Development of the chondrocranium of Ptychoglossus bicolor
In general terms, the development of the skull in Ptychoglossus bicolor follows the pattern observed in other studied lizards (de Beer, 1930; El-Toubi & Kamal, 1959; Kamal & Abdeen, 1972; Skinner, 1973; Jerez, 2007). Initially, at stage 31, some cartilages from the three regions of the chondrocranium are present as well as from the viscerocranium. The elements of the dermatocranium appear later at stage 35. Finally, in the neonate the cranium resembles the adult stage, showing a complete skull without any reduction or loss of cranial elements. The nasal capsule in the ethmoid region of the chondrocranium is complete and complex, the elements of the cartilaginous scaffolding in the orbitotemporal region are connected to each other, and in the occipital region the braincase exhibits the typical conformation in lizards. The dermatocranium is complete, and only shows the fusion of the postorbital and postfrontal bones. Thus, P. bicolor conserves the characteristics of lacertiform terrestrial lizards, which is very interesting as this species exhibits terrestrial to semi-fossorial habits, and can build galleries when put in captivity in a terrarium with soft soil and litter (pers. obs.). However, the development of some chondrocranial structures is different from other lizard species that have been well characterized. These characteristics could be common within Gymnophthalmidae, but similar studies are necessary in other species within this clade.
Below we compare the characteristics of the embryonic development of the chondrocranium of Ptychoglossus bicolor to other lizards whose development of this structure has been studied in depth.
In lizards the basipterygoid processes usually originate either from the pterygoid process of the pterygoquadrate complex, as observed in Trachylepis capensis, or from the posterior ends of the trabeculae, as observed in Lacerta agilis and Acanthodactylus boskianus (de Beer, 1930; Kamal & Abdeen, 1972; Skinner, 1973). Nonetheless, in Ptychoglossus bicolor the basipterygoid processes are formed from the acrochordal cartilage (posterior orbital cartilage). Therefore, at least three different patterns in the development of the basipterygoid processes have been described in Squamata. This is very interesting considering that this structure is always present in the squamate skull and is functionally important for cranial kinesis in protraction and retraction movements (Metzger, 2002).
Furthermore, we find variation with respect to other species in the development of the viscerocranium. In Ptychoglossus bicolor all of the elements of the viscerocranium develop concomitantly with the neurocranium, resembling the condition of the turtle Caretta caretta (Kuratani, 1999). In contrast, in Chalcides ocellatus, Acanthodactylus boskianus and Trachylepis capensis, only Meckel's cartilage and the pterygoquadrate complex develop at the same time as the neurocranium (El-Toubi & Kamal, 1959; Kamal & Abdeen, 1972; Skinner, 1973). More differences are observed in Lacerta vivipara and Calotes versicolor, in which Meckel's cartilage chondrifies much earlier than the neurocranium (de Beer, 1937; Ramaswami, 1946). There is also diversity in the timing of development of the elements of the viscerocranium. In P. bicolor, Chalcides ocellatus and Mabuya sp., the quadrate is separated from the other elements of the pterygoquadrate complex at the earliest stage analyzed (El-Toubi & Kamal, 1959; Jerez, 2007). In contrast, in other lizard taxa like Ptyodactylus hasselquistii, A. boskianus and T. capensis, the quadrate is continuous with the pterygoquadrate complex via the intermediate element from the very first stages analyzed (El-Toubi & Kamal, 1961; Kamal & Abdeen, 1972; Skinner, 1973). This indicates that there are probably some heterochronic factors affecting the ontogenetic formation of the viscerocranium with respect to the chondrocranium.
In regard to the palatoquadrate complex, in Ptychoglossus bicolor the quadrate is separated from the complex at the earliest stage analyzed, and the intermediate element is greatly reduced. This situation is similar to the one observed in Mabuya sp. and Chalcides ocellatus (El-Toubi & Kamal, 1959; Jerez, 2007). In contrast, in Ptyodactylus hasselquistii, Acanthodactylus boskianus and Trachylepis capensis (El-Toubi & Kamal, 1961; Kamal & Abdeen, 1972; Skinner, 1973), the quadrate is continuous with the pterygoquadrate complex via the intermediate element from the first stages analyzed.
Bellairs & Kamal (1981) suggested that generally in lizards the taenia marginalis arises from the back of the planum supraseptale and then elongates to contact the otic capsule. Conversely, de Beer (1930) observed the taenia marginalis projecting rostrally from the otic capsule to the planum supraseptale in Lacerta vivipara. However, in P. bicolor the taenia marginalis originates independently near the planum supraseptale, and then elongates posteriorly and rostrally to contact the otic capsule and the planum supraseptale. This pattern is somewhat similar to Trachylepis capensis (Skinner, 1973). This diversity indicates that there is more than one pattern regarding the development of the taenia marginalis. Further evidence may throw light on the variations of this pattern in other lizards, given that we see such great variation in the morphogenesis of the orbital cartilages within the chondrocranium. This condition becomes relevant when observing burrowing species, i.e. Anguis fragilis, Aniella pulchra and Acontias meleagris (Bellairs & Kamal, 1981), where reductions in the chondrocranium compromise primarily cartilages from the orbitotemporal region.
Bellairs & Kamal (1981) suggested that frequently the cochlear part of the otic capsule is attached to the basal plate in lizards. This is observed in the development of the chondrocranium in Acanthodactylus boskianus and Trachylepis capensis (Kamal & Abdeen, 1972; Skinner, 1973). However, in Ptychoglossus bicolor the otic capsule originates independently, starting with the cochlear portion, which is formed from a chondrification center separated from the basal plate (parachordal). It would be interesting to investigate whether this pattern is observed in other gymnophthalmid species, which would indicate a diagnostic character.
The gymnophthalmid chondrocranium
In general terms, the gymnophthalmids included in this study exhibit a complete chondrocranium without any reduction or loss of elements associated with microhabitat use, unlike burrowing lizards such as Anguis fragilis, Aniella pulchra and Acontias meleagris (Bellairs & Kamal, 1981). Therefore, the gymnophthalmid chondrocranium more resembles the chondrocranium of terrestrial lacertiform lizards. Despite this similarity, the gymnophthalmid lizards also exhibit unique characteristics. First, the nasal capsule is characterized by more developed nasal cartilages like the parietotectal and paranasal cartilages, which widen anteriorly and laterally; second, the presence of a large fenestra superior in the anterior dorsal region; third, the presence of a complete zona annularis formed ventrally by a large lamina transversalis anterior; and fourth, a well-developed cartilage of Jacobson's organ. Additionally, the gymnophthalmid nasal capsule is wider than long, in comparison with Cnemidophorus lemniscatus, which shows a simplified nasal capsule without a fenestra superior similar to Tupinambis (Bellairs & Kamal, 1981). The greater development of the nasal cartilages in gymnophthalmids (see above) is probably related to the well-developed olfactory bulbs and vomeronasal organ. This characteristic might play an important role in exploiting different microhabitats like the leaf litter and the underground, which in turn may have favored the great diversification observed within gymnophthalmids inhabiting the Andean and Amazonian forests. Other chondrocranial features, like the nasal septum, are more developed in gymnophthalmids than in teiids. For example, the rostral process of the nasal septum observed in Cnemidophorus lemniscatus, Ameiva undulata, Teius teyou and Aspidoscelis sexlineata (Malan, 1946; Bellairs & Kamal, 1981) is much more simplified than in gymnophthalmids where it is more developed and is the largest in the fossorial lizard Bachia bicolor (Table 1). The presence of a large rostral process in the snake-like B. bicolor probably has no functional role as it is absent in other snake-like lizards such as Aniella pulchra, Anguis fragilis and Acontias meleagris (Bellairs & Kamal, 1981), although biomechanical studies are necessary to directly test this hypothesis.
Within Gymnophthalmidae, the size of the fenestra olfactoria (advehens and evehens) is generally larger than in Cnemidophorus lemniscatus. This is consistent with the large size of the main and accessory olfactory bulbs of the gymnophthalmid species studied here. The fenestra olfactoria is smallest in Echinosaura horrida, due to the comparatively smaller olfactory bulbs in this species than in the other gymnophthalmid taxa. Scleroglossans are generally active foragers, which use a combination of vision and vomerolfaction (Cooper, 1994; Vitt & Pianka, 2005). In addition to the gymnophthalmids, this group includes lacertids, scincids, anguids, helodermatids and varanids, which all show a medium to large fenestra olfactoria and probably exhibit well-developed olfactory bulbs (Bellairs & Kamal, 1981; Schwenk, 1993; Bernstein, 1999). In contrast, ambush-foraging iguanians like chamaeleonids, polychrotids, iguanids and agamids generally use visual detection to locate prey (Cooper, 1994; Vitt & Pianka, 2005). As a consequence, the sensory olfactory apparatus in this latter group is poorly developed and the chondrocranium exhibits a fenestra olfactoria that is comparatively very small or absent (Bellairs & Kamal, 1981).
In the orbitotemporal region of the gymnophthalmid chondrocranium, we find some generalized features as well. The planum supraseptale is very thin anteriorly and posteriorly it is wide, forming a shield. This condition is also observed in C. lemniscatus and in scincids (Bellairs & Kamal, 1981). There are reductions of some orbitotemporal cartilages in Bachia bicolor including a very thin planum supraseptale, and the absence of the sphenethmoid commissures and interorbital septum. Similar reductions are observed in other serpentiform lizards like Anguis fragilis, Aniella pulchra and Acontias meleagris (Bellairs & Kamal, 1981).
The orbitosphenoid of Ptychoglossus bicolor is formed by endochondral ossification and begins to ossify during late embryonic development (Stage 40). This process starts with the ossification of the anterior surface of the pila metoptica and then extends into a portion of the taenia medialis and pila accesoria, forming a small triradiate bone. The orbitosphenoid of Bachia bicolor, Elgaria coerulea, Liolaemus scapularis and L. quilmes also appears at late embryonic stages (Good, 1995; Lobo et al. 1995; Abdala et al. 1997; Jerez, 2007; Tarazona & Ramírez-Pinilla, 2008).
There is significant variation concerning the orbitotemporal cartilages involved in the development of the orbitosphenoid. For instance, the orbitosphenoid is formed by ossification of the pila metoptica in some terrestrial lizards such as Trachylepis capensis, Liolaemus scapularis, Stenocercus guentheri and Mabuya sp. (de Beer, 1937; Skinner, 1973; Lobo et al. 1995; Torres-Carvajal, 2003; Jerez, 2007). Within the gymnophthalmid species studied here (Table 1; traits 24, 28 and 37), there is great variation in the orbitosphenoid and we observed four different patterns of development.
In the first type, the orbitosphenoid is formed by ossification of the anterior surface of the pila metoptica, and then extends into a portion of the taenia medialis and pila accesoria, forming a small triradiate bone. This is observed in 50% of the species studied here, including Ptychoglossus bicolor, P. vallensis, P. festae, Alopoglossus copii, Anadia ocellata, A. bogotensis, Cercosaura argulus and Cnemidophorus lemniscatus. This has also been observed in the fossorial snake-like gymnophthalmids Nothobachia ablephara and Scriptosaura catimbau (Roscito & Rodrigues, 2010). The fact that this condition is observed in the three Ptychoglossus species, Alopoglossus copii and C. lemniscatus might indicate that this feature is observed within Gymnophthalmidae due to common ancestry and that it evolved independently in different clades like Anadia. This pattern of development is probably not associated with ecology due to the fact that these species are generally found inhabiting the ground, leaf-litter, buried and under or in rotting logs, while at the same time arboreal taxa like Anadia ocellata exhibit the same pattern (Oftedal, 1974; Castro-Herrera et al. 2007; Anaya-Rojas et al. 2010; Layche et al. 2010).
On the other hand, 40% of the taxa showed a second pattern in which the orbitosphenoid forms by ossification of a portion of the pila metoptica and taenia medialis. These species include Cercosaura ampuedae, Echinosaura horrida, Riama striata, Pholidobolus montium, Leposoma rugiceps, Leposoma southi, Iphisa elegans and Tretioscincus bifasciatus. These terrestrial species generally inhabit the leaf-litter and some exploit semi-aquatic microhabitats (Ruthven & Gaige, 1924; Avila-Pires, 1995; Ortega-Andrade, 2006; pers. obs.). It would be interesting to evaluate whether the condition of the orbitosphenoid in these species is related to their life habits, as they belong to non-basal groups within gymnophthalmids.
A third type of orbitosphenoid formation corresponds to the condition of the orbitosphenoid in Vanzosaura rubricauda, which is formed only by the ossification of the taenia medialis (Guerra & Montero, 2009). This condition is interesting as this species is widely distributed in open areas within various habitats and substrates. Nevertheless, because only a few gymnophthalmid species have been studied, it remains unclear whether this condition in Vanzosaura rubricauda is common or rare in this family.
The last type of orbitosphenoid development we observed was found in the fossorial snake-like Bachia bicolor. In this species the orbitosphenoid is markedly broader than that of all other gymnophthalmids studied, as it involves the ossification of the taenia medialis and pila metoptica, as well as two–three additional orbitotemporal cartilages. These latter cartilages are hardly comparable to orbitotemporal cartilages such as the pila accesoria and pila antotica (Tarazona & Ramírez-Pinilla, 2008). The broad orbitosphenoid of B. bicolor resembles both structurally and ontogenetically the orbitosphenoid of amphisbaenians, and thus it has been hypothesized that the morphology and origin of this bone is related to fossorial habits (Tarazona & Ramírez-Pinilla, 2008). Nevertheless, the orbitosphenoid of other snake-like fossorial gymnophthalmids such as Scriptosaura catimbau and Nothobachia ablephara resembles that in Ptychoglossus bicolor. This is probably because these two species are fossorial but show adaptations to life in sand (Roscito & Rodrigues, 2010).
The condition of the pila antotica within Gymnophthalmidae is also variable. This cartilage can be long or short, and it is always separated from the basal plate. However, it may also be absent, as in Bachia bicolor and Ptychoglossus bicolor. In the latter species, the pila antotica is absent even in early stages of development. This cartilaginous bar is also absent in Plestiodon fasciatus, Cordylus sp., Acontias meleagris, Bradypodion pumilum, Anguis fragilis, Aniella pulchra, Agama mutabilis and Ptyodactylus hasselquistii (Rice, 1920; Brock, 1941; Van-Pletzen, 1946; Bellairs & Kamal, 1981). Furthermore, the pila antotica may appear early during embryonic development and disappear in later stages, as is the case in Plestiodon latiscutatus (Rice, 1920). This situation indicates that the condition of the pila antotica is variable among groups and is subject to change during ontogeny.
Ossification sequence of Ptychoglossus bicolor
The ossification of the skull of Ptychoglossus bicolor consists of two different processes: the development of the dermatocranium and the ossification of the chondrocranium. In Ptychoglossus bicolor the pterygoid is the first or among the first bones to ossify in the skull. This occurs also in Trachylepis capensis, Liolaemus scapularis, L. quilmes, Tupinambis rufescens, T. merianae, Lacerta agilis, Elgaria coerulea and Liopholis whitii (Skinner, 1973; Rieppel, 1994; Good, 1995; Lobo et al. 1995; Abdala et al. 1997; Arias & Lobo, 2006; Hugi et al. 2010). On the other hand, the lacrimal, postorbital and postfrontal bones are among the last elements of the dermatocranium to start to ossify during embryonic development, similar to the condition in Tupinambis merianae, T. rufescens, Liolaemus scapularis and L. quilmes (Lobo et al. 1995; Abdala et al. 1997; Arias & Lobo, 2006). The last dermal bones to fully differentiate are generally those from the frontoparietal region, which remain incompletely differentiated in the neonate (Maisano, 2001; Jerez, 2007; Tarazona et al. 2008). This suggests that the sequence of onset and termination of the ossification of the dermatocranium is somewhat conserved among the lizards studied so far. In P. bicolor the postorbitofrontal ossifies as a single bone. This is also the case in the gymnophthalmid Calyptommatus nicterus (Roscito & Rodrigues, 2010), whereas in other species like Euspondylus acutirostris, Potamites ecpleopus, Vanzosaura rubricauda, Nothobachia ablephara and Scriptosaura catimbau, the postfrontal and postorbital bones are distinct (Montero et al. 2002; Bell et al. 2003; Guerra & Montero, 2009; Roscito & Rodrigues, 2010).
The size of the frontoparietal fontanelle in the neonate lizard is variable, and depends upon the differentiation of the frontals and parietals. Thus, this fenestra may be large, small or closed at the time of birth (Maisano, 2001). Herein, we found that a large frontoparietal fontanelle was characteristic of Ptychoglossus bicolor neonates. This condition is similar to the neonate skull of Bachia bicolor (Tarazona et al. 2008). However, in Potamites ecpleopus the ossification of the frontals and parietals is more advanced (Maisano, 2001). Within Scleroglossa, the size of the neonate frontoparietal fontanelle varies from small to large, and closes early during postnatal ontogeny, unlike in Iguania where the frontoparietal fenestra closes in adult stages (Maisano, 2001; Torres-Carvajal, 2003). Particularly, the neonates of serpentiform burrowing species (Acontias meleagris, Aniella pulchra and Bipes biporus) exhibit a fully differentiated skull roof, apparently without any traces of the frontoparietal fontanelle (Bellairs & Kamal, 1981; Maisano, 2001; Torres-Carvajal, 2003). In this sense, the accelerated ossification of the dermatocranium in specialized burrowers is characteristic of scleroglossans, and represents an association between variation in body form at the cranial level and microhabitat use (Bellairs & Kamal, 1981). Nonetheless, this is not the pattern observed in Ptychoglossus bicolor and the fossorial snake-like gymnophthalmids Calyptommatus nicterus, Scriptosaura catimbau and Nothobachia ablephara (Tarazona et al. 2008; Roscito & Rodrigues, 2010). This may be due to the fact that these snake-like gymnophthalmid species inhabit soft sandy soils, and exhibit a small and complete skull, without drastic transformations.