Development of the Skull of the Pantropical Spotted Dolphin (Stenella attenuata)

Authors

  • Meghan M. Moran,

    Corresponding author
    1. Department of Anatomy and Neurobiology, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, Rootstown, Ohio
    • Department of Anatomy and Neurobiology, 4209 State Route 44, P.O. Box 95, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, Rootstown, OH 44272. Fax: +1-330-325-5911
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  • Sirpa Nummela,

    1. Department of Biosciences, University of Helsinki, Helsinki, Finland
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  • J.G.M. Thewissen

    1. Department of Anatomy and Neurobiology, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, Rootstown, Ohio
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Abstract

We describe the bony and cartilaginous structures of five fetal skulls of Stenella attenuata (pantropical spotted dolphin) specimens. The specimens represent early fetal life as suggested by the presence of rostral tactile hairs and the beginnings of skin pigmentation. These specimens exhibit the developmental order of ossification of the intramembranous and endochondral elements of the cranium as well as the functional and morphological development of specific cetacean anatomical adaptations. Detailed observations are presented on telescoping, nasal anatomy, and middle ear anatomy. The development of the middle ear ossicles, ectotympanic bone, and median nasal cartilage is of interest because in the adult these structures are morphologically different from those in land mammals. We follow specific cetacean morphological characteristics through fetal development to provide insight into the form and function of the cetacean body plan. Combining these data with fossil evidence, it is possible to overlie ontogenetic patterns and discern evolutionary patterns of the cetacean skull. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

The development of the Cetacea skull was studied in embryos (de Burlet, 1913a, 1913b, 1914a, 1914b; Schreiber, 1916; Honigmann, 1917; Rauschmann et al., 2006; Thewissen and Heyning, 2007) and fetuses (Schulte, 1916; Ridewood, 1923; Eales, 1950). Cetacean research focused on specific biological systems to understand differences within Mammalia. Comtesse-Weidner (2007), Miller (1923) and Kellogg (1928a, 1928b) studied morphological elements including telescoping. Oelschläger and Buhl (1985), Klima and van Bree (1990), and Klima (1995, 1999) studied nasal anatomy and development. Oelschläger (1986, 1990), Solntseva (1990, 1999, 2002), and Kinkel et al. (2001) concentrated on hearing reception and sound emission while Mead and Fordyce (2009) focused on general skull anatomy. Although comparative embryological studies on cetaceans were rare, developmental studies were mostly nonexistent. Such studies (e.g., Thewissen et al., 2006, Armfield et al., in press) allow for a deeper understanding of the ontogenetic constraints on the evolution of the cetacean body plan.

Habitat changes alter adaptations for specific cetacean body plans. These modifications include those of anatomical function and body plan from land mammals to fully aquatic, air breathing marine mammals. Our study focuses on anatomical structures of five Stenella attenuata (pantropical spotted dolphin) fetuses. Here we describe bony and cartilaginous structures of the cranium of these fetuses to elucidate telescoping, nasal anatomy, and ear anatomy.

MATERIALS AND METHODS

We describe the skulls of five S. attenuata fetuses from the Natural History Museum of Los Angeles County (LACM) in Los Angeles, CA (Fig. 1). These fetuses are staged using the adapted and expanded Carnegie system from Thewissen and Heyning (2007). Ages of fetuses are based on Sˇteˇrba et al. (2000). The five specimens are: LACM 94671, LACM 94592, LACM 94310, LACM 94285, and LACM 94382 (Fig. 1 and Table 1).

Figure 1.

Five cleared and stained pantropical spotted dolphin (Stenella attenuata) fetuses. Alcian blue stains the cartilage and Alizarin red stains the bone. Specimen numbers are listed to the left of each fetus. LACM 94671 is early stage 20, TL: 85 mm. LACM 94592 is late stage 20, TL: 155 mm. LACM 94310 is stage 21/22, TL: 185 mm. LACM 94285 is stage 23, TL: 213 mm. LACM 94382 is stage 23, TL: 225 mm. Scale bar = 1 cm.

Table 1. Stenella attenuata fetal specimen details
Specimen numberStageApproximate ageTotal length (TL)Skull length
  1. Specimen numbers, expanded Carnegie stage, associated ages, as well as average body length and average skull length in millimeters (based on Stěrba et al., 2000).

94671C20∼ 70 days8520
94592C2080–110 days15537
94310C21/2280–110 days18545
94285C23110–120 days21356
94382C23110–120 days22558

Each fetus is measured by placing a piece of string along the dorsum of the fetus from the rostral tip to the tail tip for a total length (TL) measurement (Table 1). The skull length is measured using calipers (Table 1) from the tip of the rostrum to the caudal most extent of the occipital bones and cartilage. Each measurement is taken three times and the average is listed in this table. No linear measurements of individual bones are presented because the vast shape change of cranial bones during development makes it difficult to take measurements consistently.

Each Stenella fetus is cleared and stained (Wassersug, 1976) to show only bone and cartilage structures. Other soft tissue structures are obliterated during the staining process. The specimen is skinned, eviscerated, and washed in water for about 7 days to remove fixative. The head is bisected to allow for better visualization of the cranial bones and cartilaginous areas after staining. This also allows for further molecular study to be completed on the contralateral side of each fetal skull specimen. Symmetry was not addressed during cranial development because this technique made it impossible. The water is changed every day to maximize the washing process. The fetus is then put into a 40% acetic acid/60% alcohol solution for 7 days. Again, the solution is changed every day. After 7 days in the 40% acetic acid/60% alcohol solution, the solution is replaced and Alcian blue stain is added to the solution. The Alcian blue stain/acetic acid/alcohol solution is checked every day; the solution is replaced every 2−3 days for anywhere between 5 days to 4 weeks depending on how well the specimen absorbs the stain. The Alcian blue stains the cartilaginous structures of the specimen.

Once the blue stain is absorbed by the cartilage, the specimen is placed in a 3–5% potassium hydroxide/Alizarin red stain solution for 1 day or until the bone absorbs the red stain. This solution is checked every hour to prevent any damage the specimen because this solution is extremely caustic and tissue quickly deteriorates. The Alizarin red stains the bone red. The double stained dolphin fetus is placed into a series of rinses of increasing concentrations of glycerol from 25% to 100%. Each fetus is stored in 100% glycerol in the refrigerator (4°C). Slight differences in the timing of these steps may occur because of the duration of fixation of the tissue.

RESULTS

Plates for LACM 94671 are provided in Figs. 1, 2, 3, and 4; LACM 94592 in Figs. 1, 3, 4, and 5; LACM 94310 in Figs. 1 and 6; LACM 94285 in Figs. 1 and 7; and LACM 94382 in Fig. 1.

Figure 2.

Head of fetus LACM 94671 (TL: 85 mm), A: median. B: lateral. C: before clearing and staining; the soft tissue structures are in situ such as the brain, epiglottis, and the tongue. D: Close up of the cranium from an inferior–lateral orientation. E: Superior–medial view of the median cranial structures. Specific cartilaginous and bony features are labeled in the figure. Scale bar = 5 mm for A–C and E. Scale does not apply to D which is foreshortened. Abbreviations in Figs. 2–7: Acc, accessory ossicle; Alg, alveolar groove; Alo, ala orbitalis; Alt, Ala temporalis; Bas crt, basihyoid cartilage; Boc, basioccipital bone; Boc crt, basioccipital cartilage; Bsp, basisphenoid bone; Bsp crt, basisphenoid cartilage; Cbp, cribriform plate; Crb, crus breve of the incus; Crl, crus longum of the incus; Den, dentary bone; Ebn, external bony nares; Ect, ectotympanic bone; End for, endolymphatic foramen; Epi, epiglottis; Exo, exoccipital bone; Fro, frontal bone; Hyp, hypoglossal canal; Iam, internal auditory meatus; Inc, incus; Inp, interparietal bone; Jfr, jugular foramen; Jug, jugal bone; Lac, lacrimal bone; LSoc, left ossification of the supraoccipital bone; Mal, malleus; Man, manubrium of the malleus; Max, maxillary bone; Mec, Meckel's cartilage; Nas, nasal bone; Nas crt, nasal cartilage; Occ fis, occipitocapsular fissure; Opt for, optic foramen; Orb fis, orbitonasal fissure; Orb sph, orbitosphenoid; Otc, otic capsule; Pal, palatine bone; Par, parietal bone; Pmx, premaxillary bone; Pmx crt, premaxillary cartilage; Pop, posterior orbital process; Pre, presphenoid bone; Pre crt, presphenoid cartilage; Prl, pars lateralis of the squamosal bone; Prm, pars medialis of the squamosal bone; Ptg, pterygoid bone; RSoc, right ossification of the supraoccipital bone; Soc, supraoccipital bone; Sph fis, sphenorbital fissure; Squ, squamosal bone; Sth, stylohyoid bone; Sth crt, stylohyoid cartilage; Stp, stapes; Thh, thyrohyoid bone; Thh crt, thyrohyoid cartilage; Ton, tongue; Vom, vomer bone.

Figure 3.

Comprehensive black and white line drawings of the developing skulls of LACM 94671. A: Lateral, and B: medial views. LACM 94592, C: lateral and D: medial views. Some structures drawn here are not represented in the photographs but were observed through the microscope. The course stipples represent cartilage and the fine stipples represent bone.

Figure 4.

Close up views of the middle ear structures including ossicles. A: LACM 94671, B: black and white line drawing of LACM 94671, C: LACM 94592, D: black and white line drawing of LACM 94592. The course stipples represent cartilage and the fine stipples represent bone.

Figure 5.

The head of LACM 94592 (TL: 155 mm). A: Medial. B: Lateral. C: Ventral oblique. D: Superior–lateral views. Scale bar = 5 mm for A–D.

Figure 6.

The head of LACM 94310 (TL: 185 mm). A: Medial, B: Lateral, C: Dorsal, D: Superior–lateral orientation. Scale bar = 1 cm for A–D.

Figure 7.

The head of LACM 94285 (TL: 213 mm). A: Median. B: Lateral. C: Dorsal. D: Inferior–lateral orientation. Scale bar = 1 cm for A–C. Scale bar = 2 cm for D.

Early Carnegie Stage 20

Intramembranous elements.

The premaxilla (Pmx) is an elongated bone, constricted to a narrow waist near its middle (Figs. 2A,D and 3A,B). The left and right premaxillae (Fig. 2A and D) are wedged between the left and right maxillae (Max, Figs. 2B,D and 3A,B) and do not extend into the rostrum. The rostrum lacks much ossification at this time. The premaxilla reaches farther rostrally than the maxilla. A small and dense piece of premaxillary cartilage (Pmx crt) is located at the tip of the rostrum (Figs. 2A,B and 3A,B). The external bony nares (Ebn, Fig. 2A) is caudal to the premaxilla, high on the forehead. Medial to the premaxilla, a number of cartilaginous structures are associated with the nasal cartilage and vomer. The development of these structures has been described in some detail by Klima (1999) and will not be discussed here.

The maxilla (Figs. 2B,D and 3A,B) is a triangular bone on the lateral side of the face and forms a narrow portion of the palate. The maxilla forms most of the rostrum at this stage. The caudal aspect of the maxilla is located lateral to the cribriform plate (Cbp, Figs. 2A and 3B) and the nasal cartilages. The maxilla extends dorsally but not as far as the frontal (Fro) and parietal (Par) bones (Figs. 2B and 3A). Medially, the maxilla is gently concave and helps support the well-developed median nasal cartilage (Nas crt, Figs. 2A and 3B) or mesorostral cartilage (see Mead and Fordyce, 2009).

Posterior to the maxilla, the lacrimal (Lac, Figs. 2D and 3A) is a narrow splint of bone in the anterior edge of the orbit. In the wall of the pharynx, two ossification centers are posterior to the maxilla. The more anterior of the two ossifications is the palatine (Pal, Figs. 2A and 3B), which makes up the lateral wall of the nasopharyngeal duct. Caudal to the palatine is the pterygoid bone (Ptg, Figs. 2A and 3B), which extends more ventrally than the palatine. The vomer (Vom) is located medially to the palatine and the pterygoid (Fig. 3B).

The frontal bone (Figs. 2B,D,E and 3A,B) is a flat, rectangular bone that forms the ventrally concave edge of the dorsal orbit. The frontal bone does not extend into the roof of the orbit and does not overlap with any other bones in this specimen (Figs. 2B and 3A). The parietal bone (Par, Figs. 2B,D and 3A) is a flat, teardrop-shaped bone immediately caudal to the frontal bone and dorsal to the otic capsule (Otc, Figs. 2A,B,E and 3A). The squamosal bone (Squ, Figs. 2B,D and 3A) is a small bone located over the posterior end of Meckel's cartilage, adjacent to the otic capsule (Mec, Figs. 2A,B,D and 3A,B).

The dentary at this stage is ossified from the ramus through the central part of the dentary (Den, Figs. 2A,B,D and 3A,B). This ossification starts at the anterior edge of Meckel's cartilage near the tip of the rostrum and ends near the middle of the orbit. The dentary is concave medially, enveloping Meckel's cartilage and wrapping around it ventrolaterally. The ectotympanic (Ect) is a very thin horseshoe-shaped ossification located ventral to the squamosal bone (Fig. 2D) and inferior to the posterior end of Meckel's cartilage.

Endochondral elements.

The nasal capsule forms the rostral wall of the cranial cavity. The cribriform plate is perforated by a large bilateral foramina for cranial nerve I (CN I) immediately lateral to the midline. Projecting rostrally in the midline is the large median nasal cartilage (Nas crt, Figs. 2A and 3B), rostrum nasi cartilagineum of Klima (1995, 1999). The lateral wall of the nasal capsule is barely developed, consisting only of a few isolated pieces of cartilage. The ethmoid is bounded caudally by the large orbitonasal fissure (Orb fis, Figs. 2A,B and 3B). Dorsal to the orbitonasal fissure, a thin cartilaginous process connects the nasal capsule to the ala orbitalis (Alo, Figs. 2A,B,E and 3B). Such a process is not present in a 48 mm Phocoena (de Beer, 1937) but is present in a 92 mm Megaptera (Honigmann, 1917). The ala orbitalis appears as a cartilaginous ring and is perforated by the optic foramen (Opt for, Figs. 2A,B,E and 3A,B). This foramen is laterally placed and large as in the 48 mm Phocoena (de Beer, 1937), unlike the 92 mm Megaptera (Honigmann, 1917). Caudally, the ala orbitalis is separated from the ala temporalis (Alt, Fig. 2E) by the wide sphenorbital fissure (Sph fis, Figs. 2A,E and 3B). The ala temporalis extends far dorso-laterally to make up the lateral wall of the braincase.

The median bones of the chondrocranium are not ossified at this stage. The presphenoid cartilage (Pre crt, Figs. 2A and 3B) is thick, short, and fused along its entire length with the ala orbitalis. The basisphenoid cartilage (Bsp crt, Figs. 2A and 3B) is thinner dorsoventrally and longer than the presphenoid cartilage. It is continuous with the basioccipital cartilage (Boc crt, Figs. 2A and 3B). The median cartilages can also be seen in the median view of the head before any staining was completed (Fig. 2C).

Dorsal to the foramen magnum is the supraoccipital bone (Soc, Figs. 2A,B and 3A,B). The supraoccipital bone is a small kidney-shaped ossification of the chondrocranium at the most posterior portion of the skull. The supraoccipital, exoccipital, and basioccipital are not ossified, but their cartilaginous precursors form a complete caudal wall to the cranial cavity.

The otic capsule is large and reaches nearly to the midline (Otc, Figs. 2A,B,E and 3A). On the endocranial side, it has a separate foramen (internal auditory meatus) for the facial and statoacoustic nerves. The internal auditory meatus (Iam, Fig. 2E), and the large basal whorl of the cochlea can be seen inside the otic capsule. A small foramen pierces the otic capsule laterally, and may represent the endolymphatic duct (End for, Fig. 2E), as previously identified by de Beer (1937) in Phocoena. The occipital arch, the cartilaginous precursor to the exoccipital and basioccipital bones, makes up the caudal part of the chondrocranium. The occipital arch extends far dorsally and connects to the ala temporalis in the lateral wall of the braincase. A large occipitocapsular fissure (de Beer, 1937) is located superior to the otic capsule and ventral to the supraoccipital bone separating the ala temporalis and the occipital arch (Occ fis, Fig. 2A,E). The large, slit-like jugular foramen (Jfr, Fig. 2E) separates the otic capsule from the occipital arch. Immediately caudal to the jugular foramen is the hypoglossal canal (Hyp, Fig. 2E), and immediately medial to the jugular foramen is a small foramen, possibly for the ventral petrosal sinus.

The middle ear ossicles are located caudal to Meckel's cartilage (Fig. 4A,B). The manubrium of the malleus (Man) is continuous with Meckel's cartilage (Fig. 4A,B). The manubrium of the malleus is large, well-developed, and points ventrally. The incus (Inc) is caudal to the malleus (Figs. 3A and 4A,B). The crus longum (Crl) of the incus points ventrally, and the crus breve (Crb) points caudally (Fig. 4A,B). The crus longum is slightly thicker than the crus breve. The stapes is faintly visible oriented mediolaterally between the crus longum and otic capsule.

Meckel's cartilage (Mec) is a solid structure surrounded by the dentary (Figs. 2A,B,D, 3A,B, and 4A,B). Meckel's cartilage projects rostrally and ventrally to the squamosal bone (Fig. 2B). Near its rostral extremity, the cartilage fades where it is in close contact with the ossifying dentary.

The stylohyoid (Sth crt) and the thyrohyoid (Thh crt) cartilages are two bars of cartilage ventral to Meckel's cartilage (Figs. 2B,D, 3A,B, and 4A,B). The stylohyoid projects as a straight cartilage bar from the chondrocranium. Ventrally, the stylohyoid cartilage is connected to the basihyoid cartilage, which was damaged during preparation of this specimen. The thyrohyoid is a narrow bar of cartilage that projects caudally from this area toward the laryngeal cartilages. No ossification centers are present in the hyoid at this stage.

Late Carnegie Stage 20 and Stage 21/22

Intramembranous elements.

The premaxilla (Pmx, Figs. 3D, 5A,D, and 6D) extends into the rostrum but does not reach into the tip as a small remnant of the premaxillary cartilage remains (Pmx crt, Figs. 5B and 6A,B,D). Caudally, the dorsal aspect of the premaxilla is perforated by a foramen, presumably for a branch of the infraorbital nerve and its associated vessels. Caudally the premaxilla ends in a narrow process, rostral to the external bony nares (Ebn, Figs. 5A and 6A). The premaxilla is not fused to the maxilla but these two bones are adjacent to each other.

The maxilla (Max) is expanded rostrally and caudally (Figs. 3C,D, 5A–D, and 6B,D). Rostrally, the maxilla reaches nearly as far as the premaxilla and forms most of the rostrum overlapping the premaxilla in lateral view. The maxilla also forms most of the palate, leaving a long alveolar groove (Alg, Figs. 3C and 5B) laterally to house the developing teeth. Tooth buds are not visible and were probably not mineralized at this stage.

The vomer is ossified at this stage (Vom, Figs. 3D and 5A,C); it is a long, narrow bone in the median plane, wedged between the nasal cartilage dorsally, and the maxilla, palatine, and pterygoid bones ventrally. The vomer does not extend as far rostrally as the maxilla, but it reaches beyond the basisphenoid (Bsp) caudally (Figs. 3D, 5A, and 6A). The nasal bone (Nas) is a small circular ossification just medial to the anterior part of the frontal and caudal to the external bony nares (Figs. 5B and 6B).

The lacrimal (Lac) is a small square bone in the rostral rim of the orbit (Figs. 3C, 5D, and 6D). The lacrimal process is located at the corner of the lacrimal bone and projects into the orbit. The lacrimal is fused with the jugal (Jug), a needle-shaped bone that extends along the entire ventral side of the orbit (Figs. 3C, 5B,D, and 6B,D).

The palatine (Pal) contributes to the caudal section of the palate (Figs. 3D, 5A,C, and 6A). At midline, the palatine is caudal to the maxilla and rostral to the pterygoid (Ptg, Figs. 3D, 5A,C, and 6A). In the infraorbital region, the palatine forms the lateral wall of the nasal cavity. The pterygoid forms the caudal portion of the palate and forms a hook similar to that in the adult (Mead and Fordyce, 2009).

The frontal bone (Fro) is considerably larger than in the previous stage and extends medially (Figs. 3C,D, 5A,B,D, and 6A,B,D). Rostrally, the frontal bone is overlapped by the maxilla (Figs. 3C, 5D, and 6D). The frontal bone also forms a distinct supraorbital ridge (Mead and Fordyce, 2009), and this ridge ends as the sharp posterior orbital process (Pop, Figs. 3C, 5B, and 6B,D), which is already developing in these Stenella fetuses. Caudally, the frontal is adjacent to the parietal (Par), but it does not overlap the frontal bone (Figs. 3C,D, 5A,B,D, and 6A–D). The parietal is an oval bone and makes up most of the lateral side of the braincase. Ventral to the parietal is the squamosal (Squ, Figs. 3C, 5B,C, and 6B,D). The squamosal consists of a pars medialis (Prm, Fig. 5D) that will develop into the lateral wall of the braincase, and a pars lateralis (Prl, Fig. 5D) that will form the zygomatic process of the squamosal bone. The interparietal bone (Inp) stretches toward the medial plane, making up part of the dorsal roof of the braincase (Figs. 3C,D, 5A,B,D, and 6A–D). There are large fontanelles between the interparietal and frontal bones (Figs. 5D and 6D) and also between the parietal and supraoccipital bone (Figs. 5D and 6C).

The dentary (Den) is ossified almost to the rostral tip but does not reach caudally to the squamosal bone (Figs. 3C,D, 5A–D, and 6A,B,D). The dentary, in its rostral two-thirds, covers the diminishing Meckel's cartilage medially, laterally, and ventrally; it only covers the lateral side of Meckel's cartilage for its caudal third (Figs. 3D, 5A, and 6A).

The ectotympanic (Ect) is undergoing ossification (Figs. 3C,D, 5A–C, and 6B). It is horseshoe-shaped and its medial edge is expanding into the ventral middle ear wall. This medial edge of the ectotympanic has a rostral process. The caudal limb of the ectotympanic terminates in a semicircular and flat piece of bone. There is no sign of a sigmoid process or an involucrum.

Endochondral elements.

The chondrocranium is disappearing at this stage; cartilage is retained mostly in the ventral midline. The cribriform plate is cartilaginous at this stage, but lacks the foramen for CN I present in the previous stage. The nasal cartilage (Nas crt) or mesorostral cartilage of Mead and Fordyce (2009) is large and triangular (Fig. 6A). It covers the vomer in the median plane of Fig. 6, however, the vomer is visible in Figs. 3D and 5A where the skull is cut more laterally exposing structures lateral to midline. The nasal cartilage continues caudally to the presphenoid (Pre), which is a large ossification center present in this area (Figs. 3D, 5A, and 6A). The ossification center for the presphenoid is continuous with that for the orbitosphenoid (Orb sph) in the ala orbitalis (Figs. 3C,D, 5A,B, and 6B). The ventral part of the orbitosphenoid is ossified and the optic foramen remains large (Opt for, Figs. 3D, 5A,B, and 6B). The dorsal part of the ala orbitalis remains cartilaginous and is surrounded by the semicircular rim of the orbit, as made by the frontal bone.

The ossification center of the basisphenoid (Bsp, Figs. 3D, 5A, and 6A) is separated by cartilage from the presphenoid rostrally and is distinct from the basioccipital bone (Boc) caudally (Fig. 6A). The ala temporalis is present, but faint and its connections to the ala orbitalis and the occipital arch are not visible. An oval ossification center for the ali temporalis is present (Alt, Fig. 6B).

Laterally, the exoccipital (Exo) is ossified (Figs. 3C,D, 5A,B, and 6B,D) and the rest of the occipital arch is cartilaginous and surrounds the foramen magnum. There are clear cartilaginous occipital condyles present at this stage. Dorsally, the supraoccipital bone (Soc) is ossified (Fig. 3C,D, 5A,B,D, and 6A–D). It is a diamond-shaped, bilateral ossification in LACM 94592. In LACM 94310, the supraoccipital bone is slightly larger and consists of three parts. Right (RSoc, Fig. 6C) and left (LSoc, Fig. 6C) ossifications are bilaterally paired dorsally and a single ventral ossification is present caudally (Soc, Fig. 6C). The supraoccipital bone stretches from the foramen magnum to where the interparietal and parietal bones meet. The supraoccipital bone does not overlap with any other bone (Fig. 6C). The supraoccipital is not fused to the interparietal bone at this time. The otic capsule is faint and displays no morphological details.

Only the most caudal portion of Meckel's cartilage remains continuous with the cartilaginous malleus (Mal) of the middle ear (Fig. 4C,D). The manubrium (Man) of the malleus is thick and points ventrocaudally. The accessory ossicle (Acc, Fig. 4C,D) is a densely ossified, circular structure that overlies, and is fused to, Meckel's cartilage (Mec, Fig. 4C,D). It is not fused to the ectotympanic as in adult odontocetes (Luo, 1998). The head of the malleus is distinct and separate from Meckel's cartilage and is located slightly ventral to Meckel's cartilage (Fig. 4C,D). The incus (Inc, Fig. 3C) is cartilaginous, with the crus longum (Crl, Fig. 4C,D) directed ventrally and articulating with the faintly visible cartilaginous stapes (Stp, Fig. 4C,D). The crus breve (Crb) points caudally (Fig. 4C,D). The crus longum is still more robust than the crus breve.

The hyoid is connected to the chondrocranium caudally. The bar-shaped stylohyoid (Sth) is ossifying and is between two cartilaginous sections of the hyoid (Figs. 3C,D, 4C,D, 5B, and 6B,D). The distal cartilage bar connects to the oval basihyoid cartilage (Bas crt), from which the thyrohyoid cartilage (Thh crt) extends caudally (Figs. 5B and 6B). The basihyoid and thyrohyoid are not ossified.

Carnegie Stage 23

Intramembranous elements.

The premaxilla (Pmx) and maxilla (Max) are in close contact in this stage of development and extend rostrally the same amount (Fig. 7B). Caudally, the premaxilla widens against the orbit and forms the lateral edge of the external bony nares (Ebn, Fig. 7A). The maxilla has a long alveolar groove (Alg, Fig. 7B), and tooth buds are visible in the soft tissue of this specimen near this groove. The maxilla overrides most of the frontal bone (Fro) as well as the lacrimal bone (Lac) and nearly reaches to the edge of the orbit (Fig. 7B). Medially, the maxilla touches the nasal bone. The vomer has a dorsal groove in which the nasal cartilage is located. The vomer extends caudally as far as the basisphenoid (Bsp, Fig. 7A). The lacrimal is distinct from, and in contact with, the frontal and maxilla bones (Lac, Fig. 7B,D).

The palatine (Pal) is separated from the pterygoid (Ptg) by the premaxilla (Pmx, Fig. 7A). The pterygoid is caudal to the palatine bone and is longer in the rostro-caudal dimension as well as in the dorso-ventral dimension compared to the palatine bone (Fig. 7A). The palatine has a limited distribution on the caudal palate, wedged between the maxilla and pterygoid bones. The pterygoid projects ventrocaudally, as in the adult skull. The pterygoid does not have any visible airsacs at this stage. No medial or lateral pterygoid plates are present.

The frontal bone is expanded to form most of the rostral wall of the braincase (Fro, Fig. 7B,D). Laterally, the frontal bone forms the dorsal edge of the orbit. The parietal (Par) forms the lateral wall of the braincase (Fig. 7B–D). The interparietal (Inp) is large and square-shaped and forms the roof of the braincase (Fig. 7B,C). The frontal, parietal, and interparietal bones are separated by narrowing sutural zones and do not overlap (Fig. 7B). The parietal is overlaid to a small extent by the pars medialis (Prm) and pars lateralis (Prl) of the squamosal (Squ, Fig. 7B,D). The supraoccipital (Soc) forms a large part of the caudal wall of the braincase and is not fused to interparietal or parietal bones (Fig. 7B,C). Both the interparietal and supraoccipital bones are fused across the midline to their contralateral bone. The fontanelle is narrower than in the previous stage rostral to the interparietal bone adjacent to the frontal bone and between the frontal and parietal bones (Fig. 7A–C).

The jugal bone (Jug) is a narrow, well-ossified bar that forms most of the ventrolateral edge of the orbit (Fig. 7B,D). Rostrally, it is fused firmly with the lacrimal bone (Fig. 7B,D). The pars lateralis (Prl) of the squamosal bone is greatly expanded and nearly touches the jugal (Fig. 7D).

The body of the dentary (Den) is ossified, but the caudal and rostral parts of it are not ossified (Fig. 7A,B,D). Caudally, the dentary leaves a large mandibular foramen through which Meckel's cartilage emerges. The mandibular condyle is not ossified (Fig. 7D). The mandibular foramen (Man for), where the acoustic fat pad is located in adult odontocetes, is visible in Fig. 7A.

The horseshoe-shaped ectotympanic bone is ventral to the squamosal bone (Ect, Fig. 7B,D). The ventral side of the ectotympanic is expanding medially forming the ventral wall of the middle ear cavity, but no involucrum is present.

Endochondral elements.

Little remains of the chondrocranium at this stage. The cribriform plate is not perforated as in the youngest stage. The nasal cartilage (Nas crt), is thick and triangular, filling the entire median plane of the rostrum, and is continuous with the medial portion of the chondrocranium (Fig. 7A). The presphenoid bone (Pre) is firmly fused to the orbitosphenoid (Orb sph, Figs. 7A,B). The optic foramen, which previously perforated the orbitosphenoid, now notches this bone from the caudal side. The optic foramen is thus fused with the sphenorbital fissure as in the adult (Mead and Fordyce, 2009). The frontal bone extends ventrally in the wall of the braincase, but wide gaps remain between it and the orbitosphenoid. As such, the orbitosphenoid is surrounded by unossified fontanelles rostrally, dorsally, and caudally.

The ala temporalis (Alt, Fig. 7A,B) is a thick oval bone, connected by a thin cartilaginous bridge to the basisphenoid (Bsp, Fig. 7A). The basioccipital (Boc) is much larger than the basisphenoid and has a short flange that is directed ventrolateral (Fig. 7A). This is the falcate process or basioccipital crest of the adult basioccipital bone (Mead and Fordyce, 2009).

The middle ear ossicles and otic capsule are not clearly visible at this stage and are probably close to the stage where they undergo ossification. The accessory ossicle (Acc) is heavily ossified (Fig. 7D) and remains attached to an ossified part of Meckel's cartilage. The occipital condyles remain cartilaginous, and so does the dorsal part of the occipital arch adjacent to the foramen magnum.

The exoccipital (Exo) forms part of the caudal wall of the braincase (Fig. 7B). A process from the exoccipital extends into the hyoid arch. The hyoid arch contains ossification centers in the stylohyoid (Sth) and thyrohyoid (Thh, Fig. 7B,D), but the basihyoid cartilage (Bas crt) is not ossified at this stage.

DISCUSSION

This study allows us to discuss in detail some adaptations that are of general interest in the development of the skull in S. attenuata from stage 20 to stage 23 of Thewissen and Heyning (2007). These adaptations relate to key cranial anatomical elements of the cetacean skull. Here, we will discuss telescoping of the skull, nasal anatomy, and the development of the middle ear and ear anatomy.

Telescoping

Telescoping in odontocetes is defined by the positioning of the maxilla and premaxilla in the skull (Miller, 1923; Kellogg, 1928a, 1928b). The premaxilla and the ascending process of the maxilla override the frontal and the parietal bones pushing dorsally and caudally (Miller, 1923; Oelschläger, 1990; Comtesse-Weidner, 2007). This dorsal movement of anterior skeletal elements alters the location of the sutures between specific bones (Miller, 1923). The premaxilla touches the supraoccipital bone and the nasal, premaxilla, maxilla, parietal, and frontal bones are all in close contact (Miller, 1923). The result of telescoping in odontocetes is the reduction in the intertemporal region of the skull and it has been suggested that this facilitates anatomical adaptations for echolocation (Kellogg, 1928a; Oelschläger, 1990). Telescoping results in altering the shape of the anterior cranium and flattening of the cranial bones (Reidenberg and Laitman, 2008), to form a cradle or basin for the melon (Miller, 1923). The initial phase of telescoping during development can be seen in four (LACM 94592, 94310, 94285, 94382) of the five Stenella fetuses presented in this article.

In addition to telescoping, external bony nares position changes. This is directly and functionally related to the environment in which these animals live (Miller, 1923; Howell, 1930; Klima, 1995; Rommel et al., 2009). The external bony nares moves its position from the tip of the rostrum to the top of the forehead (Klima, 1995; Thewissen et al., 2009). The dorsal movement of the external bony nares appears gradually in evolution in protocetids and basilosaurids and throughout modern whales (Thewissen et al., 2009). As the premaxilla and maxilla extend dorsally, the external bony nares is pushed dorsally to the top of the cranium leaving a thin sliver of frontal bone exposed in adult odontocetes (Miller, 1923).

The shifting of cranial bone position is unique to odontocete telescoping (Miller, 1923; Kellogg, 1928a). This relative displacement of cranial bones is not present in Eocene whales or mysticetes (Miller, 1923; Kellogg, 1928a) The mysticete maxilla cannot override the frontal bone due to its two bony processes (Kellogg, 1928a). The ascending process of the maxilla overlaps the frontal bone while the infraorbital process of the maxilla lies under the frontal bone securing the maxilla in position (Kellogg, 1928a). Rostral movement of cranial bones occurs in mysticetes with the posteriorly located occipital bone pushing rostrally (Kellogg, 1928b). This rostral shift in mysticetes is also called telescoping (Miller, 1923).

In S. attenuata, the maxilla does not override the frontal and parietal bones in the early stage 20 fetus (Figs. 2A,B and 3A,B), but the later fetuses do show evidence of telescoping (Figs. 3C,D and 5–7). Specifically, the late stage 20 (LACM 94592, Fig. 5) and the stage 21/22 (LACM 94310, Fig. 6) fetuses exhibit the beginning of telescoping (Figs. 3C, 5D,and 6D). The premaxilla is elongated rostrally in LACM 94592 (Figs. 3 and 5). The maxilla reaches dorsally as far as the middle of the orbit. The maxilla and the premaxilla are located dorsally and partly overlap the parietal and frontal bones (Figs. 3C,D, 5A,B, and 6A,B). In stage 21/22, the gap between the frontal bone and the maxilla is not visible (Figs. 5D and 6D). Even less of the frontal bone is exposed in the older fetuses (LACM 94285 and 94382, stage 23) as telescoping is well underway.

Nasal Anatomy

The nasal anatomy of cetaceans is unique among mammals (Klima, 1995). The median nasal cartilage, as well as the bony elements of the rostrum, acts as a conduit for echolocation emissions (Cranford et al., 1996; Klima, 1999). Klima (1999) suggested the median nasal cartilage aids the growth of the embryonic rostrum in length before the bony elements are in place. Histologically, the median nasal cartilage is different from the cartilaginous nasal septa of land mammals due to the high amount of fibrous cartilage and interwoven fiber bundles (Klima, 1999).

The vomer is a thin triangular wedge of bone in the palate of LACM 94592 (Fisg. 3D and 5A,C). The vomer is rostrally almost as long as the maxilla and flares dorsoventrally near the inferior edge of the orbit (Fig. 5A). The vomer provides a cradle (the mesorostral furrow), along with the paired premaxillae, for the median nasal cartilage. The vomer grows in length as the maxilla and premaxilla lengthen and the rostrum elongates.

The median nasal cartilage has the shape of an equilateral triangle and is one of the most conspicuous parts of the dolphin skull in the early stage 20 fetus, LACM 94671 (Figs. 2A and 3B). The median nasal cartilage flares dorsally and is elongated with the outgrowth of the rostrum in late stage 20 and stages 21/22. This lengthening can be seen in LACM 94310 (Fig. 6A). The median nasal cartilage is continuous with the chondrocranium (Fig. 6A). The median nasal cartilage does not completely reach to the rostral tip as the premaxillary cartilage is still present at the very tip of the rostrum even at stage 23 of development (Fig. 7A,B). There is minor change in the median nasal cartilage after late stage 20. The monkey lip dorsal bursae or phonic lips (Au, 1993; Cranford et al., 1996; Berta et al., 2006) are not visible in any of the fetal stages discussed here.

Ear Anatomy

The three middle ear ossicles (the malleus, incus, and stapes) transmit sound from the tympanic membrane to the inner ear (Williams et al., 1995). Lancaster (1990) made theoretical predictions of the position of the middle ear ossicles in transitional cetacean ears based on the fossil record. Thewissen and Hussain (1993), Thewissen et al. (2009), and Nummela et al. (2004, 2006) documented transitional morphologies in fossils such as pakicetids, remingtonocetids, protocetids, and basilosaurids.

Sound transmission characteristics differ between air and water prompting evolutionary change in the anatomy and morphology of the middle ear of cetaceans (Nummela et al., 2007). Fifty million years ago, pakicetids had middle ear anatomy that was more similar to land mammals than modern cetaceans (Nummela et al., 2004). Pakicetids have a small mandibular foramen and lacked a mandibular fat pad, suggesting that these early whales did not hear well in water (Thewissen and Hussain, 1993; Nummela et al., 2007). Ambulocetus, remingtonocetids, and protocetids have a large mandibular foramen and, where known, middle ear ossicles that morphologically are more similar to modern cetaceans (Nummela et al., 2004, 2007). The 35 million year old middle ear of basilosaurids is considered fully modern (Nummela et al., 2004).

Echolocating cetaceans have a pachyosteosclerotic tympanoperiotic complex that is isolated from the skull by air-filled sinuses (Purves, 1966; Oelschläger, 1990; Nummela et al., 2007; Cranford et al., 2010; Hemilä et al., 2010). This isolation of the ear from the skull allows for directional hearing in water and the sinuses change volume during pressure changes when diving (Oelschläger, 1990; Reidenberg and Laitman, 2008). The isolation of the tympanoperiotic complex in archaeocetes through modern cetaceans provides more movement of the tympanic plate. This relays sound from the water, through the mandible, to the middle ear ossicles for hearing (Fleischer, 1978; Luo, 1998; Nummela et al., 2007; Cranford et al., 2010; Hemilä et al., 2010). Isolation of the tympanoperiotic complex is not definitively exhibited in the Stenella fetuses.

The involucrum is the thickened medial wall of the tympanic bone in adults; the lateral wall is thin enough to see light shine through (Nummela et al., 2007). This morphology is present in the earliest whales, back to pakicetids, and is characteristic of cetacean ears (Nummela et al., 2007; Thewissen et al., 2009). The small tympanic ring is a U-shaped ridge of bone located on the thin lateral wall of the tympanic bone. It is where the tympanic ligament attaches (Oelschläger, 1990). None of our five embryos have an involucrum and the tympanic ring is not visible. The pharyngotympanic tube is not visible in any of the fetal stages discussed here.

The middle ear ossicles of cetaceans form a chain within the tympanic bone, as in land mammals, but the orientation of the ossicles is different (Nummela et al., 2007; Cranford et al., 2010). Fleischer (1973, 1976, 1978) noted the unusual position of the auditory ossicles in cetaceans. The manubrium of the malleus is reduced in length (Fraser and Purves, 1960; Purves, 1966; Oelschläger, 1990) and points ventrally extending in a parasagittal plane in land mammals. The cetacean incus is orientated so the crus breve and crus longum are rotated and point medially (Fleischer, 1978; Oelschläger, 1990; Kinkel et al., 2001). Kinkel et al. (2001) described the embryology of S. attenuata ossicles based on histological sections of specimens. The cetacean malleus and incus were found to be rotated approximately 90 degrees around the physiological axis of rotation (Thewissen, 1994; Kinkel et al., 2001). This is considerably more rotation than seen in Pakicetus, which is considered to be the intermediate between land mammal ossicle orientation and cetacean orientation (Thewissen and Hussain, 1993).

The rotation of the middle ear ossicles can be seen in Fig. 4. The crus longum (Crl) of the incus has started to turn medially and elongate in LACM 94592 (Fig. 4C,D) exposing the stapes. The malleus is also continuing to develop and separate from Meckel's cartilage in Fig. 4C and D.

The accessory ossicle is a middle ear structure distinct, but synostosed in mysticetes and odontocetes, as well as some fetal artiodactyls (Luo, 1998; Mead and Fordyce, 2009). The accessory ossicle is homologous with the embryonic accessory ossicle in artiodactyls, which develops into the processus tubarius and merges into the bulla (Oelschläger, 1990; Luo, 1998). Embryonically in odontocetes, the accessory ossicle changes in only minor ways from its fetal shape (Fig. 4) to the adult shape (Luo, 1998). The adult odontocete accessory ossicle is actually more similar to a fetal mysticete accessory ossicle (Luo, 1998). In adult mysticetes, the accessory ossicle fuses with the anterior process of the petrosal forming a gracile pedicle connecting but not synostosing with the bulla or processus tubarius (Luo, 1998). The accessory ossicle provides an anterior junction between the tympanic bone and the periotic bone (Oelschläger, 1990) and is visible just rostral to the malleus in LACM 94592 (Fig. 4C,D). The function of this structure is not part of the middle ear model as outlined by Nummela et al. (2004, 2007) or Cranford et al. (2010).

CONCLUSION

Cleared and stained specimens provide a great resource for studying morphological developmental changes in vertebrates. Direct interpretation of anatomy from bony and cartilaginous structures is imperative for ontogenetic comparisons. Cleared and stained specimens are more easily interpreted than serial histological sections and allow for morphological comparisons to fossils. These morphological studies can help guide molecular techniques, such as immunohistochemistry and in situ hybridization, in understanding the protein and mRNA expression in cartilage and bone.

Acknowledgements

We would like to thank Sharon Usip for all her help in the lab and in the staining process.

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