Thecodont tooth attachment and replacement in bolosaurid parareptiles

Bolosaurus grandis

The lingual side of a right dentary fragment of Bolosaurus grandis [ROMVP 83327 (Fig. 1)] is damaged, exposing the trabecular structure of the alveolar bone. Three teeth are present and show enamel ridging that radiates from the apex in all directions, extending as far down the crown to the greatest horizontal extent of the tooth. This ridging is described extensively and is present on both Bolosaurus grandis and Bo. striatus, but is more pronounced in B. grandis (Reisz, 2006). Comparisons with the holotype of Bo. grandis reveals that the three teeth represent tooth loci 10, 11, and 12 in the dentary series. The first preserved tooth (tooth 10) has visible wear and past the apex of the cusp. The thickness of the enamel varied with a maximum extent of 205 µm related to the amount of visual occlusion and could be seen through histology and CT. The second tooth is broken on its mesial side, exposing the pulp cavity. This tooth did not have an extensive enamel covering, but this may have been the result of breakage. The final and most massive tooth is in the best condition, and also shows evidence of light wear. Neither method was able to reveal tissue that could confidently be associated with cementum in ROMVP 83327.

The isolated left dentary OMNH 52311 (Fig. 2) is the holotype for Bolosaurus grandis (Reisz, Barkas & Scott, 2002), and it possesses 14 tooth loci. Seven anterior teeth are broken near the bases of the crowns but still have well-preserved roots. The tooth crowns are ridged from their apex to their greatest lateral extent before the crown-root junction. As suggested in the original description, CT data show that the roots of the three anteriormost teeth are procumbent within the dentary, as seen in other bolosaurids (Reisz et al., 2007). The cheek teeth become more bulbous posteriorly along the dentary, but the most posterior tooth located at the base of the coronoid eminence in locus 14 is very small, unridged and peg-like. The digitally segmented roots of the teeth are proportional to the size of the crowns. Resorption pits are present on the dentary only at loci 12 and 13, which house developing successional teeth. No other tooth positions appear to have replacement teeth at any stage of development. The roots of the functional teeth at these loci show modest resorption. Locus 11 is a large, exposed resorption pit. The lack of any dentine, even at depth, suggests the tooth was shed and not broken. Locus 10 contains a small resorption pit directly below the pulp cavity; however, anterior positions are lacking these sites.

OMNH 15104 is a left maxillary posterior fragment, also included in the original description of Bo. grandis (Reisz, Barkas & Scott, 2002). It contains five complete teeth with no tooth gaps (Figs. 2D–2E). Rather than the typical posterolateral tooth cusps on the dentary, these teeth feature posteromedially directed cusps to support occlusion. The three anterior teeth show extensive lingual wear, appearing as a nearly flat surface compared to the concave shape present in the two posterior teeth. CT examination shows that a significant portion of the maxillary bone is missing, exposing the tooth and the alveolar bone.

Belebey chengi

IVPP V 15907 is a posterior right dentary fragment of Belebey chengi (Müller, Li & Reisz, 2008: Figs. 3A–3B). An additional resorption pit appears behind the final tooth (Fig. 3B); however, there is no evidence of an extra tooth developing inside the dentary within this locus. Every locus contains a replacement tooth associated with a large resorption pit. While not as bulbous as Bolosaurus grandis, the teeth are expanded laterally along the tooth row and still have an occlusal surface at different stages of wear from the cusp to the lingual side along the mesial face.

IVPP V 12007 is a right dentary of B. chengi (Müller, Li & Reisz, 2008: Figs. 3C–3F). This is a well-preserved specimen containing 11 teeth, and there are no gaps along the tooth row. Recurved cusps are heavily worn on the anterior five teeth, but posteriorly from locus six, the teeth show only slight wear on their crowns, and they form a pattern of successively increasing lateral size similar to that seen in Bolosaurus. In contrast to the condition seen in Bolosaurus grandis, the teeth are vertically oriented around the symphyseal area, rather than being inclined anteriorly. Resorption pits on the medial face of the bone are present at each tooth position, and they are relatively shallow and circular in outline. Heterodonty follows the same trend as in Bolosaurus grandis, but it is more gradual. Each tooth generally follows the same morphology as its predecessor along the jaw, with only slight modifications to their shape. CT scan data show that spacing between the developing teeth increases posteriorly along the dentary as the functional teeth occupy a greater surface area of the dentary bone (Fig. 4).

Bolosaurid tooth attachment and implantation

The CT scan data show that the teeth have deep roots that are ankylosed to the jawbone (there is no space for a ligamentous connection between the teeth and the tooth sockets). Both the labial and lingual walls of the tooth roots are equally long, in contrast to the asymmetry between the labial and lingual sides commonly found in early amniotes (Gordon Edmund, Edmund & Museum, 1960; LeBlanc & Reisz, 2015). All the functional teeth have roots that extend far into the jaw but do not come into direct contact with the maxillary or dentary bone itself, remaining completely encased within the trabecular alveolar bone.

In histological sections, the alveolar bone surrounding each tooth is separated from the jawbone by a distinct reversal line (Figs. 5A and 5C), indicating that it is resorbed and redeposited with each tooth replacement event (LeBlanc & Reisz, 2013). The alveolar bone at each locus forms a circular layer of trabecular bone around the dentine root of the tooth. The alveolar bone from younger teeth crosses through the alveolar bone of older neighbouring tooth loci, even causing partial resorption of the dentine of older teeth in some cases (Figs. 5A and 5C). Under cross-polarized light, the alveolar bone is usually optically extinct, suggesting that it consists of a woven bone matrix (Fig. 5B). However, closer inspection reveals a network of parallel fibers within the alveolar bone that are interpreted as Sharpey’s Fibres extending towards the root surfaces (Fig. 5D). These partly mineralized fibers cross through the alveolar bone towards the tooth root surfaces. These fibers are small, but are visible in the alveolar bone of neighboring teeth (Fig. 5D). The dentine and surrounding alveolar bone do not make direct contact. The histological sections reveal a layer of acellular cementum around each tooth root (Figs. 5C and 5D). This comes into direct contact with the surrounding alveolar bone, supporting an ankylosis model of attachment. This is similar to tooth attachment in other early amniotes (LeBlanc & Reisz, 2015; LeBlanc et al., 2018).

Enamel thickness and ornamentation

Contrast and resolution in the CT data were such that it was not possible to differentiate the cementum layer coating the tooth roots, but the thickest sections of enamel did appear in the CT. The combination of CT data and histological thin sections did permit a complete differentiation of dental and mandibular tissues of Bo. grandis (Fig. 1), including relatively thick layers of enamel. In the sagittal section, the enamel is non-uniform in thickness, showing occlusal wear on the labial face as well as ornamentation (Fig. 1K). The enamel-dentine junction (EDJ) is a smooth line throughout the crown, whereas the external surface of the overlying enamel is jagged, forming the external striations visible in many specimens. The differences in shape between the EDJ and the enamel surface indicate that the striations are formed by the enamel-producing cells and are not influenced by the underlying dentine whatsoever. At its thickest points, the enamel measures 205 µm thick measured perpendicular to the EDJ towards the outer surface of the enamel seen in Fig. 1K (Sander, 1999; Hwang, 2011). For comparative context, the enamel in bolosaurid teeth is much thicker than that of captorhinids, but is slightly thinner or comparable to the enamel of diadectids and many herbivorous dinosaurs. However, the teeth are much larger in diadectids and herbivorous dinosaurs.

Bolosaurid tooth replacement

In the holotype of Bolosaurus grandis, there is a single locus, position 11, where the tooth is absent. The empty space is a rounded depression lined by alveolar bone (Fig. 2C). We interpret this as the penultimate stage of the tooth shedding process when the root of the functional tooth has been shed, but the replacement tooth has not yet fully erupted and fused in place. The replacement tooth likely fell out post-mortem before the specimen was buried.

The Bolosaurus grandis specimens all had deep roots surrounded by supporting tissues. Of the three specimens, only a single dentary, the holotype, had any unerupted teeth. The other two specimens, while still containing deep sockets, had no indications of developing teeth from the scans (Figs. 1G and 2D). In OMNH 52311, the developing teeth at the 12th and 13th tooth positions had already begun formation of the crown and are the two largest tooth crowns in the jaw (Figs. 2A–2C). The functional teeth show some resorption probably caused by their immediately growing replacement teeth in the same locus. As initially described for Bo. striatus (Watson 1954), the 14th tooth in Bo. grandis is significantly smaller and thinner with a different morphology from the neighboring teeth (Fig. 2A).

Contained within both of the Belebey specimens were complete sets of replacement teeth and resorption pits along the dentary (Figs. 3A, 3C, 3E and 4). Underneath each tooth locus, an unerupted replacement tooth is preserved at varying stages of dentine and enamel development. The degree of development of the replacement teeth suggests that they represent slightly different stages of tooth development along the jaw, with more developed replacement teeth located anteriorly. This suggests that new teeth formed in a sequential pattern from anterior to posterior in Be. chengi, unlike the alternating replacement waves that characterize most reptilian dentitions (Gordon Edmund, Edmund & Museum, 1960; Reisz, Haridy & Müller, 2016). The thickness of their enamel also gives an indication of their age, indicating that the more posterior replacement teeth along the dentary were less developed than those in the front of the dentary. Compared to both the anterior-most replacement tooth as well as the functional tooth in the same locus, the posterior-most developing tooth is the smallest and the youngest in the series in both specimens of Be. chengi.

The prevalence of replacement teeth in the complete dentary of Belebey (IVPP V 12007) also allows us to reconstruct the dynamics of tooth replacement in bolosaurids (Fig. 4). The youngest replacement tooth, at locus 11, is a thin cap of dentine and enamel positioned and angled at the opening of the resorption pit (Fig. 4K). This suggests that new teeth first formed near the alveolar margins of the jawbones, indicating that the tooth producing organ, the dental lamina, was situated on or near the surface of the alveolar bone, lingual to the tooth row. The replacement teeth then stimulated resorption of the alveolar bone and dental tissues along the lingual surface of functional teeth to create a resorption pit and eventually a foramina. Given the small size of the earliest-staged replacement teeth and the abundance of developing teeth within the pulp cavities of the functional teeth, (Fig. 4), this would suggest that new teeth spent significant proportions of their formative time nested deep within the jaws at a subdental position. This is unlike the condition in many other early amniotes (Gordon Edmund, Edmund & Museum, 1960). The developing teeth quickly migrated to a subdental position where they continued to develop and resorb the bases of the functional teeth, before eventually erupting and causing the functional tooth to be shed from below.

Bolosaurid teeth were anchored via a thecodont ankylosis

This study utilized a combination of CT scanning and histology, applying both techniques to the same specimen to understand the limits of each. Thin sections clearly still revealed the microanatomical details of the teeth bones that were too fine even for CT scans (Fig. 5). However, for the distribution of major tissues, such as alveolar bone, dermal jawbone, enamel, and dentine, the CT data were more useful. As seen in Figs. 1E and 1F, the presence and even the thickness of the enamel on the cusps can adequately be determined. The thickness of the enamel could not be used reliably to determine the age of teeth due to differences in occlusional wear throughout the heterodont dentition (Reisz, 2006). The only expected tissues that were not found in CT were the cementum and the Sharpey’s fibers within the alveolar bone, which were visible in histological section (Figs. 5C and 5D). Utilizing CT is completely non-destructive and allows for unlimited views in all three axes. When a 3D scan is uploaded, a virtual copy is available for anyone, who can then manipulate or view the data; however they wish, in perpetuity. However, for detailed histological descriptions, incorporating both thin section and CT data are essential for highlighting a larger range of structures and tissue types.

Tooth implantation in bolosaurids is most similar to thecodonty (sensu LeBlanc et al., 2017; Bertin et al., 2018). The roots extend deep into the socket, surpassing the height of the crown (see Fig. 2). However, bolosaurids differ from many other thecodont taxa in the lack of unmineralized periodontal ligaments around the functional teeth. Once fully erupted, bolosaurid teeth rapidly fused to the jaw, mineralizing all of the surrounding attachment tissues (Fig. 5). Bolosaurids share the same implantation and attachment features with early Permian diadectids (LeBlanc & Reisz, 2013). Many other middle-to-late Permian amniotes, particularly therapsids, show similar thecodont implantation to bolosaurids, but the surrounding soft tissues do not mineralize, leaving a periodontal ligament space between the tooth root and the alveolar bone (Rybczynski & Reisz, 2001; LeBlanc et al., 2018; Whitney & Sidor, 2020). In bolosaurids, the teeth are firmly attached to the jawbone through the presence of alveolar bone. In CT, each socket appears to blend into the next with no distinct jawbone boundary between the walls continuously; however, in histological section, reversal lines exhibit a well-defined socket. The roots in bolosaurids are clearly defined within the jawbone; however, the alveolar bone shows previous dentine resorption of even other tooth loci, as divided by reversal lines (Fig. 5A). This permits a sequential interpretation of tooth replacement as the younger material crosscuts the older attachment tissues. This appears to be universal to all early amniotes, appearing in synapsids, captorhinids and diadectids (LeBlanc & Reisz, 2015).

This deep implantation of the teeth is unique among early parareptiles, as is the novelty of dental occlusion (Reisz, 2006; MacDougall, LeBlanc & Reisz, 2014). The wear created by occlusion may have been mitigated by the tooth implantation. Deep implantation of teeth is relatively rare among Permo-Carboniferous tetrapods. One comparable taxon are the diadectids. Dentitions in both diadectids and bolosaurids show heterodonty, thecodont implantation, as well as evidence of occlusal wear (LeBlanc & Reisz, 2013; MacDougall, LeBlanc & Reisz, 2014). The molariform cheek teeth of Diadectes are mediolaterally expanded compared to the more teardrop-shaped of bolosaurids, but like bolosaurids, they also frequently possess numerous replacement pits along the length of their jaws, suggesting a similar origin of tooth buds in both taxa.

Heterodonty, as well as the novel methods of tooth implantation and replacement, make the dentition of bolosaurids unique among early Permian parareptiles. The attachment tissues themselves match those of other early stem and crown amniotes (LeBlanc & Reisz, 2013; LeBlanc & Reisz, 2015; LeBlanc et al., 2018). The appearance of Sharpey’s fibers perforating the alveolar bone indicates that at some point in replacement, the teeth remain suspended by periodontal ligaments. During the development of a tooth underneath the functional tooth, the resorption required for growth removes hard calcified elements and replaces them with soft tissue. This soft tissue well is open to the surface through resorption pits until the functional tooth is shed, exposing the much greater extent of the tissue underneath. This large replacement pit supports the developing tooth, attached by soft tissues allowing for the movement into a functional position. Eventually, with tooth maturation, these soft elements undergo centripetal mineralization as the now harder alveolar bone grows into direct contact with the cementum and the tooth is fully ankylosed to the jaw. These ligamentous channels become surrounded by bone, preserved as Sharpey’s Fibres, existing as a few remnants in the resorbed material. Teeth are inferred to undergo direct ankylosis to the tissues resulting in firm anchoring to the jaw. This is in contrast to gomphosis, the primary method of attachment for later thecodonts such as mammals. It has been inferred that these soft-tissue attachments for teeth evolved in conjunction with occlusion to limit mechanical stress (Gaengler & Metzier, 1992). Bolosaurids require a separate, possibly different explanation as intensive dental occlusion wears down the teeth (Reisz, 2006), but no analogous supporting periodontal ligamentous tissues are present in functional tooth attachment.

Mode and timing of tooth replacement in Bolosauridae minimized gaps in their occluding dentition

Thecodont taxa display two different types of tooth replacement. The first occurs in modern mammals and crocodilians, wherein a replacement tooth develops from a dental lamina that is dissociated from the oral epithelium and is buried deep within the jaw, almost underneath the functional teeth (Gordon Edmund, Edmund & Museum, 1960). The second replacement type is found in most other thecodont amniotes, where the dental lamina is still located along the lingual margins of the jaws, an ancestral feature for amniotes (Gordon Edmund, Edmund & Museum, 1960; LeBlanc et al., 2017; Leblanc, Mohr & Caldwell, 2019; Whitney & Sidor, 2020). Because new teeth still form far away from the pulp cavity of the preceding functional teeth, they first form resorption pits along the lingual surfaces of the teeth that are visible externally in fossils and skeletons. These resorption pits are the entry points for replacement tooth buds, which then mature beneath the functional tooth. This is the condition we observed in bolosaurids. However, despite the prevalence of replacement teeth in our bolosaurid sample, vacant tooth positions are rare compared to other polyphyodont amniotes (Gordon Edmund, Edmund & Museum, 1960).

One specimen of Belebey (IVPP V 15907) shows replacement pits along the lingual side of every single tooth locus, but no shed teeth (Figs. 3C–3E). The least developed replacement teeth in IVPP V 15907 are close to the opening of the small replacement pit (Figs. 3F and 4K). This is strong evidence that the dental lamina existed along the lingual margin of the tooth row, periodically initiating a new tooth that quickly formed a replacement pit along the lingual surface of the functional tooth root and alveolar bone. The developing teeth would drop to a subdental position and continue to develop for some time before causing the functional teeth to be shed. Tooth shedding may have been a relatively quick process in the tooth replacement cycle, given that we only observed one empty tooth locus in our bolosaurid sample (Figs. 2A–2C). The prevalence of replacement teeth deep in the jaws in bolosaurids also suggests that replacement teeth spent a great deal of time developing in these subdental crypts before finally erupting and replacing a functional tooth. This mode of tooth replacement, where teeth rapidly descended into the jaws and spent significant time developing underneath the functional tooth row, would have extended the life of functional teeth, allowing them to remain in the mouth for a more extended amount of time without compromising precise occlusion.

The presence of replacement teeth in Belebey (IVPP 15104) and Bolosaurus (OMNH 52311) revealed an unusual tooth replacement pattern for bolosaurids. The presence of regularly arranged tooth gaps in fossil reptiles represent a period in the replacement cycle in which the functional tooth has been shed, but the replacement tooth had not fully erupted at the time of death (Gordon Edmund, Edmund & Museum, 1960). At first glance, the dentary of Belebey (IVPP 15104) shows replacement teeth at nearly the same stage of development along the jaw (Fig. 3). It is possible that bolosaurids might have replaced all tooth positions rapidly and simultaneously, but this is an exceptionally rare occurrence in tetrapods (Bolt & DeMar, 1983) and would be extremely detrimental to an animal that relied on dental occlusion. As opposed to showing intermittent or large gaps in the tooth row, bolosaurids usually possess a gapless dentition or only a single shed tooth position. Moreover, closer inspection of the replacement teeth in Belebey (IVPP 15104) and Bolosaurus (OMNH 52311) reveal that, unlike most other polyphyodont amniotes, bolosaurids underwent a single replacement wave at a time, passing from the front of the jaw to the back (Figs. 2–4). Each replacement tooth in Belebey is incrementally more developed from the front of the jaw to the back. This single-wave model also explains why in OMNH 52311, there is no evidence of resorption pits in the anterior 10 teeth, tooth 11 has been shed, and posterior tooth positions 12 and 13 were still undergoing replacement. The replacement wave in this specimen had already passed through the first 10 teeth, while tooth 11 had recently been shed. Meanwhile, positions 12 and 13 have very large and well-developed replacement teeth and were likely nearing the tooth shedding phase (Fig. 2). As opposed to periodically replacing the entire dentition and being unable to feed for a short time, we hypothesize that bolosaurids instead replaced a single tooth position at a time, which would minimize the impact of tooth replacement on the occlusal relationships of the teeth. This proposed method assumes a reasonably equal rate of tooth development across the jaw, but the much smaller, less complex anteriormost teeth would reach eruption before the much larger posterior teeth even reached maturation, due to the variation in size in the heterodont dentition (Reisz et al., 2007).

Bolosaurid enamel is unusually thick for a small reptile

The enamel of bolosaurid teeth is thick and well ornamented (Fig. 1K). The crests at the crown apex are the thickest regions of the enamel, attaining a maximum thickness of approximately 200 µm. These ridges are clearly produced during the later stages of amelogenesis, and not during the initial phases of tooth development. We can infer this because the underlying dentine, the first hard tissue to be produced, does not conform to the shape of the outer surface of the enamel. The crowns must initially have a smooth outer shape that is later furnished with thicker crests of enamel. Whereas bolosaurid enamel is not particularly thick in absolute standards, it is extremely thick relative to the small sizes of its teeth. Many large, herbivorous dinosaurs, for example, have only slightly thicker, or even thinner enamel than these small parareptiles (Hwang, 2011). Thickened and wrinkled enamel is also unusual in small-bodied animals and may be an adaptation for occlusion and more powerful bites (Sander, 1999).