots at the caudal margin of the
larval head (Figs. 7D and 9B and F). In some specimens, the
lateral margins of the ventral side of the larval head bear two
small humped but smooth areas each with a spine,
distinguished from the rest of the cuticle by surrounding
folds (Fig. 9E). Only the lateral constrictions separate the
larval head from the rest of the body.
Proboscis. Shape considerably changed as compared to
the previous stages; now considered as developed. Proximal
part cylindrical, but becoming slightly constricted like a
bottle from half way along its length to the distal end
(Fig. 9A and B). Proboscis arising in the mid-line of the
ventral side of the larval head and pointing frontally. Mouth
opening triangular, located on rounded tip of proboscis and
closed by the tripartite lip structure (Figs. 7C and 9C).
Trumpet-like distal rim absent now.
Appendages. All three early larval appendages absent
(Fig. 9A and C–E), whereas, three pairs of fully developed
walking legs are present in the median body region (Fig. 9A,
B, G and H). First walking leg oriented anteriorly,
consisting of nine segments (Table 4). First and second
limb segments as in Stage V in terms of shape and articu-
lations. Narrow joint membrane visible between the seg-
ments. Posterior side of second segment longer compared to
that of Stage V, causing an outward orientation of the distal
leg part. Third segment swings inward outward due to
anterior and posterior pivots, joint membrane between third
and fourth segments laterally wider than medially, facilitat-
ing a wider outward swing of the distal leg part. Joint
between these segments with lateral and median pivots,
which are slightly shifted posteriorly respective anteriorly,
resulting in the ability to rotate antero-lateral to body and
postero-median away from body. Joint membrane between
third and fourth segments only weakly developed. Fourth
segment long, cylindrical and oriented almost horizontally.
Articulations between the fourth and fifth and the fifth and
sixth segments with anterior and posterior pivots, allowing
an inward-outward swing. Insertion area of fifth segment
shifted medially, thus the leg comes to rest in plane with the
body. Joint membranes between fourth and fifth and fifth
and sixth segments medially enlarged, but laterally hardly
Table 4
Overview of the articulations of the appendages of Pycnogonum litorale (Stro ¨m, 1762) (left appendage viewed from distal to proximal). Due to the axis of
view, lateral is on top and median on the bottom of the sections. First appendage, chelicera, rotated 458 to the right. For adults, the first walking leg is shown as
an example. Appendage segments counted from proximal to distal for all stages. Newly acquired limb segments marked. Diagonal sections of joints
represented by ovals, pivots represented by dots, possible movements marked by arrows and restrictions in movement marked by curves. Question marks
indicate joints where articulation could not be determined, lines indicate missing segments
Fig. 5. Developmental Stage III of Pycnogonum litorale (Stro ¨m, 1762). A. Ventral view of a whole specimen, with buds of appendage four, the first walking
leg. B. Antero-dorsal of first segment of chelicerae and proboscis. C. Lateral view of a whole specimen. D. Dorsal view of a whole specimen. E. Appendages
two and three, showing the pivot joints (one encircled).
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visible. The newly appearing, small seventh segment, with
its triangular joint area, inserts medially between segments
six and eight. Articulation with sixth segment provided by
anterior and posterior pivots, allowing only a limited
inward-outward swing of the distal segments due to the
special shape of the seventh segment. Seventh and eighth
segments connected by a joint with lateral and median
pivots, thus the eighth segment can be moved in anterior and
posterior direction; joint membranes weakly developed. The
distally narrower eighth segment is, again, in plane with the
body. It is connected with the terminal ninth segment by
anterior and posterior pivots, allowing an inward-outward
swing of this narrow inwards curved and sharply pointed,
hook-like element. Joint membrane between eighth and
ninth segments proximally bulged on the median side and
laterally invisible (Fig. 9A, B, G and H; Table 4). Second
walking leg oriented laterally, also consisting of nine
segments, which match the preceding leg in length, shape
and articulation (Table 1). Third leg also with nine segments
and as the anterior two legs except for its smaller length and
its orientation more towards the posterior (Fig. 9A and B).
Middle region. Comprises the three segments with
developed walking legs, but the anterior border of the first
segment is rather indistinct, so pre-forming the future tagma
cephalosoma of the adults, comprising the early larval head
and the first walking-leg segment. The walking legs arise
from laterally oriented, inverted conical sockets provided by
the body, which make the narrow, tubular trunk appear
broader than it actually is. Distance between the sockets and
their diameter decreases from the first to the third walking-
leg segment. Each segment bears a strongly developed
hump mid-dorsally, which may carry wart-like protuber-
ances and a spine (individual variation; Fig. 9B).
Hind body. Composed of one segment carrying the pair
of posteriorly pointing bullet-shaped buds of the fourth pair
of walking legs, which flank the more club-shaped trunk
end. Trunk end increasing in diameter towards its end
bearing the slit-shaped anus terminally (Fig. 9A and B).
3.7. Juvenile phase (Fig. 10A–F; Tables 2 and 3)
The juvenile phase of P. litorale is distinguished from the
larval phase mainly by the full development of the fourth
and last pair of walking legs. All four legs have now nine
segments and resemble each other in shape, joint design and
axis of swing of the different segments. The proboscis is
constricted in its middle region and has a triangular mouth
opening distally (Fig. 10D and F), as described for larval
Stage VI. Since there is no further segment with immature
legs present, the remaining trunk region comprises only the
rod-shapedendpiececarryingtheanusterminally(Fig.10A,
G and H).
The juvenile phase is, in addition, characterized by an
increase in width of the anterior body portion relative to
proboscis length and an enlargement of the eye hump to
make up much of the dorsal area of the larval head region
(Fig. 10A and H; Table 2). The body cuticle becomes
progressively more sclerotized but also smoother and the
dorsal humps become progressively more pronounced.
Sexes are distinguishable from the first juvenile stage: it is
only in the males that the second pair of post-cheliceral
larval appendages reappears and develops progressively
into the characteristic ‘ovigers’, the egg carriers. Male
specimens of early juvenile stages possess only small
knoblets on the postero-ventral side of the larval-head
region, which fade out into a spike-like outgrowth
(Fig. 10C). During further development, the knoblets
develop into anteriorly pointing, externally unsegmented
rod-shaped limb buds with pointed distal ends (Fig. 10H).
Segmentation of this pair of legs is only weakly indicated,
boundaries being partly marked by short, spine-like setae
(Fig. 10E). One late juvenile male had ovigers consisting of
ten distinct segments (not figured). Some juvenile males
possessed an area of apparently thin and folded cuticle,
demarcated off from the rest of the surface on the ventral
side of the second segment of the fourth walking leg,
indicative of the future genital opening (see Fig. 11D for an
adult male).
In summary, the morphology of the juveniles does not
change significantly, throughout development except for the
features described above and a steady increase in length.
With this, the juveniles show a gradual ontogeny with
equally gradual morphogenesis of most of the structures and
no signs of metamorphic jumps. After five to seven molts,
specimens of both sexes reach maturity and are considered
as adults.
3.8. Adults (Figs. 7G and H, 10I and J and 11; Tables 2–4)
Sexual dimorphism is clearly present in P. litorale, not
least in the sizes of males and females; the latter have a
mean length of 10 mm; the former measure only 8 mm
(Table 2).
Body. Distinctly divided into three functional regions
(Figs. 7G and H and 10I and J). In sharp contrast to all
earlier semaphoronts, anterior part now including the
segment of the first walking legs and being called the
cephalosoma (already preformed larval stage VI). Sub-
sequent middle portion comprising the three remaining
walking-leg bearing segments. Males now with an extra pair
of clearly segmented legs in front of the walking legs, the
Fig. 6. Developmental Stage IV of Pycnogonum litorale (Stro ¨m, 1762). A. Ventral view showing the first pair of walking legs (app 4). B. Dorsal view showing
the incompletely developed humps. C. View of ocular tubercle. D. Antero-lateral view of head with chelicerae. E. Appendage three, showing the articulation
(encircled). F. Lateral view of specimen, with focus on the fourth appendage. G. Ventral view of head, with its three pairs of appendages.
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