THE ANATOMICAL RECORD 293:2001–2006 (2010)

Development of the DorsalCircumorbital Bones in the Leopard

Gecko (Eublepharis macularius) and ItsBearing on the Homology of These

Elements in the GekkotaPATRICK ARTHUR DAVID WISE* AND ANTHONY PATRICK RUSSELLDepartment of Biological Sciences, University of Calgary, Calgary, Alberta,

Canada T2N 1N4

ABSTRACTFive nominal elements comprise the circumorbital series of bones in

gekkotans: prefrontal, postfrontal, postorbital, jugal, and lacrimal. Deter-mination of the homology of two of these, the postfrontal and postorbital,has been particularly problematic. Two conflicting hypothesis exist relatingto these: either the postorbital is lost and the postfrontal remains or theyfuse during development to form a combined element, the postorbitofron-tal. Such a combined element apparently occurs in at least some membersof all lizard clades. There is, however, no direct developmental evidencethat supports either theory. To overcome that, we investigate the sequenceand pattern of ossification in the circumorbital region in a developmentalseries of the Leopard gecko. We posit that both the postfrontal and postor-bital appear during development. Contrary to previous predictions theyneither fuses to each other, nor do either degenerate. Instead, the postfron-tal shifts anteriorly and fuses with the frontal to become indistinguishablefrom it by the time of hatching, and the postorbital persists as a robust in-dependent element bounding the frontoparietal suture. These observationsaccord, in part, with both hypotheses of homology of these elements andresult in the recognition of a new pattern, placing in doubt the existence ofthe composite postorbitofrontal. The phylogenetic implications of thesefindings may prove to be far reaching if similar and conserved patterns ofdevelopment are encountered in other clades. Anat Rec, 293:2001–2006,2010. VVC 2010 Wiley-Liss, Inc.

Keywords: Eublepharis macularius; homology; circumorbitalbones; postfrontal; postorbital; postorbitofrontal

Recently Daza and Bauer (2010) reviewed the patternand occurrence of the circumorbital series of bones inGekkotans, recognizing five nominal elements in thiscluster (prefrontal, postfrontal, postorbital, jugal, andlacrimal). They noted that in lepidosaurs, this generalpattern of circumorbital elements is variously modifiedby integration with the tooth-bearing maxilla and thelongitudinal and dorsally situated frontal and by thevariable presence of neomorphic elements (palpebrals,supraorbital and parafrontals). Of the circumorbital ele-ments proper, Daza and Bauer (2010) reported that onlythe prefrontal is invariable in its participation in the or-bital margin.

The orbit of gekkotans can be disproportionately large,when compared with that of other squamates, and it isalways incomplete posteriorly (the outcome of a reduc-tion in relative size and shape of the postorbital and the

*Correspondence to: Patrick Arthur David Wise, Departmentof Biological Sciences University of Calgary, 2500 UniversityDr. N.W. Calgary, AB Canada T2N 1N4. Fax: 403-289-9311.E-mail: padwise@ucalgary.ca

Received 6 May 2010; Accepted 26 August 2010

DOI 10.1002/ar.21277Published online 2 November 2010 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2010 WILEY-LISS, INC.

jugal) and is confluent with the single opening resultingfrom the confluence of the supratemporal and infratem-poral fenestrae. These traits have resulted in majorchanges in both the configuration and presence of ele-ments of the circumorbital series.

Daza and Bauer (2010) not only paid particular atten-tion to the determination of the presence of the lacrimaland jugal in gekkotans but also commented on the pre-frontal and the putatively combined postfrontal andpostorbital, which they argued is primitively present inthe Gekkota as the postorbitofrontal. It was reported byDaza and Bauer (2010) that the postfrontal and postorbi-tal apparently fuse to form a single element in somemembers of all lizard clades (Fig. 1B), a tendency alsonoted by Conrad (2008). This differs from the primitivesituation in which a separate postfrontal and postorbitaloccur in the dorsal rim of the orbit (Fig. 1A). In the lat-ter case the postfrontal contacts the frontal and parietaland bridges the suture between these bones, and thepostorbital contacts the postfrontal but is excluded fromcontact with the frontal and parietal by the interveningpostfrontal (consistent with the condition figured forElgaria coerulea by Maisano, 2001). Daza and Bauer’s(2010) conclusion about the identity of the single elementthat subtends and spans the frontoparietal suture in thedorsal rim of the orbit was that it is the result of suchfusion (Daza and Bauer, 2010), in accordance with an ear-lier proposal of Daza et al. (2008). They identified thisputatively combined element as the postorbitofrontal,adopting the most conservative interpretation because itdoes not necessitate the loss of either element. Theirinterpretation was guided primarily by the observationthat basal members of the Gekkonomorpha posses bothelements (Conrad and Norell, 2006), and additionally onstatements such as that of Siebenrock (1895) that thepostorbital is absorbed by the postfrontal. Empirical evi-dence for such fusion is, however, rather slender.

Daza and Bauer (2010) surveyed 105 species of gekko-tans and reported a single element lying at the postero-dorsal corner of the orbit, clasping the frontoparietalsuture, in all species except Lygodactylus that theyexamined. This element is usually triangular in outline,with a laterally oriented vertex, but some taxa depart inmodest ways from this general configuration. Contact ofthis element with the prefrontal occurs only in Phel-suma. In some pygopodids, this putative postorbitofron-

tal is perforated by one or two foramina (Stephenson,1962; Kluge, 1976).

Evans (2008) had previously postulated that the singleossification bridging the frontoparietal suture in limbedgekkotans is the postfrontal (Fig. 1C). This accords withthe 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 pygopo-dids that results from fusion of the postfrontal andpostorbital (Fig. 1B). This would imply the absence ofpostorbital in limbed gekkotans. Reasons for interpreta-tion of Evans (2008) were that two bones are present inthis location in one species of the pygopodid genus Lia-lis, as reported by Rieppel (1984), and that the presenceof one or more perforations in the single element foundin some species of Delma, Lialis, and Pygopus belies evi-dence of fusion only in the pygopods. However, Daza andBauer (2010) argued that because pygopodids are nestedwithin limbed gekkotans, the likely primitive state forthe Gekkota in its entirety is the presence of a fusedpostfrontal and postorbital, resulting in the presence ofa postorbitofrontal (Fig. 1B).

However, there is no direct developmental evidenceavailable for the testing of these two competing hypothe-ses, each of which is based on extrapolation fromobserved adult morphology that is interpreted in differ-ent ways in a broader systematic context. Evans’ (2008)hypothesis leads to the prediction that in developmentonly a postfrontal element (Fig. 1C), represented by a sin-gle center of ossification, should appear in limbed gekko-tans and that either there should be no evidence of thepresence of a postorbital or that the latter will make onlya transient appearance and then degenerate. Conversely,the hypothesis of Daza and Bauer (2010) leads to the pre-diction that in all gekkotans ossification centers for thepostfrontal and postorbital should occur, with these cen-ters later coalescing into a single unit. In both instances,the outcome will yield a single element that bridges thesuture between the frontal and the parietal, with the con-tact being made either by the postfrontal alone or by thebase of the postfrontal to which the postorbital hasbecome fused in a lateral location, not likely participatingin the contact with the frontal and parietal.

Our investigation of the sequence and pattern of ossifi-cation of cranial elements in the eublepharid gekkotanEublepharis macularius enables us to report on

Fig. 1. Schematic representation of element identity and location inthe dorsal circumorbital region of lepidosauromorphs in left lateralview. Blocks indicate relative placement and proportions, and translu-cency indicates element overlap. Color coding of elements follows theconvention used by Daza and Bauer (2010): orange, frontal; white, pa-

rietal; yellow, postfrontal; blue, postorbital; green, postorbitofrontal.Conditions represented: A: basal pattern as seen in Sphenodon; B:the gekkotan pattern as advocated by Daza and Bauer (2010); C: thegekkotan pattern as proposed by Evans (2008); D: the pattern dis-played by Eublepharis macularius, this study.

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ossification events in the dorsal circumorbital region ata critical juncture in development and allows us toaddress the predictions consistent with the hypothesesof Evans (2008) and Daza and Bauer (2010).

MATERIALS AND METHODS

Our data are based on 32 embryos that encompass de-velopmental Stages 34–42 (Wise et al., 2009) of the Leop-ard gecko (Eublepharis macularius). These embryos werecleared and stained using methods modified from those ofFilipski and Wilson (1985), staining only with Alizarin redS (to enable detection of ossification centers in dermal ele-ments and the tracing of their development through sub-sequent stages). An additional series of double-stainedembryos was prepared, so we are able to confirm that noneof the elements reported here display any presence of car-tilage or its precursors. All elements were initially exam-ined in situ. However, for detailed examination of theskeletal structures in question, the head skeleton of eachembryo was gently teased apart to isolate individual ele-ments, permitting an unobstructed view of their anatomy.

Observations were made using a Nikon SZ800 dissect-ing microscope. Images were taken using a Nikon D200Camera and were cropped and sized in Adobe PhotoshopVersion 9.0.2. Additionally, the images were refined usingthe sharpen filter and auto levels commands in Photoshop.

RESULTS

An ossification center occupying the classical positionof a postfrontal (Fig. 1A) (Conrad, 2008; Daza andBauer, 2010) first appears as a small triangular centerin late Stage 35 (Fig. 2A). The base of this triangle isoriented medially and straddles the future suturebetween the frontal and parietal. It continues to enlargethroughout Stages 35 and 36 (Fig. 2A,B). By Stage 37(Fig. 2C) an anterior process that ultimately becomesthe part of the element that borders the frontal is evi-dent. Both the triangle and anterior process continue togrow throughout Stage 38 (Fig. 2D–F), and by mid-Stage38 (Fig. 2E), its lateral and caudal processes begin toextend. By late Stage 38, its anterior process is completein terms of its adult proportional length, and ossificationof its lateral process has extended, producing a spike(Fig. 2F), yielding a triradiate element. Stages 39 and 40witness an increase in size without any concomitantchange in form (Fig. 2G,H). In Stage 41 (not illustrated),the posterior process has widened to achieve its definitiveshape. By late Stage 42 (Fig. 2I), the anterior process ofthe postorbital is still pointed and spike-like and has notyet gained the wide, blunt form of mature specimens.

Lying slightly anterior to the aforementioned element,but still separating its rostral portion from contact withthe frontal, another ossification center that occupies aposition anterior to the triangular element lying

Fig. 2. The right postorbital of Eublepharis macularius in dorsolateral view, from the initiation of ossifi-cation in Stage 35 (A), through Stages 36 (B), 37 (C), 38 (D–F), 39 (G), 40 (H), and 42 (I) (staging accordingto Wise et al., 2009). Abbreviations: ap, anterior process; fprs, frontoparietal suture; lp, lateral process;par, parietal; po, postorbital; pp, posterior process. Scale bar in each panel is 1 mm.

EUBLEPHARIS CIRCUMORBITAL BONES 2003

alongside the frontal as described above. This, however,only has a transient independent existence. It is firstobserved late in Stage 36 (Fig. 3A) as a thin splint lyinglateral to the dorsolateral edge of the bridge of the fron-tals. By early Stage 38 (Fig. 3B), this element is still sep-arated largely from the frontal, but by mid-Stage 38 (Fig.3C), its caudal end is extensively ossified and is con-nected to the dorsolateral edge of the frontal, although itsmidsection and rostral end still remain free. From thedorsolateral edge of the caudal end of the frontal, a pro-gressive expansion of ossification begins that extendsanteriorly to meet the caudal end of the lateral splint.The dorsolateral edge of the caudal end of the frontaltakes the form of the barb of a fish-hook. Throughoutthese developmental stages, elongation of the supraorbi-tal splint continues, so that by Stage 39, it excludes thefrontal from the orbital margin and now comes to liebetween the prefrontal and the frontal as well as betweenthe triangular element described above, and the frontal(Fig. 3D). Coalescence continues, progressing from caudalto rostral, resulting in the fusion of the supraorbitalsplint to the dorsolateral edge of the frontal. By Stage 39(Fig. 3D), fusion to the frontal is almost complete, exceptfor the rostralmost portion. The prior presence of theonce-independent splint is still evident, however, as athin strut along the dorsolateral edge of the frontal. ByStage 40, complete fusion between the splint and thefrontal has occurred (Fig. 3E).

DISCUSSION

Our observations accord in part with the predictionsderived from the hypotheses of both Evans (2008) andDaza and Bauer (2010). The former predicts that thepostfrontal should appear as a single center and remainas a single center throughout development, and that thepostorbital will either not appear or will appear andthen lose its individual identity (but will not fuse to thepostfrontal). This is partly borne out. The elementregarded as the postfrontal by Evans (2008) appears anddevelops as a single center, but actually has all of themorphological characteristics of the primitive gekkotanpostorbital (Conrad and Norell, 2006) (Fig. 2) but lyingin a more dorsomedial position, bounding the frontopari-etal suture. An additional element, however, lying ante-rior to this appears, but quickly fuses to the lateral edgeof the frontal in the dorsal rim of the orbit (Fig. 3). Thiselement lies more anterior than the location of the prim-itive gekkotan position for the postfrontal (see illustra-tions in Conrad and Norell, 2006), and lacks the lateralprocess of that element, but still separates the rostralportion of the postorbital from contact with the frontal.We advocate that it is more parsimonious to propose ananterior shift of an existing element than the appear-ance of a de novo structure and the loss of another.Here, the postfrontal and the postorbital maintain aclose position relative to one another, but with the lossof the postorbital bar and upper temporal arch, charac-teristic of more derived gekkotans, there is a dorsome-dial and rostral shift with respect to their relationshipto the less labile elements comprising the skull roofingbones, namely the frontal and the parietal.

Predictions resulting from Daza and Bauer’s (2010)hypothesis indicate that ossification centers for the post-frontal and postorbital should both appear and then

should fuse to form a composite postorbitofrontal. Againthese are partially borne out. Two elements do appear(Figs. 2 and 3), but they do not fuse to each other, andthey do not exhibit the primitive relationship of the post-orbital to the postfrontal (Fig. 1A), but rather lie in se-ries. The splint-like element unites with the frontal (Fig.3), and the triangular element persists as a robust inde-pendent bone, clasping the frontoparietal suture as wellas fulfilling all of the criteria for recognition as the post-frontal (Fig. 2), if it was present alone, rather than fusedpostorbitofrontal.

Our results cannot fully resolve the competing hypothe-sis as they relate to the Gekkota in its entirety because theEublepharidae is the sister taxon to Sphaerodactylidae þ[Gekkonidaeþ Phyllodactylidae], whereas the Diplodacty-lidae þ [Carphodactylidae þ Pygopodidae] constitutes thesister taxon to that cluster (Gamble et al., 2008). Thus, thecondition advocated for the pygopods of a fused postfrontaland postorbital could still pertain and could also be a char-acteristic of the Diplodactylidae and the Carphodactylidae.However, the eublepharids are basal to the sphaerodactyl-ids, gekkonids, and phyllodactylids (Daza and Bauer,2010: Fig. 2) and, thus, can be hypothesized to exhibit acondition characteristic of this cluster, especially as itappears to represent a situation somewhat similar to thatof basal lepidosauromorphs (Daza and Bauer, 2010: Figs.1A,2) and some primitive gekkonomorphs (Conrad andNorell, 2006), with the exception that the anterior elementis fused to the frontal rather than being free, a conditionalso noted to occur in some iguanids (Norell, 1989). Euble-pharids retain many primitive features among gekkotans(Kluge, 1962; Daza, personal communication); thus thedorsal circumorbital bones could represent another exam-ple of such expression. The developmental pattern exhib-ited by Eublepharis, however, places in doubt the validityof transferring the trait of a compound postorbitofrontal toencompass all of the Gekkota (Daza and Bauer, 2010).

As a result of our observations, three alternativehypotheses can be erected. The first posits that both thepostfrontal and postorbital elements are present buttheir anatomical relationships have changed, the post-frontal undergoing reduction in size and an anteriorshift to lie alongside the frontal and to contact the pre-frontal, with the postorbital being retained (as per Riep-pel, 1992) and extending medially to contact thefrontoparietal suture (Fig. 1D). Because this necessitatesthe recognition of no additional elements, and becausewe found no evidence of fusion between developmentalcenters representing the postfrontal and postorbital, wefavor this interpretation.

Alternatively the splint-like element that abuts, andlater fuses with, the frontal could represent a de novoossification that has no homolog in other squamates.This would then leave the triangular element to be iden-tified as the postfrontal (because of its anatomical rela-tionships) 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 ele-ment and the triangular element that articulates withthe frontal and parietal as a combined postorbitofrontal(Fig. 1B, in part), but there is no developmental evi-dence to support this.

Thus, our preferred interpretation (Fig. 1D) requiresonly a shift in position of elements already known to bepresent primitively in the Squamata (Indeed, this could

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Fig. 3. The postfrontal of E. macularius from (A) initiation of ossifi-cation in late Stage 36 to complete fusion with the frontal (E) in Stage40 (staging according to Wise et al., 2009). Panels A–C are in dorso-lateral view; D and E are dorsal views, and rostral is to the right in allviews. A–C and E depict elements of the right side; D depicts ele-ments from the left. Anterior is to the right. A, later Stage 36; B and C,Stage 38; D, Stage 39; E, Stage 40. The white arrow in A–D demar-

cates the rostral end of the postfrontal and the white arrowhead thecaudal end. The white and black arrowheads demarcate the point offusion between the caudal end of the postfrontal and the ‘‘barb’’ ofthe frontal. The black arrow in panel D demarcates the overlap of theprefrontal and postfrontal. Abbreviations: fps, frontoparietal suture;par, parietal; pfr, postfrontal; po, postorbital; pref, prefrontal. Scale barrepresents 1 mm.

also be interpreted as the retention of the primitive loca-tion of the postfrontal and an anterior shift of the post-orbital—the blue and yellow color coding on Fig. 1Dwould be switched to depict this). The second interpreta-tion outlined above requires the additional developmen-tal events of a de novo ossification (the splint-likeelement) and the loss of an element (the postorbital).The third alternative would require the same de novoaddition and the fusion of two elements for which thereis no developmental evidence.

The five developmental stages (range, 36–40) overwhich the initiation of ossification of the postfrontal (ourpreferred interpretation—see above) and its subsequentfusion to the frontal occur cover a time span that canvary from 8 to 14 days (Wise et al., 2009). Dense sam-pling of embryos over these developmental stages per-mitted the observation of critical but transient events.

The phylogenetic implications of these data may bemore far reaching within the Squamata, becausedetailed information about potentially transient eventsin skull development are scarce. The widespread recog-nition of a postorbitofrontal in most squamate lineagesmay hinge upon assumptions about fusion of elements(Bellairs and Kamal, 1981) for which there is little evi-dence. This sporadic occurrence of putative fusion of thepostorbital and postfrontal is homoplasious, and thusconvergent. If the pattern observed in Eublepharis ismore widespread in its distribution, then alternateexplanations would pertain (although these may notreduce the incidence of homoplasy).

We echo the recommendations of Daza and Bauer(2010) that particular attention should be given to pat-terns of ossification in this cranial region across theSquamata. Questions of homology can only be resolvedthrough the acquisition of details of embryonic develop-ment gained from densely sampled appropriate periodsof prehatching development.

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