Download Evolutionary developmental perspective for the origin of turtles: the

EVOLUTION & DEVELOPMENT
13:1, 1 –14 (2011)
DOI: 10.1111/j.1525-142X.2010.00451.x
Evolutionary developmental perspective for the origin of turtles: the
folding theory for the shell based on the developmental nature of the
carapacial ridge
Shigeru Kuratani,a,! Shigehiro Kuraku,b and Hiroshi Nagashimaa
a
Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
Laboratory for Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Konstanz 78464, Germany
b
!Author for correspondence (email: saizo@cdb.riken.jp)
SUMMARY The body plan of the turtle represents an
example of evolutionary novelty for acquisition of the shell.
Unlike similar armors in other vertebrate groups, the turtle shell
involves the developmental repatterning of the axial skeleton
and exhibits an unusual topography of musculoskeletal
elements. Thus, the turtle provides an ideal case study for
understanding changes in the developmental program
associated with the morphological evolution of vertebrates.
In this article, the evolution of the turtle-specific body plan is
reviewed and discussed. The key to understanding shell
patterning lies in the modification of the ribs, for which the
carapacial ridge (CR), a turtle-specific embryonic anlage, is
assumed to be responsible. The growth of the ribs is arrested
in the axial part of the body, allowing dorsal and lateral oriented
growth to encapsulate the scapula. Although the CR does not
appear to induce this axial arrest per se, it has been shown to
support the fan-shaped patterning of the ribs, which occurs
concomitant with marginal growth of the carapace along the
line of the turtle-specific folding that takes place in the lateral
body wall. During the process of the folding, some trunk
muscles maintain their ancestral connectivities, whereas the
limb muscles establish new attachments specific to the turtle.
The turtle body plan can thus be explained with our knowledge
of vertebrate anatomy and developmental biology, consistent
with the evolutionary origin of the turtle suggested by the
recently discovered fossil species, Odontochelys.
INTRODUCTION
turtles have secondarily closed. For the third hypothesis, molecular phylogenetic analyses have almost unanimously concluded that the turtles have a close affinity to archosaurian
diapsids (crocodilians and avian families) (Fig. 1; Caspers
et al. 1996; Zardoya and Meyer 1998; Hedges and Poling
1999; Kumazawa and Nishida 1999; Mannen and Li 1999;
Mindell et al. 1999; Cao et al. 2000; Zardoya and Meyer 2001;
Iwabe et al. 2005; Hugall et al. 2007). This is also supported
by comparative analyses of karyotypic and genomic features
between sauropsidian lineages (Matsuda et al. 2005; Kuraku
et al. 2006; Chapus and Edwards 2009). Curiously, this view is
similar to views held by classical embryologists such as
Haeckel (1891) and de Beer (1937).
The second question, or the origin of the turtle body plan,
contains many different themes, most of which are related to
the structure and formation of the shell. The turtle shell consists of a dorsal half called the carapace, and a ventral moiety,
called the plastron (Fig. 2). The carapace is based on the
vertebrae and modified ribs (Fig. 2A). Histogenetically, the
neural arches and ribs of turtles first undergo perichondral,
then endochondral ossification, and finally an extensive
membranous ossification. Additionally, the nuchal (plus
The question of turtle evolution is 2-fold. First, the phylogenetic position of the turtle remains controversial (Lyson and
Gilbert 2009), and second, the turtle body plan is quite unique
among vertebrates and is difficult to derive from a generalized
pattern of the amniotes. Undoubtedly, these two major questions are related to each other. For the first question, three
major hypotheses have been proposed. The first and traditional view is based largely on reptilian skull morphology and
maintains that a basal position occurs in the turtle because its
cranium does not show any temporal fenestrae, classifying the
turtles as anapsida (Romer 1966; Gaffney 1980; Carroll 1988;
Laurin and Reisz 1995; Lee 1996, 1997, 2001; Reisz 1997;
Lyson et al. 2010). This position of turtles has been recently
supported by an event-pairing test using extant amniote embryos (Werneburg and Sánchez-Villagra 2009). By detailed
analysis of osteologic traits, Rieppel and deBraga suggested a
different hypothesis in which turtles belong to a sister-group
to extant lepidosaurian diapsids (Sphenodon and squamates)
(Rieppel and deBraga 1996; deBraga and Rieppel 1997; Hill
2005). This view postulates that the temporal fenestrae of the
& 2011 Wiley Periodicals, Inc.
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EVOLUTION & DEVELOPMENT
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200
100
Vol. 13, No. 1, January--February 2011
0 Million years ago
Squamata
Lepidosauria
Tuatara
Birds
Archosauria
Crocodiles
Cryptodira
(hidden-necked turltes)
Odontochelys
Proganochelys
Pleurodira
(side-necked turtles)
Mammals
Geological timescale
supramarginal plates, pygal, and all marginal plates if present)
develops from independent dermal ossification centers, unlike
the costal and neural plates. Because of the poor development
of the back muscle, the bone histology of the carapace
appears to be dermal. This, however, does not necessarily
Casichelydia
Fig. 1. A phylogenetic tree showing relationships and estimated divergence times
of sauropsid lineages. Divergence times
are based on molecular estimates by the
TimeTree project (Hedges and Kumar
2009). Fossil records for Odontochelys
and Proganochelys are dated as being
from 220 and 220–205 mya, respectively.
mean that this structure is involved any exoskeletal elements
in its evolutionary origin (for an historical review, see Goette
1899; Vallén 1942; see also Joyce et al. 2009; Lyson et al. 2010,
for fossil evidence on the exoskeletal origin of the carapace).
In the ontogenetic development, no major independent der-
Fig. 2. The turtle shell and comparison
of amniote body plans. (A) Dorsal (left)
and ventral (right) views of the carapacial
skeleton of Pelodiscus sinensis. The carapace is composed of fused ribs and vertebrae. (B) Ventral view of the plastron
(redrawn from Ogushi 1911). Schematic
drawings of avian (C) and turtle (D) skeletons showing topographical relationships between ribs and scapulae. In a
typical body plan of amniotes, as seen in
avians, the scapula is found outside the
rib cage, whereas in the turtle, the scapula
is located inside the rib cage. cos, costal
plate; dr, dorsal ribs; ent, entoplastron;
epi, epiplastron; hyo, hyoplastron; hypo,
hypoplastron; neu, neural plate; nu, nuchal plate; r, rib; sc, scapula; T, dorsal
vertebrae; xiphi, xiphiplastron.
Kuratani et al.
mal ossification centers appear to take part in the carapacial
development, except for the above-noted elements that are
unique to the turtle lineage (Gilbert et al. 2001, 2008). In
reference to the above inconsistency between the embryogenetic and histogenetic features of the turtle carapacial ribs,
Gilbert et al. (2008) pointed out that a heterotopic shift
(change in position of development in evolution) of the rib
primordium would have resulted in a shift in the mode of
ossification. As a result of this shift, a new interaction took
place between the dermis and the rib primordium, leading to
the ectopic dermal ossification in the superficial mesenchyme
(Cebra-Thomas et al. 2005; reviewed by Gilbert et al. 2008).
More profound questions arise from comparative morphological viewpoints. For example, unlike the condition
normally found in amniotes, the scapula of turtles is encapsulated in the shell (Fig. 2, C and D). The turtle scapula is
located underneath the carapace, unlike in most other vertebrates in which the scapula is found outside the rib cage
(Fig. 2C). Thus, the turtle specifically appears to have broken
the basic rules of the vertebrate body plan. The presence of
the dermal plastron, or the ventral half of the shell, is also
mysterious. The origin and homology of this structure are not
well understood. As will be discussed below, the carapace and
plastron may not have arisen simultaneously in evolution.
Mainly because of the reasons indicated above, the turtle shell
is regarded as an evolutionary novelty, and not simply arising
from a conspicuously modified reptile based on the same basic
body plan (Burke 1989, 1991, 2009; Hall 1998; Rieppel 2001,
2009), or even a hopeful monster, an organism with a profoundly mutant phenotype that has the potential to establish
a new evolutionary lineage (Rieppel 2001; Thei!en 2006,
2009). One of the methods used to explain such a profound
change in morphology is to assume that alterations have
occurred in the developmental program, producing the type
of changes that would lead to enormous changes in topographical relationships among skeletal and muscular elements. The present review is thus intended to analyze the
turtle body plan anatomically and developmentally, with the
aim of providing a better understanding of the nature of
turtle evolution.
THE CARAPACE
Morphologically, the position of the turtle scapula is problematic because the morphological homology is primarily determined by the ‘‘relative positions’’ of elements and the body
plan of tetrapods is based on such a homological identification of each anatomical element (Woodger 1945; Eldredge
1989; reviewed by Hall 1998). Two hypotheses, which are not
entirely mutually exclusive, have been proposed to explain the
cause of the encasement of the scapula. The first argues that
the pectoral girdles moved backward into the rib cage from its
Turtle shell evolution
3
anterior side during evolution (Ogushi 1911; Watson 1914;
Lee 1996). This is mainly assumed in the ‘‘composite model’’
of turtle shell origin, in which the turtle shell is thought to have
derived from fusion of the internal skeleton and preexisting
osteoderms, the bony plates developing within the dermis that
can be observed in crocodiles and many lizards (Lee 1996;
Joyce et al. 2009). The other hypothesis proposes that the
turtle ribs are deflected to a more superficial position outside
the scapula (Ruckes 1929; Burke 1989, 1991; Cebra-Thomas
et al. 2005). This hypothesis is supported by the ‘‘de novo
model,’’ in which ectopically positioned ribs cause dermal
ossification around them, leading to emergence of the carapace
(Gilbert et al. 2001, 2008; Rieppel 2001, 2009). The latter theory in particular is suggestive of saltatorial evolution because it
is difficult to advance plausible sequential changes from a potential ancestor because the scapula can only be situated outside or inside the rib cage and there are no topological
intermediates (Rieppel 2001). In fact, stem turtles appear
abruptly in the fossil record. Until recently, the oldest wellknown stem turtle, Proganochelys, had a fully formed carapace that provided no clue about the origin of the carapace.
By observing the relationships between the pectoral girdle
and body cavity in the embryos of several species of turtle,
Ruckes (1929) suggested that the backward shift of the pectoral girdle does not occur during development because the
ribs are prevented from growing in a normal ventral direction,
and are instead displaced far laterally and dorsally outside the
pectoral girdle by remaining in the carapacial dermis. This
view is rather similar to our ‘‘folding theory’’ explained below.
Burke (1989, 1991) attributed this displacement of turtle ribs
to the lateral attraction of rib progenitor cells by the inducible
function of the carapacial ridge (CR) (Burke 2009).
CR AND THE ARCHITECTURE OF AMNIOTE
EMBRYOS
A unique feature of the turtle embryo is the appearance of a
longitudinal ridge in the flank, called the CR. This turtlespecific structure has long been suspected to be able to induce
a turtle-specific pattern of rib growth, probably by attracting
rib progenitor cells, and thereby regulating the morphological
patterning of rib growth in the lateral and dorsal directions
(Burke 1989, 1991; Cebra-Thomas et al. 2005). Histological
images of the CR are also thought to support the hypothesis
described above. The CR consists of an accumulation of undifferentiated mesenchyme and a thickened surface ectoderm
covering the mesenchyme. Such a configuration is highly
reminiscent of certain tissue interactions, as found in the
apical ectodermal ridge (AER) of the limb bud, which is
responsible for the growth and patterning of the limbs and
has a histological composition similar to the CR (Burke 1989,
1991; Cebra-Thomas et al. 2005). Moreover, it is important to
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Vol. 13, No. 1, January--February 2011
identify the origin of the CR to determine whether it truly
represents a turtle-specific novelty; that is, a similar longitudinal ridge appears in most other amniotes. The latter has
been called the Wolffian ridge, after the name of the discoverer (for the embryological works of Wolff 1759).
Since Wolff’s (1759) work, a longitudinal ridge has been
recognized along the flank of the pharyngula of amniotes
(Fig. 3). This ridge, called the ‘‘Wolffian ridge,’’ has often
caused confusion among embryologists (summarized by
Stephens 1982; Stephens et al. 1992). Historically, the
Wolffian ridge was sometimes explained as the source of
hypaxial myoblasts, which may not be entirely wrong because
at these developmental stages the ventrolateral lip of the
dermomyotome (source of the so-called hypaxial muscles;
Gros et al. 2004) is temporarily located in this ridge (see Froriep 1885). The ridge has also been described as the site of
limb bud development (O’Rahilly and Gardner 1975; reviewed by Christ 1990; Carlson 1996), or even the origin of
the urogenital system. The Wolffian ridge appears on the lateral surface of the embryo (Fig. 3) and differs from the mesonephric ridge that lies far more medially in the embryo, and
it does not represent epidermal thickening along the body axis
(Stephens et al. 1992).
The Wolffian ridge appears only transiently in all amniote
embryos, including turtles, and represents the root of the lateral body wall, which attaches to the axial part of the embryonic body; namely, the dorsal half of embryos that was
originally occupied by the early somites (Fig. 3; Nagashima
et al. 2007). Later in development, this ridge flattens out in
most amniotes, including chicken embryos (Fig. 3). This
morphology and developmental sequence (appearance and
disappearance) of the Wolffian ridge is commonly recognized
in various vertebrate embryos, whether the animal develops a
carapace or not (Keibel 1906).Thus, the overt Wolffian ridge
defines the boundary between the axial domain and the lateral
body wall by its longitudinal indentation, corresponding to
the dermal ‘‘lateral somitic frontier’’ defined by Nowicki et al.
(2003). The Wolffian ridge is also present in turtles, as seen in
embryos at stage 13 of the Chinese soft-shelled turtle,
Pelodiscus sinensis (Tokita and Kuratani 2001), and the CR
and Wolffian ridge coexist as separate entities (on both sides
of the axial–lateral body wall boundary) in a small window of
the developmental timetable (Fig. 3).
The CR is not equivalent to the Wolffian ridge because the
latter appears in the medial-most part of the body wall,
whereas the former develops in the lateral-most part of the
axial domain (Fig. 3). For this topographical relationship, the
CR can be regarded as specific to turtle embryos. The rib
primordium in turtles, which grows toward the CR, remains
dorsal to the body wall, and is thus an axial structure
throughout the developmental period (Burke 1989; Nagashima et al. 2007). Our labeling using CM-DiI (C-7000,
Molecular Probes, Eugene, OR, USA) is consistent with the
Fig. 3. Schematic diagrams showing developmental sequences at
the trunk level in chicken (left) and Pelodiscus sinensis (right). Both
embryos show similar initial developmental patterns with the proximal part of the body wall swelling to form the Wolffian ridge
(WR) at the junction with the axial part of the body (arrows, top).
The junction is represented by a notch on the surface of the embryo, as commonly seen in the early amniote pharyngula. At the
next stage (middle) in the chicken embryo, sclerotome (sc)-derived
rib primordia (r) and muscle plate (mp) invade the body wall, and
in a later stage (bottom), the muscle plate also invades the lateral
body wall. Note, in the final stage, that the Wolffian ridge is flattened on the embryonic surface. In P. sinensis, in contrast, only the
poorly developed muscle plate invades the body wall, and ribs
remain axially. Uniquely in the turtle, the ventrolateral part of the
axial domain secondarily swells to form the CR, dorsal and parallel
to the Wolffian ridge. ax, axial domain; lbw, lateral body wall; n,
notochord; nt, neural tube.
Kuratani et al.
morphological evaluation described above and shows that the
indentation on the flank surface, located between the CR and
the Wolffian ridge, represents the axial–lateral body wall
boundary (Fig. 3; Nagashima et al. 2007), which is the lateral
limit of the dermatome-derived dermis as opposed to the
lateral mesoderm-derived dermis at the cell lineage level.
Through the persistent growth of the CR, the flank indentation is visible for an exceptionally long period of development
in turtles, whereas in other amniotes, the flank indentation
disappears (see Fig. 3; Keibel 1906). Studies on chicken–quail
chimeras have shown that a similar dermal boundary exists at
the junction of the body wall in avian embryos (Burke and
Nowicki 2003; Nowicki et al. 2003; Nagashima et al. 2005).
FUNCTION OF THE CR
If the CR is a turtle-specific novelty (at the embryogenetic
level), does the CR function in the turtle-specific pattern of rib
growth and explain the mechanical basis that brings about the
turtle body plan? There have been several studies conducted
to date that address this question at experimental embryological and molecular levels.
The experimental methods have removed, arrested, or
added the CR to determine the change in rib growth in the
turtle embryo. By eliminating the CR, Burke (1991) showed a
changed pattern of rib growth; however, histological analyses
were not conducted. The CR is very easy to regenerate, and
simple ablation of the CR will result in restoration of the CR
in the same position (Burke 1991; Nagashima et al. 2007).
This suggests that the CR is established as a typical ‘‘field,’’
constantly induced by the surrounding embryonic environment. In our experiments, the early CR was microcauterized
so that the cells at the site of the wound were unable to
respond to the inductive activities from the environment
(Nagashima et al. 2007). Using this method, the rib growth
did not change its dorsoventral pattern as observed in histological preparations and the ribs were arrested axially as they
are in normal development, and grew laterally. At the site of
cauterization, however, the characteristic fan-shaped growth
of the ribs was arrested, and the ribs grew with their distal
ends close to each other.
When an ectopic CR was added to the turtle embryo, there
was no change in the rib growth pattern. Thus, it appears that
the CR does not directly regulate the axial arrest or the lateral
growth of the ribs by providing the source of upstream factors
to induce such patterns. Rather, the CR apparently functions
in the flabelated pattern of rib formation, which is characteristic of the turtle carapace. This is consistent with the active
proliferation of the mesenchyme in the CR (Burke 1989;
Nagashima et al. 2007).
At the histological and molecular level, the function of the
CR has been compared with that of the AER in the vertebrate
limb or fin buds (Burke 1989). The expression of Fgf10 and
Turtle shell evolution
5
Msx1 reportedly occur in the CR of Trachemys and Emys
embryos, respectively (Loredo et al. 2001; Vincent et al. 2003),
and the expression of Msx2 and Shh in the epidermis and
Gremlin, Bmp4, and Pax1 in the mesenchyme have been
reported to occur in the late stages of CR development of
Trachemys (Moustakas 2008); however, expression of these
genes was not confirmed in P. sinensis (Kuraku et al. 2005;
data not shown). By comprehensive cDNA screening for
CR-specific genes in P. sinensis, we have identified four
genes, cellular retinoic acid-binding protein (Crabp)-I, Sp-5,
lymphocyte enhancer factor (Lef)-1, and Apcdd-1 (Kuraku
et al. 2005). These genes are not additional paralogues generated by Pelodiscus- or turtle-specific gene duplications; they
are orthologs of the genes universally found in amniotes. In
situ hybridization confirmed their CR-associated expression,
specifically in turtle embryos, and it is likely that alteration in
the regulation of these genes, which is unique to the turtle
lineage, resulted in their CR-associated expression. The technology used in the cDNA screening involved in vitro cloning
of cDNA fragments on microbeads (massively parallel signature sequencing; Brenner et al. 2000; Reinartz et al. 2002;
Torres et al. 2008; Wold and Myers 2008). This approach,
independent of the availability of large-sequence transcriptomic and genomics resources, is a powerful tool that enables
compact surveys of gene expression to be conducted in any
given tissue in nonmodel organisms.
Of the four genes indicated above, the developmental function of Crabp-I is still not well understood (Kuraku et al. 2005).
Among the other three, Sp-5 and Apcdd-1 are regulated by
Lef-1, downstream of the so-called canonical Wnt-signaling
pathway (Takahashi et al. 2002, 2005; Weidinger et al. 2005;
Shimomura et al. 2010). In the latter case, a nuclear localization is expected for b-catenin, as the cofactor of Lef-1, to enable it to function as a transcriptional factor (reviewed by
Novak and Dedhar 1999). Immunolocalization of b-catenin
showed that this was the case, and overexpression of a dominant-negative form of Lef-1 (lacking the b-catenin-binding
domain) led to the arrest of carapace formation, a pattern
similar to that seen after CR microcauterization (Nagashima
et al. 2007). Thus, the CR-specific expression of Lef-1 is necessary for normal growth of the carapacial margin and carapacial patterning is likely to be regulated by Wnt molecules.
We have examined as many Wnt cDNAs in P. sinensis as
possible to see whether any of them were differentially expressed between the CR and its adjacent region of the embryo. However, we have not successfully identified the
responsible Wnt gene (unpublished data). It is possible that
Lef-1 in the CR could be regulated downstream by different
signals, such as hepatocyte growth factor (HGF) (Danilkovitch-Miagkova et al. 2001; Monga et al. 2002; Nelson and
Nusse 2004; Rasola et al. 2007).
Some genes related to limb bud development (Capdevila
and Izpisua Belmonte 2001) were expressed in the CR of
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EVOLUTION & DEVELOPMENT
Vol. 13, No. 1, January--February 2011
P. sinensis; however, there were also genes that were not
expressed (Kuraku et al. 2005). Thus, even if the CR or
carapacial patterning per se resulted from the co-option of a
limb developmental program, it would be at most partial and,
therefore, not by a simple evolutionary event. Our current
understanding of the CR, therefore, is that it does not simply
function as an inducer of the turtle-specific rib growth pattern
(axial arrest and lateral growth), but rather as the marginal
growth center of the carapace, assisting the fan-shaped
growth of the ribs. Nevertheless, the appearance of the CR
and the axial arrest of the rib primordium are coextensive and
these two phenomena are tightly linked to each other developmentally. Thus, we are tempted to assume that the CR and
the axial arrest of the ribs are induced together by an as-yet
unknown, identical upstream factor, or that the axially arrested ribs induce or maintain the CR, which further induces
the fan-shaped growth of the ribs. Turtle embryo-based experiments to test these scenarios have not yet been conducted.
In addition, a more exhaustive molecular search will be
needed to understand the development and function of the
CR because this turtle-specific embryonic structure is highly
relevant to the anatomical architecture of this animal, as will
be shown below.
SCAPULAR ENCAPSULATIONFTHE FOLDING
THEORY
To resolve the questions surrounding turtle-specific anatomy,
an understanding of the embryonic developmental patterns
and processes of turtles is needed. In particular, in association
with the position of the scapula encapsulated in the shell, we
need to observe the muscle connecting the scapula or forelimb
and the trunk. Below, we illustrate how our ‘‘folding theory’’
explains the evolutionary and developmental origins of the
unique turtle body plan.
In the deeper part of the shoulder of amniotes, there are
several muscles connecting the scapula and the back: the
serratus anterior (AS), rhomboid, and levator scapulae muscles. The latter two muscles form the levator scapulae–rhomboid (LSR) complex because they are innervated commonly
by the dorsal scapular nerve, arise from a common precursor,
and occupy a similar position (Nagashima et al. 2009). The
AS muscle is innervated by the long thoracic nerve. These
muscles are commonly found in the shoulder in various
amniotes (Fürbringer 1874, 1875, 1900, 1902). Other muscles,
such as the latissimus dorsi muscle and the pectoralis, are
found in the more superficial layer of the shoulder and commonly connect the forelimb (humerus) and the trunk.
Because of the formation of the shell and encapsulation of
the scapula in the turtle, the muscles described above have
also changed their shapes or connectivities. For example, the
AS muscle of the turtle is found beneath the carapace, and the
LSR complex is shifted rostrally into the neck. The connectivities of the pectoralis and latissimus dorsi muscles have also
changed conspicuously: the pectoralis attaches onto the dorsal
aspect of the dermal plastron in the turtle, not the ventral
aspect of the sternum, and the latissimus dorsi muscles connect onto the neck, not onto the back as in other amniotes.
Of the muscles described above, the alteration of deeper
muscles can be easily explained by the folding of the turtle
embryonic body. In the early stages of development, the
scapula of P. sinensis lies rostral to the ribs and at the junction
of the forelimb bud and the lateral body wall, as in other
amniote embryos. There are two factors in the turtle embryo
that shift the scapula beneath the ribs: the axial arrest of the
ribs and the poor development of the blade in the turtle
scapula. In most amniotes, the scapula grows a caudal process
called the blade, which lies lateral to the rib cage. This blade
specifically arises from thoracic somites in the avian embryo
(Chevallier 1977; Huang et al. 2000). Thus, the AS muscle
usually stretches more or less dorsoventrally in avian families
and nonturtle reptiles, which is also seen in mammalian embryos and adults (Fürbringer 1875, 1902; Ribbing 1931).
In the early turtle embryo, the AS anlage arises from a part
of the muscle plate connecting between the dorsal portion of
the scapula and the distal part of the second rib, a connectivity that is common in all amniotes (Fig. 4). Because of the
topographical position of the scapula, this muscle stretches
anteroposteriorly, probably representing the original developmental position of the muscle. Because the rib growth is
axially arrested, the rostral ribs in the turtle grow laterally and
anteriorly over the scapula, corresponding to the fan-shaped
patterning process induced by the CR (Nagashima et al.
2007). Interestingly, during this process of carapace formation, the connectivity of the AS muscle does not alter and
follows the shifts of the skeletal elements. Thus, the muscle
rotates inward to assume a position underneath the carapace
(Fig. 4). The developmental changes of LSR muscles are more
straightforward. They are connected to the dorsoanterior part
of the shoulder girdle and vertebrae, and this relationship is
again maintained through the carapace formation (Fig. 4).
Thus, the deeper muscles simply follow the topographical
changes of the skeletal elements during development, and
their connectivities or morphological homologies are preserved through this process. This shift can be seen as the
inward folding of the lateral body wall. The original body wall
can be sought to the folded muscle plate, the direct derivative
of the myotome, which is bent strongly inward with the fanshaped growth of the ribs. With respect to this muscle plate,
the scapula anlage is found as laterally as it first appeared,
and the CR is found along the line of the folding. In other
words, the carapacial margin represents the place where the
body wall was folded during development.
Development of the superficial muscles is slightly different
from the deeper muscles. The latissimus dorsi, for example,
Kuratani et al.
Turtle shell evolution
7
Fig. 4. Development of AS and LSR
muscles. Schematic representation of the
muscle development in chicken (left) and
Pelodiscus sinensis (right). Both animals
develop from a nearly identical embryonic morphology (top), except the ribs of
the P. sinensis embryo are comparatively
shorter than those in the chicken. At this
stage, the AS anlage is found on the
ventroposterior aspect of the scapula, and
the LSR is found dorsoanteriorly. Note
that the AS connects the scapula to the
anterior ribs. Later in the turtle development, the ribs grow laterally and anteriorly by folding the dorsal part of the
lateral body wall inward, encapsulating
the scapula in the rib cage. This growth
does not alter connectivities between the
ribs, AS and LSR muscles. as, serratus
anterior; lsr, levator scapulae–rhomboid
muscle complex; mp, muscle plate; r, rib;
sc, scapula.
which usually connects to the back and the humerus, connects
the humerus and the ventral aspect of the nuchal plate in
turtles, a turtle-specific novelty (Figs. 2A and 5). During development, the superficial muscles first arise from the
dermomyotome and migrate for a long distance, without
establishing any connectivity to the skeletal anlagen, and into
the limb bud (Fig. 5A). During this process, the myoblasts
characteristically express a homeobox gene, Lbx-1, in addition to Pax3, and their migration is dependent on HGF
signaling (Jagla et al. 1995; Mennerich et al. 1998; Dietrich
1999; Alvares et al. 2003). A similar behavior for the
dermomyotome-derived myoblasts has been found for the
tongue, infrahyoid, mammalian diaphragm, and cucullaris
(trapezius and sternocleidomastoid muscles), in addition to
the limb muscles, and these cells have been collectively called
the migrating myogenic precursor cells (MMP cells) (reviewed
by Birchmeier and Brohmann 2000). Some of the MMP cells
in the limb bud secondarily grow out again to establish connections to the back as latissimus dorsi and pectoralis muscles
(Fig. 5, B and C). They are specifically called ‘‘in-and-out’’
muscles and they are thus classified as limb muscles from their
developmental patterns and mechanisms (Evans et al. 2006).
The development of MMP muscles in the turtle is fundamentally similar to that in other amniotes: the anlagen for
tongue, infrahyoid, cucullaris, and limb muscle precursors
have been observed to express Lbx-1 (Nagashima et al. 2009,
and H. Nagashima and S. Kuratani, unpublished observation). Curiously, however, the direction of the growing
latissimus dorsi muscle anlage in the turtle differs from that
in other amniotes. That is, instead of growing dorsally and
posteriorly and expanding to cover the back of the embryo, in
the turtle embryo the latissimus dorsi muscle anlage grows
more anteriorly to find connection in the nuchal plate (Fig. 5,
B and D). Thus, the shift of connectivity for this muscle is to
be ascribed to the late (getting ‘‘out’’ of the limb bud) phase of
patterning, whose developmental mechanism remains unclear.
The strange growth pattern of this muscle in the turtle may be
again related to the CR; that is, no muscles can invade the
carapacial primordium, the latissimus dorsi, or the cucullaris.
As far as the developmental pattern suggests, the CR appears
to inhibit the immigration of myoblasts in late development.
Thus, the developmental behaviors of superficial and deep
muscles differ from each other quite conspicuously in the
turtle embryo. The deeper muscles primarily belong to the
trunk muscles, connecting the trunk and shoulder girdle, and
establish early connectivities that do not alter in later development. The superficial muscles, in contrast, are categorized
into limb muscles and find their connectivities rather late in
development, which apparently gives the muscles a ‘‘flexibility,’’ mainly because of a low level of developmental burden
(Riedl 1978) derived from the late-developmental timing.
Thus, turtle evolution has used changes in the connectivities
of these flexible muscles, not the solid trunk muscles, to attain
the carapace. Even when the connectivities are topologically
changed, the homologies of these flexible limb muscles, such
as the latissimus dorsi and pectoralis, can be discerned by the
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EVOLUTION & DEVELOPMENT
Vol. 13, No. 1, January--February 2011
Fig. 5. Development of the latissimus
dorsi and pectoralis muscles. (A) In the
early development of amniotes, MMPs
expressing Lbx-1 migrate from the ventrolateral aspect of somites to the forelimb
bud to differentiate into mature myoblasts. (B) MMPs differentiate into dorsal
and ventral muscle masses in the forelimb
bud to form extensor and flexor muscles,
respectively. The proximo-caudal part of
the dorsal and ventral muscle masses give
rise to the latissimus dorsi and pectoralis
muscles, respectively. In the chicken (C),
the latissimus dorsi arises from the forelimb buds and grows posteriorly to expand over the back. In Pelodiscus sinensis
(D), the anlage grows dorsally and anteriorly to circumvent the carapace, and to
connect inside the nuchal plate. The pectoralis in P. sinensis attaches to the dorsal
aspect of the plastron, not the ventral
aspect of the sternum as in the chicken.
h, humerus; dm, dorsal muscle mass; fl,
forelimb bud; ld, latissimus dorsi; mmp,
migrating myogenic precursors; mp, muscle plates; nu, nuchal plate; p, pectoralis;
pl, plastron; s, somites; st, sternum; v,
vertebrae; vm, ventral muscle mass.
relative positions of their anlagen when they come out of the
limb bud (Fig. 5, B and C). Thus, these muscles are developmentally constrained in their primary morphological patterns and positions.
FOSSIL EVIDENCE
Until recently, the oldest well known fossil turtle was Proganochelys, which lived 220–205 mya in Germany (Fig. 1;
Gaffney 1990). Although this animal retained ancient traits,
such as the supratemporal bone, lacrimal bone and duct,
moveable basipterygoid articulation, a middle ear without a
bony lateral wall, paired vomer, the paroccipital process of the
opisthotic bone attached to the braincase only at its distal end,
a nonretractable neck, palatine teeth, and cervical ribs (Romer
1956; Gaffney 1990), its shell was already very similar to that
found in modern turtles. Namely, it consisted of a carapace
and plastron, each composed of comparable skeletal elements
found in modern turtles. Evidently, this animal represented a
basal lineage with respect to the common ancestor of the
Cryptodira and the Pleurodira (Fig. 1). However, because of
the morphology of the shell, it does not help our understanding of the origin of the basic body plan of the turtles.
Recently, Li et al. (2008) discovered another important
fossil called Odontochelys, which lived 220 mya in China (Fig.
1). Interestingly, this animal possessed a dermal plastron,
again very similar to that found in modern turtles, but no
carapace. Because the plastron of this animal represents a
solid plate of dermal complex, it is unlikely that the condition
of the back of this animal represents a secondary reduction as
has been assumed (Reisz and Head 2008). Most curiously, the
dorsal ribs of this animal have not grown in the fan shape that
is characteristic of those seen in modern turtle carapaces. Because of the latter feature, the scapula is located rostral to the
ribs, not underneath them. The axial arrest of the ribs is apparent because they are short and do not bend ventrally.
The morphology of the Odontochelys appears to offer a
hint as to the evolutionary origin of the turtle body plan (Fig.
6; Nagashima et al. 2009). First, the embryos of Odontochelys
are likely to have developed the CR at certain developmental
stages as far as their ribs are arrested axially. As noted above,
development of the CR is coextensive with the arrested rib
growth, if not the CR functions in the arrest itself (Nagashima
et al. 2007). However, we assume that a hypothetical CR in
the Odontochelys would not have persisted to grow rostrally
and caudally to complete a circle representing the carapacial
margin. This assumption is also likely and consistent with the
absence of the carapace and lack of a fan shape in the ribs in
this fossil species; the fan-shaped growth of the ribs depends
on the late function of the CR (Nagashima et al. 2007). Thus,
the morphology of the shoulder region of Odontochelys resembles that in the TK stage 16 of P. sinensis embryos, in
which the scapula is clearly located rostral to the dorsal ribs
(Fig. 4; see also Sánchez-Villagra et al. 2009, Fig. 4, J and K).
Indeed, the scapula is morphologically always lateral to the
Kuratani et al.
Turtle shell evolution
9
Fig. 6. A hypothetical scenario for the evolution of the turtle body plan. A phylogenetic tree shows the probable timing for the acquisition
of the major morphological characteristics of turtles. In Odontochelys, the carapacial ridge (CR) (red broken line) may have developed only
temporarily and incompletely in the embryo. In Proganochelys, the CR (red solid line) forms a complete circle, inducing the fan-shaped
growth of the ribs. The morphology of Odontochelys and Proganochelys are referred from Li et al. (2008) and Gaffney (1990).
body wall and rostral to the first rib, even in modern adult
turtles, as has been shown anatomically (Ogushi 1911; Nagashima et al. 2009), which is secondarily shifted by the abovementioned folding, and in turn is based on the fan-shaped
growth in the late developmental stages.
Thus, we can imagine that the AS muscle of the
Odontochelys would have stretched anteroposteriorly between
the rostral scapula and caudal ribs (Fig. 6), more or less
assuming the shape of the scalenus muscles in amniotes. The
affinity of the AS and scalenus has already been indicated by
anatomists (Nishi 1931; Romer and Parsons 1977). The
position of the latissimus remains questionable. In modern
turtles, the growth of the in-and-out muscles is apparently
arrested by the CR, and the muscle growth circumvents the
carapace, because no muscles extend over the carapace of
modern turtles. Because the carapace does not exist in
Odontochelys, and a CR in this animal would not have grown
anteroposteriorly but vanished earlier in development, the latissimus of this animal could have found its connection onto
some dorsal vertebrae. The pectoralis, in contrast, may have
inserted onto the dorsal aspect of the plastron, which was
already present in Odontochelys (Fig. 6; Nagashima et al.
2009). Thus, as far as the fundamental anatomical pattern is
concerned, turtle evolution appears to have proceeded in a
sequence that involves a folding of the body wall, which is
parallel to the embryonic development of the modern turtle.
Although this evolutionary change can be explained as a developmental repatterning, the evolutionary process of the
turtle may not be saltatory as has been assumed because the
anatomical pattern of Odontochelys can reasonably be regarded as an intermediate.
RIDDLE OF THE PLASTRON
The evolution of the turtle plastron remains enigmatic. It appears that this structure arose as an entirely novel structure or
as a modification of an ancestral skeletal element found in
some reptiles (Romer 1956; Claessens 2004). The plastron in
modern turtles is generally made up of nine bones (Fig. 2B),
of which the anterior-most pair, the epiplastron, are thought
to be homologous to the clavicle in other amniotes. Medial to
the epiplastron is an entoplastron, homologous to the interclavicle and posteriorly positioned to the remaining gastralia,
the original scales found in the ventral dermis (Romer 1956;
Gaffney 1990; reviewed by Gilbert et al. 2001).
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EVOLUTION & DEVELOPMENT
Vol. 13, No. 1, January--February 2011
It has recently been suggested that the turtle shell has been
acquired as an evolutionary novelty, differentiated from the
neural crest (Clark et al. 2001; Pennisi 2004; Cebra-Thomas
et al. 2007; Gilbert et al. 2007, 2008). This assumption is
supported by the expression of the HNK-1 antigen, PDGF
receptor, and other proteins related to neural crest cells, as
well as vital dye-based cell labeling in late-stage turtle embryos. This idea is based on the hypothetical co-option of the
cephalic crest-like potency of differentiation to the trunk crest
during turtle development.
As emphasized mainly in experimental avian embryology,
the skeletogenic differentiation is restricted to the cephalic
neural crest, which is capable of producing the major part of
the cranial skeletal elements, including cartilage and dermal
bones, and various types of connective tissue, the repertoire of
which is not usually seen in the trunk crest (reviewed by Le
Douarin 1982; Noden 1988; also see Kuratani 2005). However, to date, the skeletogenic potential has not been strictly
associated with the cephalic crest. The trunk crest-derived
cells are also capable of differentiating into chondrocytes
when they are grown in long-term culture or exposed to a
cephalic skeletogenic environment (McGonnell and Graham
2002; Abzhanov et al. 2003; Ido and Ito 2006). Thus, the
difference between cephalic- and trunk-crest cells may not be
as large as we have imagined. Gilbert and colleagues (Clark
et al. 2001; Pennisi 2004; Cebra-Thomas et al. 2007; Gilbert
et al. 2007) emphasize the similarity between the dermal ossification seen in the turtle shell (carapace and plastron) and
the amniote calvarium (dermal skull roof).
Before asking about the turtle plastron, it has to be remembered that even the developmental origin of the vertebrate dermal skull roof remains controversial (reviewed by
Gross and Hanken 2008). Some studies support a crest origin
for the entire skeletal complex, and others support a mesodermal and cephalic neural crest origin, with different investigators finding different boundaries in different animals (Le
Lièvre 1974, 1978; Noden 1982, 1984; Couly et al. 1993; Le
Douarin and Kalcheim 1999; Chai et al. 2000; Jiang et al.
2002; Matsuoka et al. 2005). It is unanimously recognized
that the cephalic crest cells are more or less involved in the
formation of the dermal skull roof. However, it remains unknown whether the entire dermal bone should be regarded as
having a crest origin. The local embryonic environment is
equally as important for the proper differentiation of the cephalic mesenchyme (Schneider 1999; reviewed by Kuratani
2005).
The trunk crest in the turtle may have acquired a cephalic
crest-like potential. This is an attractive idea; however, it is
equally plausible to assume that the lateral mesoderm of the
turtle embryo has acquired a cephalic crest-like capability to
form the carapace, and this has not yet been ruled out. In that
case, the lateral mesodermal cells would also show a gene
expression profile similar to that of the cephalic crest cells.
The enigma of the turtle plastron alerts us to the evaluation of so-called ‘‘marker genes’’ in studies of evolutionary
developmental biology. As yet, there is no simple rule or
principle to explain the relationships between homologous
genes and homologous anlage. It would be particularly risky
to homologize a certain cell population by the expression of a
single gene; gene regulation can evolve and shift (see Hall
1998; Locascio et al. 2002; Shigetani et al. 2002, for the case of
heterotopy in gene regulation). As a typical case, HNK-1 has
been used as a ‘‘crest marker’’ only for animals in which the
immunoreactivity of the HNK-1 antibody has been shown to
colocalize, at single-cell resolution, with migrating crest cells
at certain limited developmental stages. A good example of
fair evaluation of HNK-1 immunoreactivity can be found in a
classical article by Rickmann et al. (1985). The antibody primarily recognizes an epitope on some carbohydrate molecules
that may be present in migrating crest cells and a number of
cell types including neuroblasts, early neurons, or supporting
cells (Tucker et al. 1984; Vincent and Thiery 1984). Moreover,
to visualize HNK-1 immunoreactivity, the carbohydrate molecules should be stabilized using fixatives containing acetic
acid; however, immunohistochemistry based on this antibody
can produce different results when using different types of
fixatives (Rickmann et al. 1985, and references therein). In our
experiments in P. sinensis, HNK-1 did not react with cells
other than peripheral nerves when embryos were fixed with
Bouin’s fixative (Fig. 7), unlike results reported by Gilbert’s
group (Clark et al. 2001; Cebra-Thomas et al. 2007; Gilbert
et al. 2007).
Because a part of the clavicle in mammalian embryos is
derived from the most caudal population of cephalic neural
crest cells (Matsuoka et al. 2005), the plastron may also be
derived partially from the neural crest. However, to show the
origin of the entire plastron, or to detect the distribution and
differentiation of turtle crest cells, a new long-term labeling
method (preferably with a genetic marker that is not diluted
by cell division and that is free from contamination) is needed.
Popular vital dyes, such as DiI or DiO, are not suitable for
these types of long-term experiments. It will also be necessary
to label the lateral mesoderm of the turtle embryo to show
whether it gives rise to the plastron or not. Overall, the embryonic origin of the turtle plastron remains enigmatic.
CONCLUSIONFEVOLUTIONARY SCENARIO OF
THE TURTLES AND HOMOLOGY
From the discussions above, we can depict a scenario for the
evolution of a turtle body plan as shown in Fig. 6. In the
common ancestor that gave rise to Odontochelys and modern
turtles (the latter also includes fossil species such as Proganochelys), the scapula would have been located rostral to
the dorsal ribs. In these animals, the axial arrest of the ribs
Kuratani et al.
Turtle shell evolution
11
Fig. 7. Expression of the HNK-1 epitope
in a stage 17 Pelodiscus sinensis embryo.
A transverse section of a P. sinensis
embryo fixed with Bouin’s fixative was
stained with hematoxylin and eosin (A
and C) or HNK-1 antibody (B and D).
(A and B) The dorsal part of the embryo
showing distribution of the HNK-1 antigen. Note that the HNK-1 antigen is
restricted to the dorsal root ganglia (drg)
and dorsal part of neural tube (nt) and
that the dermis dorsal to the neural tube
is not stained with HNK-1. The neural
arch and vertebral body (vb) are also
HNK-1-negative. (C and D) The ventral
part of the embryo showing the anlage of
the plastron (pl). Note that the primordium of the plastron is completely negative for HNK-1 antibody whereas the
peripheral nerves (pn) stained positive.
Scale bar 5 100 mm. em, epaxial muscles.
would have taken place along with the transitional development of the CR in the embryos. This allows the plastron to
arise dermally, in the absence of ventral ribs. However, this
condition is not sufficient to create a dorsal carapace.
Odontochelys would have been one of the lineages that arose
among the animals with such a developmental program.
Because of the persistence and encircling of the CR in
certain lineages among the above-noted ancestral animals, the
dorsal ribs were induced to grow in a fan shape that did not
exist in Odontochelys. This developmental movement encapsulated the scapula, folded the body wall inward together with
the AS muscle, which is now rotated underneath the carapace,
and inhibited the invasion of limb bud-derived dorsal muscles
such as the latissimus dorsi. The latter muscle circumvented
the CR and found a new attachment in the rostral aspect of
the carapace, or the nuchal bone, using its developmental
flexibility such that its attachment is not specified early, as in
the trunk muscles (such as the AS or the LSR muscle complex). Thus, the body plan of the modern turtle can be
understood with our current knowledge of developmental biology, by assuming an intermediate (basal) state as seen in
Odontochelys. The synapomorphy in the developmental program for modern turtles would now involve completion of the
CR with the function of fan-shaped rib growth induction, the
resultant folding of the body wall and muscles, encapsulation
of the scapula, and the formation of new connectivities of
forelimb bud muscles.
In his monumental text, Philosophie anatomique, the
French anatomist Geoffroy Saint-Hilaire (1818) put forth a
rule of morphological homology: principe des connexions.
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EVOLUTION & DEVELOPMENT
Vol. 13, No. 1, January--February 2011
According to this rule, the animal body is composed of a set
of morphological elements that are connected together in an
identical manner. Thus, morphologically homologous elements in different animals can be found in equivalent positions in the shared body plan, although Saint-Hilaire himself
used the term ‘‘analogie’’ to mean the homology we use today. In the evolution of the turtle body plan, we can continue
to apply this criterion to the homologization of the AS muscle
and the LSR muscle complex. They have apparently changed
their positions; however, their connectivities to the skeletal
elements are morphologically unchanged. The most conspicuous example, in this context, can be found in the AS muscle
that is found beneath the carapace. The strange position of
this muscle in the turtle is now understood to be the result of
inward folding of the body wall with the AS maintaining the
same connectivities to the scapula and ribs, which are established early in development (Nagashima et al. 2009). SaintHilaire was absolutely correct in the homology of this muscle.
However, in other aspects of the turtle evolution, we can
also find a shift of connectivities, such as those in the limb
bud-derived muscles. For example, in tongue and cucullaris
muscles, the muscle precursor cells migrate long distances
based on HGF-mediated signaling, to establish connections to
the skeletal elements derived from a different mesenchymal
anlagen to their own (occipital somite-derived tongue muscles
attached to the crest-derived hyoid bone, somite-derived limb
muscle attached to the lateral mesodermal limb skeletons,
etc.). Evolutionarily, these muscles have more flexibility in
anatomical positions and, as a result, Saint-Hilaire’s connectivities or segmental assignments tend to be lost. This is also
where the nerve plexus tends to appear.
It is easy to conceive that the above developmental flexibility potentially results in ‘‘evolutionary novelty’’ quite frequently. These muscles are unbound, at least from the
conservative connectivities, with a certain fixed repertoire of
skeletal elements sharing the same developmental origins.
Evolution is thought to seek out chances of heterotopic shift
(coupling and decoupling) in the connectivities among developmental modules to establish novel morphological patterns,
and thereby traditional morphological homologies are often
disturbed or even lost (Müller and Wagner 1991; Shigetani
et al. 2002). The turtle body plan appears to offer a good
example with which to study coupling and decoupling in
evolution. Thus, the position of the AS muscle should not be
regarded as a novelty per se; it is rotated because it maintained the same ancestral connectivity (under the same
ancestral developmental constraint). Rather, the novelty can
be found in the latissimus dorsi and pectoralis, which have
established nonequivalent, turtle-specific connectivities. It has
not escaped our attention that the appearance of the CR is
behind this. The novel nature of the CR has been discussed
above. To complete the evolution of the body plan for Proganochelys and modern turtles, the dermally shifted ossifica-
tions of the carapace primordia (ribs) and the associated
reduction of the back muscle need to be considered.
Acknowledgments
We thank Marcelo R. Sánchez-Villagra for critical reading of the
manuscript.
REFERENCES
Abzhanov, A., Tzahor, E., Lassar, A. B., and Tabin, C. J. 2003. Dissimilar
regulation of cell differentiation in mesencephalic (cranial) and sacral
(trunk) neural crest cells in vitro. Development 130: 4567–4579.
Alvares, L. E., et al. 2003. Intrinsic, Hox-dependent cues determine the fate
of skeletal muscle precursors. Dev. Cell 5: 379–390.
Birchmeier, C., and Brohmann, H. 2000. Genes that control the development of migrating muscle precursor cells. Curr. Opin. Cell Biol. 12:
725–730.
Brenner, S., et al. 2000. Gene expression analysis by massively parallel
signature sequencing (MPSS) on microbead arrays. Nat. Biotechnol. 18:
630–634.
Burke, A. C. 1989. Development of the turtle carapace: implications for the
evolution of a novel bauplan. J. Morphol. 199: 363–378.
Burke, A. C. 1991. The development and evolution of the turtle body plan.
Inferring intrinsic aspects of the evolutionary process from experimental
embryology. Am. Zool. 31: 616–627.
Burke, A. C. 2009. Turtles . . . again. Evol. Dev. 11: 622–624.
Burke, A. C., and Nowicki, J. L. 2003. A new view of patterning domains in
the vertebrate mesoderm. Dev. Cell 4: 159–165.
Cao, Y., Sorenson, M. D., Kumazawa, Y., Mindell, D. P., and Hasegawa,
M. 2000. Phylogenetic position of turtles among amniotes: evidence from
mitochondrial and nuclear genes. Gene 259: 139–148.
Capdevila, J., and Izpisua Belmonte, J. C. 2001. Patterning mechanisms
controlling vertebrate limb development. Annu. Rev. Cell Dev. Biol. 17:
87–132.
Carlson, B. M. 1996. Patten’s Foundations of Embryology. 6th Ed. McGraw-Hill, New York.
Carroll, R. L. 1988. Vertebrate Paleontology and Evolution. Freeman,
New York.
Caspers, G.-J., Reinders, G.-J., Leunissen, J. A. M., Wattel, J., and de Jong,
W. W. 1996. Protein sequences indicate that turtles branched off from
the amniote tree after mammals. J. Mol. Evol. 42: 580–586.
Cebra-Thomas, J., et al. 2005. How the turtle forms its shell: a paracrine
hypothesis of carapace formation. J. Exp. Zool. 304B: 558–569.
Cebra-Thomas, J. A., Betters, E., Yin, M., Plafkin, C., McDow, K., and
Gilbert, S. F. 2007. Evidence that a late-emerging population of trunk
neural crest cells forms the plastron bones in the turtle Trachemys scripta.
Evol. Dev. 9: 267–277.
Chai, Y., et al. 2000. Fate of the mammalian cranial neural crest during
tooth and mandibular morphogenesis. Development 127: 1671–1679.
Chapus, C., and Edwards, S. V. 2009. Genome evolution in Reptilia: in
silico chicken mapping of 12,000 BAC-end sequences from two reptiles
and a basal bird. BMC Genomics 10 (suppl 2): S8.
Chevallier, A. 1977. Origine des ceintures scapulaires et pelviennez chez
l’embryon d’oiseau. J. Embryol. Exp. Morph. 42: 275–292.
Christ, B. 1990. Entwicklung der Extremitäten. In K. V. Hinrichsen (ed.).
Humanembryologie. Springer-Verlag, Berlin, pp. 838–862.
Claessens, L. P. A. M. 2004. Dinosaur gastralia; origin, morphology, and
function. J. Vertebr. Paleontol. 24: 89–106.
Clark, K., et al. 2001. Evidence for the neural crest origin of turtle plastron
bones. Genesis 31: 111–117.
Couly, G. F., Coltey, P. M., and Le Douarin, N. M. 1993. The triple origin
of skull in higher vertebrates: a study in quail–chick chimeras. Development 117: 409–429.
Danilkovitch-Miagkova, A., Miagkov, A., Skeel, A., Nakaigawa, N., Zbar,
B., and Leonard, E. J. 2001. Oncogenic mutants of RON and MET
receptor tyrosine kinases cause activation of the b-catenin pathway. Mol.
Cell. Biol. 21: 5857–5868.
Kuratani et al.
de Beer, G. R. 1937. The Development of the Vertebrate Skull. Oxford
University Press, London.
deBraga, M., and Rieppel, O. 1997. Reptile phylogeny and the interrelationships of turtles. Zool. J Linn. Soc. 120: 281–354.
Dietrich, S. 1999. Regulation of hypaxial muscle development. Cell Tissue
Res. 296: 175–182.
Eldredge, N. 1989. Macroevolutionary Dynamics: Species, Niches, and
Adaptive Peaks. Mcgraw-Hill Co, New York.
Evans, D. J. R., Valasek, P., Schmidt, C., and Patel, K. 2006. Skeletal
muscle translocation in vertebrates. Anat. Embryol. 211: S43–S50.
Froriep, A. 1885. Über Anlagen von Sinnesorganen am Facialis, Glossopharyngeus und Vagus, über die genetische Stellung des Vagus zum
Hypoglossus, und über die Herkunft der Zungenmusculatur. Arch. Anat.
Physiol. 1885: 1–55.
Fürbringer, M. 1874. Zur vergleichenden Anatomie der Schultermuskeln.
Jenaische Zeitschr. 8: 175–280.
Fürbringer, M. 1875. Zur vergleichenden Anatomie der Schultermuskeln.
Morph. Jahb. I: 637–816.
Fürbringer, M. 1900. Zur vergleichenden Anatomie der Brustschulterapparates und der Schultermuskeln. Jenaische Zeitschr. 34: 215–718.
Fürbringer, M. 1902. Zur vergleichenden Anatomie der Schultermuskeln.
Ebendaselbst 36: 289–736.
Gaffney, E. S. 1980. Phylogenetic relationships of the major groups
of amniotes. In A. L. Panchen (ed.). The Terrestrial Environment
and the Origin of Land Vertebrates. Academic Press, London,
pp. 593–610.
Gaffney, E. S. 1990. The comparative osteology of the triassic turtle Proganochelys. Bull. Am. Mus. Nat. Hist. 194: 1–263.
Geoffroy Saint-Hilaire, E. 1818. Philosophie Anatomique, Tome Premiere.
J.B. Baillière, Paris.
Gilbert, S. F., Bender, G., Betters, E., Yin, M., and Cebra-Thomas, J. A.
2007. The contribution of neural crest cells to the nuchal bone and
plastron of the turtle shell. Integ. Comp. Biol. 47: 401–408.
Gilbert, S. F., Cebra-Thomas, J. A., and Burke, A. C. 2008. How the turtle
gets its shell. In J. Wyneken, M. H. Godfrey, and V. Bels (eds.). Biology
of Turtles. CRC Press, Boca Raton, pp. 1–16.
Gilbert, S. F., Loredo, G. A., Brukman, A., and Burke, A. C. 2001.
Morphogenesis of the turtle shell: the development of a novel structure in
tetrapod evolution. Evol. Dev. 3: 47–58.
Goette, A. 1899. Über die Entwicklung des knöchernen Ruckenschildes
(Carapax) der Schildkröten. Z. wiss. Zool. 66: 407–434.
Gros, J., Scaal, M., and Marcelle, C. 2004. A two-step mechanism for
myotome formation in chick. Dev. Cell 6: 875–882.
Gross, J. B., and Hanken, J. 2008. Review of fate-mapping studies of
osteogenic cranial neural crest in vertebrates. Dev. Biol. 317: 389–400.
Haeckel, E. 1891. Anthropogenie oder Entwickelungsgeschichte des Menschen.
Keimes- und Stammesgeschichte. 4th Ed. Wilhelm Engelmann, Leipzig.
Hall, B. K. 1998. Evolutionary Developmental Biology. 2nd Ed. Chapman &
Hall, London.
Hedges, S. B., and Kumar, S. 2009. The Timetree of Life. Oxford University
Press, New York.
Hedges, S. B., and Poling, L. L. 1999. A molecular phylogeny of reptiles.
Science 283: 998–1001.
Hill, R. V. 2005. Integration of morphological data sets for phylogenetic
analysis of Amniota: the importance of integumentary characters and
increased taxonomic sampling. Syst. Biol. 54: 530–547.
Huang, R., Zhi, Q., Patel, K., Wilting, J., and Christ, B. 2000. Dual
origin and segmental organisation of the avian scapula. Development 127:
3789–3794.
Hugall, A. F., Foster, R., and Lee, M. S. Y. 2007. Calibration choice, rate
smoothing, and the pattern of tetrapod diversification according to the
long nuclear gene RAG-1. Syst. Biol. 56: 543–563.
Ido, A., and Ito, K. 2006. Expression of chondrogenic potential of mouse
trunk neural crest cells by FGF2 treatment. Dev. Dyn. 235: 361–367.
Iwabe, N., et al. 2005. Sister group relationship of turtles to the birdcrocodilian clade revealed by nuclear DNA-coded proteins. Mol. Biol.
Evol. 22: 810–813.
Jagla, K., et al. 1995. Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes.
Mech. Dev. 53: 345–356.
Turtle shell evolution
13
Jiang, X., Iseki, S., Maxson, R. E., Sucov, H. M., and Morriss-Kay, G. M.
2002. Tissue origins and interactions in the mammalian skull vault. Dev.
Biol. 241: 106–116.
Joyce, W. G., Lucas, S. G., Scheyer, T. M., Heckert, A. B., and Hunt, A. P.
2009. A thin-shelled reptile from the Late Triassic of North America and
the origin of the turtle shell. Proc. Biol. Sci. 276: 507–513.
Keibel, F. 1906. Die Entwickelung der äusseren Körperform der Wirbeltierembryonen, insbesondere der menschlichen Embryonen aus den
ersten Monaten. In O. Hertwig (ed.). Handbuch der Entwickelungslehre I.
Verlag von Gustav Fischer, Jena. Vol. 2. pp. 1–176.
Kumazawa, Y., and Nishida, M. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence
for archosaurian affinity of turtles. Mol. Biol. Evol. 16: 784–792.
Kuraku, S., Ishijima, J., Nishida-Umehara, C., Agata, K., Kuratani, S., and
Matsuda, Y. 2006. cDNA-based gene mapping and GC3 profiling in the
soft-shelled turtle suggest a chromosomal size-dependent GC bias shared
by sauropsid. Chrom. Res. 14: 187–202.
Kuraku, S., Usuda, R., and Kuratani, S. 2005. Comprehensive survey of
carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution. Evol. Dev. 7: 3–17.
Kuratani, S. 2005. Craniofacial development and evolution in vertebrates:
the old problems on a new background. Zool. Sci. 22: 1–19.
Laurin, M., and Reisz, R. R. 1995. A reevaluation of early amniote phylogeny. Zool. J. Linn. Soc. 113: 165–223.
Le Douarin, N. M. 1982. The Neural Crest. Cambridge University Press,
Cambridge, UK.
Le Douarin, N. M., and Kalcheim, C. 1999. The Neural Crest. 2nd Ed.
Cambridge University Press, Cambridge, UK.
Le Lièvre, C. S. 1974. Rôle des cellules mesectodermiques issues des crêtes
neurales céphaliques dans la formation des arcs branchiaux et du skelette
viscéral. J. Embryol. Exp. Morphol. 3: 453–577.
Le Lièvre, C. S. 1978. Participation of neural crest-derived cells in the
genesis of the skull in birds. J. Embryol. Exp. Morphol. 47: 17–37.
Lee, M. S. 1996. Correlated progression and the origin of turtles. Nature
379: 812–815.
Lee, M. S. Y. 1997. Pareiasaur phylogeny and the origin of turtles. Zool. J.
Linn. Soc. 120: 197–280.
Lee, M. S. Y. 2001. Molecules, morphology, and the monophyly of diapsid
reptiles. Contrib. Zool. 70: 1–22.
Li, C., Wu, X., Rieppel, O., Wang, L., and Zhao, L. 2008. An ancestral
turtle from the Late Triassic of southwestern China. Nature 45: 497–501.
Locascio, A., Manzanares, M., Blanco, M. J., and Nieto, M. A. 2002.
Modularity and reshuffling of Snail and Slug expression during vertebrate evolution. Proc. Natl. Acad. Sci. U.S.A. 99: 16841–16846.
Loredo, G. A., et al. 2001. Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle
embryos. J. Exp. Zool. 291B: 274–281.
Lyson, T., and Gilbert, S. F. 2009. Turtles all the way down: loggerheads at
the root of the chelonian tree. Evol. Dev. 11: 133–135.
Lyson, T. R., Bever, G. S., Bhullar, B. A., Joyce, W. G., and Gauthier, J. A.
2010. Transitional fossils and the origin of turtles. Biol. Lett. 23: 830–833.
Mannen, H., and Li, S. S. 1999. Molecular evidence for a clade of turtles.
Mol. Phylogen. Evol. 13: 144–148.
Matsuda, Y., et al. 2005. Highly conserved linkage homology between birds
and turtles: bird and turtle chromosomes are precise counterparts of each
other. Chrom. Res. 13: 601–615.
Matsuoka, T., et al. 2005. Neural crest origins of the neck and shoulder.
Nature 436: 347–355.
McGonnell, I. M., and Graham, A. 2002. Trunk neural crest has
skeletogenic potential. Curr. Biol. 12: 767–771.
Mennerich, D., Schäfer, K., and Braun, T. 1998. Pax-3 is necessary but not
sufficient for lbx1 expression in myogenic precursor cells of the limb.
Mech. Dev. 73: 147–158.
Mindell, D. P., Sorenson, M. D., Dimcheff, D. E., Hasegawa, M., Ast, J.
C., and Yuri, T. 1999. Interordinal relationships of birds and other
reptiles based on whole mitochondrial genomes. Syst. Biol. 48: 138–
152.
Monga, S. P., et al. 2002. Hepatocyte growth factor induces Wnt-independent nuclear translocation of b-catenin after Met-b-catenin dissociation
in hepatocytes. Cancer Res. 62: 2064–2071.
14
EVOLUTION & DEVELOPMENT
Vol. 13, No. 1, January--February 2011
Moustakas, J. E. 2008. Development of the carapacial ridge: implications
for the evolution of genetic networks in turtle shell development. Evol.
Dev. 10: 29–36.
Müller, G. B., and Wagner, G. P. 1991. Novelty in evolution: restructuring
the concept. Annu. Rev. Ecol. Syst. 22: 229–256.
Nagashima, H., Kuraku, S., Uchida, K., Ohya, Y. K., Narita, Y., and
Kuratani, K. 2007. On the carapacial ridge in turtle embryos: its developmental origin, function, and the chelonian body plan. Development
134: 2219–2226.
Nagashima, H., et al. 2009. Evolution of the turtle body plan by the folding
and creation of new muscle connections. Science 325: 193–196.
Nagashima, H., Uchida, K., Yamamoto, K., Kuraku, S., Usuda, R., and
Kuratani, S. 2005. Turtle–chicken chimera: an experimental approach
to understanding evolutionary innovation in the turtle. Dev. Dyn. 232:
149–161.
Nelson, W. J., and Nusse, R. 2004. Convergence of Wnt, b-catenin, and
cadherin pathways. Science 303: 1483–1487.
Nishi, S. 1931. Muskeln der Rumpfes. In L. Bolk, E. Göppert, E. Kallius,
and W. Lobosch (eds.). Handbuch der vergleichenden Anatomie der Wirbeltiere. Vol. 5. Urban, Berlin, pp. 351–446.
Noden, D. M. 1982. Patterns and organization of cranial skeletogenic and
myogenic mesenchyme: a perspective. In B. G. Sarnat (ed.). Progress in
Clinical and Biological Research: Factors and Mechanism Influencing
Bone Growth. Alan R. Liss Inc, New York, pp. 167–203.
Noden, D. M. 1984. The use of chimeras in analyses of craniofacial development. In N. M. Le Douarin and A. McLaren (eds.). Chimeras in
Developmental Biology. Academic Press, London, pp. 241–280.
Noden, D. M. 1988. Interactions and fates of avian craniofacial mesenchyme. Development 103 (suppl.): 121–140.
Novak, A., and Dedhar, S. 1999. Signaling through b-catenin and Lef/Tcf.
Cell Mol. Life Sci. 56: 523–537.
Nowicki, J. L., Takimoto, R., and Burke, A. C. 2003. The lateral somitic
frontier: dorso–ventral aspects of anterior–posterior regionalization in
avian embryos. Mech. Dev. 120: 227–240.
Ogushi, K. 1911. Anatomische Studien an der japanischen dreikralligen
Lippenschildkröte (Trionyx japonicus). Morpholog. Jahrbuch. 43: 1–106.
O’Rahilly, R., and Gardner, E. 1975. The timing and sequence of events in the
development of the limbs in the human embryo. Anat. Embryol. 148: 1–23.
Pennisi, E. 2004. Neural beginnings for the turtle’s shell. Science 303: 951.
Rasola, A., et al. 2007. A positive feedback loop between hepatocyte growth
factor receptor and b-catenin sustains colorectal cancer cell invasive
growth. Oncogene 26: 1078–1087.
Reinartz, J., et al. 2002. Massively parallel signature sequencing (MPSS) as
a tool for in-depth quantitative gene expression profiling in all organisms.
Brief Funct. Genomic Proteomic 1: 95–104.
Reisz, R. R. 1997. The origin and early evolutionary history of amniotes.
Trends Ecol. Evol. 12: 218–222.
Reisz, R. R., and Head, J. J. 2008. Turtle origins out to sea. Nature 456:
450–451.
Ribbing, L. 1931. Die Muskeln und Nerven der Extremitäten. In L. Bolk,
E. Göppert, E. Kallius, and W. Lubosch (eds.). Handbuch der
vergleichenden Anatomie der Wirbeltiere. Vol. 5. Urban & Schwarzenberg, Berlin, pp. 543–656.
Rickmann, M., Fawcett, J., and Keynes, R. J. 1985. The migration of
neural crest cells and the growth of motor axons through the rostral half
of the chick somite. J. Embryol. Exp. Morphol. 90: 437–455.
Riedl, R. 1978. Order in Living Organisms. Wiley Press, Chichester.
Rieppel, O. 2001. Turtles as hopeful monsters. BioEssays 23: 987–991.
Rieppel, O. 2009. How did the turtle get its shell? Science 325: 154–155.
Rieppel, O., and deBraga, M. 1996. Turtles as diapsid reptiles. Nature 384:
453–455.
Romer, A. S. 1956. Osteology of the Reptiles. University of Chicago Press,
Chicago.
Romer, A. S. 1966. Vertebrate Paleontology. 3rd Ed. University of Chicago
Press, Chicago.
Romer, A. S., and Parsons, T. S. 1977. The Vertebrate Body. Saunders,
Philadelphia.
Ruckes, H. 1929. Studies in chelonian osteology part II, The morphological
relationships between the girdles, ribs and carapace. Ann. N.Y. Acad. Sci.
31: 81–120.
Sánchez-Villagra, M. R., Müller, H., Sheil, C. A., Scheyer, T. M., Nagashima, H., and Kuratani, S. 2009. Skeletal development in the Chinese
soft-shelled turtle Pelodiscus sinensis (Testudines: Trionychidae).
J. Morphol. 270: 1381–1399.
Schneider, R. A. 1999. Neural crest can form cartilages normally derived
from mesoderm during development of the avian head skeleton. Dev.
Biol. 208: 441–455.
Shigetani, Y., Sugahara, F., Kawakami, Y., Murakami, Y., Hirano, S., and
Kuratani, S. 2002. Heterotopic shift of epithelial-mesenchymal interactions in vertebrate jaw evolution. Science 296: 1316–1319.
Shimomura, Y., et al. 2010. APCDD1 is a novel Wnt inhibitor mutated in
hereditary hypotrichosis simplex. Nature 464: 1043–1047.
Stephens, T. D. 1982. The Wolffian ridge: history of a misconception. ISIS
73: 254–259.
Stephens, T. D., Sanders, D. D., and Yap, C. Y. F. 1992. Visual demonstration of the limb–forming zone in the chick embryo lateral plate.
J. Morph. 213: 305–316.
Takahashi, M., et al. 2002. Isolation of a novel human gene, APCDD1,
as a direct target of the b-catenin/T-cell factor 4 complex with probable
involvement in colorectal carcinogenesis. Cancer Res. 62: 5651–5656.
Takahashi, M., Nakamura, Y., Obama, K., and Furukawa, Y. 2005. Identification of SP5 as a downstream gene of the b-catenin/Tcf pathway
and its enhanced expression in human colon cancer. Int. J. Oncol. 27:
1483–1487.
Thei!en, G. 2006. The proper place of hopeful monsters in evolutionary
biology. Theory Biosci. 124: 349–369.
Thei!en, G. 2009. Saltational evolution: hopeful monsters are here to stay.
Theory Biosci. 128: 43–51.
Tokita, M., and Kuratani, S. 2001. Normal embryonic stages of the
Chinese softshelled turtle Pelodiscus sinensis (Trionychidae). Zool. Sci.
18: 705–715.
Torres, T. T., Metta, M., Ottenwälder, B., and Schlötterer, C. 2008. Gene
expression profiling by massively parallel sequencing. Genome Res. 18:
172–177.
Tucker, G. C., Aoyama, H., Lipinski, M., Tursz, T., and Thiery, J.-P. 1984.
Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and
on some leukocytes. Cell Differ. 14: 223–230.
Vallén, E. 1942. Beiträge zur Kenntnis der Ontogenie und der vergleichenden Anatomie des Schildkrötenpanzers. Acta Zool. 23: 1–127.
Vincent, C., Bontoux, M., Le Douarin, N. M., Pieau, C., and MonsoroBurq, A. H. 2003. Msx genes are expressed in the carapacial ridge of
turtle shell: a study of the European pond turtle, Emys orbicularis. Dev.
Genes Evol. 213: 464–469.
Vincent, M., and Thiery, J. P. 1984. A cell surface marker for neural crest
and placodal cells: further evolution of the peripheral and central nervous system. Dev. Biol. 103: 468–481.
Watson, D. S. M. 1914. Eunotosaurus africanus Seeley and the ancestors of
the Chelonia. Proc. Zool. Soc. Lond. 11: 1011–1020.
Weidinger, G., Thorpe, C. J., Wuennenberg-Stapleton, K., Ngai, J., and
Moon, R. T. 2005. The Sp1-related transcription factors sp5 and sp5like act downstream of Wnt/b-catenin signaling in mesoderm and
neuroectoderm patterning. Curr. Biol. 15: 489–500.
Werneburg, I., and Sánchez-Villagra, M. R. 2009. Timing of organogenesis
support basal position of turtles in the amniote tree of life. BMC Evol.
9: 82.
Wold, B., and Myers, R. M. 2008. Sequence census methods for functional
genomics. Nature Methods 5: 19–21.
Wolff, C. F. 1759. Theorie von der Generation. Georg Olms Verlagsbuchhandlung, Heldesheim.
Woodger, J. H. 1945. On biological transformations. In W. E. Le Gros
Clark and P. B. Medawar (eds.). Essays on Growth and Form Presented to
D’Arcy Thompson. Cambridge University Press, Cambridge, UK, pp.
95–120.
Zardoya, R., and Meyer, A. 1998. Complete mitochondrial genome
suggests diapsid affinities of turtles. Proc. Natl. Acad. Sci. USA 95:
14226–14231.
Zardoya, R., and Meyer, A. 2001. The evolutionary position of turtles
revised. Naturwissenschaften 88: 193–200.