J. Zool., Lond.

(2004) 264, 333–354

C 2004 The Zoological Society of London Printed in the United Kingdom DOI:10.1017/S0952836904005837

Studies of embryonic development and the reproductive cycle

in ovoviviparous Australian Onychophora (Peripatopsidae)

Muriel H. Walker1 * and Noel N. Tait2

1 Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, U.K.
2 Department of Biological Sciences, Macquarie University, Sydney 2109, New South Wales, Australia
(Accepted 14 April 2004)

Abstract
Embryos of four ovoviviparous species of Australian onychophorans were examined to establish the process of
their development, their reproductive cycle and to estimate the gestation period. The process of development is the
same in all four species with, in the early embryo, segment halves separated by broad bands of extra-embryonic
ectoderm and the embryos growing by the posterior addition of segments. Seven developmental stages (I–VII) can
be clearly identified from external morphology. Analysis of the developmental stages of the embryos present in
females collected throughout a calendar year has allowed estimation of the time taken for each developmental stage
and demonstrated that there is an annual reproductive cycle with a total gestation period of c. 12 months. Stages I
and VII are prolonged, the latter related to the late completion of midgut development. The simultaneous presence
of stage I and stage VII embryos at certain times of year indicates an overlap of generations within the uteri, which
is more extensive for some species than others. As the stage VII embryos of one cohort complete their development,
enlarged oocytes are released from the ovaries to become fertilized as they pass the seminal receptacles and enter
the uteri to start their embryonic development to form the next cohort. Cephalofovea clandestina, Phallocephale
tallagandensis and Ruhbergia bifalcata have head structures in males, Euperipatoides rowelli does not. The presence
of embryos at all times of year in the uteri of mature females has implications for mating and sperm storage that
are discussed.

Key words: Onychophora, embryology, reproduction, gestation period

INTRODUCTION Embryonic development in the Peripatidae was first

The diversity of reproductive strategies displayed by ony-
chophorans is remarkable for such a small phylum whose
members are otherwise conservative in their morphology
and general way of life. In neotropical members of
the Peripatidae, development is viviparous with embryos
attached to a placenta for part of their development
(Anderson & Manton, 1972; Campiglia & Walker, 1995).
The eggs of these peripatids are small, yolk-free and lack
enveloping membranes (Huebner & Lococo, 1994). On
the other hand, species of Peripatidae from south-east
Asia are ovoviviparous with large, yolky eggs surrounded
by membranes (Evans, 1901). In the Peripatopsidae,
development is oviparous or ovoviviparous and the eggs
are usually large and yolky and surrounded by an inner
vitelline and an outer chorionic membrane (Ruhberg,
1985; Reid, 1996), while in South African Peripatopsidae,
the eggs are less yolky (Anderson, 1973).
∗All
correspondence to: M. H. Walker.
E-mail: muriel.paterson2@btopenworld.com
studied in Epiperipatus trinidadensis and Macroperipatus
torquatus (Kennel, 1884, 1885) and later Anderson &
Manton (1972) described development of the placenta in
these species and suggested it was analogous to the mam-
malian yolk-sac placenta. More recently, development
in the viviparous Peripatus acacioi has been described
(Walker & Campiglia, 1988; Campiglia & Walker, 1995).
These studies demonstrated that the structure of the
placenta in viviparous onychophorans is not uniform. In
P. acacioi, the placenta is analogous to the mammalian
non-invasive epitheliochorial placenta (Walker &
Campiglia, 1995). They also noted cyclic changes in the
uterus and, depending on the time of year, one or two
cohorts of embryos were present. Development in South
African Peripatopsidae was observed by Sedgwick (1885–
1888) who defined a series of developmental stages for
Peripatopsis capensis. Later, Manton (1949) provided a
detailed description of embryonic development in four
species of Peripatopsis from South Africa, P. balfouri,
P. capensis, P. moseleyi and P. sedgwicki. Studies of
early development in Peripatoides novaezealandiae were
334 M. H. WALKER AND N. N. TAIT
undertaken by Sheldon (1888, 1889) but the very yolky
eggs were not amenable to 19th century histological
technique (Anderson, 1973). With the exception of
the South African peripatopsid Opisthopatus cinctipes
(Walker, 1992b, 1995), the process of segment formation
in both families follows a similar pattern. As the mouth
and anus form, thickened lateral bands of the blastoderm,
separated dorsally and ventrally by extra-embryonic
ectoderm, develop down each side of the embryo. These
bands differentiate paired, segmental swellings with inter-
segmental grooves (Anderson, 1973). The body elongates
by the proliferation of segments from a posterior growth
zone until the characteristic number of segmental swell-
ings for the species has formed. Variations in the
development of the external form relate to the degree
of separation of the segment halves by extra-embryonic
ectoderm (Anderson, 1973). In P. novaezealandiae, which
has large, yolky eggs, the anterior segment halves are
widely separated (Sheldon, 1888), but in the viviparous
P. acacioi, which has small yolk-free eggs, the segment
halves are separated by only narrow bands of extra-
embryonic ectoderm (Walker & Campiglia, 1988;
Campiglia & Walker, 1995). In O. cinctipes, the embryo
elongates without any visible external or internal segment
boundaries, with only ectoderm and endoderm present.
Mesodermal somites are not formed until elongation is
complete (Walker, 1992b, 1995).
Onychophorans also display different methods of in-
semination (for review see Tait & Norman, 2001). Within
the Peripatidae, it has been assumed that sperm are
introduced into the female vagina by direct gonopore/
gonopore contact (Walker & Campiglia, 1998). On the
other hand, dermal insemination has been documented as
a common mode of insemination in the Peripatopsidae
(Manton, 1938a,b; Walker, 1992a; Walker & Campiglia,
1998). However, a number of Australian species of peri-
patopsids display unusual structures on the heads of males
(head structures) that are known to deliver sperm to the
female genital opening in two species (Tait & Briscoe,
1990; Tait & Norman, 2001).
In the past decade, there has been renewed interest in
onychophorans and particularly in the Australian fauna,
including studies on aspects of development in both
oviparous (Brockmann, Mesibov & Ruhberg, 1997) and
ovoviviparous species (Brosius-Roggenbuck & Ruhberg,
2000; Sunnucks et al., 2000). Sunnucks et al. (2000)
have undertaken an extensive study of the reproduc-
tive biology of Euperipatoides rowelli. Recent studies
by Eriksson & Budd (2000) and Eriksson, Tait & Budd
(2003) have investigated the structure and development
of the head in Euperipatoides kanangrensis, with
particular emphasis on the central nervous system. These
studies have clarified our understanding of the homo-
logies of the head appendages of onychophorans and
arthropods and provide further evidence for the clade
Ecdysozoa (Aguinaldo et al., 1997; Schmidt-Raesa et al.,
1998).
Previous detailed studies of embryonic development in
onychophorans have each focused on one or two species
(Anderson & Manton, 1972; Walker & Campiglia, 1988;
Campiglia & Walker, 1995). Studies by Campiglia &
Walker (1995) demonstrated the importance of examining
the uteri of onychophorans at different times of the
year to establish the full process of development and
also to estimate the gestation period. In the present
study, embryos from four species, each from separate
genera, of Australian Peripatopsidae, were examined
from females collected at intervals throughout a calendar
year. Morphological (Reid, 1996) and molecular (Gleeson
et al., 1998) analyses indicate that although the three
species that possess head structures in males seem to be
closely related, the fourth species lacking this structure
belongs to a clade that is widely divergent. From the
material studied here, a series of developmental stages
based on external morphology is defined and estimations
of the gestation period have been made.
MATERIALS AND METHODS
Females from 4 species of Onychophora were collected at
intervals throughout a calendar year. At least 4 mature
females of each species were hand collected on each
sampling date from decaying eucalypt logs at the fol-
lowing locations: Euperipatoides rowelli, Reid 1996,
and Phallocephale tallagandensis, Reid 1996, from South
Forest Way, Tallaganda State Forest, near Captains Flat
(35◦ 38 S, 149◦ 31 E); Ruhbergia bifalcata, Reid 1996,
from the Tinderry Mountains near Michelago (35◦ 44 S,
149◦ 31 E), both localities south of Canberra: Cephalo-
fovea clandestina, Reid 1996, from Mini Mini Road,
near Hampton in the Great Dividing Range (33◦ 43 S,
150◦ 03 E) west of Sydney. Cephalofovea clandestina,
P. tallagandensis and R. bifalcata possess head structures
in males (Reid, 1996).
Females were weighed, relaxed and killed with ethyl
acetate vapour and the reproductive tract dissected in
phosphate buffer. The lengths of the paired uteri and the
number of embryos that each contained were recorded.
The embryos were removed from each uterus in sequence
and left in buffer for c. 10 min. The 2 membranes
surrounding each embryo at stage III (see below) of
development or older were removed, using mounted
entomological pins, before fixation. It was not possible
to remove the membranes from earlier stages without
damage to the embryo. These embryos were placed in
fixative for 1 h and then the membranes pierced with
entomological pins to assist penetration of the fixative.
All embryos were fixed in half strength Karnovsky’s
fixative (Karnovsky, 1965) for 24 h, dehydrated through
an ethanol series and stored in 70% ethanol at 4 ◦ C.
Embryos were further processed for scanning electron
microscopy (SEM). Where possible, the membranes were
removed from the fixed stage II embryos, although this
did result in some damage. Specimens were further
dehydrated to 100% ethanol and passed through an
ethanol/acetone series to 100% acetone before drying
using a Samdri critical point drier. For each species at least
10 embryos at each stage of developmental stages II–VII
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora
were examined in detail using SEM. Embryos were
mounted on aluminium stubs using double-sided sticky
tabs, sputter-coated with gold/palladium and examined
using a Cambridge S100 or an Hitachi S-3000H scanning
electron microscope.
Some embryos were processed for ultramicrotomy,
and subsequent light microscopy (LM) and transmission
electron microscopy (TEM). Specimens were rehydrated
to water and placed in 1% osmium tetroxide in phosphate
buffer for 1 h. They were then dehydrated through an
ethanol series and surface-embedded in Spurr’s low-
viscosity embedding resin (Walker & Roberts, 1982).
Semi-thin sections (1 µm) were cut with a diamond knife
using a Reichert Ultracut E microtome. Sections were
mounted on glass slides, stained with 1% toluidine blue
in 1% borax and examined using an Olympus BHS
microscope. For TEM, ultra-thin sections were mounted
on copper grids, stained with uranyl acetate and lead
citrate and examined using a Jeol 100CX transmission
electron microscope.
To make a qualitative assessment of the timing of
development throughout the year, the developmental
stages (I–VII) present in the uteri of all females were
recorded: C. clandestina (n = 826 embryos), E. rowelli
(n = 784 embryos), P. tallagandensis (n = 787 embryos)
and R. bifalcata (n = 500 embryos). The state of develop-
ment of the oocytes attached to the paired but fused ovaries
was also noted for most females.
Four to 6 females of each species were maintained
in the laboratory. Each female was housed in a 50 ml
specimen container with a base of packed, damp peat and
fed on isopods. They were examined at approximately
weekly intervals and dates of births to each female
recorded.
RESULTS
Stages of embryonic development
Seven stages of embryonic development, I–VII, are des-
cribed based on the external morphology of the embryo.
These stages are the same for all four species examined.
While the features that identify each stage are illustrated
here using SEM images, embryos can be assigned to a
developmental stage using a dissecting microscope. The
sex of embryos could not be determined.
Fresh material
The appearance of the developing embryos, through their
transparent surrounding membranes, provided an indica-
tion of their stage of development. When several stages of
development were present within the uteri, the embryos
were always arranged in sequence with the earliest stages
located proximally and the most mature distally towards
the vagina. Stage I embryos are oval and a uniform yellow
colour, indicative of a large amount of yolk. At stage II, the
335
preoral segment is seen as a slight anterior protrusion from
the oval shape of the embryo and two bands of blastoderm
form along the sides of the embryo. These structures can
be identified by their white colour, compared to the rest
of the embryo, which is filled with yellow yolk. In stage
III, the embryo has elongated and is flexed ventrally with
the mouth level with the anus. As the embryos increase
in length, they remain flexed and, when all the segments
have formed, the body coils to accommodate it within
the surrounding membranes, a thicker outer chorion and
a very thin inner vitelline membrane. Older embryos
usually straighten when removed from the confines of their
surrounding membranes. In a few early embryos, there
seemed to be small amounts of yolk granules beneath the
egg membranes but external to the embryo, but this was
not a consistent feature.
Stage I embryos
Development from fertilized egg to the formation of the
preoral segment is defined as stage I (not illustrated).
These embryos could not be removed intact from their
surrounding membranes.
Stage II embryos
Stage II embryos can be removed from the surrounding
egg membranes, but are very fragile and most suffered
some damage. The early stage II embryo is oval with
bands of blastoderm on either side separated by dorsal
and ventral extra-embryonic ectoderm covering the yolk
mass (Fig. 1a). The developing preoral segment consists
of two areas raised up from the anterior end of the yolk
mass with a groove in the mid-line where the two halves
of the segment meet (Fig. 1a). The mouth is located on the
ventral surface below the fusion between the two halves
of the preoral segment (Fig. 1a). Posterior to the preoral
segment, the two bands of blastoderm are raised from the
surface of the yolk mass. They extend along the sides of the
embryo and curve around to terminate and fuse ventrally
midway along the length of the yolk mass (Fig. 1a).
The anus is located at the posterior junction of the two
bands where they are flattened against the yolk mass.
Shallow grooves in the blastoderm bands posterior to
the preoral segment indicate the boundaries of the jaw
and oral papilla segments (Fig. 1a). In a slightly later
stage II embryo, the yolk mass and the blastoderm bands
have increased in length from the posterior growth zone
(Fig. 1b). The antennae start to form as small protrusions
from the dorsal surface of the preoral segment (Fig. 1b).
The boundaries between the preoral, jaw, oral papilla and
first pair of walking leg segments are more clearly marked
and the positions of the next four segments are also visible
as shallow grooves in the blastoderm bands. The posterior
ends of the blastoderm bands are wrapped around the yolk
mass. The dorsal and ventral extra-embryonic ectoderm
is very thin and outlines of yolk granules are seen beneath
it (Fig. 1b).
336 M. H. WALKER AND N. N. TAIT

(b) dee
(a)
wl
op
op
j
ym vee
j an

po

A gz

M A

an
(c) (d) an
e po

j M
gz
op an
A

dee vee gz
dee j

op

vee

Fig. 1. SEM micrographs. (a) Stage II Cephalofovea clandestina embryo with blastoderm bands on either side of the embryo separated
by the yolk mass (ym). The mouth (M) is located mid-ventrally behind the preoral segment ( po). The boundaries of the future jaw ( j)
and oral papillae (op) segments are marked by shallow grooves in the blastoderm (arrow). A, anus. Scale bar = 250 µm. (b) Later Stage II
C. clandestina embryo in which the antennae (an) have started to form. The boundaries of the jaw ( j), oral papilla (op) and first pair
of walking legs (wl) are more clearly marked and indications (arrows) of the next four segment boundaries are present. No segment
boundaries are seen on the blastoderm in the posterior growth zone (gz). Profiles of yolk granules are seen through the thin dorsal and
ventral extra-embryonic ectoderm (dee, vee) separating the blastoderm bands. A, anus. Scale bar = 250 µm. (c) Stage III Euperipatoides
rowelli embryo that is folded mid-ventrally and the oral papillae (op) and leg pairs 1–7 are starting to elongate. The eye (e) is a small
pit located laterally on the preoral segment ( po) below the base of the antenna (an). The ectoderm (arrows) is extending out from the
blastoderm bands and obscuring the outlines of yolk granules that are seen below the dorsal and ventral extra-embryonic ectoderm
(dee, vee). gz, growth zone; j, jaw; M, mouth. Scale bar = 0.5 cm. (d) Stage III E. rowelli embryo. The antennae are triangular and flattened
over the dorsal surface of the preoral segment. The eye pit (e) is located laterally on the preoral segment. The ectoderm (arrows) is
extending out from the blastoderm bands obscuring the profiles of yolk granules beneath the extra-embryonic ectoderm (dee, vee). A,
anus; gz, growth zone; j, jaw; op, oral papilla. Scale bar = 0.5 cm.

Stage III embryos
The stage III embryo has elongated further and is
folded mid-ventrally. Whereas in the stage II embryo the
blastoderm bands curved around the yolk mass, the yolk
mass has now extended with the blastoderm bands on
either side of it (Fig. 1c). The jaw, oral papilla and first six
pairs of walking leg segments are seen as a graded series
of lateral protrusions from the blastoderm bands (Fig. 1c).
The boundaries of the segments that bear walking leg
pairs 6–10 are indicated by small indentations across the
blastoderm bands (Fig. 1c). At the posterior growth zone,
segment boundaries are not visible on the blastoderm
bands (Fig.1d). The anus is seen at the posterior end
where the two blastoderm bands meet (Fig. 1d). The
two halves of the preoral segment are now more swollen
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora
and the antennae are triangular and flattened over the
dorsal surface of the preoral segment (Fig. 1d). The
developing eye is a small pit located laterally below the
base of the antenna on each half of the preoral segment
(Fig. 1c,d). The embryonic ectoderm immediately
adjacent to the blastoderm bands is now thicker and
profiles of yolk granules are not detectable beneath it
although they are seen beneath the rest of the extra-
embryonic ectoderm (Fig. 1c,d).
Stage IV embryos
In the stage IV embryo, further segments are added at
the posterior growth zone and, by the end of this stage,
all segments are present. The segment halves are still
separated by extra-embryonic ectoderm, an extensive band
on the highly convex dorsal surface and a narrower band
on the concave ventral surface (Fig. 2a). In the embryo
illustrated in Fig. 2a, oral papillae and leg pairs 1–7
are elongating in an anterior–posterior sequence, with
leg pair 1 longer than leg pair 7. Leg pairs 8–14 are
present as lateral conical bulges that diminish in size
towards the posterior end, and leg pair 15 is not yet
formed (Fig. 2a). Leg appendages have a conical distal
end and increase in length by the proximal addition of
annuli. The future openings of the nephridiopores are seen
on the fourth and fifth pairs of walking legs by the time
they have elongated and there are four distinct annuli on
each leg (Fig. 2b). The antennae, composed of a series of
annuli, are elongated and curl over the preoral segment
(Fig. 2a,c). The preoral segment halves have increased
in size and overhang the mouth opening (Fig. 2a,c). The
pit marking the position of the developing eye is now
represented by a small indentation (Fig. 2d). The future
jaws have become rounded and the first two lobes of the
oral lips start to form laterally between the base of the
preoral segment halves and the jaws (Fig. 2a). The jaws
start to migrate towards the ventral mid-line and become
distinct rounded structures lacking the annuli seen in other
anterior appendages at this stage (Fig. 2a,c). The number
of lobes in the oral lips increase and they extend back
laterally along the sides of the jaws (Fig. 2c). The base
of each jaw becomes narrowed to a stalk that appears to
pass down into the body of the embryo (Fig. 2c,d) as
the lips extend below the jaws (Fig. 2d). The tongue is
first seen above the mouth between the two halves of the
preoral segment as the lips extend backwards around each
jaw (Fig. 2c). The openings of the slime glands are first
seen as indentations at the distal ends of the oral papillae
(Fig. 2a) that subsequently deepen (Fig. 2c). The openings
of the salivary glands appear laterally on the ventral
body surface level with the bases of the oral papillae
(Fig. 2a,c,f ). The frontal processes, which will form the
anteriormost oral lips, appear as rounded protrusions from
the anterior dorsal aspect of each half of the preoral
segment (Fig. 2e). Subsequently, as the antennae and
anterior walking legs continue to elongate, a groove
develops in each jaw starting to distinguish the two jaw
blades (Fig. 2f ).
337
Stage V embryos
The stage V embryo is more elongated and adopts a coiled
posture to accommodate itself within the surrounding
membranes (Fig. 3a). The body is usually folded between
walking leg pairs 3 and 6 and again at the level of walking
leg pairs 10 and 11, with the posterior end of the body
folded over the head. In the early stage V embryo, there
is a distinct band of ventral extra-embryonic ectoderm
(Fig. 3b), and a broader band of dorsal extra-embryonic
ectoderm that both diminish in width during this stage of
development as the ectoderm extends towards the dorsal
and ventral mid-lines. In the early stage V embryo, a small
indentation appears on the ventral surface of the last pair
of legs even before they begin to elongate (Fig. 3b). As
the last pair of legs starts to elongate, these indentations
become small pits, the future openings of the genital ducts
(Fig. 3c). Each walking leg elongates by the addition of
annuli and, when fully elongated, each leg has six annuli
and a distal foot (Fig. 3b). The central lumen of the
body in the stage V embryo is still packed with yolk and
there is no evidence of a lumen in the developing midgut
(Fig. 3b). The jaws have migrated further towards the
ventral mid-line (Fig. 3d). The lips extend below the jaws
and start to curve in towards the mid-line (Fig. 3d). The
openings of the salivary glands, still level with the bases of
the oral papillae, have also moved closer to the mid-line
and become crescent-shaped (Fig. 3e). During stage V,
the body lengthens as the distance between the bases of
the walking legs increases (Fig. 3d). In the later stage V
embryo, the ventral extra-embryonic ectoderm is virtually
absent (Fig. 3d,e) but some of the dorsal extra-embryonic
ectoderm still persists (Fig. 3e). In the late stage V embryo,
the cerebral grooves start to form. They appear first as
small anterior indentations on the preoral segment halves
(Fig. 3d) which deepen (Fig. 3e). At this stage, there are
microvilli on the outer surface of the embryonic ectoderm
and an embryonic cuticle starts to form over the surface
of the embryo attached to spot desmosomes at the tips of
the microvilli (not illustrated).
Stage VI embryos
In the early stage VI embryo, ventral extra-embryonic
ectoderm is no longer present although there is still a nar-
row band of dorsal extra-embryonic ectoderm (Fig. 3f ).
Papillae start to form on the body surface, seen as small
swellings that first appear laterally on the dorsal surface
of the body beneath the embryonic cuticle (Fig. 4a). The
mid-ventral surface of the body is smooth in comparison
(Fig. 4b). The papillae increase in number and eventually
form linear arrays on the dorsal and lateral surfaces of
the body (Fig. 4b). The smooth area on the mid-ventral
surface decreases in size as the lateral annuli between
the leg pairs extend further towards the ventral mid-line
(Fig. 4c,d), and becomes restricted to a series of rounded
masses, the ventral organs (Fig. 4b). Where the specimens
have been fractured in the mid-region, the body cavity is
still filled by the developing gut, which is packed with
338 M. H. WALKER AND N. N. TAIT

(a) (b) n
an
po A f

M
l (d) an

j sg

op
vee

dee j

(f)
(c) t (e)
an
an

po
po fp l
j
l
an

vee j sg
op dee op

sg

Fig. 2. SEM micrographs. (a) Stage IV Euperipatoides rowelli embryo in which the oral papillae (op) and anterior 7 pairs of walking
legs are elongating and the next 7 pairs of legs are indicated by conical projections from the blastoderm bands. The ectoderm (arrows) is
extending towards the mid-line although distinct bands of extra-embryonic ectoderm (dee, vee) remain. The antennae (an) have elongated.
The oral lips (l) have started to form laterally below the preoral segment ( po). An indentation at the end of the oral papilla (op) marks the
future opening of the slime gland. The openings for the salivary glands (sg) are located laterally, level with the bases of the oral papillae.
A, anus; j, jaw; M, mouth. Scale bar = 500 µm. (b) Annuli (arrows) on an elongating fourth walking leg of a stage IV E. rowelli embryo.
The opening of the nephridiopore (n) is located proximal to the developing foot (f ). Scale bar = 25 µm. (c) Ventral view of the anterior
end of a stage III E. rowelli embryo. The preoral segment halves ( po) overhang the mouth with the developing tongue (t) between them.
The oral lips now extend from the base of the preoral segment to below the jaws ( j). The base of the jaw is narrowed to form a stalk. The
future opening for the slime gland has deepened (black arrow) at the end of the oral papilla (op). The band of ventral extra-embryonic
ectoderm (vee) is narrower as the ectoderm (white arrows) spreads towards the mid-line. an, antenna; sg, salivary gland openings. Scale
bar = 250 µm. (d) Head of a stage IV E. rowelli embryo where a small lateral indentation (arrow) on the preoral segment marks the position
of the eye. an, antenna; j, jaw; l, oral lips. Scale bar = 100 µm. (e) Frontal processes (fp) on the anterior dorsal surface of the preoral
segment halves of a stage IV Phallocephale tallagandensis embryo. an, antenna; dee, dorsal extra-embryonic ectoderm. Scale bar = 100 µm.
(f ) Ventral surface of the head of a stage IV E. rowelli embryo. There is a groove on the surface of the jaw ( j) indicative of the formation
of the two jaw blades. an, antenna; l, oral lips; op, oral papilla; sg, salivary gland opening. Scale bar = 250 µm.

yolk and still lacks a lumen (Fig. 4d). In the early stage
VI embryo, claws start to develop on the feet indicated
by a groove seen beneath the embryonic cuticle at the end
of the conical-shaped foot (Fig. 4a). This occurs in an
anterior–posterior sequence and, in the embryo illustrated
in Fig. 4a, claws have still to form on the last three pairs
of walking legs, but in the late stage VI embryo all the
feet and claws are formed. The body papillae continue to
develop and profiles of developing sensory spines can be
seen outlined beneath the embryonic cuticle (Fig. 4d,f ).
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora 339

(a) dee (c) (e)

dee

an

an l
em

cg

(b)
y sg
j
op

vee
(d) (f) fp cg
cg

t
l
f

a sg
j
l

sg j

op

dee

Fig. 3. SEM micrographs. (a) Stage V Cephalofovea clandestina embryo in the coiled position adopted within the egg membranes. The
dorsal extra-embryonic ectoderm (dee) is narrower as the ectoderm (arrows) spreads towards the mid-line. an, antenna; em, remnant of egg
membranes) Scale bar = 250 µm. (b) Fractured early stage V Ruhbergia bifalcata embryo showing the midgut region packed with yolk (y).
All legs, apart from the last pair, are elongating and each has a series of annuli (a) and a distal conical foot (f ). At the bases of the last pair
of walking legs small indentations mark the position of the genital duct openings (black arrows). The ventral extra-embryonic ectoderm
(vee) is decreasing as the ectoderm spreads towards the mid-line (white arrows). Scale bar = 0.5 cm. (c) Ventral surface of the posterior
end of a stage V Phallocephale tallagandensis embryo. The last pair of walking legs is now elongating, the openings for the genital ducts
(white arrows) are now distinct pits and the ectoderm has spread nearer to the ventral mid-line (black arrows). A, anus. Scale bar = 100 µm.
(d) Ventral surface of the anterior end of a later stage V C. clandestina embryo. The body has lengthened due to an increase in distance
between bases of walking legs. The cerebral grooves (cg) start to appear as indentations at the anterior of each half of the preoral segment.
Ventral extra-embryonic ectoderm is absent (arrow). j, jaw; l, lips; op, oral papilla; sg, salivary gland openings. Scale bar = 250 µm.
(e) Later stage V Euperipatoides rowelli embryo in which the cerebral grooves (cg) are forming. The oral lips (l) have extended below
the jaws ( j) towards the crescent-shaped salivary gland openings (sg). Extra-embryonic ectoderm is virtually absent on the ventral surface
although a band of dorsal extra-embryonic ectoderm (dee) remains. an, antenna; t, tongue; op, oral papilla. Scale bar = 250 µm. (f ) Stage VI
C. clandestina embryo in which the cerebral grooves (cg) are now longitudinal slits on the surface of the preoral segment with the frontal
processes (fp) located above them. The crescent-shaped opening of the salivary gland (sg) are closer to the ventral mid-line and are level
with the lower margins of the oral lips (l). The ventral surface is smooth but annuli are now present on the lateral surface of the body
(arrow). dee, dorsal extra-embryonic ectoderm; j, jaw; t, tongue. Scale bar = 250 µm.

Using TEM, the developing adult cuticle is seen below
the embryonic cuticle, separated from it by a layer of
exuvial fluid (Fig. 4e). At the posterior end of the body, the
openings of the genital ducts continue to migrate towards
the ventral mid-line, level with the bases of the last pair of
walking legs (Fig. 4d,f ). In the late stage VI embryo,
they merge adjacent to the ventral mid-line, which is
marked by a longitudinal fold in the embryonic cuticle
(Fig. 4g). This ventral fold of the embryonic cuticle starts
in the mouth region (Fig. 4b,c) and eventually extends the
340 M. H. WALKER AND N. N. TAIT

A (b)
(a) op l

sg
vo
c

dee p
fp
t
cg j

c p

fp
(c) (d)

cg y
t
A
l

gd vo ec
sg

(e) (f) (g)

ss
ec

vo gd
n vo A

ec gd
ec

Fig. 4. (a–d,f,g) SEM micrographs; (e) TEM micrograph. (a) Dorsal surface of an early stage VI Phallocephale tallagandensis embryo.
Developing body papillae ( p) are seen as a series of small swellings on lateral aspects of the dorsal surface. The claws (c) are starting to form
on all but the last three pairs of legs. A, anus; dee, dorsal extra-embryonic ectoderm. Scale bar = 250 µm. (b) Stage VI P. tallagandensis
embryo with rows of developing papillae (p) on the dorsal and lateral surfaces of the body. The ventral organs (vo) in comparison are smooth
and the ventral mid-line is marked by a fold in the embryonic cuticle (arrow). cg, cerebral grooves; fp, frontal processes; j, jaw; l, lips;
t, tongue; op, oral papilla; sg, salivary gland openings. Scale bar = 0.5 cm. (c) Anterior end of an early stage VI P. tallagandensis embryo.
The smooth area on the ventral surface is restricted by lateral annuli between leg pairs (white arrows). Cerebral grooves (cg) form on the
preoral segment, the jaws (j) continue to migrate towards the mid-line that is marked by a fold in the embryonic cuticle (black arrow).
The oral lips (l) extent towards the salivary gland (sg) openings. fp, frontal processes; t, tongue. Scale bar = 250 µm. (d) Posterior end of
a stage VI Cephalofovea clandestina embryo. The openings to the genital ducts (gd) have migrated towards the ventral mid-line that is
marked by a fold in the embryonic cuticle (ec) which bisects the ventral organs (vo). The midgut still lacks a lumen and is still packed
with yolk (y) and profiles of developing sensory spines are seen on the body surface (arrows). A, anus. Scale bar = 100 µm. (e) Cuticle
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora 341

length of the body (Fig. 4d). During stage VI, the oral
lips continue to expand in a posterior direction and start
to curve inwards towards the ventral mid-line level with
the bases of the oral papillae (Figs 4c & 5a). The now
crescent-shaped openings for the salivary glands migrate
nearer to the ventral mid-line (Fig. 4c). As the mouth
starts to close, and the base of the lips become closer to
the mid-line, the openings to the salivary glands move
into the mouth opening (Fig. 5a). The jaws also sink
further into the mouth as they come closer to the mid-line
(Figs 4b,c & 5a). The tongue is located anteriorly in the
mouth between the two halves of the preoral segment
(Fig. 4c). The cerebral grooves deepen and form a slit
on the ventral surface of the preoral segment halves
(Fig. 3f). These slits deepen further as the ventral organ
of the preoral segment is moved into the interior (Fig. 5a),
and the two halves of this segment start to fuse. As
this takes place, the frontal processes that were located
at the anterior of the preoral segment halves (Fig. 4c)
move to a more ventral position at the anterior limits of
the cerebral grooves (Fig. 5a). As the jaws move nearer
to the mid-line, the developing tongue is more clearly
distinguished (Fig. 5a). In the later stage VI embryo,
additional swellings appear associated with the frontal
processes (Fig. 5a) as they migrate towards the top of
the mouth. The frontal processes are still separated from
the upper margin of the mouth (Fig. 5c) but eventually
join the anterior margin of the oral lips above the tongue
(Fig. 5b). The jaws and tongue still protrude slightly from
the mouth (Fig. 5b). The trunk ventral organs become
progressively smaller and the oral lips approach the ventral
mid-line (Fig. 5c). In species displaying a head structure,
the beginnings of development of this structure can be
seen in the stage VI embryo as indentations on the dorsal
surface of the head. In P. tallagandensis, there is an area on
the dorsal surface of the head where developing papillae
are absent with a longitudinal depression at its centre
(Fig. 5d).
(Fig. 5g). In species with head structures, these are clearly
visible as distinct structures on the dorsal surface of
the head (Fig. 6a–c). In R. bifalcata (Fig. 6a), the head
structure at this stage is in the form of a slight depression
at the base of which is a pair of lobes. In C. clandestina
(Fig. 6b), the head structure is in the form of a deeper
depression surrounded by a ring of large papillae bearing
sensory spines. In P. tallagandensis (Fig. 6c), the head
structure forms a central disc of lobe-like papillae sur-
rounded by a ring of enlarged papillae bearing sensory
spines. Even at this stage it is not possible to sex embryos
as the head structures are not yet fully developed. In
the stage VII embryo, the eye is fully formed and is
distinguishable from the surrounding body papillae by
its smooth surface (Fig. 6d). The nephridiopores are
clearly seen on the fourth and fifth leg pairs proximal
to the spine-covered foot pads (Fig. 6e). Anaesthetizing
the mother with ethyl acetate frequently results in slime
being extruded from the oral papillae of the stage VII
embryos in her uterus (Fig. 6d) indicating that the slime
glands are fully functional in the embryo at this stage
of development. At the posterior end, the single genital
opening is located mid-ventrally between the last pair
of legs (Fig. 6f ). The remnants of the trunk ventral
organs are seen as small smooth areas on the mid-ventral
surface (Fig. 6f ). Although externally the embryo seems
to be fully formed, development is not yet completed.
In early stage VII embryos, 1 µm sections indicate that
there is a distinct lumen in both the foregut and hindgut
that is lined with cuticle (Fig. 6g). The midgut region
in the early stage VII embryo is, however, still packed
with yolk and no clear lumen is visible (Fig. 6h). In
very late stage VII, embryos there is a distinct lumen
that is not lined by cuticle and only remnants of yolk
are present within the epithelial cells lining the midgut
(Fig. 6i).
Ovaries and size of oocytes

Stage VII embryos
In the stage VII embryo, the papillae on the body surface
are fully developed (Fig. 5e) and the sculpturing of the
scales on the papillae is evident (Fig. 5f). The embryonic
cuticle is frequently shed when the embryos are removed
from the uterus and their surrounding membranes, but
some remnants of it are seen over the claws (Fig. 5f).
The oral lips and the frontal processes now completely
surround the mouth and the jaws are no longer visible,
but the tongue can be seen at the back of the open mouth
Examination of the ovaries, of females used in this study,
revealed variation in the size of oocytes. All ovaries bore
small (5 × 7 µm) transparent oocytes that lacked yolk and
were located close to the ovarian epithelium (Fig. 7).
Oocytes increase in size as yolk starts to accumulate and,
as they do so, they are raised on follicular stalks above the
ovarian surface (Fig. 7). For the purposes of this study, the
oocytes that were in the size range 1500–1750 µm × 750–
1000 µm were recorded as large oocytes (see Tables 2–5).
Usually only two or three oocytes of this size were present
in any female.

layers of a stage VI P. tallagandensis embryo. A layer of exuvial fluid (*) separates the relatively smooth embryonic cuticle (ec) from
the more sculptured adult type cuticle (arrow) covering the developing papillae on the body surface. n, nucleus. Scale bar = 1 µm.
(f ) Posterior end of a later stage VI C. clandestina embryo. The openings of the genital ducts (gd) are now close to the ventral mid-line, the
ventral organs (vo) are reduced in size and the sensory spines (ss) more developed. A, anus; ec, embryonic cuticle. Scale bar = 100 µm.
(g) Late stage VI C. clandestina embryo in which the openings to the genital ducts (gd) have fused in the mid-line and the ventral organs
(vo) are further reduced in size. ec, embryonic cuticle. Scale bar = 100 µm.
342 M. H. WALKER AND N. N. TAIT

(a) fp fp
(b)

cg

t
t

j
l
l

sg
j
vo

fp
(c) t (d)

vo

ec
(f) (g) op
(e)

t
l

fp
vo

Fig. 5. SEM micrographs. (a) Stage VI Phallocephale tallagandensis embryo. The cerebral grooves (cg) bisect the two halves of the
preoral segment which have now fused in the mid-line. The posterior margins of the oral lips (l) are close to the mid-line and enclose the
salivary gland openings (sg) within the mouth. The frontal processes (fp) are still located anterior to the cerebral grooves. j, jaw; t, tongue;
vo, ventral organs. Scale bar = 250 µm. (b) Stage VI Cephalofovea clandestina embryo. The cerebral grooves are closed and the frontal
processes (fp) are now in closer proximity to the oral lips (l) which surround the jaws ( j) and tongue (t). Scale bar = 125 µm. (c) Dorsal
view of the anterior end of a stage VI P. tallagandensis embryo. The frontal processes (fp) are approaching the oral lips (l) that enclose
the jaws ( j) and tongue (t). The ventral organs (vo) are reduced in size. Scale bar = 100 µm. (d) Developing head structure (arrow) of
a stage VI P. tallagandensis embryo. Scale bar = 500 µm. (e) Stage VII Euperipatoides rowelli embryo in which the body papillae and
sensory spines are fully developed. Scale bar = 100 µm. (f ) Foot of a stage VII E. rowelli embryo showing the sculpturing of the scales
of the papillae (arrow) and with remnants of embryonic cuticle (ec) over the claws. Scale bar = 50 µm. (g) Dorsal view of the head of a
stage VII R. bifalcata embryo. The tongue (t) is seen within the mouth and the frontal processes (fp) are fused with the anterior ends of
the oral lips (l) surrounding the mouth. The ventral organs (vo) are greatly reduced in size. op, oral papilla. Scale bar = 250 µm.
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora 343

(b) (c)
(a)

fp
(d) (e) (f) A

e
an

op vo

(h) wl (i)
(g)

lu

Fig. 6. (a–f,h) SEM micrographs; (g, i) light micrographs. (a) Head structure of a stage VII Ruhbergia bifalcata embryo. Scale
bar = 250 µm. (b) Head structure of a stage VII Cephalofovea clandestina embryo. Scale bar = 100 µm. (c) Head structure of a stage
VII Phallocephale tallagandensis embryo. Scale bar = 125 µm. (d) Head of a stage VII Euperipatoides rowelli embryo showing the eye
(e), antennae (an) and an oral papilla (op) from which slime (arrow) has been extruded. Scale bar = 250 µm. (e) Nephridopore opening
(arrow) on the ventral surface of the fourth walking leg of a stage VII E. rowelli embryo. fp, foot pad. Scale bar = 50 µm. (f). Genital
aperture (arrow) and remnants of a ventral organ (vo) at the posterior end of a stage VII R. bifalcata embryo. A, anus. Scale bar = 100 µm.
(g) 1 µm transverse section of the foregut of an early stage VII P. tallagendensis embryo showing the lumen (lu) lined with cuticle (arrow).
Scale bar = 10 µm. (h) Early stage VII P. tallagendensis embryo showing the midgut packed with yolk granules (arrow). wl, walking leg.
Scale bar = 250 µm. (i) 1 µm transverse section of the midgut of a late stage VII P. tallagendensis embryo. There is no cuticle lining the
lumen (*) and only small residues of yolk remain within the gut epithelium (arrow). Scale bar = 10 µm.

Reproductive cycle
Summary data concerning weight, uterus length and
numbers of embryos contained in the females examined
are provided in Table 1. Specimens that did not contain
developing embryos were in the smallest range of body
weight and uterine length, indicating that they were
reproductively immature. All other individuals contained
embryos in their uteri regardless of the time of year that
they were dissected. The stages of development (I–VII)
present within the uteri of each female and the presence
of large oocytes in their ovary were recorded throughout
a calendar year. These data are presented for each species
along with the months in which births occurred in the
laboratory in Tables 2–5. Numbers of births and dates
they were recorded are given in Table 6.
In the majority of females examined, the number of
embryos present in each horn of the uterus was equal ± 3
(Tables 2–5). At no time of year did any female contain
the complete range of developmental stages. Although
relatively few females were sampled each month and there
are variations among individuals and species, patterns in
the presence or absence of different developmental stages
throughout the year are seen. The presence of both stages
I and VII indicates an overlap of embryo cohorts within
the uterus of the female. In C. clandestina, overlapping
344 M. H. WALKER AND N. N. TAIT
Table 1. Summary data
Cephalofovea
Species clandestina
Uterus length in gravid females (cm) 1.0–4.0 (n = 13)
Uterus length in juvenile females (cm) 0.5 (n = 1)
Weight gravid females (g) 0.079–0.222 (n = 16)
Weight juvenile females (g) 0.037 (n = 1)
Total numbers of embryos per gravid 3–55 (n = 26)
female
Average/mean embryo nos per gravid 32.0 (n = 26)
female
s In all four species, arrays of embryos between stages
Fig. 7. SEM micrograph of an Ruhbergia bifalcata ovary bearing
small yolkless oocytes (arrow), some attached to stalks (s) starting
to accumulate yolk and others that are much larger. Scale bar =
500 µm.
cohorts were noted between February and June but, in the
females of this species examined, stage VII embryos were
recorded in relatively few individuals. In P. tallagandensis
an overlap of cohorts was seen between April and July. In
R. bifalcata the overlap was noted between February and
May, but in E. rowelli both stages I and VII were present
in uteri in all sampled months except between September
and December.
In C. clandestina, P. tallagandensis and R. bifalcata,
large yolk-filled oocytes were present on the ovaries in
the month preceding the appearance of stage I embryos in
the uterus and during the first month that stage I embryos
were present. In E. rowelli, stages I and VII or I or VII
were recorded between February and September and large
oocytes were noted between November and April. When
only stage I and/or stage VII embryos were present in
the uterus and their total number was below the mean for
the species, it would be expected that large oocytes would
complete their growth, be released, fertilized and enter the
uterus as stage I embryos; e.g. females C24 (Table 2) and
P42 (Table 3). In juvenile females, identified by their lack
of embryos and short uteri, e.g. P18 (Table 3) and E23
Euperipatoides Phallocephale Ruhbergia
rowelli tallagandensis bifalcata
1.3–4.8(n = 24) 1.4–2.6 (n = 26) 1.0–2.4 (n = 15)
1.0–1.7 (n = 3) 1.0 (n = 1)
0.27–0.463 (n = 27) 0.058–0.161 (n = 30) 0.065–0.122 (n = 20)
0.203–0.240 (n = 3) 0.057 (n = 1) 0.061 (n = 1)
1–49 (n = 38) 4–41 (n = 42) 1–29 (n = 32)
20.6 (n = 38) 19.0 (n = 42) 15.6 (n = 32)
(Table 5), large oocytes were present indicating that these
females were about to enter their first reproductive cycle
and their uteri would soon contain stage I embryos.
II–VI were recorded in November and December, with
these arrays also recorded in E. rowelli and R. bifalcata
in January. Stages IV–VII were recorded in some
P. tallagandensis in February and in C. clandestina some
embryos at stages II and III were recorded between
June and October. Stage VII embryos were recorded for
C. clandestina in February, March and December, for
the majority of P. tallagandensis between February and
July, for R. bifalcata between January and May and for
E. rowelli in all sampled months excluding September
and December.
Cephalofovea clandestina females gave birth in the
laboratory between October and February with the number
of embryos born on each occasion varying between one
and 11, with numbers over five being unusual (Table 6).
Phallocephale tallagandensis gave birth normally to one
or two young on each occasion. They were recorded
mainly in May, June and December, although one female
gave birth in July (Table 6). Ruhbergia bifalcata females
gave birth between May and June and between February
and April and usually gave birth to young one at a time
(Table 6). Euperipatoides rowelli births occurred between
June and August and in February and April with the
number of young born on each occasion usually between
one and three (Table 6).
The morphology of the different developmental stages
is identical in the four species studied (see above). There
is also good synchronization among the females of each
species in terms of the stages present within the uterus at
any time of the year, apart from R. bifalcata where this
is more patchy. Taking into account the time when stage
I embryos are first seen in the uterus and the timings of
births in the laboratory, for all species the total gestation
time is estimated to be c. 12 months. It is probable
that development through stages II–VI takes around c. 3
months in total. As a result of the process of midgut
formation (see above) stage VII is more prolonged and
may take c. 4–6 months, likewise stage I is also prolonged
over a 4–5 month period.
Table 2. Stages of embryos present in female C. clandestina collected at different times of year. ∗ , Juvenile; LH, left hand; RH, right hand

Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora

Female Weight Uterus length Stage No. Stage Stage Stage Stage Stage Stage Stage No. embryos No. embryos Total no. of Large oocytes Births in the
Month no. (gm) (cm) I I II III IV V VI VII LH uterus RH uterus embryos present laboratory

January
February C24 0.131 2.0 ✓ 14 7 7 14 ✓ ✓
C25 0.16 2.5 ✓ 17 ✓ 11 13 24 ✓
C26 0.107 2.0 ✓ 11 5 6 11
C27 0.093 1.0 ✓ 3 1 2 3
March
April C1 – – ✓ 30 17 13 30
C2 – – ✓ 34 18 16 34
C3 – – ✓ 55 27 28 55
May
June C4 – – ✓ 18 ✓ ✓ 14 12 26
C5 – – ✓ 25 ✓ 19 16 35
C6 – – ✓ 8 ✓ 2 8 10 ✓
C7 – – ✓ 55 ✓ 32 29 61
July C8 – – ✓ 35 ✓ ✓ 29 26 55
C9 – – ✓ 28 ✓ 15 19 34
C10 – – ✓ 26 ✓ 18 15 33
August
September
October C11 0.138 – ✓ 14 ✓ ✓ 19 17 36 ✓
C12 0.084 – ✓ 17 ✓ ✓ 14 19 33
C13 0.079 – ✓ 14 ✓ ✓ 16 16 32
November C14 0.117 2.5 0 ✓ ✓ 20 21 41 ✓
C15∗ 0.037 0.5 0 0 0 0
C16 0.087 3.0 0 ✓ ✓ ✓ 12 11 23
C17 0.290 3.5 0 ✓ ✓ ✓ 20 23 43
C18 0.122 3.0 ✓ 7 ✓ ✓ ✓ ✓ 18 17 35 ✓
December C19 0.134 2.4 0 ✓ ✓ ✓ ✓ ✓ 10 9 19 ✓
C20 0.115 2.8 0 ✓ ✓ ✓ ✓ ✓ 15 14 29
C21 0.111 3.1 0 ✓ ✓ ✓ 15 16 31
C22 0.150 4.0 0 ✓ ✓ ✓ 26 23 49
C23 0.091 3.0 0 ✓ ✓ ✓ ✓ 15 15 30

345
Table 3. Stages of embryos present in female P. tallagandensis collected at different times of year. ∗ , Juvenile; LH, left hand; RH, right hand

346
Female Weight Uterus length Stage No. Stage Stage Stage Stage Stage Stage Stage No. embryos No. embryos Total no. of Large oocytes Births in the
Month no. (gm) (cm) l I II III IV V VI VII LH uterus RH uterus embryos present laboratory
January
February P36 0.112 1.3 0 ✓ ✓ ✓ ✓ 9 9 18 ✓
P37 0.115 1.5 0 ✓ ✓ ✓ 0 9 9 ✓
P38 0.100 2.0 ✓ 1 ✓ 9 11 20 ✓
P39 0.090 1.2 0 ✓ ✓ 5 7 12
P40 0.069 1.2 0 ✓ 5 4 9
P41 0.058 1.3 0 ✓ ✓ 3 3 6
P42 0.064 2.6 0 ✓ 3 6 9 ✓
P43 0.058 1.7 0 ✓ 4 4 8 ✓
March
April P1 – – ✓ 12 ✓ 15 14 29
P2 – – ✓ 27 ✓ 20 19 39
P3 – – ✓ 32 ✓ 21 20 41
May P4 – – ✓ 29 ✓ 16 18 34 ✓
P5 – – ✓ 22 11 11 22
P6 – – ✓ 20 ✓ 17 14 31

M. H. WALKER AND N. N. TAIT

P7 – – ✓ 15 ✓ 15 14 39
June ✓
July P8 – – ✓ 27 ✓ ✓ 18 16 34 ✓
P9 – – ✓ 16 ✓ ✓ 13 11 24
P10 – – ✓ 18 ✓ ✓ 13 11 24
P11 – – ✓ 21 ✓ 11 10 21
August
September P12 0.092 – ✓ 8 ✓ ✓ ✓ 13 12 25
P13 0.080 – ✓ 2 1 3 4
P14 0.070 – 0 ✓ ✓ ✓ 7 9 16
P15 0.052 – ✓ 4 ✓ ✓ 4 5 9
October
November P16 0.074 1.2 ✓ 14 7 7 14
P17 0.087 1.8 0 ✓ ✓ ✓ 9 3 12
P18∗ 0.57 1.0 0 0 0 0 ✓
P19 0.087 2.5 0 ✓ ✓ ✓ 11 13 24
P20 0.108 2.1 ✓ 2 ✓ ✓ ✓ 8 10 18
P21 0.084 1.7 0 ✓ ✓ ✓ ✓ 6 5 11
P22 0.111 2.2 0 ✓ ✓ ✓ 11 9 20
P23 0.089 – 0 ✓ ✓ 8 8 16
P24 0.092 2.0 0 ✓ ✓ ✓ 7 9 16
P25 0.103 1.8 0 ✓ ✓ ✓ 8 7 15
P26 0.070 1.5 0 ✓ ✓ ✓ 5 6 11
P27 0.076 2.2 0 11 11 22
P28 0.064 1.8 0 ✓ 6 7 13
December P29 0.121 2.1 ✓ 2 ✓ ✓ ✓ ✓ 11 9 20 ✓
P30 0.117 1.7 0 5 5 10
P31 0.120 2.5 0 ✓ ✓ ✓ ✓ 17 12 29 ✓
P32 0.161 2.4 0 ✓ ✓ ✓ 10 12 22
P33 0.060 1.4 0 ✓ ✓ ✓ 3 3 6
P34 0.098 1.4 0 13 1 14
P35 0.117 2.5 0 ✓ ✓ ✓ ✓ 5 6 11
Table 4. Stages of embryo present in female R. bifalcata collected at different times of year. ∗ , Juvenile; LH, left hand; RH, right hand

Female Weight Uterus length Stage No. Stage Stage Stage Stage Stage Stage Stage No. embryos No. embryos Total no. of Large oocytes Births in the
Month no. (gm) (cm) I I II III IV V VI VII LH uterus RH uterus embryos present laboratory

Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora

January T27 0.103 1.3 0 ✓ ✓ ✓ 5 6 11
T28 0.094 1.4 ✓ 2 ✓ 3 4 7 ✓
T29 0.089 1.7 0 ✓ ✓ ✓ ✓ 7 7 14
T30 0.065 1.3 0 ✓ ✓ ✓ ✓ 1 4 5 ✓
February T31 – 2.4 ✓ 12 6 6 12 ✓ ✓
T32 0.076 1.5 ✓ 1 ✓ ✓ 1 4 5 ✓
T33 0.066 0.9 ✓ 2 1 1 2 ✓
T34 0.067 1.5 0 ✓ 6 4 10 ✓
March
April T1 – – 0 ✓ 12 6 18 ✓ ✓
T2 – – ✓ 1 ✓ ✓ ✓ ✓ ✓ 14 15 29
T3 – – ✓ 26 14 12 26 ✓
T4∗ – – 0 0 0 0
May T5 – – ✓ 6 ✓ 11 9 20 ✓
T6 – – ✓ 4 ✓ 6 4 10
T7 – – ✓ 10 ✓ 10 12 22
T8 – – ✓ 7 ✓ 8 6 14
June ✓
July T9 – – ✓ 20 ✓ ✓ 17 13 30
T10 – – ✓ 7 ✓ 10 8 18
T11 – – ✓ 17 7 10 17
T12∗ – – 0 0 0 0
August
September T13 0.061 – ✓ 18 ✓ 11 13 24 ✓
T14 0.098 – ✓ 17 ✓ 13 11 24
T15 0.084 – ✓ 8 ✓ ✓ 5 10 15
T16∗ 0.061 – 0 0 0 0
October
November T17 0.098 – 0 ✓ ✓ 17
T18 0.077 2.0 0 ✓ ✓ ✓ 11 11 22
T19 0.82 2.1 ✓ 4 ✓ ✓ ✓ 11 6 17
T20 0.107 – 0 ✓ ✓ ✓ 23
T21 0.064 1.0 0 ✓ 1 1
T22 0.090 2.4 0 ✓ ✓ ✓ 13 12 25
December T23 0.131 2.5 ✓ 2 ✓ ✓ ✓ ✓ 12 11 23
T24 0.103 2.1 ✓ 5 ✓ ✓ 9 10 20
T25 0.084 1.1 ✓ 1 ✓ 2 1 3
T26 0.073 1.9 0 ✓ ✓ ✓ ✓ 8 8 16

347
Table 5. Stages of embryos present in female E. rowelli collected at different times of year. ∗ , Juvenile; LH, left hand; RH, right hand

348
Female Weight Uterus length Stage No. Stage Stage Stage Stage Stage Stage Stage No. embryos No. embryos Total no. of Large oocytes Births in the
Month no. (gm) (cm) I I II III IV V VI VII LH uterus RH uterus embryos present laboratory
January E28 0.463 4.0 ✓ 4 ✓ ✓ ✓ ✓ 14 15 29 ✓
E29 0.306 4.5 ✓ 10 ✓ ✓ ✓ ✓ ✓ 9 14 23 ✓
E30∗ 0.223 1.7 0 0 0 0 ✓
E31 0.261 3.8 ✓ 9 ✓ ✓ 10 10 20 ✓
E32 0.273 2.5 ✓ 11 ✓ 6 6 12 ✓
February E33 0.388 4.0 ✓ 39 20 19 39 ✓ ✓
E34 0.359 4.0 ✓ 11 ✓ 14 15 29
E35 0.270 3.0 ✓ 15 13 28
E36 0.227 4.0 ✓ 3 ✓ 7 7 14
March
April E37 0.431 4.8 ✓ 40 ✓ 25 22 47 ✓
E38 0.309 4.5 ✓ 16 ✓ 14 10 20 ✓
E39 – 1.3 ✓ 2 1 1 2 ✓
E40 0.259 2.9 ✓ 19 9 10 19 ✓
E41∗ 0.203 1.3 0 0 0 0

M. H. WALKER AND N. N. TAIT

E42 0.180 2.4 ✓ 17 9 8 17
E1 – – ✓ 8 3 5 8
E2∗ – – 0 0 0 0 ✓
E3 – – ✓ 1 1 1
May E4 – – ✓ 17 ✓ 22 20 42
E5 – – ✓ 17 ✓ 24 25 49
E6 – – ✓ 11 6 5 11
E7 – – 0 ✓ 3 3 6
June ✓
July E8 – – ✓ 19 5 14 19 ✓
E9 – – ✓ 21 ✓ 18 21 39
E10∗ – – 0 0 0 0
E11 – – ✓ 22 ✓ 21 18 39
August ✓
September E12 0.399 – ✓ 16 9 7 16
E13 0.277 – ✓ 12 ✓ 12 9 21
E14 0.208 – ✓ 4 2 2 4
E15 0.383 – ✓ 9 12 8 20
0ctober
November E16 0.346 3.6 0 ✓ ✓ 6 8 14
E17 0.233 4.8 0 ✓ 13 8 21
E18 0.253 2.5 0 ✓ 1 0 1 ✓
E19 0.227 3.2 0 ✓ ✓ ✓ ✓ 7 7 14
E20 0.237 3.5 0 ✓ ✓ ✓ ✓ 11 6 17
December E21 0.341 4.5 0 ✓ ✓ 19 25 44 ✓
E22 0.361 3.5 ✓ 14 ✓ ✓ 13 11 24 ✓
E23∗ 0.240 1.0 0 0 0 0 ✓
E24 0.354 2.6 ✓ 2 ✓ 5 5 10 ✓
E25 0.318 1.5 ✓ 2 1 1 2 ✓
E26 0.572 4.4 ✓ 6 ✓ ✓ 23 20 43 ✓
E27 0.303 2.5 ✓ 3 ✓ ✓ 6 14 20 ✓
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora 349

Table 6. Recorded births to females maintained in the laboratory. ∗ , Female died; ∗∗ , embryos aborted; ∗∗∗ , embryos aborted and female
died

Month Oct Nov Dec Feb Apr Total

Date 24 27 30 5 12 20 25 4 11 19 3
Cephalofovea
∗
clandestina No. 1 1 1 3 1 4 3 8 2 7 30
∗
No. 2 11 11
∗
No. 3 3 5 3 2 4 4 5 3 2 31
∗
No. 4 1 1 4 2 3 10 21
∗
No. 5 1 1 1 2 8 13
No. 6 0
Month June July Aug Feb Apr
Date 23 30 4 8 14 18 21 24 28 31 6 8 14 19 3
Euperipatoides
rowellia No. 1 1 2 1 1 5
No. 2 1 1 4 2 4 2 1 1 2 2 1 9 3 33
∗
No. 3 3 2 2 7
∗
No. 4 4 3 3 1 1 12
Month May June July Nov Dec Feb
Date 7 9 12 14 16 19 20 23 26 30 4 10 17 23 27 14 18 21 20 4 11 19
Phallocephale
tallagandensis No. 1 1 1
∗∗∗
No. 2 2 1 1 1 2 1 2 4 2 16
∗∗ ∗
No. 3 1 1 1 1 1 5
No. 4 1 2 1 1 1 1 1 1 2 1 3 15
Month May June Feb Apr
Date 7 9 12 14 16 19 20 28 30 10 13 19 3
Ruhbergia
bifalcata No. 1 0
No. 2 1 1 1 1 1 5
No. 3 1 1 2
No. 4 2 1 1 1 1 1 1 7 15
No. 5 0
No. 6 4 4

a
Data from Sunnucks et al. (2000).

DISCUSSION ectoderm separating the segment halves (Walker &

Embryonic stages
Embryonic development, in the species examined in
this study, follows the pattern normally seen within the
Onychophora (Anderson, 1973) with segments added
sequentially from a posterior growth zone. In the earliest
stage II embryos examined, the future mouth and anus are
present. Sheldon (1888) suggested that in Peripatoides
novaezealandiae the stomodaeum is formed earlier than
the proctodaeum, with the latter not being formed until
the equivalent of a late stage II embryo. Evidence of an
elongating ventral blastopore, ‘the primitive groove’, that
subsequently closes except to leave openings for the mouth
and anus, as noted in some species of onychophorans
(Balfour, 1883; Sedgwick, 1885; Evans, 1902; Bouvier,
1905; Manton, 1949) was not observed. In the early
embryos of the viviparous Peripatus acacioi, where yolk
is absent, there are only narrow bands of extra-embryonic
Campiglia, 1988; Campiglia & Walker, 1995). In stage
II–IV embryos described here, the segment halves are
separated by broad bands of extra-embryonic ectoderm, a
consequence of the large amount of yolk present within the
embryos confirming the observations of Sheldon (1888)
for Peripatoides novaezealandiae. Sheldon (1888, 1889)
also described the presence of ‘peripheral yolk’ between
the early developing embryo of P. novaezealandiae and
its surrounding membranes and noted that the amount of
peripheral yolk diminished and was absent by the time
the appendages started to form. She suggested that this
yolk had a nutritive function, which was absorbed by
the embryo by some unknown mechanism. The presence
of a thick layer of yolk between the egg membranes
and immature embryos of P. novaezealandiae, was also
noted by Brosius-Roggenberg & Ruhberg (2000). Evans
(1902) also noted the presence of ‘external’ yolk on the
ventral surface of early Eoperipatus weldoni embryos,
but commented that the quantity of this yolk was highly
350 M. H. WALKER AND N. N. TAIT
variable and sometimes was absent. Small accumulations
of yolk in this location were noted in some of the embryos
examined here, but this was not a common feature of all
embryos. It is possible that, in some specimens, a small
amount of yolk could escape from the embryo at stage
I before it is enclosed by the extra-embryonic ectoderm
and developing ectoderm. Embryos at this stage are very
fragile and rupture very readily.
The developing eye first appears in the stage III embryo
as an ectodermal invagination and by stage IV is marked
only by a shallow depression as the ectoderm covers the
initial opening to the exterior. Similar observations were
noted in Euperipatoides kanangrensis (Eriksson et al.,
2003). The eye only becomes distinguishable again on the
body surface in the stage VII embryo. The salivary glands
are the modified nephridia of the third body segment,
the oral papilla segment (Storch & Ruhberg, 1993). In
the adult, the paired salivary glands unite to form a
common duct that opens into the ventral side of the
foregut (Storch & Ruhberg, 1993). In the embryo, the
openings of the salivary glands first appear as indentations
on the lateral ventral surface, level with the bases of
the oral papillae in the stage IV embryo, also noted in
E. kanangrensis (Eriksson et al., 2003) and Peripatopsis
capensis (Sedgwick, 1885). As development proceeds and
the lower lips come together, the openings of the salivary
glands have migrated towards the ventral mid-line and in
the stage VII embryo they are enclosed within the mouth.
Also during stage IV, indentations first appear that mark
the positions of the future openings of the nephridia at
the base of the foot pads of the fourth and fifth pairs
of walking legs. Paired openings for the future genital
ducts first appear as indentations at the bases of the last
pair of walking legs as these start to elongate in the
stage V embryo. As indicated by Snodgrass (1938) and
illustrated here, the openings for the genital ducts migrate
towards the ventral mid-line during development of the
embryo and fuse to form the ejaculatory duct or vagina.
This is similar to the formation of the common salivary
duct, but the genital openings first appear in stage V
rather than stage VI as a result of the anterior–posterior
sequence of segment addition and segmental appendage
elongation.
The frontal processes, that will form the anterior lips,
first appear as conspicuous structures during stage IV.
Eriksson et al. (2003) suggested, from their study of
E. kanangrensis, that the migration of the frontal processes
from an anterodorsal to a ventral position, and their
innervation from the dorsal part of the brain (Eriksson &
Budd, 2000) supports the view that the ancestral position
of the mouth in onychophorans was terminal. Eriksson
et al. (2003) noted that frontal processes have not been
reported in the embryos of the viviparous P. acacioi.
Indeed, frontal processes, which are so conspicuous in
the stage IV embryos of Australian onychophorans, seem
to be absent in P. acacioi (M. H. Walker, pers. obs.;
S. Campiglia, pers comm.) and also the South African
peripatopsids P. capensis and Opisthopatus cinctipes
(Sedgwick, 1885–1888; M. H. Walker, pers. obs.;
B. Sherbon, pers. comm.).
Ventral organs, as segmentally arranged thickenings
of the mid-ventral ectoderm, become conspicuous in the
stage VI embryo and diminish in size during subsequent
stages and are not evident in the newborn. Anderson
(1973) stated that the ventral nerve cords arise from a
mid-ventral thickening of embryonic ectoderm that is
continuous from segment to segment. The median part
of this thickening forms the distinctive ventral organs
of each trunk segment. Kennel (1885) and Sedgwick
(1887) claimed that the ventral organs do not contribute
neurons to the central nervous system. This is refuted
by Pflugfelder (1948) and Eriksson et al. (2003), hence
details of the formation of the central nervous system of
the trunk of onychophorans requires further investigation.
The only ventral organs that have been identified in the
developing head of onychophorans are those of the preoral
segment (Eriksson et al., 2003). These begin to invaginate
to form the conspicuous cerebral grooves. The completely
invaginated ventral organs of the head persist in the adult
as a pair of vesicles, the hypocerebral (or infracerebral)
organs attached to the ventral surface of the brain (Dakin,
1922; Snodgrass, 1938; Pflugfelder, 1948; Eriksson et al.,
2003). The function of the hypocerebral organs is
unknown although it has been suggested that they have
an endocrine function (Pflugfelder, 1948). The cerebral
grooves first appear in the stage V embryo and their
invagination is complete by the end of stage VI, when
the lips close around the mouth. In the newborn animal,
it is from the region where the trunk ventral organs were
located that papillae are added to the body surface as
the animal grows at successive moults (Campiglia &
Lavallard, 1969). In species with head structures, these
first appear at stage VI as an indentation on the dorsal
surface of the head. While these head structures are as
yet not fully developed as in the sexually mature male,
at stage VII they display species-specific variation and
their form can be interpreted in terms of the structure
that they will develop into on maturity. Observations of a
number of neonates of R. bifalcata failed to identify any
dimorphism of the structure among specimens. Hence, it
is assumed that, at this stage of development, the structure
is comparable in form between males and females. In
R. bifalcata, it is possible that the paired lobes later
develop in males into the pair of curved spines that is
characteristic of the everted head structure of this species
(Reid, 1996). In C. clandestina, the sexually mature
male possesses an eversible pit unadorned with hardened
structures (Reid et al., 1995). In P. tallagandensis, the head
structure of adult males is an eversible cylindrical structure
with a domed tip, a form from which the generic name
Phallocephale was derived (Reid, 1996). This structure is
possibly developed from the outer circle of lobed papillae
which will form the side walls of the structure while the
central lobe forms the domed tip.
A variety of different simplistic terminologies has been
used to describe embryonic stages in Australian Onycho-
phora (Leishman & Eldridge, 1990; Brockmann et al.,
1997; Brosius-Roggenbuck & Ruhberg, 2000; Sunnucks
et al., 2000). More recently, Eriksson et al. (2003) have
categorized 10 developmental stages in E. kanangrensis
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora
Table 7. Comparison of stages I–VII described here with stages A–G in P. capensis by Sedgwick (1885–1888). Times of year when
different stages of P. capensis embryos were observed by Sedgwick are in italic
Stage I From fertilized egg to the formation of the pre-oral
segment
Stage II Pre-oral segment formed anterior to the mouth, two
bands of blastoderm separated by the yolk mass fuse at the
posterior anus. Indications of jaw and oral papilla segment
boundaries, antennae start to develop
Stage III Embryo increases in length from posterior growth
zone and is folded mid-ventrally. Indications of segment
boundaries occur along > 50% of blastoderm bands. Eyes
start to develop
Stage IV All segment boundaries indicated on the
blastoderm bands. Antennae and anterior appendages
elongating. Lips start to form, jaws start to migrate,
openings of salivary glands visible, eye pit now covered by
ectoderm
Stage V All the walking legs elongating, openings to genital
ducts form at bases of last pair of walking legs, cerebral
grooves start to form. Embyronic cuticle forms
Stage VI All legs fully elongated, claws form on the feet.
Papillae develop on the body surface. Ventral organs form.
The genital openings fuse in the mid-line. Cerebral grooves
close, lips surround mouth enclosing salivary duct openings
Stage VII Externally the embryo appears fully developed
but gut formation is incomplete until the end of this stage
when the embryos are ready for birth and pigmented
with particular reference to the central nervous system
and primarily based on head development. The definitions
of some of their stages are reliant on observation of the
internal anatomy of the embryo and the first stages of
development were not included. The stages I–VII pro-
posed here are based on the external morphology of the
embryo and can be identified by examination using a dis-
secting microscope or hand lens and can, therefore, readily
be applied to other onychophoran embryos develop-
ing from yolk-filled eggs. These stages are comparable
to those given for P. capensis by Sedgwick (1885–1888;
see Table 7).
Reproductive cycle
Analysis of the embryonic stages in specimens of onycho-
phorans examined in this study suggest that all four
species have an annual reproductive cycle with a total
351
Stage A Segmentation of egg and formation of ‘primitive
streak’
June and August U.K.
Stage B Blastopore divides to form mouth and anus into two
parts. Anterior somites move to anterior to form mesoderm
of pre-oral segment July August
Stage C Hind end curved ventrally – growth from posterior end
July August Cape
Stage D Appearance of appendages in anterior posterior
sequence but not all appendages formed at this stage.
Eye pits form and lips enclose jaws and buccal cavity. Pre-oral
segment bilobed
Stage E Embryo folded all somites now present.
Closure of eye pit, growth of lips.
Ectoderm invagination forms stomodaeum.
Pit at end of oral papillae + perforation at base of oral
papillae (=salivary duct openings)
October U.K.
Stage F Similar to previous stage – main changes
lips fuse in midline and enclose salivary glands,
cerebral grooves deepen, mouth opening complete –
approximation of ventral swellings at base of
jaws Two halves of preoral segment merge in mid-line
October U.K.
Stage G Greater transparency of embryo.
All appendages present – all ringed + claw rudiments on feet,
jaws hidden by lips start of body papillae.
Integument ringed appearance
December U.K.
Other changes through to birth in May – merely those of
growth
gestation period estimated to be c. 12 months. This
is comparable with the gestation time for the viviparous
P. acacioi (Campiglia & Walker, 1995) and the
ovoviviparous P. capensis (Sedgwick, 1885) and implied
for P. leuckartii by Steel (1896), but longer than other
estimates for the gestation time in Australian peripatopsids
(Hardie, 1975; van der Lande, 1978). Stage I is protracted
and may last 4–5 months, stages II–VI are estimated to
take c. 3 months and stage VII is again protracted and
takes 4–6 months. The extended time taken for stage I
to be completed can be explained by the requirement
for early embryonic development to take place without
much observable surface differentiation. The extensive
time taken in stage VII is a result of the considerable
morphological changes still required for completion of
embryonic development even though the early stage VII
embryo may seem to be fully formed from its overall
external appearance. In the early stage VII embryo,
the midgut is still packed with yolk and there is no
352 M. H. WALKER AND N. N. TAIT
distinct gut cavity in this region. In the late stage VII
embryo, there is a distinct midgut cavity and very little
yolk remains associated with the cells lining the gut.
At this stage, the antennae are usually pigmented. The
extended time for development at stage VII noted in
this study is similar to the c. 6 month development
time of the foetus in P. acacioi (Campiglia & Walker,
1995). Moreover, Sedgwick (1885) noted that embryos of
P. capensis reached their fully formed stage G in December
but females did not give birth until May, with growth
of the embryo the only change occurring during this
period (Table 7). Further evidence for the necessity for
developmental changes to occur during stage VII comes
from the observation that females following stress of
collection or during maintenance in culture may give
birth to stage VII embryos that do not survive. Steel
(1896) also reported the non-viable abortion of embryos
from females when ‘distressed by close confinement or
uncomfortable conditions’. He also noted that most young
were born fully extended but others were ‘doubled up’ in
their enclosing membranes and considered that the latter
could be premature. It is evident that, in the embryos
described here, the large amount of yolk probably provides
all the nutrients required by the embryos throughout their
year-long gestation. From the data presented here, it is
evident that yolk persists within the midgut region until
the late stage VII embryo. It would seem that embryos
that are born prematurely are unable to survive, because
the gut will not be a completely functional organ until
just before birth. In E. rowelli, Sunnucks et al. (2000)
showed a 10% increase in the average dry weight of
‘developed’ compared to ‘undeveloped’ embryos from
the same female. They suggested that this could indicate
some provisioning of embryos with nutrients other than
yolk, but did not indicate how this could be achieved. In
P. acacioi, where the developing embryos are not enclosed
in surrounding membranes, it is known that the gut is a
functional organ in the maturing foetus (Campliglia &
Walker, 1995). In the uterus, the mouths of these foetuses
are open and nutrients enter the gut by this route and are
digested.
The presence, during certain months of the year, of
embryos at only stages I and VII indicates there is
an overlap of cohorts within the uteri. This overlap of
cohorts was noted in P. acacioi (Campiglia & Walker,
1995). Sunnucks et al. (2000) also noted that, in their
study of E. rowelli, many females contained offspring that
were clearly divided into more developed and less well-
developed batches, although there may be gradations of
development within a batch.
The fact that the morphological stages of development
are identical in the four species examined here is not
unexpected. Embryonic development in all onychophor-
ans that have been studied to date, with the notable
exception of a single genus of the Peripatopsidae from
South Africa, Opisthopatus, (Walker, 1995), exhibit the
same pattern with the body growing by the addition of
segments from a posterior growth zone. It is also not
unexpected that the total gestation time for all four species
is c. 12 months, again similar to P. capensis (Sedgwick,
1885–1888) and P. acacioi (Campiglia & Walker, 1995).
This study suggests that the bulk of the morphological
stages of development (stages II–VII) occur relatively
quickly. In all species, births occur predominantly in the
winter months. However, the extended period of time
that there are overlapping cohorts of embryos within the
uterus and mature oocytes on the ovaries in E. rowelli
compared with the other three species may be owing to the
mechanism and timing of insemination. Since E. rowelli
is a dermal inseminator, sperm can reach the seminal
receptacles for storage at any time independently of the
presence of embryos in the reproductive tract. The other
species studied here have head structures in males and
hence are presumed to be vaginal inseminators (see below
for elaboration).
In the vast majority of specimens, embryos were
present in the uteri of females collected throughout the
year. The few specimens that did not contain embryos
were at the smallest size range for that species and
hence were presumably reproductively immature. This
is compatible with reproductive data for E. rowelli
(Sunnucks et al., 2000). In this species, males become
reproductively mature in their first year and mate with
females. Females carrying sperm stored in their seminal
receptacles do not become sexually mature until their
second or possibly even third year. After maturity, there is
a linear relationship between body mass and the number
of developing embryos. A similar delayed sexual maturity
in females compared with males and relationship between
size and fecundity has been reported in C. tomahmontis
and E. leuckartii (Leishman & Eldridge, 1990) and
Plicatoperipatus jamaicensis (Havel, Wilson & Hebert,
1989). The presence of embryos at all times of the
year, in females that have reached sexual maturity, has
implications for mating and sperm storage. The presence
of embryos in the uteri would presumably prevent the
passage of sperm up the reproductive tract for storage
in the seminal receptacles. Mating has been observed
in Florelliceps stutchburyae, a species of onychophoran
with a head structure in males (Tait & Norman, 2001).
In this species, the male head carrying a spermatophore
was observed wedged into the female genital opening and
sperm were delivered into the vagina. Dissection of the
female revealed the absence of developing embryos in
her uteri. It is presumed that the same mechanism of
insemination occurs in the three species in this study that
possess a head structure in males, i.e. C. clandestina,
P. tallagandensis and R. bifalcata. This suggests that
mating in species with a head structure occurs early in life,
followed by long-term storage of sperm for subsequent
fertilizations. A study of seminal receptacles and their
accessory pouches in the species examined in this study is
in progress to elucidate the possible role of the accessory
pouches in sperm storage (M. H. Walker, pers. comm.).
The possibility that vaginal mating could take place
when the uterus is empty after a breeding cycle is not
substantiated by data presented here. In the species with
head structures, there is no time during the year when
the uteri are empty. In another species that possesses a
head structure, C. tomahmontis, Leishman & Eldridge
 Embryonic development and reproductive cycle in ovoviviparous Australian Onychophora
(1990) reported that upon dissection the uteri of females
that had given birth to numerous young in laboratory
culture were empty. From the data presented here, the
presence of some stage I embryos in the uteri of these
females would be expected. In contrast, E. rowelli does
not possess a head structure in males and spermatophores
have been observed deposited on the body surface of
females maintained in culture (B. Sherbon, pers. comm.).
Dermal insemination has also been reported in species of
African peripatopsids (Manton, 1938a; Walker, 1992a).
This strategy circumvents the problem of access to the
seminal receptacles via the female reproductive tract.
Acknowledgements
We gratefully acknowledge the following people and
organizations. The National Parks and Wildlife NSW
and State Forests of NSW for permits to collect onycho-
phorans.The Royal Society, London, for a grant to MHW.
Debra Birch and Jenny Norman (Microscopy Unit, De-
partment of Biological Sciences, Macquarie University),
Evie Roberts, Stefan Hyman and Natalie Allcock (E. M.
Laboratory, School of Biological Sciences, University of
Leicester) for technical asistance. James Forber (project
student, University of Leicester) for preparation of the LM
sections used. Dr Paul Sunnucks (La Trobe University) for
his constructive criticism of the manuscript.
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