Individuals in the cadaveric sample used in this study were obtained after death by natural causes in captivity (Table 1). All specimens were fixed in formalin by immersion, or were frozen and then subsequently formalin-fixed. In sum, 39 cadavers were studied, some of which were previously sectioned (e.g., Smith et al. 2015, 2021a), and some newly prepared for the present study. In addition, one sectioned prenatal loris (Nycticebus coucang), estimated to be at an early fetal stage of development, was available for study from the Hubrecht collection (Table 1). The prenatal specimens examined are not stage-matched among the species, a difficult aim even with more common primates. The youngest prenatal primate in our sample, Nycticebus, has a fully fused palate, but little ossification of the chondrocranium, and the body (based on museum photographs) remains highly flexed. This specimen is judged to be early fetal. The fetal Loris specimens are considerably more advanced in basicranial ossification and may be midgestation. Prenatal Lemur and Saguinus were assessed to be fetal based on relative head size compared to known newborns (Table 2). Furthermore, the Lemur specimen was recorded stillborn at “12–18 days preterm” at Duke Lemur Center, Durham, NC, U.S.A. Throughout the text, “newborns” or “neonates” are specimens of known age or similar to specimens of known age up to 7 days postnatal (see Smith et al. 2020 for further discussion on aging). “Infants” are also of known age, and in this sample all are between 19 and 30 days postnatal age (Table 1; Suppl. Tables S1, S2).

Most specimens were scanned using microcomputed tomography (µCT). Scanning was conducted at Northeast Ohio Medical University (NEOMED) using a Scanco vivaCT 75 scanner (scan parameters: 70 kVp; 114 mA; 380 ms exposure time). The volumes were reconstructed using 20.5–30 µm cubic voxels (depending on head size) and exported as 2048×2048 8-bit TIFF stacks for three-dimensional reconstructions (DeLeon and Smith 2014). All three-dimensional reconstructions were carried out using Amira 2019.1 software (Thermo Fisher Scientific). Segmentations were carried out automatically via the magic wand tool, to establish basicranial contours for measurements, and then separately to isolate the sphenoid bone. PLY files of selected specimens can be viewed at the following https://www.morphosource.org/projects/000367594?locale=en.

A subset of the sample was serially sectioned, including an age range (based on known ages or surmised based on varying head sizes) for Saguinus spp. (9 specimens), Saimiri boliviensis (8), Loris tardigradus (1), Otolemur crassicaudatus (2) and Varecia spp. (4). In most cases, one half of the head was sectioned in a sagittal plane in order to examine the midline synchondroses. The contralateral side was sectioned in either a coronal or horizontal plane, which enabled a better understanding of the orientation of the ABS. Routine paraffin embedding followed decalcification in a formic acid-sodium citrate solution. Sections were at 10 µm thickness, and every fourth to tenth section was mounted on glass slides. Slides were alternately stained using Gomori trichrome or hematoxylin-eosin procedures (for more details see DeLeon and Smith 2014). Serial sections were examined by light microscopy using a Zeiss stereo microscope (X0.64 to X1.6 magnification) or a Leica DMLB photomicroscope (X25 to X630). Selected sections were photographed using an Axiocam MRc 5 Firewire camera attached to the Leica microscope) or a MRc 150 Firewire camera (attached to the Zeiss microscope).

Fetal and newborn specimens of Saguinus spp. (5), fetal Lemur catta (1), and newborn Varecia variegata (1) were immunohistochemically studied to establish the mitotic characteristics of chondrocytes within sphenoidal synchondroses. The specimens were prepared using immunohistochemistry to detect proliferating cell nuclear antigen (PCNA), a marker of mitotic cells. Briefly, sections were deparaffinized and rehydrated to water. A short antigen retrieval step was accomplished in boiling Sodium Citrate Buffer for 2 minutes followed by cooling. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol, and then 1% Goat Serum was used to block non-specific binding. Sections were then incubated with PCNA primary antibody (AbCam, Cambridge, MA, USA, ab18197) diluted 1 to 3000 in Goat Serum for 2 hours at room temperature. After three washes with Phosphate-Buffered Saline (PBS) sections were incubated with Goat Anti-Rabbit Secondary Antibody conjugated for HRP (AbCam, ab6721) for 1 hour at room temperature and were subsequently washed again with PBS three times. Finally, sections were exposed to 3,3’-diaminobenzidine (DAB) (Vector Laboratories, Bullingame, CA, USA) for 3 minutes, the reaction was stopped with water. Most sections were also counterstained with Fast Green 0.1% solution diluted 1:10 in water and the sections were dehydrated, cleared, and mounted with permount (Fisher Scientific, Waltham, MA, USA). Selected sections of each specimen were also prepared as negative controls, in which the primary antibody was omitted. In most negative controls, DAB staining was barely detected; only one control slide revealed reactivity to nuclei, but the reactivity was still far less than in the slides for which the primary antibody was present (Suppl. Fig. S1).

Figure 1. 

Histology of prenatal Nycticebus coucang sphenoid bone, as seen in coronal sections. A) Anterior aspect of developing sphenoid region, where the Vidian nerve (vn) passes medially and superiorly to ala temporalis (at). B) Magnified view of AT, same section as plate a, revealing densely-packed chondrocytes, and newly formed perichondrial bone (*). C) A more posterior section shows the alatemporalis to be much smaller, and further posteriorly (D) it connects to the alar process (ap) of the lamina hypophyseos (lh); here, the Vidian nerve passes inferiorly. Further abbreviations: hy, hypophysis. Scale bars: A, 250 µm; B, 30 µm; C, D, 150 µm.

In selected cases, µCT scan volumes were manually rotated until the slice planes were aligned to histology sections of the same specimen. This was accomplished using Amira software. Approximate matches were sought, focusing on the sphenoid bone and in particular the contours of developing foramina. Subsequently, every 4th to 5th histology section was matched to corresponding µCT slices. Matching histology and µCT sections/slices were then opened in Photoshop, and the histology was resized and pasted onto the CT slice, made partially transparent, manually rotated into alignment with the µCT slice, and then made opaque and saved as a new file name. This was done for all µCT slices containing the sphenoid bone, and some space anteriorly and posteriorly. Then, using Amira, segmentation of the sphenoid bone was accomplished, using the µCT slices overlaid with aligned histology (see below).

Trigeminal nerve branches were traced to infer the extent to which foramina are formed by the cartilaginous template of the sphenoid as opposed to expansion of appositional bone. Across vertebrates, the relationship of the basicranium to the branches of the trigeminal nerve and associated ganglia have drawn attention for decades (e.g., de Beer 1929; Maier 1987; Presley 1993; Yamamoto et al. 2018). A major distinction between reptiles and mammals is the location of the initial roots of primary trigeminal branches and the trigeminal ganglia, which are housed completely within the skull of mammals, owing to a secondarily formed neurocranial wall. In reptiles, by contrast these neural elements are outside of the neurocranium (Maier 1987). The specific relationship of trigeminal branches to parts of the sphenoid anlagen have been revealed to vary, even within mammals (Maier 1987). Thus, while the trigeminal nerve branches exit in identically named foramina in many mammals, it may not develop in precisely the same way. Foramina may or may not be surrounded by cartilage during development (membranous bone completes the boundary of some foramina). Further complicating the nerve-to bone topography is that whereas a nerve branch may initially be encircled by cartilage of a chondrocranial element, portions of cartilage may regress during development, and be replaced by de novo membranous bone (e.g., Zeller 1987). In addition, the nerve to the pterygoid canal (Vidian nerve) was traced based on its position close to the interface of the basi- and alisphenoid elements. This nerve passes close to the basitrabecular process of the basisphenoid (de Beer 1929), to which the ABS is connected (Maier 1993; Smith et al. 2021a). Thus, the Vidian nerve serves as a useful landmark to identify the alibasisphenoidal synchondrosis before or after it fuses.

Cranial measurements were made on two primate genera (Saguinus geoffroyi, S. oedipus and Lemur catta) for which we had prenatal and early postnatal stages (Table 2; Suppl. Table S1). The sample sizes are small and thus interpreted with caution. To bolster samples of Lemur catta, we used one specimen of unrecorded age, which matched the size and dental eruption status of a fetal specimen that was of known gestational age. Some previously sectioned specimens had been measured using calipers to obtain cranial (prosthion-inion) and palatal (prosthion-posterior midpalatal point) length (Smith et al. 2015). For most specimens these measurements were acquired digitally using Amira, after segmentation and reconstruction. In addition, sphenoid, basisphenoid, presphenoid, and ISS lengths were measured on the ectocranial sides of the bones. Width of the presphenoid and basisphenoid were measured at the anterior and posterior ends. These measurements were used to track differences in proportions at fetal, newborn and infant ages (Tables 3–4).

The early fetal slow loris (Nycticebus coucang) has little ossification of the chondrocranium, which still possesses the cartilaginous precursors for the sphenoid bodies and for the alisphenoid (Fig. 1A). The cartilage is dense with chondrocytes, virtually devoid of extracellular matrix. Small patches of perichondrial bone can be seen bordering parts of the ala temporalis (Fig. 1B). Posteriorly the ala temporalis becomes smaller (Fig. 1C) and is connected to the alar process of the lamina hypophyseos (Fig. 1D). The Vidian nerve passes in proximity to the alar process and the medial margin of the ala temporalis. Posteriorly, near its origin at the intersection of the deep and greater petrosal nerves, it passes inferior to these structures (Figs 1C, D). Anteriorly, at the level of the lamina trabecularis (site of future presphenoid body) the nerve penetrates the ala temporalis from beneath, and courses posteriorly before exiting on the superior side (Fig. 1A).

In a later stage, fetal slender loris (Loris tardigradus), the sphenoid has three separate ossified components, the basisphenoid body, the alisphenoid-pterygoid complex, and the fused presphenoid-orbitosphenoid (Fig. 2). The alisphenoid is extensively ossified but has persisting cartilage at the base of the lateral and medial pterygoid plates (Fig. 2A). The basisphenoid and alisphenoid are united by an elongated ABS, which is the remnant of the alar process of the hypophyseal plate, as indicated by the persisting relationship with the Vidian nerve (Fig. 2B). By tracing the lateral edge of the ABS (Fig. 2C), one can also observe bone of perichondrial origin that spreads laterally as the membranous part of the alisphenoid.

Figure 2. 

Histology of three components of sphenoid bone in fetal Loris tardigradus, as seen in the coronal plane. A) Cartilage shown at bases of medial pterygoid (mpp), lateral pterygoid plates (lpp), and the alisphenoid (alis). B) The alisphenoid projects anterior to the basisphenoids, adjacent to the presphenoid (ps). Here the Vidian nerve (Vn) can be seen near its departure from the pterygoid canal. C) The alibasisphenoidal synchondrosis (abs), with membranous expansion of the alisphenoid emanating from its lateral limit. The Vidian nerve is inferior to the abs. Further abbreviations: cat, cartilage of the auditory tube. Stains: a, c, hematoxylin and eosin; b, Gomori trichrome. Scale bars: A, 150 µm; B, 300 µm; C, 75 µm.

The ABS is widely patent in the fetal Lemur catta, connecting the basitrabecular process of the basisphenoid with the alisphenoid (Fig. 3A–B); its breadth is easier to appreciate in histological sections (Fig. 3C) than in µCT reconstructions. The axis of chondrocyte columns in the ABS of the fetus lies within the coronal plane. It has a bipolar organization with proliferating chondrocytes (Fig. 3C), most of which are PCNA positive (not shown). In the fetus, histological sections indicate that no cartilage remains in the orbitosphenoid.

Figure 3. 

Fetal (A, B, C) compared to newborn (D) Lemur catta. A) The sphenoid bones are in the dorsal view, with ghosted skull for context. B) A more magnified view of the fetus reveals the basitrabecular process (btp) of the basisphenoids, positioned adjacent to the alibasisphenoidal synchondrosis (abs). The gap at the abs is difficult to discern osteologically, but histology (C) reveals it clearly, bridging the basiphenoid (bs) and alisphenoid. Note the Vidian nerve (Vn) approaching the posterior side of the pterygoid canal from below. D) In the newborn, histology (not shown) reveals no vestige of the cartilage of the abs. The basitrabecular process is still apparent. Note that the intrasphenoidal synchondrosis (iss) is proportionally reduced in the newborn compared to the fetus. Further abbreviations: bo, basioccipital; ps, presphenoid. Scale bars: A, 1.5 mm; B, 0.5 mm; C, 200 µm; D, 1.5 mm.

Compared to the late fetal Lemur catta, in the newborn both the ISS and ABS become reduced; no trace of cartilage can be found at the ABS in one serially sectioned newborn (histology not shown). At birth, the alisphenoid bone is greatly expanded anteroposteriorly (Fig. 3D). The alisphenoid, basisphenoid, and presephenoid all become proportionally more elongated anteroposteriorly in the newborn. The orbitosphenoid enlarges less by comparison. Organization of midline synchondroses in Varecia spp. was previously described (Smith et al. 2021a); here we confirm PCNA-reactivity in the proliferating chondrocytes zone of all midline synchondroses at birth. A newborn Varecia exhibited PCNA-reactive chondrocytes within SOS (Suppl. Fig. 2A). The chondrocytes were lightly-labeled, perhaps due to lengthy fixation in formalin over decades. However, the chondrocytes were above the background level in control slides (Suppl. Fig. S2B).

In Saguinus geoffroyi, there is remarkable reduction of intrasphenoid synchondrosis and ABS from fetal to newborn stages of development. Because the Saguinus fetus is not a late stage, the magnitude of reduction is much greater than observed in fetal vs. newborn Lemur catta. Ratios of ISS length to total anteroposterior sphenoid length reveal a large difference in the ISS between ages. In the fetus, the ISS comprises 41% of the midline length of the sphenoid (Table 3), whereas in the newborn the ISS is reduced to ~ 7% of sphenoid length. The proportions of the pre- and basisphenoid bodies also differ greatly, with the width/length ratio of each being far smaller in the newborn, especially for the presphenoid (Table 4). This reflects a great elongation of each bone proportionally. The ABS is oriented anterolaterally, and across age, the growth of the alisphenoid bone expands in all directions, but notably in anteroposterior breadth (Fig. 4).

Figure 4. 

Fetal (A, C) and newborn (B, D) specimens of Saguinus geoffroyi. A, B) The sphenoid bone is shown in ventral view, with the whole skull in ghosted view. Note the marked decreased distance between the basisphenoid (bs) and presphenoid (ps). Also note that the alibasisphenoidal synchondrosis (abs), between the basisphenoid and the alisphenoid (alis), oriented laterally but also anteriorly in the fetus, appears fused or nearly so at birth. C, D) Enlarged dorsal views show the degree of ossification of the sphenoid overall. The fetal specimen has a more precociously ossified basisphenoid and alisphenoid relative to the presphenoid and orbitosphenoid (os). In the enlarged view of the fetus (C), the orbitosphenoid is shown to be in a very early point in ossification; the ossified element is the inferior (metoptic) root. Also note the position of the basitrabecular process (btp), located at the posterior margin of the abs. This landmark can also be seen in the newborn (D, arrows), but the synchondrosis is greatly reduced at birth. Further abbreviations: bo, basioccipital. Scale bars: A, 1 mm; B, 1.5.mm; C, 750 µm; D, 1.5 mm.

In the fetal Saguinus geoffroyi, the presphenoid, basisphenoid, alisphenoid, and orbitosphenoid are separate elements (Figs 4C, 5A), the latter represented only by a metoptic root. By comparison to the newborn (Fig. 4D), the alisphenoid is anteroposteriorly reduced in the fetus. The basitrabecular process is a prominent spike, directed laterally (Fig. 4C) as observed in the late fetus as well as in the newborn specimen. Both the intrasphenoid and ABS are greatly reduced in the newborn (Figs 4B, D).

Based on a comparison of earlier to later stages, the sphenoid bone of Saguinus spp. is proportionally longer in the older specimens with a relatively reduced ISS (Table 2). The ISS is narrower in the newborn (Fig. 5B, D) compared to the fetus (Fig. 5A, C). Histology indicates it remains an active growth center at birth. There are a greater number of cells and absolute length in the proliferating and hypertrophic zones in the fetus (Fig. 5E) as opposed to the newborn (Fig. 5F). In both the ISS and PSept, the proliferating zone is shorter by more than 50% in absolute length in newborns (Figs 5F, H) compared to the fetus (Figs 5E, G). In the fetus, PCNA is expressed by cells throughout the proliferating zone and by chondrocytes in numerous rows of the hypertrophic zone (Figs 5E, G). In the newborn, proliferating chondrocytes and only a restricted number of hypertrophic chondrocytes, nearest to the proliferating zone, are PCNA positive (Figs 5F, H).

Figure 5. 

Fetal (left side) and newborn (right side) midline synchondroses in Saguinus spp. A, B) Midline segments of Saguinus geoffroyi crania are shown at the two ages revealing the decreased space between the presphenoid (ps) and basisphenoid (bs) bones in the newborn (B) compared to the fetus (A). C, D) low magnification views of the midline synchondroses in the fetus (C, same specimen as A) and a newborn (D, S. oedipus used instead due to better preservation); both stained with Gomori trichrome procedure. Indicated are the intrasphenoidal (iss) and presphenoseptal (psept) synchondroses. Below, higher magnifications of the iss (E, F) and PSept (G, H) at the same ages, prepared using PCNA immunohistochemistry, with fast green counterstain. Since all higher magnification images are at the same magnification, it is clear that the absolute length of the zones of proliferating (pz) and hypertrophic (hz) chondrocytes is reduced in the newborn compared to the fetus. Further abbreviations: fr, frontal; os, orbitosphenoid; bo, basioccipital. Scale bars: A, 1 mm; B, 2 mm; C, D, 1 mm; E–H, 30 µm.

The ABS was PCNA-reactive at birth as well, but the organization of proliferating chondrocytes was more difficult to visualize in newborns (Fig. 6E, inset) than in the fetus (Fig. 6C). Older infant specimens were not studied here using immunohistochemistry, pending a broader cross-sectional age study.

When histologically sectioned in the coronal plane, the ABS of fetal Saguinus geoffroyi appears as a roughly oval cartilaginous mass (Suppl. Fig. S3), in which organization of the chondrocytes is not discernable. Sagittal sections reveal the ABS in a longitudinal view (Suppl. Fig. 3; Figs 6A, B). Chondrocytes are clearly organized into proliferating and hypertrophic columns; proliferating chondrocytes and the first rows of hypertrophic chondrocytes are PCNA-reactive (Fig. 6C). At birth, the ABS of Saguinus geoffroyi or Saguinus oedipus has fewer rows of proliferating chondrocytes than in the fetus. It is also redirected to be oblique, oriented anteroinferiorly, within the sagittal plane (Fig. 6D). The organization is bipolar. However, proliferating chondrocytes are no longer well-organized into columns, and fewer hypertrophic chondrocytes are visible (Fig. 6E). PCNA reactivity is still visible (Fig. 6E, inset), but not consistently near the ossification front.

Figure 6. 

The alibasisphenoidal synchondrosis (abs) in late fetal Saguinus geoffroyi (A–C) and newborn Saguinus oedipus (D, E). A) The approximate level of the sagittal histological section of the fetal sphenoid bone is shown as a dashed line over the basal view of the skull (sphenoid emphasized, remainder of skull ghosted in black). B) a parasagittal section revealing an elongated abs. C) a section near the one shown in B shown at higher magnification, prepared using immunohistochemistry to PCNA, counterstained with fast green. Note chondrocytes in the reserve (rz) and proliferating zones (pz) are PCNA+, but most chondrocytes in the hypertrophic zone (hz) are PCNA-negative. D, E) low and higher magnifications of abs in newborn S. oedipus. Note the growth is now re-directed slightly inferiorly from the basitrabecular process (btp) of the basisphenoid bone, and the abs is proportionately shorter in anteroposterior breadth. Inset: A preparation of a nearby section to that in e (see box for specific location), made using immunohistochemistry to PCNA, counterstained with fast green. Multiple chondrocytes are PCNA + (arrows), but few chondrocytes are organized into columns. Note the close relationship of abs to the Vidian nerve (Vn). Further abbreviations: alis, alisphenoid; at, auditory tube; bo, basioccipital; bs, basisphenoid; cat, auditory tube cartilage; cnVg, trigeminal ganglion; ps, presphenoid. Stains: Gomori trichrome, except for inset (see above). Scale bars: A, 1mm; B, 100 µm; C, 40; D, 250 µm; E, 80 µm; inset, 20 µm.

Perinatally, the ISS is widely patent in all strepsirrhines examined here (Fig. 8). In Lemur, the ISS comprises nearly 20% of the anteroposterior length of the sphenoid bone in the smallest (late fetal) specimens (see Table 3). This percentage decreases in newborns (~13%) and still more in infants (~8%) (Table 3). A stark difference exists in absolute dimensions of the sphenoid bone in fetal compared to newborn Saguinus geoffroyi, and this reflects changing proportions of the bone. The length/width ratios of pre- and basisphenoids are near 1.2 in the fetus, but the relative length of the presphenoid drastically increases in the newborn (Table 4). In length, the increase in presphenoid length (361%) far exceeds that of other cranial measurements, including basisphenoid and overall cranial length (Table 3).

It should be noted that the comparison of fetal to newborn sphenoid dimensions are not analogous in the Saguinus spp. and Lemur catta. The prenatal Saguinus geoffroyi specimen is clearly a much earlier stage fetus than the prenatal Lemur catta; this is reflected in the % increase in cranial length (Table 3). However, unlike Lemur catta, in Saguinus spp. the % changes in presphenoid length scale much differently than % changes for basisphenoid length (Fig. 7). This implies growth of the sphenoid segments is more closely isometric in Lemur.

Figure 7. 

Length-width ratios of the presphenoid and basisphenoids shown across ages in subadult lemurs (Lemur catta) and tamarins (Saguinus spp.). The fetal to newborn data are from Saguinus geoffroyi, whereas the newborn to infant data are from Saguinus oedipus. Because the data are from two different species; there is some discrepancy between newborn data points for the presphenoid (by chance, this is not the case for the basisphenoid). The presphenoid stands out as a segment that is elongated (higher length-width ratio) compared to the basisphenoid; these preliminary data indicate this may be a proportional change during later fetal development

The sphenoid bone of the newborn Varecia rubra is relatively narrower in width and anteroposteriorly longer than the late fetal Varecia rubra, and especially so compared to the older infant Varecia variegata (Fig. 8). This same contrast applies to the presphenoid and basisphenoid bodies as well. This is quantifiable in the cross-sectional age series of Lemur. The width/length ratio of the pre- and basisphenoid bodies decreases progressively in newborn compared to fetal specimens, and to a lesser extent in infant compared to newborn specimens (Table 2). Across age, the orbitosphenoids increase greatly in breadth, whereas the alisphenoids expand superiorly at the anterior and anterolateral sides (Fig. 8).

Figure 8. 

The sphenoid bone in two specimens of Varecia spp, revealing perinatal transformation of the different components of the bone. A, C) a stillborn specimen of V. rubra that was undersized compared to newborn specimens, and presumably at a late fetal stage. B, D), a 24-day-old infant V. variegata. The top row shows a ventral view, the bottom row shows an anterior view, slightly lateral to the left side. Further abbreviations: alis, alisphenoid; bs, basisphenoid; fr, foramen rotundum; mpp, medial pterygoid plate; lpp, lateral pterygoid plate; os, orbitosphenoid; ph, pterygoid hamulus; pc, pterygoid canal; ps, presphenoid. Scale bars: A, C, 1.5 mm; B, D, 1 mm.

Neither of the lemurids has a widely patent ABS at birth. In contrast, the ABS is widely patent in late fetal Otolemur (Fig. 9). This specimen, at ~ 90 % of average head length for newborn Otolemur crassicaudatus, has well-organized columns of proliferating and hypertrophic chondrocytes, with some chondrocytes expressing reactivity to PCNA antibodies (Figs 9E, F). A newborn Otolemur crassicaudatus also possesses a wide ABS. In both specimens, which are close in cranial length (Table 1), the average (right and left) transverse breadth of the ABS is just less than 1 mm. In an infant (not histologically sectioned), the gap at ABS has narrowed considerably, measuring an average of 0.2 mm in transverse breadth (Suppl. Fig. S4).

Figure 9. 

Late fetal cranium of Otolemur crassicaudatus, showing the synchondrosis (*) between the alisphenoid (alis) and basisphenoid (bs). The slice plane in CT at top left (A) is indicated by a red dashed line through the endocranial view of the skull (B). C) Histology from a twin sibling, closely matching the CT slice plane. D) A magnified view shows this is a bipolar synchondrosis, with a zone of hypertrophic chondrocytes (hz) adjacent to the alisphenoid and basisphenoid. E) A higher magnification view, enlarged from boxed area in D reveals rows of proliferating chondrocytes (pz) adjacent the hypertrophic zone. F) A higher magnification view in a nearby section is prepared with PCNA immunohistochemistry. Note PCNA-reactive chondrocytes (arrows), which are strongly reactive in proliferating chondrocytes and moderately in hypertrophic chondrocytes (box in (E) shows the approximate location of plate f). Further abbreviations: bo, basioccipital. Scale bars: A, 1.5 mm; B, 4 mm; C, 0.5 mm; D, 100 µm; E, 50 µm; f, 10 µm.

The proportions of the bodies appear to differ little between newborns and the older infant Saguinus oedipus, and this is supported by the slight difference in width to length ratios of the pre- and basisphenoid (Table 4). The ISS decreases slightly, as a percentage of total sphenoid length (Table 3). Across age, the orbitosphenoid increases in breadth and the anterolateral sides of the alisphenoid expand superiorly. Ossification is more progressed overall at one-month, including anterior and lateral expansion of the alisphenoid and orbitosphenoid. Additionally, the pterygoid canal of the newborn is not fully enclosed, but is more fully enclosed at one-month (Fig. 10). Correspondingly, cartilage within the ABS synchondrosis is markedly reduced at one-month of age, appearing to consist only of small islands of cartilage at the joint site.

Figure 10. 

Transformation of Saguinus oedipus sphenoid bone comparing newborn (A, C) and one-month-old (B, D) displaying different components of bone. Images presented in ventral (top row) and slight anterolateral view (bottom row). Further abbreviations: alis, alisphenoid; amfo, anterior margin foramen ovale; bs, basisphenoid; mpp, medial pterygoid plate; lpp, lateral pterygoid plate; ph, pterygoid hamulus; ps, presphenoid; oc: optic canal; os, orbitosphenoids; pc, pterygoid canal; fr, foramen rotundum. Scale bars, 1.5 mm.

The sample of Saimiri boliviensis is a narrower age range, and midline synchondroses are similar to those of Saguinus (e.g., bipolar ISS), which were previously described (Smith et al., 2021a). However, the ABS is variable in our sample. Cartilage remains in the ABS in 5 of 8 specimens that were serially sectioned. The specimen with the smallest head size also has the widest separation between the alisphenoid and basisphenoid (Figs 11A–C). As in perinatal Saguinus, the cartilaginous ABS has a slightly downward and lateral trajectory. Yet, when histology is superimposed over CT slices and three-dimensionally reconstructed (Figs 11D-E), the ABS is shown to primarily orient anteroposteriorly, as in newborn Saguinus spp. The spatial relationships to branches of the trigeminal and Vidian nerves (Figs 11C, 11F) and auditory tube (Fig. 11C) are precisely the same as in Saguinus (Fig. 6D).

Figure 11. 

Histology of the alibasisphenoidal synchondrosis (abs) in a perinatal Saimiri boliviensis, shown in coronal sections. A–C) anterior to posterior sections, showing that the abs connects to the alisphenoid (alis) anteriorly (A), and to the basitrabecular process (btp) of the basisphenoid (bs) posteriorly (C). D-F) Reconstruction of the same stillborn Saimiri boliviensis based on aligned CT and histology slices. D) Sphenoid shown in dorsal view with the cartilaginous abs shown in red. The white dashed lines indicate the levels of section A and C. E) Magnification of the same view, with the path of the maxillary (V2) and Vidian nerve (Vn) indicated by green dashed lines. F) Sphenoid, in anterior view, shows all three major trigeminal nerve branches and the course of each nerve passing through its respective foramina. Further abbreviations: cat, cartilage of auditory tube; fo, foramen ovale; os, orbitosphenoid; fr, foramen rotundum. oc, orbital canal; ps, presphenoid; iss, intrasphenoid synchondrosis; sof, superior orbital fissure; V1, ophthalmic division of trigeminal nerve; V3, mandibular divisions of trigeminal nerve. Scale bars: A–C, 0.5 mm; D, 1 mm; E, F, 1.5 mm.

The most detailed report on sphenoid development in a non-human primate focused on Loris tardigradus (Ramaswami 1957). Earlier stages of development have been described in Galago senegalensis (Kanagasuntheram and Kanan 1964), but these stages precede ossification. Likewise, embryonic stages of cranial development are well described in some Old World monkeys (e.g., Hendrickx 1971), but later (fetal) stages that reveal patterns of ossification have been virtually ignored in the literature.

In Loris tardigradus, the basisphenoid ossifies prior to the presphenoid, as in humans. There is a single center described for the BS body, and paired alar centers. As in humans, the posterior crus of the orbitosphenoid and presphenoid body appear as separate centers and fuse; the anterior crus fuses later (Ramaswami 1957). From our observations, we can confirm that the presphenoid and orbitosphenoid have independent ossification centers in Saguinus geoffroyi, and it is most certainly the posterior crus of the latter which ossifies earliest (Fig. 5C). This may indicate a common pattern among primates, as we now have examples in both suborders. However, Maier (2000) interpreted that the presphenoid bone of papionins may form by spreading from the orbitosphenoid and into the presphenoid body. Thus, more studies may be required to establish the degree of variation in how ossification centers emerge.

Our sectioned Loris tardigradus specimen establishes that the membranous portion of the bone initiates ossification as a perichondrial extension of the incipient ABS (Fig. 2C), which develops within the alar process of the ala temporalis. This mode of enlargement is already well known based on Maier’s (1987) study of Monodelphis. The Loris fetus also reveals that endochondral growth of the alisphenoid begins at the medial side of the ABS, where it connects to the basitrabecular process of the basisphenoid. This assumes the slender loris goes on to form a bipolar organization of the ABS, as seen in all other primates described here and previously (Smith et al. 2021a).

Regarding the sequence of fusion of portions of the sphenoid, all species fuse the presphenoid/orbitosphenoid complex prior to the basisphenoid and alisphenoid. The former pair may fuse closer to mid gestation, whereas the ABS persists in later fetal (e.g., Fig. 3B), or at least to perinatal age. After the four parts of the sphenoid have formed, the sphenoid participates in at least five active growth centers, the SOS, ISS, PSept, and ABS (paired). We have too few specimens to quantify prenatal changes in synchondroseal growth. But our qualitative and immunohistochemical observations imply that proliferation of chondrocytes in all of these synchondroses is likely very rapid prenatally. In the earliest stage fetus we studied (fetal Saguinus geoffroyi), the numerous rows of proliferating and hypertrophic chondrocytes and widespread PCNA activity are impressive compared to newborns. This likely reflects the rapid establishment of pre- and basisphenoid bodies, as well as midline growth of the entire cranium. In midline synchondroses, PCNA activity remains ubiquitous in the proliferating zone at birth, but becomes more restricted in the hypertrophic zone. The implied age-related reduction in mitosis is also characteristic of rodents (Dai et al. 2017). Our report adds some new depth to our understanding of the ABS, which has rarely been discussed comparatively, although it has long been known to exist where the basitrabecular process of the alisphenoid meets the alisphenoid (see Maier 1987, and references therein for further discussion). The reason for this may be its comparatively small size, which means it appears in only a small space even among serial sections, and thus visualization is difficult. However, it has been clearly pictured by authors such as Spatz (1964) who revealed that in the tree shrew (Tupaia glis), it appears as a transversely oriented bar connecting the basi- and alisphenoids (fig. 23, therein). In addition, the synchondrosis is clearly indicated as a transverse connection between these two parts of the sphenoid in mice (Mus musculus) by McBratney-Owen et al. 2008, see fig. 9B, therein). Thus, in a rodent, a scandentian, most strepsirrhines we studied here (e.g., Figs 3C, 8) the ABS, and its growth potential, is directed primarily in a lateral direction. Moreover, it is more lateral in mid-fetal than perinatal Saguinus geoffroyi (Fig. 6). In light of this, it appears to be the case that a lateral orientation of the ABS is primitive for eurchontoglirans.

Maier (1993) discusses the ontogenetic shift of the site of ABS prenatally. It begins as a cleft between the hypophyseal plate (which gives rise to the basisphenoid) and the ala temporalis (forms the base of the alisphenoid). Later it forms a synchondrosis between these cartilages and then transitions to a suture. Maier (1993) refers to the site as the “junctura basopalatini” after Gaupp (1910, cited therein), but notes that it has not been given an official anatomical name (we have suggested alibasisphenoidal synchondrosis or suture for its later stages). Based on Maier’s illustration of the chondrocranium of a marsupial (Monodelphis) the joint (1987, fig. 3) is laterally and slightly anteriorly oriented. This could indicate that a lateral orientation is plesiomorphic for all therians.

Monkeys, in contrast including the platyrrhines described here as well as at least one catarrhine (Papio anubis) have an ABS that is directed more anteriorly and inferiorly from the basisphenoid. Further observations are clearly needed to assess the broader therian pattern on the one hand, and on the other hand whether all of Anthropoidea are similar in a derived anteroinferiorly directed ABS. Here we also confirm the ABS bears characteristics of an active growth center at fetal stages, including well-organized proliferating and hypertrophic chondrocytes, and PCNA reactivity indicating ongoing mitoses, as discussed more fully below.

Although the late fetal Lemur catta of known age is only estimated to be ~2 weeks premature (see above) by comparison to average newborn cranial length, a 24% increase is indicated (Table 3). The same age comparison suggests a modest decrease in ISS diameter, and that the sphenoid length increases in tandem with skull length (26%, Table 3). A comparison of infants to newborns suggests the sphenoid may grow more rapidly than the skull as a whole during infancy, and the ISS diminishes (~20%) during this interval (Table 3). The disparity of the presphenoid and basisphenoid length between age groups is not as stark as in the Saguinus comparisons (Table 3; also see Fig. 7 regarding proportional changes).

Fetal and newborn Saguinus geoffroyi are markedly different in cranial length (Table 2). Cranial length differs between the fetus and average newborn cranial length by 64% (Table 3). A comparison of ISS diameter reveals a 65% decrease suggesting this synchondrosis may be adding bone to adjacent surfaces of the basisphenoid and presphenoid rapidly during fetal growth. However, the sphenoid differs by 112% in length, suggesting other sphenoidal synchondroses lead to non-isometric growth. This is supported by percentage increase in presphenoid length (361%), which far exceeds that of the basisphenoid, which differs by ~100% in length between the fetus and newborn (Table 3). Assuming the fetal specimen is typical for its stage, these differences suggest the presphenoid outpaces the basisphenoid in fetal longitudinal growth, and that of cranial length. This certainly agrees with qualitative impressions (Fig. 5), and observations that the anterior limit of the presphenoid projects within the midface at birth (Smith et al. 2017). A comparison of the average cranial and sphenoid dimensions in newborns compared to a single one-month-old suggests a more modest pace of growth during early infancy. If the one-month-old is typical for its age, our findings suggest a modest change (~6 %) in cranial length during the first month. But again, the sphenoid, and especially the presphenoid, are outpacing growth of the cranium as a whole (Table 3; 34% increase in presphenoid length, versus 6% for the cranial length).

Our analyses suggest the sphenoid bones have differing midline growth trajectories in platyrrhines and strepsirrhines. Newborn platyrrhines have proportionally longer presphenoids than basisphenoids and our measurements hint that this manifests prenatally, because it is rostrally projected toward the septal cartilage at birth. Proportionally large presphenoids also typify Old World monkeys (Smith et al. 2021a) and this suggests that in monkeys growth in this bone occurs at a faster rate than other sphenoid parts, perhaps especially at the rostral end. Although this may seem counterintuitive in light of limited facial projection in most monkeys, endochondral growth is also redirected more inferiorly in monkeys (Smith et al. 2017, 2021a). Strepsirrhines, by comparison, have a sphenoid growth pattern that is closer to isometric within (i.e., basi- versus presphenoid) and relative to the skull. This may also mean that interstitial chondral growth, as opposed to that which occurs during endochondral ossification, contributes greatly to length increase in the septal cartilage of the strepsirrhines. Similarly, Wealthall and Herring (2006) clearly showed that proliferating chondrocytes of PSept were insufficient to fully explain septal cartilage growth in mice, and that interstitial chondral growth throughout the entire septum explained the remaining growth.

Another synchondroseal growth pattern that differs between strepsirrhines and monkeys occurs at the ABS. Here, we confirm our previous observations (Smith et al. 2021a) that organization of chondrocyte columns in ABS is directionally different in these primate groups, being relatively more laterally directed in strepsirrhines and more anteriorly directed in monkeys. Yet, the findings herein suggest the most significant timeframe in which the ABS might affect the middle cranial fossa occurs prenatally. PCNA reactivity supports a hypothesis that it is active in fetal specimens, and may be present but quiescent or with limited growth potential at birth. However, the evidence also confirms the synchondrosis is greatly diminished in dimensions by the perinatal age, with reduced PCNA reactivity. Therefore, we postulate its primary importance as an active growth center is prenatal. A subtle but notable difference exists between fetal and newborn Saguinus. The ABS is directed more laterally in the fetus than in the newborn. In newborns, the ABS is directed both anteriorly and ventrally. The same trajectory is seen in the ABS of newborn Saimiri, described above, and in Papio (Smith et al. 2021a). This may signify a redirection of interstitial growth late in ontogeny. As this site has been studied little, future work must consider whether important growth events occurring in the middle cranial base affect overall form, as suggested recently by Bastir and Rosas (2006). The implied inferior redirection of the ABS coincides with the same altered trajectory at PSept: initially it orients anteriorly, but in newborns PSept is inclined downward. One possible role of ABS is to ensure the alisphenoid coordinates with downward facial orientation in anthropoids, which is critical because the sphenoid is a key interface between the developing facial skeleton and cranial vault.

The bilateral elements of the sphenoid rely more on appositional bone growth. We should note this begins even prior to endochondral ossification of the ala temporalis in Loris tardigradus. The alisphenoid expands within perichondrium of the unossified tip of the ala, with appositional bone extending laterally in the form of a thin plate. This phenomenon was described in detail also in Monodelphis domestica by Maier (1987; termed Zuwachsknochen). In Lemur, the orbitosphenoid ossifies before birth. Thus, perinatal expansion of the orbitosphenoid, like the alisphenoid, is entirely via appositional expansion. Our older infant samples show this continues, and may accelerate, during early infancy.