Skeletal Development in the Fossorial Gymnophthalmids Calyptommatus

Zoology 115 (2012) 289–301

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Zoology

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Skeletal development in the fossorial gymnophthalmids Calyptommatus

sinebrachiatus and Nothobachia ablephara

∗

Juliana G. Roscito , Miguel T. Rodrigues

◦

Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Trav. 14, n 321, Cidade Universitária, São Paulo, SP 05508-090, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The development of the cartilaginous and bony elements that form the skull and axial and appendic-

Received 7 December 2011

ular skeleton is described in detail for the post-ovipositional embryonic development of the fossorial

Received in revised form 26 January 2012

gymnophthalmid species Calyptommatus sinebrachiatus and Nothobachia ablephara. Both species have a

Accepted 2 February 2012

snake-like morphology, showing an elongated body and reduced or absent limbs, as well as modiﬁcations

in skull bones for burrowing, such as complex articulation surfaces and development of bony extensions

Keywords:

that enclose and protect the brain. Similar morphological changes have originated independently in sev-

Squamate embryonic development

eral squamate groups, including the one that led to the snake radiation. This study characterizes the

Chondrogenesis

Osteogenesis patterns of chondrogenesis and osteogenesis, with special emphasis on the features associated with

the burrowing habit, and may be used for future comparative analyses of the developmental patterns

Limb reduction

Fossoriality involved in the origin of the convergent serpentiform morphologies.

1. Introduction eyes, loss of external ear openings, and speciﬁc features of the skull

skeleton related to the burrowing habit, such as strong articula-

Squamate reptiles (lizards, snakes and amphisbaenians) com- tions between some bones and the presence of bony expansions in

prise a vast array of species distributed throughout the most particular bones forming a solid protection for the brain (Roscito

diverse habitats (Vitt et al., 2003). Such ecological diversity, which and Rodrigues, 2010).

implies variations in habitat use, diet, locomotion, and behavior, To investigate the development of these morphological features

is reﬂected in morphological specializations that are often simi- associated with fossoriality, we analyzed the post-ovipositional

lar among species that show similar lifestyles. One example is the patterns of chondriﬁcation and ossiﬁcation of the skull and the axial

convergent evolution of morphological features associated with a and appendicular skeleton, with special focus on the appearance of

“head-ﬁrst” burrowing habit, such as a stout skull protecting the cartilage condensations and on the timing of ossiﬁcation of limb

brain and sense organs, and a snake-like body form, with an elon- elements.

gated body and reduced or absent limbs.

Such changes are similar among several non-related squamate

2. Materials and methods

groups, including the one from which the snakes have diversiﬁed

(Lee, 1998; Wiens et al., 2006; Brandley et al., 2008). One such

C. sinebrachiatus and N. ablephara are both endemic to a Quater-

group is the Gymnophthalmidae (after Estes et al., 1988), which

nary sandy dune area in the State of Bahia, Brazil, in the margins

has its evolutionary history marked by several independent events

of the São Francisco River; C. sinebrachiatus is found in the right

of limb reduction and body elongation, and also by other changes

margin (Rodrigues, 1991), in the Xique-Xique dune ﬁeld, while

associated with fossoriality, such as eye reduction and loss of the

N. ablephara is found in the left margin (Rodrigues, 1984), in the

external ear (Pellegrino et al., 2001). The gymnophthalmid species

Alagoado dune ﬁeld.

Calyptommatus sinebrachiatus and Nothobachia ablephara are both

52 embryos of C. sinebrachiatus, distributed in two ontogenetic

fossorial, head-ﬁrst burrowers that have extremely reduced limbs

series representing each day of development from the 2nd day after

(N. ablephara has a two-digit hindlimb and a one-digit forelimb; C.

oviposition up to the 34th day, and 13 embryos of N. ablephara, dis-

sinebrachiatus has a one-digit hindlimb and the forelimb is absent), nd

tributed along 2-day intervals from the 2 day after oviposition

elongated bodies with the trunk being longer than the tail, reduced

up to the 26th day, were ﬁxed in absolute ethanol or 10% neutral-

buffered formalin and stained for cartilage and bone following a

combination of procedures described in the literature (Potthoff,

∗

1983; Taylor and Van Dyke, 1985; Song and Parenti, 1995; Springer

Corresponding author.

E-mail address: [email protected] (J.G. Roscito). and Johnson, 2000).

290 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301

The stained embryos were examined in an Olympus SZX12 three hyplogossal nerve foramina are easily observed at the base

stereomicroscope (Olympus, Tokyo, Japan) with a digital camera of the occipital arch (Fig. 1D and F).

attached for photographic documentation. The otic capsule is larger, but not yet differentiated into prootic

and opisthotic regions. The anterior, posterior and horizontal semi-

circular canals are distinct, as well as a short alar process directed

3. Results

anteriorly, approaching the epipterygoid (Fig. 1I). The posteroven-

tral margin of the horizontal semicircular canal is formed by a

In the following we provide a detailed description of the post-

slightly thicker cartilage, representing the future paroccipital pro-

ovipositional embryonic development of cartilaginous and bony

cess. The facial foramen is seen at the ventral margin of the otic

elements of the skull and axial and appendicular skeleton of N.

capsule, and the small cartilaginous columela rests in the fenes-

ablephara and C. sinebrachiatus, stating speciﬁc differences when

tra ovalis (Fig. 1E and G), which is located posteriorly to the facial needed.

foramen.

The epipterygoid and quadrate are longer than in previous

3.1. Skull stages, but still slender (Fig. 1I). The posterior quadrate process of

the dermal pterygoid is also longer (Fig. 1G).

At oviposition and during the ﬁrst days of development, no The hyoid elements are more easily observed (Fig. 1I): the tri-

skeletal structure is present in the embryos. Skeletal development angular basihyal and the short glossohyal are located medially;

in the skull begins in the chondrocranium, with the cartilaginous the paired hyoid cornua, projecting anteriorly, is connected to the

basal plate being the ﬁrst structure to develop around 8–9 days anterolateral margin of the basihyal; the epihyal projects posteri-

after oviposition. This short cartilage forms at the posterior region orly from the hyoid cornu; and the ﬁrst ceratobranchial, connected

of the skull ﬂoor, ﬂanking the notochord, and embedding it (Fig. 1A) to the posterior ends of the basihyal, projects posteriorly.

The slender V-shaped trabecula also develops around this stage; The second dermal bone to develop in the skull is the suran-

its posterior paired ends are associated with the lateral ends of the gular, forming around 20–21 days after oviposition; it is located

transverse acrochordal cartilage (represented in an older embryo in posterodorsally to Meckel’s cartilage and anteriorly to the articu-

Fig. 1E), delimiting a large hypophysial fenestra, and extend anteri- lar region (Fig. 2A). Soon after, a thin dermal pre-articular develops

orly in a single cartilaginous rod (the trabecula communis) forming at the lingual surface of the mandible, ventrally to the surangu-

the interorbital and nasal septum. Short basipterygoid processes lar (Fig. 2A). Meckel’s cartilages start to fuse at the mandibular

develop at the lateral extremities of the acrochordal cartilage (rep- symphysis.

resented in an older embryo in Fig. 1E). By this stage, the basicapsular commissure establishes the ﬁrst

Soon after, three short cartilaginous condensations are connection between the basal plate and the otic capsule, uniting

observed, united ventrally to the basal plate and located posteriorly them anteroventrally. The dorsal end of the metotic ﬁssure is nar-

to it, representing the pre-occipital (the two posteriormost con- rower (Fig. 2B) due to a slight constriction between the dorsal tip

densations) and occipital (the anteriormost condensation) arches; of the occipital arch and the posterior margin of the otic capsule.

these cartilages are very similar to vertebral neural arches. The The notochord is still present, extending up to the anterior margin

nasal and otic capsule cartilages also appear around this stage; the of the basal plate and approaching the acrochordal cartilage. The

shell-shaped otic capsule (Fig. 1B) has a small ventral pit represent- lagenar (ventral) and vestibular (dorsal) regions of the prootic are

ing the fenestra ovalis. distinct in the otic capsule cartilage.

The pre-occipital and occipital arches soon fuse into the occipital The pterygoid is wider and longer: its anterior process reaches

arch, which is incorporated to the basal plate; this happens after 12 the level of the middle of the eye (Fig. 2B), and the posterior

days of development (Fig. 1A). The otic capsule is widely separated quadrate process extends to reach the quadrate.

from the basal plate and occipital arch through the metotic ﬁssure. The basipterygoid processes are long and relatively stout. The

At around 14–15 days, the quadrate develops anteriorly to the notochord starts to regress from the middle of the basal plate, being

otic capsule and dorsally to the articular region. Meckel’s carti- slender anteriorly. Quadrate and epipterygoid are stouter, but still

lage forms at the posterior region of the mandibular bud, and soon short.

stretches anteriorly. Other dermal bones appear at this phase: the triangular-shaped

Approximately 16 days after oviposition, two foramina for the palatine develops anteriorly to the pterygoid; frontal and parietal

hypoglossal nerve are observed at the base of the occipital arch. The form as thin and short paired strips at the lateral edges of the skull;

dorsal extremity of the arch is short, reaching the level of the middle the small and slender jugal is located posterior and ventral to the

of the otic capsule cartilage. The horizontal semicircular canal is eye (Fig. 2C); and the maxilla, also small but somewhat longer than

visible at the otic capsule. The epipterygoid develops as a small and the jugal, is located in front of the eye. In the mandible, the dentary

very slender rod of cartilage, next to the quadrate (Fig. 1C). The develops at the anterior tip of Meckel’s cartilage, and the small coro-

ﬁrst dermal bone to appear is the pterygoid, located posteriorly noid and splenial appear posteriorly to the dentary, located dorsally

to the eye. Meckel’s cartilage is stouter and grows longer as the and midventrally relative to Meckel’s cartilage, respectively.

mandibular bud stretches (Fig. 1C). In the hyoid, the glossohyal extends up to the anterior of the

As for the hyoid apparatus, the ﬁrst elements to develop are the snout, approaching the mandibular symphysis. The paired second

basihyal and the short glossohyal fused to it, the hyoid cornu, the ceratobranchial develops as a small cartilage in between the ﬁrst

epihyal, and the ﬁrst ceratobranchial, the latter three represented ceratobranchials; the second epibranchial also develops around

by short and slender cartilages. this stage.

Development of the cartilaginous structures continues: around The quadrate is the ﬁrst endochondral bone to start ossiﬁcation,

18 days after oviposition, the basal plate is wider and extends which begins medially (Fig. 2D); the ossiﬁcation of the epipterygoid

through the posterior chondrocranium ﬂoor, but does not close is delayed but also starts at the medial portion of the bone.

the basicranial fenestra (Fig. 1D–F). The metotic ﬁssure narrows as While the dermal bones already present continue to develop,

the basal plate/occipital arch complex and otic capsule cartilages other dermal elements form later in development, after 26 days

grow larger, although they remain completely separated (Fig. 1F (Fig. 2E–H). The thin and small premaxilla forms at the tip of the

and H) since the basicapsular commissure, which is the ﬁrst snout, with the small egg tooth soon observed. The maxilla (Fig. 2E

connection between these two cartilages, is not yet present. The and F) is larger and divided into an irregularly ossiﬁed alveolar

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301 291

Fig. 1. Skull development in cleared and stained embryos of Calyptommatus sinebrachiatus (A, B and D–I) and Nothobachia ablephara (C). (A) Detail of notochord and basal

plate in lateral view in a 12-day embryo; anterior to the top. (B) Chondrocranium of a 13-day embryo in right lateral view. (C) Skull of a 16-day embryo in lateral view.

(D–F) Skull of an 18-day embryo in ventral view, with details of the posterior (D) and medial (E) portions, and a schematic representation of both (F); anterior to the left.

(G and H) Photograph (G) and drawing (H) of the posterior portion of the skull in a 20-day embryo in lateral view. (I) Hyoid apparatus in latero-ventral view in a 20-day

embryo. Abbreviations: 1ctb, ﬁrst ceratobranchial; ac.c, acrochordal cartilage; asc, anterior semicircular canal; bh, basihyal; bpt.p, basipterygoid process; bsp, basal plate;

cm, columella; eph, epihyal; ept, epipterygoid; fo, fenestra ovalis; gh, glossohyal; hc, hyoid cornu; hn.f, hypoglossal nerve foramen; hsc, horizontal semicircular canal; M.c,

Meckel’s cartilage; mf, metotic ﬁssure; nt, notochord; oc.a, occipital arch; ot.c, otic capsule; psc, posterior semicircular canal; pt, pterygoid; q, quadrate; tr, trabecula. Scale

bars for A, C–E, G and H = 0.25 mm; B, F and I = 0.5 mm.

portion and a smaller dorsal portion representing the facial pro- the anterior portion of the mandible (Fig. 2E and H) and reaches

cess. The prefrontal (Fig. 2F) is represented by a faint and shapeless the splenial posteriorly; the ossiﬁcation in the upper and anterior

ossiﬁcation anterior to the eye. In C. sinebrachiatus, the posterior end of the dentary, where the teeth will form, is quite irregular.

and temporal processes of the jugal are distinct (Fig. 2E and G), The splenial, pre-articular, coronoid and surangular are larger, but

but the anterior maxillary process is not present, while in N. able- they do not contact each other (Fig. 2H). The large alveolar fora-

phara the jugal is very slender and semilunar in shape (Fig. 2C). The men is seen at the anterior portion of the splenial. The surangular

post-orbital, frontal and parietal are elongated antero-posteriorly; is the most developed element in the mandible and forms the pos-

a short descending process of the parietal is present in both C. terior portion of the labial surface; the large surangular foramen is

sinebrachiatus (Fig. 2E and G) and N. ablephara. The short and thin observed dorsally (Fig. 2H). The pre-articular is also well developed

squamosal and supratemporal are located posteriorly to the pari- and covers most of the posterior lingual surface.

etal and near the cephalic condyle of the quadrate (Fig. 2E). In the With the growth of the otic capsule and the basal plate/occipital

palatal region, pterygoid and palatine are more developed, and the arch complex, two other connection points between them are

vomer appears as a small and triangular ossiﬁcation anterior to the observed. The apposition of the posterior margin of the otic cap-

palatine. sule to the dorsal extremity of the occipital arch closes the metotic

In the mandible, the small angular forms between the splenial ﬁssure dorsally, and an additional connection between these carti-

and the pre-articular (Fig. 2H). The dentary extends through all lages divides the metotic ﬁssure into two openings, one anterior

292 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301

Fig. 2. Skull development in cleared and stained embryos of Calyptommatus sinebrachiatus (A and D–I) and Nothobachia ablephara (B and C); anterior to the right in all images.

(A) Mandibular dermic bones of a 25-day embryo in latero-ventral view developing at the posterior region of Meckel’s cartilage. (B) Chondrocranium of a 22-day embryo in

lateral view. (C) Jugal in a 26-day embryo. (D–I) Photographs (D, F–I) and drawing (E) of a 26-day embryo detailing the otic capsule (D), the anterior (F), posterior (G), and

mandibular (H) dermic bones, and the metotic ﬁssure subdivision (I); (E) gives a representation of the entire skull. Abbreviations: 1ctb, ﬁrst ceratobranchial; al.p, alar process;

ang, angular; bh, basihyal; bsp, basal plate; cm, columella; crn, coronoid; d, dentary; dp.p, descending process of the parietal; eph, epihyal; ept, epipterygoid; f, frontal; gh,

glossohyal; j, jugal; m, maxilla; M.c, Meckel’s cartilage; mf, metotic ﬁssure; n, nasal; oc.a, occipital arch; or, occipital recess; ot.c, otic capsule; p, parietal; pa, prearticular; pl,

palatine; pm, premaxilla; po, postorbital; pt, pterygoid; ptf, posttemporal fenestra; q, quadrate; spl, splenial; sq, squamosal; sra, surangular; st, supratemporal; v, vomer; vf,

vagus foramen. Scale bars = 0.25 mm, except for E = 0.5 mm.

and ventral to the recessus scalae tympani (the future occipi- the base of the basipterygoid processes and then extends medially

tal recess), and the other one posterior and dorsal; the vagus to enclose the basisphenoid, forming the parabasisphenoid. The

nerve passes through this opening (the future vagus foramen) basicranial fenestra and the hypophysial fenestrae remain open

(Fig. 2I). until the ﬁnal stages of development when, in juveniles, these

Ossiﬁcation in the chondrocranium is followed by the occipi- fenestrae are closed by the growth of the basioccipital and the

tal arch, which ossiﬁes into the exoccipital; its ossiﬁcation starts parabasisphenoid. The otic capsule, the tectum synoticum (the car-

in its medial region, slightly above the posteriormost hypoglossal tilage that connects the otic capsules dorsally) and the basal plate

foramen. start ossiﬁcation soon after the basisphenoid and develop into the

In the dermatocranium, at around 33–35 days of development, prootic and opisthotic (both derived from the otic capsule carti-

small teeth develop in the maxilla, and the alveolar portion and lage), the supraoccipital and the basioccipital, respectively (Fig. 3B

facial process fuse posteriorly. In the jugal of C. sinebrachiatus, a and C1). In C. sinebrachiatus, the shaft of the columella is shorter and

small maxillary process projects anteriorly; the posterior and dor- stouter, and its base is wider than that of N. ablephara. The ossiﬁca-

sal processes are longer. The triangular-shaped postorbital is small tion of the columella is also delayed, starting from its base and later

and has a long posterior process. progressing to the shaft. The distal margin of the cephalic condyle of

In pre-hatching stages, ossiﬁcation has progressed greatly in the quadrate remains cartilaginous until ﬁnal pre-hatching stages,

the chondrocranium. The acrochordal cartilage and the basiptery- as do the articular surface of the mandibular condyle, the tips of the

goid processes ossify into the basisphenoid, while the tips of the epipterygoid and the paroccipital process.

basipterygoid processes remain cartilaginous (Fig. 3A) until early The short cartilaginous ascending process of the tectum syn-

post-hatching stages. The dermal parasphenoid first ossifies around oticum is observed only at the final stages. The chondrocranial

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301 293

Fig. 3. Skull development in cleared and stained pre-hatching embryos of Calyptommatus sinebrachiatus (A–D and F–G) and Nothobachia ablephara (E); anterior to the left in

all images except in (C1–4). (A and B) Chondrocranium in ventral (A) and dorsal (B) views, showing the ossiﬁcation patterns of the endochondral bones. (C) Drawings of the

skull in dorsal (C1), lateral (C3) and ventral (C4) views, and a photograph of the skull in lateral view (C2); dark areas in the drawings correspond to cartilaginous tissues. (D)

Triradiate left jugal in lateral view. (E) Formation of the dermic roof of the skull (dorsal view) with development of the frontal and parietal bones. (F) Detail of the posterior and

lateral end of the frontal and its articulation with the parietal in dorsal view. (G) Formation of the compound postorbitofrontal in lateral view. Abbreviations: al.p, alar process;

asc, anterior semicircular canal; bo, basioccipital; bpt.p, basipterygoid process; cs, crista sellaris; ecp, ectopterygoid; exc.a, exoccipital area; f, frontal; fpt, frontoparietal tab; j,

jugal; m, maxilla; m.ps, maxillary palatal shelf; mp.j, maxillary process of the jugal; n, nasal; np.pm, nasal process of the premaxilla; oc, occipital condyle; or, occipital recess;

p, parietal; pbs, parabasisphenoid; pf, postfrontal; plp.f, posterolateral process of the frontal; pm, premaxilla; po, postorbital; pof, postorbitofrontal; pp.j, posterior process of

the jugal; prf, prefrontal; psc, posterior semicircular canal; pt, pterygoid; ptf, posttemporal fenestra; q, quadrate; qp.pt, quadrate process of the pterygoid; so, supraoccipital;

sq, squamosal; st, supratemporal; tp.j, temporal process of the jugal; tr, trabecula; v, vomer. Scale bars for A, B, D, F, and G = 0.25 mm; C = 1.0 mm; E = 0.5 mm.

bones are separated by large strips of cartilaginous tissue that gets (Fig. 3C2) than in N. ablephara. The fronto-parietal tabs are already

narrower as growth progresses. formed before hatching (Fig. 3F).

The articulation between maxilla and premaxilla forms at later Nasal, septomaxilla, ectopterygoid, and the posterodorsal pala-

stages, with the development of the premaxillary and maxillary tine and anterior processes of the prefrontal also form in late stages.

processes of the maxilla and premaxilla, respectively (Fig. 3C2 and The postfrontal develops late and, in C. sinebrachiatus, soon fuses to

C3). The nasal process of the premaxilla forms completely at the the postorbital to form the compound postorbitofrontal (Fig. 3G).

ﬁnal stages of development (Fig. 3C1). The jugal shows an irregu- In N. ablephara these two bones remain separate.

lar ossiﬁcation, but is relatively well developed (Fig. 3D). Frontal In the palate of C. sinebrachiatus, the growth of the bony exten-

and parietal start growing toward the midline, with the frontal sions of the palatine, pterygoid, and ectopterygoid to close the

being more developed than the parietal (Fig. 3C1). The fusion of suborbital fenestra starts at the ﬁnal stages (Fig. 3C4), and the clo-

the frontals progresses antero-posteriorly and the fusion of the sure is completed after hatching. N. ablephara also shows some

parietals occur in the opposite direction, starting posteriorly and growth of the palate bones, but the suborbital fenestra remains

progressing anteriorly to meet the frontal. At hatching, the fusion of open.

the frontals is almost complete but not that of the parietals (Fig. 3E), The development of the bones in the mandible is completed

and thus the closure of the parietal fontanelle ends in the post- during embryonic development; cartilaginous tissue remains at the

hatching stages, as well as the growth of the descending process dorsal surface of the articular bone and in the posterior extremity

of the parietal, which is much more developed in C. sinebrachiatus of the retroarticular process; both ossify after hatching.

294 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301

In the hyoid, the ﬁrst ceratobranchial is completely ossiﬁed posterior the vertebra, the more extensive the cartilaginous tissue

(Fig. 3C2); the other elements are mineralized through the calci- in this region: in the anteriormost vertebra the only cartilaginous

ﬁcation of the cartilage, a process that occurs after hatching. tissue left is found in the articular surfaces of the zygapophyses

and in the dorsal tip of the arch; in the posteriormost vertebrae,

3.2. Axial and appendicular skeleton next to the sacral region, the dorsal half of the neural arches is

entirely cartilaginous.

3.2.1. Vertebrae The ossiﬁcation of the intercentrum of the atlas is delayed in

All stages of vertebral development, from the formation of carti- relation to the other intercentra.

lages to the ossiﬁcation process, progress in an antero-posterior and The ossiﬁcation of the transverse processes progresses proximo-

temporal gradient, with development starting in the most anterior distally, and in the vertebral series, in an antero-posterior direction,

region of the trunk and progressing, over time, to posterior ones. with the processes of the posteriormost vertebra showing more

Thus, all descriptions are based on the ﬁrst appearance of structures extensive areas of cartilage than those of the anterior ones.

anteriorly. At the ﬁnal stages of development, the sternal and xiphister-

At early stages of development (10–12 days after oviposition), nal ribs are united to the sternum and xiphisternum, respectively,

the vertebral series is formed by the short and cartilaginous neural and the inscriptional ribs join with their opposites to form the

arches connected to the vertebral centra, which are represented post-xiphisternum. These structures mineralize through cartilage

by U-shaped cartilages embracing the notochord ventrally, with calciﬁcation, which starts at pre-hatching stages and ends only after

the dorsal portion forming later (Fig. 4A). By this stage, the axis hatching.

is already larger than the atlas. The cartilages of the mid-trunk

vertebrae are less developed than the anterior ones and the most 3.2.2. Pectoral girdle and forelimb

posterior ones are completely absent (Fig. 4B). The pectoral girdle and the anterior limb start to develop rel-

As development proceeds, the neural arches elongate and their atively late when compared to the skull. In N. ablephara and C.

distal tips enlarge. Short transverse processes develop laterally to sinebrachiatus, the ﬁrst elements to form in this region are the

the centra around the 16th–18th day of development, but ribs form scapulocoracoid, the suprascapular cartilages and the dermal clav-

later, around 20 days after oviposition. icle, at around 16 days of development for N. ablephara, and around

Small intercentra united to the ventral margins of the most ante- 20 days for C. sinebrachiatus (Fig. 5A). When they are ﬁrst formed,

rior vertebral centra develop around this stage; 4 intercentra are the scapular portion of the scapulocoracoid and the suprascapula

observed in the 16th-day embryo of N. ablephara (Fig. 4C), and in the are represented by a single and short cartilaginous plate, while the

20th-day embryos of C. sinebrachiatus and N. ablephara this num- coracoid portion is distinct from it; in N. ablephara, the coracoid

ber rises to 8 intercentra. The atlas intercentrum is smaller than foramen is opened dorsally, but in C. sinebrachiatus no such foramen

that of the axis, and the ones subsequent to it show a progressive is present. The short and slender clavicle is located midventrally

reduction in size. and anterior to the girdle cartilages. The short and cartilaginous

The short transverse processes of the sacral vertebrae are humerus is the only limb element present in N. ablephara (Fig. 5B);

present and directed toward each other, but not yet fused distally. in C. sinebrachiatus the humerus develops later.

The pre- and post-zygapophyses become distinct in the neu- In N. ablephara, the coracoid foramen soon closes dorsally (at

ral arches after approximately 24 days of development, and are around 18–20 days of development), and a small radius and ulna

completely formed only at late embryonic stages. The dorsal fusion develop in the limb, as well as a cartilaginous condensation in the

of each pair of neural arches occurs at late stages of develop- autopodial region probably corresponding to the ulnare (Fig. 5C).

ment, being ﬁrst observed in the anteriormost vertebrae around In the 26-day embryo of N. ablephara, the girdle and the limb

26–27 days after oviposition; the fusion also progresses antero- elements are more developed. The clavicle is stouter and longer

posteriorly. and the dermal interclavicle develops as a pair of short ossiﬁca-

In the 26-day embryos of both species, the ribs are longer and tions orientated antero-posteriorly and close together medially; in

present in all vertebrae (Fig. 4D). One and two pairs of sternal ribs C. sinebrachiatus the interclavicle is only observed later, around 33

are observed in N. ablephara and C. sinebrachiatus, respectively. days of development, also as two short, slender and transverse bars

In both species only one pair of xiphisternal ribs is present. The (Fig. 5D) and shorter lateral processes not connected to the medial

inscriptional ribs form posterior to the xiphisternal ribs, also in an body.

antero-posterior gradient, with the posteriormost ones being slen- The ossiﬁcation of the humerus, radius and ulna in N. ablephara

der and shorter; N. ablephara shows 7 pairs of inscriptional ribs, and also starts around this stage (26 days of development), progressing

C. sinebrachiatus shows 5 pairs. proximo-distally with the perichondral ossiﬁcation of the humerus

At the same stage, the distal tip of the transverse processes of (Fig. 5E) being more advanced than that of the radius and ulna. A

the sacral vertebrae fuse with each other, but a proximal foramen small metacarpal and one small phalanx, both cartilaginous, form

remains (Fig. 4E). in the autopodial region of the limb. In C. sinebrachiatus the ossiﬁca-

Vertebral ossiﬁcation starts late in development; this process tion of the vestigial humerus starts at the ﬁnal pre-hatching stages

was only observed in C. sinebrachiatus specimens. In the 30-day of development; it is completely ossiﬁed only at juvenile stages

embryo, the bases of the neural arches and of the vertebral centrum (data not shown).

of the atlas (which is incorporated into the centrum of the axis as Also at this stage, the paired sternal plates are observed medi-

the odontoid process) are still cartilaginous. The centrum of the ally, with one pair of sternal ribs connected to their lateral margins

axis is ossiﬁed and the neural arches are slightly stained red, but in N. ablephara and two pairs in C. sinebrachiatus (Fig. 5F). The fusion

the dorsal tip is still formed by cartilage. In the neural arches of of the sternal plates occurs soon after, at around 27–28 days. Pos-

the subsequent vertebrae an ossiﬁcation pattern similar to the one terior to the sternum, another pair of xiphisternal ribs and 5 (in

described for the neural arches of the axis is observed, with the base C. sinebrachiatus) and 7 (in N. ablephara) pairs of inscriptional ribs

of the arches ossifying earlier than their dorsal extremity. Given the will eventually join medially, in an anterior–posterior progression,

anterior–posterior progression of development, the more posterior to form the xiphisternum and the post-xiphisternum, respectively

the vertebra, the more extensive the cartilaginous tissue. (Fig. 5G).

The articular surfaces of the pre- and post-zygapophyses remain The ossiﬁcation of the endochondral scapular portion of

cartilaginous until late stages of development. Again, the more the scapulocoracoid is delayed with respect to the limb. In C.

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301 295

Fig. 4. Vertebral development in cleared and stained embryos of Calyptommatus sinebrachiatus (A and E) and Nothobachia ablephara (B–D). (A) Neural arches, vertebral centrae

and notochord in a 12-day embryo; anterior to the right. (B) Vertebral series of a 16-day embryo, showing the difference between the degree of development in anterior and

posterior regions of the trunk. (C) Anterior vertebrae and corresponding intercentra in a 16-day embryo; anterior to the left. (D) Anterior portion of the trunk of a 24-day

embryo showing well-developed vertebrae and associated ribs; anterior to the left. (E) Sacral vertebrae of a 26-day embryo showing the transverse processes fused distally;

anterior to the top. Abbreviations: ic, intercentrum; na, neural arch; nt, notochord; vc, vertebral centrum. Scale bars = 0.25 mm, except for B = 1.0 mm.

sinebrachiatus the ossiﬁcation begins around the 33 day of devel- the 16-day embryo of N. ablephara, the femur cartilage is clearly

opment from a center of ossiﬁcation in the middle of the scapular distinguished in the hindlimb.

plate; ossiﬁcation in the coracoid portion is delayed. The miner- The pelvic elements develop around 18–20 days after oviposi-

alization of suprascapula, epicoracoid, sternum, xiphisternum and tion in both species; short pubis, ischium and ilium form apparently

post-xiphisternum (and the associated ribs) occurs through carti- as a single cartilaginous condensation at the base of the limb. At this

lage calciﬁcation, a process that starts late in development and ends stage, short tibia and ﬁbula cartilages can be clearly distinguished

only after hatching. At pre-hatching stages in both species, the two in both species (Fig. 6B). Also, in C. sinebrachiatus, a metatarsal is

pieces of the medial body of the interclavicle fuse and the lateral observed in the autopodium (Fig. 6B).

processes fuse, although incompletely, to its medial body. The pelvic girdle elements become longer and stouter as devel-

In the juvenile, the clavicle and interclavicle are much more opment proceeds, with the ilium being longer than the pubis and

developed, but the latter is still not completely formed since ischium. At around 26 days of development, perichondral ossiﬁca-

its lateral processes are not entirely fused to its body (Fig. 5H). tion starts at the femur, tibia and ﬁbula both in C. sinebrachiatus and

The scapulocoracoid is completely ossiﬁed, but the region of the N. ablephara, while the pelvic bones are still cartilaginous (Fig. 6C).

glenoid fossa is still formed by cartilaginous tissue. In N. ablephara, Two metatarsals (III and IV) and one phalanx at digit IV develop in

the cartilaginous epicoracoid region delimits the three scapu- the autopodium of N. ablephara; in C. sinebrachiatus no cartilages

locoracoid fenestrae (posterior coracoid, anterior coracoid, and are observed in the autopodium.

scapulocoracoid fenestrae) (Fig. 5H); there are no fenestrae in the In C. sinebrachiatus, the ossiﬁcation of girdle elements starts at

scapulocoracoid of C. sinebrachiatus (Fig. 5I). The calcification of the late stages of development, with the first signs of ossification in

sternum cartilage starts from its posterior portion and progresses the pubis, ischium and ilium being observed in the 33-day embryo;

anteriorly; the same is true for each individual post-xiphisternal ossiﬁcation in the ilium seems to progress more rapidly than in

plate, but the calciﬁcation process progresses antero-posteriorly. the other two elements. In this embryo, the astragalus–calcaneum

In the anterior limb of N. ablephara, the ossiﬁcation of the long is still cartilaginous, as well as the small metatarsal and the ﬁrst

bones progresses rapidly, while the ossiﬁcation in the carpal region phalanx.

is delayed (Fig. 5J); the ossiﬁcation process in the carpal region The astragalus–calcaneum cartilage starts ossiﬁcation at pre-

could not be clearly observed due to weak staining in the available hatching stages through two ossiﬁcation centers, one in the

material. astragalus region and one in the calcaneum region (Fig. 6D and

E). In N. ablephara, distal tarsal IV is the most developed element

(after the astragalus–calcaneum), followed by distal tarsal III; the

3.2.3. Pelvic girdle and hindlimb ossiﬁcation process is, thus, more advanced in the ﬁrst element

The ﬁrst cartilage to appear in this region is a Y-shaped car- (Fig. 6E).

tilage representing the femur, tibia and ﬁbula primordia, which A small cartilage forms distal to the astragalus–calcaneum and

is observed in a 13-day embryo of C. sinebrachiatus (Fig. 6A). In lateral to the single metatarsal in C. sinebrachiatus (Fig. 6F) and

296 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301

Fig. 5. Anterior limb and scapular girdle development in cleared and stained embryos of Calyptommatus sinebrachiatus (A, D, F, G, I and J) and Nothobachia ablephara (B, C, E,

and H). (A) Photograph and schematic representation of an initial stage of girdle development in a 20-day embryo of C. sinebrachiatus, both in lateral view; anterior to the

top. (B) Initial stage of girdle and limb development in a 16-day embryo of N. ablephara, in lateral view; anterior to the top. (C) Scapular girdle and anterior limb in a 20-day

embryo; anterior to the top. (D) Dermic clavicle and interclavicle; anterior to the top. (E) Anterior limb of a 26-day embryo and the beginning of perichondral ossiﬁcation in

the humerus. (F) Photograph and schematic representation of the scapular girdle and anterior limb in a 26-day embryo; anterior to the top. (G) Post-xiphisternum plates and

inscriptional ribs in a pre-hatching embryo, showing the beginning of cartilage calciﬁcation; anterior to the left. (H and I) Scapular girdles of pre-hatching embryos of (H) N.

ablephara and (I) C. sinebrachiatus. (J) Anterior limb of a pre-hatching embryo. Abbreviations: aut, autopodium; cl, clavicle; crc, coracoid; dc, distal carpal; epc, epicoracoid;

hu, humerus; i-rb, inscriptional rib; icl, interclavicle; mc, metacarpal; p.xph, post-xiphisternum; ph, phalanx; ra, radius; rb, rib; rdl, radiale; sc, scapula; scc, scapulocoracoid;

spc, suprascapula; str, sternum; str-rb, sternal rib; ul, ulna; uln, ulnare; xph, xiphisternum; xph-rb, xiphisternal rib. Scale bars = 0.25 mm, except for I = 1.0 mm.

might correspond to a distal tarsal IV or to a vestigial metatarsal. and Kamal (1961a,b,c); Rieppel (1992a,b, 1993a,b,c, 1994); Rieppel

The last regions to ossify are the epiphyses of the limb bones, the and Zaher (2001); Hanken and Hall (1993); Abdala et al. (1997);

tips of the girdle bones, and the tarsal elements. Maisano (2000, 2001); and others.

The ossiﬁcation of limb and girdle elements progresses in juve- The literature on the osteology of the Gymnophthalmidae is rep-

nile phases. In N. ablephara, a small ossiﬁcation ventral to the 4th resented by extensive comparative studies focused primarily on

metatarsal probably corresponds to the 5th metatarsal. general aspects of adult morphology and on the evolution of the

group, such as those by MacLean (1974), Presch (1980, 1983), Estes

4. Discussion et al. (1988), Hoyos (1998), Lee (1998), Evans (2008), and Russel and

Bauer (2008). Anatomical studies describing in detail the arrange-

Descriptions of the osteological arrangement of living and fossil ment of the cranial and axial skeleton of adult gymnophthalmids

adult squamate specimens are quite frequent in the anatomi- are more recent: Montero et al. (2002 – skull of Euspondylus acu-

cal literature; this large amount of data reﬂects the wide use tirostris), Bell et al. (2003 – skull of Neusticurus ecpleopus), Rodrigues

of osteological characters in phylogenetic systematics. However, et al. (2005 – Dryadosaura nordestina), Rodrigues et al. (2007 –

descriptions of the developing cartilage and bone elements are Alexandresaurus camacan), Tarazona et al. (2008 – skull of Bachia

still scarce for Squamata. The ﬁrst records of extensive embryolog- bicolor), Guerra and Montero (2009 – skull of Vanzosaura rubri-

ical studies date back to the comparative morphology period, at cauda), Jerez and Tarazona (2009 – axial skeleton of B. bicolor),

the beginning of the 20th century, developed mainly by Goodrich Rodrigues et al. (2009 – Caparaonia itaiquara), and Roscito and

(1930) and de Beer (1937). Since that time, other studies have con- Rodrigues (2010 – skull of C. sinebrachiatus, Scriptosaura catimbau,

tributed signiﬁcantly to the knowledge of embryonic development and N. ablephara). Still, only a couple of studies deal with the embry-

of cartilage and bone in lizards and snakes, such as those of Bellairs onic development of the skeleton in gymnophthalmids (Bell et al.,

(1949, 1965); Bellairs and Kamal (1981); Romer (1956); el Toubi 2003; Tarazona et al., 2008); such studies report only the post-natal

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301 297

Fig. 6. Posterior limb and pelvic girdle development in cleared and stained embryos of Calyptommatus sinebrachiatus (A–D, F) and Nothobachia ablephara (E). (A) Cartilage

condensation in the posterior limb bud of a 13-day embryo and its representation in the lower left corner. (B) Pelvic girdle and limb elements in a 23-day embryo. (C)

Ossiﬁcation progresses in the long bones of the limb while pelvic girdle elements are still cartilaginous. (D–F) Ossiﬁcation patterns in the tarsal region of pre-hatching

embryos. Abbreviations: ac, astragalus–calcaneum; ast, astragalus; calc, calcaneum; dg, digit; dt, distal tarsal; fe, femur; ﬁ, ﬁbula; il, ilium; is, ischium; mt, metatarsal; ph,

phalanx; pu, pubis; ti, tibia; ts region, tarsal region. Scale bars = 0.25 mm, except for B = 0.5 mm.

development of a few elements of the skull. The present work rep- exoccipital, is formed by the fusion of three pre-occipital arches,

resents, thus, the ﬁrst detailed analysis of skeleton development in which are serially homologous to vertebrae and are believed to have

pre-hatching gymnophthalmid embryos. originated from a fusion of the anterior vertebrae (Gladstone and

The embryonic development of the skull and the axial and Erichsen-Powell, 1915; Romer, 1956). The hypoglossal nerve roots

appendicular skeletons is quite similar in both C. sinebrachiatus and emerge between the arches, being enclosed in separated foramina

N. ablephara. Also, the general pattern of development of skull ele- after the fusion of the arches.

ments observed in these gymnophthalmids – development of the The serial homology of the pre-occipital arches to the verte-

posterior and basal elements of the chondrocranium followed by brae is an old idea proposed by early morphologists (see short

formation of Meckel’s cartilage and of elements derived from the review in Noden and Schneider, 2000), and new evidence based

splanchnocranium (such as quadrate and epipterygoid) and of the on experimental embryologial data, mainly related to the analysis

dermic pterygoid, then by the remaining elements of the braincase of axial patterning along the vertebral column by the Hox genes,

and ﬁnally by the dermic covering of the skull – follows that of are shedding light on this issue.

other lizards, snakes, amphisbaenians, turtles, and Sphenodon (de In chicks, for example, it was demonstrated experimentally that

Beer, 1937). the ﬁrst ﬁve somites contribute to form the occipital region of the

The rapid and pronounced growth of the brain in early stages of braincase (although this fact had already been shown by Goodrich

organogenesis is not accompanied by the development and growth in 1930): cells from the ﬁrst somite form the exoccipital and cells

of the cartilaginous and bony elements that support it. The ﬁrst car- derived from the others (2nd to 5th, the last one contributing only

tilaginous structures to develop in the skull are the basal plate, the with its anterior portion) form the basioccipital and the condyles

occipital arches and the trabeculae forming the initial chondrocra- (Couly et al., 1993). The remaining somites give rise to the verte-

nium ﬂoor, which are soon followed by the otic capsules forming brae, which are formed by the fusion of the posterior half of one

the lateral protection for the brain. The dorsal covering forms only somite to the anterior half of the subsequent somite. It is, thus,

at late stages with the development of the synotic tectum posteri- proposed that the occipital region of the braincase be considered

orly and of the dermic parietal and frontal bones anteriorly. Such a a “giant vertebra” formed by the fusion of the 5 ﬁrst somites; also,

pattern is common for reptiles, as described by Romer (1956). the presence of the multiple hypoglossal nerve roots provide fur-

ther evidence to such a developmental scenario, since dorsal root

4.1. Occipital arch sensory ganglia normally develop at the anteriormost part of each

somite (Teillet et al., 1987). In addition, transgenic studies in mice

In vertebrates in general, including the gymnophthalmids involving a shift in the expression of Hox-4.2 (Hoxd4) showed

described, the occipital arch, which later ossiﬁes into the that when this gene was expressed at a more anterior domain

298 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301

than its normal expression pattern, the supraoccipital and exoc- different character state if during embryonic development the spe-

cipital bones were underdeveloped, or even absent, and ectopic ciﬁc element is formed but then subsequently reduced; similarly,

structures that resembled neural arches from the vertebrae formed an element that is assumed to be formed by a fusion between two

attached to the basioccipital; also, the basioccipital acquired verte- or more bones might, in fact, have a single origin. These problems

bral characteristics (Lufkin et al., 1992). In an evolutionary scenario highlight the importance and utility of using developmental

proposed by de Beer (1937), the occipital region of the vertebrates features to polarize series of transformations (reviewed by Kluge

is considered the neocranium, formed after the assimilation of ver- and Strauss, 1985; de Queiroz, 1985).

tebral elements posterior to the vagus nerve to the palaeocranium The postorbitofrontal of C. sinebrachiatus is a good example of

(formed by the trabecular and parachordal cartilages) of the ances- such a problem: this element is formed by the fusion of a very

tral agnathes. reduced postfrontal to the well-developed postorbital, and by look-

ing at the adult condition, one would be left to wonder whether

4.2. Other skull elements such a condition could be the result of a reduced postfrontal fused

to a well-developed postorbital (or vice versa), or whether it rep-

The jugal of C. sinebrachiatus is unique among Gymnophthalmi- resents a single element with the other having been lost at some

dae for the presence of a well-developed posterior free process point of the evolutionary history of the species. The identity of the

(Roscito and Rodrigues, 2010). By the time the jugal is ﬁrst vis- posterior circumorbital elements is particularly difﬁcult to deter-

ible this process is already present, as is the dorsal process; the mine in fossil forms, and the postorbitofrontal has been interpreted

jugal, therefore, assumes a pronounced “>” shape. Müller (2003) as a postorbital, postorbitofrontal, jugal, or some fusion of these

has observed that in some taxa of modern lizards (he did not state (see Haas, 1979, and Lee and Caldwell, 1998 for Pachyrhachis prob-

which taxa, though), the posterior process of the jugal ossiﬁes dur- lematicus, for example; or the discussion in Zaher and Scanferla,

ing postnatal ontogeny from a ligament that replaces the lower 2011).

temporal arcade, but C. sinebrachiatus shows a different develop- The lacrimal bone is another example: as discussed previously

mental pattern in which the posterior free process forms at early (Roscito and Rodrigues, 2010), the absence of the lacrimal in the

stages, at the moment of the ﬁrst appearance of the jugal. A tri- adults of N. ablephara and C. nicterus – and of C. sinebrachiatus

radiate jugal is common among basal diapsid reptiles and extant (which shows the same condition as its sister species; J.G. Roscito,

squamate groups (Gauthier et al., 1988; Evans, 2003; Conrad, 2008), personal observation) might be considered a loss or an early

but among the extant forms, the posterior free process is rarely as fusion to neighboring elements during embryonic stages. Again, the

developed as it is in C. sinebrachiatus. The anterior maxillary pro- analysis of embryonic development showed that this bone never

cess of the jugal forms only at later stages. The jugal of N. ablephara develops in N. ablephara and C. sinebrachiatus and, thus, is not fused

does not have a posterior free process, being slender and slightly to other bones.

sigmoidal in shape; as in C. sinebrachiatus, the maxillary process

forms later. 4.3. Pectoral and pelvic girdle

Adult specimens of C. sinebrachiatus do not have an open sub-

orbital fenestra, while adults of N. ablephara have a small opening The development of girdles and limbs also follows a similar gen-

(Roscito and Rodrigues, 2010). The closure of this fenestra occurs at eral pattern in both species here analyzed, with a few differences

the ﬁnal stages of development due to the increased growth of the being observed in the timing of development of some structures,

pterygoid, ectopterygoid, and palatine, which overlap the palatal mainly those of the pectoral girdle and forelimb.

shelf of the maxilla to form a stiff bony plate. A narrower sub- The reduced dermal interclavicle of N. ablephara is ﬁrst

orbital fenestra is characteristic of Xantusidae and Teidae species represented by two thin dermal membranes elongated antero-

(Estes et al., 1988), and the narrowing occurs through the medial posteriorly. At the ﬁnal stages of development the primordia fuse

expansion of the pterygoid; this is similar to that observed for C. with each other and the lateral processes form separately from the

sinebrachiatus and N. ablephara, the difference being that the fen- medial body; development of the interclavicle is completed only in

estra is further closed by the palatine and ectopterygoid. juvenile phases. In C. sinebrachiatus, the interclavicle follows this

The skull of both C. sinebrachiatus and N. ablephara is structured same pattern of development, with the formation of paired pri-

to serve as a burrowing tool (Roscito and Rodrigues, 2010), showing mordia of its medial body and of the lateral processes, which fuse

features that strengthen the skull and protect the brain and sense with each other in late stages of embryonic development and in

organs from the impact of the head-ﬁrst burrowing movements early juvenile life.

(Rieppel, 1984, 1996). Therefore, features such as complex artic- Development of the sternum, xiphisternum and post-

ulations between skull elements – for example, the articulation xiphisternum follows an antero-posterior sequence, with the

between maxilla and premaxilla; a greater overlapping between anteriormost elements forming earlier than posteriormost ones.

bones, especially the frontal and parietal, and those that close the All develop from paired structures that form on either side of

suborbital fenestra; and extended growth of some bones, such as the body: two sternal plates form ﬁrst, followed by one pair of

the palatal elements, or the ventral process of the parietal – are clear xiphisternal ribs that soon fuse to each plate’s posterior extremity

evidence of the existence of evolutionary pressures imposed by a to form the xiphisternum, and a number of inscriptional ribs (as in

fossorial and burrowing habit that have shaped the developmental Etheridge, 1965) fuse to form the post-xiphisternum. The fusion

processes. process of these structures also occurs in an antero-posterior

Fusion or loss of elements is a frequent outcome of structural sequence. These structures undergo mineralization through carti-

simplification (Hanken and Hall, 1993; Lee, 1998) and reflects, in lage calcification, a process that also progresses antero-posteriorly,

conjunction with skull consolidation, morphological changes in but the calciﬁcation within each plate begins from its posterior

response to body size and/or lifestyle; furthermore, lost or fused margin and progresses anteriorly.

elements are frequently used in systematic studies to establish

evolutionary relationships between groups. However, inferring 4.4. Limbs

that one speciﬁc bone or cartilage was either lost or fused to some

other element, based only on adult morphologies, may not always The ossiﬁcation process of the long bones of the limbs follows

be correct and may lead to phylogenetic inconsistencies and even the sequence of development of their cartilaginous precursors;

wrong topologies. Losses, for example, might be considered a thus, humerus/femur are the ﬁrst elements to start perichondral

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 289–301 299

ossiﬁcation, followed by radius/ulna and tibia/ﬁbula, and later of amniote limb development (following the primary axis/digital

by metacarpal/metatarsal and phalangeal ossiﬁcation (these last arch model), and thus may be regarded as truncations of putative

ones also ossify in an anterior to posterior sequence). Carpal/tarsal ancestral sequences. The identity of the only digit of the forelimb

elements, although being formed following the proximo-distal of N. ablephara can be assigned to digit IV, considering that the

sequence of development, are the last limb elements to start the developmental pattern follows the primary axis/digital arch model,

ossiﬁcation process, which begins at the ﬁnal stages of develop- while in the hindlimb some morphological features and the rela-

ment and ends in early juvenile life; therefore, there is a decoupling tions between skeletal elements (metatarsal and tarsal elements)

of cartilage formation and ossiﬁcation process (in agreement with are still conserved, allowing for a clear identiﬁcation of the two

observations by Rieppel, 1992a,b; Shapiro, 2002 and others). In digits as digits III and IV.

the forelimb of N. ablephara, ossiﬁcation is ﬁrst seen in the distal The analysis of gene expression during limb development can

carpal IV. The remaining elements, the identity of which could not add some new and important insights into the question of digit

be properly assigned and, hence, could be either distal carpals or identity in these lizards. Knowledge of abnormal limb bud pat-

metacarpals, are still cartilaginous. This delay in the ossiﬁcation of terning is derived from extensive experimental studies, performed

these elements may suggest that they are distal carpals rather than mainly in chick and mouse embryos, that involve the disruption of

metacarpals, since the metacarpal of the only digit is in a much normal development through either mutational or misexpression

more advanced stage of ossiﬁcation, while the distal carpal IV is assays. Such studies have revealed major signaling cascades that

starting to ossify; also, the fact that the carpal/tarsal region is the are involved in patterning the developing limb. Among them are

last to ossify provides additional support for the identiﬁcation of the FGF family of genes, involved in patterning the proximo-distal

these elements as distal carpals. Nevertheless, the possibility that axis via the apical ectodermal ridge (Saunders, 1948; Summerbell

this delay in the ossiﬁcation of these elements could be a conse- et al., 1973; Lewandoski et al., 2000; Mariani et al., 2008; among

quence of the structural reduction of the limb cannot be excluded, others) and in promoting limb growth through the Shh/Grem1/FGF

which would make the identiﬁcation of these elements difﬁcult. feedback loop (Panman et al., 2006). The gene sonic hedgehog (Shh)

Two ossiﬁcation centers form in the astragalus–calcaneum plays a fundamental role in patterning the anterior-posterior axis

cartilage in both species, the largest one corresponding to the of the limb and in establishing the number and identity of dig-

astragalus and the smaller one to the calcaneum. These ossiﬁca- its (Riddle et al., 1993; Harfe et al., 2004) through a complex set

tion centers fuse in post-hatching stages to form the compound of interactions with several other signaling molecules (for further

astragalo-calcaneum (as described by Mathur and Goel, 1976; details, see Tickle, 2006). Downregulation or loss of Shh signaling

Rieppel, 1992a,b; Shapiro, 2002); in N. ablephara, ossiﬁcation then leads to limb truncation, which involves the reduction or loss of dis-

progresses with distal tarsal IV, followed by distal tarsal III. The tal elements, including the digits (Chiang et al., 2001; reviewed by

same ossiﬁcation sequence for the tarsal elements was observed in Tickle, 2006). Although such knowledge is important for providing

Lacerta vivipara (Rieppel, 1992a), Cyrtodactylus pubisculus (Rieppel, insights into the developmental basis of some limb abnormalities,

1992b), Lacerta agilis (Rieppel, 1994), and Hemiergis (Shapiro, it may not reﬂect the patterning mechanisms involved in the evo-

2002). lution of limb-reduced phenotypes among all vertebrates.

Limb reduction is clearly more pronounced in C. sinebrachiatus So far, the developmental mechanisms involved in limb reduc-

than in N. ablephara. From the morphogenetic model proposed by tion in lizards have only been studied in Anguis fragilis (Raynaud,

Shubin and Alberch (1986), it follows that limb cartilaginous con- 1990; Raynaud and Kan, 1992; Raynaud et al., 1998) and in species

densations form in a proximo-distal sequence, following a primary of the genus Hemiergis (Shapiro, 2002; Shapiro et al., 2003). In the

axis of development which progresses through the ulna/ﬁbula, case of A. fragilis, it was demonstrated that an apical ectodermal

ulnare/calcaneum, distal carpal/tarsal IV, and digit IV. Digital con- ridge (AER) develops in the embryonic limb but later degener-

densations form, then, in a posterior-anterior sequence from digit ates, leading to cell death and limb degeneration; the processes

IV, and digit V is formed independently (Shubin and Alberch, 1986; responsible for this degeneration were not investigated but such

Rieppel, 1992a; Shapiro, 2002). Phalanges are also formed proximo- deﬁciency results in a lack of FGF expression and that of other genes

distally. The proposed digit developmental sequence for anuran that act in response to signals from the AER, including Shh (see ref-

amphibians and amniotes, based on the primary axis/digital arch erences above). As for Hemiergis, it was shown that a heterochronic

model (Shubin and Alberch, 1986), is IV > III > II > V > I; in cases of change in Shh expression results in the formation of different limb-

limb reduction, digits are lost in an opposite sequence from that of reduced morphologies; thus, the longer Shh is expressed, the more

normal development, with elements that were formed ﬁrst being complete is the limb (i.e., more digits formed; and vice versa).

the ones that are lost last (Greer, 1987; Oster et al., 1988). Such a Although somewhat preliminary (when compared to the degree

conserved pattern might reﬂect either a simple evolutionary trun- of knowledge available for chick and mouse), both studies provide

cation of the developmental trajectories of these elements, or the important information on some of the developmental mechanisms

increased function of digits III and IV in autopod stability in locomo- that are involved in specifying limb-reduced morphologies and

tion, and thus would be constrained under stronger evolutionary should deﬁnitely be taken into consideration when analyzing other

pressures acting upon their maintenance (Greer, 1991). cases of limb reduction/loss among natural populations, such as

Limb reduction, which is deﬁned as the loss of one or more limb the limb-reduced Calyptommatus and Nothobachia species. Thus, in

elements (even of single phalanges) when compared to an ances- parallel with the condition observed in Hemiergis (Shapiro et al.,

tral condition, reaches extremes in some lizards, as is the case in 2003), an earlier interruption of Shh expression in the hindlimb of

N. ablephara and C. sinebrachiatus. Extreme reduction frequently Calyptommatus, when compared to the duration of expression of

involves the loss of morphological information, which may ren- Shh in its sister species Nothobachia (or in the lacertiform, closely

der the identiﬁcation of cartilaginous or bony elements through a related Vanzosaura, Psilophthalmus and Procellosaurinus), might be

simple anatomical analysis difﬁcult. However, if one assumes that the main factor involved in the further reduction/loss of the digits.

limb development in both gymnophthalmids follows the primary Several types of limb-reduced morphologies, as compared to

axis/digital arch pattern, then it might be reasonable to identify, a pentadactyl condition, have independently evolved among the

in the hindlimb of C. sinebrachiatus, the tarsal element distal to most diverse lineages of terrestrial vertebrates. During embryonic

the astragalus–calcaneum as distal tarsal IV, and the only digit development, digits are formed ﬁrst by the condensation of undif-

as digit IV; this conﬁguration, just like that of the forelimb (a ferentiated mesenchymal cells, forming pre-chondrogenic anlagen

humerus rudiment), closely resembles an early intermediate stage that later differentiate into the cartilaginous primordia of digits

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