Zoology 115 (2012) 289–301 Contents lists available at SciVerse ScienceDirect Zoology journa l homepage: www.elsevier.com/locate/zool 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. © 2012 Elsevier GmbH. All rights reserved. 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). 0944-2006/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.zool.2012.02.004 290 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