Extreme Cranial Ontogeny in the Upper Cretaceous Dinosaur Pachycephalosaurus

Background Extended neoteny and late stage allometric growth increase morphological disparity between growth stages in at least some dinosaurs. Coupled with relatively low dinosaur density in the Upper Cretaceous of North America, ontogenetic transformational representatives are often difficult to distinguish. For example, many hadrosaurids previously reported to represent relatively small lambeosaurine species were demonstrated to be juveniles of the larger taxa. Marginocephalians (pachycephalosaurids + ceratopsids) undergo comparable and extreme cranial morphological change during ontogeny. Methodology/Principal Findings Cranial histology, morphology and computer tomography reveal patterns of internal skull development that show the purported diagnostic characters for the pachycephalosaurids Dracorex hogwartsia and Stygimoloch spinifer are ontogenetically derived features. Coronal histological sections of the frontoparietal dome of an adult Pachycephalosaurus wyomingensis reveal a dense structure composed of metaplastic bone with a variety of extremely fibrous and acellular tissue. Coronal histological sections and computer tomography of a skull and frontoparietal dome of Stygimoloch spinifer reveal an open intrafrontal suture indicative of a subadult stage of development. These dinosaurs employed metaplasia to rapidly grow and change the size and shape of their horns, cranial ornaments and frontoparietal domes, resulting in extreme cranial alterations during late stages of growth. We propose that Dracorex hogwartsia, Stygimoloch spinifer and Pachycephalosaurus wyomingensis are the same taxon and represent an ontogenetic series united by shared morphology and increasing skull length. Conclusions/Significance Dracorex hogwartsia (juvenile) and Stygimoloch spinifer (subadult) are reinterpreted as younger growth stages of Pachycephalosaurus wyomingensis (adult). This synonymy reduces the number of pachycephalosaurid taxa from the Upper Cretaceous of North America and demonstrates the importance of cranial ontogeny in evaluating dinosaur diversity and taxonomy. These growth stages reflect a continuum rather than specific developmental steps defined by “known” terminal morphologies.


Introduction
Pachycephalosaurids are a group of ornithischian dinosaurs united by the presence of cranial ornamentation and an enlarged frontoparietal dome, a unique morphology among vertebrates. The history and description of Pachycephalosaurus involved many prominent dinosaur paleontologists of the early 20 th century. A review of their early observations and taxonomy, together with the systematic paleontology of Pachycephalosaurus proposed in this study, is provided in Supporting Information [Text S1].
A great many papers have been written about pachycephalosaurid crania, in particular concerning the variability of the frontoparietal domes [1,2 and references cited therein], but there are few studies that have attempted to understand the development of these unusual cranial structures. Interestingly, even though ontogeny was discussed in a few of these evaluations [3][4][5][6][7][8], it was not considered an important variant beyond the relative inflation of the dome, apparently because of the relatively similar sizes of the different individuals from time-equivalent strata. The presumption that individuals possessing juvenile characteristics are expected to be much smaller than adults is not corroborated in hadrosaurs [9] and ceratopsian dinosaurs [10][11][12] with a sufficient sample size. Prior evaluations of cranial variation in pachycephalosaurids did not consider earlier studies that demonstrated several dinosaur taxa showed allometric growth of the skull and a significant expression of horns, domes and bony ornaments when the skulls reached approximately 50% size. Now, with a greater number of pachycephalosaurid skulls from the Upper Cretaceous of the Western Interior of North America available for study, and the use of comparative cranial morphology, histology and computer tomography, multiple lines of evidence support our alternative hypothesis that Dracorex hogwartsia and Stygimoloch spinifer represent earlier growth stages in a single taxon, Pachycephalosaurus wyomingensis.
Relative, or proportional growth (allometry of Huxley [13]), refers to shape change with regard to an increase in size. Brown and Schlaikjer [10] first reported this relative growth in dinosaur crania in the Cretaceous Mongolian ceratopsid Protoceratops. Later studies by Rozhdesvensky [14] found the phenomenon not only in Protoceratops, but also in the Mongolian hadrosaurid Saurolophus and theropod Tarbosaurus. Allometric growth was apparent in these taxa, in part, because of the large number of specimens representing a range of ontogenetic transitions collected from the Mongolian strata. Dinosaurs of the Upper Cretaceous of Mongolia are tremendously abundant and this region is recognized around the world as one of the planet's most fertile collecting grounds. Due to relatively lower specimen density in the Western Interior of North America, ontogenetic transformational representatives were difficult to distinguish until Dodson [9] performed statistical studies on a group of ''closely related'' lambeosaurine hadrosaurs from Upper Cretaceous strata in Alberta, Canada, in 1975. Dodson's study revealed that many hadrosaurids previously reported to represent relatively small lambeosaurine species were instead juveniles of the larger taxa. These lambeosaurine taxa experienced cranial allometric growth and retained juvenile characters (neoteny) until the skulls reached approximately 50% adult size. Dodson [9] compared the retarded development of the characteristic hollow narial crest in lambeosaurines with the expansion of the casque in the avian taxon Casuarius. Casque development begins after the cassowary skull reaches 65% to 80% adult size. Dodson showed that extended neoteny and late stage allometry increased the morphological disparity between particular growth ''stages'' within the lambeosaurine taxa. In 1976, Dodson [11] published a related study on Protoceratops, and provided statistical data to quantify the earlier qualitative observations on allometric growth in these dinosaur skulls [10,14].
Morphogenetic and osteohistogenetic evaluations of dinosaur genera from the Upper Cretaceous Hell Creek Formation of Montana, North and South Dakota, and equivalent age sediments in adjacent states and Canadian Provinces, are providing evidence that extreme modification of dinosaur skulls occurs when these skulls approach $50% adult size in many dinosaur groups, particularly the Marginocephalia. Marginocephalian dinosaur growth and histology of their cranial ornamentations follow the predicted trend of osteohistogenesis. A Triceratops ontogenetic series described by Horner and Goodwin [15,16] revealed major ontogenetic modifications to cranial ornamentations on various parts of the skulls. The epiparietals and episquamosals bordering the edge of the parietalsquamosal frill are initially diminutive before expanding to larger, taller equilateral triangular bones, and later in ontogeny, flatten dorsoventrally and elongate as they merge with the edge of the frill. The smallest epiparietals, episquamosals and postorbital horns reveal the youngest histological tissues. Postorbital horns that grew straight at first, arc strikingly backward in younger individuals then recurve forward in later stages of ontogeny. Forward horn orientation and expression of the largest epiparietals and episquamosals occur in crania that are about 65% the length of the largest known specimens. We confirm a similar pattern of development and osteohistogenesis in the cranial morphology of the squamosal horns, nodes and frontoparietal dome in the skulls of the pachycephalosaurids Dracorex hogwartsia, Stygimoloch spinifer and Pachycephalosaurus wyomingensis.

Results and Discussion
Comparative Cranial Morphology of Pachycephalosaurus, Stygimoloch and Dracorex In the following section, we begin with Pachycephalosaurus and demonstrate that the nominal pachycephalosaurid genera, Dracorex and Stygimoloch, represent the same taxon at an earlier ontogenetic stage. We use comparative cranial histology and morphology to confirm how these ontogenetic transformations developed in a single taxon, Pachycephalosaurus (Table 1). These growth stages reflect a growth continuum rather than specific developmental steps defined by ''known'' terminal morphologies. This is important because currently we do not know the ultimate size or maximum age of any dinosaur species and an adult growth stage assignment purports a potentially false terminal morphological state. The frontoparietal and lateral cranial elements are highly inflated in the largest, and presumably oldest domed Pachycephalosaurus and follow the morphological sequence proposed for Stegoceras [3,17]. The frontoparietal dome was not preserved with the isolated left squamosal and holotype of S. spinifer, UCMP 119433 ( Figure 1A), but a dome is present in more complete pachycephalosaurid skulls with identical squamosals and squamosal horns referred to S. spinifer such as UCMP 131163 ( Figure 1B Figure 4) that is evidence of cranial vault expansion during postnatal craniofacial growth [18]. The intrafrontal suture functions as an intramembranous bone growth site and remains unossified as new bone is formed [18] in this subadult ontogenetic stage. The absence of a visible intrafrontal suture on the dorsal skulls of ''Stygimoloch'' previously obscured their relative age.
In Figure 3, morphological landmarks are colored and evident on the skulls of Pachycephalosaurus, Dracorex and Stygimoloch. Inflation of the frontoparietal dome and corresponding closure of the temporal fenestrae are two major morphological changes that occur between Dracorex and Pachycephalosaurus with concurrent lengthening of the skull that ontogenetically separates the three pachycephalosaurids older than ''Dracorex'' shown in Figure 3A-F (see Table 1). A composite image of the holotype of Dracorex    Figure 5. The combined cranial morphology of this subadult ontogenetic stage was verified by the authors and Sullivan [8:359] in two skulls from the Hell Creek Formation, Garfield County, Montana, that are privately held and currently unavailable for study. UCMP 556078 ( Figure 3C,D) is a slightly more advanced subadult stage of ontogeny, compared to the composite skull in Figure 5, with an inflated frontoparietal and lateral elements that are enlarged but not yet incorporated into the dome as they are in Pachycephalosaurus. We predict this ontogenetic trend, if projected backward toward an even younger end member than ''Dracorex'', would result in a flat-headed morph ,43 cm long ( = midline length of ''Dracorex'') with a slightly thickened (or incipiently domed) frontoparietal region, open supratemporal fenestrae and dorsal skull covered with emerging nodal ornamentation with clusters of relatively smaller diameter pyramidal nodes on the squamosals. A slightly less robust, narrower skull compared to the holotype of ''Dracorex'', with these precise morphological characteristics, is figured in the literature [19:6] and discussed by Sullivan [8]; however it is privately held.
A series of prominent pyramidal nodes on the anterodorsal surface of the nasals in the holotype of Dracorex hogwartsia ( Figure 3G,H) match the nasal ornaments preserved in the AMNH 1696 skull of Pachycephalosaurus wyomingensis ( Figure 3A,B) [8] and a slightly younger P. wyomingensis skull ( Figure 3C,D). We concur with Bakker et al. [20:331] that the ''long snout, two halfrings of pyramidal spikes on the snout…'' of ''Dracorex'' are nearly identical to the arrangement observed in Pachycephalosaurus and ''Stygimoloch''. The distinctive cluster of squamosal horns undergoes allometric growth before they are reduced in subadults. The slightly curved squamosal horns and nodes increase in length ontogenetically from ''D.'' hogwartsia ( Figure 3G,H) to ''S.'' spinifer ( Figure 3E,F) before the horns erode and nodes are modified into their blunt but robust pyramidal shape in the ontogenetically most advanced skulls of P. wyomingensis ( Figure 3A,B). The nodes form asymmetrical clusters on the dorsoposterior surface of the squamosals in the adult Pachycephalosaurus ( Figure 6, Figure 7). Just as the number, arrangement and shape of the epiparietals and episquamosals vary between individual Triceratops [16], the variability in the arrangement, dimensions and symmetry of the nodal ornamentation on the posterior skull margin in Pachycephalosaurus is neither unexpected nor unique within the Marginocephalia [8,21]. While a strict 1:1 correspondence between the squamosal ornamentation in ''Dracorex'' and Pachycephalosaurus is not exact, it is clearer between ''Dracorex'' and ''Stygimoloch'' before the frontoparietal undergoes extreme ontogenetic modification, particularly in the posterior-most region of the skull with the expansion of the cranial vault. Individual variation within this ontogenetic continuum plays a major role in the morphology of this highly modified region of the skull. Alternatively, these ''morphs'' could represent different taxa, but evidence from comparative cranial histology, computer tomography, morphology, and similar patterns of growth observed in Stegoceras [17] and Triceratops [15,16] does not support this interpretation.  Giffin's study [7] of seven pachycephalosaurids assesses geometric growth and relative frontoparietal dome shape using two landmarks restricted to the frontoparietal dome in a geographically diverse sample too small for statistical testing.

Cranial Histology
Bone histology is a valuable tool for assessing various growth parameters including relative growth rate and general bone ontogeny. Radial vascularization, for example, ''…is functionally associated with very fast deposition of relatively modest amounts of new compact bone'' [22:512]. Additionally, woven bone with minimal osteonal deposition is indicative of early osteogenesis, whereas dense Haversian or reconstructed tissues are indicative of later osteogenesis [17]. We utilize these general histological observations to ontogenetically order the various pachycephalosaurid cranial characteristics described in this study (Table 1,  Table 2).
Histological examination of the cranial ornamentations of dinosaurs reveals complex morphologies composed primarily of tissue formed during metaplasia. Metaplasia is the process in which dense fibrous connective tissues are transformed directly into bone without the intervention of a periosteum or the presence of osteoblasts [23]. Metaplastic bone, particularly as seen in dinosaur crania, has a wide variety of forms, from highly porous to extremely dense. These tissues can appear very similar to bone deposited by a periosteum in that they contain vascular canals, osteocyte-like lacunae and can be reworked by Haversian-like innervations. Although similar in appearance, the structures that look like osteocytes lack canaliculi, and instead possess stubby lateral processes that do not connect to adjacent cells [24]. These structures are herein referred to as fibrocytes and apparently represent trapped fibroblasts. Metaplastic bone is common in  reptiles [25], but generally described in association with osteoderms [25][26][27][28][29] and ossified tendons [30]. The metaplastic tissues in the skulls of pachycephalosaurids vary greatly in gross appearance depending on the cranial structure in which they form. As observed for other bone tissues, these metaplastic tissues undergo a consistent, predictable development in that the younger tissues are much more vascularized compared to increasingly mature tissues [17].
A coronal histological section of a pachycephalosaurid skull previously referred to ''Stygimoloch'' (UCMP 128383) shows an open intrafrontal suture in coronal view ( Figure 8A). The highly vascularized nature of the frontoparietal dome showing vascular spaces oriented primarily in a radial pattern ( Figure 8B) suggests that the dome was actively growing at a rapid pace [22:512]. Abundant fibers and fibrocytes located at the dorsal surface of the dome indicate an area of apparent inflation and ossification ( Figure 8C). A fully formed dome referable to Pachycephalosaurus ( Figure 8D) is much denser with comparatively less vascularization suggesting that growth has subsided [17]. Extending over the entire outer-most margin of the dome interior are the ends of fibers that apparently connected to a fibrous epidermal covering the dome ( Figure 8E). The presence of these exposed fibers suggests the dome was still inflating as there is no evidence of an erosional surface. A node on the postorbital bar ( Figure 8F), by contrast, possesses a surface that is erosional ( Figure 8G), bluntshaped and lacks any evidence of attachment or growth. An overlying matrix preserves this degraded surface. The most interesting and controversial aspect of our hypothesis, that ''Dracorex'' and ''Stygimoloch'' represent younger ontogenetic stages of Pachycephalosaurus, relates to the squamosal horns. They are medium to large pointed structures in ''Dracorex'' [20] and ''Stygimoloch'' (Figure 1, Figure 2), but are relatively shorter, blunt to lightly pointed and robust in Pachycephalosaurus (Figure 6,  Figure 7). Three squamosal horns were sectioned to establish this sequence, including the holotype of ''Stygimoloch'' (UCMP 119433). If ''Dracorex'' and ''Stygimoloch'' represent ontogenetic stages of Pachycephalosaurus, then it must be shown that these horns grow and elongate in ''Dracorex'' and ''Stygimoloch'' before the onset of erosion when they are reduced in size and modified in shape. We do not know at what growth stage maximum squamosal horn length is achieved, but it appears to occur between the holotype of ''Dracorex'' and some stage of ''Stygimoloch''.
The smallest horn (UCMP 128383) was found associated with the ''Stygimoloch'' dome in Figure 8A, revealing evidence of rapid dome growth. This horn also reveals highly vascularized tissue ( Figure 9A) indicative of rapid expansion in size and an extremely irregular exterior surface ( Figure 9B) that is very similar to bone that contacts a periosteum. The absence of osteocytes and abundance of fibers indicates the bone is expanding through metaplasia rather than periosteal ossification. A cross section through the largest central horn (#1 sensu Galton and Sues [31]) from the holotype of ''Stygimoloch'' ( Figure 1A) reveals metaplastic tissue with an embayed exterior surface ( Figure 9C) that is being eroded ( Figure 9D). There is no evidence that metaplasia was occurring at the time of the animal's death; however, there is an indication histologically that the horn underwent an earlier period of erosion followed by a phase of metaplasia, and subsequently a final period of erosion that was occurring when the animal died. Evidence for this sequence is the erosional unconformity indicated by the arrows in Figure 9C.
Another horn (MOR 560; found isolated) cut both longitudinally ( Figure 10A) and transversely ( Figure 10B) is also in an erosion mode as evidenced by a surface that is degrading with no signs of metaplasia ( Figure 10C). The primary metaplastic veneer is very thin, and most of the bone has been reconstructed by secondary innervations. Sharpey's fibers near the exterior surfaces and indented vessel channels on the exterior surfaces of each of the sectioned horns suggest that they were keratin covered regardless of whether they were undergoing metaplasia or erosion.

Conclusions
We propose that Dracorex hogwartsia and Stygimoloch spinifer are growth stages of Pachycephalosaurus wyomingensis and represent an ontogenetic series of P. wyomingensis united by shared morphology and increasing skull length. The youngest and most complete skull of Pachycephalosaurus yet known belongs to the growth stage best illustrated by ''Dracorex''. This synonymy significantly reduces the number of Upper Cretaceous pachycephalosaurid taxa.
Timing of dome inflation is probably highly variable based on the cranial patterns preserved in Stegoceras [4,32] and Triceratops [15,16]. Like some hadrosaurids and ceratopsids, these pachycephalosaurids retain their juvenile cranial characters until they are at least 50% grown. This ontogenetic pattern contributed to the naming of some new pachycephalosaurid taxa based on diagnostic characters (large supratemporal fenestrae, squamosal horns, nodal cranial ornamentation, inflated frontoparietal dome) that we demonstrate in this study are, in fact, ontogenetic features that undergo extreme modification as the skull length increases, the cranial vault enlarges and the frontoparietal dome expands. The squamosal horns and nodes of Pachycephalosaurus underwent a similar transformation to the epiparietal and episquamosal elements of Triceratops [16: figure 2] that (1) grew from diminutive to large triangular-shaped ornaments; (2) eroded as they reduced dorsoventrally and (3) flattened and lengthened as they merged onto the edge of the parietal-squamosal frill. Pachycephalosaurid horns originated ontogenetically as nodelike structures, some of which expanded into horns and bony spikes and then became modified as blunt, nodal structures in late stage ontogeny. The various shapes of the frontoparietal dome, from the characteristic tall broad dome of the Pachycephalosaurus wyomingensis (AMNH 1696) to the relatively flattened morphology of Pachycephalosaurus ''reinheimeri'' (DMNS 469) also reflect a comparable morphological transformation that occurs ontogenetically.
Marginocephalian dinosaurs employ metaplasia to grow their horns, cranial ornaments, domes and shields as they rapidly remodel their skulls. Any hypothesis testing of head butting in pachycephalosaurids, combat in ceratopsids or similar agonistic encounters must take into account the fact that the skulls of these dinosaurs are composed largely of metaplastic tissue.

Materials and Methods
Twenty-one pachycephalosaurid skulls and cranial elements examined in this study are listed in Table 2. Four casts were included to confirm external morphologies and supplement fossil specimens used for histological analysis. Cut blocks of fossils were embedded in Silmar-40 polyester resin prior to histological sectioning and mounted to glass slides with epoxy resin. Archival casts were made of the specimens before cutting and are catalogued in the collections of the MOR and UCMP. The thin sections were ground on a Buehler Ecomet grinder and sections were studied by light microscopy under normal and polarized light. Histological sections from this study are catalogued into the slide libraries of the MOR and UCMP. Some of the histological slides were prepared for a previous study by the authors [17].