Dental eruption relative to skull size

Absolute age data for dental eruption in tenrecs, golden moles and sengis are not yet available in the literature. As an alternative, we compared the proportion of erupted permanent cheek teeth with metrics of skull size. We expect that if these taxa show delayed eruption of the permanent dentition, then some or all of their permanent teeth should finish erupting after the completion of growth, leading to specimens at or near adult size without a full complement of permanent teeth. Anecdotal observations of this nature (Figure 1) are in part what led Leche [8] to draw the conclusion that tenrecs and golden moles show delayed eruption.

This previously unquantified observation is consistent with the data collected here. In general, afrotherians without a full complement of permanent teeth are significantly larger (as a proportion of median adult jaw length) than non-afrotherians (such as didelphids, strepsirhines, hominoids, platyrrhines, scandentians, canids and erinaceids) at a comparable stage (Figure 2). Among the 22 genera sampled in this study (11 afrotherians, 4 strepsirhines, 2 gibbons, 1 tree shrew, 1 carnivoran, 2 erinaceids and 1 didelphid), all of the afrotherian genera exhibited specimens with 95% median adult jaw length that had less than 60% of their permanent, occluding premolars and molars erupted (Table 1). Stated differently, adult size among afrotherians is frequently reached prior to the eruption of many permanent teeth. Non-afrotherians showing less than 60% erupted cheek teeth were typically well under 90% median adult jaw length, except for one specimen of Eulemur fulvus (ZMB 4915) which reached 95% (Table 1). Two afrotherian groups (macroscelidids and chrysochlorids) had specimens that reached, or exceeded, median adult jaw length with less than 40% of their permanent cheek teeth erupted (Figure 2). Another index of skull size, condylobasal length, also shows that most afrotherians reach adult-proportions with fewer permanent cheek teeth erupted. However, because jaws are better represented in museum collections (particularly for the growth series of the taxa we sampled), we focus on the latter measurement in this paper.

One-third erupted	Chrysochloridae, Macroscelididae
One-half erupted	Potamogale , Oryzorictes , Setifer , Procavia , Tenrec , Eulemur
Two-thirds erupted	Didelphis, Erinaceus, Echinosorex, Hylobates, Perodicticus, Lemur, Tupaia
Fully erupted	Otocyon, Otolemur, Symphalangus

Despite these qualifications for T. ecaudatus, the afrotherians measured here with incompletely erupted permanent teeth, particularly sengis and golden moles, show adult proportions more frequently than non-afrotherians at a comparable stage of eruption (Table 1 and Figure 2). Non-afrotherians with unerupted cheek teeth are generally smaller than conspecifics with full, permanent dentitions. We interpret this to indicate that unlike afrotherians, most other mammals erupt the permanent dentition early enough in ontogeny so as to overlap extensively with growth.

Absolute age at eruption of permanent cheek teeth

For hyraxes, elephants, sea cows and many other mammals, absolute age at eruption of the complete, permanent dentition is already known. Figure 3 summarizes these data, standardized by age at female sexual maturity across mammals. Both sexual maturity and alternative scaling factors such as longevity may be skewed by outliers, such as unusually long-lived zoo specimens. Because data on sexual maturity are more widely available for wild populations than data on longevity, the former are used here.

Shigehara [15] noted that the eruption of the full permanent dentition may postdate sexual maturity in some mammals, including most old world primates, Didelphis, Bos, Sus, Equus, Felis and Canis familiaris (but not C. lupus or C. mesomelas). Among the taxa he studied, only perissodactyls showed completion of dental eruption after both sexual maturity and fusion of longbone epiphyses. However, some terrestrial artiodactyls show considerably later eruption of permanent cheek teeth than perissodactyls. In particular, Hippopotamus does not fully erupt all of its permanent cheek teeth until approximately 16 years of age [27]. The white rhinoceros shows the latest appearance of the complete permanent dentition among the perissodactyls sampled here, erupting all cheek teeth at around 8 years [28]. Some marsupials tend to show complete eruption of cheek teeth shortly after sexual maturity, a pattern greatly exaggerated in the dentally manatee-like wallaby Petrogale concinna, in which the seemingly endless eruption of supernumerary molars continues throughout life [14]. Hence, some artiodactyls, perissodactyls, carnivorans, anthropoid primates and marsupials show complete eruption of permanent cheek teeth at or near adulthood, ranging from slightly after to twice the age of female sexual maturity. Other non-afrotherian mammals show complete eruption prior to female sexual maturity (Figure 3).

The three afrotherians for which dental ontogeny is best known, Loxodonta, Procavia and Trichechus, show ages at complete cheek-tooth eruption that are, respectively, 2.6, 3.7 and more than 10 times the age of female sexual maturity. As mentioned above, Trichechus erupts an apparently unlimited number of cheek teeth throughout life [10]. In contrast, the highly-derived, peg-like cheek teeth of the sirenian Dugong dugon erupt between 6 and 9 years, at or shortly before sexual maturity at around 9 years; the prominent tusks of the dugong erupt later at around 15 years [29]. Compared with other major clades of mammals, and except for the cheek teeth of the dugong (but not its incisors), paenungulate afrotherians show late eruption of permanent teeth, postdating sexual maturity by a wide margin (Figure 3).

CCD-like characters across mammals

In addition to humans, Runx2 sequence variation has also been documented in mice [30–32] and domestic dogs [33]. Homozygous mouse mutants lacking Runx2 fail to ossify membrane or endochondral bones [34]. These mutants exhibit initiation of tooth morphogenesis, but further development is incomplete [35]. Owing to an overall lack of bone development, they asphyxiate at birth from an unossified ribcage. Heterozygous mutant mice survive birth and show many of the same skeletodental anomalies as humans with CCD, including poor ossification of membrane bones such as the clavicle and elements of the braincase [34]. However, they develop relatively normal molars [32] possibly because mice have just a single dental generation, not two as in most other mammals; and in human CCD it is only the second generation of teeth that is affected [18, 23]. Molars in mice and other mammals are commonly regarded among paleontologists as part of the same generation as permanent premolars, since both form the permanent dentition and partly overlap in terms of eruption. However, molars share a developmental origin from the primary dental lamina with 'first-generation' deciduous teeth, distal to the site of origin of the latter and buccal to the embryonic 'lingual successional lamina' (see [36], p. 186) which gives rise to permanent antemolars. Wang et al [31] observed that successionary cheek tooth buds, normally absent or very small in wild-type mice, were much more commonly observed in Runx2 mutant mice, further indicating that this locus may contribute to the morphogenetic pathway of the post-primary generation of teeth. They also noted greater expression of Runx2 protein in the lower than in the upper dentition. Our observations do not indicate different timing of eruption of the upper and lower teeth. Indeed, such a pattern would be problematic for mammals with functional occlusion. However, eruption of some lower tooth loci (e.g., p4) prior to their upper counterparts is not uncommon among mammals [37].

Sequences of afrotherian Runx2 available on public databases are limited, and at the time of writing include only partial sequences derived from ongoing genomes of Loxodonta and Echinops [38]. Only the former samples the DNA binding region, mutations to which may be linked to expression of CCD in humans [19]. Sequences of Runx2 from Loxodonta and other mammals such as Pan, Macaca, Rattus, Mus, Equus and Canis are easily alignable to human Runx2 for at least part of their length. These non-human mammals generally showed the conserved nucleotides present in the human wild type, not the mutations to Runx2 concentrated in the DNA binding domain among human CCD patients (see Tables 1 and 2 in [19]), except for a sequence of Canis. This specimen (GenBank XM_532158) [39] exhibits many changes to its Runx2 sequence, including a mutation commonly present among genetically documented human patients with CCD [19]: instead of the 'CGG' codon for arginine (R) at codon 225, they exhibit a 'CAG' codon for glutamine (Q). While hypodontia among domestic dogs (and other mammals) is known, they do not regularly show delayed eruption of the adult dentition (Figure 2).

At first glance the distribution of CCD-like characteristics across mammals, including delayed dental eruption, vertebral anomalies, clavicle reduction and testicondy (Figure 3), appears to show some co-occurrence. In particular, members of Afrotheria, Perissodactyla and terrestrial Cetartiodactyla lack clavicles and show some delay in the eruption of permanent teeth; and afrotheres and perissodactyls exhibit an increased number of thoracolumbar vertebrae. Examined quantitatively, however, and including other characters observed among CCD patients, this covariation is not significant (Figure 4). The distribution of these characters on a plot of the three principal coordinates that explain just over half of the variation does not place them in close proximity (Figure 4A), as would be expected if these features consistently occurred together (see Figure 1S in [40]). Nor is there a significant correlation between vertebral number and delay in eruption (Figure 4B). Whether or not this signal changes with consideration of a more complete morphological dataset, including ontogenetic data for small afrotheres as well as their other recently identified synapomorphies [3, 6, 7], remains to be seen.

Ontogeny of dental eruption

Based on these observations, we conclude that Leche was correct in his assessment that like large afrotherians in which dental ontogeny is well documented, sengis, tenrecs and golden moles also show relatively late eruption of the permanent dentition. Compared to non-afrotherians, permanent cheek teeth finish erupting only after these taxa have reached adult body size. Considered in isolation, this could mean that growth occurs faster and earlier in these afrotherians than other mammals, rather than the converse of late-erupting permanent teeth. However, although some afrotherians have relatively and/or absolutely long gestation times (e.g., hyraxes and elephants), which could be interpreted to support the hypothesis that growth takes place early, this is not true of Afrotheria as a whole. Tenrec, for example, has a shorter gestation time than Cavia (Rodentia) and Saimiri (Primates), although it can reach a larger body size than either taxon (Additional file 1). The sengi Rhynchocyon (with a mass of about 540 g and gestation of around 42 days) compares well with the rodent Sciurus (with a mass of about 530 g and gestation of around 44 days; see Additional file 1).

Notably, and in contrast to other afrotherians, the shrew-like tenrecid Microgale appears to erupt its molars (but not its premolars) relatively early during its development. Few museum collections have specimens of Microgale that exhibit fewer than three fully erupted molars [41]. In fact, the difficulty in distinguishing deciduous from permanent premolars in 'adult'-sized specimens of this taxon [8] has contributed to an unusually high level of species-level synonymy within this genus throughout much of the 20th century [41, 42]. We assume that the frequent confusion of deciduous and permanent premolars in the literature results from the frequent retention of the former and late eruption of the latter, as in other small afrotherians examined here. In order to test this assumption, and include this taxon in our sample, we would need more sophisticated techniques to determine the deciduous versus permanent identity of premolars e.g., by using radiographs or computed tomography images, a sampling strategy that will be employed in a future study.

A further caveat is the dentition of the highly derived aardvark. Growth in this taxon is poorly known, and the last comprehensive summary of its dental development was published in 1934 (see [12]). From this we know that Orycteropus appears to lack functional deciduous teeth, although some may persist as rudiments in the gums well into adulthood. A summary in preparation (Lehmann, submitted) enables us to tentatively conclude that its permanent cheek teeth are erupted at a relatively early age, a result that is not surprising given its lack of functional deciduous teeth. Hence, as with the molar teeth of Microgale and Dugong, this too qualifies as an exception to what we believe otherwise to be a rule among afrotherians: that their permanent dentition finishes erupting relatively late during ontogeny, after sexual maturity and the occurrence of most growth.

CCD character covariation

The apparent lack of character covariation (Figure 4) results in part from the fact that wild-type mammals represent ecologically viable populations, capable of successfully perpetuating their lineage, and should not be expected to mirror pathologies in a different species (Homo sapiens) with which they may share a developmental program. Regarding the genetic control of tooth succession, there is a complex interplay among genes expressing a number of morphogenic proteins, including Shh, BMP and FGF, which are mediated at least in part by Runx2 [18, 30] and that relate to the development of teeth [30–32, 35, 43] and bone [20, 23, 34]. The pathologies of CCD-afflicted humans are a result of the decreased functionality of the DNA binding region in the regulatory locus Runx2 [19, 20]. Depending on the extent of this decreased functionality, CCD humans show a mix of phenotypes, often (but not always) including features exhibited in afrotherians. Imperfect covariation among these characters among human CCD patients does not negate their relatively well-established link to Runx2 [19]. Similarly, imperfect correlation of CCD-like characters among mammals does not in itself negate the potential influence of Runx2 in their development.

Hence, a better understanding of Runx2 variation among afrotherians may yet provide insight into the mechanisms behind delayed eruption of the permanent teeth. We propose that this feature comprises an additional, shared characteristic of the newly recognized afrotherian clade.