Development of the Skull of the Hawksbill Seaturtle, Eretmochelys imbricata

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1 JOURNAL OF MORPHOLOGY 274: (2013) Development of the Skull of the Hawksbill Seaturtle, Eretmochelys imbricata Christopher A. Sheil* Department of Biology, John Carroll University, North Park Boulevard, University Heights, Ohio ABSTRACT The embryonic development of the cranium of the Hawksbill Seaturtle (Eretmochelys imbricata) is described on the basis of cleared and doublestained specimens. Illustrations and descriptions of the chondrocranium at Stage 25 form the basis of comparison with similar data for the Loggerhead Seaturtle, Caretta caretta. Morphological changes through prehatching ontogeny are described for all dermal and endochondral elements of the skull and jaws, and the sequence of appearance and ossification of these elements are compared with similar data for Chelonia mydas, Natator depressa, and Dermochelys coriacea. Comparisons among suggest that a possible remnant of the dorsal edge of the orbital cartilage remains and represents a portion of the posterior orbital cartilage in C. caretta, representing a novel pattern of development for the posterior orbital cartilage for this species. Anatomy of cartilages of the orbitotemporal region are described for E. imbricata and C. caretta. J.Morphol. 274: , VC 2013 Wiley Periodicals, Inc. KEY WORDS: Eretmochelys imbricata; chondrocranium; ossification; development; anatomy INTRODUCTION Numerous studies of embryology in turtles have contributed to the understanding of developmental biology in reptiles (e.g., Rathke, 1848; Parker, 1880; Voeltzkow, 1903; Fuchs, 1915; Deraniyagala, 1933, 1939, 1953; Domantay, 1968; Blanck and Sawyer, 1981; Crastz, 1982; Miller, 1982; also for detailed review prior to 1985 see Miller 1985: ). However, the majority of these studies describe external morphological variation through ontogeny in a framework that provides criteria for ordering developmental progress, or for assessing primary homology of individual skeletal elements (Burke and Alberch, 1985; Gilbert et al., 2001; Sheil and Portik, 2008; Fabrezi et al., 2009). The morphology of the complete embryonic chondrocranium has been described and illustrated for several of the nearly 300 described species of turtles (Iverson, 1992), representing 6 of 14 recognized families: Chelidae [Emydura subglobosa (Paluh and Sheil, 2013) and Phrynops hilarii (Bona and Alcalde, 2009)]; Cheloniidae [Caretta caretta (Kuratani, 1999) and Eretmochelys (Chelone) imbricata (Fuchs, 1915)]; Chelydridae [Chelydra serpentina (Rieppel, 1976; Sheil and Greenbaum, 2005) and Macrochelys temminckii (Sheil, 2005)]; Emydidae [Chrysemys picta (Shaner, 1926), Emys orbicularis (Kunkel, 1912) and Trachemys scripta (Tulenko and Sheil, 2006)]; Testudinidae [Dipsochelys gigantea (Gerlach, 2012)]; and Trionychidae [Apalone spinifera (Sheil, 2003)] and Pelodiscus sinensis (Sanchez-Villagra et al., 2009). Additionally, among seaturtles, the complete embryonic neurocranium has been described for Eretmochelys (Chelone) imbricata (Fuchs, 1915), and Caretta caretta (Kuratani, 1999) and individual structural units of the embryonic and adult chondrocranium have been described for C. caretta (Kuratani, 1987, 1989) and Dermochelys coriacea (Nick, 1912); prehatching sequences of ossification of cranial elements have been described (though superficially) for only the Loggerhead Seaturtle (C. caretta; Kuratani, 1999). Comparisons among all these studies show that considerable differences exist in the developmental morphology and sequence of formation and ossification of skeletal elements of these species and demonstrate that additional studies are needed to describe variation in these processes. Paluh and Sheil (2013:fig. 5) proposed a hypothesis to explain the changes in chondrocranial anatomy observed among extant turtles relative to other amniote clades, and suggested that additional data from other species of turtles are necessary to understand how changes have occurred within Testudines. The Hawksbill Seaturtle (Eretmochelys imbricata) is a medium-sized, omnivorous turtle that has a circum-tropical distribution throughout much of the Atlantic, Pacific, and Indian Oceans (Ernst and Barbour, 1989). It can be found in a diversity of habitats across both hemispheres, from rocky shores and coral reefs to shallow coastal waters characterized by soft-bottom *Correspondence to: Christopher A. Sheil; Department of Biology, John Carroll University, North Park Boulevard, University Heights, OH csheil@jcu.edu Received 6 January 2012; Revised 26 April 2013; Accepted 3 May Published online 30 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI /jmor VC 2013 WILEY PERIODICALS, INC.

2 substrates and mudflats. This species is representative of a monotypic genus, and is listed as critically endangered on the IUCN Red List (Mortimer and Donnelly, 2008; Blumenthal et al., 2009; Gaos et al., 2010; ver.3.1, accessed 27 February 2013). A recent phylogenetic analysis of turtles (Thomson and Shaffer, 2010:fig. 5) suggests the following relationships within Cheloniidae: [(Natator depressus 1 Chelonia mydas) 1 [E. imbricata [Caretta caretta 1 (Lepidochelys olivacea 1 L. kempii)]]], and a molecular divergence study (Duchene et al., 2012) estimates that E. imbricata shares a most recent common ancestor with C. caretta (a closely related seaturtle for which some embryological and developmental data have been collected) approximately 30 million years ago. Here, baseline ontogenetic data from embryonic specimens of E. imbricata are presented and expand the body of data for turtle developmental cranial morphology of the skull. The mature, latestage chondrocranium and relative timing of formation and ossification of bones of the skull are described for this species. The chondrocranium of E. imbricata was compared with that of another cheloniid, C. caretta (Kuratani, 1987, 1989, 1999), and briefly with data reported for ossification sequences in the cranium of two other seaturtles (Chelonia mydas and Dermochelys coriacea). MATERIALS AND METHODS DEVELOPMENT OF THE SKULL OF HAWKSBILL SEATURTLE Twenty-one cleared and double-stained embryos and hatchlings of E. imbricata were examined (Appendix A); though this now is a IUCN Red-Listed species, these specimens were collected in 1966, prior to listing. Relative developmental stages of all embryos were assigned according to the external morphological criteria of Miller (1985). Observations and illustrations were made with a stereo dissection microscope equipped with a camera lucida. Techniques for clearing and double-staining with Alcian Blue and Alizarin Red follow Wassersug (1976) and Taylor and Van Dyke (1985); stainable calcium salts still remained in these bones because the specimens originally were fixed in buffered formalin, then rinsed in water before longterm storage in 70% ethanol. The onset of ossification (Table 1) was considered to be the earliest developmental stage at which the retention of Alizarin Red stain was apparent in a particular element; however, indication of ossification without retention of Alizarin Red stain was identified on the basis of surface texture that indicated osteogenesis within a specified blastema (Rieppel, 1993a). Terminology of skeletal elements and structures is based on that of Kunkel (1912) and Bellairs and Kamal (1981) for the chondrocranium, Gaffney (1972, 1979) for the skull and lower jaw, and Schumacher (1973) for the hyoid apparatus. The stage-25 embryo of E. imbricata (Fig. 1; KU ) was chosen for descriptions of the mature chondrocranium because it exhibits maximum chondrification with no apparent modification associated with ossification (Kunkel, 1912); however, the entire prehatching series was examined (Appendix A). These data were compared with similar data for another cheloniid turtle, C. caretta (Kuratani, 1987, 1989, 1999). The sequence and relative timing of ossification of cranial elements and is compared with similar data for C. caretta, C. mydas, and D. coriacea (Rieppel, 1993a). TABLE 1. Ossification sequence of Eretmochelys imbricata 1125 Stage Cranium Lower jaw 24 Prefrontal Maxilla Jugal Dentary Vomer Surangular Palatine Prearticular Pterygoid Squamosal 25 Frontal Dentary Premaxilla Surangular Columella Prearticular Parietal Angular Prefrontal Coronoid Postorbital Quadratojugal Jugal Maxilla Palatine Pterygoid Squamosal 25/26 Frontal Coronoid Quadrate Quadratojugal Exoccipital Vomer Basisphenoid Premaxilla Columella 26 Supraoccipital Exoccipital Basioccipital 27/28 Supraoccipital (paired) Prootic Basioccipital 28 Quadrate Supraoccipital Hatching Prootic Opisthotic Nonbold text indicates that ossification is inferred to have commenced on the basis of texture of prepared tissues but without retention of Alizarin Red stain, or that one or more of the specimens examined did not indicate any ossification at this stage; bold indicates elements that retain Alizarin Red stain and appear well ossified in most specimens of a given stage. Elements are listed alphabetically within a stage. RESULTS Fully Formed Chondrocranium of Eretmochelys imbricata (Miller Stage 25) The chondrocranium is approximately twice as long as wide and longer than tall, and as seen in dorsal view (Fig. 1A) is wider posteriorly than anteriorly. In lateral aspect (Fig. 1C), the anterior one-third of the chondrocranium is directed ventrally, and the ventral margin of the entire chondrocranium is arched dorsally. The nasal capsules are slightly compressed anteroposteriorly; the rostrum is very short and not tube-like. As seen in dorsal view (Fig. 1A), the ectochoanal cartilages (ec) do not extend laterally beyond the planum supraseptale (ps) or otic capsules (oc). In lateral aspect (Fig. 1C), the orbitotemporal region is composed of the planum supraseptale (ps), interorbital

3 1126 C.A. SHEIL Fig. 1. Chondrocranium of late Stage 25 (KU ) embryo of E. imbricata exhibiting maximum chondrification with minimal modification of chondrocranium from ossifying elements. (A) Dorsal view. (B) Ventral view. (C) Lateral view. Abbreviations see Appendix B. Scale bar 5 1 mm. septum (is), and pila metoptica (pm); this region is considerably longer than the otico-occipital region, and approximately three times longer than the nasal region. Each planum supraseptale (ps) is thin, occupies more than half of the orbitotemporal region, and extends dorsolaterally over the medial half of the orbit (Fig. 1A). The otico-occipital region is robust and forms the posterior two-fifths of the chondrocranium. As seen in lateral view (Fig. 1C), the quadrate cartilage (q) is prominent and crescentric, and the pterygoquadrate cartilage is relatively long and slender. The occipital arches (oa) are approximately one-third the length of the otic capsule (Fig. 1B). In dorsal aspect (Fig. 1A), the tectum synoticum (ts) is short (anteroposteriorly) and arches dorsally over the concave cavum cranii (Fig. 1). Nasal capsule. As seen in lateral view (Fig. 1C), each is directed ventrally relative to the primary axis of the chondrocranium. The margins of the fenestra narina (fn) are visible in lateral view; therefore, the anterior margin of the nasal capsule is concave. The zona annularis (zn) is broad and the lateral margin of each is nearly vertical; as seen in dorsal and lateral views (Figs. 1A,C), the midbody of the zona annularis is pierced by a small, circular foramen epiphaniale (fe). Ventral to the foramen epiphaniale, the lateral margin of each zona annularis is convex. The planum antorbitalis (not labeled in Fig. 1) is concave and bears a short, thin ectochoanal cartilage (ec) that extends posterolaterally beyond the sphenethmoid commissura (sc). The fenestra septi nasi are not apparent in any available specimens. The

4 DEVELOPMENT OF THE SKULL OF HAWKSBILL SEATURTLE sphenethmoid commissura is prominent, short, and unites the nasal capsules and anterior margins of the planum supraseptale (ps); each encloses the orbitonasal fissure. The fenestra olfactoria (fo) are relatively large, conspicuous, and broadly separated along the midline by the nasal septum. The orbitonasal fissure (onf) is apparent in dorsal and lateral views and inscribes the posterodorsal margin of the nasal capsule. As seen in ventral view (Fig. 1B), each nasal capsule bears a thin, conspicuous labial ridges (i.e., occlusal margin) that converge anteriorly to the midline. At the anteroventral terminus, the nasal septum bears a short, cylindrical rostral cartilage (rc). Posterior to the labial ridge (Fig. 1B), the floor of each nasal capsule is broad and slightly concave, and the lamina transversalis (lta) is longer than wide and bears a pair of small, circular, anterodorsally directed foramen praepalatinum (fp); these foramina are separated by the nasal septum (ns), and each pierces the lamina transversalis (lta) at the level of posterior margin of the ectochoanal cartilage (ec). Posterolateral to each foramen praepalatinum, the nasal capsules bear a long, slender paraseptal cartilage (psc) that extends beyond the posterior terminus of the ectochoanal cartilages; each paraseptal cartilage forms the lateral wall of a short, narrow channel that directs the anterior nasal artery from the palate to the fossa nasalis. The paired fenestrae narina (fn) are circular, and the choanae (ch) are relatively large, directed anteriorly, and form the passage between the palatal and nasal regions (Fig. 1A). Internal anatomy of the nasal capsules was not studied. Orbitotemporal region. This region is composed of a conspicuous, thin planum supraseptale (ps), a relatively low interorbital septum (is), and a pair of free pila antotica (paa; Fig. 1C). Each planum supraseptale is perforated by several irregular foramina and extends laterally over the anterodorsal portion of the orbit. The dorsal margin of the planum supraseptale is irregular but convex and extends dorsally beyond the margin of the otic region (Fig. 1A), and only slightly beyond the level of the lateral margin of the ectochoanal cartilage (ec; Fig. 1A); it does not reach the level of the medial margin of the quadrate cartilage (q). As seen in dorsal and lateral views (Figs. 1A,C, respectively), the plana supraseptale fuse medially with the interorbital septum (is), and anteriorly, each tapers to contact the nasal capsules via the sphenethmoid commissura (sc), which forms the lateral margin of the orbitonasal foramen (onf; Fig. 1C). Small, triangular processes extend from the posterior margin of the planum supraseptale, and are assumed here to be the taenia marginalis (tma; Fig. 1C); these are well separated from the pila metoptica (pm) and pila antotica (paa). Each pila metoptica (pm) is thin, poorly chondrified, and relatively short; contact between these bilateral 1127 structures forms the subiculum infundibuli (si; Fig. 1A). Each pila metoptica extends dorsolaterally and is broadly separated from the planum supraseptale (ps); a complete foramen for the ophthalmic artery (foa) is not complete in any specimens. The midbody of each planum supraseptale is deeply inscribed by a large, irregular foramen for the optic nerve (2, Fig. 1C) that is nearly three times longer than tall; this foramen is incomplete posteriorly and joins the foramen for the ophthalmic artery (foa). The subiculum infundibuli (si) is relatively tall and unites the pila metoptica, medially (Fig. 1A); anteromedially, the subiculum infundibuli bears a midline ridge that extends anteriorly and is continuous with the posteroventromedial margins of the plana supraseptale. Ventrally, the subiculum infundibuli is thick and fused with the pilae metoptica (pm) and trabecula communis. Each pila antotica (paa) is approximately four times longer than wide, well separated from the taenia marginalis (tma) anteriorly, and only narrowly separated from the otic capsule (oc) posteriorly (Fig. 1C); the pilae antotica are not bilobed distally. Each pila antotica also separates the foramen for the oculomotor nerve (3, Fig. 1) from the foramen nervi trigemini (5, Fig. 1), and extends dorsally nearly to the level of the dorsal margin of the foramen for the optic nerve. The foramina nervi abducentis are not apparent in any of the specimens examined. In dorsal aspect (Fig. 1A), much of the posterior orbital cartilage remains at Stage 25, and the pilae metoptica (pm) are joined ventromedially by a low, thin crista sellaris (cs) that narrowly separates the pituitary (pf) and basicranial fenestrae (bf). The foramen nervi trigemini (5) is open dorsally and smaller than the foramen for the oculomotor nerve (Fig. 1C); additionally, the interorbital septum (is) is relatively long, slender, and nearly twice as tall anteriorly (at the anterior margin of the foramen for the optic nerve) as posteriorly (at the anterior margin of the pila metoptica). The length of the pituitary fenestra (pf 5 fenestra hypophyseos; Gaffney, 1979:136) is less than half that of the basicranial fenestra (bf, Fig. 1A). The posterior margin of each trabecula is fused to the ventral margin of the posterior orbital cartilage (i.e., pilae antotica), at the level of the lateral margins of the crista sellaris (cs). The basicranial fenestra is longer than wide. The notochord extends anteriorly through the crista sellaris and terminates within the pituitary fenestra. There is no indication of basitrabecular processes (5 basipterygoid processes; Gaffney, 1979) in any specimens. Otic capsule. The otic capsules (oc) join dorsomedially by a thin, conspicuous tectum synoticum (ts), which bears a prominent anterior process that extends anterodorsally beyond the level of the dorsal margin of the planum supraseptale (ps), and a posterior process that extends beyond the occipital

5 1128 C.A. SHEIL condyle (c) and occipital arches (oa) (Fig. 1C). Each otic capsule is about twice as long as wide, and slightly longer than tall. As seen in dorsal and ventral aspects (Figs. 1A,B), a small, ovoid foramen nervi facialis (7, Fig. 1) is present between the anteroventral margin of the otic capsule and basal plate. Additionally, a pair of circular auditory nerve foramina (8, Fig. 1) are present in the medial wall of each otic capsule; the diameter of the posteriormost foramen is twice that of the anteriormost foramen. Laterally, each otic capsule contacts a large, semilunate quadrate cartilage (q) that is longer than tall and inscribed ventrally by the cavum tympanii (cv) (Fig. 1C). The anterior margin of each quadrate cartilage bears a robust, anteromedially directed pterygoquadrate cartilage that bears a short, triangular ascending process (pas), and a relatively long pterygoid process (ppr). The pterygoid process curves anterolaterally and dorsally and is longer than wide; the long axis of the ascending process (pas) is about one-sixth that of the pterygoid, and is broadly separated from the pila antotica (paa). The area articularis of the quadrate is distinctly bilobed and medially directed (Fig. 1C) and the lateral lobe is slightly larger than the medial lobe. The fenestra ovalis is circular and pierces the midbody of the ventrolateral margin of each otic capsule. Each subcapsular process is large, stout, and extends posterolaterally from the posteroventral margin of the otic capsule (oc). The metotic fissure narrowly separates from the anterior and dorsal margins of the occipital arches (oa). The external plate of the columella (ca) occupies nearly onefourth of the cavum tympani. Basal plate and occipital region. The basal plate is nearly twice as long as wide and only slightly wider anteriorly and posteriorly than at midbody. The basicranial fenestra (bf) is longer than wide and well separated from the pituitary fenestra (pf) by the crista sellaris (cs; 5 ventral margin of the posterior orbital cartilage). The notochord (n) occupies the midline of the basal plate and extends from the occipital condyle (c), through the crista sellaris (cs), and terminates in the pituitary fenestra. Laterally, the basal plate broadly contacts the otic capsules (oc) and a large foramen nervi facialis (7, Fig. 1A) is present in the margin forming the basal plate-otic capsule suture (Fig. 1A); the prefacial commissura is thick and Fig. 2. Development of the embryonic dermato- and endocranium of E. imbricata at Stage 24 (KU ), early Stage 25 (KU ), and late Stage 25 (KU ). (A) Dorsal view. (B) Ventral view. (C) Lateral view. Dermal elements are removed from the right side of the skull to expose the underlying chondrocranium (Fig. 39 for details of chondrocranium). Abbreviations see Appendix B. Scale bar 5 1mm.

6 DEVELOPMENT OF THE SKULL OF HAWKSBILL SEATURTLE 1129 Fig. 3. Development of the embryonic dermato- and endocranium of E. imbricata at late Stage 25 early Stage 26 (KU ), late Stage 27 early Stage 28 (KU ), and in a posthatching specimen with carapace length of 45.6 mm (KU ). (A) Dorsal view. (B) Ventral view. (C) Lateral view. Dermal elements are removed from the right side of the skull to expose the underlying chondrocranium (Fig. 39 for details of chondrocranium). Abbreviations see Appendix B. Scale bar 5 1 mm separates the foramen nervi facialis from the foramen nervi trigemini. The basal plate arches slightly dorsally and forms the floor of the cavum cranii, and the anterolateral margins diverge to the level of the crista sellaris. The base of the each pila antotica (paa), has a tiny, anterolaterally directed foramen for the cerebral branch of the internal carotid artery (not illustrated). Posteriorly, the basal plate is fused synchondrotically with the base of the occipital arches (oa). Though obscured in lateral view by the otic and quadrate cartilages (q; Fig. 1C), the occipital arches are nearly twice as tall as long and slightly expanded distally. The occipital arches curve dorsomedially and are widely separated along the dorsal midline. The bases of the occipital arches separate pairs of foramen nervi hypoglossi (12); anteriorly, each occipital arches are is separated from the posterior margin of the otic capsule by a narrow metotic fissure. Sequence of Ossification Neurocranium. Basioccipital. The median basioccipital first appears at Stage 26 (KU ) and retains little Alizarin Red stain; it is only slightly longer than wide and is separated from all neighboring bony elements. Ossification of the basioccipital progresses relatively slowly, and by late Stage 27 early Stage 28, the basioccipital remains weakly ossified and is only slightly longer than wide. In a single late Stage-27 to early Stage-28 specimen (KU ), the basioccipital is well ossified and lies anterior to the condylus occipitalis; the foramen posterius canalis carotici interni are not apparent in any prehatching specimens. After hatching, the basioccipital ossifies rapidly, and in all posthatching specimens (Fig.3)thisboneishighlyossified,occupiesthe posterior half of the basal plate, and forms the ventromedial one-third of the occipital condyle. The basioccipital extends laterally beyond the margins of the exoccipitals and articulates broadly with the opisthotics (Fig. 3C); anteriorly, the basioccipital is widely separated from the posterior margin of the parabasisphenoid and prootic. Posterodorsally, the basioccipital articulates broadly with the exoccipitals and forms the ventral margin of the foramen magnum. (Figs. 2, 3; Table 1)

7 1130 C.A. SHEIL Basisphenoid. The parabasisphenoid is a compound bone that forms from the fusion of the dermal parasphenoid anteriorly and endochondral basisphenoid, posteriorly (Gaffney, 1979:136). Pehrson (1945) presented an extensive review of the literature and provided compelling evidence that the dermal parasphenoid, which is present in virtually all turtles, is conspicuously absent in turtles of the family Cheloniidae (i.e., all seaturtles except Dermochelys; Thomson and Shaffer, 2010). According to Gaffney [1979: ; summarized from Pehrson (1945)], the anterior and posterior parasphenoid blastema of turtles form anterior and posterior of the pituitary fenestra (5 fenestra hypophyseos); the anterior blastema eventually degenerates, whereas the posterior blastema floors the pituitary fenestra and forms the sella turcica of adults. In Cheloniidae, the posterior parasphenoid blastema (if present) is transitory in ontogeny and does not ossify in adults. According to Gaffney (1979), the rostrum basisphenoidale of cheloniids is thought to ossify from the taenia intertrabecularis (Pehrson, 1945). Because cheloniids lack the dermal anterior and posterior parasphenoid blastema, the complex endochondral element that forms posteromedial to the basicranial fenestra is identified here as the basisphenoid, rather than the parabasisphenoid; the term parabasisphenoid is reserved for those taxa that have a dermal parasphenoid and endochondral basisphenoid elements that fuse during ontogeny. Pehrson s (1945) anterior and posterior parasphenoid blastema are not apparent in any specimens involved in this study; however, the anterior parasphenoid blastema is known to degenerate early in ontogeny, whereas the posterior parasphenoid blastema appears only briefly in ontogeny and is transitory when present (Gaffney, 1979: ; also see Pehrson, 1945). A late Stage-25 to early Stage-26 specimen (KU ) indicates endochondral ossification of the basisphenoid ventral to the crista sellaris and posterior-most portion of the taenia intertrabecularis. The wellossified basisphenoid is generally V-shaped, extends posterolaterally, and ventrally invests the anterolateral margins of the basicranial fenestra (Fig. 3B). The basisphenoid does not contact the medial margins of the pterygoid; however, by late Stage 27 early Stage 28, this bone is highly ossified and its lateral margins are invested ventrally by (and fused to) the medial margin of the pterygoid. Posteriorly, the basisphenoid is deeply concave and the basicranial fenestra is visible; only the posterior margin of the basicranial fenestra is unossified. Ossification of the basisphenoid follows the anterior margin of the basal plate (at the level of the prefacial commissura) and the posterior half of each trabeculum is ossified; however, the pituitary fenestra is not invested by the basisphenoid. In dorsal and ventral aspects (Figs. 3A,C), the crista sellaris and posterior half of each trabecula are well ossified (endochondrally). The degree of ossification of the basisphenoid progresses rapidly after hatching, and in KU , ossification of the trabeculae extends to the base of the pila metoptica; the crista sellaris is completely ossified and, with the trabeculae, forms the lateral and posterior margins of the sella turcica. The posterior margin of the basisphenoid is bilobed and widely separated from the basioccipital, whereas the lateral margins are ventrally invested by the medial margin of the quadrate ramus of the pterygoid. Columella. The columella begins to ossify at midshaft by late Stage 25 (Fig. 3B). Ossification progresses medially and laterally, and by middle Stage 26, only the lateral and medial portions remain cartilaginous; the basal plate and extracolumella are cartilaginous in all specimens. Exoccipital. By Stage 26, each exoccipital forms the lateral margin of the foramen magnum and posterior margin of the posterior-most foramen nervi hypoglossi; the exoccipitals and opisthotics form the margins of a conspicuous foramen postotica plus foramen jugulare posterius. By late Stage 27 early Stage 28 (Fig. 3B), the exoccipitals are robust, form the ventral half of the occipital arch, and contribute to the posterior margin of the metotic fissure; the anterolateral margin of each forms the medial margin of the foramen postotica plus foramen jugulare posterius. Ossification progresses slowly, and by hatching, each exoccipital is highly ossified and articulates broadly with the basioccipital (ventromedially), supraoccipital (dorsally), and opisthotics (posterolaterally). Posteromedially, each exoccipital bears a relatively short, robust condylar process that forms the lateral one-third of the occipital condyle; medially, the condylar processes are separated broadly from one another, however, each articulates with the condylar process of the basioccipital. In all specimens, the exoccipitals form all but the ventral margins of the foramen nervi hypoglossi and posterior margins of the metotic fissure. Opisthotic. Opisthotics are not apparent in any prehatching specimens; however, these paired elements ossify rapidly after hatching, and in all posthatching specimens the opisthotics are relatively large and conspicuous. In ventral aspect (Fig. 3B), each articulates broadly with the anterior margin of the exoccipital, medial margin of the quadrate, and posterior terminus of pterygoids quadrate ramus. Prootic. Prootics are not apparent in any prehatching specimens; however, these paired elements ossify rapidly after hatching, and in all posthatching specimens, the well-ossified prootics articulate broadly with the quadrate, quadratojugal, and processus inferior of the parietal. Each prootic forms the posterior margin of the foramen nervi trigemini.

8 DEVELOPMENT OF THE SKULL OF HAWKSBILL SEATURTLE Supraoccipital. Paired supraoccipitals are weakly ossified with little retention of Alizarin Red stain in a single Stage-26 specimen (KU ), and are located in the lateral one-third of the tectum synoticum. By late Stage 27 early Stage 28, the supraoccipitals are highly ossified and occupy all but the dorsomedial one-fifth of the tectum synoticum (Figs. 3A,C). The supraoccipitals form the posteromedial walls of the fossa temporalis superior and are narrowly separated from the dorsal margin of the exoccipital, prootic, and opisthotic. Dermatocranium. Premaxilla. These bones first ossify by middle Stage 25 (Fig. 2) and are relatively large; the dorsal surface of each ventrally invests the anterior half of the nasal capsule and forms the floor of the fossa nasalis. At middle Stage 25, the premaxillae are narrowly separated along the midline, and each has a distinct labial ridge that defines the lateral margin of the triturating surface, which bears many tiny nutritive foramina; the triturating surface is slightly concave. In lateral aspect (Fig. 2C), the ventral margin of the triturating surface is irregular and directed slightly ventrally relative to that of the maxilla. The posterolateral surface of each premaxilla is invested by the anterior margin of the maxilla by late Stage 25 (Fig. 2B) and each is broadly separated from the anterior margin of the vomer. By late Stage 25 early Stage 26, the premaxillae ventrally invest the anterior two-thirds of the nasal capsules, and each articulates broadly with the anterior margin of the maxilla; these paired bones articulate medially. Anteromedially, the dorsal surface of each premaxilla is slightly concave and the fossa nasalis accommodates the rostral cartilage of the septum nasalis. By late Stage 27 early Stage 28, (Fig. 3B), the triturating surface of each premaxilla is deeply concave posterior to the labial ridge, and a small, circular foramen praepalatinum (which allows for the passage of the anterior nasal artery) is present in the posterior third of the palatal process. Little shape change occurs after hatching; however, as seen in ventral view (Fig. 3B), the posterior margin of each articulates with the anterior margin of the anteroventral plate of the vomer. Maxilla. The maxillae are among the first elements of the skeleton to ossify, and by Stage 24 exhibit weak ossification as long, slender bones located ventral to the posterior half of the nasal capsule and planum supraseptale (Figs. 2B,C). Each is approximately eight times longer than tall, approximately six times longer than wide, and more robust anteriorly than posteriorly; and the posterior terminus is narrow and thin. In ventral aspect (Fig. 2B), each maxilla is inscribed deeply by a long, narrow canalis alveolaris superior that occupies the posterior two-thirds of the element; at this stage, the maxillae do not contact 1131 neighboring bony elements. Ossification progresses rapidly (anteriorly and posteriorly), and by early Stage 25 each is well ossified. In ventral and lateral aspects (Figs. 2B,C), the ventrolateral margin of each maxilla bears a shallow, V-shaped labial ridge (in cross section) that is most prominent anteriorly and diminishes in height posteriorly. By Stage 25, the dorsal surface of each maxilla is gently concave and forms the ventral margin and ventrolateral wall of the presumptive fossa orbitalis. In lateral aspect (Fig. 3C), the prefrontal process of each maxilla is prominent, extends dorsally beyond the level of the ventral margin of the interorbital septum, laterally invests the ventrolateral terminus of the vertical plate of the prefrontal, and forms the lateral wall of the fossa nasalis. The palatine process of each maxilla is broad and thin, but does not contact the palatine or vomer; however, at late Stage 25, the anteromedial margin of each maxilla articulates broadly with the lateral margin of the premaxilla. The dorsal surface of each bears small, circular foramen superius maxillaris and foramen alveolare superius. By late Stage 25 early Stage 26, the palatine process of each maxilla is subhorizontal and articulates broadly with the lateral margin of the palatine. Additionally, the large prefrontal process of each maxilla is laterally invested by the ventral terminus of the vertical plate of the prefrontal; the internal surface of the prefrontal process bears a shallow channel that accommodates the ectochoanal cartilage of the nasal capsule. By late Stage 27 early Stage 28 (Fig. 3), the maxillae are heavily ossified and have the adult configuration; however, articulation with neighboring elements is limited and the palatal process of each is separated narrowly from the lateral margin of the anteroventral plate of the vomer (Fig. 3B). Though the overall shape of this bone changes little after hatching, these elements enlarge rapidly and in all posthatching specimens the maxillae articulate tightly with the premaxillae, vomer, palatines, jugals, and prefrontals. Prefrontals. The prefrontals are among the first cranial elements to ossify (Stage 24), are located dorsolateral to the posterior margin of the nasal capsule, follow the contour of the planum antorbitalis, and are nearly four times taller than long (Fig. 2C). By early Stage 25, each bears a vertical plate that extends ventrally and forms the anterior margin of the orbit; the posterior margin is slightly concave. By late Stage 25, each prefrontal bears a dorsal plate that is broad, conspicuous, and extends medially; however, the prefrontals do not articulate along the midline. The posterior margin of each prefrontal is slightly concave and the vertical plate is large and forms the anteromedial margin of the fossa orbitalis (Fig. 2C); additionally, the ventral terminus of the vertical plate is invested laterally by the dorsal margin of the

9 1132 C.A. SHEIL prefrontal process of the maxilla, whereas the posterior terminus of the horizontal plate articulates broadly with the anterior margin of the frontal. The ventromedial margin of the vertical plate is concave and forms the dorsal margin of the foramen orbitonasalis (Fig. 3C), whereas the posterior margin is slightly concave and forms the anterior margin of an incomplete foramen interorbitalis. At late Stage 25 early Stage 26, the ventromedial margin of each vertical plate is not in contact with the prefrontal process of the vomer. The medial margins of the vertical plates are broadly separated from one another and form the lateral walls of a relatively wide fissura ethmoidalis, which passes between the fossa nasalis and cavum cranii. By late Stage 27 early Stage 28 (Fig. 3B), the ventromedial margin of each vertical plate articulates with the prefrontal process of the vomer and forms all of the lateral margin of the fissura ethmoidalis; additionally, the vertical plates form the dorsal, medial, and lateral margins of the foramen orbitonasalis. In dorsal aspect (Fig. 3A), the horizontal plates are large and articulate broadly along the midline. In all posthatching specimens, the ventromedial margin of each vertical plate is separated narrowly along the midline, and articulates tightly with the prefrontal process of the vomer; the medial margin of each vertical plate is thin and continuous with the parasagittal ridge of the frontal. Frontals. Long, slender frontals first appear without retention of Alizarin Red stain by early Stage 25. By middle Stage 25, the well-ossified frontals are longer than wide, form the dorsal margin of the orbit, and articulate broadly with the posterior margin of the horizontal plate of the prefrontal. Posteriorly, each frontal tapers to a narrow point that approaches, and is narrowly separated from, the anterolateral margin of the parietal. The ventral surface of each has a thin, inconspicuous parasagittal ridge that runs nearly the entire length of the element; the parasagittal ridges are subparallel. By late Stage 25 (Fig. 2A), each frontal articulates broadly with the prefrontal and parietal, and the anterior terminus of each converges toward the midline; the conspicuous parasagittal ridge of each frontal follows the contour of its corresponding planum supraseptale, and separates the fossa orbitalis (laterally) from the sulcus olfactorius (medially). By late Stage 25 early Stage 26 (Fig. 3A), the lateral margin of each frontal is prominently concave, whereas the medial margin is convex and irregular. Anteriorly, each frontal tapers to a narrow point and is invested dorsally by the posterior margin of the horizontal plate of the prefrontal (Jones et al., 2011; Type 5 Scarf Joint); the posterior terminus of each is invested dorsally by the anterior terminus of the parietal. At late Stage 25 early Stage 26, the parasagittal ridge is large and the frontals remain broadly separated along the midline. Each frontal ossifies anteriorly, posteriorly, and medially, and by Stage 26, the anterior one-fourth of each articulates along the dorsal midline, whereas the posterior terminus of each articulates with the anterior terminus of the postorbital and eliminates the parietal from the margin of the orbit. By late Stage 27 early Stage 28, the frontals articulate along the midline anteriorly, and remain narrowly separated posteriorly (Fig. 3A). Additionally, each frontal articulates broadly with the anterior margin of the parietal; the parasagittal ridges are relatively thin and continuous with the processus inferior parietalis and form the dorsal margin of the foramen interorbitalis. Postorbital. Postorbitals first indicate ossification at early Stage 25 (Fig. 2A,C). The anterior margin of each is relatively smooth and forms the posterior margin of the orbit. By middle Stage 25, each postorbital is highly ossified and the dorsal margin of each extends slightly medially and articulates broadly with the lateral margin of the parietal, thereby forming the anterodorsolateral roof of the presumptive fossa temporalis superior (Fig. 3A). By late Stage 25, each postorbital articulates broadly with dorsolateral surface of the parietal and forms the lateral and most of the dorsal walls of the fossa temporalis superior; the postorbitalparietal suture extends nearly the entire length of the element. By late Stage 25 early Stage 26, the postorbitals compose much of the temporal region of the skull, and the anterior margin of each is prominently concave and forms the posterior margin of the orbit. The ventral terminus of each postorbital is invested laterally by the posterodorsal terminus of the jugal, and each articulates broadly with the anterior margins of the quadratojugal and squamosal. The internal surface of each postorbital is relatively smooth, concave, and bears a thin vertical ridge that separates the fossa orbitalis and fossa temporalis. By Stage 26, this vertical ridge is conspicuous and separates the lateral portions of the fossa temporalis inferior and fossa orbitalis. By late Stage 27 early Stage 28, the roof of the fossa temporalis superius is complete and each postorbital articulates broadly with the parietal, quadratojugal, squamosal, and jugal. Parietals. These paired elements are conspicuous by early Stage 25 (Fig. 2), and each is prominently concave medially and located lateral to the planum supraseptale and otic capsule (Figs. 2A,C); the triangular vertical plate of each is more ossified than the horizontal plate. The lateral surface of each parietal bears a short, narrow, longitudinal ridge that contributes to the lateral wall of the fossa temporalis superior. The margins of the parietal are irregular, and each is nearly five times longer than tall (Fig. 2C). By middle Stage 25, the anterior margin of the horizontal plate of each parietal extends anteriorly to the level of the

10 DEVELOPMENT OF THE SKULL OF HAWKSBILL SEATURTLE posterior margin of the frontal, whereas the posterior margin is long, thin, and dorsally invests the lateral two-thirds of the tectum synoticum (Fig. 3A). Additionally, thin, triangular processus inferior parietalis extend ventrally to the level of the pterygoquadrate cartilage; however, these processes do not contact, and each lacks a sulcus cartilaginis epipterygoideus. By late Stage 25 early Stage 26, each processus inferior parietalis extends ventrally to the level of the ascending process of the pterygoquadrate cartilage, and bears a weak sulcus cartilaginis epipterygoideus; however, the ascending process of each pterygoquadrate cartilage does not insert into this sulcus. Additionally, the ventral margin of the processus inferior parietalis is separated only narrowly from the crista pterygoideus by the pterygoquadrate cartilage. By late Stage 27 early Stage 28, the parietals articulate with one another along the midline and are only narrowly separated posteriorly. In dorsal aspect (Fig. 3A), the posteromedial portion of each parietal dorsally invests the anterior half of the tectum synoticum and a portion of the supraoccipital. Posterolaterally, each parietal articulates with the dorsomedial margin of the squamosal. The roof of the fossa temporalis superior is complete and highly ossified, and the posterior margin of each parietal is slightly concave. The sulcus cartilaginis epipterygoideus of each processus inferior parietalis accommodates a short, robust ascending process of the pterygoquadrate cartilage (5 presumptive epipterygoid). The posterior margin of the foramen interorbitale and anterior margin of the foramen nervi trigemini are ossified; the sulcus cartilaginous epipterygoideus shows weak ossificaion. Squamosal. These are among the first elements of the skull to ossify, and by Stage 24, each is relatively thin and covers the posterolateral wall of the quadrate cartilage. At Stage 24, each triangular squamosal forms the posterior margin of the cavum tympani (Fig. 2C). Ossification progresses dorsally, and by early Stage 25, each bears a thin, vertical crista paraoccipitalis that extends beyond the dorsal margin of the quadrate cartilage and forms the posterolateral margin of the presumptive fossa temporalis superior. By late Stage 25, each squamosal forms the posterodorsal margin of the cavum tympani and the crista paraoccipitalis extends dorsally nearly to the level of the dorsal margin of the otic capsule (Fig. 3C). By late Stage 25 early Stage 26, the crista paraoccipitalis of each squamosal forms the posterolateral wall of the fossa temporalis superior and articulates broadly with the postorbital and parietal. By late Stage 27 early Stage 28, the dorsomedial margin of each squamosal articulates broadly with the posterolateral margin of the parietal and forms the posterolateral wall of a complete fossa temporalis superior Palatine. The palatines exhibit weak ossification without retention of Alizarin Red stain by Stage 24, and first appear as thin, slender bones that do not articulate with neighboring elements. By early Stage 25 (Fig. 2C), each triangular palatine is nearly twice as wide anteriorly as posteriorly. The posterolateral margin of each is smooth and forms the anteromedial margin of the fenestra subtemporalis. By middle Stage 25, each palatine forms much of the palate and the medial one-third of each is directed dorsomedially towards the interorbital septum; along the midline the roof of the palate is concave; each bears a small, circular foramen nervi vidiani. By late Stage 25 early Stage 26, each palatine articulates broadly with the palatine process of the maxilla (laterally), and is narrowly separated from the anterodorsal margin of the pterygoid (posteriorly). Additionally, each palatine is narrowly separated from the anterior terminus of the jugal, lateral margin of the vomer, and interorbital septum. By late Stage 27 early Stage 28, the palatine process of each jugal extends medially and articulates with the posterolateral margins of the palatines. After hatching, the degree of ossification of the palatines progresses rapidly, and in all posthatching specimens, the lateral third of each palatine is heavily ossified, robust, and contributes to the triturating surface. Vomer. An unpaired, median vomer first ossifies without retention of Alizarin Red stain by Stage 24, and is located ventral to the interorbital septum, at the level of the anterior margin of the foramen for the optic nerve. The margins of the vomer are irregular and this bone is more than twice as long as wide (Fig. 2B). By late Stage 25, this bone bears a prominent, broad, ventromedial ridge that extends nearly the entire length of the element; the posterior terminus is thin and deeply concave, whereas the anterior terminus is robust and bears a conspicuous ventromedial plate (Fig. 3B). The anterolateral margins of the vomer are prominently concave and form the medial margins of the apertura narium interna, whereas the anteroventral margin is wide and ventrally invests the posterior one-third of the nasal septum and half of the paraseptal cartilages. Dorsolaterally, the vomer bears a pair of small prefrontal processes that define the lateral margins of a large, longitudinal, V-shaped sulcus septum nasi, which accommodates the septum nasalis. By late Stage 25 early Stage 26, this large, highly ossified element is composed of two distinct regions an anteroventral plate, and a long, slender posterior region. As seen in ventral view (Fig. 3B), the anteroventral plate is robust, nearly as long as wide, and more than twice as wide as the posterior portion. By Stage 26, the anteroventral plate of the vomer is broad, contacts the anterior margin of the palatine and anteromedial margin of the maxilla, and contributes to the triturating surface (Fig. 3B).

11 1134 C.A. SHEIL Pterygoid. At Stage 24, each pterygoid is nearly six times longer than wide, and located ventral and slightly medial to the pterygoquadrate cartilage, and the dorsal surface of each is unornamented. By early Stage 25, each pterygoid extends from the level of the pila metoptica (anteriorly) to the level of the foramen nervi facialis (posteriorly), and by middle Stage 25, each is distinctly tripartite and composed of a quadrate ramus, transverse flange, and corpus. The quadrate ramus is six times longer than the transverse flange and corpus and is considerably longer than wide and follows the contour of the pterygoquadrate cartilage to the level of the area articularis of the quadrate (Fig. 2C). The posterior margin of the transverse flange and the lateral margin of the quadrate ramus are confluent, concave, and form the anterior and medial margins of the fenestra subtemporalis. By late stage 25, the transverse flange is robust and bears a small, conspicuous, nearly vertical processus pterygoideus externus. The dorsal surface of each bears a short crista pterygoidea that laterally invests the pterygoquadrate cartilage; the height of the crista pterygoidea is greatest at midbody and diminishes anteriorly and posteriorly. At late Stage 25, the posterior half of the quadrate ramus of each bone extends posteriorly nearly to the level of the columella, and invests the anteroventral margin of the otic capsule. By late Stage 25 early Stage 26, each pterygoid occupies much of the palate and in ventral aspect (Fig. 3B), the lateral surface of each is deeply concave and forms the medial margin of the fenestra subtemporalis and ventrally invests the pterygoquadrate cartilage. The crista pterygoidea is large, prominent, and extends nearly the entire length of the dorsal surface of the element. At late Stage 25 early Stage 26, the processus pterygoideus externus of each is relatively short, robust, and prominent, and is located at the level of the anteromedial margin of the fenestra subtemporalis. By late Stage 27 early 28, the posteromedial portion of each pterygoid ventrally invests (and fuses with) the lateral third of the basisphenoid. In ventral aspect (Fig. 3B), each pterygoid follows the embayment of the lateral margin of the pterygoquadrate, and the quadrate ramus of each pterygoid extends posteriorly beyond the columella auris. The transverse flange of each pterygoid is nearly onethird the length of the quadrate ramus, and ventrally invests the posterior margin of the palatine. Additionally, the processus pterygoideus externus of each is relatively large. The large crista pterygoidea of each pterygoid articulates broadly with the processus inferior parietalis and forms the ventral margin of the foramen nervi trigemini. The sulcus cartilaginous epipterygoideus inscribes the dorsal margin of the crista pterygoidea, at the level of the pterygoid-parietal suture; however, the pterygoid and ascending processes of the pterygoquadrate cartilage are not ossified. Jugal. Weakly ossified jugals appear by Stage 24 and are considerably longer than tall, triangular, and located between the posterior terminus of the maxilla and anterior margin of the quadrate cartilage (Fig. 2C). The dorsal margin of each jugal is relatively smooth and forms the posteroventral margin of the orbit; all other margins are irregular. By early Stage 25, each jugal forms the lateral margin of the fenestra subtemporalis and the internal surface of each is slightly concave (Fig. 2C). The anterior terminus of each jugal dorsally and laterally invests the posterior terminus of the maxilla, and approaches the lateral margin of the palatine. By late Stage 27 early Stage 28, the palatine process of the jugal extends medially and articulates with the posterolateral margin of the palatine. Quadratojugal. Thin, weakly ossified, and triangular quadratojugals are present by middle Stage 25. As seen in lateral view (Fig. 3C), each is located lateral to the anterior margin of the quadrate cartilage, at the level of the base of the pterygoquadrate cartilage. By late Stage 25, each quadratojugal laterally invests the anterolateral margin of the quadrate cartilage and the posterior margin of each is prominently concave and forms the ossified anterior margin of the cavum tympani (Fig. 3C). By late Stage 25 early Stage 26, the anterior terminus of each quadratojugal is invested laterally by the posterior terminus of the jugal and each articulates broadly with the postorbital and squamosal, and bears a prominent, thin ventral process that laterally invests the quadrate cartilage to the level of the area articularis mandibularis (Fig. 3C). In ventral aspect (Fig. 3C), the thin ventral margin of each quadratojugal forms the posterolateral margin of the fenestra subtemporalis. Splanchocranium. Epipterygoid. In all specimens examined, the ascending and pterygoid processes of the pterygoquadrate are weakly chondrified and thin. Though the epipterygoids do not ossify, the ascending processes articulate with the ventral terminus of the processus inferius parietalis; the pterygoids bear relatively shallow, inconspicuous sulci cartilaginous epipterygoideus. Quadrate. The quadrates ossify by late Stage 25 early Stage 26, at which point the medial wall of the cavum tympani and margins of the incisura columellae auris are well ossified (Figs. 3B,C). After Stage 26, endochondral ossification progresses rapidly, and by late Stage 27 early Stage 28, the entire medial margin of each is well ossified. Additionally, the pterygoquadrate cartilage is highly ossified to the level of the ascending process, and the area articularis mandibularis and lateral portions of the quadrate remain cartilaginous. After hatching, ossification of the quadrate progresses slowly, and in all posthatching specimens the proximal one-third of the pterygoquadrate is highly ossified to the level of the base