An early bothremydid from the Arlington Archosaur Site of Texas

Four turtle taxa are previously documented from the Cenomanian Arlington Archosaur Site (AAS) of the Lewisville Formation (Woodbine Group) in Texas. Herein, we describe a new side-necked turtle (Pleurodira), Pleurochayah appalachius gen. et sp. nov., which is a basal member of the Bothremydidae. Pleurochayah appalachius gen. et sp. nov. shares synapomorphic characters with other bothremydids, including shared traits with Kurmademydini and Cearachelyini, but has a unique combination of skull and shell traits. The new taxon is significant because it is the oldest crown pleurodiran turtle from North America and Laurasia, predating bothremynines Algorachelus peregrinus and Paiutemys tibert from Europe and North America respectively. This discovery also documents the oldest evidence of dispersal of crown Pleurodira from Gondwana to Laurasia. Pleurochayah appalachius gen. et sp. nov. is compared to previously described fossil pleurodires, placed in a modified phylogenetic analysis of pelomedusoid turtles, and discussed in the context of pleurodiran distribution in the mid-Cretaceous. Its unique combination of characters demonstrates marine adaptation and dispersal capability among basal bothremydids.

Diagnosis: Pleurochayah appalachius gen. et sp. nov. is diagnosed as a pleurodire by its quadrate with ventral process extending medially to the braincase below the cranioquadrate space, and a condylus mandibularis positioned anterior to the basioccipital-basisphenoid suture. We attribute it to Pelomedusoides by the absence of parietal-squamosal contact, and to Podocnemidoidea by a quadrate-basioccipital contact, and a prootic that is covered in ventral view. P. appalachius gen. et sp. nov. is a representative of Bothremydidae based on its basisphenoid-quadrate contact, a (short) quadrate-supraoccipital contact, a large exoccipital-quadrate contact, and the absence of a fossa precolumellaris. P. appalachius gen. et sp. nov. has the following unique combination of primitive and derived cranial characters: moderate antrum post-oticum (shared with Kurmademydini and Cearachelyini); incisura columella auris fully open and confluent with sulcus eustachii; present, but incipient, supraoccipital-quadrate contact; subtriangular, anteriorly-pointing basisphenoid; moderate posterior temporal emargination; foramen stapediotemporale located anteriorly on otic capsule, but not adjacent to foramen nervi trigemini; moderately-sized fossa pterygoidea; short parietals with uniform width; undivided fenestra postotica; photograph, (f) ventral drawing, (g) right lateral photograph, (h) right lateral drawing, (i) posterior photograph, and (j) posterior drawing. bo basioccipital, bs basisphenoid, cm condylus mandibularis, co condylus occipitalis, cs crista supraoccipitalis, ex exoccipital, fjp foramen jugulare posterius, fnt foramen nervi trigeminale, fp fossa pterygoidea, fpcci foramen posterius canalis caroticus internus, fr frontal, fst foramen stapediotemporale, ica incisura columella auris, iof interorbital foramen, mx maxilla, op opisthotic, or orbit, pa parietal, pal palatine, pf prefrontal, pm premaxilla, po postorbital, pr prootic, pt pterygoid, qu quadrate, so supraoccipital, sq squamosal, to tympanic opening, tb tuberculum basioccipitale, XII nervi hypoglossi. Red lines indicate visible sutures.  Histology. See "Supplementary Information" for full histological description. The results of our histological analysis are largely consistent with other known bothremydid taxa, which have a significantly thicker external than internal cortex 45,46 . The predominance of woven bone suggests fast skeletal growth, a derived characteristic  Phylogenetic placement. In the phylogenetic analysis, twenty minimum length trees of 1513 steps were obtained. In the strict consensus tree, P. appalachius was positioned at the base of the Bothremydidae in an unresolved polytomy with Kinkonychelys rogersi 54 , Sankuchemys sethnai 55 , and Kurmademys kallamedensis 56 (Fig. 6). In 16 of the minimum trees, P. appalachius fell as the basal-most member of the Bothremydidae; while in the other four trees, it fell within the Kurmademydini as its basal-most member. See "Supplementary Information" for Character State Analysis and full character matrix. The fossa precolumellaris is absent as in other early bothremydids. The quadrate process of the pterygoid is reduced compared to later bothremydids. The interorbital distance is wide, similar to that of bothremydines and Galianemys spp. The tympanic opening is elongated and oval. It lacks the slit-like fenestra postotica that characterizes cearachelyines. Pleurochayah appalachius gen. et sp. nov. shares some traits with the contemporaneous A. peregrinus; however, most shared characters are plesiomorphic for bothremydids. The two taxa differ in many other phylogenetically significant characters. In general, the crania of both taxa are wide relative to their lengths, although this trait is common among bothremydids, and P. appalachius is closer in proportions to the slightly narrower Cearachelys than to the wider A. peregrinus. Prefrontal-parietal contacts are absent (as in many bothremydids). Both exhibit www.nature.com/scientificreports/ a short supraoccipital-quadrate contact, such that the prootic does not contact the opisthotic, as in many bothremydids. The two taxa lack a fossa precolumellaris and possess an incisura columella auris that is not completely closed, although the feature is substantially more open in P. appalachius gen. et sp. nov. Unlike A. peregrinus, the orbits in P. appalachius gen. et sp. nov. are more dorsally than laterally positioned. A lateral, more vertical orientation of the orbits is considered primitive among turtles, and the most common state for bothremydines 10 . However, dorsal and more horizontally oriented orbits are a synapomorphy for Bothremys spp. and Chedighaii sp. 10 . There is not a clear functional distinction between the lateral and dorsal positions of the orbits, though in chelonioids it has been hypothesized that dorsal orientation is associated with shallow-water and/or benthic habits, and vertical orientation with pelagic adaptation 57 . The fossa orbitalis is posteriorly enlarged in A. peregrinus, but not in P. appalachius gen. et sp. nov., in which the septum orbitale is only slightly posterior to the orbital rim. There is extensive contact between the postorbital and frontal in A. peregrinus, but only a minimal point of contact in P. appalachius gen. et sp. nov. The foramen stapediotemporale is located further from the foramen nervi trigemini in P. appalachius gen. et sp. nov., although in both taxa the foramen stapediotemporale is positioned anteriorly on the otic chamber. Pleurochayah appalachius gen. et sp. nov. has a short crista supraoccipitalis, as opposed to the long crista supraoccipitalis in A. peregrinus.

Discussion
Using ratios of basal skull width to other measures, Fig. 7 compares general skull proportions of a selection of bothremydid taxa. The ratio of the overall width of the skull to basal skull width in P. appalachius gen. et sp. nov. is 0.87, similar to the cearachelyine C. placidoi, which falls between the narrow Taphrosphyini (< 0.82) and broader Bothremydini and K. kallamedensis (> 0.97) (Fig. 7). The preorbital length to basal skull width ratio of P. appalachius gen. et sp. nov. is also quite low, embedded in a cluster with cearachelyines, K. kallamedensis, and F. mechinorum (Fig. 7). In the ratio of interorbital width to basal skull width, P. appalachius gen. et sp. nov. falls within the known taphrosphyine and cearachelyine ranges (Fig. 7). A similar intermediate position within the bothremydine spectrum of variation, is repeated in the height to basal skull width ratio (Fig. 7). However, P. appalachius gen. et sp. nov. could not be included in the final comparison due to its height being diagenetically altered by crushing. In summary, compared to other bothremydids known from skulls, P. appalachius gen. et sp. nov. has a moderately narrow skull with a very short face and moderately spaced orbits (Fig. 7).
Humeral comparison between Pleurochayah appalachius gen. et sp. nov. and other pleurodirans. Previous studies used humeral morphology to infer locomotor modes and performance 41,[59][60][61][62] and paleoecology 63 in turtles. Figure 4 provides a comparison of all published fossil pleurodiran humeri, along with a few extant taxa. The proximal humerus of pleurodires generally has a hemispherical head that is slightly separated from the lateral and medial processes, but varies in proportional length and width, and in orientation relative to the humeral shaft 10 (Fig. 4). A more spherical humeral head is considered the derived state in bothremydids 10 (Fig. 4). The relative proportions of the humeral head (i.e., length to width) are nearly equal in P. appalachius gen. et sp. nov., similar to most specimens of Stupendemys spp., Taphrosphys sulcatus, and Hydrome- www.nature.com/scientificreports/ dusa tectifera (Fig. 4). In contrast, in Bothremys barberi, Bothremys sp., Chupacabrachelys complexus, and the extant Podocnemis expansa, the humeral head is slightly longer than wide, while in Notoemys laticentralis, the humeral head is wider than long (Fig. 4). The longitudinal axes of the humeral heads of P. appalachius gen. et sp. nov. and Taphrosphys sulcatus are parallel with the humeral shaft (Fig. 4). However, in Bothremys barberi, Bothremys sp., Chupacabrachelys complexus, Notoemys laticentralis, Podocnemis expansa, Hydromedusa tectifera, and Stupendemys spp., the longitudinal axis is tilted slightly laterally and the difference between the heights of the processes is generally greater, echoing this tilt (Fig. 4). The lateral and medial humeral processes are homologous with the trochanter major and minor, respectively 61 . Usually, the medial process is taller and more robust than the lateral, and the difference in height varies significantly between taxa (Fig. 4). The medial process is only slightly taller than the lateral process in P. appalachius gen. et sp. nov., similar to Taphrosphys sulcatus, but the difference in heights is more notable in the remainder of the comparative sample (Fig. 4). The lateral process is shifted significantly distally in sea turtles and to a lesser degree in Carettochelys insculpta 34 . The humeral head of P. appalachius gen. et sp. nov. is taller than both processes, as in all compared taxa except the chelid Hydromedusa tectifera, Notoemys laticentralis, and most humeri of Stupendemys spp. (Fig. 4). The neck of the medial process is slightly depressed, forming a sloping saddle in all compared taxa except Notoemys laticentralis (Fig. 4).
The large proximal processes of semiaquatic turtle humeri increase the mechanical advantage of both the m. pectoralis major and m. latissimus dorsi supporting more powerful stroke and recovery during both aquatic rowing and terrestrial walking [64][65][66] . Muscles inserting on the proximal aspects of the lateral and medial processes act primarily to adduct and retract the humerus, while the more distal insertions are associated with protraction and abduction. In general, the lateral process tends to be associated with anterior movement of the limb, and the medial with posterior movement 34 . The lateral process is also relatively proximal compared to other sampled turtles, and is most similar to the marine-adapted Taphrosphys sulcatus (Fig. 4). Lateral and medial processes of similar height and size, as in P. appalachius gen. et sp. nov., suggests that adduction and retraction of the forelimb www.nature.com/scientificreports/ may be comparably distributed between the anterior and posterior directions, resulting in more equally powerful stroke and recovery motions during rowing. This is in contrast to an emphasis on posterior forelimb movement such as the flapping motion of many marine turtles usually associated with an enlarged medial process 34 . The proximal humeral morphology of P. appalachius gen. et sp. nov., including a derived spherical head and robust medial and lateral processes similar to marine-adapted Taphrosphys sulcatus, suggests well-developed near-marine aquatic capabilities. Recovery of more skeletal material is needed to further test this intriguing hypothesis. While it should be noted that the described postcranial material was not found in direct association with the P. appalachius DMNH 2013-07-1782 cranium, we believe it to be the most parsimonious explanation that they belong to the same taxon, rather than the alternative that two previously undescribed pleurodiran species were present at AAS.

Histological comparison of Pleurochayah appalachius gen. et sp. nov. with fossil and extant pleurodires.
In his seminal work on turtle shell histology, Scheyer 45 developed a system of categories to quantify and relate histological characteristics and trends to the degree of aquatic adaptation of fossil and recent turtles. These categories are based on published ecological data of recent turtles [67][68][69][70] , and though a strict adherence to categorization should be treated with caution, a consideration of P. appalachius gen. et sp. nov. in the context of the analysis of Scheyer 45 yields possible paleoecological insights into the new taxon. Figure 8 compares the percentage thickness of the external and internal cortices relative to total shell bone thickness. P. appalachius gen. et sp. nov. falls closest to the marine-adapted bothremydids Chedighaii barberi and Taphrosphys sulcatus (Fig. 8). Comparative turtle taxa included here fall within Category II (semiaquatic to mainly aquatic) and Category III (fully aquatic) in degree of aquatic adaptation 45 . Categories II and III were combined into the Freshwater Histological Type of Jannello et al. 71 , which includes marine coastal environments. Turtles in Category II spend much of their lives in the water, often going onto land to migrate, forage, and bask 45 . They all have shell bones with a diploë microstructure, in which internal and external cortical bone layers frame interior cancellous bone 45 (Fig. 8). The cortices are well developed, and the histological profile of the bone has a generally compact appearance 45 . Vascularization of the cortical bone is higher than terrestrial (Category I) turtles due to more primary vascular canals and secondary osteons 45 . Also, the transitions between the cortices and interior cancellous bone remain conspicuous 45 . Among the currently sampled taxa, the bothremydid Foxemys cf. mechinorum and chelid Emydura subglobosa belong to Category II, as do the sampled Podocnemididae (Stupendemys geographicus and Podocnemis erythrocephalica) and the pelomedusid Pelomedusa subrufa, though the latter also has tendencies toward Category I (Fig. 8). Turtles in Category III (the bothremydids Chedighaii barberi and Taphrosphys sulcatus) are characterized by: rarely leaving an aquatic environment to bask or lay eggs; www.nature.com/scientificreports/ reduction in compact bone layers in the shell; strong reduction in thickness of the internal cortex compared with the external cortex; strongly vascularized external cortex that is rarely reduced in thickness; and highly organized trabeculae in the cancellous bone 45 . A well-developed external cortex is hypothesized to ensure structural stability of the shell bones 45 . The clustering of P. appalachius gen. et sp. nov. with the Category III bothremydids (Fig. 8) is intriguing and suggests a highly aquatic, possibly semi-marine lifestyle. This functional interpretation is consistent with the morphology of the humerus. Unlike the consistent account of aquatic adaptation shared by the sampled Podocnemididae, the bothremydid Foxemys cf. mechinorum shows less aquatic adaptation in its microanatomy than the marine bothremydid taxa Bothremys barberi and Taphrosphys sulcatus 45 (Fig. 8). Also, P. appalachius gen. et sp. nov. has similar cortex to full shell thickness ratios as the marine taxa, further suggesting that it may also have been highly adapted to aquatic life (Fig. 8). Additional discoveries of shell and postcranial material may reveal further paleoecological insights 63,73 on P. appalachius gen. et sp. nov. and related taxa. In particular, histological sampling of Algorachelus spp. to measure relative cortical thickness and other relevant characteristics may be useful in assessing the paleoecology of these taxa.
Pleurodiran distribution and southern Appalachian biogeography. As Kurmademydini is not retrieved in the current phylogenetic analysis, Pleurochayah appalachius gen. et sp. nov. is part of an unresolved basal bothremydid polytomy along with Sankuchemys sethnai 55 , Kinkonychelys rogersi 54 , and Kurmademys kallamedensis 56 (Fig. 6). Basal bothremydids are known from Africa, South America, India, and now North America. The group originated in Gondwana during the early Cretaceous and secondarily colonized Europe and North America in the late Cretaceous 13 . The analysis of Joyce et al. 13 implies that at least five separate clades invaded North America from Gondwana. In addition to the new taxon described herein, these migrations include the newly established ancestors of Paiutemys tibert prior to the Cenomanian, the previously established ancestors of Chedighaii and newly established ancestor of Chupacabrachelys complexus prior to the Campanian, the previously established ancestors of Bothremys cooki prior to the Maastrichtian, and the newly established ancestor of Taphrosphys sulcatus prior to the Paleocene 13 .
The vast majority of bothremydines are preserved in near-shore marine deposits surrounding the Atlantic Ocean 10 , while basal bothremydids are recovered from continental sediments 2,10,13 . Trans-Tethyan dispersal events (from northern Gondwana to the European archipelago) appear to be responsible for the movement of many faunal groups during the early Upper Cretaceous, particularly during the Cenomanian-Santonian interval 74 . The southwest coast of Appalachia has a similarly complex biogeographical system and lies along a land connection and migration route from Laramidia 75 . The AAS also occupies a unique geographical location near the opening of the Western Interior Seaway, which connected the Arctic to the Gulf of Mexico beginning in the late Albian 31 . As such, it was situated near a confluence of brackish and near-shore marine waters with continental drainages from the Ouachita Mountains to the north 17 . Marine adaptation was previously known as early as the Santonian among the Bothremydini, as Bothremys arabicus and Chedighaii sp. or Bothremys barberi likely had near-shore marine lifestyles 76 . Freshwater pleurodiran taxa experienced reduced diversity in the Albian to Campanian interval, while the littoral and marine taxa showed a trend of steadily increasing diversity over the same period 77 . The shell histology of P. appalachius gen. et sp. nov. provides an example of a basal bothremydid with comparable marine adaptation to bothremydines (Fig. 8), occurring near the beginning of the trend of increased marine diversity.
A partial adaptation of some bothremydid subclades to brackish and marine waters facilitated their dispersal and the invasion of different niches. More than vicariance or large-scale extinctions, dispersal events are considered the most important influence on biogeography in pleurodires 77 . Oceanic dispersals are known to be a significant cause of biogeographic range changes for other groups such as tortoises 78,79 , lizards 80 , amphibians 81 , and invertebrates 82 . Some pelomedusids and chelids have island distributions that suggest short dispersals 77 , and extant freshwater turtles Chelodina expansa and Emydura macquarii have been exposed to saline conditions for long periods (50 days) without physiological problems 83 . Consistent with this pattern of increased marine adapation in the mid-Cretaceous, Pleurochayah appalachius gen. et sp. nov. demonstrates gross morphological and paleohistological signatures of marine or highly aquatic brackish adaptations. This suggests that marine adaptations among stem-pleurodires were earlier and more widespread than previously thought 77 . In the context of the AAS paleoenvironment, P. appalachius gen. et sp. nov. joins a number of previously described brackish or marine-tolerant taxa including the crocodyliform Terminonaris, hybodont sharks, and multiple invertrebrates that are consistent with a costal setting where terrestrial, freshwater, and marine taxa intermingle 20,84,85 .

Conclusions
The current study places Pleurochayah appalachius gen. et sp. nov. at the base of the Bothremydidae, in an unresolved polytomy with the members of Kurmademydini (Fig. 6). It is the oldest known crown pleurodire and bothremydid from North America. This study also extends the geographic range of basal bothremydids to North America, which is significant since they were previously known only from South America prior to the Cenomanian (Fig. 9). Pleurochayah applachius gen. et sp. nov. is known from the lower middle Cenomanian, predating Algorachelus peregrinus in the uppermost middle-lowermost upper Cenomanian 9 , and Pauitemys tibert in the uppermost Cenomanian 13

Materials and methods
Fossil specimens were collected and prepared by researchers, staff, and volunteers of the Perot Museum of Nature and Science. There were no definitive associations between the cranium and postcranial material. Specimens were measured with 6″ Mitutoyo Absolute Digimatic calipers to the nearest 0.01 mm and rounded to the nearest 0.1 mm. Some distances and angles were measured from high quality digital photographs using ImageJ 88 . For our phylogenetic analysis of P. appalachius gen. et sp. nov., we used the recent character matrix of 285 characters from Hermanson et al. 14 designed to reveal phylogenetic relationships within Pleurodira. Characters following a morphocline were ordered according to Hermanson et al. 14 . Phylogenetic analyses were performed in TNT 1.5 89 . We conducted a traditional search using 1000 replicates of Wagner trees and tree bisectionreconnection (TBR) saving 100 trees per replication. The full character matrix derived from Hermanson et al. 14 is available in "Supplementary Information".
We apply the taxonomic scheme of turtles presented by Joyce 90 unless otherwise specified. Phylogenetically defined clade names are used in accordance with PhyloCode guidelines 91,92 .
For histological thin-sectioning, DMNH 2013-07-0525 was left undecalcified and embedded in plastic resin following the protocol of Lee and Simons 93 . Slides were imaged using a motorized light microscope (Ni-U; Nikon, Tokyo, Japan, USA) with a strain-free long working distance objective (10× Plan Fluor: numerical aperture of 0.3, resolvable size ≈ 1 μm). Focus and stitching of histological montages were controlled by software (NIS Elements D; Nikon, Tokyo, Japan, USA). The montages were sharpened using Photoshop (CC; Adobe, San Jose, California, USA) with the "Unsharp Mask" filter set at 10 px and are high resolution (2.1 μm per pixel). To comply with major grant funding agencies and promote data transparency, we provide the images as freely accessible digital slides at the Paleohistology Repository (Lee and O'Connor 94 : http:// paleo histo logy. appsp ot. com).