• staging system (SES);
  • heterochrony;
  • embryogenesis;
  • Parsimov;
  • skeletogenesis;
  • Pleurodira;
  • Cryptodira;
  • skull, skeleton


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Using the Standard Event System (SES) to study patterns of vertebrate development, we describe a series of 17 embryos of the pleurodire turtle Emydura subglobosa. Based on a sequence heterochrony analysis including 23 tetrapod taxa, we identified autapomorphic developmental shifts that characterise Testudines, Cryptodira, and Pleurodira. The main results are that Testudines are characterised by an autapomorphic late neck development, whereas pleurodires and cryptodires show a different developmental timing of the mandibular process. Additionally, we described the ossification pattern of E. subglobosa and compared the data to those of five other turtles. Pleurodires show the epiplastron to ossify before or simultaneously with maxilla and dentary. In contrast, cryptodires show a later ossification of this bone. Because evolutionary developmental studies on turtles have previously focused only on “model organisms” that all belong to Cryptodira, we underline the necessity to include a pleurodire taxon for a more comprehensive, phylogenetically more informative approach. Developmental Dynamics 238:2770–2786, 2009. © 2009 Wiley-Liss, Inc.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Several methods have been developed to analyse developmental patterns within a phylogenetic framework (Smith, 1997; Richardson et al., 2001; Jeffery et al., 2005; Colbert and Rowe, 2008; Harrison and Larsson, 2008). As a basis for comparison in both molecular and morphological studies, a reference system is necessary to describe the developmental features of an embryo, which is usually done by focusing on only a few selected species, or “model organisms.” However, there are changes in the timing of developmental characters (Haeckel, 1896), so-called sequence heterochronies (Smith, 1997; Velhagen, 1997), that become obvious when embryos of different species are compared to each other. This makes it difficult to use a staging system developed for a species A to analyse the development of a species B. As a result, the use of “model organisms” has recently been questioned (Mitgutsch, 2003; Sellier et al., 2006; Jenner and Wills, 2007) and new “staging tables” were developed for many additional species (Cretekos et al., 2005; Boughner et al., 2007; Sanger et al., 2008; Nolte et al., 2009; de Jong et al., 2009). Because all these are based on a different breadth of sampled specimens, a certain degree of simplification, and a somewhat typologically confined morphological scope (Hopwood, 2005, 2007), Werneburg (2009) developed a standard system that explicitly considers variation to describe developmental characters in a comparable and traceable way that may serve as reference in Evo-Devo research. This new system avoids typologisation and simplification as seen in many “staging tables,” and includes an expandable standard reference list of vertebrate developmental characters to document both inter- and intraspecific variability.

The growing research on turtle embryology has been triggered by an interest in the evolutionary and developmental origin of the unique turtle shell (Nagashima et al., 2005, 2007, 2009; Gilbert et al., 2001, 2008; Scheyer et al., 2008; Li et al., 2008), and the singular skull anatomy among living reptiles lacking any fenestrations (Rieppel, 1990; Müller, 2003). Although the phylogenetic position of turtles within Amniota (see Rieppel, 2004, 2008) and the phylogenetic relationships within turtles (Gaffney and Meylan, 1988; Joyce, 2007; Scheyer, 2007) remain unresolved, insights from embryology have been successfully employed to address questions on turtle evolution (Goodrich, 1930; Burke, 1991; Eßwein, 1992; Nagashima et al., 2005, 2007; Sánchez-Villagra et al., 2007; Werneburg and Sánchez-Villagra, 2009).

For Testudines, two standard “staging tables” have been traditionally used. Miller (1985) described a “31 staged embryology” for marine turtles (Chelonioidea), which was of interest mostly for biological conservation research (Bell et al., 2003), whereas in many other laboratories the common snapping turtle Chelydra serpentina (Cryptodira) became a popular “model organism” based on Yntema's (1968) “27 staged embryology: (Galbraith et al., 1989; Rieppel, 1990, 1993; O'Steen, 1998; Packard et al., 2000; Sheil and Greenbaum, 2005; Franz-Odendaal, 2006). In recent years, the prehatching development of additional turtle species was analysed (Guyot et al., 1994; Beggs et al., 2000; Tokita and Kuratani, 2001; Greenbaum and Carr, 2002). Nevertheless, all these observations used either Miller's (1985) or Yntema's (1968) “staging tables” as a reference, and were of a categorical kind obviating information on intraspecific variability (Werneburg, 2009).

Extant turtles can be subdivided into two monophyletic clades, Pleurodira and Cryptodira (Gaffney and Meylan, 1988). Depending on the phylogenetic hypothesis, the clades either diverged from each other as early as 220 Million years ago, in the Late Triassic, or at the latest in the Middle Jurassic, around 165 Million years ago (Danilov and Parham, 2008; Scheyer and Anquetin, 2008). Pleurodires and cryptodires can be mainly distinguished from each other by the mechanism of head retraction. While the former group, the side-necked turtles, put their neck/head sideward under the anterior edge of the shell, the latter group, hidden-necked turtles, retract their neck/head in an S-shape inside their shell. In addition, there are several further cranial and postcranial characters that characterise the two groups, such as the position of the trochlear process in the jaw adductor chamber (Schumacher, 1973; Gaffney and Meylan, 1988) or the connection mode between the pelvis and the shell (summarised, e.g., by Mickoleit, 2004).

In the present contribution, we provide for the first time a case study of the Standard Event System (SES) (Werneburg, 2009) to describe an embryonic series. While traditional turtle “model organisms” all belong to Cryptodira, species of which possess highly derived characters (Joyce, 2007; Scheyer, 2007), only few studies in comparative embryology have so far considered pleurodires (Eßwein, 1992; Sánchez-Villagra et al., 2007; Vieira et al., 2007, 2009; Scheyer et al., 2008; Fabrezi et al., 2009; Bona and Alcalde, 2009). Here we describe and analyse the early development of the red-bellied short-necked turtle Emydura subglobosa (Krefft, 1876), which is a common carnivore species living in freshwater environments of Northern Australia and Papua New Guinea (Legler and Georges, 1993). Tzika and Milinkovitch (2008) have already proposed E. subglobosa to be a suitable pleurodiran species for evolutionary developmental studies due to its easy keeping and breeding requirements (Nicol, 1993; Highfield, 1996; Hennig, 2001; Pawlowski, 2001; Schwarz, 2006).

The sequence of developmental events in Emydura subglobosa, here described in detail, was previously included in the developmental data set of Werneburg and Sánchez-Villagra (2009), in which the development of 15 turtle species and 8 tetrapod taxa were comparatively analysed using the Parsimov-approach (Jeffery et al., 2005). The authors tested alternative hypotheses for the position of turtles within Amniota as well as hypotheses for turtle ingroup relationships (Fig. 1). A total of 56 heterochronic shifts of developmental events supported a sister group relationship of Testudines to all remaining extant reptiles (crocodiles + birds and lizards/snakes + tuatara) when Chelonioidea (marine turtles) were assumed to be basal within cryptodires (Fig. 1B). By contrast, in the present study we use the heterochrony data from Werneburg and Sánchez-Villagra (2009) to focus on the temporal shifts of external morphology development characterising Testudines, Pleurodira, and Cryptodira. Furthermore, we describe the pattern of ossification of E. subglobosa and compare it to the timing of ossification of five other turtle species (Supp. Table S1, which is available online).

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Figure 1. Tested topologies for the position of Testudines relative to extant reptiles (Sauria, Archosauria, or Lepidosauria) and alternative hypotheses for turtle ingroup relationships. Emydura subglobosa represents the only pleurodire species opposing Cryptodira either with (a) snapping turtles (Chelydridae), (b) marine turtles (Chelonioidea), or (c) soft shell turtles (Trionychia) as basal taxa. For details and references, see text. Figure modified from Werneburg and Sánchez-Villagra (2009).

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  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Staging System of Emydura subglobosa (Figs. 2 and 3, Supp. Figs. S1–S18)

For illustrations, see Figures 2 and 3; additional figures and depictions can be found in Supp. Figures S1–S18, which are available online. Abbreviations: AER = apical epidermal ridge, Cat. No. = Catalogue number, CB = carapace width, CL = carapace length, CRL = crown-rump length, CPH = carapace-plastron height (total height of shell), d = days of incubation, PL = plastron length, PB = plastron width, SES = Standard Event System (Werneburg, 2009).

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Figure 2. Developmental series of Emydura subglobosa showing the whole body in lateral view. Numbers 1–18 refer to the specimens as described in the text. For detailed illustrations and depictions, see Supp. Figs. S1–18 and Table S1. Scale bars = 3 mm.

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Figure 3. Developmental series of Emydura subglobosa showing the heads (in lateral view) and the left forelimbs (lower arm in dorsal view). Numbers 1–18 refer to the specimens as described in the text. For detailed illustrations and depictions, see Supp. Figs. S1–18 and Table S1. Scale bars = 3 mm.

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E. subglobosa 1.

Twenty-three somite pairs are visible. The external nares and the otic vesicle are developed. The eye is characterised by the presence of an optic fissure and a clear contour of the lens/iris. The forelimb ridge has formed. The maxillary process has reached the midline level of the eye and the mandibular process has reached the posterior level. The 2nd to 5th pharyngeal arches as well as the 1st to 4th pharyngeal slits are visible. Age: 6d, CRL: 5.42 mm, Cat. No.: PIMUZ lab No. 2009.01.

E. subglobosa 2.

Thirty-two somite pairs are visible. The forelimb bud consists of a terminal paddle. The maxillary process has reached the anterior level of the lens while the mandibular process has reached its posterior level. The urogenital papilla bud is developed. Age: 7d, CRL: 13.5 mm, Cat. No.: PIMUZ labNo. 2009.02.

E. subglobosa 3.

Thirty-nine somite pairs are visible and the pupil has started to form. The forelimb is elongated, forming an elbow, and an apical epidermal ridge has formed. The hind limb is paddle-shaped and also shows an AER. The maxillary process has reached the anterior level of the eye while the mandibular process has reached the midline level. Age: 9d, CRL: 15.8 mm, Cat. No.: PIMUZ labNo. 2009.03.

E. subglobosa 4.

The exact somite number is now difficult to discern. The otic capsule has become inconspicuous. The forelimb and the hind limb have developed digital plates. The maxillary and the frontonasal process are fused. The mandibular process has reached the anterior level of the lens. The 2nd pharyngeal arch (the hyoid arch) has formed an opercular flap. The carapacial ridge is now visible. Age: 12d, CL: ca. 3.5 mm, Cat. No.: PIMUZ labNo. 2008.19.

E. subglobosa 5.

The thoracal bulbus comprising heart and liver has disappeared. The mandibular process has reached the level of the frontonasal process and simultaneously the point of occlusion with the upper jaw. All pharyngeal slits are closed. A cervical flexure of 90° can be recognised. The lower eyelid has formed and already started overgrowing the eye, reaching the ventral level of the lens. The caruncle is visible. The carapace is clearly delimited around its periphery and projects beyond the root of the tail. Pigment cells are visible on the carapace, the tail, and the limbs, but not yet on the plastron and the throat. A few cells have already reached the digital plate. The neck and the dorsum of the head also show several scattered pigment cells. Lateral to the neck and on the buccal/ear region, a distinct dark horizontal streak-like cluster of pigment cells has formed. On the dorsolateral face of the snout, anteroventral to the eye, a small concentration of pigment cells is visible. Age: 13d, CL: ca. 9 mm, CB: ca. 7 mm, Cat. No.: PIMUZ labNo. 2008.23.

E. subglobosa 6.

The fingers are longer than wide. Scales are visible on the neck, and scutes have developed on the carapace. Pigment cells on the carapace are widespread and the translucent costal and neural sutures leave the impression of a rough orientation of the cells along the sutures of the scutes. The pigment streak lateral to the neck has spread towards the dorsum of the neck. The pigment streak lateral to the buccal/ear region has a horizontal expansion but its posterior-most part is bent dorsally. Together with the dorsal head pigment cells, the buccal/ear streak circumvents a white non-pigmented region posterior to the eye. Age: 21d, CL: ca. 12 mm, CPH: ca. 7.5 mm, PL: ca. 9 mm, Cat. No.: PIMUZ labNo. 2008.24.

E. subglobosa 7.

All 13 scleral papillae are visible in the eye and the first claws occur as horny, clearly distinct structures. Pigmentation has increased considerably, particularly in the digital region, the carapace, and the dorsum of the head. In part, the cells are no longer delineable. The pigment cell streak lateral to the neck has completely spread into the dorsal neck pigmentation and is no longer distinguishable either. The claws are not pigmented yet. Pigmentation along the sulci (the sutures) of the carapacial scutes has increased. The buccal/ear streak and the pigment spot dorsolateral to the snout have fused ventral to the eye to form a frontomaxillary streak, the pigmentation of which has expanded onto the ventral part of the lower eyelid. Age: 24d, CL: 13.5 mm, CPH: ca. 7 mm, PL: ca. 9 mm, Cat. No.: PIMUZ labNo. 2008.25.

E. subglobosa 8.

The occipital head projection has disappeared and a clearly delineated rhamphotheca has formed. The white area dorsal to the buccal/ear streak has extended caudad and forms a white temporal streak that from now on is more distinct than the buccal/ear streak. Verrucas occur on the dorsum of the neck. A few pigment cells are detectable on the throat. Age: 26d, CL: 11.08 mm, CB: 8.5 mm, CPH: 8.15 mm, PL: 6.95 mm, PB: 6.75 mm, Cat. No.: PIMUZ labNo. 2009.04.

E. subglobosa 9.

Scales are now visible on the dorsum of the head and on the throat. Limb scales occur up to the digital region. Scales are also visible on the tail. The urogenital papilla has become inconspicuous and the periphery of the carapace is irregular. The pigmentation of the carapace has increased and a white non-pigmented border remains around its periphery. Pigment cells are visible on the plastron. The pigmentation of the whole body has extremely increased and pigment cells are only visible on the plastron. The rhamphotecae of both jaws have been entered by pigment cells where they attach the frontomaxillary pigment streak. The rhamphoteca of the upper jaw is also pigmented along its ventral border. The frontomaxillary streak only includes parts of the lower eyelid and the white temporal streak shows a rostrad extension over the upper lid, over parts of the lower eyelid, and over the dorsum of the snout towards the nose. It forms a white frontotemporal streak. Neck and head are peculiarly flexed left laterally and remain in this position until shortly before hatching. Age: 28d, CL: 13.74 mm, CB: 11.26 mm, CPH: 7.19 mm, PL: 10.15 mm, PB: 5.38 mm, Cat. No.: PIMUZ labNo. 2008.28.

E. subglobosa 10.

The scleral papillae have become inconspicuous. With an enlarged number of scales, the pigmentation of the whole body has increased. The white frontotemporal streak is sharply contoured and pigment cells are not scattered any longer; its temporal part has moved more dorsal to surround the posterodorsal edge of the eye formed by the eyelids. The digital plates clearly show a web-like shape. Age: 33d, CL: min. 19.1 mm, CB: 16.47 mm, CPH: 9.02 mm, PL: 14.32 mm, PB: 8.6 mm, Cat. No.: PIMUZ labNo. 2009.05.

E. subglobosa 11.

The pigmentation of the claws has increased. Age: 35d, CL: 13.62, CB: min. 10.75 mm, CPH: 7.21 mm, PL: 10.59 mm, PB: 7.61 mm, Cat. No.: PIMUZ labNo. 2008.29.

E. subglobosa 12.

The upper eyelid has enlarged and covers the eye dorsally up to the level of the upper pupil border. Age: 36d, CL: 20.26 mm, CB: min. 14.61 mm, CPH: 11.64 mm, PL: 15.7 mm, PB: 8.45 mm, Cat. No.: PIMUZ labNo. 2008.73.

E. subglobosa 13.

No distinct changes can be noted. Age: 38d, CL: 22.93 mm, CB: 17.56 mm, CPH: 10.89 mm, PL: 19.29 mm, PB: 9.75 mm, Cat. No.: PIMUZ labNo. 2009.06.

E. subglobosa 14.

The lower eyelid covers more than half of the eye. Age: 43d, CL: 21.8 mm, CB: min. 15.44 mm, CPH: 11.17 mm, PL: 15.71 mm, PB: 9.17 mm, Cat. No.: PIMUZ labNo. 2008.74.

E. subglobosa 15.

The pigmentation of the inframarginal shields (between carapace and plastron) has increased. Age: 50d, CL: 23.03 mm, CB: 18.15 mm, CPH: 11.56 mm, PL: 18.54 mm, PB: 11.73 mm, Cat. No.: PIMUZ labNo. 2009.07.

E. subglobosa 16.

No distinct changes. Age: 55d, CL: 20.32 mm, CB: 14.28 mm, CPH: 10.02 mm, PL: 16.85 mm, PB: 8.09 mm, Cat. No.: PIMUZ labNo. 2008.75.

E. subglobosa 17.

The animal has hatched and the cervical flexure of 90° has disappeared. No specific coloration except for the brownish primary colour. Age: 65d, CL: 24.08 mm, CB: 19.66 mm, CPH: 12.61 mm, PL: 20.47 mm, PB: 11.43 mm, Cat. No.: PIMUZ labNo. 2009.08.

E. subglobosa 18.

Specific coloration is visible: a reddish periphery of the carapace and a reddish plastron. The frontomaxillary streak remains but is constricted by the light upper rhamphotheca. The white frontotemporal streak has become much darker. Age: subadult, CL: 79.2 mm, CB: 70.95 mm, CPH: 30.36 mm, PL: 60.96 mm, PB: 49.95 mm, Cat. No.: PIMUZ lab No. 2009.09.

Heterochronic Shifts (Tables 1, 2)

Independent of cryptodire ingroup relationships, the shifted events characterising Testudines include the character complexes (as defined by Werneburg, 2009) of general head, eye, and scale development (Table 1a–c), whereas only three neck-related developmental features are common to all tested topologies (Table 1d). When assuming Sauria to be the sister group of turtles (Fig. 1B, Werneburg and Sánchez-Villagra, 2009), Testudines are supported by eight characters (Table 1a), whereas two other sister-group arrangements include fewer shifts for Testudines (Table 1b,c; Archosauria hypothesis: 5; Lepidosauria hypothesis: 6).

Table 1. Number of Heterochronic Shifts That Characterise Testudinesa
EventMovedRelative to event(s)
  • a

    The heterochronic shifts for all nine tested hypotheses were compared against each other. First only the three saurian-related topologies (a), then only the three archosaur-related topologies (b), the lepidosaur-related topologies (c), and finally a consensus of all hypotheses (d) were compared and the consensus of each was calculated. Results of the tie-included analysis are shown. Results of the tie-excluded analysis overlapping with the tie-included analysis are marked with asterisks. For details see text. The nomenclature of events and their abbreviations follow Werneburg (2009).

a. Testudines (consensus of all Sauria hypotheses, Fig. 1A–C)
Head projection disappearedEarlyThroat scales, whole forelimb scales*
Pupil formsEarlyHind limb paddle
Cervical flexure 90°LateForelimb AER
Cervical flexure disappearedLateFirst claw, whole forelimb scales*, eyelid begun overgrow*, lower half eye
b. Testudines (consensus of all Archosauria hypotheses, Fig. 1D–F)
Head projection disappearedEarlyWhole forelimb scales
Pupil formsEarlyMand midline eye
Cervical flexure 90°LateForelimb AER
Cervical flexure disappearedLateFirst claw, whole forelimb scales
c. Testudines (consensus of all Lepidosauria hypotheses, Fig. 1G–I)
Tail scalesLateFinger
Cervical flexure 90°LateForelimb AER*
Cervical flexure disappearedLateScleral papillae inconspicuous, first claw, whole forelimb scales*, lower half eye*
d. Testudines (consensus of all hypotheses, Fig. 1A–I)
Cervical flexure 90°LateForelimb AER
Cervical flexure disappearedLateFirst claw, whole forelimb scales
Table 2. Number of Heterochronic Shifts That Characterise Emydura subglobosa and Cryptodiraa
EventMovedRelative to event(s)
  • a

    Number of heterochronic shifts that characterize Emydura subglobosa and Cryptodira when calculating a consensus of all nine test hypotheses (Fig. 1A–I). Tie-including shifts are listed. Results of the tie-excluded analysis overlapping with the tie-included analysis are marked with asterisks. For details see text.

a. Emydura subglobosa (consensus of all hypotheses, Fig. 1A–I)
External naresEarlyOptic fissure, 4th arch, 5th arch, 3th slit, 4th slit
Scleral papillaeLateFinger*, first claw
Max midline eyeEarlyOptic fissure, 5th arch, 3th slit
Mand posterior eyeEarlyRelative to optic fissure, contour lens/iris
Mand posterior lensEarlyForelimb paddle, urogenital papilla bud
Mand level frontonasalEarlyCaruncle, carapace beyond tail
b. Cryptodira (consensus of all hypotheses, Fig. 1A–I)
Mand posterior lensLateHind limb digital plate

For Emydura subglobosa, we found 16 terminal shifts common to all tested topologies, which all are in the head and refer to the character complexes of nose, eye, maxillary and mandibular process (Table 2a). For Cryptodira, only one temporal shift is common to all topologies, which is associated with the development of the mandibular process (Table 2b).

Adult Limb Anatomy (Figs. 4 and 5: box)

The adult limb anatomy of Emydura subglobosa has not been described so far. Based on the architecture of chondral precursors, we anticipate that the carpus in the adult consists of three carpal rows plus one postaxial element, the pisiform. The proximal carpal row consists of the ulnare and intermedium, the central carpal row of centrale 1 and 2, and the distal row of five carpals named distal carpal 1 to 5. The phalangeal formula for the manus is 2-3-3-3-3, the plesiomorphic formula for pleurodirans (Boulenger, 1889; Sánchez-Villagra et al., 2007, Fig. 5). The pes shows a reduced phalangeal formula of 2-3-3-3-2. The tarsus is formed by two tarsal rows. The proximal row consists of the intermedium and fibulare. The distal row is made of five distal tarsals, named distal tarsal 1 to 5. All epiphyses and all tarsals are ossified in the subadult specimen of E. subglobosa (specimen 18). In the subadult carpus, the pisiform and the ulnare are still cartilaginous (Fig. 5E).

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Figure 4. Cleared and stained specimens of Emydura subglobosa. Red, calcified structures (alizarin red); dark blue, cartilage (alcian blue); light blue, connective tissue. Skull (A) and whole skeleton (B) in lateral view. The left manus (C) and pes (D) are shown in dorsal view. Row-numbers refer to the specimen numbers as described in the text. Scale bar = (A, B) 2 mm, (C, D) 1 mm.

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Ossification Pattern of Emydura subglobosa (Figs. 4, 5, Table 3)

The recorded ossification sequence of Emydura subglobosa is summarised in Table 3 and illustrated in Figure 4, the ossification of the forelimb and hind limbs is shown in Figure 5. Note that the younger E. subglobosa specimen No. 9 was found to be more advanced in ossification than the older specimen No. 11. These two specimens were not distinguishable by SES-characters when considering the external development. Hence, the order in Table 3 is based on the progress of ossification and not on the age of the specimens (as for the description of external development).

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Figure 5. General anatomy of the limb (grey box) and a diagram of the sequential ossification of the zygopodial and autopodial bones in Emydura subglobosa (A–E). Elements shown in white are still cartilaginous, processed ossification is marked by different degrees of grey scale (light grey corresponds to early ossification, dark grey corresponds to advanced ossification), and well-ossified elements are marked in black. c, centrale; dcI-IV, distal carpal 1 to 4; i, intermedium; mc, metacarpal; dt, distal tarsal; dt 5, distal tarsal 5 (hooked fifth element); mt, metatarsal; pi, pisiform; r, radius; u, ulna; ul, ulnare; IV + c, distal tarsal 4 fused with centrale. Numbers of specimens refer to the numbers used in the text. Scale bars = 3 mm.

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Table 3. Ossification Sequence of Emydura subglobosaa
Specimen no.Skeletal regionSkeletal elements that start ossification
  • a

    The sequence is based on the very first onset of ossification in every skeletal element. The signs (−) and (+) indicate the degree of ossification compared in all new occurring elements in the respective specimen. Hence, elements marked with (−) are less advanced in ossification. Elements marked with (+) are more advanced in ossification than other elements that occur in the same specimen. When no (+) or (−) is presented, the elements do not differ conspicuously in their degree of ossification. Note that the progress of ossification was found to be far more developed in specimen 9 than in the older specimen 11.

5DermatocraniumDentary, maxilla
 Pectoral girdleHumerus (−)
 PlastronEpiplastron (+)
6Dermatocranium PlastronAngular, frontal, jugal, palatine, parietal, postorbital, premaxilla, prefrontal, pterygoid, surangular, squamosal
  Entoplastron, hyoplastron, hypoplastron, xiphiplastron
 SplanchnocraniumCornu branchiale I
 Shoulder girdleRadius, ulna
 Pelvic girdleFemur (−), tibia (−), fibula (−)
 Cervical vertebraeCentra (+)
 Dorsal vertebraeCentra, ribs
 NeurocraniumBasioccipital, basisphenoid, exoccipital, opisthotic, supraoccipital
 SplanchnocraniumColumnella, quadrate
 Shoulder girdleDorsal process of the scapula (+)
 Dorsal vertebraeNeural arches (−)
9DermatocraniumPrearticular, vomer
 Shoulder girdleMetacarpals III, IV (+)
 Pelvic girdleMetatarsals II (−), III (+), IV
 Cervical vertebraeNeural arches (+)
 Sacral vertebraeCentra
 Caudal vertebraeCentra (−)
 Shoulder girdle“Acromion” process of the scapula (+), coracoid (+), metacarpal I (−), metacarpal II, metacarpal V (−), phalange 1 digit I (−), phalange 2 digit I (−), phalange 2 digit II (−), phalange 3 digit II (−), phalange 2 digit III (−), phalange 3 digit III (−), phalange 2 digit IV (−), phalange 3 digit IV (−), phalange 2 digit V (−), phalange 3 digit V (−)
 Pelvic girdleIlium (+), ischium (+), pubis (+), metatarsal I, metatarsal V (−), phalange 1 digit I (−), phalange 2 digit I (−), phalange 2 digit II (−), phalange 3 digit II (−), phalange 2 digit III (−), phalange 3 digit III (−), phalange 2 digit IV (−), phalange 3 digit IV (−), phalange 1 digit V (−)
 Sacral vertebraeNeural arches (−)
 Caudal vertebraeNeural arches
14Shoulder girdlePhalange 1 digit II, phalange 1 digit III, phalange 1 digit IV, phalange 1 digit V
 Pelvic girdleDistal tarsal 5 (−), phalange 1 digit II, phalange 1 digit III, phalange 1 digit IV
 Sacral vertebraeRibs
 Caudal vertebraeRibs (+)
 CarapaceNuchal plate (+)
16 [subadult]CarapaceCostal plates
 SplanchnocraniumCorpus hyoidis, cornu branchiale II
 Shoulder girdleIntermedium, centrale 1, centrale 2, distal carpal 1, 2, 3, 4, 5
 Pelvic girdleIntermedium, fibulare, distal tarsal 1, 2, 3, 4, phalange 2 digit V
 CarapacePeripheral plates, neural plates, suprapygal plate

The very first onset of ossification was recorded in two cranial and two postcranial elements of specimen 5, showing the fine-grained sequence epiplastron > maxilla > dentary > humerus. As soon as all plates of the plastron, most parts of the dermatocranium, and all cervical centra start ossification (specimen 6), skeletogenesis starts perichondrally at mid shaft of the cornu branchiale I (specimen 7). The cornu branchiale I is the first element of the splanchnocranium starting ossification. In the same specimen, ossification occurs at the mid shafts of the hind limb stylopods and sequentially at the same region of both the forelimb and hind limb zeugopods. The fine-grained sequence (see Experimental Procedures section) is as follows: radius, ulna > femur > fibula > tibia. In E. subglobosa, the timing of the onset of ossification of the forelimb precedes the hind limb in stylopodial and zeugopodial regions. The humerus, radius, and ulna start ossification before corresponding elements of the hind limb. However, first retention of Alizarin Red is visible simultaneously in the metapodials of the forelimbs and hind limbs (specimen 9).

Conversely, the first sign of ossification in the phalanges is visible earlier in the hind limbs than in the forelimbs. In general, ossification starts perichondrally at mid shaft of the diaphyses in every long bone of the limbs and proceeds from proximal to distal with a postaxial dominance.

Anterior dorsal centra as well as anterior dorsal ribs show onset of ossification at the same time as the ulna and radius (specimen 7), closely preceding timing of beginning ossification of the femur. Then, tibia and fibula are next to show perichondral ossification at mid shaft.

All structural elements of the cervical, dorsal, sacral, and caudal vertebrae (i.e., centra, neural arches, and ribs) start ossification independently from each other. Onset of ossification of centra starts cranially (specimen 7) and proceeds in an intervallic anteroposterior direction along the axial skeleton (specimen 11, 9). The centra are the first vertebral structures to show onset of ossification. Onset of ossification of the centra starts first in anterior cervical vertebrae. Later, onset of ossification of the centra is sequentially visible in anterior dorsal vertebrae, middle cervical vertebrae, middle dorsal vertebrae, anterior sacral vertebrae, anterior caudal vertebrae, posterior cervical vertebrae, posterior dorsal vertebrae, and so on.

Neural arches also show an anterior to posterior ossification sequence (specimens 11, 12) with the exception of the sacral neural arches, which are the last in this order (specimen 12). The dorsal spines of the neural arches are mostly reduced in the cervical region. However, they can be seen in the dorsal, sacral, and caudal vertebrae.

Ribs generally ossify first at their mid-shaft (specimen 7), before expanding in proximal and distal directions. Ribs sequentially display ossification in the dorsal, caudal (transverse process sensu Rieppel, 1993), and last at the sacral region.

After the onset of ossification of the tibia and fibula, the dorsal process of the scapula starts ossification (specimen 11), closely followed by anterior dorsal neural arches. The dorsal process of the scapula is the first to show beginning ossification among all other pectoral and pelvic girdle elements. The ilium, ischium, pubis, “acromion” process of the scapula and coracoid start ossification at the same time as the articular (specimen 12) only slightly preceding the first sign of ossification in the sacral and caudal neural arches.

Some regions of the neurocranium (basisphenoid, basioccipital, exoccipital, opisthotic, supraoccipital) and one part of the splanchnocranium (quadrate) exhibit red staining (specimen 11), all preceding the sacral and caudal centra as well as the metapodials of the manus (metacarpals III, IV) and pes (metatarsals II, III, IV).

Sacral and caudal neural arches and all remaining elements of the pectoral girdle (“acromion” process of the scapula, coracoid) and pelvic girdle (ilium, ischium, pubis) show ossification after onset of ossification in metacarpals III, IV, and metatarsals II to IV (specimen 12). The manus reveals the following sequence of ossification: metacarpal IV > metacarpal III > metacarpal II > metacarpal I > metacarpal V. The sequence ossification in the pes is: metatarsal III > metatarsal IV > metatarsal II > metatarsal I > metatarsal V. The fifth metapodial of both the manus and pes start perichondral ossification soon after onset of metapodial I.

The ossification patterns of the phalanges (specimens 12, 14) vary in their direction: digit I of both the manus and pes start ossification in the first and second phalanges in a proximodistal sequence, whereas digits II, III, and IV show a distoproximally directed sequence. Additionally, the fifth digit of the manus shows another, alternative sequence: intermediate phalange > terminal phalange > proximal phalange. The terminal phalange of digit V of the pes starts ossification only after hatching.

The nuchal plate begins ossification simultaneously with the caudal ribs (specimen 14) and shortly before the proximal phalanges of digits II to IV and the terminal phalange of digit V, i.e., shortly before hatching. The costal plates are the next and last carapace elements, which start ossification before hatching (specimen 16), and distal tarsal 5 is the last element of the whole limb to start ossification in prehatching stages (specimen 14). By the oldest prehatching specimen, distal tarsal 5 shows a small, medial center of endochrondral ossification.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Grundmuster of Turtle External Development

Our analysis of external developmental characters suggests that Testudines are characterised by two features related to neck development, namely a delayed cervical flexure of 90° and a delayed disappearance of this flexure (Table 1d). Next to the ossification mode of cervical vertebrae (Werneburg and Sánchez-Villagra, 2009), this phenomenon may be correlated with an elongated neck in turtles in comparison to the outgroup species.

In the present study, we did not find temporal shifts in the neck development distinguishing Cryptodira and Pleurodira. However, Werneburg and Sánchez-Villagra (2009) used a different consensus approach than we did (see below). They found the delay of the cervical flexure as one autapomorphic shift in the solely used pleurodire species E. subglobosa, as discussed in the following. Because both longer and shorter necks can be observed in cryptodires and pleurodires, the adult neck length may not be correlated with the embryonic shifts, but rather related to head retraction. Whereas cryptodires pull their head into the shell by S-shaping their neck, pleurodires lay their neck/head sideward under the anterior edge of the shell. Different head muscles underlie these movements (Shah, 1963; Herrel et al., 2008) and the duration of their ontogeny, which has not been studied yet, may influence temporal shifts in external development. Werneburg and Sánchez-Villagra (2009) focused only on one single phylogeny (Fig. 1C) to record the neck-related shift in E. subglobosa. Here we eliminated topological constraints and found no neck-related shift, which indicates the strong influence of the underlying phylogenetic hypothesis on the occurrence and distribution of heterochronic temporal shifts.

When assuming a Sauria- or Archosauria-relationship of Testudines (Table 1a,b), the early disappearance of the posterodorsal head projection is an autapomorphic character for turtles, independent of cryptodire relationships. This feature may be correlated with the early ossification of the occipital/parietal region of the skull of turtles. This ossification pattern is also present in other amniote groups (Sánchez-Villagra et al., 2008; Hugi et al., 2009).

One character complex, the onset of mandibular process characters, seems to be of phylogenetic relevance and distinguishes the two major turtle clades. Whereas in pleurodires an accelerated development of the lower jaw can be observed (Table 2a), in cryptodires there is only a single delayed mandibular process character (Table 2b). In the context of jaw muscle development, this finding is potentially important because the development of the jaw musculature is directly related to the development of the mandibular process (Edgeworth, 1935; Ziermann, 2008). However, the different patterns could also be related to different feeding modes. While both adult and juvenile pleurodires show the same type of fast prey capture behaviour (Lemell et al., 2002; Schwarz, 2006), some cryptodires change their feeding behaviour during life (Bonin et al., 2006). The accelerated development of the mandibular process in pleurodires may be necessary to enable this fast prey capture.

Werneburg and Sánchez-Villagra (2009) reconstructed a late occurrence of the lower eyelid when compared to the development of the mandibular process. The development of the trigeminal nerve-innervated m. levator bulbi, which connects the upper with the lower eyelid (Ogushi, 1913; Lakjer, 1926), may be associated with the development of the m. adductor mandibulae, which is also innervated by the trigeminal nerve and associated with the mandibular process (Rieppel, 1990). A late differentiation of the m. levator bulbi from the m. adductor mandibulae anlage and/or the small extent of the former muscle in turtles (Lakjer, 1926) may explain the delayed development of the lower eyelid.

Variability Versus “Stage”

To describe the external development, we ordered the investigated E. subglobosa specimens chronologically using an average age interval of 3.5 days, which was determined by the availability of specimens. By comparing the length of the embryos, we observed a decrease in carapace length in specimens 14 to 16, which may be interpreted as a case of intraspecific variability but could also be correlated to the flexible shell of turtle embryos (Cherepanov, 1995; Alibardi and Thomson, 1999; Gilbert et al., 2008) that deforms by embryonic movements, cervical flexure, and internal organ development.

The older the specimens are, the more intraspecific variation is detectable. In early development, slight differences seem to occur only in somite count, whereas other parts of the embryo are rarely affected. Comprehensive studies on intraspecific variation during external organogenesis are still lacking. Several “staging tables” refer to scale and pigmentation development to describe reptile embryo stages. However, these descriptions are all of a synoptic, typological style (Werneburg, 2009) and neglect both intraspecific variability and the comparability of the examined characters. For instance, many authors describe pigmentation patterns (Beggs et al., 2000) but do not describe their shape or explain whether they are visible as pigment cells (“tail has pale pigmentation”). Another example is limb scale development, which was used to describe very advanced stages of Chelydra serpentina (Yntema 1968); the poor applicability of these characters to stage other species was often criticised (Beggs, 2000; Tokita and Kuratani, 2001; Werneburg, 2009). Detailed descriptions and depictions of pigmentation development and its intraspecific variation may help to evaluate more comprehensive studies of neural crest cell distribution and pattern formation (Olsson, 1993).

Patterns of Turtle Ossification (Figs. 4–6, Table S1)

Based on double-stained whole-mounted specimens, the ossification patterns of six turtle species were compared against each other, representing two Pleurodira, E. subglobosa (this study), Phrynops hilarii (Fabrezi et al., 2009; Bona and Alcalde, 2009), and four Cryptodira, the trionychids Apalone spinifera (Sheil, 2003) and Pelodiscus sinensis (Sánchez-Villagra et al., 2009) as well as the chelydrids Chelydra serpentina (Sheil and Greenbaum, 2005) and Macrochelys temminckii (Sheil, 2005). All studied turtles show more or less the same adaptations to feeding mechanisms (snapping), locomotion (paddling), and habitat (fresh water). Tables 3 and S1 summarise the information on the ossification pattern of E. subglobosa. In Figure 6, major trends of ossification for Testudines, Pleurodira, and Cryptodira are listed (note that not all listed characters necessarily indicate autapomorphic features because no outgroup comparison was performed in this study).

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Figure 6. Trends of ossification within turtles based on the comparison of two pleurodiran and four cryptodiran species. Each character refers to the onset of ossification in the respective element. Note that all characters represent only the distribution of characters, no autapomorphies, because no outgroup comparison or sequence analysis was performed. Some of the trends noted in turtle ossification have previously been identified in other reptiles (e.g., Sánchez-Villagra et al., 2008; Hugi et al., 2009). Thus, some of the characters listed may actually represent reptilian plesiomorphies. For details see text.

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In E. subglobosa, the rough sequence of ossification in the cranium is dermatocranium > splanchnocranium > neurocranium. Dentary and maxilla are first to start ossification in the dermatocranium. The cornu branchiale I is the first splanchnocranial element that begins to ossify. The articular ossifies later, showing onset of ossification simultaneously with the relatively late onset in the pelvic girdle in specimen 12. All neurocranial elements start ossification in the same specimen (specimen 11). The rough sequence of ossification in the cranium of E. subglobosa is the same in Pelodiscus sinensis. Both turtles differ from the reported sequences of the other studied turtles. Apalone spinifera shows the dermatocranial parts to ossify first, sequentially followed by neurocranial and splanchnocranial elements. In Chelydra serpentina, Phrynops hilarii, and Macrochelys temminckii also parts of the dermatocranium start ossification first, followed by a simultaneous onset of ossification in both the neuro- and splanchnocranium. In the pleurodires and partly in the cryptodires (C. serpentina, M. temminckii, P. sinensis), the articular shows ossification shortly before or even after hatching. For example, in P. hilarii the articular begins ossification shortly after the onset in the nuchal plate, whereas in E. subglobosa, the articular starts ossification shortly before. As an exception, A. spinifera shows the articular relatively early to retain Alizarin Red, which is approximately in the middle of its ossification sequence. However, A. spinifera still displays a later timing in the onset of ossification of the articular than in the quadrate, like almost all of the studied turtles. As an exception, C. serpentina displays a simultaneous onset of ossification in the articular and quadrate only in hatched specimens. All studied turtles show the dentary and the maxilla to be among the first cranial elements to ossify. The Pleurodira show at least the epiplastron to start ossification even earlier or simultaneously with the dentary and the maxilla. Conversely, the Cryptodira display onset of ossification of at least the epiplastron later than the onset of ossification in the dentary and maxilla. The maxilla and the dentary start ossification simultaneously with the first retention of Alizarin Red in the axial skeleton (cervical and dorsal centra) and the plastron (hyo-, hypo-, and xiphiplastron) of P. sinensis. Then, other parts of the dermatocranium and the remaining plastron elements begin ossification simultaneously with the first onset in the anterior and posterior stylopods. In A. spinifera, stylopodial regions start to ossify as soon as most dermatocranial elements show calcification. In M. temminckii, the ossification sequence proceeds to the stylopodial region after the onset in the maxilla. There are no heterochronic shifts of external morphology, as referred to in Table 1 and 2, that seem to correlate with the ossification pattern of the studied turtle species. One may expect a different timing of maxilla and dentary ossification in pleurodires and cryptodires, because both groups are distinguished from each other by the timing of ossification of the maxillary and mandibulary process. In this regard, it may be worthwhile for future studies to also investigate the relative influence of the developing jaw musculature.


In E. subglobosa and Pelodiscus sinensis, the vertebral sequence of ossification is centra > ribs > neural arches. The overall ossification gradient of these single vertebral elements is not strictly anteroposteriorly directed. Only the dorsal, sacral, and caudal centra show a strict overall and region-referred anteroposterior sequence in both species. The neural arches subsequently display ossification in the cervical, dorsal, and caudal regions. The ossification sequence of the ribs is first seen in the dorsal portion, then subsequently in the cervical, caudal, and sacral region. Chelydra serpentina, Macrochelys temminckii, and Phrynops hilarii show the following vertebral ossification sequence: centra, ribs > neural arches, whereas Apalone spinifera displays centra to begin ossification before the simultaneous onset of the ribs and the neural arches. In P. sinensis and M. temminckii, ossification proceeds in an anteroposterior gradient in all centra as seen in E. subglobosa. All other studied cryptodire turtles show partial anteroposterior gradients in single vertebral regions with an overall anteroposterior ossification gradient. P. hilarii shows a simultaneous onset of ossification in the anterior half of the vertebral column (cervical and dorsal vertebrae), which later continues to the posterior half (sacral and caudal vertebrae). Neural arches start ossification at the same time in the cervical, sacral, and caudal vertebrae, whereas dorsal neural arches are slightly delayed. In P. hilarii, only the dorsal ribs start ossification before hatching; all other ribs tend to ossify only thereafter. In A. spinifera and the Chelydridae, every single rib starts ossification before hatching. All compared Testudines studied show a relative early onset of ossification in their vertebral centra, which provide a strong vertebral column, and their overall anterior-posterior gradient of ossification may be correlated with the delayed development of the cervical flexure (Table 1). Werneburg and Sánchez-Villagra (2009) already pointed out the correlation of neck flexure and ossification patterns. Based on its strong vertebral column, the neck may not flex until the spatial limitations in the egg force it to move. Finally, the general architecture of the vertebral column and adjacent soft tissues may also render the axis inflexible.

Pectoral and pelvic girdle.

The dorsal process of the scapula is the first element of the pectoral girdle that shows ossification in E. subglobosa. It starts to ossify slightly before the quadrate. All other pectoral and pelvic girdle elements show the beginning of ossification at the same time with the onset of the articular. In Phrynops hilarii, the dorsal process of the scapula is also the first element of the pectoral girdle to ossify. In Pelodiscus sinensis, the dorsal process and the “acromion” process of the scapula begin to ossify at the same time, preceding the coracoid. In Apalone spinifera and Chelydra serpentina, the dorsal process of the scapula starts to ossify simultaneously with its “acromion” process and the coracoid. In Macrochelys temminckii, the dorsal process of the scapula starts ossification clearly before its “acromion” process, both preceding the coracoid.

Forelimb and hind limb.

In E. subglobosa (Figs. 4, 5), stylopodial and zeugopodial elements of the forelimb start ossification before their counterparts of the hind limb (forelimb acceleration, Richardson et al., 2009). In contrast, the tarsal region shows either a simultaneous or earlier onset of ossification than the carpal region. The sequence of onset of limb ossification proceeds proximodistally with a postaxial predominance in the zeugopodial and autopodial region. Postaxial predominance in the ossification sequence is considered a characteristic feature of amniotes (Fröbisch, 2008). The phalanges of E. subglobosa show ossification sequences either in a proximodistal or distaloproximal direction depending on their digit position. Any acceleration of the forelimb compared to the hind limb is rather ambiguous in trionychids and chelydrids. However, two unambiguous ossification sequences can be determined for Phrynops hilarii and Macrochelys temminckii. In the former, metatarsals I and IV begin to ossify before metacarpals II to IV. In the latter, metatarsals II, III, and IV start ossification before any metacarpal. The timing of onset of ossification of the ungual phalange of digit V clearly differentiates pleurodires from cryptodires. The ungual phalange of digit V starts ossification after hatching in the studied pleurodires, whereas in all studied cryptodires this element ossifies before hatching. The hooked element in the pes of Apalone spinifera, P. hilarii, and Pelodiscus sinensis has been described as metatarsal V. However, its ossification is delayed relative to other metatarsals and it ossifies after or shortly prior to hatching. Fabrezi et al. (2009) studied the identity of this hooked tarsal element in P. hilarii and other pleurodire turtles. They stated that this element is a distal tarsal rather than a metatarsal bone due to its tarsal morphology. This bone shows a late onset of ossification in an endochondral ossification center. In contrast, other limb bones ossify earlier and show perichondral ossification first. These arguments are also supported by the hooked element of E. subglobosa, which determine it as distal tarsal 5 and not as metatarsal V.

Plastron and carapace.

The epiplastron is the first element of the whole skeleton that starts ossification in E. subglobosa. The nuchal plate is the first element of the carapace to ossify, but at the same time also among the latest skeletal elements to start ossification in prehatchings. Only in the largest prehatching specimen, the costal plates begin to ossify, whereas the peripheral, neural, and suprapygal ossify only in posthatchings. Pleurodires show at least one plastron element, the epiplastron, to begin ossification before or simultaneously with the very first onset in the dermatocranium. In cryptodires, the epiplastron starts to ossify relatively later than dentary and maxilla do. In addition, the nuchal plate starts to ossify relatively early in the cryptodires and very late (i.e., shortly before hatching) in the pleurodires. Thus, the timing of onset of ossification of the nuchal plate differentiates Pleurodira from Cryptodira. Vallois (1922), Shah (1963), and Herrel et al. (2008) already pointed out major differences in the neck musculature in Pleurodira and Cryptodira due to the diverging behaviour of neck/head retraction in both groups. Especially the mm. corticocervicalum (Herrel et al., 2008), which originate at the nuchal plate and are inserted into the posterior-most cervical vertebrae, seem to develop large stresses onto the nuchal plate. The m. testooccipitis (Scanlon, 1982) and testocapitis (Hoffmann, 1890), which originate from the anterior lower surface of the carapace, do not appear in trionychids (Rathke, 1848; Ogushi, 1913), whereas in other cryptodiran species such as the leatherback turtle Dermochelys coriacea (Schumacher, 1972), these muscles may have an additional influence on the early onset of nuchal plate ossification. Cryptodires show a retraction of the neck/head into the shell in late embryology (e.g., Tokita and Kuratani, 2001). Neck retraction may rather be the result of limited space of the egg than of an active retraction mechanism of the musculature. However, early existing neck musculature and also the nuchal plate may underlie indirect stresses that lead to an early onset of ossification. At least the epiplastron ossifies earlier in pleurodires than in cryptodires. Also here the configuration and development of neck/head musculature should be taken into account: The m. plastrosquamosus originates directly from the plastron, and the m. coracohyoideus (Scanlon, 1982) is attaching to the shoulder girdle, which in turn is also connected to the plastron (Fürbringer, 1874; Ogushi, 1913). Detailed anatomical observations are necessary to clarify which elements of the plastron provide the origin site for those muscles and tendons, and hence to understand the ossification pattern of the epiplastron in both turtle groups.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information


Fertilised eggs of E. subglobosa were obtained from a private breeder. The embryos came from different clutches and were incubated at a temperature of 29–30° C from February 11th until May 11th 2007. At an average interval of about 3.5 days (beginning at day 6 of incubation and ending at hatching day 65), 17 specimens were intoxicated and mortified in chlorobutanol and fixed in a 4% formalin solution. The subadult specimen was obtained from the pet trade. The crown-rump length (CRL) was measured only for the first three specimens. Thereafter, the anteroposterior expansion of the carapace and other shell dimensions were measured, since the actual CRL became obscured by retraction of the neck under the carapace or by neck torsion (Fig. 1). Specimens are currently deposited at the Paläontologisches Museum und Museum der Universität Zürich (PIMUZ), and are intended to be integrated into the embryological collection of the Museum für Naturkunde Berlin.

Description of External Development (Figs. 2, 3, S1–S18)

Following Werneburg (2009), we refrained from categorising our specimens into “stages” and ordered them by days of incubation. For each of the 18 specimens, we assigned a progressional number. In the following, we protocol the developmental events occurring in each specimen referring to the SES-characters of Werneburg (2009). In Werneburg (2009), several staging systems were compared and an overall comparable set of 104 characters was developed, which are easy to recognise in each vertebrate embryo. The characters are clearly defined and prevent any ambiguous interpretation. The SES provides a guideline of documenting and presenting embryological data. Herein only new occurring characters (events) in particular specimens/stages are documented. If those characters are retained in older specimens, they will not be re-described. Because of the coding of individual specimens with a standard system of development characters and an explicit and reproducible consideration of species variation, the SES system of Werneburg (2009) avoids typological features of standard staging systems.

In our descriptions, we also list characters that do not fit to the SES-characters but are assumed to be of a species-characteristic value (Giannini et al., 2006), such as coloration and proportions (italicised letters in Results/Staging system section). Two overview plates are presented picturing lateral views of the whole bodies (Fig. 2), as well as the head and the left forelimb (Fig. 3). In the supplementary information, more detailed illustrations (Figs. S1–S18) are presented following the SES-guide.

Photographs of all specimens were made with a digital camera (Leica DFC420 C) mounted on a stereomicroscope at a resolution of 2592 × 1944 pixels.

Analysis of Temporal Shifts in External Development (Tables 1, 2)

We used the developmental data and the protocol of Werneburg and Sánchez-Villagra (2009) to detect heterochronic shifts for the nine most-discussed topologies of turtle phylogeny (see Introduction section, Fig. 1A–I). We ran Parsimov (Jeffery et al., 2005) to calculate Acctran and Deltran optimisations (Maddison and Maddison, 2001) for each shifting element and, focusing on the branch leading to Testudines, we first calculated the consensus of both optimisation models for each tree separately. Our approach was to summarise all temporal shifts that autapomorphically characterise Testudines, both independent of any cryptodiran subgroup arrangements (Fig. 1a–c) and of the position of turtles within reptiles (Fig. 1A–I). We created a temporal shift consensus of all Sauria-topologies (Fig. 1A–C), all Archosauria-topologies (Fig. 1D–F), and all Lepidosauria-topologies (Fig. G–I; Table 1a–c). Finally, by creating a consensus of tree topologies, we calculated a conservative list of temporal shifts for Testudines (Table 1d).

Werneburg and Sánchez-Villagra (2009: Supplement) presented 94 temporal consensus shifts for E. subglobosa under their favoured topology (Fig. 1B). For Cryptodira the authors found eleven developmental consensus shifts. Here we present the consensus shifts for E. subglobosa and Cryptodira common to all tested topologies (Table 2).

For each analysis, we calculated temporal shifts using the data (event pair) matrix including all ties (simultaneously occurring events). The results of the tie-included analysis are shown in Tables 1 and 2. As proposed by different authors (Weisbecker et al., 2008; Ziermann 2008) we also calculated temporal shifts using the same data matrix excluding all ties (not shown). We calculated a consensus of both approaches and marked the overlapping shifts with asterisks (Tables 1 and 2).

Interpretation of Temporal Shifts and Evolutionary Scenarios

Studies on sequence heterochrony developed within the last 20 years (summarised by Ziermann, 2008; Werneburg, 2009) and recent approaches use a parsimony algorithm to estimate developmental shifts for particular nodes in a given phylogeny (Jeffery et al., 2005).

Werneburg and Sánchez-Villagra (2009) adverted to problems of interpreting heterochronic developmental shifts and evolutionary scenarios. For example, accelerated forelimb development has been compared to eye and somite development (Werneburg and Sánchez-Villagra, 2009: Supplement, Shift No. 438), whereas there is no obvious biological reason, at the utmost a shared up-regulation of similar transcription factors, why fast forelimb development, which could be interpreted as necessary to create a good basis for long arms in the adult, should be related to the eye and somite development. Thus temporal shifts are interpreted as simply existing character states and, without overinterpreting their biological significance, they can be taken to support dichotomies when comparing different topologies. Further Evo-Devo studies on molecular features or the onset of hormone control may be able to interpret findings like limb bud/tail bud interferences.

Another problem when analysing sequence heterochrony is that of pseudo shifts. These are shifts Parsimov (Jeffery et al., 2005) reports when the resolution of embryonic stages in one species is higher than in another, making it likely that the characters displayed by the youngest specimen of the less-resolved species have developed earlier. In the Parsimov results of Werneburg and Sánchez-Villagra (2009), there are several delayed (pseudo) shifts in early development that can be interpreted as derived because of the low resolution of the underlying embryonic series. In turtles, for example, several developmental “staging tables” begin when most cranial structures have already started to form, a character set that is mostly comparable to that of Yntema's (1968) stage 12 (Beggs et al., 2000; Greenbaum, 2002; Greenbaum and Carr, 2002; Tokita and Kuratani, 2001; this study). Such pseudo shifts should be excluded when discussing developmental shifts.

Clearing and Staining

In order to study the ossification sequence, we cleared and double-stained eight prehatchlings and the subadult specimen following standard protocols (Taylor and Van Dyke, 1985). Red = calcified structures (alizarin red), dark blue = cartilage (alcian blue), light blue = connective tissue (Fig. 4). The sequence of ossification, as documented in Table 3, is based on the first marking with Alizarin red.

Furthermore, the sequence of ossification is based on the very first visible retention of Alizarin Red in each skeletal element of the embryo. If possible, a fine-grained sequence (Hugi et al., 2009) of ossification is provided, when more than one skeletal element displays the first uptake of Alizarin Red in an embryo. The relative retention of Alizarin Red (i.e., area of the element showing retention of Alizarin Red compared to its adult absolute ossified size) is the basis for this fine-grained sequence. We interpreted an element with a higher degree of ossification to occur earlier than an element with a lower degree. However, the value of the fined-grained sequence to define the onset of ossification of elements should be interpreted with caution. Apparently, an element showing a larger degree of ossification could have been ossified earlier, but it also could have been ossified later and have shown a faster overall ossification.

Nomenclature follows Gaffney (1977, 1979) for the skull bones, and Schumacher (1973) for the hyoid apparatus. Terminology of shell elements corresponds to Zangerl (1969), and that of the remaining postcranial elements to Sheil and Greenbaum (2005).


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

We thank Christian Mitgutsch, Torsten Scheyer, and Laura A.B. Wilson for discussion, technical support, and useful comments on the manuscript. Two anonymous reviewers provided useful suggestions. Bernd Wolff kindly made the embryos of Emydura subglobosa available. This work was supported by Swiss National Fond grant 3100A0-116013 (to M.R.S.-V.), Forschungskredit der Universität Zürich grant 3772 (to I.W.), and Deutsche Forschungsgemeinschaft grant Mu 1760/2-3 (to J.M.)


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  2. Abstract
  7. Acknowledgements
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

DVDY22104SuppFigsS1-S8.pdf34363KSupporting Figures S1 to S8.
DVDY22104SuppTableS1.pdf1657KTable S1.Comparison of the onset of ossification of Emydura subglobosa (this study), Phrynops hilarii (Bona and Alcalde, 2009), Pelodiscus sinensis (Sánchez-Villagra et al., 2009), Apalone spinifera (Sheil, 2003), Chelydra serpentina (Sheil and Greenbaum, 2005), and Macrochelys temminckii (Sheil, 2005).

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