Recently Daza and Bauer (2010) reviewed the pattern and occurrence of the circumorbital series of bones in Gekkotans, recognizing five nominal elements in this cluster (prefrontal, postfrontal, postorbital, jugal, and lacrimal). They noted that in lepidosaurs, this general pattern of circumorbital elements is variously modified by integration with the tooth-bearing maxilla and the longitudinal and dorsally situated frontal and by the variable presence of neomorphic elements (palpebrals, supraorbital and parafrontals). Of the circumorbital elements proper, Daza and Bauer (2010) reported that only the prefrontal is invariable in its participation in the orbital margin.
The orbit of gekkotans can be disproportionately large, when compared with that of other squamates, and it is always incomplete posteriorly (the outcome of a reduction in relative size and shape of the postorbital and the jugal) and is confluent with the single opening resulting from the confluence of the supratemporal and infratemporal fenestrae. These traits have resulted in major changes in both the configuration and presence of elements of the circumorbital series.
Daza and Bauer (2010) not only paid particular attention to the determination of the presence of the lacrimal and jugal in gekkotans but also commented on the prefrontal and the putatively combined postfrontal and postorbital, which they argued is primitively present in the Gekkota as the postorbitofrontal. It was reported by Daza and Bauer (2010) that the postfrontal and postorbital apparently fuse to form a single element in some members of all lizard clades (Fig. 1B), a tendency also noted by Conrad (2008). This differs from the primitive situation in which a separate postfrontal and postorbital occur in the dorsal rim of the orbit (Fig. 1A). In the latter case the postfrontal contacts the frontal and parietal and bridges the suture between these bones, and the postorbital contacts the postfrontal but is excluded from contact with the frontal and parietal by the intervening postfrontal (consistent with the condition figured for Elgaria coerulea by Maisano, 2001). Daza and Bauer's (2010) conclusion about the identity of the single element that subtends and spans the frontoparietal suture in the dorsal rim of the orbit was that it is the result of such fusion (Daza and Bauer, 2010), in accordance with an earlier proposal of Daza et al. (2008). They identified this putatively combined element as the postorbitofrontal, adopting the most conservative interpretation because it does not necessitate the loss of either element. Their interpretation was guided primarily by the observation that basal members of the Gekkonomorpha posses both elements (Conrad and Norell, 2006), and additionally on statements such as that of Siebenrock (1895) that the postorbital is absorbed by the postfrontal. Empirical evidence for such fusion is, however, rather slender.
Daza and Bauer (2010) surveyed 105 species of gekkotans and reported a single element lying at the posterodorsal corner of the orbit, clasping the frontoparietal suture, in all species except Lygodactylus that they examined. This element is usually triangular in outline, with a laterally oriented vertex, but some taxa depart in modest ways from this general configuration. Contact of this element with the prefrontal occurs only in Phelsuma. In some pygopodids, this putative postorbitofrontal is perforated by one or two foramina (Stephenson, 1962; Kluge, 1976).
Evans (2008) had previously postulated that the single ossification bridging the frontoparietal suture in limbed gekkotans is the postfrontal (Fig. 1C). This accords with the interpretation given by El-Toubi and Kamal (1961), who posited that the postorbital is absent. Evans (2008) indicated that there is a compound bone only in pygopodids that results from fusion of the postfrontal and postorbital (Fig. 1B). This would imply the absence of postorbital in limbed gekkotans. Reasons for interpretation of Evans (2008) were that two bones are present in this location in one species of the pygopodid genus Lialis, as reported by Rieppel (1984), and that the presence of one or more perforations in the single element found in some species of Delma, Lialis, and Pygopus belies evidence of fusion only in the pygopods. However, Daza and Bauer (2010) argued that because pygopodids are nested within limbed gekkotans, the likely primitive state for the Gekkota in its entirety is the presence of a fused postfrontal and postorbital, resulting in the presence of a postorbitofrontal (Fig. 1B).
However, there is no direct developmental evidence available for the testing of these two competing hypotheses, each of which is based on extrapolation from observed adult morphology that is interpreted in different ways in a broader systematic context. Evans' (2008) hypothesis leads to the prediction that in development only a postfrontal element (Fig. 1C), represented by a single center of ossification, should appear in limbed gekkotans and that either there should be no evidence of the presence of a postorbital or that the latter will make only a transient appearance and then degenerate. Conversely, the hypothesis of Daza and Bauer (2010) leads to the prediction that in all gekkotans ossification centers for the postfrontal and postorbital should occur, with these centers later coalescing into a single unit. In both instances, the outcome will yield a single element that bridges the suture between the frontal and the parietal, with the contact being made either by the postfrontal alone or by the base of the postfrontal to which the postorbital has become fused in a lateral location, not likely participating in the contact with the frontal and parietal.
Our investigation of the sequence and pattern of ossification of cranial elements in the eublepharid gekkotan Eublepharis macularius enables us to report on ossification events in the dorsal circumorbital region at a critical juncture in development and allows us to address the predictions consistent with the hypotheses of Evans (2008) and Daza and Bauer (2010).
MATERIALS AND METHODS
Our data are based on 32 embryos that encompass developmental Stages 34–42 (Wise et al., 2009) of the Leopard gecko (Eublepharis macularius). These embryos were cleared and stained using methods modified from those of Filipski and Wilson (1985), staining only with Alizarin red S (to enable detection of ossification centers in dermal elements and the tracing of their development through subsequent stages). An additional series of double-stained embryos was prepared, so we are able to confirm that none of the elements reported here display any presence of cartilage or its precursors. All elements were initially examined in situ. However, for detailed examination of the skeletal structures in question, the head skeleton of each embryo was gently teased apart to isolate individual elements, permitting an unobstructed view of their anatomy.
Observations were made using a Nikon SZ800 dissecting microscope. Images were taken using a Nikon D200 Camera and were cropped and sized in Adobe Photoshop Version 9.0.2. Additionally, the images were refined using the sharpen filter and auto levels commands in Photoshop.
An ossification center occupying the classical position of a postfrontal (Fig. 1A) (Conrad, 2008; Daza and Bauer, 2010) first appears as a small triangular center in late Stage 35 (Fig. 2A). The base of this triangle is oriented medially and straddles the future suture between the frontal and parietal. It continues to enlarge throughout Stages 35 and 36 (Fig. 2A,B). By Stage 37 (Fig. 2C) an anterior process that ultimately becomes the part of the element that borders the frontal is evident. Both the triangle and anterior process continue to grow throughout Stage 38 (Fig. 2D–F), and by mid-Stage 38 (Fig. 2E), its lateral and caudal processes begin to extend. By late Stage 38, its anterior process is complete in terms of its adult proportional length, and ossification of its lateral process has extended, producing a spike (Fig. 2F), yielding a triradiate element. Stages 39 and 40 witness an increase in size without any concomitant change in form (Fig. 2G,H). In Stage 41 (not illustrated), the posterior process has widened to achieve its definitive shape. By late Stage 42 (Fig. 2I), the anterior process of the postorbital is still pointed and spike-like and has not yet gained the wide, blunt form of mature specimens.
Lying slightly anterior to the aforementioned element, but still separating its rostral portion from contact with the frontal, another ossification center that occupies a position anterior to the triangular element lying alongside the frontal as described above. This, however, only has a transient independent existence. It is first observed late in Stage 36 (Fig. 3A) as a thin splint lying lateral to the dorsolateral edge of the bridge of the frontals. By early Stage 38 (Fig. 3B), this element is still separated largely from the frontal, but by mid-Stage 38 (Fig. 3C), its caudal end is extensively ossified and is connected to the dorsolateral edge of the frontal, although its midsection and rostral end still remain free. From the dorsolateral edge of the caudal end of the frontal, a progressive expansion of ossification begins that extends anteriorly to meet the caudal end of the lateral splint. The dorsolateral edge of the caudal end of the frontal takes the form of the barb of a fish-hook. Throughout these developmental stages, elongation of the supraorbital splint continues, so that by Stage 39, it excludes the frontal from the orbital margin and now comes to lie between the prefrontal and the frontal as well as between the triangular element described above, and the frontal (Fig. 3D). Coalescence continues, progressing from caudal to rostral, resulting in the fusion of the supraorbital splint to the dorsolateral edge of the frontal. By Stage 39 (Fig. 3D), fusion to the frontal is almost complete, except for the rostralmost portion. The prior presence of the once-independent splint is still evident, however, as a thin strut along the dorsolateral edge of the frontal. By Stage 40, complete fusion between the splint and the frontal has occurred (Fig. 3E).
Our observations accord in part with the predictions derived from the hypotheses of both Evans (2008) and Daza and Bauer (2010). The former predicts that the postfrontal should appear as a single center and remain as a single center throughout development, and that the postorbital will either not appear or will appear and then lose its individual identity (but will not fuse to the postfrontal). This is partly borne out. The element regarded as the postfrontal by Evans (2008) appears and develops as a single center, but actually has all of the morphological characteristics of the primitive gekkotan postorbital (Conrad and Norell, 2006) (Fig. 2) but lying in a more dorsomedial position, bounding the frontoparietal suture. An additional element, however, lying anterior to this appears, but quickly fuses to the lateral edge of the frontal in the dorsal rim of the orbit (Fig. 3). This element lies more anterior than the location of the primitive gekkotan position for the postfrontal (see illustrations in Conrad and Norell, 2006), and lacks the lateral process of that element, but still separates the rostral portion of the postorbital from contact with the frontal. We advocate that it is more parsimonious to propose an anterior shift of an existing element than the appearance of a de novo structure and the loss of another. Here, the postfrontal and the postorbital maintain a close position relative to one another, but with the loss of the postorbital bar and upper temporal arch, characteristic of more derived gekkotans, there is a dorsomedial and rostral shift with respect to their relationship to the less labile elements comprising the skull roofing bones, namely the frontal and the parietal.
Predictions resulting from Daza and Bauer's (2010) hypothesis indicate that ossification centers for the postfrontal and postorbital should both appear and then should fuse to form a composite postorbitofrontal. Again these are partially borne out. Two elements do appear (Figs. 2 and 3), but they do not fuse to each other, and they do not exhibit the primitive relationship of the postorbital to the postfrontal (Fig. 1A), but rather lie in series. The splint-like element unites with the frontal (Fig. 3), and the triangular element persists as a robust independent bone, clasping the frontoparietal suture as well as fulfilling all of the criteria for recognition as the postfrontal (Fig. 2), if it was present alone, rather than fused postorbitofrontal.
Our results cannot fully resolve the competing hypothesis as they relate to the Gekkota in its entirety because the Eublepharidae is the sister taxon to Sphaerodactylidae + [Gekkonidae + Phyllodactylidae], whereas the Diplodactylidae + [Carphodactylidae + Pygopodidae] constitutes the sister taxon to that cluster (Gamble et al., 2008). Thus, the condition advocated for the pygopods of a fused postfrontal and postorbital could still pertain and could also be a characteristic of the Diplodactylidae and the Carphodactylidae. However, the eublepharids are basal to the sphaerodactylids, gekkonids, and phyllodactylids (Daza and Bauer, 2010: Fig. 2) and, thus, can be hypothesized to exhibit a condition characteristic of this cluster, especially as it appears to represent a situation somewhat similar to that of basal lepidosauromorphs (Daza and Bauer, 2010: Figs. 1A,2) and some primitive gekkonomorphs (Conrad and Norell, 2006), with the exception that the anterior element is fused to the frontal rather than being free, a condition also noted to occur in some iguanids (Norell, 1989). Eublepharids retain many primitive features among gekkotans (Kluge, 1962; Daza, personal communication); thus the dorsal circumorbital bones could represent another example of such expression. The developmental pattern exhibited by Eublepharis, however, places in doubt the validity of transferring the trait of a compound postorbitofrontal to encompass all of the Gekkota (Daza and Bauer, 2010).
As a result of our observations, three alternative hypotheses can be erected. The first posits that both the postfrontal and postorbital elements are present but their anatomical relationships have changed, the postfrontal undergoing reduction in size and an anterior shift to lie alongside the frontal and to contact the prefrontal, with the postorbital being retained (as per Rieppel, 1992) and extending medially to contact the frontoparietal suture (Fig. 1D). Because this necessitates the recognition of no additional elements, and because we found no evidence of fusion between developmental centers representing the postfrontal and postorbital, we favor this interpretation.
Alternatively the splint-like element that abuts, and later fuses with, the frontal could represent a de novo ossification that has no homolog in other squamates. This would then leave the triangular element to be identified as the postfrontal (because of its anatomical relationships) and the postorbital would be absent.
The third alternative would be to recognize the splint-like element that fuses with the frontal as a de novo element and the triangular element that articulates with the frontal and parietal as a combined postorbitofrontal (Fig. 1B, in part), but there is no developmental evidence to support this.
Thus, our preferred interpretation (Fig. 1D) requires only a shift in position of elements already known to be present primitively in the Squamata (Indeed, this could also be interpreted as the retention of the primitive location of the postfrontal and an anterior shift of the postorbital—the blue and yellow color coding on Fig. 1D would be switched to depict this). The second interpretation outlined above requires the additional developmental events of a de novo ossification (the splint-like element) and the loss of an element (the postorbital). The third alternative would require the same de novo addition and the fusion of two elements for which there is no developmental evidence.
The five developmental stages (range, 36–40) over which the initiation of ossification of the postfrontal (our preferred interpretation—see above) and its subsequent fusion to the frontal occur cover a time span that can vary from 8 to 14 days (Wise et al., 2009). Dense sampling of embryos over these developmental stages permitted the observation of critical but transient events.
The phylogenetic implications of these data may be more far reaching within the Squamata, because detailed information about potentially transient events in skull development are scarce. The widespread recognition of a postorbitofrontal in most squamate lineages may hinge upon assumptions about fusion of elements (Bellairs and Kamal, 1981) for which there is little evidence. This sporadic occurrence of putative fusion of the postorbital and postfrontal is homoplasious, and thus convergent. If the pattern observed in Eublepharis is more widespread in its distribution, then alternate explanations would pertain (although these may not reduce the incidence of homoplasy).
We echo the recommendations of Daza and Bauer (2010) that particular attention should be given to patterns of ossification in this cranial region across the Squamata. Questions of homology can only be resolved through the acquisition of details of embryonic development gained from densely sampled appropriate periods of prehatching development.