Morphology and microstructure

Previous authors have noticed that the more mesially located teeth in the jaws of Hesperornis and Ichthyornis are progressively more recurved than the distal ones [6, 13, 47, 48]. This characteristic is shared by other toothed birds including Archaeopteryx [13, 49]. Hence, the isolated Hesperornis tooth YPM.1206B, which is highly recurved, is likely to derive from a mesial position within the jaw partly represented by the YPM.1206 specimen. Therefore, this isolated tooth must belong to a mesial portion of a dentary, since the premaxilla is edentulous in Hesperornis [6]. Furthermore, the tooth YPM.1206B must belong to the right dentary, i.e., precisely, YPM.1206A, judging from the lingual inclination toward the left side, visible in this tooth. Ichthyornis also exhibits toothless premaxillae [50]; this feature has been interpreted as illustrating partial dental reduction near the origin of the avian crown clade [4]. The predentaries present in Hesperornis and Ichthyornis (mesial to the dentaries) are also toothless [51, 52]. The mesial-distal gradient of tooth straightness in Ornithurae (Hesperornithiformes and Ichthyornithiformes) might reflect an adaptation to piscivory in both taxa. Conversely, in Richardoestesia gilmorei (which otherwise presents some dental similarities to toothed birds, but whose phylogenetic position within Coelurosauria is unclear) exhibits the opposite condition: the teeth become mesially straighter, and distally more recurved distad, possibly reflecting an adaptation to feeding “on insects and other soft-bodied prey” ([24]: 123). However, links between the mesial-distal gradient of tooth straightness and diet are far from obvious; for example, certain terrestrial predators (probably largely insectivorous) such as Compsognathus exhibit the same gradient [53] as the piscivorous ornithurines described here.

Our observations confirm and precise important differences in shape between Ichthyornis and Hesperornis tooth crowns, described previously [6, 47]. In Ichthyornis the crown bears very prominent ridges, mesially and distally, reaching the apex of the tooth; these ridges are sharp, cutting edges without serrations. In Hesperornis, mesial and distal ridges without serrations are present, but faint and far less developed than in Ichthyornis, and they do not reach either the apex or the neck of the tooth. In the more curved Hesperornis teeth, the distal ridge is shifted labially. At comparable mesio-distal positions along the jaw, tooth crowns are more constricted labio-lingually in Ichthyornis than in Hesperornis. The curvature of teeth in Hesperornis is distributed evenly, and affects the entire rounded crown (especially in mesial teeth). In Ichthyornis, the more mesially situated teeth are ‘recurved’ distad, but this results exclusively from a sharp angle between the root and the crown. The crown itself is therefore directed distad, but it is essentially straight. Finally, Hesperornis crowns bear fine basal-apical ridges (“fluted ornamentation” [17]), whereas Ichthyornis crowns are devoid of ornamentation. Confirming Sander’s observations on a presumed Hesperornis sp. tooth [17], we show that the fluted ornamentation of Hesperornis teeth are largely the product of thickening of the enamel layer, with no substantial visible EDJ preformation. The slight mesial and distal carinae of Hesperornis consist of a combination of moderate EDJ deformation, and moderate enamel thickening (the latter initially highlighted by O.C. Marsh [6]); enamel thickening is also observed towards the apex of the crown. In Ichthyornis as well as in the Maastrichtian tooth hereafter assigned to the Ichthyornithiformes, the well-marked mesial and distal carinae are also formed by a combination of EDJ shaping and enamel thickening.

The enamel of Hesperornis and Ichthyornis is prismless as in most non-mammalian amniotes (except agamid lizards). In both Ichthyornis and Hesperornis, the enamel consists of an extremely thin layer of columns orthogonal to the enamel surface, and each column is composed of divergent crystallites. The microstructure of the thin enamel layer (mostly <5 μm; thicker at the carinae and apex) therefore corresponds to one single basal unit layer (BUL), as suggested earlier on a presumed Hesperornis sp. tooth [17]. The microstructure of the enamel in both Ichthyornis and Hesperornis is highly simplified compared to that of the non-avian theropods illustrated by S.H. Hwang [28, 29]. This author also described the Schmelzmuster of enamel of two teeth of indeterminate Late Cretaceous birds [29], and compared them with that of the presumed Hesperornis sp. tooth [17]. Hwang interpreted a second, thinner layer over the BUL, composed of parallel crystallites [29]. Specifically, the second tooth examined was said to bear resemblance to Hesperornis as well [29], and its features were discussed in comparison with those of Sander [17] as if it was positively Hesperornis. However, we see no reason to consider that such a taxonomic assignment is supported. Hence, we interpret the lack of a second layer on top of the BUL in our specimens, as well as in the cf. Hesperornis sp. tooth [17] as a Hesperornis characteristic, and consider the teeth described in [29] to derive from probable, indeterminate Mesozoic birds. A thin BUL is thus confirmed for Hesperornis, and shown for the first time in Ichthyornis.

Contra [29] with Aves indet., no tubules originating from the EDJ and extending to the surface are observed in micrographs or in virtual sections of the enamel of Hesperornis, nor Ichthyornis. It seems that the observed tubules [29] are artifacts caused by acid etching employed to enhance surface observations. In [29], there are no transverse sections illustrated that could show the tubules originating from the EDJ and running to the surface. The ‘tubules’, which seem to be holes, are only observed in longitudinal and oblique sections.

The thinness and simplification of enamel in Hesperornis and Ichthyornis, with one BUL only, are apparently unique among studied archosaurs, and indeed reptiles [17, 28, 29]. In Ichthyornis and Hesperornis, enamel thicknesses of 4 to 10 μm (away from such zones of thickening as apex and carinae) amount to 0.27–0.30 % of crown height, whereas in the Nile crocodile for instance, which exhibits similarly-shaped teeth associated to largely piscivorous function, this percentage is 1.0 to 1.3 %. If this attests to a tendency toward reduction of enamel cover among crownward ornithurines, it might be significant that the Maastricht tooth hereafter assigned to Ichthyornithiformes exhibits a greatly reduced enamel covering, with enamel entirely absent from the basal part of the crown. An evolutionary process of enamel reduction preceding tooth loss on the line to crown birds would be in line with these observations. As a hypothesis, it would imply that the inactivation of enamel protein genes [54] was not strictly a consequence of arrested tooth development, but was perhaps already incipient in some Late Cretaceous toothed ornithurines.

Attachment and implantation

Some of the teeth preserved in situ in the Hesperornis and Ichthyornis dentaries are obviously displaced within the dentary, due to post-depositional processes (as noted in [6]). This is most obvious in several of the teeth within the Hesperornis dentary. Displacement of these teeth might have been favored by the teeth being situated in a groove, with only slight bone constrictions around each tooth root. In contrast, the teeth of Ichthyornis are set in discrete alveoli, and therefore were likely more firmly attached and less liable to become displaced in the face of post-depositional factors such as water infiltration, chemical processes of alteration, sediment compaction and deformation, and other diagenetic effects. However, in situ teeth within the Ichthyornis dentary also exhibit some post-mortem displacement, including the most mesially positioned among preserved tooth, which is slanted mesially. Incompletely erupted teeth in Ichthyornis tend to be more inclined (distad) than fully erupted ones, which are set more upright with growth [6, 47]. Here we observe the same phenomenon in Hesperornis, with in situ teeth that are not fully erupted and grown (see below). The decomposition of non-mineralized attachment tissues such as the periodontal ligament, as well as alteration processes affecting root tissues (most visible in Ichthyornis), are likely responsible for this displacement. In addition, diagenetic compression has affected several of the specimens (especially the dentaries), and tooth roots within the Hesperornis dentary are hence often fractured and slightly distorted. However, the features preserved at certain locations allow characterization of dental attachment and implantation in both taxa.

In Hesperornis, the groove constrictions are formed by the same bone tissue as the surrounding jaw bone; they are not secondarily deposited (Fig. 2e). The same applies to the formation of the septum distally separating the unique alveolus in the mesialmost part of the dentary from the groove. In contrast, true sockets are formed of primary alveolar bone, which is histologically different from the bone tissue comprising the dentary (see Ichthyornis below for more details). The histological tissue comprising the constrictions in Hesperornis is therefore different from that of the true sockets observed in crocodilians or Ichthyornis ([31, 47]; Fig. 3d, e, f; Additional file 10: Fig. S3). In places, bone adjacent to teeth in Hesperornis has been resorbed simultaneously with the shedding of the tooth, and new bone has been redeposited with the development of the new replacement tooth. This results in resorption lines and holes (see Fig. 7d, f, Additional file 8: Fig. S5). The process of reformation of bone around the teeth is probably similar to that of true alveoli made of alveolar bone, yet we demonstrate the absence of true alveolar bone in Hesperornis.

The woven-fibred bone comprising the alveolar intersepta of Ichthyornis is similar to the juvenile caiman model described in [55] (see also Additional file 10: Fig. S3). Interdental septa are formed by the fusion of several bony plates running along the jaw. In adult specimens, these woven interseptal plates are remodelled and integrated into the jaw bone. Alveolar bone is composed of woven-fibred bone when growing quickly, or lamellar bone if growing at a slower rate [56–59]. The Ichthyornis alveolar bone attests to fast growth speed. It was formed simultaneously with the formation of the last series of teeth formed at death, since alveolar bone is principally a feature of the tooth root [34, 56]. During dental replacement, as a tooth is shed, alveolar bone is resorbed partially or totally and then reformed around the root of the new tooth (including transverse septa) by ectomesenchymal cells associated with the tooth [56]. In certain paravians such as troodontids, the interdental (alveolar) septa, where formed, are made of bone tissue that differs histologically from the laminar bone of the dentary [23]. The septa appear to be formed of true alveolar bone exhibiting woven texture with large lacunae, as in Ichthyornis or crocodilians.

‘Bone of attachment’ is an ill-defined term, which corresponds to bone tissue that is undifferentiable from alveolar bone, so it is synonymous ([34, 56]; contra [13, 36, 37]). The statement that so-called ‘bone of attachment’ in theropods would be attached to the tooth without a periodontal ligament and even without cementum [13], a form of ankylosis, is contrary to observations across archosaurs and other tetrapods [34]. Attachment of the root takes place via cementum attached to alveolar bone by a periodontal ligament (unmineralized to more or less mineralized). But periodontal ligament, if unmineralized, is not preserved in vertebrate fossils. And without fine-scale histological analyses of teeth in place in jaws, cementum can be overlooked, as it is often very thin in small archosaurs, and/or easily confused with dentine or bone.

Acrodonty, pleurodonty, subthecodonty, and thecodonty are terms describing gross morphology, but only histology analyses can properly differentiate among pertinent categories [56]. If these terms are practical descriptors of the depth of implantation, they are of limited interest in terms of phylogeny, because they appear highly subject to homoplasy; they are determined by the interplay of different amounts and arrangements of various attachment tissues, which can differ even within a single jaw [34]. True thecodonty has been known to characterize a minima archosaurs, mammals, mosasaurs (with a mineralized ligament) and some snakes (gomphosis, i.e., attachment through an unmineralized periodontal ligament) [34, 56, 60]. Attachment in thecodont snakes is hinged (i.e., the ligament is present on one side only; [56]). True thecodonty by gomphosis is furthermore exhibited by diadectids, which are early representatives of the amniote stem group, and its loss in some amniote groups appears to be secondary and derived from true thecodonty [34]. Acrodonty or pleurodonty (in extant squamates for instance) would therefore be secondary, and derived from thecodonty, again, not representative of the primitive state as previously thought [34]. Hence, thecodonty appears to be primitive for the amniote crown together with some stem amniotes (Cotylosauria), along with numerous associated characteristics: alveolar bone, cellular and acellular cementum, Sharpey’s fibers, lingual tooth replacement via the tooth germ entering the root through a resorption pit, and loss and resorption of most of attachment tissues including some alveolar bone during replacement. The longstanding restriction of thecodonty to crocodilians, mammals, marine reptiles, some Cretaceous snakes and certain dinosaurs is therefore obsolete [34].

Although the mode of tooth implantation in Hesperornis differs in some respects from classic thecodonty, the attachment mode is similar (despite the lack of alveolar bone). Implantation in a groove is presumably autapomorphic of Hesperornis (and possibly some other Hesperornithiformes). We propose that similar attachment attests to close homology, despite different implantation, of Hesperornis and typically thecodont taxa. A contrario, the superficially similar implantation in a socket of mosasaurs and crocodiles appears to be merely analogy, since the homologous attachment tissues involved exhibit very different tissue arrangements and amounts, and in mosasaurs the periodontal ligament is mineralized whereas crocodiles exhibit gomphosis [34].

The very thin space between root cementum and bone (locally < 50 μm) in Hesperornis could be interpreted as resulting from diagenetic compaction. Therefore, this space could have been wider in life, and could have accommodated a periodontal ligament. However, Sharpey’s fibers in the cementum of Hesperornis teeth can be traced in continuity through both cementum and adjacent bone. This indicates that a periodontal ligament was indeed absent and that cementum and bone were linked rather firmly via Sharpey’s fibers directly. Despite the absence of true alveolar bone, there is reworking of the dentary bone adjacent to the teeth. Hence, Hesperornis exhibits most features of thecodonty through gomphosis, albeit with secondary implantation in a groove. The thin spacing, and firm attachment via Sharpey’s fibers but without periodontal ligament, between cementum and bone might be an apomorphic, secondary feature compensating for a lack of sockets in maintaining firm tooth implantation in a groove. In Hesperornis, the cementum is well developed and rather thick, with a cellular layer and an acellular layer, comparable to the condition exhibited by crocodilians [16, 40], mammals [61, 62] and fossil marine reptiles [63, 64].

The presence of cementum has only rarely been observed in small non-mammalian amniotes [56], and has even been said to be unknown in non-avian theropods [65], making a supposed difference with birds. However, we report two types of thickened cementum at least in the non-avian theropod Richardoestesia isosceles. We therefore suspect that the perceived absence of cementum in non-avian theropods may reflect, firstly, the relative rarity of well preserved roots on isolated non-avian theropod teeth, as well as the near absence of data on attachment tissues from fine-scale investigations. Isolated teeth of Hesperornis, Richardoestesia, and an indeterminate (archosaurian, presumably theropod) tooth of comparable size, all exhibit well-developed cementum, as well as Sharpey’s fibers distributed within the cementum. Lower density in the distribution of cementocyte lacunae in the latter, indeterminate isolated tooth might reflect a slower rate of deposition (the density of entombed cementocytes in cellular cementum is hypothesized to be proportional to the speed of tissue formation; [61]). These shared features indicate thecodonty comparable to that in birds and crocodilians, in non-avian theropods such as Richardoestesia. Comparable features include cementum (acellular and cellular) overlying the root dentine, and Sharpey’s fibers, possibly with a periodontal ligament attaching cementum to bone in life. Following [34], this condition represents thecodonty through gomphosis, since these isolated teeth fell out of the jaw and the roots became detached from the bone, post-mortem, without damaging any part of the cementum or dentine. It implies that the periodontal ligament was non- or slightly mineralized (gomphosis), as opposed to mineralized (‘ankylosis’, where teeth generally do not fall out post-mortem fully intact with roots). Interestingly, the different orientations of the Sharpey’s fibers in the Hesperornis tooth vs. the two other of these three isolated teeth (strongly oblique Sharpey’s fibers with a strong basal-apical component, and fibers diverging basally around the root, in Hesperornis) suggest differences in attachment. These differences are possibly linked with differences in the constraints undergone by the teeth in life, driven by diet and/or feeding mode.

The groove with constrictions in Hesperornis is different from the groove in the distal part of juvenile crocodilian jaws, which does not exhibit constrictions (Additional file 10: Fig. S3; [40]). Septa form mesio-distally during development in the crocodilians, and are formed of alveolar bone [55]. In contrast, the constrictions in Hesperornis are not composed of alveolar bone, but are simply outgrowths of the main jaw bone, as is the separation of the unique mesialmost small ‘alveolus’. The groove in Hesperornis only superficially resembles the state in juvenile crocodilians. Therefore, this original autapomorphy cannot be interpreted simply as a neotenic characteristic. The groove observed in [47] within the distal portion of some Ichthyornis dentaries (inferred to belong to juvenile individuals) is very similar to that of Hesperornis, and exhibits constrictions. In Ichthyornis the rest of the teeth are inserted in sockets (and in presumed adults all teeth reside in sockets). The attachment is thecodont, with alveolar bone and alveolar septa that appear to grow in a similar manner as in the juvenile crocodilians (e.g., [31]). The presence of the groove, the derived loss of alveolar septa, and loss of alveolar bone, appear to represent hesperornithiform autapomorphies. Indeed, uniquely in Hesperornis, the groove extends nearly along the whole dentary (to the exclusion of the small, single, mesialmost alveolus revealed here). This appears to be the adult condition, and indeed no evidence of juvenile remains is known. Hesperornis can therefore be viewed as having secondarily lost thecodonty. The mesialmost individual small alveolus in the Hesperornis dentary might be a remnant of the thecodont condition of Hesperornis ancestors, now formed in the absence of alveolar bone. The reacquisition of a near-complete groove at adult stage effectively resembles the opposite of the progression of alveoli during crocodilian development (a common extant model for all archosaurs). This ontogenetic progression is apparently visible in early ontogenetic representatives of Ichthyornis, exhibiting a groove with constrictions similar to the juvenile crocodilian condition. Hence, the evolution of the groove in Hesperornis would have taken a path reminiscent of a neotenic character, but leading to a groove non-strictly homologous with that of juvenile crocodilians or even Ichthyornis. As for non-avian theropods, it remains to be seen whether or not a comparable groove exists in juveniles, with septa forming mesio-distally later in development (as might be expected in the hypothesis that this pattern would be plesiomorphic at least for theropods). Yet another type of pseudo-groove is known in adult troodontids [23]. Constrictions mark the delimitations of spaces for tooth implantation, on the sides and bottom of the groove, as in Hesperornis. But contrary to other archosaurs, the groove in Troodontidae is progressively better defined mesially, whereas sockets are formed in the distal part of jaw. The opposite condition is exhibited in juvenile crocodilians and juvenile Ichthyornis —where septa form first mesially and later toward the distal end. Even in Hesperornis, the single small alveolus is located at the mesialmost portion of the tooth row, as opposed to the troodontid condition. The constrictions hence would be shared with the condition in some juvenile archosaurs (probably convergent), while the precise aspect, or position (and presumably ontogeny) of the groove would represent a troodontid autapomorphy. The mesial absence (or nearly so) of alveolar septa in troodontids is hypothetically related to the crowding of teeth in this region in this group relative to other theropods [23].

To summarize, Ichthyornis and Hesperornis are thecodont through gomphosis (despite the presence of a secondary groove in Hesperornis). In Hesperornis, the tooth root is covered by two types of thick cementum (alveolar and non-alveolar), which is attached to alveolar bone via Sharpey’s fibers but at least partly without a periodontal ligament. This is also the case (cementum, fibers) in some isolated comparative teeth from indeterminate Mesozoic birds or non-avian theropods. Even though the preservation state of Ichthyornis did not allow recognition of cementum and Sharpey’s fibers, alveolar bone delimiting true sockets is recognized.

Tooth growth and replacement

The in situ teeth of both Ichthyornis and Heperornis lack a large portion of their root. We interpret this as suggesting that these teeth were not fully developed, since tooth growth starts from the crown apex and completes with the root growing in the basal direction. Obviously, (1) the size of functional teeth in situ with incomplete roots (even considering displacement) leaves no place for a root if virtually replaced at its original, life location at the time of death; (2) the teeth that have apparently been displaced the least are generally small, with crowns only beginning to emerge from the occlusal border of the dentary, indicating growing replacement tooth stage. The larger tooth in our studied Hesperornis dentary, for instance, was presumably at a more advanced growth stage, although it was still not fully erupted. At this growth stage, these replacement teeth exhibited fully formed crowns, but not yet fully formed roots, which grew later. Fully-grown teeth (fully functional or even about to be replaced) are not preserved in the Hesperornis dentary for a variety of possible taphonomic reasons. For instance, such teeth, more prominently protruding from the jaw, were possibly more prone to falling out following death, especially with roots starting to be resorbed in the groove without alveoli. Alternatively, mature teeth may have been more prone to shedding; this may have been the case if they were fully emerged and close to being replaced and expelled by replacement teeth growing within their root. In the Ichthyornis dentary fragment, similarly, even the larger teeth present are not fully grown; they are much smaller than their alveoli and are not yet erupted. Their roots were only at the initial stage of growth at time of death, and would have presumably grown further only with the crowns erupting into their functional position.

Geometry of replacement in Hesperornithiformes

In Hesperornis, an isolated functional tooth with a lingual, oval resorption pit in its root has been described [6]. This resorption pit includes a tooth germ in place. A similar condition has been described in Parahesperornis [13, 48], a close relative of Hesperornis [8]. Tooth germs are observed here in the Hesperornis dentary. These are slightly displaced post-mortem, but nevertheless are situated under larger teeth, or lingually inside the groove against the lingual groove wall. They exhibit the previously described geometry of dental replacement of Parahesperornis and Hesperornis. YPM.1206B seems to show a possible lingual resorption pit in the root (Fig. 1a), which again is in line with the previously characterized tooth replacement in the Hesperornithiformes, whereby the tooth germ enters the root lingually and then develops under the functional tooth.

Geometry of replacement in Ichthyornis

The resorption pit observed on the side of one Ichthyornis tooth (Figs. 1e and 8c) is the first identified in this taxon, and reveals a similar dental replacement geometry to that observed in Hesperornis [6], Parahesperornis [13, 36, 37, 48], Archaeopteryx [31], crocodilians (e.g., [40, 66–68]; Additional file 10: Fig. S3), and stemward non-avian theropods (see below). The alleged differences in replacement geometry between non-avian theropods and the other taxa cited in [13, 36, 37, 48] are disproven. On the contrary, in all of these groups dental replacement proceeds on the lingual side by a small, growing replacement tooth germ entering the root cavity of the functional tooth through a lingual resorption pit, which is due to the activity of odontoclasts. Later, the replacement tooth grows under the functional one and finally expells it. Our observations therefore contradict the view of supposed ‘vertical’ tooth replacement in Ichthyornis proposed in [9], where the tooth germ would purportedly enter the functional tooth root from under its base without forming a resorption pit (or scar). This assumption was apparently motivated by the prior lack of data regarding resorption pits or scars in Ichthyornis. However, not all teeth preserving roots illustrate resorption pits, even in Hesperornis. In fact, teeth with resorption pits are very scarce among ornithurine teeth preserving roots (both isolated and in place within jaws). By comparison, in juvenile crocodiles, nearly all functional teeth exhibit root resorption at the same time (Additional file 10: Fig. S3). The replacement rate diminishes with age in crocodiles [69], and we lack data on growth series of Ichthyornis and Hesperornis. But our observations suggest that in these birds the frequency of dental replacement might have been markedly lower than in Crocodylia. As a result, we suggest that the number of ornithurine tooth generations during life would have been correspondingly lower, as well. Marsh ([6]: 125) was the first to claim that vertical replacement would characterize both Ichthyornis and crocodilians, as opposed to the horizontal (lingual) replacement of Hesperornis and mosasaurs. However, our study illustrates that all of these taxa, as well as all archosaurs in general, display horizontal, lingual tooth replacement, including Ichthyornis (this study) as well as crocodilians (e.g., [66]). Tooth replacement becomes vertical once the tooth germ has acquired its position in the functional tooth root; however, the geometry of replacement setup indicates that it is most accurate to characterize this pattern as lingual replacement. There is presently no evidence to suggest that vertical replacement ever existed among archosaurs.

Paleobiology

Diet

Piscivory has long been assumed for both Hesperornithiformes and Ichthyornithiformes, based on distally recurved/hooked or distally slanting crown shape (increasingly towards the mesial end), presumably adapted for holding fish and other slippery prey. Marine depositional settings where the fossils are found are in accordance with this interpretation, both for Hesperornithiformes (flightless, foot-propelled divers adapted to pursuing fish underwater) and Ichthyornis (volant marine birds that likely acquired prey at the water’s surface). The basal-apical crown ridges exhibited by Hesperornis are also observed in several presumably piscivorous marine reptiles, suggesting an association with a piscivorous diet [17]. However, the functional and adaptive significance of these ridges remains unclear. Ichthyornis teeth, with their sharp cutting carinae, may suggest that these birds used their teeth to cut pieces apart before ingesting, whereas Hesperornis presumably swallowed prey whole more generally.

Growth dynamics

Our analyses of von Ebner increment lines indicate that one Hesperornis regalis tooth formed in 66 days, to be compared to what was observed [20, 21] for a crocodilian (110 days for juvenile Caiman) or a large theropod (264 days for juvenile Tyrannosaurus). Smaller teeth (from smaller species) are formed and replaced more quickly than larger ones (from larger species), for instance in sauropod dinosaurs [35]. But this size effect alone does not seem to explain a faster rate of tooth growth in Hesperornis regalis, especially given that Hesperornis was an extremely large bird, compared with other stem birds (almost the size of a human). Moreover, as was noticed earlier [70], the increment lines observed in several studies have an anomalously large width (from 10 to roughly 20 μm in [20, 21], and 15 μm in [35]), and may in fact correspond to Andresen lines instead of daily increments (intervals between Andresen lines corresponding to several days). Thus, the relatively high estimates of tooth replacement rates based on those figures should be considered with caution.

The rate of crown and root extension in Hesperornis (calculated here at the cervix level) is especially rapid compared with non-avian teeth studied here, and previous studies; however, crown extension rates for reptiles were not provided in [20, 21]. The rate of human premolar tooth extension has been shown to be variable: it begins slowly (at a value of 4 μm/day), rises (to a maximal rate of 8 to 18 μm/day) and reduces again as the apex of the root closes [71]. Our extension rates, calculated at the cervix level, are higher than these and other primate tooth measurements (e.g., different Proconsul teeth yield values from 6 to 34 μm/day; [72]). High extension rates in Hesperornis teeth might correspond to a functional need for the rapid installation and anchorage of new teeth when preceding teeth had fallen out. In addition, elongated and thin teeth are functionally linked to piscivory, which might explain part of the rate of crown extension (crown height vs. width or length).

Our suggestion of a lower number and frequency of dental replacements in Hesperornis (see above in ‘tooth growth and replacement’) than in non-avian theropods or Crocodylia (see [24]) might be tested in future studies of dentine increment of functional teeth with associated replacement teeth, in ornithurines. Incidentally, based on bone histology, ornithurines such as Hesperornis and Ichthyornis have been shown to exhibit body growth rates and inferred metabolic levels similar to those of modern birds. These rates are comparable to those of eutherian mammals —also fully homeothermic— and higher than those of comparatively stemward birds and dinosaurs, extant crocodilians, and other archosaurs [4, 73–76]. If Hesperornis exhibited growth rates similar to those of modern birds, adult size might have been attained within the first several weeks or months following hatching. As such, fewer dental replacements in Hesperornis might be expected, since turnover in the dentition would not be necessary to accommodate any additional growth of the jaws. This situation would contrast with that exhibited by most polyphyodont reptiles, in which continuous dental replacement throughout life allows shed teeth to be replaced with slightly larger ones in order to accommodate the continuous, slow body growth typical of poikilothermic organisms such as crocodiles [22].

Dental characters and bird origins

Several alleged characteristics of the dentitions of Hesperornis and Ichthyornis have been cited in support of a non-dinosaurian or a non-theropod origin of birds [13, 36, 37, 48]. According to these authors, as summarized by Feduccia ([37]: 79), these features include, for birds and differing from ‘typical’ theropods: peg-like teeth without ornamentation or serrations; distal teeth with expanded roots; ‘subthecodont insertion’, teeth developing in a groove and septa forming around roots later; dental replacement from lingual side of roots, then ‘vertical’; oval resorption pit closed at base, on lingual side of the root of teeth being replaced; attachment with cementum and periodontal ligament. Our new observations allow us to critically evaluate these features.

Some Troodontidae have been shown to display many alleged ‘avian’ dental features [23, 77, 78]. This has led authors to acknowledge that these troodontids share homologous dental characters with birds [37]. These features include, for some or all troodontids: peg-like teeth without serrations; teeth without ornamentation on enamel; large root; constriction between crown and root; oval resorption pit closed at tooth base and germ tooth growing inside root of functional tooth; and absence of lingual interdental plates. Incidentally, peg-like teeth are not especially widespread in birds, and numerous species exhibit other morphologies (tooth crowns that are, for example, recurved, or bulbous). Even within the same individual, peg-like teeth may occur distally, but mesially the teeth can become highly recurved, such as in Hesperornis and Ichthyornis. This feature is even observed in Archaeopteryx [79]. Beyond troodontids, however, all of the characters cited as discriminating birds (and troodontids) from ‘typical’ theropods fall into one of two categories: 1) characters once thought to be exclusively avian and now known in ‘typical’ theropods, or 2) characters once thought to be unknown in birds but now reported among their representatives.

Features described previously as ‘avian’ but now known in ‘typical’, non-avian theropods

Serrations on tooth crowns are absent in most teeth of Compsognathus (except the distalmost ones), as well as on some of the teeth in Ornitholestes and probably Coelurus [80]. Serrations are also lacking in some troodontids such as Byronosaurus jaffei (including juveniles), Mei long, Archaeornithoides deinosauriscus, and Urbacodon itemirensis [81–86], as well as Anchiornis —which is either avian or troodontid— [78]. Serrations are lacking similarly on the teeth of Buitreraptor and Rahonavis (unenlagiine dromaeosaurs; [87, 88]), Sinornithosaurus milleni and Microraptor (microraptorine dromaeosaurs; [77]), dromaeosaurid hatchlings [89], probable deinonychosaurian teeth of uncertain affinity such as some “Paronychodon” types [90], as well as some Alvarezsauridae (Shuvuuia, Mononykus; [91, 92]), ornithomimosaurs, and therizinosaurs [87, 88]. Constriction between crown and root exists in some members of the Troodontidae such as Troodon, Saurornithoides mongoliensis and Byronosaurus jaffei [23, 77, 82, 83, 85, 93], Microraptor (Dromaeosauridae; [94, 95]), Alvarezsauridae (Shuvuuia, Mononykus) and some ornithomimosaurs and therizinosaurs [77, 87, 88, 91, 92]. Constriction between crown and root has been considered to be a derived condition within theropods [23]. Furthermore, absence of denticles and the constriction between the crown and the root are symplesiomorphic characters of the Maniraptoriformes and Maniraptora [87, 88]. Under this scenario, the denticles and absence of constriction in most dromaeosaurids, for instance, would be considered to be apomorphic. Incidentally, in birds with well-known dentitions (e.g., certain ornithurines and Archaeopteryx), constriction between the crown and root is not always pronounced; it is most prominent in distally positioned teeth (which tend to be straighter), and becomes faint to absent for mesial teeth (which tend to be more recurved, or slanted). Absence of interdental plates is probably a derived condition within theropods [23]. Interdental plates are absent in troodontids [23], which show jaw edges that are broadly similar to those of crownward toothed birds such as the ornithurines examined here. Expanded tooth roots are another supposedly ‘avian’ feature, but these are known in a number of non-avian theropods [23, 24, 65]. Taphonomic biases are certainly responsible for previously obfuscating the prevalence of expanded tooth roots in these stemward, non-avian theropods since, in the majority of cases, theropod teeth are found isolated, without most of the root. However, the same holds true for the fossil teeth of birds; these are often preserved in isolation without their roots (see many of the Aves indet. isolated teeth studied or cited here). In birds and some troodontids, the root has been described as being covered with cementum and held in place by a periodontal ligament. Post-mortem decomposition of the ligament would therefore cause the tooth to become detached. In ‘typical’ theropods, the teeth have been described as attached directly to cancellous bone designated under the term ‘bone of attachment’, via ‘sub-pleurodonty’ [13, 36, 37]. This would strengthen the root implantation after decay, and only the crown would therefore be expected to break off, and, hence, be found preferentially as isolated fossil remains. But attachment through cementum and periodontal ligament is widespread, including in dinosaurs, which are fully thecodont, not subpleurodont, and cancellous ‘bone of attachment’ is synonymous with alveolar bone (see Discussion-Implantation, attachment). The strength of tooth implantation in most avian and non-avian theropod taxa is likely comparable, since all exhibit a similar thecodont attachment with cementum, periodontal ligaments and alveolar bone.

We find the claim that isolated fossil bird teeth, in contrast to those from non-avian dinosaurs, should be expected to preferentially preserve the root (and that this would reflect a different mode of implantation and attachment; [13]) to be unfounded. The few known ‘isolated’ avian teeth that have been diagnosed to species (the material belonging to the Hesperornithiformes, Ichthyornithiformes, and Archaeopteryx discussed by these authors) are teeth that fell out of the jaw after death, and were generally found in close association with other cranial remains. These associations enable species-level identifications. However, a number of isolated bird teeth are known in the same state of preservation as isolated non-avian theropod teeth from a number of Mesozoic localities (e.g., Judith River Fm.). Unfortunately, the difficulty of positively identifying these to species casts doubt on the very identification of these remains as bird teeth. Hence, identification biases may help explain the false impression that the ratio of teeth preserved with and without roots differs between birds and ‘typical’ theropods. Conversely, a few typical isolated theropod teeth with their roots do exist in the published record; they have generally undergone little transport compared with shed teeth [96], but are otherwise preserved as well as shed teeth in proportion to their respective numbers produced until an individual’s death. Whereas isolated teeth bearing roots are teeth that have fallen out after death (due to decomposition of the periodontal ligament), isolated teeth without roots are shed during life, thanks to continuous replacement. Species with prolonged and/or frequent replacement also will naturally produce more shed teeth. They generally lack roots because the root is almost completely resorbed when a functional tooth is expelled by a growing replacement tooth, and because roots are rarely preserved in this manner. Continuous replacement yields a much greater quantity of shed teeth (all generations before death for a given individual) than the quantity one would expect for root-bearing teeth (originating from only one generation of teeth, at the time of death). The preservation state of avian vs. non-avian theropod teeth therefore provide no evidence pertaining to supposed differences in root attachment (contra [13]).

Features previously described as ‘non-avian’ but actually found in birds

As we highlight above, although isodonty has been cited as an avian feature, several birds exhibit highly recurved mesialmost teeth, and straight distalmost teeth, with a gradient in between. These taxa therefore qualify as exhibiting heterodonty. Describing avian teeth as peg-like is overly simplistic (see above); furthermore, we highlight the labio-lingual compression of avian teeth even in comparison with the non-avian coelurosaur Richardoestesia. These avian teeth do not qualify as peg-like, except possibly the distalmost teeth in heterodont taxa. Surface enamel ornamentation is often cited as a ‘non-avian’ feature, but is extremely well-marked in Hesperornis (‘fluted’ ornamentation, i.e., ridges of enamel), a fact often overlooked despite initially being described by Marsh [6]. More recently, well-marked basal-apical grooves were described in tooth crowns of an enantiornithine bird [97]. Serrations are cited as ‘non-avian’, but recently a Mesozoic enantiornithine bird from China has been shown to display tooth crowns with ‘crenulations’ (even though these differ somewhat from the serrations seen in many non-avian theropods in their shape and in their arrangement in two parallel basal-apical rows along the distal edge of tooth crowns) [98]. The alleged difference between a closed pit in birds and crocodilians, and a ‘scar’ open toward the basal direction of the root in theropods [13, 36, 37], is also not concordant with our observations, nor with the available, published data. In Ichthyornis we see a resorption pit that is ovoid, on the lingual side of the root, and open at the basal edge of the preserved root. Depending on the degree of resorption (and preservation) of the functional tooth root, and the degree of penetration of the tooth germ at time of death, there is a range of degrees of extension of resorption pits, even within a given species. Hence, contra [13], the resorption pit is not always closed at its base in birds, nor in Crocodylia (Additional file 10: Fig. S3). Conversely, a dromaeosaur tooth has been reported to exhibit a closed resorption pit at its root [65]. ‘Interdental plates’ are situated between teeth but lingual to the tooth row. Though superficially individualized, these plates are integrally part of the jaw bone, and are histologically continuous with the adjacent bone (be it the dentary, maxilla, or premaxilla; [99]; contra [13]; contra [36, 37]). Interdental plates are absent in Hesperornis and Ichthyornis; the lingual edge of the jaw maintaining the teeth is totally continuous with the rest of the jaw bone. Archaeopteryx, however, possesses interdental plates similar to those of most ‘typical’ theropods, consistent with its stemward position with respect to Hesperornis and Ichthyornis [49, 79]. This character, which is widespread and primitive among archosaurs, was independently lost several times throughout archosaur evolutionary history, perhaps through fusion and a smoothing of the grooves delimiting the plates in lingual view. Disparity in the number and degree of individualization of the plates indicates considerable plasticity across their evolutionary history, with possible occurrences of re-evolution of plate individualization.

‘Vertical’ vs. ‘horizontal’ families of replacement teeth

Contra [6] and contra [9], Ichthyornis shows the same kind of lingual replacement as Hesperornithiformes, Archaeopteryx, some troodontids, and many ‘typical’ theropods (contra [37]). ‘Vertical’ replacement appears completely absent in archosaurs, whereas lingual replacement appears to be the rule. Another purported difference between the avian condition and that of ‘typical’ theropods was that, in the latter group, the replacement tooth would grow lingual to the functional tooth, without migrating within its root —at most making a scar in the lingual side of the root— before the shedding of the functional tooth [13, 36, 37]. This would represent a difference between non-avian theropods on the one hand, and birds and crocodilians on the other hand; in the latter two groups most of the growth of a replacement tooth, from germ stage to the stage where the functional tooth sheds, takes place inside the functional tooth root after having migrated there through a resorption pit created by odontoclasts around the tooth germ. Observations in one troodontid [77] indicate that the tooth germ makes an ovoid, closed resorption pit in the lingual side of the functional tooth root, and grows inside the latter root, which is considerably expanded. It seems that simultaneously the tooth germ makes a resorption hole in the bone wall (not observed in birds or crocodilians), a possible consequence of there being less space around the teeth in troodontids. The geometries of tooth replacement in birds and troodontids, vs. most other theropods, may be seen as the two ends of a continuum with many intermediate geometries not well documented due to the rarity of sufficiently well preserved series of replacement teeth. It is conceivable that the avian and crocodilian replacement geometries evolved from a plesiomorphic geometry retained by stemward theropods. Indeed, the typical theropod geometry is widely seen in other dinosaurs, such as sauropods with their batteries of replacement teeth lingual to functional ones [35]. Moreover, the ‘typical’ theropod condition is probably not uniform. Currie and Zhao [65] reported a drawing of a dromaeosaurid tooth with a closed ovoid resorption pit in the side of its expanded root. These authors ascribed the comparative rarity of such teeth in theropods as being due to tooth replacement occurring at a higher angle in relatively narrow jaws, resulting in that stage of replacement being “more transitory” ([65]: 2245).

An apparently real difference between many non-avian theropods, other (non-avian) dinosaurs, and archosaurs in general (e.g., [31]), vs. birds and some troodontids, consists of the existence in the former of variable ‘batteries’ of replacement tooth germs lingual to a functional tooth, whereas in birds (and some troodontids) a maximum of one replacement germ is hitherto observed lingual to a functional tooth or inside its root. Again, this decrease of the number of replacement teeth at a time is probably in line with a lower number and frequency of dental replacements (oligophyodonty) in birds in general, as already hypothesized in Archaeopteryx [31, 100].

To summarize, Hesperornis and Ichthyornis, the most crownward toothed birds known, show numerous derived dental features. These include extremely thin and simplified enamel in both taxa, and the presence of a groove housing the teeth in Hesperornis. Additionally, numerous features of their dental biology have been erroneously characterized in existing literature (ranging from geometry of dental replacement, form of resorption pit, and similarity of implantation between Hesperornis and Ichthyornis). We provide evidence for accurate assessment of these and other features. Furthermore, we demonstrate that many dental features do not radically differ between theropods and several other dinosaur groups, birds, and crocodilians (including geometry of dental replacement, and presence or not of interdental lingual plates). Indeed, the supposedly ‘avian’ condition appears to be much more phylogenetically widespread than previously reported. Arguments suggesting that the dentitions of Hesperornis and Ichthyornis provide evidence for a non-theropod, or non-dinosaurian origin of birds are therefore in error. Additionally, we confirm that some of the so-called ‘avian’ dental characteristics are only shared by certain non-avian theropod subclades, such as troodontids. These homologous characters add to the great phylogenetic proximity between troodontids and birds, now acknowledged to such an extent that one of these two clades is probably a subclade of the other [37, 101–103]. Finally, we contribute to document that some characters generally assumed to be absent in birds (e.g., ornamentation of the enamel, serrations) are occasionally present.

Toward more precise identification of late cretaceous isolated avian teeth

Distinguishing morphologically between possible isolated avian teeth and Richardoestesia isolated teeth has already been shown to be difficult [25]. On a graph showing crown base width vs. height (Fig. 5), isolated TMP teeth studied here (Fig. 4) plot together with Ichthyornis, Hesperornis and other avian teeth studied, as well as the teeth identified as avian by Sankey et al. [25] (a sample that includes some of our TMP teeth; see Material and Methods): compared to Richardoestesia the avian tooth crowns seem to be proportionately larger at base, relative to their height; this trend is accentuated by the two larger of TMP teeth in our sample (Fig. 5). All of these isolated teeth are found in the same, or contemporaneous, localities in North America, where important numbers of such fossils are found in diverse localities [25]. Hence, it is useful to find further criteria for identification of isolated teeth as avian or non-avian. The two larger TMP teeth that also stand out in terms of their relative crown width vs. height, actually display additional characteristics, and altogether this questions their supposed avian status. One (TMP 1989.103.0025) bears a well-marked constriction between crown and root, and was re-identified recently as Richardoestesia isosceles (Coelurosauria incertae sedis; [27, 38]), contra [25]. The other large tooth (TMP 1996.012.0040) bears no clear constriction, is rather straight and bears no serrations, but exhibits a wear facet at the tip of the crown. We concur with [27, 38] that TMP 1989.103.0025, with its peculiar small serrations limited to a small part of both the mesial and distal carinae, does not belong to a bird. TMP 1996.012.0040 might be avian, but in the absence of associated skeletal remains, no precise identification is possible. Interestingly, wear facets are scarce, but not unknown in bird teeth (some are observed in presumably insectivorous birds, like Archaeopteryx [104]; or Pengornis [105]), despite the general inference that birds do not process food with their jaws [4]. We consider that TMP 1996.012.0040 is better assigned to cf. Aves indet. These two larger teeth might not belong with the avian teeth as they plot in a different portion of morphospace (Fig. 5). We consider that TMP 1989.103.25 is more likely to be compatible with Richardoestesia isosceles [27, 38] despite its marginal position, also relative to the other Richardoestesia teeth. In that case, the labio-lingual slight crown enlargement (relative to height) would appear to discriminate only marginally the ornithurine teeth from most contemporaneous non-avian North American theropod teeth. One TMP tooth crown (TMP 1986.052.0054) bears no serrations, and would fit well with birds on the basis of its ornithurine-like morphology; we concur with [25] in assigning it to Aves indet. Finally, two tooth crowns (TMP 1986.030.0039 and TMP 1994.031.0032) bear serrations recalling non-avian theropods. A single Mesozoic bird is now known to have crenulated teeth, reminiscent of serrations, albeit with unique characteristics of shape and distribution [98]. As a result, an avian status for those two serrated crowns is not totally excluded, but it is more likely that they belong to non-avian theropods. We consider it most appropriate that these teeth are referred to Theropoda indet. in the absence of associated remains.

The Maastricht tooth (Fig. 1h) is shown here to be positively identifiable as either belonging to Ichthyornis sp., or to a closely related taxon within the Ichthyornithiformes. It shows the characteristic triangular, labio-lingually compressed crown shape, with sharp, unserrated mesial and distal ridges, and very thin enamel (mostly around 5 μm in thickness). The whole preserved tooth shows the characteristic ichthyornithiform angle between crown and root (the root is visibly well developed despite the fact that it lacks most of its basal portion). The tooth size is also compatible with its diagnosis as Ichthyornis. The associated elements preserved alongside the tooth also allowed Dyke et al. [106] to recognise a closer affinity of the specimen with Ichthyornis than with any other known ornithurine taxon, despite the Maastricht specimen’s relatively larger size. Hence, we consider that an assignment of the specimen to the Ichthyornithiformes is pertinent. This represents the only record of Ichthyornithiformes outside North America, and the expanded distribution of this clade parallels that of the Holarctic Hesperornithiformes. This specimen not only represents one of the youngest known ichthyornithiforms [10], but indeed one of the youngest known non-neornithine ornithurines. The extension of the crownward-most portion of the avian stem towards the Cretaceous-Paleogene boundary supports the idea that the proximal avian stem survived until, and perished in, the end-Cretaceous mass extinction event [10, 107].