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Research
Cite this article: Janssen R, Jörgensen M,
Lagebro L, Budd GE. 2015 Fate and nature of
the onychophoran mouth – anus furrow and its
contribution to the blastopore. Proc. R. Soc. B
282: 20142628.
http://dx.doi.org/10.1098/rspb.2014.2628
Received: 27 October 2014
Accepted: 17 February 2015
Subject Areas:
developmental biology, evolution, genetics
Fate and nature of the onychophoran
mouth– anus furrow and its contribution
to the blastopore
Ralf Janssen, Mette Jörgensen, Linda Lagebro and Graham E. Budd
Department of Earth Sciences, Palaeobiology, Uppsala University, Villavägen 16, 75236 Uppsala, Sweden
The ancestral states of bilaterian development, and which living groups have
conserved them the most, has been a controversial topic in biology for well
over a hundred years. In recent years, the idea that gastrulation primitively
proceeded via the formation of a slit-like blastopore that then evolved into
either protostomy or deuterostomy has gained renewed attention and
some molecular developmental support. One of the key pieces of evidence
for this ‘amphistomy’ theory comes from the onychophorans, which form
a clear ventral groove during gastrulation. The interpretation of this structure has, however, proved problematic. Based on expression patterns of
forkhead ( fkh), caudal (cad), brachyury (bra) and wingless (wg/Wnt1), we show
that this groove does not correspond to the blastopore, even though both
the mouth and anus later develop from it. Rather, the posterior pit appears
to be the blastopore; the posterior of the groove later fuses with it to form
the definitive anus. Onychophoran development therefore represents a
case of ‘concealed’ deuterostomy. The new data from the onychophorans
thus remove one of the key pieces of evidence for the amphistomy theory.
Rather, in line with other recent results, it suggests that ancestral bilaterian
development was deuterostomic.
Keywords:
Onychophora, early development,
gene expression, amphistomy
1. Introduction
Author for correspondence:
Ralf Janssen
e-mail: [email protected]
The phylum Onychophora is closely related to the Arthropoda [1]. Because of
the phylogenetic position of the onychophorans at the base of the highly divergent arthropods, understanding of onychophoran development is critical for
the unravelling of ancestral developmental traits of the common ancestor of
onychophorans and arthropods. However, it is still the case that relatively
little is known about onychophoran biology, including their development.
Despite their close relationship to arthropods, the development of onychophorans is in some aspects crucially different from arthropod development. One
such difference concerns the fate of the blastopore and the development of
mouth and anus. Because arthropods belong to the protostomes the blastopore
should in theory [2] only develop into the mouth. Careful studies of early
arthropod development, however, revealed that a number of arthropods
undergo deuterostomic development [3–5]. As a probable adaptation to
yolk-rich eggs, the arthropod mouth and anus often form secondarily after
complete closure of the blastopore [3,6,7]. This developmental mode can also
be observed in some onychophorans [8,9]. In other onychophorans, as for
example in Euperipatoides kanangrensis, the model organism of this study, the
blastopore is believed to first develop into one single opening, a slit-like
furrow along the anterior–posterior axis. After median closure, the remnants
of this furrow give rise to mouth and anus [9–12].
The nature of the onychophoran blastopore is thus, despite several studies,
still not fully resolved. While some authors believe indeed that the so-called
mouth –anus furrow represents the blastopore [10–13], others suggest that
the pit posterior to the mouth– anus furrow represents the blastopore [8,9].
These diverging views are owing to different opinions on whether the furrow
is involved in endoderm formation [9].
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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(a) Embryo collection, fixation and embryonic staging
Specimens of the onychophoran E. kanangrensis [20] were
collected in New South Wales, Australia. The embryos were collected, staged and prepared for in situ hybridization experiments
according to previously published methods [21,22].
(b) Gene cloning
Total RNA was isolated from E. kanangrensis embryos of different
stages using TRIZOL (Invitrogen). In a subsequent step, poly-A
RNA was extracted from total RNA (PolyATtract mRNA isolation system III, Promega) and reverse transcribed into cDNA
blastopore
2
anus (a)
both
mouth (m)
deuterostomy
amphistomy
protostomy
m
m
m
a
a
a
(b)
amphistomy theory
m
a
anterior
closure
median
closure
posterior
closure
m
m
a
a
anus
(deuterostomy)
anus and mouth
(amphistomy)
mouth
(protostomy)
Figure 1. The fate of the blastopore during development. The blastopore
either gives rise to the anus (a; deuterostomy), the mouth (m; protostomy)
or both, the anus and the mouth (amphistomy). (b) The amphistomy theory
is based on the idea that a slit-like furrow was present in the last common
ancestor of all bilaterians. This furrow then gave rise either to the anus of
deuterostomes (via anterior closure), the mouth of protostomes (via posterior
closure) or both, mouth and anus (via median closure). Grey ovals represent
embryos. The blastopore and its remnants are in white. Closure of the
blastopore is indicated in black. a, anus; m, mouth.
(SuperscriptII first strand synthesis system for RT-PCR, Invitrogen). Fragments of onychophoran fkh, bra and cad were isolated
by means of RT-PCR with gene-specific primers based on
sequence information found in a sequenced embryonic transcriptome [22]. Sequences of all amplified fragments were determined
from both strands on a 3100 automated sequencer, terminator
cycle sequencing kit (Perkin-Elmer Applied Biosystems, Foster
City, CA) using Big Dye dye-terminators v. 3.1 (Big Dye terminator
cycle sequencing kit; Perkin-Elmer Applied Biosystems), and
cloned into pCR-II vectors (TA cloning kit dual promoter,
Invitrogen, Carlsbad, CA). Isolated sequences are available
under accession nos. LN812024 (Ek-cad), LN812023 (Ek-bra) and
LN812025 (Ek-fkh).
(c) Whole mount in situ hybridization and nuclear
staining
In situ hybridization was performed as described in [23]. Digoxigenin-labelled RNA probes for all genes were transcribed from
Proc. R. Soc. B 282: 20142628
2. Material and methods
(a)
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There is consensus about the position and origin of the onychophoran mouth; it is believed to originate from the anterior
part of the furrow, regardless of the furrow’s nature [9–12].
Most authors agree with the idea that the anus is formed from
the posterior opening of the furrow [9–12]. A recent study by
Eriksson & Tait [13], however, showed that in E. kanangrensis
the mouth–anus furrow is initially continuous with the posterior
pit, the blastopore sensu [8,9]. It suggests further that the onychophoran anus forms posterior to the mouth–anus furrow [13].
The authors argue that the posterior opening of the furrow
does not form the anus, but that this opening disappears at
some point during embryogenesis. This conclusion is mainly
based on the expression of the segment polarity gene orthologue
wingless (wg/Wnt1) that is used to homologize the position of the
anus of arthropod embryos with that of onychophoran embryos.
These varied forms of gastrulation and gut opening development can be set in a broader picture of how bilaterian
development as a whole has developed. The puzzling presence
of classical ‘protostomes’ and ‘deuterostomes’, in which the
blastopore develops either into the mouth (or mouth and
anus) or anus, respectively (figure 1a), led some authors to
conclude that the through gut evolved at least twice in bilaterians [2]. More recently, the repeated observation of slit-like
blastopores in a variety of organisms, such as annelids [14],
onychophorans [9], arthropods [15] and nematodes [16], has
led to the ‘amphistomy’ theory [17] in which the protostomous
and deuterostomous states are both derived from a primitive
slit-like blastopore that closed centrally to leave the mouth at
one end and the anus at the other (figure 1b) (e.g. fig. 22.2 of
[18]). Nevertheless, others have argued that the reported
examples of ‘amphistomy-like’ development in fact represent
special cases of protostomy or deuterostomy rather than true
amphistomy as seen in annelids [5,19]. The (disputed) case in
onychophorans has been suggested to have arisen as a result
of terrestrialization [5].
In order to clarify these issues, we here document the fate
of the onychophoran mouth –anus furrow in a series of successive embryonic stages. Our data show that the posterior
of the furrow moves into a position very close to the posterior
pit region (i.e. the position of the anus), and that the indentation of the posterior pit disappears. The proctodaeal marker
genes forkhead ( fkh) and caudal (cad) are not expressed in the
posterior pit but in the posterior of the mouth –anus furrow.
Exclusive expression of the blastoporal marker genes
brachyury (bra), and indeed also wg/Wnt1, in the posterior
pit suggests that this region represents the blastopore. This
result contradicts the assumption that the ventral furrow is
derived from the blastopore.
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3
fkh
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pp
pp
(a¢)
(d) stage 10
(b) stage 3
(b¢)
(e)stage 12
(d¢)
(e¢¢)
a
m–a
(c¢)
(c) stage 5
(e¢)
m
a
( f ¢¢)
( f ) stage 16
a
m
*
(g) stage 19
Figure 2. Expression of E. kanangrensis forkhead (fkh). For all panels, anterior is to the left representing ventral views. Panels (a 0 – e 0 ) show DAPI-stained embryos
corresponding to embryos shown in (a – e). Panels e 0 0 and f 0 0 represent close-up photographs of the anal region of embryos shown in e and f. Red arrows point to
the position of the posterior pit, which is free from expression. Green arrows point to expression in the anus ( posterior of the mouth – anus furrow), which expresses
fkh. Green arrowheads point to expression in the mouth. Black arrows point to expression along the ventral rim(s) of the split germ band. White arrows correspond
to black arrows in DAPI-stained embryos. The black arrowhead points to a common staining artefact in L15. Asterisk in panel (g) marks expression inside the head
bulbs. a, anus; m, mouth; m – a, mouth – anus furrow; pp, posterior pit.
the entire cloned fragments. Cell nuclei were stained by incubation
of the embryos in 1 mg ml21 of the fluorescent dye 4-6-diamidin2-phenylindol (DAPI) in phosphate-buffered saline with 0.1%
Tween-20 (PBST) for 30 min followed by extensive washes in PBST.
(d) Data documentation
Embryos were analysed under a Leica dissection microscope
equipped with a Leica DC100 digital camera. The image processing
software Adobe PHOTOSHOP CS2 (v. 9.0.1 for Apple Macintosh) was
used for linear corrections of brightness, contrast and colour values
in all images.
3. Results
(a) Expression of marker genes
Gene expression analysis in E. kanangrensis represents a wellestablished technique. The level of background is usually
Proc. R. Soc. B 282: 20142628
(a) stage 1/2
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4
bra
(b) stage 6
(c¢)
proct
(e) stage 18
Figure 3. Expression of E. kanangrensis brachyury (bra). For all panels, anterior is to the left and ventral view. Panels (a 0 – c 0 ) represent DAPI-stained embryos
corresponding to embryos shown in (a –c). Green arrows point to the position of the anus. Abbreviations as in figure 2; proct, proctodaeum.
low, and detectable expression patterns are clear. For the genes
investigated in this study, we do not find any significant variation: in all embryos of a given stage, the pattern is the same for
a given gene.
Euperipatoides kanangrensis forkhead (Ek-fkh) is expressed
strongly in the germ disc around the mouth–anus furrow,
but not the posterior pit (figure 2a). When the two halves of
the embryo grow towards the anterior on either side of the
mouth–anus furrow, transcripts of fkh are detected in all
tissue in between (figure 2b,c). Stronger signal, however, is
along the ventral margins of the germ band(s) and close to
the furrow (figure 2b,c). When the mouth–anus furrow closes,
expression disappears from the tissue that has grown together,
but remains in the openings, i.e. the mouth and the posterior
part of the mouth–anus furrow (figure 2d,e,e 00 ). Anterior
expression is at later stages, after the mouth opening has disappeared behind the outgrowing head bulbs, at either side of the
mouth in the posterior rim of these bulbs (figure 2d–f). At stage
16, the posterior signal is in the anus, but does not extend into
posterior adjacent tissue of the posterior pit (figure 2f,f 00 ). At
later developmental stages, fkh is also transcribed inside the
head bulbs. Expression in and around the mouth, in the anus
and along the ventral margin of the split germ band is present
at all developmental stages, but never extends into the posterior
pit (figure 2, all panels).
At all investigated stages, E. kanangrensis brachyury
(Ek-bra) is expressed in the posterior pit, but is absent from
the mouth –anus furrow (including the anus; figure 3).
At later stages, Ek-bra expressing cells contribute to the
developing hindgut (figure 3d,e). No other signal of Ek-bra
has been observed at any of the investigated developmental
stages.
Euperipatoides kanangrensis caudal (Ek-cad) is expressed in
the posterior part of the developing embryo, but not in the
centre of the posterior pit (figure 4a–d) (cf. [22] (supplement)).
Its expression surrounds the pit and extends anteriorly into
the posterior of the mouth–anus furrow, and a few lastformed posterior segments (figure 4a–d). Expression of cad in
the posterior segments is strongest in the dorsal edge of the
germ band and as transverse stripes between the coelomic
pouches (figure 4b,c). The segmental expression may form a
short-range gradient, but this is unclear from the available
data. At later stages, Ek-cad is expressed in the ventral portion
of the anal valves and also in tissue that possibly turns into the
hindgut (figure 4e,f).
(b) Successive developmental stages showing origin
and migration of the anus
At approximately stage 7/8, the mouth–anus furrow splits into
mouth and anus; this is the result of median closure of the furrow.
The part of the mouth–anus furrow that develops into the mouth
soon after appears as a small round structure. The part of the
furrow that develops into the later anus remains for some time
as a slit-like structure (figure 5a). The suture of the closing central
part of the mouth–anus furrow is still visible in figure 5a. At this
stage (stage 9), the pit that is located posterior to the furrow
appears as a round anus-like indentation (figure 5a). At the subsequent stage 10, the pit is compressed. Now, the posterior part of
the mouth–anus furrow is round and looks like a typical anus
(figure 5b). At stage 11, the anus ‘moves’ further towards the posterior pit. The latter does not appear as a typical anus anymore.
This becomes clear at the subsequent stages 12–14 (figure 5d,e).
The former indentation has fully disappeared, whereas the opening of the anus (the former posterior part of the mouth–anus
furrow) persists. At stage 15, the anal valves form, and the
anus is now in a position posterior and between these structures
(figure 5f). This is the position of the anus as seen at later
developmental stages and adult onychophorans.
4. Discussion
(a) The onychophoran anus forms from the posterior of
the mouth –anus furrow
We carefully observed formation and fate of the mouth and
the anus in E. kanangrensis. Our observations reveal that the
Proc. R. Soc. B 282: 20142628
(b¢)
(d ) stage 18
a pp
m–a
ma
(a¢)
(c) stage 11
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(a) stage 2/3
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cad
(a) stage 4
(b) stage 8
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* *
*
*
(c) stage 10
(d) stage 11
a
m–a
pp
* *
(a¢)
(b¢)
(c¢)
*
a
*
(d¢)
L14
av
(e) stage 17
av
( f ) stage 17
Figure 4. Expression of E. kanangrensis caudal (cad). For all panels anterior is to the left and ventral view, except panels (c,f; lateral view). Panels (a 0 – d 0 ) represent
DAPI-stained embryos corresponding to embryos shown in (a – d). Green arrows point to the position of the anus. White arrows point to the position of the posterior
pit. Asterisks in panels (c) and (c0 ) mark transverse segmental stripes of expression. Abbreviations as in figure 2; av, anal valve.
slit-like groove which forms early during development
[13,22] closes from its centre to the outer edges leaving an
anterior opening and a posterior opening (figure 5). On
purely morphological grounds, this could be considered an
instance of amphistomy. However, our gene expression
results do not support this interpretation (discussed below).
Posterior to the posterior remnant of this furrow is a region
of high cell density. In the centre of this area an indentation
forms. This is the blastopore sensu [9] (discussed below).
The anterior opening of the furrow forms the future mouth
as concordantly suggested by authors [8 –13]. The posterior
opening remains throughout development and does not, as
previously suggested by Eriksson & Tait [13], disappear at
later developmental stages. By contrast, the indentation in
the centre of the posterior pit region disappears. Eriksson &
Tait [13] suggested that this latter structure turns into the
anus, but our observations show that this is not the case.
The main argument that led [13] to suggest that the anus
forms in the region posterior to the mouth –anus furrow is
that the ostensible anus-marker wg/Wnt1 is expressed in
this region. Although it is true that wg/Wnt1 serves as an
anus marker in arthropods (and other bilaterians), this is
merely, because, in these animals, the anus forms together
with the invaginating proctodaeal tissue. It has to be taken
into account that wg/Wnt1 (as a member of the Wnt pathway)
is rather a blastopore marker, or a marker of invaginating
cells, than a marker of the anus per se [24]. We investigated
the expression of two molecular markers, fkh and cad. In
arthropods, these genes are regularly associated with the formation of the anal valves and the anus [25– 33]. As expected,
cad is strongly expressed in the onychophoran anal valves. In
addition to that, and in contrast to wg/Wnt1, it is also
expressed in the tissue surrounding the posterior opening
of the mouth –anus furrow. The fkh gene is exclusively
expressed in the posterior of the slit anterior to the posterior
pit (figure 2). At later developmental stages, fkh is clearly
expressed in the anus, and because this expression can
easily be followed throughout development, it can unambiguously be associated with the posterior of the furrow.
A look at early stages reveals that neither cad nor fkh are
expressed in the centre of the posterior pit, the structure
that, according to Eriksson & Tait [13], would form the
anus. This shows that the onychophoran anus originates
from the posterior opening of the mouth –anus furrow and
not the region posterior to it.
(b) Where is the blastopore?
The blastopore is generally referred to as the site of endomesoderm formation (reviewed in [5]). It has been argued that the
mouth–anus furrow in onychophorans may represent the blastopore, or at least part of it [9–13]. The early claims for this view
were eventually grounded on the findings of [34,35], who
claimed that cells which originated from the mouth–anus
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a
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m
a pp
m–a
(a) stage 9
m
a pp
a
Euperipatoides
kanangrensis
Platynereis
dumerilii
Figure 6. Expression of the blastopore marker brachyury (bra) in an onychophoran
and an annelid. Note that the onychophoran mouth–anus furrow including its
anterior extension (the mouth (m)) and its posterior extension (the anus (a)) do
not express bra; bra is expressed in the posterior pit posterior to the furrow (the
putative blastopore (bp)). In an annelid, bra is expressed in the complete furrow
indicating that this structure represents the blastopore. While the situation in annelids supports the presence of amphistomy theory, data from the onychophoran
show that its development is rather deuterostomic. a, anus; bp, blastopore; m,
mouth; m–a, mouth–anus furrow. (Online version in colour.)
pp
a
(c) stage 11
a (pp?)
mouth–anus furrow implies that this structure is not part of the
blastopore. Therefore, we suggest that the blastopore sensu [9],
i.e. the posterior pit, indeed, represents the onychophoran blastopore. It is, however, possible that endodermal tissue that
comes from the edges of the furrow is not under control of
the canonical endomesodermal gene regulatory network, and
does for this reason not express bra and/or wg/Wnt1 (or any
other Euperipatoides Wnt gene) [39].
(d) stage 12/13
a
(pp?)
(e) stage 14
a
av
( f ) stage 15
Figure 5. Morphological evidence for the origin of mouth and anus in
E. kanangrensis. Developmental series showing that the anus originates
from the posterior remnant of the mouth – anus furrow. The indentation
in the posterior pit area disappears. For all panels, anterior is to the left
and ventral view. The arrow in (a) points to the suture of the closing
mouth – anus furrow. Arrows in (c – f ) point to the position of the mouth.
Abbreviations as in figure 2; av, anal valve. (Online version in colour.)
furrow would represent endomesoderm and thus contribute
to the midgut. Support also came from the recent work of Eriksson & Tait [13], who suggested that both the mouth–anus
furrow and the region posterior to the furrow should be
considered the onychophoran blastopore.
Endomesoderm formation in Bilateria is under control of
a conserved gene regulatory network (reviewed in [36]). At
the top of this network acts beta-catenin (aka armadillo
(arm)), a target of the Wnt signalling pathway [37]. Nuclearization of arm activates a number of endomesoderm-specific
target genes such as bra. Wnt genes and bra are thus plausible
blastopore marker genes (reviewed in [38,39]).
In onychophorans, both wg/Wnt1 [13] and bra (figure 3) are
strongly expressed in the region posterior to the mouth–anus
furrow. It appears thus plausible from morphological observations (invagination of mesodermal and endodermal tissue;
summarized in [3]) and the presence of blastopore marker
genes ([13] and this study) that this region is part of the blastopore. The fact that neither of the two genes is expressed in the
(c) Onychophoran development: another case of
‘concealed’ deuterostomy
The evidence we present here shows that the ventral slit which
appears in onychophoran development is not the blastopore,
even though the mouth and anus both develop from it. The blastopore (as defined in this article) exclusively develops into
proctodaeal (hindgut) derivatives. In addition, none of the typical mouth or anus marker genes are expressed in the complete
mouth–anus furrow in onychophorans. This differs from an
annelid, where for example bra is at least weakly expressed
along an elongate blastopore that develops into both mouth
and anus ([40], but see [41] for different expression patterns in
another annelid).
The onychophorans thus have neither protostomous nor
amphistomous development. If only the region posterior to
the mouth–anus furrow represents the blastopore in onychophorans, then the developmental mode is deuterostome-like.
Although the blastopore does not develop into the anus,
the fact that it gives rise to the hindgut suggests that onychophoran ancestors developed their anus from the blastopore
(deuterostomy), and that the blastopore-independent formation
of the anus (and the mouth) evolved secondarily, possibly as an
adaptation to yolk-rich embryos. Protostomy and deuterostomy may thus represent a similar case of adaptive variation
in early development that has also been suggested for other
features such as the varying origin of the coelom [42].
The developmental mode observed in E. kanangrensis may
be called ‘concealed deuterostomy’ in order to distinguish it
from true deuterostomy and amphistomy. ‘Amphistomy’ in
onychophorans and annelids, in the sense that both mouth
and anus develop from a ventral slit-like structure, is thus not
Proc. R. Soc. B 282: 20142628
(b) stage 10
a
bp
6
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m
m
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D
Deuterostomia
P/A/D
Arthropoda
A/D
Onychophora
D
P/D
Priapulida
P/A/D
Mollusca
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a
zo
so
dy
Ec
lia
ra
pi
S
ia
m
to
os
Figure 7. Putative cases of deuterostomy, protostomy and supposed amphistomy are plotted on a simplified animal phylogeny. Note that deuterostomic
development (D) is widespread, while amphistomy (A) and protostomy (P) are restricted to ‘protostomes’. The ‘X’ indicates that amphistomy is not present in
onychophorans (this study). Information was taken from table 4.1 of [5]). (Online version in colour.)
only morphologically different, but also partially involves
different genetic factors (figure 6). It is therefore rather a
convergent than a homologous (and possibly ancestral) developmental mode. Plotting supposed cases of amphistomy
against their newer interpretations and other new investigations of taxa such as priapulids [5,19,43] on a phylogeny
(figure 7) reveals that deuterostomy, not amphistomy, is the
ancestral state of bilaterian development.
Acknowledgements. We gratefully acknowledge the support of the NSW
Government Department of Environment and Climate Change by
provision of a permit SL100159 to collect onychophorans at Kanangra
Boyd National Park, and to the Australian Government Department of
the Environment, Water, Heritage and the Arts for export permits
WT2009-4598 and WT2012-4704. We wish to thank Jean Joss, Robyn
Stutchbury, Rolf Ericsson and especially Noel Tait for their help during
onychophoran collection. The onychophoran transcriptome was
analysed with the kind support of Nico Posnien and Alistair McGregor.
Funding statement. Financial funding was provided to G.E.B. and L.L. by
the Swedish Natural Science Council (VR) and the Swedish Royal
Academy of Sciences (KVA), and to G.E.B. and R.J. by the European
Union via the Marie Curie Training networks ‘ZOONET’ (MRTNCT-2004-005624).
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