Embryonic Development of the Fossorial Gymnophthalmid Lizards Nothobachia

Home , Nothobachia, Pharyngeal slit

Zoology 115 (2012) 302–318

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Embryonic development of the fossorial gymnophthalmid lizards Nothobachia

ablephara and Calyptommatus sinebrachiatus

∗

Juliana G. Roscito , Miguel T. Rodrigues

Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Cidade Universitária, Rua do Matão, Trav. 14, no. 321, São Paulo, SP, CEP 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 evolutionary history of the lizard family Gymnophthalmidae is characterized by several independent

Received 7 December 2011

events of morphological modiﬁcations to a snake-like body plan, such as limb reduction, body elongation,

Received in revised form 19 March 2012

loss of external ear openings, and modiﬁcations in skull bones, as adaptive responses to a burrowing and

Accepted 20 March 2012

fossorial lifestyle. The origins of such morphological modiﬁcations from an ancestral lizard-like condition

can be traced back to evolutionary changes in the developmental processes that coordinate the building

Keywords:

of the organism. Thus, the characterization of the embryonic development of gymnophthalmid lizards is

Gymnophthalmidae

an essential step because it lays the foundation for future studies aiming to understand the exact nature

Lizards

of these changes and the developmental mechanisms that could have been responsible for the evolution

Fossorial lifestyle

of a serpentiform (snake-like) from a lacertiform (lizard-like) body form. Here we describe the post-

Limb reduction

Staging system ovipositional embryonic development of the fossorial species Nothobachia ablephara and Calyptommatus

sinebrachiatus, presenting a detailed staging system for each one, with special focus on the development

of the reduced limbs, and comparing their development to that of other lizard species. The data provided

by the staging series are essential for future experimental studies addressing the genetic basis of the

evolutionary and developmental variation of the Gymnophthalmidae.

1. Introduction lacertiform genera Tretioscincus, Micrablepharus, Gymnophthalmus,

Procellosaurinus, Vanzosaura and Psilophthalmus are lizard-like in

Squamata is one of the most diverse groups among vertebrates, shape, have a tail that is longer than the body, and have well devel-

comprising approximately 9500 species including lizards, snakes, oped limbs and digits, with 5 or 4 digits in the forelimb (the three

amphisbaenians, turtles, crocodiles, and tuatara (Uetz, 2012) dis- former genera have 5 digits while the three latter have 4) and 5 dig-

tributed along a wide range of habitats and niches (Vitt et al., 2003). its in the hindlimb. Nothobachia, Scriptosaura and Calyptommatus

A common trait among squamate reptiles is the repeated evolution are fossorial and psammophilic (adapted to life on sand) and show

of a snake-like morphology in distantly related groups (Wiens et al., morphological features related to this lifestyle, such as loss of exter-

2006; Brandley et al., 2008; Skinner et al., 2008), involving mor- nal ear openings, an elongated body, and reduced or absent limbs.

phological changes associated with body elongation and with the Nothobachia has a styliform forelimb and a small hindlimb with 2

reduction or the complete loss of limbs. Such an evolutionary pat- digits, and Scriptosaura and Calytpommatus have no forelimbs and

tern is clearly seen in the lizard group Gymnophthalmidae (Estes the hindlimb is styliform.

et al., 1988), which is characterized by the convergent occurrence The origins of such morphological modiﬁcations that charac-

of morphological adaptations to a fossorial lifestyle, such as reduc- terize the evolutionary history of the Gymnophthalmini can best

tion and/or loss of limbs, body elongation, and loss of external ear be understood by studying the embryonic development of these

openings. species, since changes in the developmental processes that coordi-

The Gymnophthalmini, a monophyletic group of nine genera nate the patterning and growth of the embryos are the main sources

(Rodrigues, 1991; Pellegrino et al., 2001; Rodrigues and dos Santos, of evolutionary variation in phenotypes (Raff, 2000).

2008; Rodrigues et al., 2009) within the Gymnophthalmidae, is The knowledge of how development is organized is fundamental

one of the groups in which the evolutionary transition from a for understanding the causes underlying variation in the devel-

lizard-like to a snake-like morphology is clearly recognizable. The opmental processes. Thus, detailed descriptions of the phases of

embryonic development are essential references for evolutionary

and developmental studies aiming to address how development

∗ evolves and how evolutionary processes are driven by changes in

Corresponding author.

E-mail address: [email protected] (J.G. Roscito). development.

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 303

Developmental biologists are more familiar with embryonic maintained in small rounded plastic boxes (20 cm diameter, 8 cm

staging tables of vertebrates such as the chicken Gallus gal- high) containing slightly moist ﬁne sand. Immediately after ovipo-

lus (Hamburger and Hamilton, 1951), the frog Xenopus laevis sition, clutches were transferred to and maintained in photographic

(Nieuwkoop and Faber, 1967), the ﬁsh Danio rerio (Kimmel et al., ﬁlm tubes with moist sand to prevent drying, at environmental

◦

1995), and the mouse Mus musculus (Kaufman, 1992), and to a lesser temperatures (28–35 C). As in all gymnophthalmids, clutch size

degree, those of Eleutherodactylus coqui (Townsend and Stewart, was invariably two eggs (Rodrigues, 1991). Females were released

1985), Ambystoma mexicanum (Schreckenberg and Jacobson, back to the ﬁeld after egg laying.

1975), and the medaka ﬁsh Oryzias latipes (Iwamatsu, 2004), for 52 eggs of C. sinebrachiatus and 13 eggs of N. ablephara were

example. obtained and were opened at regular intervals throughout devel-

As for turtles, lizards, and snakes, descriptions of embry- opment, which lasts approximately 45 days from oviposition to

onic development are widespread in the literature (Dufaure and hatching in both species. The ontogenetic series of C. sinebrachia-

Hubert, 1961; Zehr, 1962; Dhouailly and Saxod, 1974; Bellairs tus consists of two embryos representing each day of development,

and Kamal, 1981; Guyot et al., 1994; Rieppel, 1992, 1994; el from the second day after laying up to the 34th day, and two older

Mouden et al., 2000; Jackson, 2002; Shapiro, 2002; Greenbaum, embryos representing the pre-hatching stage. For N. ablephara, the

2002; Boughner et al., 2007; Sanger et al., 2008; Noro et al., 2009; ontogenetic series is distributed with two-day intervals through-

Wise et al., 2009; Boback et al., 2012; among others) and pro- out development, from the second day after egg laying up to the

vide valuable information on the ontogeny of these species. Also, 26th day, with only one embryo for each stage. These intervals

over the last years a few studies addressing the regulation of were chosen in order to be able to observe early organogenesis,

some aspects of developmental processes of lizards and snakes which is the period where most features are appearing or rapidly

(Raynaud et al., 1998; Cohn and Tickle, 1999; Shapiro et al., 2003; changing shape; later on in development, growth is the dominant

Gomez et al., 2008; Vonk and Richardson, 2008; Vonk et al., 2008) process (Andrews, 2004). Developmental time, measured in days,

have enriched the knowledge on the evolution of the processes was counted from the moment of egg laying.

involved in the formation of new body forms within squamate Embryos were removed from the egg, carefully dissected from

groups. However, the data available is still unsatisfactory consider- the embryonic membranes and ﬁxed immediately in 100% ethanol

ing the diversity of forms and habitats occupied, and the adaptive or 10% neutral-buffered formalin; after a week, the ﬁxed embryos

responses in body form, reproduction, locomotion, physiology and were transferred to 70% ethanol.

behavior. The material was examined with an Olympus SZX12 stereomi-

Given the evolutionary history of the Gymnophthalmini and croscope (Olympus, Tokyo, Japan), and digital pictures were taken

their well-established phylogenetic relationships (see Pellegrino with a digital camera attached to it.

et al., 2001; Castoe et al., 2004; but for further discussion on the

relationships within the gymnophthalmids, see Castoe et al., 2004;

2.1. Staging criteria

Rodrigues et al., 2007, 2009), the group has the potential to become

a good model for researchers aiming to comprehend the variation

The phases of development were determined based on key diag-

in developmental pathways that could have led to the modiﬁca-

nostic features of external morphology, such as those deﬁned by

tion of a lacertiform to a serpentiform body form. Therefore, in this

the Standard Event System (SES; Werneburg, 2009); this system

paper we present a detailed description of the post-ovipositional

establishes morphological criteria for staging embryos and facili-

embryonic development of Nothobachia ablephara and of Calyptom-

tates comparisons between taxa, allowing more reliable analysis

matus sinebrachiatus, and thus establish a working ground for future

of variation of developmental trajectories. The features analyzed

experimental studies.

included somite number, development of structures such as the

otic capsule and the endolymphatic duct, including calcium depo-

sition (the endolymphatic duct is not considered in the SES), eyes

2. Materials and methods

C. sinebrachiatus (Fig. 1A; Rodrigues, 1991) is a small fossorial

and nocturnal lizard endemic to a semiarid dune ﬁeld region on the

right margin of São Francisco River, in the region of Santo Inácio,

in Bahia, Brazil (Fig. 1C). Its average snout–vent length is approx-

imately 60 mm, with the tail shorter than the body. The eyelids

are fused and covered by a modiﬁed scale, there is no external

ear opening, and the limbs are greatly reduced, with the fore-

limb being absent externally and the hindlimb having only one

digit.

N. ablephara (Fig. 1B; Rodrigues, 1984) is a diurnal and also fos-

sorial lizard, endemic to the same geographical region where C.

sinebrachiatus is found, but, differently from the latter, N. ablephara

is found in the dune ﬁelds on the left margin of São Francisco River

(Fig. 1C), in the region of Alagoado. Its average snout–vent length

is 55 mm, with the tail slightly longer than the body. The eyelid

is fused but the eye is not covered by a modiﬁed scale as in C.

sinebrachiatus, there is no external ear opening, and the limbs are

reduced, with a one-digit forelimb and a two-digit hindlimb.

Field expeditions to the Quaternary dune region of the São Fran-

cisco River took place in February 2001, March 2005, and January

2006. Pregnant females were obtained from the region of Santo

Fig. 1. Adult specimens of (A) Calyptommatus sinebrachiatus and (B) Nothobachia

Inácio, and from the Xique-Xique and Alagoado dune ﬁelds, in

ablephara. (C) Location of Xique-Xique and Alagoado dune ﬁelds, at the Quaternary

the state of Bahia, Brazil (Fig. 1C). Until oviposition, females were sand dune region of the São Francisco River; river ﬂows to the northeast.

304 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

(lens, choroid ﬁssure and pigmentation), nervous system (distinct Facial region: Maxillary process of the ﬁrst pharyngeal arch

telencephalon hemispheres, pineal gland, “ﬂattening” of the mes- reaching the level of the middle of the eye.

encephalic bulge), fronto-nasal, maxillary and mandible processes, Pharyngeal arches: Mandibulary process of the 1st and 2nd

pharyngeal arches and opening and closure of pharyngeal slits, pharyngeal arch (hyoid arch) buds distinct and already fusing at

heart, limbs, and external genital structures; see Werneburg (2009) the ventral midline (Fig. 3, St1b); ﬁrst pharyngeal slit (hyoideo-

for a description of such features. Development of late features, mandibular slit) is open, formed by the fusion of the ﬁrst two

such as scale formation and body pigmentation, were not analyzed pharyngeal arches; second pharyngeal slit also open and third slit

because late stages were underrepresented in the sample; thus, noticeable through a groove (Fig. 3, St1c).

the ﬁrst events of scale formation and body pigmentation could Heart: Tubular-shaped.

not be observed in N. ablephara and only poorly analyzed in C. sine- Limbs: Fore- and hindlimb buds prominent, hindlimb bud

brachiatus. The number of days after oviposition (dao) is given for slightly larger (Fig. 4, St1f and St1h).

reference. Stage 2: 6–8 dao, approx. SVL size: 8.0 mm (Fig. 2, St2).

The number of somites is difﬁcult to ascertain from a certain Somite pairs: 48–49.

phase of development until hatching. At the beginning of develop- Brain and otic region: Optic vesicle prominent; endolymphatic

ment, the embryos are more transparent and the determination of duct is longer, ending in a sac with calcium deposits.

the number of somite pairs is possible (at these stages somite num- Eye: Pigmentation ﬁrst visible (Fig. 2, St2); pigment distributed

ber was counted from the ﬁrst somite to the posterior region of the laterally to the lens and concentrated posteriorly.

cloaca); however, at later stages the somites are no longer distin- Facial region: Maxillary process reaching the level of the anterior

guishable. Also, embryos become opaque when ﬁxed and, hence, margin of the eye; the large fronto-nasal process is still distinct from

somite number can no longer be determined. The analysis of a few the maxillary process.

embryos does not exclude individual variation in somitogenesis, Pharyngeal arches: Further medial fusion of the mandibulary

which is one of the main reasons why somite number was not process of the 1st and 2nd arch (hyoid arch) buds; second and third

used for staging embryos. Therefore, stages were deﬁned based on pharyngeal slits open, a groove marks the fourth slit.

the ﬁrst appearance of a given feature, or on easily distinguished Heart: Tubular-shaped.

morphological changes of the analyzed structures; somite number Limbs: Pronounced proximo-distal growth of the limbs (Fig. 4,

was an additional but not crucial character for staging. Snout–vent St2f and St2h), limb segments are not yet distinct; AER not observed

length (SVL) of each embryo was measured from the tip of the snout in either fore- (Fig. 5A and B) or hindlimb (Fig. 5C and D); short tail,

to the cloacal region. with one coil (Fig. 4, St2h).

Stage 3: 10–12 dao, approx. SVL size: 9.3 mm (Fig. 2, St3).

2.2. Scanning electron microscopy Somite pairs: 52.

Brain and otic region: Brain well developed; telencephalon

Scanning electron microscopy (SEM) of the fore- and hindlimb hemispheres observable (Fig. 2, St3).

buds of both species was performed in order to analyze the pres- Eye: Two distinct rings of pigments in the eye, the inner one

ence of the apical ectodermal ridge (AER). Two embryos of C. with pigments more concentrated and the outer one with pigments

sinebrachiatus, one at developmental stage 3 and the other at stage sparsely distributed (Fig. 2, St3).

5 (the former representing the initial development of the forelimb Facial region: A deep groove separates the large fronto-nasal

and the latter representing the stage when the forelimb reaches processes; the maxillary process reaches beyond the anterior mar-

its maximum size, preceding the degeneration phase), and one gin of the eye (Fig. 3, St3a and St3b).

embryo of N. ablephara at stage 2 were sectioned in the sagittal Pharyngeal arches: Mandibular process and 2nd arch (hyoid

plane and both the left and the right halves were prepared for arch) fused and extending to reach the ventral ﬂoor of the skull

imaging with SEM. (Fig. 3, St13b); a slight groove still marks the place of fusion.

Heart: Complete torsion of the heart, with more deﬁned com-

3. Results partmentalization.

Limbs: Limb buds longer (Fig. 4, St3f and St3h); stylopod, zeugo-

Here we describe the post-oviposition embryonic development pod and autopod distinct, with the autopod being more ﬂattened

of N. ablephara and C. sinebrachiatus based on external morpho- than the others.

logical features and using the Standard Event System (Werneburg, Genital region: Buds of the cranial lip of the cloaca present.

2009). A summary of their embryonic development is found in Tail longer, with two coils.

Tables 1 and 2, respectively. Stage 4: 14 dao, approx. SVL size: 10.6 mm (Fig. 2, St4).

Embryos are already into early organogenesis at the moment of Somite pairs: No longer distinguishable.

oviposition, and thus the staging tables shown in this work do not Brain and otic region: Brain well developed; pineal gland

cover earlier processes such as cleavage, gastrulation and neurula- observed at the boundary between telen- and mesencephalon;

tion. Nine developmental stages for N. ablephara and twelve for C. endolymphatic sac larger, with dense calcium deposits (Fig. 2,

sinebrachiatus are described. Embryo size and corresponding time St4).

of development (incubation time measured in days after oviposi- Eye: Pigments more uniformly distributed throughout the eye

tion = dao) are shown for reference only, these variables were not (Fig. 2, St4), but still concentrated in two rings.

used for staging. Facial region: Maxillary process and fronto-nasal process fused

into a uniform structure (Fig. 2, St4).

3.1. Nothobachia ablephara Pharyngeal arches: Mandibular/hyoid arch complex extending

to the tip of the snout, reaching the anterior margin of the eye;

Stage 1: 2–4 dao, approx. SVL size: 5.6 mm (Fig. 2, St1). pharyngeal slits are closed.

Somite pairs: 46; short tail bud not segmented into somites. Limbs: Fore- and hindlimb buds are more developed, with a

Brain and otic region: Mesencephalon prominent; endolym- paddle-shaped autopod (Fig. 4, St4f and St4h).

phatic duct visible, but with no sign of calcium deposits. Genital region: Developing cranial and caudal lips of the cloaca

Eye: Optic vesicle horseshoe-shaped with a distinct lens (Fig. 3, observed around the small hemipenis buds (Fig. 6, St4).

St1a); the choroid ﬁssure is open. Stage 5: 16 dao, approx. SVL size: 9.0 mm (Fig. 2, St5).

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 305

Table 1

Summary of the principal morphological changes occurring during embryonic development of Nothobachia ablephara, from stages 1 through 9.

Structure Character Nothobachia ablephara Structure Character Nothobachia ablephara

St1 St2 St3 St4 St5 St6 St7 St8 St9 St1 St2 St3 St4 St5 St6 St7 St8 St9

Somites 35–40 somite pairs Nose Nasal buds fused at X

midline

40–45 somite pairs Ear Otic capsule distinct X

45–50 somite pairs X X Endolymphatic X

duct/sac visible

50–55 somite pairs X Calcium deposits in X

endolymphatic sac

55–60 somite pairs Heart Tubular heart X

Maxillary Maxillary bud at the level S-shaped heart X

process of the posterior margin

of the eye

Maxillary bud at the X Heart torsion

midline level of the eye

Maxillary bud at the level X Heart internal to the X

of the anterior margin of thorax

the eye

Maxillary and nasal buds X Limbs Forelimb bud X

fused

Mandibular Mandibulary and hyoid X Hindlimb bud X

and hyoid arch buds distinct

arches Mandibulary and hyoid X Stilopod-zeugopod X

arch buds fusing distinction in forelimb

- “elbow”

Mandibulary and hyoid X Stilopod-zeugopod X

arch buds fused distinction in hindlimb

- “knee”

Jaw formed and of same X Autopod distinct in X

size as maxilla (upper forelimb - “rist”

jaw)

Pharyngeal 1st arch (hyoid arch) X Autopod distinct in X

arches forelimb - “ankle”

2nd arch X Forelimb autopod X

paddle-shaped

3rd arch X Hindlimb autopod X

paddle-shaped

4th arch X Forelimb digit X

condensations visible

Pharyngeal Hyoideo-mandibular slit X Hindlimb digit X

slits condensations visible

1st slit X Degeneration of X

interdigital membrane

in forelimb

2nd slit X Degeneration of X

interdigital membrane

in hindlimb

3rd slit X Genital Cranial lip buds X

region

Slits closed X Caudal lip buds X

Eye Choroid ﬁssure closed X Hemipenis buds X

Few pigmentation X Cranial lips completely X

granules fused

Two distinct rings of X Caudal lip completely X

pigment fused

Pigmentation complete X Scales Tail and trunk (ﬂanks) X

Pharyngeal arches: Mandibular/hyoid arch complex reaching Heart: Thoracic cavity enclosing the heart.

the level of the maxilla. Limbs: Anterior limb bud slightly longer and more slender

Limbs: Anterior limb bud growing larger; autopod symmetric (Fig. 4, St6f); posterior limb bud also longer; digit condensations

(Fig. 4, Stf) with a small and subtle bulge representing the digit are clearly visible (Fig. 4, St6h).

condensation; posterior limb bud larger than the anterior limb; the Genital region: Cranial and caudal lips of the cloaca formed;

autopod is asymmetric, with two different bulges representing the hemipenis buds more developed (Fig. 6, St6).

formation of each digit (Fig. 4, St5h). Stage 7: 22 dao, approx. SVL size: 12.1 mm (Fig. 2, St7).

Genital region: Hemipenis buds slightly larger; cranial and cau- Eye: Pigmentation stronger; choroid ﬁssure still lacks pigments.

dal lips more developed (Fig. 6, St5). Facial region: Slender and longer snout.

Stage 6: 18–20 dao, approx. SVL size: 11.2 mm (Fig. 2, St6). Limbs: Anterior limb bud much more slender than the posterior

Brain and otic region: Brain well developed, with telen- and one; the digit condensations are still clearly visible on both buds

mesencephalon projecting from the top of the head (Fig. 2, St6). (Fig. 4, St7f and St7h).

Facial region: Maxilla and mandible form a uniform snout (Fig. 2, Genital region: Caudal lip of the cloaca easily distinguishable

St6). (Fig. 6, St7a); hemipenis buds slightly larger (Fig. 6, St7b).

306 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 X St11 X X

X St10 X

St9 X X

St8 X

X X X St7 X

X St6 X X X

included.

St5

not

X St4 sinebrachiatus

was

St3 X stage

St2 X X

Calyptommatus St1 X X X -

pre-oviposition

visible X

fused

the fused

thorax

midline hindlimb distinction

11; visible

in at in the

interdigital

duct/sac sac

condensations

to (ﬂanks) of

ﬁrst

hindlimb

“knee”

distinct -

forelimb completely

in fused buds

buds buds completely

through bud autopod digit

heart

bud absent X

distinct

deposits

trunk 1 heart

lip lips lip lip

torsion internal buds

capsule and

hindlimb

stages

Dorsal Forelimb Endolymphatic visible degeneration Character Nasal Otic Calcium Tubular S-shaped Heart Heart Forelimb Hindlimb Stilopod-zeugopod Hindlimb Hindlimb Degeneration Begining Cranial Caudal Hemipenis Cranial Caudal Tail Pigmentation in membrane endolymphatic “ankle” paddle-shaped

from

region

Structure Nose Ear Heart Limbs Genital Scales Body

sinebrachiatus

St11

St10

Calyptommatus

X St9 of

St8 X

St7

development

St6

X St5 X X X

embryonic

X Autopod St4 X sinebrachiatus

St3 X X X X during

X X X X

occurring X X Calyptommatus St1 St2 X X X X X

eye

eye of of

fused X changes

size

arch arch arch the

the

slit of pigment

level midline level

of buds

same granules X of

hyoid hyoid hyoid

the the the

closed arch) jaw)

at at at nasal pairs pairs pairs pairs pairs complete

margin and and and rings

margin

and

eye

bud bud bud and morphological

(hyoid ﬁssure

(upper

the

somite somite somite somite somite

distinct fusing fused of distinct

closed

pigmentation arch slit

formed

arch slit arch

posterior anterior

arch slit

principal 35–40 Mandibulary Character buds buds buds level maxilla the the Hyoideo-mandibular 2nd 4th 55–60 Choroid Mandibulary Maxillary 40–45 1st 3rd 45–50 Mandibulary Maxillary 50–55 3rd Jaw 1st Maxillary Slits 2nd Few Maxillary Two Pigmentation

the

hyoid 2

Structure Somites arches Pharyngeal slits Mandibular and Eye Maxillary process Pharyngeal arches Table Summary

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 307

Fig. 2. Developmental series of Nothobachia ablephara, stages 1–9 (images St1–St9, respectively). Scale bar = 1.0 mm.

Stage 8: 24 dao, approx. SVL size: 12.6 mm (Fig. 2, St8). Brain and otic region: Prosencephalon, mesencephalon and

Brain and otic region: The brain is not as prominent as in previ- rombencephalon distinct; otic capsule with well-deﬁned borders

ous stages, the head has reached a more uniform shape (Fig. 2, St8); (Fig. 8, St1a).

endolymphatic sacs closer at the dorsal midline. Eye: Optic vesicle horseshoe-shaped, without pigmentation

Limbs: Skeletal elements from the zeugopod (tibia/ﬁbula and (Fig. 8, St1a).

radius/ulna) and autopod (metacarpals/metatarsals and phalanges) Facial region: Maxillary processes small, reaching the level of

can be seen through the skin due to transparency (Fig. 4, St8f and the posterior margin of the eye.

St8h). Pharyngeal arches: Mandibulary process and 2nd arch (hyoid

Genital region: Cranial and caudal lips of the cloaca and hemipe- arch) distinct (Fig. 8, St1b); ﬁrst (hyoideo-mandibular) and second

nis buds well developed (Fig. 6, St8). pharyngeal slits open; a furrow marks the third slit; primordium of

Beginning of scale formation in the tail and ventrolateral portion third pharyngeal arch visible.

of the trunk. Heart: Tubular-shaped; two chambers represent the atrium and

Stage 9: 26 dao, approx. SVL size: 14.1 mm (Fig. 2, St9). ventricle.

The head is more uniform in shape and the snout is longer. Scale Limbs: Forelimb bud absent (Fig. 7, St1), the bud region is

formation is visible in the ventrolateral region of the trunk. marked by an opaque plate at the lateral side of the trunk; hindlimb

Limbs: Limb buds are much more slender (Fig. 4, St9f and St9h), bud is represented by a small bulge (Fig. 9, St1).

and the interdigital membrane starts to degenerate in the hindlimb Stage 2: 4 dao, approx. SVL size: 6.5 mm (Fig. 7, St2).

(Fig. 4, St9h). Somite pairs: 47–49.

Genital region: Distinction between males and females is clear Brain and otic region: Telencephalon easily observed, distinct

around this stage, since in females the hemipenis buds are in an from the rest of the brain; 6 rhombomeres can be distinguished

advanced stage of regression, being represented by small protu- at the rombencephalon; endolymphatic duct primordia observed

berances (Fig. 6, St9). dorsally to the optic capsule.

Eye: Without pigmentation.

Facial region: Maxillary processes reaching the level of the mid-

3.2. Calyptommatus sinebrachiatus dle of the eye.

Pharyngeal arches: Mandibular process and hyoid arch grow-

Stage 1: 2–3 dao, approx. SVL size: 4.2 mm (Fig. 7, St1). ing midventrally; third pharyngeal slit opened; a furrow marks the

Somite pairs: 36. fourth slit.

308 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

Fig. 3. Details of embryonic stages St1 and St3 of Nothobachia ablephara. (St1a–c) Pharyngeal arches and slits in a stage 1 embryo in lateral (St1a and St1c) and ventral (St1b)

views. (St3a and b) Head of a stage 3 embryo in lateral (St3a) and ventrolateral (St3b) views. Scale bar for St1a–c and St3a = 0.5 mm, for St3b = 1.0 mm. Abbreviations: 2p.s,

second pharyngeal slit; e, eye; ed.s, endolymphatic sac, fn.p, fronto-nasal process; h.a, hyoid arch (second pharyngeal arch); hm.s, hyoideo-mandibular slit (ﬁrst pharyngeal

slit); m.p, mandibular process of the ﬁrst pharyngeal arch; mx.p, maxillary process; oc, otic capsule.

Heart: Tubular heart prominent, “S”-shaped (Fig. 7, St2). Facial region: Nasal capsules large and visible (Fig. 8, St4),

Limbs: Forelimb bud small, hindlimb not much larger (Fig. 9, internal and external prominences distinct; maxillary processes

St2). distinct, reaching the level of the anterior border of the

Stage 3: 5–6 dao, approx. SVL size: 6.8 mm (Fig. 7, St3). lens.

Somite pairs: 50–53. Pharyngeal arches: Mandibulary process and hyoid arch fused

Brain and otic region: Endolymphatic duct wider posteriorly, midventrally, a vestigial groove still marks the region of fusion;

indicating the endolymphatic sac. pharyngeal slits remain open, but less distinct.

Eye: Pigmentation visible, with pigmentation granules ﬁrst Limbs: Forelimb (Fig. 9, St4f) and hindlimb (Fig. 9, St4h)

appearing dorsally (Fig. 8, St3); choroid ﬁssure closed. buds longer than in previous stage and narrower along the

Facial region: Maxillary processes reaching the midlevel of the antero–posterior axis.

eye (Fig. 8, St3; fronto-nasal processes ﬁrst visible as two protuber- Stage 5: 9–11 dao, approx. SVL size: 9.5 mm (Fig. 7, St5).

ances at the anterior region of the snout. Somite pairs: 53.

Pharyngeal arches: Mandibulary process and hyoid arch meet- Brain and otic region: Pineal gland above telencephalon; its body

ing ventrally; second and third pharyngeal slits distinct and wide and stalk are clearly visible.

open; fourth slit begins to open; primordium of 5th pharyngeal arch Eye: Pigmentation extending to the complete eye area, higher

present (Fig. 8, St3). density of granules around lens.

Heart: Beginning of the torsion of the heart (Fig. 8, St3). Facial region: The depression separating the fronto-nasal pro-

Limbs: Fore- and hindlimb buds slightly larger and growing cess is wide; maxillary processes surpass the level of the anterior

along the proximo-distal axis (Fig. 9, St3f and St3h); AER not margin of the eye.

observed in either limb bud (Fig. 5E and F). Pharyngeal arches: Pharyngeal slits are all closed, but

Stage 4: 7–8 dao, approx. SVL size: 8.19 mm (Fig. 7, St4). grooves still mark the ﬁrst (hyoideo-mandibular slit) and second

Somite pairs: 50–53. ones.

Brain and otic region: Mesencephalon more prominent Heart: Torsion almost complete; the heart is less conspicuous.

than in earlier stages (Fig. 8, St4); otic capsules still well Limbs: Forelimb reaches its maximum size at this stage

deﬁned; beginning of calcium deposition in the endolymphatic (Fig. 9, St5f), while the hindlimb bud continues to elongate

sac. antero-posteriorly (Fig. 9, St5h); AER not observed in both fore-

Eye: Pigmentation granules sparsely distributed, but mostly (Fig. 5G and H) and hindlimb (Fig. 5I and J).

concentrated in the posterior region of the eye. Stage 6: 12–14 dao, approx. SVL size: 9.5 mm (Fig. 7, St6).

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 309

Fig. 4. Limb development in Nothobachia ablephara, from embryonic stage 1 (St1) through 9 (St9). Subscript letters f and h after each stage number correspond to forelimb

and hindlimb, respectively; arrows in St6h point to digit condensations. Scale bars = 0.5 mm, except for St9f and St9h = 1.0 mm.

Somite pairs: From this stage on, somite number can no longer Limbs: Forelimb begins to degenerate; a small constriction is

be accurately estimated. seen distally (Fig. 9, St6f); hindlimb compressed dorso-ventrally

Brain and otic region: Otic capsule no longer distinct at this (Fig. 9, St6h).

stage; endolymphatic sac slightly triangular, with calcium deposits Genital region: Hemipenis buds are small (Fig. 10, St6); primor-

concentrated centrally (Fig. 8, St6a). dia of the cranial lips of the cloaca are present.

Eye: Prominent with strong pigmentation (Fig. 8, St6a). Stage 7: 15–17 dao, approx. SVL size: 10.2 mm (Fig. 7, St7).

Facial region: Maxillary processes reaching the fronto-nasal pro- Brain and otic region: Brain well developed, with both hemi-

cess (Fig. 8, St6a and St6b); they later unite at the midline and the spheres of the telencephalon distinct; pineal gland still visible,

groove between them is less evident. located above the telencephalon and anterior to the mesen-

Pharyngeal arches: Mandibular process and hyoid arch uniform, cephalon; endolymphatic duct is shorter and slender, extending

forming a distinct lower jaw which is anchored at the palate; scars towards the dorsal midline; calcium deposits are denser.

of ﬁrst and second pharyngeal slits are still observed. Facial region: Maxillary and fronto-nasal processes forming a

Heart: Formed, but not yet withdrawn into the thoracic cavity. continuous but not yet uniform structure (Fig. 8, St7).

310 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

Fig. 5. Electron microscopy of the limb buds of (A–D) Nothobachia ablephara and (E–J) Calyptommatus sinebrachiatus. (A and B) Anterior limb bud of a 6-day embryo (embryonic

stage 3) in lateral (A) and frontal (B) views. (C and D) Posterior limb bud of a 6-day embryo (embryonic stage 3) in lateral (C) and frontal (D) views. (E and F) Anterior limb

bud of a 6-day embryo (embryonic stage 2) in lateral (E) and frontal (F) views. (G and H) Anterior limb bud of a 9-day embryo (embryonic stage 2/3) in lateral (G) and frontal

␮

(H) views. (I and J) Posterior limb bud of a 9-day embryo in lateral (I) and latero-frontal (J) views. Scale bars = 100 m.

Fig. 6. Development of the genital region of Nothobachia ablephara. (St4) Stage 4 embryo; arrow points to the caudal lip of the cloaca and asterisk marks the developing

hemipenis bud. (St5) Stage 5 embryo; arrow points to the hemipenis bud. (St6) Stage 6 embryo. (St7a and St7b) Stage 7 embryo; arrow in St7a points to the caudal lip of the

cloaca. (St8) Stage 8 embryo. (St9♀) Stage 9 female embryo with a vestigial hemipenis (arrow). Scale bars for St4, St7a and b, St8, and St9♀ = 0.5 mm; scale bars for St5 and

St6 = 1.0 mm.

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 311

Fig. 7. Developmental series of Calyptommatus sinebrachiatus, stages 1–12 (images St1–St12, respectively). Scale bar = 1.0 mm.

Pharyngeal arches: Jaw growing towards the snout, reaching the of the ﬁrst pair of hemipenian spines are observable as two protu-

level of the anterior part of the eye (Fig. 8, St7). berances at the base of the organ; in female embryos, regression of

Heart: Less conspicuous, thoracic wall is closing. hemipenis buds is evident.

Limbs: Forelimb signiﬁcantly reduced; slight constrictions in the Stage 9: 20–22 dao, approx. SVL size: 11.5 mm (Fig. 7, St9).

hindlimb separate the stylopod, zeugopod and autopod; the latter Scales present at ﬂanks.

is paddle-shaped and a bit wider than the other segments (Fig. 9, Brain and otic region: Endolymphatic sacs close to each other,

St7h). characteristically triangular in shape.

Genital region: Cranial lips of the cloaca and hemipenis buds Facial region and pharyngeal arches: Snout prominent; the

are not clearly distinguished from each other, forming a continuous upper and lower jaws have nearly the same size (Fig. 8,

structure (Fig. 10, St7♂ and St7♀); the primordia of the caudal lips St9).

of the cloaca make their ﬁrst appearance; male and female embryos Heart: Already withdrawn into the body wall, which is closed

are already distinguishable, with hemipenis buds much smaller in anteriorly and open posteriorly.

females than in males. Limbs: Hindlimb slender and longer; articulations between the

Stage 8: 18–19 dao, approx. SVL size: 10.1 mm (Fig. 7, St8). segments are well marked (Fig. 9, St9h); the autopodial membrane

Eye: Eyelids ﬁrst appearing and covering the external region of is thinner, being more transparent.

the optic globe; eye pigmentation uniformly distributed. Genital region: In females, the hemipenian buds are clearly

♀

Pharyngeal arches: Jaw almost reaching the level of the maxilla regressing (Fig. 10, St9 ) as compared to the male hemipenis buds

(Fig. 7, St8). (Fig. 10, St9♂) which are much more developed.

Limbs: Forelimb absent; hindlimb autopod well differentiated, Stage 10: 23–25 dao, approx. SVL size: 11.7 mm (Fig. 7, St10).

its border being thicker than the central part; a distinct articulation, Head well developed. A slight depression posterior to the jaw

similar to a wrist, separates the zeugopod from the autopod (Fig. 9, articulation indicates the place of the opening for the external ear.

St8h). Limbs: In the hindlimb (Fig. 9, St10h), a thickening correspond-

Genital region: Genital system more developed than in previous ing to the only digit is visible at the medial region of the autopod.

stages (Fig. 10, St8a); the cranial and caudal lips of the cloaca are Genital region: Hemipenis buds larger (Fig. 10, St10).

more distinct; the caudal lip is represented by a single structure Stage 11: 26–34 dao, approx. SVL size: 13.4 mm (Fig. 7, St11).

(Fig. 10, St8b), and the groove between the caudal lip primordia is Scales are evident at the lateral and dorsal regions of the body,

no longer visible; hemipenis larger (Fig. 10, St8a); the primordia and also on the tail. Body pigmentation is ﬁrst visible dorsally.

312 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

Genital region: Hemipenal ornamentation well formed (Fig. 10, St12♂).

4. Discussion

The data presented in this work represents the two ﬁrst stag-

ing systems of gymnophthalmid species, improving our current

knowledge on squamate development. Comparative analyses of

embryonic development are important for understanding the

developmental processes that underlie evolutionary novelties,

such as the transition from a lacertiform to a serpentiform body

shape seen in several squamate lineages (Wiens et al., 2006;

Brandley et al., 2008) including the Gymnophthalmidae. Also, the

characterization of embryonic development showing the morpho-

logical changes over time is of great importance for comparative

and experimental studies.

Our results show that the initial phases of development of C.

sinebrachiatus and N. ablephara, including cleavage, gastrulation,

neurulation and the beginning of organogenesis, occur inside the

oviduct, since at the moment of oviposition the embryos already

showed more than 30 pairs of somites, maxillary and mandibu-

lar processes (derived from the 1st pharyngeal arch), the second

pharyngeal arch, and limb buds. Even though we did not observe

the initial phases, it may be assumed that the ontogenetic features

of these phases are, to some extent, conserved between squamate

taxa (Pasteels, 1970; Hubert, 1985; el Mouden et al., 2000). Further-

more, embryonic development during organogenesis is of greater

interest here because it is during this period that the morpholog-

ical changes related to speciﬁc and adaptational characters can be

observed (Billet et al., 1985; Jackson, 2002).

The embryonic development of C. sinebrachiatus and N.

ablephara is quite similar, showing some conserved tetrapod

Fig. 8. Details of embryonic stages St1, St3, St4, St6, St7, St9, and St12 of Calyptom-

matus sinebrachiatus. (St1a and St1b) Pharyngeal arches and slits in a stage 1 embryo developmental processes such as the development of paired

in lateral (St1a) and ventral (St1b) views. (St3) Pharyngeal arches and slits and the fronto-nasal, maxillary, mandibular and hyoid arch prominences.

heart in a stage 3 embryo. (St4) Head of a stage 4 embryo in lateral view; arrow

Fronto-nasal and maxillary prominences arise separately and at

points to the fronto-nasal process. (St6a and St6b) Head of a stage 6 embryo in lat-

each side of the head and later fuse to form the snout; similarly,

eral (St6a) and ventrolateral (St6b) views; asterisk marks the maxillary process of

the mandibular processes of the ﬁrst arch (anterior) and the sec-

the ﬁrst pharyngeal arch in both. (St7 and St9) Head of a stage 7 (St7) and stage 9

(St9) embryo in lateral views, showing the development of the mandible towards ond arch (hyoid arch; posterior) fuse and grow forward to reach

the snout. (St12) Head of an embryo in pre-hatching stages; arrow points to the

the snout. The cranial and caudal lips of the cloaca also form

closed external ear. Scale bars for St1a and b = 0.25 mm, St3, St4, St6a and b, and

from the fusion of separated and paired primordia. The main dif-

St9 = 0.5 mm, and for St7 and St12 = 1.0 mm. Abbreviations: 2p.s, second pharyngeal

ference between the embryonic developmental processes in both

slit; 3p.s, third pharyngeal slit; 4p.s, fourth pharyngeal slit; e, eye; h.a, hyoid arch

(second pharyngeal arch); hm.s, hyoideo-mandibular slit (ﬁrst pharyngeal slit); m.p, species concerns limb bud shape, with both limbs of C. sinebrachia-

mandibular process of the ﬁrst pharyngeal arch; oc, otic capsule. tus showing a greater degree of reduction than those of N. ablephara,

and hence attaining a smaller size and a relatively slower growth

Brain and otic region: Endolymphatic sacs are joined middor- rate.

sally, with dense calcium deposits; external ear opening is present. Formation of the hindlimb bud precedes that of the forelimb bud

Eye: The eye is almost completely covered by the eyelids. in C. sinebrachiatus; this may also be true for N. ablephara, since in

Facial region: Snout elongated; egg tooth visible at the tip of the the ﬁrst stage analyzed the hindlimb was bigger than the forelimb.

snout. This relative delay in development of the forelimb bud in relation

Limbs: Long hindlimb; digit seen due to transparency, and inter- to the hindlimb bud may be associated with the greater degree of

digital membrane degenerating (Fig. 9, St11h). structural reduction observed in the forelimb.

Genital region: Hemipenis apex slightly bifurcated (Fig. 10, Forelimb reduction achieves the greatest degree in C. sine-

♂

St11 ) with spermatic duct visible; small buds corresponding brachiatus, with the arrested development of the forelimb bud.

to the second pair of spines are visible above the ﬁrst pair of This bud reaches its maximum size around stage 5 (9–11 days),

♂

hemipenian spines (primary spines; Fig. 10, St11 ). In female and from this point on it starts to degenerate; the animal is

embryos, vestiges of the hemipenis buds are diminutive (Fig. 10, born, thus, with no vestige of an external limb. The same pat-

♀

St11 ). tern of development and subsequent reduction was observed in

Stage 12: Pre-oviposition stage, approx. SVL size: 19.3 mm Anguis fragilis (Raynaud, 1962, 1963), in the forelimb rudiments

(Fig. 7, St12). of the glass snake Ophisaurus apodus (Rahmani, 1974), in python

Scales differentiated and pigmentation complete (Fig. 7, St12). (Cohn and Tickle, 1999), and in several cetacean species (Lande,

Brain and otic region: External ear opening covered by an 1978; Thewissen et al., 2006). In the degeneration phase, the fore-

enlarged scale (Fig. 8, St12). limb bud shows a small distal constriction that might be related

Eye: Eye reduced, covered by an ocular scale (Fig. 8, St12). to the degeneration of the distal mesenchyme (as Raynaud and

Facial region: Sharp and elongated snout (Fig. 8, St12). Kan, 1992, observed for A. fragilis). In the tail of both gymnoph-

Limbs: Hindlimb slender and styliform (Fig. 9 St12h). thalmid embryos, a similar distal constriction is associated

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 313

Fig. 9. Limb development in Calyptommatus sinebrachiatus, from embryonic stage 1 (St1) through 12 (St12). (St1 h) Hindlimb bud (arrow) of a stage 1 embryo. (St2) Fore-

and hindlimb buds of a stage 2 embryo; arrow points to the forelimb bud. For subsequent stages, subscript letters f and h after each stage number correspond to forelimb

and hindlimb, respectively. Arrow in St6f points to the distal constriction seen in the degeneration phase of the forelimb bud. Scale bars = 0.5 mm, except for St4 h and

St9 h = 0.25 mm, and for St12 h = 1.0 mm.

314 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

Fig. 10. Development of the genital region of Calyptommatus sinebrachiatus. (St6) Stage 6 embryo; arrow points to hemipenis bud. (St7♂ and St7♀) Sexual differences between

male (St7♂) and female (St7♀) embryos at stage 7. (St8a and St8b) Stage 8 embryo; arrows point to the cranial (St8a) and caudal (St8b) lips of the cloaca. (St9♂ and St9♀)

Sexual differences between male (St7♂) and female (St7♀) embryos at stage 9. (St10) Well-developed hemipenis in a stage 10 embryo. (St11♂ and St11♀) Sexual differences

between male (St11♂) and female (St11♀) embryos at stage 11; arrow in St11♂ points to the hemipenian spine bud. (St12♂) Hemipenis in a pre-hatching embryo. Scale

bars = 0.5 mm.

with the apoptotic activity modeling the distal extremity and reg- and Ornitz, 2008; Zeller et al., 2009). AER signaling is not neces-

ulating the size of the tail (Hirata and Hall, 2000). sary, though, for limb budding (as reviewed by Stopper and Wagner,

Despite the degeneration of the forelimb bud in C. sinebrachiatus, 2005).

the short development of this bud is sufﬁcient to form a vestigial In chicks, Xenopus frogs, and mice, for example, the AER is a

humerus, which, in the adult, is represented by a small ossiﬁcation thickened layer of apical ectodermal cells at the distal margin of

closely associated to the scapulocoracoid (J.G. Roscito, pers. obs.). the limb between the dorsal and ventral sides. However, organi-

Several experimental studies (Saunders, 1948; Summerbell, 1974; zation of these apical cells into a distinct ridge is not essential

Rowe and Fallon, 1982) revealed that the duration of development for normal AER patterning activity, at least in the frog Eleuthero-

of the limb buds is proportional to the proximo-distal differenti- dactylus (Fang and Elinson, 1996; Richardson et al., 1998; Hanken

ation of mesenchymal cells into the speciﬁc structures that form et al., 2001). Therefore, the absence of a morphologically distinct

the limbs (Tickle, 2006); therefore, any event that interrupts limb AER in the limb buds of C. sinebrachiatus and N. ablephara, as

bud development results in the formation of incomplete limbs. In seen through SEM imaging, does not necessarily imply absence

cases of limb reduction in natural tetrapod populations, the reduc- of signaling function; it is likely that these cells do promote limb

tion/loss of elements occurs in a distal–proximal sequence that is patterning through actively signaling to the mesenchyme under-

the inverse sequence of the order in which these elements develop, neath it, since both fore- and hindlimbs of N. ablephara and the

with the most distal elements being lost ﬁrst (Lande, 1978; Rieppel, hindlimbs of C. sinebrachiatus develop normally. Future histological

1992). and gene expression studies in the limbs are necessary for investi-

Limb formation and patterning is coordinated by several signal- gating the presence and role of AER in limb development in these

ing factors integrated into complex and interdependent networks. animals.

Signaling from the AER coordinates proximo–distal development The truncation of forelimb development in C. sinebrachiatus, on

and is fundamental for promoting mesenchyme cell prolifera- the other hand, may be due to several factors, including absence

tion (Sun et al., 2002) and hence, distal growth of the limb, and of AER signaling. In the lizards A. fragilis, O. apodus and Scelotes

also interacts with other signaling centers, such as the zone of inornatus, limb bud degeneration is suggested to be the result of AER

polarizing activity (ZPA; Riddle et al., 1993; Laufer et al., 1994; degeneration (Raynaud, 1990; Rahmani, 1974; Vasse et al., 1974;

Towers and Tickle, 2009), to establish limb patterning. Deﬁcien- Raynaud et al., 1975). Studies in python snake embryos also showed

cies in these regulatory networks may lead to limb bud reduction that the absence of AER signaling may be the reason for limb bud

or degeneration (Mariani and Martin, 2003; Sun et al., 2002; Yu degeneration (Cohn and Tickle, 1999).

J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318 315

Delimiting criteria for embryonic staging is of great importance animal and egg breeding, allows more reliable comparative anal-

for comparisons between taxa (Andrews, 2004; Werneburg, 2009). yses. Variations in temperature and breeding conditions for

Having a “common language” (Werneburg, 2009) for describing females and eggs must be taken into account when analyzing and

embryonic development, as well as establishing guidelines for determining stages of embryonic development: small changes in

Podarcis Agama Liolaemus Eublepharus Anolis sagrei Paroedura Species Nothobachia Calyptommatus muralis macularius impalearis pictus tenuis tenuis

ablephara sinebrachiatus (Lemus and

(Dhouailly and (Wise et al.,2009) (el Mouden (Sanger et al.,2010) (Noro et al.,2009) Saxod,1974) et al.,2000) Duvauchelle,1966)

o o o o o o o o room Temperature 28-35 C 28-35 C 28 C 28 C ± 1 C 28 C 28 C 28 C temperature Duration of ~45 days ~45 days 46 days 55 days 90 days 52 days in ovo 22-27 days in ovo 60 days in ovo development in ovo in ovo in ovo in ovo inside oviduct

St 25-26

St St 27 26-27 26-27 St1-2 St 27 St 28 St 3 Oviposition St 29 0-1 0-1 St 28 dpo St 28 1 St 29 1dpo St 4-5 2

St 1

3 St 1 3d St 30

3-4

dpo

4 St 2 St 30

5 St 29 St 6 5dpo Days after oviposition

St 3

6 St 31

7 St 2 7d

St 4 St 4 St 8 8 St 9 8dpo

9 St 32

St 10

10 St 5

11 St 3

12 12d St 33

St 31

St 6

13 St 32

14 St 4

15 St 34

St 33

16 St 5 St 7

17 St 34

18 St 35

St 8

19 St 6 19d

35- 36

21 St 9 21d

22 St 7

St 35

24 St 8 St 10

25 25d

26 St 9

27 St 38

38- 39 29

30 St 11

Fig. 11. Comparison between developmental stages established for the lizard species Nothobachia ablephara, Calyptommatus sinebrachiatus, Podarcis muralis, Eublepharus

macularius, Agama impalearis, Anolis sagrei, Paroedura pictus and Liolaemus tenuis tenuis. Incubation temperatures and duration of embryonic development from oviposition

to hatching are indicated, except for L. tenuis tenuis, for which development was described until oviposition only. Stage naming follows the nomenclature used in the original

references. The number of days after oviposition is indicated in the left column, and all stages shown in the comparison are aligned with the respective period in which

they are reported to take place. Boxes colored with the same color indicate overall morphological similarity, inferred from the descriptions and illustrations available in the

original references. Box height corresponds to the duration of each stage, measured in days. Pre-hatching stages were not included in the comparison.

316 J.G. Roscito, M.T. Rodrigues / Zoology 115 (2012) 302–318

temperature can accelerate or slow down embryonic development digit condensations of all other lizards compared; again, this later

(Fowler, 1970; Hubert, 1985; Deeming and Ferguson, 1991); also, appearance could be related to the morphological reduction of the

females under stressful conditions can respond by either retaining limb.

the eggs inside the oviduct or releasing them earlier than normal Comparative analyses are only validated, though, when backed

(J.G.R., pers. obs. for Tropidurus sp.), which would retard or accel- up by measurable data sufﬁcient to support a phylogenetic

erate oviposition, a stage frequently used as departure point for framework, which allows the polarization of developmental trans-

developmental tables (Andrews, 2004). formations and, hence, the establishment of an evolutionary

While some characters, such as the pharyngeal arches and slits, scenario for embryonic modiﬁcations. However, the lack of a care-

show a well conserved developmental pattern, others, such as limb ful character standardization for establishing reptile embryonic

morphology and somite number, show great variation between staging tables makes any attempts to infer phylogenetic meaning

species and also within the same species: different degrees of limb preliminary. Still, the inclusion of the closely related C. sinebrachia-

reduction can be seen among different species, and also within tus and N. ablephara as the only limb-reduced lizards most likely

the same species (different degrees of reduction in the fore- and introduces a bias into the comparisons since it does not exclude the

hindlimbs of C. sinebrachiatus, for example), and intraspeciﬁc vari- possibility of phylogenetic constraints driving the observed simi-

ation in vertebral number (somite number) is frequent (e.g., Greer, larities. Nevertheless, interspeciﬁc comparisons such as this one

1987). are important to provide preliminary data for studies that attempt

Comparisons between developmental stages in different to investigate the developmental patterns of evolutionary transfor-

squamate taxa are somewhat complicated due to the great mor- mations.

phological diversity within the group, which is often the result of An important variable that can affect embryonic development

relative changes in the timing of development of structures (het- is temperature, and although interspeciﬁc comparisons as such are

erochronies; de Beer, 1951; Gould, 1977; Hall, 1984). The limited valuable, it should be noted that the differences in the temper-

number of embryos analyzed in this study does not allow for an atures in which development occurred for each species analyzed

assessment of intraspeciﬁc variability, which would be important might inﬂuence the timing of embryonic development (Hubert,

for establishing heterochronic processes. Also, when establishing 1985; Deeming and Ferguson, 1991; Andrews, 2004): for example,

◦

developmental stages, it is possible that the assessment of character a 4 C rise in incubation temperature for the eggs of Dipsosaurus dor-

variability is neglected by the subjective typiﬁcation of the “stage” salis is responsible for a 27-day acceleration in development (Muth,

(Werneburg, 2009). Nevertheless, interspeciﬁc comparisons 1980). Thus, it is impossible to determine to which extent these dif-

can provide important clues to the evolutionary variation of ferences in temperature may account for differences in the times of

development. oviposition, or for differences in the duration of stages (measured

The data available for embryonic development of other in days in Fig. 11), but still, comparative data are important to direct

lizards was used for a comparison between the development of future investigations.

N. ablephara and C. sinebrachiatus and that of six other lizard Differences in developmental time and rate are frequent among

species (Fig. 11). The elaboration of this comparative chart took into animal species, and, therefore, detailed data on morphological

account descriptions and illustrations of the structures that were modiﬁcations that take place throughout development are essen-

used in the present study to stage the embryonic development of tial for the establishment of developmental patterns and for

the two gymnophthalmids analyzed). The days of development for the analysis of the processes that generate variations in body

which the stages were determined, and the temperature in which morphology. Staging systems are, therefore, a primary and essential

development occurred is shown for all species in the comparison. step for experimental and evolutionary studies.

This comparative table is a preliminary attempt to show the pro- The data presented for the development of the fossorial and

gression of embryonic development, i.e., the development of the limb-reduced lizards C. sinebrachiatus and N. ablephara offers sup-

major morphological features, in the six lizard species included in port for future studies addressing the developmental mechanisms

the analysis in relation to that of C. sinebrachiatus and N. ablephara. underlying the evolutionary processes involved in the evolution of

Features such as pharyngeal arches, slit openings and closures, otic fossorial squamates.

development, heart formation, and others, tend to occur at simi-

lar times and follow a similar developmental progression among Acknowledgments

the lizards analyzed. Differences are reﬂected in the variability in

timing of the development of some characters, including those We thank the Fundac¸ ão de Ampparo à Pesquisa do Estado de

that are less subjected to developmental constraints and, hence, São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento

more likely to undergo morphological modiﬁcations, such as the Cientíﬁco e Tecnológico (CNPq) for funding, and F.F. Curcio, D.

limbs. Pavan, R.M.L. Santos and R.V. Villela for help in the ﬁeld.

With the exception of C. sinebrachiatus and N. ablephara, all

other lizard species analyzed have pentadactyl limbs. In all species, References

limbs are formed in early development, but there is a difference

Andrews, R.M., 2004. Patterns of embryonic development. In: Deeming, D.C. (Ed.),

in the sequence in which the limb buds are formed: in both

Reptilian Incubation Environment Evolution and Behaviour. Nottingham Uni-

gymnophthalmids and in Agama impalearis the hindlimb forms

versity Press, Nottingham, pp. 75–102.

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