Equatorial pliosaurid from Venezuela marks the youngest South American occurrence of the clade

Pliosaurids were the dominant macropredators in aquatic environments at least since the Middle Jurassic until their extinction in the early Late Cretaceous. Until very recently, the Cretaceous record of Pliosauridae has been poor and difficult to interpret from the taxonomic and phylogenetic perspective. Despite that the knowledge of Cretaceous pliosaurids improved in recent years, numerous aspects of their evolutionary history still remain only poorly known. Here, we report the first pliosaurid material from Venezuela. The taxon is most likely earliest Cenomanian in age, thus representing the youngest occurrence of Pliosauridae from South America. The Venezuelan taxon is based on a well-preserved tooth crown whose morphology and outer enamel structural elements appear to resemble especially those observable in the giant pliosaurid Sachicasaurus vitae from the Lower Cretaceous of Colombia. The new discovery extends the pliosaurid record on the continent by more than 10 million years and likely marks the southernmost Upper Cretaceous occurrence of Pliosauridae, worldwide. We also briefly discuss the affinities of the enigmatic Venezuelan elasmosaurid Alzadasaurus tropicus and highlight similarities to elasmosaurids from the Western Interior Seaway.

Geological and stratigraphic setting. The specimen MCNC-1830 originates from the La Aguada Member of the La Luna Formation at the "Cementos Andinos" quarry, where calcareous rocks are mined for cement production. The quarry is located in the Andes range (Cordillera de Mérida), east of Lake Maracaibo, 10 km to the northeast of Monay city, Candelaria Municipality, Trujillo state, western Venezuela (Fig. 1A). MCNC-1830 was collected in situ in 2014 by one of the authors (JDCB), at the top of the quarry (9° 36′ 52″ N, 70° 24′ 3″ W), in the same outcrop of the La Aguada Member shown by 37 (Fig. 3A in 37 ) (Fig. 1).
The La Luna Formation is the most prolific petroleum source rock in western Venezuela and part of eastern Colombia [54][55][56][57] , and represents a marine sequence deposited under anoxic-dysoxic conditions along the passive margin of northern South America during the Cenomanian-Campanian 57 . The La Luna Formation is an extensive geological unit that spans the foreland of the southern Caribbean Ridge, including a large part of northwest of Venezuela (Sierra de Perijá to the Mérida Andes) and to Colombia, and gradually transitions east into the contemporaneous Rio Querecual Formation (eastern Venezuela) which is equivalent in facies 55 . These concretions range from a few centimeters to well over a meter in length (e.g., 37 , Fig. 3C,D). In the southeast of the Maracaibo basin in the Lara and Trujillo states, the La Luna Formation is divided into three members (Fig. 1B):   Table 1 37,39,56 and references therein). The thickness of the La Luna Formation ranges from 100 to 300 m, generally increasing northwards ( 56,58 and references therein).
In the Lara and Trujillo states, the La Aguada Member reaches a thickness of ~ 60 m 39 . The outcrops of the La Aguada Member, as exposed at the top of the "Cementos Andinos" quarry, consist of dense dark-grey limestones (less than ~ 60-70 cm thick), intercalated by compact and laminated black/dark-grey shales, and abundant calcareous concretions. MCNC-1830 derives from a black shale horizon that has produced ichnofossils, molluscs, chondrichthyans 40 , abundant osteichthyans 59 , and a marine snake 37,60 . The base of the La Aguada Member at the "Cementos Andinos" quarry overlays a fossiliferous dark-grey sandy limestone (personal observation, Fig. 1C) that has been identified as the top of the upper Albian Maraca Formation in the Andes of Trujillo and Lara states 38 . Other authors (e.g., 61,62 ) have used the term the 'La Puya member' to refer to a thin section (< 30 m) at the top of the Peñas Altas Formation in the Andes of Lara and Trujillo (Fig. 1). Therefore, the discrepancy between the use of the Maraca Formation or the 'La Puya Member' for the thin sequence under the La Aguada Member is still unresolved 37 .
The precise age of the La Aguada Member and its corresponding sections across Venezuela and Colombia remains uncertain, ranging from Albian-Cenomanian 55,61,63-65 , lower-upper Cenomanian 39,63-69 and even Cenomanian-Santonian 70,71 . Most relevant, perhaps, is the dating of the La Peña/San Felipe Sections by 72 , located in the eastern part of the Maracaibo Basin on the eastern edge of the village of Chejendé, Trujillo, which is less than 10 km from the "Cementos Andinos" quarry. Based on nannofossils, the La Aguada Member of Chejendé was deposited no earlier than in the latest Albian to middle Cenomanian interval 72 . However, only the base of the La Aguada Member was exposed, thus strongly suggesting an earliest Cenomanian age for MCNC-1830 that was recovered much higher in the section ( 72 , p. 352 and Fig. 3A). Despite the debate on the exact age of the base of the La Luna Formation east of Lake Maracaibo (e.g., La Aguada Member), it seems that, based on the ammonite record, it becomes progressively younger westward [63][64][65] . For a detailed discussion on the age of the La Aguada Member see Supplementary Information 1.
Paleoenvironment and other vertebrates. During much of the Early Cretaceous, what is today Venezuela was covered by an epicontinental sea that rapidly transgressed during the latest Albian and Cenomanian towards the craton followed by a period of re-oxygenation 55 . The La Aguada Member has often been considered to cover a transitional environment between the shallow water conditions of the Maraca Formation (La Puya Member) and the pelagic low energy conditions of the La Luna Formation; however, water depths never exceeded 50 m 38,40,55,73 . The La Aguada Member has intervals rich in organic matter which have been suggested to be of algal origin 40 . Sedimentological and invertebrate (micro)fossil proxies indicate a shallow water environment (presence and abundance of globigerinid and a scarcity of globotruncanid foraminifera) with oxygenated and generally nutrient-rich surface waters and a stratified water column seem to have been present 40,55 . The La Luna Formation is associated with an outer shelf/upper slope paleoenvironment with a high diversity of medium to large marine vertebrates (see Supplementary Table 1) that would have served as ample food resources for opportunistic predators 55 . Nonetheless, the vertebrate record of the La Aguada Member remains fairly limited. A wide variety of bony fish remains have been uncovered, including scales, isolated and semi-articulated cranial and postcranial remains of Xiphactinus 59 , other ichthyodectiforms, enchodontids, and small indeterminate fishes 40,64 . A high diversity of lamniform sharks (at least 12 taxa in five clades, eight of which are anacoracids), have been described from the La Luna Formation, representing active pelagic predators and scavengers of large vertebrates and small nektobenthic predators feeding on small bony fish and invertebrates 40 . The new plesiosaur specimen adds to the diversity of large marine reptiles from the La Luna Formation and represents the largest predator described from the strata so far ( 74,75 , Supplementary Table 1).
Bottom water conditions were predominantly anoxic or suboxic, as indicated by the scarcity of benthic invertebrates with only rare occurrences of small bivalve moulds in the limestones (and undetermined ammonites), and some inoceramids in the calcareous concretions and a lack of reworking by bioturbation and/or high-water energy conditions (for more details see the Supplementary Information 1; 40,55 ). It seems that anoxic sedimentation, possibly related to upwelling along the northwestern coast of South America, was widespread across the Venezuelan and Colombian platform and possibly even spanning a major part of northern South America and the southern Caribbean during the Cenomanian-Santonian 55 . Towards the top of the Chejendé Member oxygen and nutrient conditions improve and pelecypods and ammonites are more frequent in the concretion-rich portion of the section 55 .

Material and methods
Material. The study is based on an isolated tooth crown belonging to a brachauchenine pliosaurid of probable early Cenomanian (early Late Cretaceous) age. The specimen originates from the La Aguada Member of the La Luna Formation, Candelaria Municipality, Trujillo state, western Venezuela. It is housed at the Museo de Ciencias Naturales de Caracas in Caracas, Venezuela (MCNC) under the catalog number MCNC-1830 (Fig. 3).
The specimen (MCNC-1830) was found as part of a larger collection of fossil vertebrates at the "Cementos Andinos" quarry with the support of the authorities of the mining company. Legal authorization was issued by the Instituto del Patrimonio Cultural de Venezuela (IPC) through the collection permit No. 000327/2013, and through permission for mobilization and study No. 071/2015.

Multivariate analyses.
To further assess the taxonomic affinities of MCNC-1830 and to explore its morphospace occupation among thalassophonean pliosaurids, we performed cluster and principal coordinates analyses using the dataset of 5 . This dataset was constructed to collectively summarize the dental morphological We replicated the protocol of 5 ; we applied a 50% completeness threshold to remove the influence of taxa based on insufficiently complete/preserved material, scaled the data to equal variance and a mean of zero through subtraction of the mean value for each character and then divided it by the standard deviation. A distance matrix was created using the Gower metric, that is well suited for datasets that comprise both continuous and discrete variables 76 . We used the cluster v2.1.0 package in the R statistical environment (RStudio Version 1.2.5033 77 ); from the resulting distance matrix a cluster dendrogram analysis using the stats base package and the Ward. D2 method was produced.
The same matrix was used to explore the dental morphospace occupation of particular thalassophonean taxa through a principal coordinates analysis, using ape v5.3 78 . We again used the Gower metric and applied the Cailliez correction for negative eigenvalues. See Supplementary Information 2 for the R code.
The terminology of tooth crown orientation and morphology. We follow the crown orientation terminology of 79 : apical, toward the crown apex; basal, toward the cervix dentis; distal, away from the tip of the snout; labial, toward the lips; lingual, toward the tongue; mesial, toward the tip of the snout. The morphological traits exposed on the outer enamel surface are described using the nomenclature as adopted by 5,6 : apicobasal ridges, longitudinally running enamel ridges of variable apicobasal extent that can be developed around the entire crown circumference and are approximately semicircular or triangular in cross-section; ridglets, subtle apicobasally-expressed enamel structures that are often developed between adjacent apicobasal ridges or on an unridged enamel surface; the ridglets may be very indistinct as well as produce a distinct vermicular pattern (see 4 : Fig. 7).    are approximately semicircular in cross-section and are developed around the entire circumference though they are most densely packed linguodistally. All of the ridges appear to reach the base of the crown, as is widespread among brachauchenines 5 . Some are very pronounced and likely reached the apex though due to the lack of the apical part, this cannot be confirmed. Some of the ridges are approaching each other on the linguodistal part of the crown, around the mid-section, but no ridges have been observed to branch, unlike in Brachauchenius lucasi, 'Polyptychodon' hudsoni, and Megacephalosaurus eulerti that typically show clear branching ridges around the mid-sections of tooth crowns. Mesiolabially, the enamel surface exposed between the apicobasal ridges shows well-pronounced ridglets, forming a vermicular pattern, similar to the state observable in Sachicasaurus vitae and some specimens from the 'Polyptychodon' assemblage, such as CAMSM B 75754.

Systematic paleontology.
Assessment through multivariate analyses. The results of our multivariate analyses are broadly similar to those of 5 . The principal coordinates analysis (PCoA) as well as the cluster analysis recognize the presence of two general tooth crown 'morphogroups' in pliosaurids, one comprising the crowns with a conical shape (subcircular cross-section) and the other one including those with the trihedral/subtrihedral morphology (triangular/subtriangular cross-section) (Fig. 4). As in 5 , PCoA largely separates the two 'morphogroups' by the first principal coordinate axis (Fig. 4A). A similar result, to that of the PCoA, was obtained through the cluster analysis that placed MCNC-1830 in a cluster with S. vitae and further with 'Polyptychodon' type 1 and Megacephalosaurus eulerti (Fig. 4B), within the 'conical' part of the cluster dendrogram.

Discussion
Dental disparity of the Cretaceous pliosaurids. The results of our multivariate analyses differ in some aspects from those of 5 , which warrants some discussion. The addition of the late Barremian brachauchenine Sachicasaurus vitae expands the crown morphospace occupation of the pre-Aptian taxa towards the positive side of the second coordinate axis, further supporting the hypothesis that the latest brachauchenines experienced a substantial decrease in their dental disparity. The most significant difference is the placement of GFMSU h-216 ('Crimean pliosaurid') within the 'trihedral' cluster. The analysis of 5 placed the specimen among the conical-toothed taxa, in a cluster together with the Callovian (Middle Jurassic) taxon 'Pliosaurus' andrewsi and the Cenomanian (Late Cretaceous) specimen MWGUW 009761 (' Annopol pliosaurid'), none of which is particularly reminiscent of the trihedral-to-'trapezoid' morphology of GFMSU h-216. It is worth noting that MWGUW 009761 shows a cross-section somewhat resembling a triangular shape, and may thus be characterized as being gently subtrihedral 31 . However, considering its overall morphology and its enamel character state distribution, we did not alter any scores for this specimen in the current version of the dataset.
The difference in the placement of GFMSU h-216 between 5 and our study clearly stems for the correction of the carinal score for the specimen (0 → 2). 'Pliosaurus' andrewsi and MWGUW 009761 still cluster together among the conical-toothed pliosaurids.
Remarks on the Venezualan elasmosaurid Alzadasaurus tropicus. The most complete plesiosaur find from Venezuela is a partial postcranial skeleton (AMNH 6796) that was discovered by a Venezuelan oil company near the vicinity of Altagracia de Orituco, eastern Venezuela and established as Alzadasaurus tropicus by Colbert 41 . Preserved are the posteriormost cervical vertebra, four pectoral and eight dorsal vertebrae, parts of associate ribs, the left scapula, a nearly complete left and parts of the right coracoid, a left humerus as well as parts of the left radius, ulna and carpus ( 41 , p. 4). A. tropicus was considered a 'nomen vanum' by 44 (p. 54), a taxon that was adequately described but lacks sufficient diagnostic characters (nomen dubium of current use). Following Colbert's ( 41 , p. 5) diagnosis for A. tropicus, the taxon is characterized by vertebrae with round centra and rather high, compressed, neural spines; scapula that has a broad dorsal process and a fairly broad ventral plate, not contributing to a median midline bar; an elongated coracoid that is expanded along the posterior margin, having a long posterior coracoid blade; coracoids which meet along a median symphysis anteriorly, being separated by an elongated median vacuity posteriorly; and humerus that is elongated and distally expanded.
Similar posterior cervical, pectoral or dorsal vertebrae with roundish centra and high, transversely compressed neural spines are present in various elasmosaurid taxa, including Callawayasaurus colombiensis ( 44 , Plate 3, Fig. C), Thalassomedon haningtoni (S. Sachs pers. obs. November 2015), Futabasaurus suzukii ( 83 , Fig. 5H), or Elasmosaurus platyurus ( 84 , Fig. 5). A scapula with a broad dorsal process and an anteriorly broad ventral plate, lacking a pectoral bar, is a condition reminiscent of T. haningtoni (see 85 ,Fig. 14). Transversely expanded anterior scapulae are present, e.g., in Libonectes morgani ( 86 , Fig. 2) or Elasmosaurus platyurus ( 44 , Fig. 14) where the scapulae form a posteromedial link to the anteromedial coracoids, called the pectoral bar. The coracoid of A. tropicus is expanded along the posterior margin, having a long posterior coracoid blade. The post-symphyseal coracoid, also called the coracoid blade, is usually shorter relative to the complete length of the coracoid and wider posteriorly (see e.g., 41 , Fig. 8, 45 , Fig. 6 for comparison). A similar elongate and narrow coracoid blade is present in the latest Cretaceous elasmosaurids Hydrotherosaurus alexandrae where the coracoid blade is, however, less elongate relative to the complete length (see 85 Fig. 23, 88 , Fig. 6G). The Turonian Libonectes morgani has clearly shorter coracoid blades ( 86 , Fig. 2). The condition in the potentially coeval Cenomanian Thalassomedon haningtoni is unknown as the post-symphyseal coracoids of the holotype specimen (DMNS 1588) are not preserved. The type specimen of Alzadasaurus (FMNH 12009), described as Al. riggsi by 85 , was assigned to Thalassomedon by 45 . However, this specimen is an immature individual and most of the remains are fragmentary, heavily distorted and therefore insufficient for a confident diagnosis. The material, currently under study by SS & DM, therefore cannot be unambiguously assigned to T. haningtoni and is best considered a nomen dubium, following 89 . The coracoids typically connect anteriorly along their medial symphysis, and also the posterior intercoracoid vacuity is a condition characteristic for elasmosaurid plesiosaurs (see discussion in 90 ). Humeri that are elongated and distally expanded are likewise found in several elasmosaurid taxa, such as Thalassomedon haningtoni ( 85 , Fig. 15), Libonectes morgani ( 91 , Fig. 7), Futabasaurus suzukii ( 83 , Fig. 8A,B) or Aphrosaurus furlongi ( 88 , Fig. 8).
In summary, all characters considered diagnostic for Alzadasaurus tropicus, as discussed by 41 , are also present in the potentially coeval Cenomanian elasmosaurid Thalassomedon haningtoni, known from the midwestern USA (see discussion in 92 ). Only the shape of the coracoid blade cannot be confirmed as the post-symphyseal coracoid parts are missing in the Thalassomedon holotype specimen (DMNS 1588) and the pectoral girdles are not preserved in the referred specimen (UNSM 50132) either. Several characters considered diagnostic for A. tropicus by 41 are typical for elasmosaurids in general, such as the intercoracoid vacuity.
Even though we have not observed any characters that could be used to diagnose Alzadasaurus tropicus, confirming its status as a nomen dubium or its similarity to Thalassomedon haningtoni, which would be suggested based solely upon Colbert's 41 publication, is beyond the scope of this study.
Other potential pliosaurid specimens from the Upper Cretaceous of South America. It  www.nature.com/scientificreports/ representing a caudal and a sacral or proximal caudal centrum, has not been supplemented with detailed comparisons of contemporary taxa. A morphometric analysis of the specimens placed them outside the Elasmosauridae which led to the conclusion that pliosaurids are the most plausible group. However, similar proportions and a similar morphology (centra that are wider than long/high and higher than long, having amphicoelous articular faces) are also known for coeval early Late Cretaceous polycotylids (see e.g. , 93 ). This group was not considered in the comparisons by Meza-Velez and O'Gorman 48 . For that reason, and owing to the apparent homoplasy in plesiosaur vertebral characters (e.g., 94 ), the taxonomic assignment is questionable. Pending more detailed assessment of the material and vertebral character distribution within Plesiosauria, the vertebrae are probably best interpreted as Plesiosauria indet.

Conclusions
With the notable exception of the pliosaurid and elasmosaurid specimens originating from the upper Aptian (Lower Cretaceous) strata of the Paja Formation in Colombia, the plesiosaur record from the mid-Cretaceous (approximately encompassing the Aptian-Turonian interval) of South America is based on fragmentary remains of indeterminate phylogenetic affinities.
Here, we report the first pliosaurid material from Venezuela. The specimen originates from the La Aguada Member of the La Luna Formation in the Andes range (Cordillera de Mérida), east of Lake Maracaibo, 10 km to the northeast of Monay city, Candelaria Municipality, Trujillo state, western Venezuela. It was discovered in strata most likely deposited in the early Cenomanian (earliest Late Cretaceous). Despite comprising a single tooth crown, the preservation of the specimen allows for a detailed description, comparisons to teeth of other Cretaceous pliosaurids, and an assessment through multivariate analyses of data that have become available recently.
The overall morphology of the Venezuelan specimen and the distribution of its outer enamel structural elements indicate affinities to late-diverging brachauchenines and appear to resemble especially those observable in Sachicasaurus vitae, a recently described giant pliosaurid from the upper Barremian (Lower Cretaceous) of Boyacá, Colombia.
The most complete plesiosaur material from Venezuela described to date includes a partial postcranial specimen, established as the type of Alzadasaurus tropicus. The taxon is usually considered to lack diagnostic features and is treated as a nomen dubium. Our preliminary assessment of the specimen concurs with this though we have also observed characters that are shared with the middle Cenomanian (lower Upper Cretaceous) elasmosaurid Thalassomedon haningtoni from the midwestern USA.
In turn, the Venezuelan pliosaurid represents the youngest South American representative of the clade, over 10 Ma younger than the second youngest South American record ('Kronosaurus' boyacensis). Additionally, if the early Cenomanian age for the deposition of the fossil-bearing strata proves correct, the newly described specimen also marks the southernmost Upper Cretaceous occurrence of Pliosauridae, worldwide. Regardless, the Venezuelan pliosaurid represents a significant addition to the scarce record of the mid-Cretaceous plesiosaurs of South America and is another indicator of the potential and abundance of marine vertebrates from the Cretaceous of Venezuela.