A Complete Skull of an Early Cretaceous Sauropod and the Evolution of Advanced Titanosaurians

Advanced titanosaurian sauropods, such as nemegtosaurids and saltasaurids, were diverse and one of the most important groups of herbivores in the terrestrial biotas of the Late Cretaceous. However, little is known about their rise and diversification prior to the Late Cretaceous. Furthermore, the evolution of their highly-modified skull anatomy has been largely hindered by the scarcity of well-preserved cranial remains. A new sauropod dinosaur from the Early Cretaceous of Brazil represents the earliest advanced titanosaurian known to date, demonstrating that the initial diversification of advanced titanosaurians was well under way at least 30 million years before their known radiation in the latest Cretaceous. The new taxon also preserves the most complete skull among titanosaurians, further revealing that their low and elongated diplodocid-like skull morphology appeared much earlier than previously thought.


Introduction
Titanosaurians are known as the most diverse group of sauropod dinosaurs, including one-third of all known genera of sauropods [1,2]. Although their characteristic wide-gauge trackways have already been recorded in the Middle Jurassic [3,4], titanosaurians are mostly known from Late Cretaceous postcranial remains [2]. Their abundance in Upper Cretaceous sediments has been regarded as a result of the successful radiation of a clade herein referred as the ''advanced titanosaurians''. This radiation includes saltasaurids, nemegtosaurids and the closely related Isisaurus and Diamantinasaurus (but not the basal lithostrotian Malawisaurus), and was possibly triggered by the Cenomanian-Turonian global extinction of diplodocoid sauropods [5][6][7][8]. The presence of advanced titanosaurians in the Early Cretaceous, however, rests only on a few questionable fragmentary remains [9,10]. The lack of an adequate sampling in the known fossil record has limited our understanding of the early evolution and initial diversification of advanced titanosaurians. Additionally, with the exception of a few well-preserved skulls from the Campanian and Maastrichtian of Madagascar and Asia [11,12], little is known about the origin of their highly-modified skull anatomy, which shows remarkable convergences with diplodocids [8,13], as illustrated by their posteriorly displaced nares, forward leaning quadrates, and narrow-crowned cylindrical teeth restricted to the front of the snout. Preservation of a nearly complete skull among sauropods is rare, probably because of their delicate construction. Our knowledge of titanosaurian skull anatomy, in particular, is mostly restricted to Nemegtosaurus [11] and Rapetosaurus [12], which are among the youngest records for this group. The absence of well-preserved titanosaurian cranial remains from the Early Cretaceous presents a major hurdle to understanding the fossil record of this group. Here we report on a new advanced titanosaurian sauropod discovered in outcrops of Aptian age in the Quiricó Formation of the Sanfranciscana Basin [14] (Figures S1, S2, and S3). Its discovery fills an important temporal gap and provides new information on the initial changes that led to the cranial anatomy of more derived titanosaurians. The present discovery also represents the first described titanosaurian skull for the South American continent.

Data matrix construction
The data matrix used in the phylogenetic analysis is based on a published phylogenetic analysis [15], with the addition of Tapuiasaurus and the recently published character scorings for three other Early Cretaceous titanosaurians (Phuwiangosaurus, Tangvayosaurus, Diamantinasaurus) [10,16]. Nine skull characters were added to this dataset [2,11,13], resulting in a data matrix of 246 characters scored across 33 taxa (Text S2). Five of the multistate characters were treated as ordered, as in the original phylogenetic analysis [15].

Heuristic tree search and support measures
The dataset was analyzed using equally weighted parsimony in TNT [17,18] with a heuristic search of 1,000 replicates of Wagner trees followed by tree bisection-reconnection (TBR) branch swapping.
Two alternative support measures, Bremer support [19] and bootstrap resampling, were used to evaluate the robustness of the nodes of the most parsimonious trees. Bremer support [19] in TNT v.1.1 uses a combination of heuristic searches that save suboptimal trees with constraints for non-monophyly. One thousand bootstrap replicates were made using a heuristic tree search of 10 wagner trees (with random addition sequences) followed by TBR. Results of these replicates were summarized using absolute frequencies for each group.

Nomenclatural Acts
The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLoS by sending a request to PLoS ONE, Public Library of Science, 1160 Battery Street, Suite 100, San Francisco, CA 94111, USA along with a check for $10 (to cover printing and postage) payable to ''Public Library of Science''.

Systematic Paleontology
Holotype. MZSP-PV (Museu de Zoologia da Universidade de São Paulo) 807, consists of an articulated partial skeleton composed of an almost complete skull and mandible, hyoid apparatus, atlas, axis, five cervical and five dorsal vertebrae and ribs, left sternal plate, right coracoid, right humerus, left radius, ulnae, metacarpals, femora, left fibula, and an almost complete left pes.
Horizon and locality. The skeleton was found in outcrops from the Quiricó Formation (Sanfranciscana Basin), at Embira-Branca Hills near Coração de Jesus City, in northern Minas Gerais, Brazil.
Age. The age of deposition in the Sanfranciscana Basin is well constrained by two magmatic events to the Lower Cretaceous. It postdates the eruption of the Paraná continental flood basalts dated at 138-128 Ma [20], and for the most part predates alkalic lavas and volcaniclastic rocks dated at 95-76 [21], which are intercalated with sandstones of the upper part of the basin fill. The lacustrine deposits of the Quiricó Formation, which are in the lower part of the Sanfranciscana sequence, are constrained to the Aptian based on the presence of sarcopterygian fishes [22], ostracods [23], and palynomorphs [24] (see Text S1 and Figures S1, S2, and S3).
Diagnosis. An advanced titanosaurian diagnosed by the following autapomorphies: hook-shaped posteroventral process of the quadratojugal; anterior process of the jugal tapering and forming most of the ventral margin of the antorbital fenestra; anterolateral tip of the pterygoid contacts the medial surface of the ectopterygoid. The new taxon is also diagnosed by the following unique combination of characters: deep fossa on the lateral surface of the maxilla between the antorbital fenestra and the subnarial foramen; elongated middle cervical vertebrae; posterior dorsal vertebrae with well-developed prespinal lamina and absence of hyposphene-hypantrum; deep fossae located below intraprezygapohyseal lamina; crescentric-shaped sternal plate; proximodistally long coracoid; elongated ulna and distally expanded radius.

Description and comparisons
The skull of Tapuiasaurus (Figure 1), as in Rapetosaurus and Nemegtosaurus, has an elongated rostrum with narrow premaxillae that are not broadly exposed laterally, cylindrical teeth extending up to the level of the preantorbital fenestra, forward leaning quadrates, and external nares retracted to the level of the orbits [15,25]. The antorbital fenestra is larger than in most macronarians (including Nemegtosaurus) but not as elongated as in Rapetosaurus. The premaxilla projects posterodorsally along the dorsal surface of the rostrum, as in Nemegtosaurus [11]. Tapuiasaurus also shares with Rapetosaurus and Nemegtosaurus an elongated post- dentigerous process of the maxilla. This process of the maxilla tapers posteriorly instead of forming a robust contact with the jugal, as in Rapetosaurus. A unique feature of Tapuiasaurus is the presence of a long anterior process of the jugal that covers the dorsal edge of the maxilla and forms most of the ventral margin of the antorbital fenestra. The lacrimal has a broad ventral process and a remarkably long anterior process on its dorsal extremity, a condition otherwise only known in Rapetosaurus [12]. The prefrontal has a short transverse articulation with the nasal and bears an anterior process as in nemegtosaurids [11], which is extremely long and thin as in Rapetosaurus [12].
As in most neosauropods the postorbital bears a posterior process and its jugal process is elongated and anteroposteriorly flattened. The postorbital of Tapuiasaurus contacts the parietal, excluding the frontal and squamosal from the supratemporal margin. The exclusion of the squamosal from the supratemporal fenestra also occurs in Nemegtosaurus, but contrasts with the more generalized condition of other titanosaurians in which the squamosal participates from the margins of this opening (including Rapetosaurus). The parietals have a broad surface separating the supratemporal fenestrae (as in other titanosaurids [15]). The occipital portion of the parietals and the supraoccipital are dorsoventrally low, as in Nemegtosaurus and Rapetosaurus. However, in contrast to these two taxa [15], the squamosal and postorbital of Tapuiasaurus are not ventrally shifted respect to the parietal so that the supratemporal fenestra is visible in lateral view. The squamosal participates on the margin of the supratemporal fenestra as in most titanosaurids, except for Rapetosaurus [12]. The quadrate projects anteroventrally and bears a deep fossa, but the lateromedial crushing of the specimen precludes determining if this was exposed posterolaterally as in nemegtosaurids. The quadratojugal bears an acute posteroventral process that directs (but fails to reach) the quadrate condyles, a feature that may have been present in Rapetosaurus given the articular facet for this bone preserved in the quadrate [12]. The anterior process of the quadratojugal expands ventrally so that the ventral margin of the quadratojugal is markedly concave as in Nemegtosaurus.
Tapuiasaurus also resembles nemegtosaurids in the presence of a reduced quadrate flange of the pterygoid [15] but bears a remarkably modified anterolateral process that contacts the medial surface of the ectopterygoid, as in Diplodocus [15]. The supraoccipital is low and resembles the condition of nemegtosaurids and some basal titanosaurians. Tapuiasaurus shares with advanced titanosaurians the presence of an acute non-articular ventral tip on the paroccipital process [15,26]. The basal tubera are robust as in Rapetosaurus [12] and basal neosauropods. The basipterygoid processes are short, cylindrical shaped, and bear a sagittal ridge between them, closely resembling the condition in Rapetosaurus [25]. The basisphenoid contacts the medial surface of the quadrate and the quadrate flange of the pterygoid is reduced, as in nemegtosaurids [15].
The lower jaw of Tapuiasaurus (Figure 1) also shows derived features shared with Nemegtosaurus and Rapetosaurus, such as an unexpanded symphyseal region that is oriented perpendicular to the mandibular ramus and a smoothly curved tooth row in dorsal view. However, unlike Nemegtosaurus, the Meckelian groove does not reach the symphyseal region. The angular of Tapuiasaurus, however, is well exposed on the lateral surface of the posterior region of the mandible, distinguishing this taxon from the condition of most macronarians [15].
The toothrow extends up to the level of the preantorbital fenestra as in non-diplodocoid sauropods (Figure 1). All the upper and lower teeth of Tapuiasaurus are cylindrical and bear thin, regular carinae on their mesial and distal edges (Figure 2) that extend to the apex of each tooth. The enamel surface is slightly wrinkled, with diminute grooves that extend obliquely with respect to the apicobasal axis of the crown on the lingual and labial surfaces of the carinae. Older and more worn teeth tend to lack these grooves, presumably due to tooth-food abrasion.
The crowns of the upper tooth row are comparably broader mesiodistally than the crowns of the lower tooth row, a feature also present in Nemegtosaurus [11]. The upper teeth have slenderness index (SI; from [27]) values that range between SI 5.9 (in the 3 rd tooth) and SI 4.1 (in the 13 th tooth), whereas this index ranges between SI 4.9 (in the 2 nd tooth) and SI 3.4 (in the 9 th tooth) for the lower teeth. The upper teeth are also apicobasally longer than the lower teeth and the apicobasal length of the entire dentition decreases towards the posterior end of the tooth row. Up to three replacement teeth can be seen in the premaxilla and the maxilla. The crowns of Tapuiasaurus bear both planar high-angled and V-shaped wear facets on upper and lower teeth (Figure 2). The Vshaped wear facets are labiolingually narrow and only slightly developed and they only occur in a few teeth. The high-angled wear facets are much more extensive and are present in most teeth of the anterior region of the toothrow. Given the high-angled wear facets are more extensive and occur in highly worn crowns they probably occur in a final stage of the ontogeny of the teeth. The unusual combination of high-angled and V-shaped wear facets has also been described for Nemegtosaurus [11], whereas most other sauropods have wear facets of either one type or the other.
The hyoids are two long and curved, boomerang-like bones ( Presacral vertebrae are opisthocoelous and highly pneumatized, with camellate internal structure and large pleurocoels, as in other titanosauriforms (Figure 4). The mid-cervical centra are more than four times as long as high, as in non-saltasaurid titanosaurians [15]. Mid-dorsal vertebrae have a large diapophyseal lamina [28] that meet the spinopostzygapophyseal laminae along the neural spine, and share with advanced titanosaurians an extensive prespinal lamina and the absence of a hyposphene-hypantrum [2,15,28,29]. The dorsal ribs are plank-like with a large proximal pneumatopore as in titanosauriforms.
The coracoid is proximodistally long (Figure 5), and the distal end of the radius is expanded as in Rapetosaurus and saltasaurids [15], but differs from the derived condition of the latter group in being anteroventrally rounded. Titanosaurian characters present in the appendicular skeleton include a crescentric-shaped sternal plate ( Figure 5) and a well-developed olecranon process on the ulna [15]. Tapuiasaurus shares with Rapetosaurus and saltasaurids an expanded distal end of the radius, but lacks the robust ulnar proportions of saltasaurids ( Figure 6). The fragmentary hindlimb elements have a combination of characters supporting titanosaurian affinities such as lateromedially broad femoral shaft (Figure 6), broad pedal phalanges, laterally deflected unguals, and ungual I and II subequal in size.

Phylogenetic analysis
Two most parsimonious trees of 445 steps (CI = 0.613, RI = 0.789) were found in all replicates, using the collapsing rule 3 for zero-length branches [30], the strict consensus of which is shown in Figure 7 (see also Figure S4). Bremer and bootstrap support values for the nodes of the consensus tree are given in Figure 7 for selected nodes (see Figure S5 for other support values and Figure S6 for support values on the reduced consensus tree). A list of unambiguous synapomorphies supporting the nodes of the strict consensus of Figure S4 is shown in Text S3.
The branch support values for several nodes within Lithostrotia are moderate to low, with Bremer support values ranging between 1 and 3 and bootstrap frequencies ranging between 55 and 76 ( Figure S5). These relatively low values are caused because several incomplete taxa (i.e., Diamatinasaurus, Isisaurus, and Nemegtosaurus; see results below) can be placed in alternative positions without producing a marked increase in tree length [31]. In order to evaluate this issue, several runs were performed to test the degree of character support for positioning Tapuiasaurus deeply nested within advanced titanosaurians.
Constrained searches. Using constrained searches in TNT to find the most parsimonious trees that depict selected taxa in alternative positions (using the force command before the heuristic tree searches), we found that the three most feebly supported taxa within Lithostrotia are the fragmentary Early Cretaceous Diamantinasaurus, the latest Cretaceous Isisaurus, and Nemegtosaurus (which lacks postcranial elements). These taxa can be placed in alternative positions within Lithostrotia (including at the base of this clade), increasing the tree in only one to three steps.
However, placing Tapuiasaurus at the base of Lithostrotia requires six extra steps if it is forced to be only in a slightly more basal position, but more derived than Malawisaurus (i.e., as the sister group of the node of advanced titanosaurians, see Figure 7). Several derived characters of the skull and mandibles of Tapuiasaurus are responsible for this marked increase in tree length. In particular, the derived characters shared by Tapuiasaurus and Rapetosaurus that are absent in Nemegtosaurus are the ones that increase the tree length if Tapuiasaurus is placed more basally in the tree. These include the presence of an antorbital fenestra that is subequal or larger than the maximum orbital diameter (character 6), posteriorly tapering jugal process of the maxilla (character 235), narrow and elongated prefrontals (character 239), presence of a sagittal ridge between the basipterygoid processes (character 242), robust basal tubera (character 48), and unexpanded dentary at the mandibular symphysis (character 55).  Reduced consensus for Bremer support and Bootstrap/ Jackknife analyses. Reduced consensus can be used to reveal common phylogenetic information ignoring the position of unstable taxa. This is usually done in the set of most parsimonious trees. However, it can be applied to other sets of topologies, such as the suboptimal trees found during a Bremer analysis or to the set of trees found during bootstrap/jackniffe pseudoreplicates (as implemented in TNT). In this way, support values can be calculated for a subset of the taxa present in the data matrix, ignoring the effect that highly unstable or incomplete taxa can have in the support measures [31]. The result of this type of analysis shows higher character support for the inclusion of

Discussion
The phylogenetic analysis shows that Tapuiasaurus is deeply nested within an advanced titanosaurian clade formed by nemegtosaurids and saltasaurids (as well as Isisaurus and Diamantinasaurus), as the sister taxon of Rapetosaurus (Figure 7; Figure S4). The affinities between these two genera are supported by several cranial features that are absent in Nemegtosaurus, including the length of the antorbital fenestra subequal or larger than the orbit, robust basal tubera, unexpanded mandibular symphysis, maxillary jugal process tapering posteriorly, and prefrontal with narrow and elongated anterior process. The advanced titanosaurian clade is supported by the presence of postcranial features such as a proximodistally elongated coracoid, procoelous middle and posterior caudal vertebrae, and distal condyles expanded on the anterior surface of the humerus [15], only the first one being preserved in Tapuiasaurus. The placement of Tapuiasaurus among advanced titanosaurians is however robustly supported, given that its placement as the sister group of that clade requires seven extra steps in the parsimony analysis. This result also holds for other advanced titanosaurians, suggesting that the derived position of Tapuiasaurus can be considered as robust as that of other taxa belonging to that clade [ Figures S5 and S6].
The derived position of Tapuiasaurus and its Aptian age reveal the existence of multiple and extensive ghost lineages [5], which lengthen to 30 million years the diversification of advanced titanosaurians (Figure 7). Early Aptian diversification of advanced titanosaurians may explain their global distribution on landmasses that were comparatively isolated by the Late Cretaceous [32].
The complete skull of Tapuiasaurus indicates that the basic cranial morphology of advanced titanosaurians (narrow crowns, elongate rostrum, retracted naris, and an anteroventrally inclined quadrate), previously known only in the latest Cretaceous Rapetosaurus and Nemegtosaurus, was acquired at the initial diversification of the group during the Early Cretaceous. Furthermore, the discovery of Tapuiasaurus in Aptian rocks of South America demonstrates that these advanced titanosaurians with a derived skull morphology coexisted with other lineages of large herbivores, such as the more plesiomorphic broad-crowned titanosauriforms and diplodocoid sauropods, during a period of major changes in terrestrial ecosystems that involved the diversification of flowering plants and appearance of several modern lineages of vertebrates [6]. The long period of coexistence of these sauropod lineages suggests that the evolutionary success of advanced titanosaurians after the Cenomanian-Turonian extinction is better explained by an opportunistic radiation rather than by competitive replacement. Text S1 Geological Setting.

(DOC)
Text S2 Character list and data matrix used in phylogenetic analysis. Character definitions 1 to 234 are from [15] and have the same numeration as in the original publication. The additional characters are either new or taken from [2] and their respective sources are cited along with the character number of the original publication. Characters 8, 37, 64, 66, and 198 were set as ordered. The data matrix corresponds to the phylogenetic analysis published in [15] with the following modifications. Character scorings for Euhelopus were taken from the corrected list provided in [34]. Character scorings for Phuwiangosaurus and Tangvayosaurus were taken from [16] and those of Diamantinasaurus follow those given by [10].