Microanatomy of the stem-turtle Pappochelys rosinae indicates a predominantly fossorial mode of life and clarifies early steps in the evolution of the shell

Unlike any other tetrapod, turtles form their dorsal bony shell (carapace) not from osteoderms, but by contribution of the ribs and vertebrae that expand into the dermis to form plate-like shell components. Although this was known from embryological studies in extant turtles, important steps in this evolutionary sequence have recently been highlighted by the Triassic taxa Pappochelys, Eorhynchochelys and Odontochelys, and the Permian Eunotosaurus. The discovery of Pappochelys shed light on the origin of the ventral bony shell (plastron), which formed from enlarged gastralia. A major question is whether the turtle shell evolved in the context of a terrestrial or aquatic environment. Whereas Odontochelys was controversially interpreted as aquatic, a terrestrial origin of turtles was proposed based on evidence of fossorial adaptations in Eunotosaurus. We report palaeohistological data for Pappochelys, a taxon that exemplifies earlier evolutionary stages in the formation of the bony shell than Odontochelys. Bone histological evidence reveals (1) evolutionary changes in bone microstructure in ribs and gastralia approaching the turtle condition and (2) evidence for a predominantly amphibious or fossorial mode of life in Pappochelys, which support the hypothesis that crucial steps in the evolution of the shell occurred in a terrestrial rather than fully aquatic environment.

The origin of the turtle shell has remained controversial for centuries. Recent finds of fossil stem-turtles have expanded our knowledge on the origin of the turtle skeleton and in particular the structure of its shell [1][2][3][4][5] . Turtles are unique among tetrapods in the possession of a bony shell that is integrated with the axial skeleton. The dorsal portion of this shell (carapace) is formed by both endoskeletal and exoskeletal components ( Fig. 1): broadened ribs and neural spines are combined with secondary metaplastic ossifications, which are sutured to form a rigid shell 6 . Together, they encompass continuous structures reaching from endoskeletal layers well into the dermis 7 . In contrast to other reptiles, the trunk ribs are immobile and extend dorsolaterally into the dermis, where they are covered by dermal bone to form composite elements (costals). By contrast, the ventral portion of the bony shell (plastron) forms without any endoskeletal contribution but its origin has remained an open question until recently [6][7][8][9][10][11] . It has long been assumed that much of the plastron formed through fusion of the gastralia except for the anterior portion, which comprises ventral bones of the pectoral girdle 7,11 .
The recent discoveries of three stem-turtles, Odontochelys and the slightly older Eorhynchochelys from the Late Triassic (Carnian) of China 1,2 and Pappochelys from the Middle Triassic (Ladinian) of Germany 3 , have added crucial palaeontological evidence concerning the evolution of the turtle shell. These stem-turtles share broadened, T-shaped ribs (in transverse section) that do not form a complete carapace, but they differ in the formation of the plastron, revealing two steps in the evolutionary sequence of the formation of this structure. Unlike the (2019) 9:10430 | https://doi.org/10.1038/s41598-019-46762-z www.nature.com/scientificreports www.nature.com/scientificreports/ fully-formed turtle-like plastron of Odontochelys 1 , the venter of Pappochelys retains a full complement of paired, unusually large and diverse gastralia 3,12 . Eorhynchochelys also has gastral ribs but their arrangement is unclear 2 . Pappochelys, Eorhynchochelys, and Odontochelys fill the gap between fully shelled stem-turtles (e.g., Keuperotesta 13 , Proterochersis 14 , Proganochelys 15 ) and the oldest known putative stem-turtle Eunotosaurus, which has broadened but still very long trunk ribs that are T-shaped but simple paired gastralia 4,8 . The broadened ribs of Eunotosaurus and Odontochelys have also been a topic in the debate concerning the aquatic vs. terrestrial origin of turtles 8,9 . Although the detailed description of Pappochelys 3,12 revealed many transitional aspects of the osteology between the former mentioned taxa, two major questions in the origin of turtles remain: (a) what was the evolutionary sequence leading to the development of the bony shell in turtles and (b) what was the ancestral mode of life for Pan-Testudines?
As a first step toward addressing these questions, we examined the bone microstructure of Pappochelys rosinae. To this end, we have sectioned limb-bones, vertebrae, ribs, and gastralia of this taxon and also employed micro-CT-scanning data. Microanatomy of Pappochelys has then been compared with data for other tetrapods, as well as other representatives of the turtle clade. We hypothesise that the evolution of habitat preference among early stem-turtles did not occur in a clean "step-wise" manner.

Results
Limbs. Femur and humerus in Pappochelys rosinae both display a very small medullary cavity (~3% of the surface ratio) ( Fig. 2A,B) surrounded by a thick compact periosteal cortex, revealing osteosclerosis. The central medullary cavity is surrounded by a narrow zone of cancellous bone in the humerus (Suppl. Fig. S1A) but not in the femur ( Fig. 2A,B). The parallel-fibred matrix is only poorly vascularized by small longitudinal simple canals. Osteosclerosis solely results from the thickened cortex. Bone compactness of femur SMNS 91357 is 96.8%. The analysis of this femur with Bone Profiler 16 and including the resulting values of this analysis into the Supplementary Excel Sheet (SOM4) of 17 revealed parameters that suggests an amphibious mode of life.
When compared to terrestrial amniotes 18 as well as to some marine sauropterygians 19,20 , the medullary cavity in long bones of Pappochelys is rather small. It differs even more from aquatic turtles, which have no clear-cut medullar cavity but spongiosa in the medullar region 18 . Only some placodonts, pistosaurs, and a few large nothosaurs show a similar reduced medullary cavity combined with a thickened cortex 20,21 . However, most aquatic amniotes achieve osteosclerosis by completely different patterns: by incomplete endochondral ossification (i.e., retainment of calcified cartilage) or by intensive endosteal deposits (i.e. filling the medullary region by endosteal bone) 22 .
Microanatomy of long bones of Pappochelys differs from that in extant turtles 23,24 as well as from the microstructure revealed by micro-CT-scans in Proganochelys (Suppl. Fig. S1B). In contrast to turtles, femur and humerus of Pappochelys lack inner spongiosa but have a tubular structure with an open, although rather small cavity. Such a tubular www.nature.com/scientificreports www.nature.com/scientificreports/ inner structure is common in most extant terrestrial and semi-aquatic amniotes [25][26][27][28] . Interestingly, unlike in many other tetrapods (e.g. 18 ), the microanatomy of turtle humeri does not reveal a clear signal reflecting the preferred mode of life of the respective taxa 24 . The cortex of the femur of Pappochelys has in general a much lower vascular density compared to turtles. www.nature.com/scientificreports www.nature.com/scientificreports/ Vertebrae. The micro-CT scanned dorsal vertebra was heavily crushed mediolaterally by compaction of the surrounding sediment. The anterior and posterior part of the amphicoelous centrum and the centre and dorsal portion of the neural spine are cancellous (Fig. 2K,L). These cancellous areas have been identified as endochondral territories. In general, the cavities in the endochondral part are small (except for the antero-ventral part of the centre of the neural spine) and connected by thick trabeculae, resulting in a tight network similar to that of terrestrial amniotes [29][30][31][32] . The endochondral areas are surrounded by a compact, locally thick, periosteal bone ( Fig. 2K,L). However, a distinct separation into an inner bony ring surrounding the neural canal (as described for squamate vertebrae 31,32 ) is not visible. In longitudinal view, the neural arch is heavily fragmented (Fig. 2K) but the transversal view ( Fig. 2L) reveals compact periosteal tissue. The anterior and posterior endochondral part of the centrum are separated by a pillar of compact periosteal bone. The overall impression of the dorsal vertebra is osteosclerotic.
Amniote vertebrae display a high degree of morphological and microstructural variability, and interpretation of this variation remains difficult 33 . Preliminary analyses testing possible associations between vertebral structure and mode of life suggest that vertebrae of fossorial taxa are denser than those of terrestrial taxa (both sharing a small number of relatively thick trabeculae) and those in aquatic taxa are intermediate in density as expressed by a large number of relatively thin trabeculae 33,34 . This evidence renders an aquatic life style-based on the microstructure of the dorsal vertebra-unlikely for Pappochelys but suggests a fossorial life style.
The microstructure of dorsal vertebrae in turtles is difficult to compare due to their morphological changes in the course of shell development. In a previous study of turtle shell bones 35 a vertebra of the aquatic Platemys platycephala ( 35 fig. 31a) and a neural of the terrestrial Terrapene carolina triunguis ( 35 fig. 54e) with the corresponding vertebral centrum attached, among others, were figured. Both differ in structure: Terrapene has a rather large vertebral canal surrounded by thin trabeculae in thin neural arch pedicels and vertebral centra, whereas the vertebral canal of Platemys is also extensive, but surrounded by thick trabeculae in rather stout pedicels and a less reduced centrum. Thus, the vertebral microstructure of both turtles differs from that of Pappochelys.  S1E). Despite the presence of a medullary cavity the overall impression of the ribs is osteosclerotic, as well (compare to 34,36 ).

Ribs. The thoracic ribs of
The upturned and downturned formation of the processes reflect either a slight overlap with the adjacent ribs or at least a musculotendinous connection between adjacent processes (as is the case of broadened ribs in some edentates 37 ). The processes are not as broad and widely imbricating as in Eunotosaurus, but more closely resemble those of Odontochelys, especially in the asymmetric outline of the flanges in ventral view 9 .
In Pappochelys, as in Eunotosaurus, the cortex lacks interwoven structural fibres or other structures that would indicate metaplastic ossification of dermis. However, incorporation of anchoring fibres is found in the transversal process of two ribs (SMNS 91115, SMNS 92069; Fig. 2D-H) of Pappochelys, which may form an early stage in the evolution of metaplastic ossification. The presence of numerous short and angled fibres (SMNS 91115) and locally Sharpey's fibres (SMNS 92069) in the processes suggest a strong fibrous connection to those of neighbouring ribs. Thus, in Pappochelys, the anterior and posterior processes develop as outgrowths of the rib periosteum. In turtles, this connection is present in the sutural margins of the shell bones laterally. The ribs of Pappochelys differ from those of Eunotosaurus in lacking a woven-fibred portion within the ventral bulge of the T-shaped cross-section (Suppl. Fig. S1C-E), in a distinctly higher compactness, and morphologically they did not overlap as extensively.
The costalia, which make up a large portion of the carapace in turtles, are homologous with the amniote ribs; they combine costal periosteum with an additional layer of metaplastic bone 7 . This is added dorsal to the original rib anlage and develops from interwoven structural fibres. Pappochelys and extant turtles share a (sub)circular cartilage anlage of the rib, which becomes surrounded by a layer of periosteal bone. From this periosteal layer, early outgrowths of bony spiculae grow into the surrounding dermal tissue, as is also the case in Eunotosaurus 5 . The vascular cavities surrounding the rib anlage are formed in the same way in Pappochelys and crown turtles 5 .
Ribs of Pappochelys are unique considering their shape, inner structure, and tissue, although some extinct taxa share comparable broadening of their ribs (see 5 ).
Gastralia. The large gastralium SMNS 91895 has a smooth medial margin, whereas the lateral margin is increasingly lobate from the interior to the external bone surface, leading to the formation of several pronounced prongs/ridges interspersed with valleys (Fig. 2I,J). The gastralium has a large, central medullary cavity that roughly matches the shape of the cross-section and is partially lined by a thin layer of endosteal bone. Two more, smaller and not ornamented, gastralia were sectioned along with the rib from SMNS 91115. The larger one (SMNS 91115a) has an open medullary cavity, whereas the smaller one (SMNS 91115b) shows a medullary region that contains endosteal bone. The latter might be related to a more distal sampling location. The low vascularized (SMNS 91895) and avascular (SMNS 91115a, b) cortices of gastralia are made of parallel-fibred matrix. The cortex of the large ornamented gastralium SMNS 91895 contains numerous large, globular osteocytes, whereas osteocytes are far less numerous and flat in the other two gastralia. The lateral portion of the ornamented large gastralium (SMNS 91895) contains numerous prominent Sharpey's fibres and shorter fibres in the inner and outer cortex, whereas the medial portion shows no distinct fibres (Fig. 2J). The shorter fibres might also have anchored soft-tissue to the bone. SMNS 91115a and b, both lack any kind of fibres. (2019) 9:10430 | https://doi.org/10.1038/s41598-019-46762-z www.nature.com/scientificreports www.nature.com/scientificreports/ Gastralia of Pappochelys are superficially pachyostotic 3,12 , but retain a hollow internal structure, which make them less osteosclerotic when compared to aquatic amniotes. In plesiosaurs, gastralia have a cancellous internal structure 38 whereas those of the ichthyosaur Mixosaurus 39 , eosauropterygians 34 , and the rhynchocephalian Palaeopleurosaurus 40 are compact without any internal spaces. Instead, gastralia of present-day alligators show diffuse mineralisation 41,42 and another gastralium has a small cancellous centre 43 . The gastralia of Pappochelys with open medullary cavities more closely resemble those of terrestrial taxa, such as two paracrocodylomorph archosaurs from the same deposit (Batrachotomus 44 and a yet undescribed small rauisuchian).
The presence of a large central cavity, the development and density of fibres and the ornamented margin in large gastralia of Pappochelys are unique among tetrapod gastralia reported to date.

Discussion
Life style. Morphology and microanatomy do not always correlate directly to habitat preference in many species, as aquatic and terrestrial species often share similar histological and morphological features owing to frequent evolutionary reversals in habitat preference 33,34 . This is especially true for turtles 24 . In Pappochelys, the histology and microanatomy of limb-bones, vertebrae, ribs and gastralia reveals a complex picture, which is in this combination-for each bone as well as in sum-unique. Although all bones of Pappochelys are osteosclerotic, microanatomical patterns and processes involved differ from that of what is known for aquatic amniotes and a clear identification of life style for Pappochelys is hampered.
However, the simple presence of an increase in cortex thickness accompanied by a reduction in medullary cavity size need not indicate aquatic dispositions: the same features have been reported for the terrestrial lepidosaur Sceloporus 30 as well as the burrowing potential stem-turtle Eunotosaurus 8 . In combination with numerous osteological correlates 3,12 , we argue (on the basis of the microanatomy of the vertebra) that a terrestrial (i.e. fossorial) or modest amphibious mode of life (based on the analysis of the femur with bone profiler) is much more plausible for this taxon than a fully aquatic one. turtle shell development. The unique histology (i.e. presence of fibres) of the ribs and gastralia combined with their specialized morphology 3,12 gives insights into the development of the turtle carapace and plastron. Pappochelys thus exemplifies an important step in the evolution of the turtle shell (Figs 3 and 4). This is because its short and broadened ribs were already confined to the dorsal part of the trunk and located in a superficial position to extend well into the dermis. The dorsal surface of the ribs was heavily ornamented, consistent with a shallow position within the dermis but also indicating that they were not yet covered by keratinous scutes. Ventrally, the gastralia were greatly thickened to form a rigid basket in Pappochelys. The ribs were still somewhat moveable, as indicated by the joints as well as the abundance of fibres in the horizontal 'wings' of the ribs. These fibres suggest that strain acted on them, presumably from musculature ventilating the lungs. At the same time, other fibres indicate that successive ribs were already interconnected to form a protocarapace. This is consistent with the curved, wave-like cross-section of the ribs, which indicates slightly imbricating flanges. The articulation of free ribs might explain why the carapace evolved more slowly than the plastron along the turtle stem, as some mobility of the rib cage continued to be required for respiration.
The incipient dorsal shell in Eunotosaurus (Fig. 4) was recently studied in detail 5 . It consisted of greatly expanded thoracic ribs that had started to expand into the dermis. As in other amniotes, these ribs were strongly curved and elongate, probably still associated with a cartilaginous sternum ventrally. Pappochelys may well have elaborated on such an early stage by separating the ribs from the sternum and adding enlarged gastralia. In contrast to Eunotosaurus, the ribs of Pappochelys are shorter and less ventrolaterally curved (Fig. 4). Recently, the suppression of the sternum in early development has been demonstrated to be an essential prerequisite for the formation of the turtle plastron -this involves reprogramming of cartilage-producing chondroblasts into bone-forming osteoblasts 10 .
The transformation of modified gastralia into part of the turtle plastron is of particular interest. Eunotosaurus had thin rod-like gastralia much as in most amniotes, but they were reduced to two elements per transverse row, lacking the medial element present in most reptiles 5 . Pappochelys also has two gastralia per row but the individual elements are much larger than in Eunotosaurus and regionally differentiated 12 . They are heavily ornamented with ridges on the ventral side and some are twisted, indicating a more complex three-dimensional arrangement than in the primitive amniote condition 9 . Unlike many other reptiles, Pappochelys has only one row of gastralia per vertebral segment. The finger-like projections at the distal ends of these gastralia closely resemble the distal bifurcations and projections on the plastral elements of Odontochelys 3 .
Most gastralia of Pappochelys bear parallel ridges, especially along their ventral surfaces. These ridges merge into finger-like projections at the distal ends of some gastralia 12 . Like the intensity of ornamentation, the presence and density of fibres in gastralia of Pappochelys are unique among tetrapod gastralia reported to date. They probably indicate an expansion of gastralia from the layer of abdominal musculature well into the dermis. Although of different embryological origin, the gastralia thus parallel the tendency of ribs to expand into layers of the dermis, which were first steps toward the formation of carapace and plastron.
How did these enlarged gastralia transform into the plastron? In Pappochelys, fusion of neighbouring gastralia has not been confirmed by bone histology, because even the broadest elements with notable bifurcations have single medullary cavities. Instead, single gastralia appear to have split at various levels, starting from distal levels near the tips up to about midlength of the element. Hence, the next step in the evolution of the plastron may have involved (1) large-scale fusion of adjacent gastralia to form plates (as is indicated by early development of extant turtles 11 and (2) metaplastically ossifying preformed dermal tissue around the gastralia. The latter occurs mainly during posthatching development in the plastron formation of both, extant hard-shelled and soft-shelled turtles, as has been shown by shell bone histology 35,45,46 . The evolutionary sequence and timing leading to a fully ossified plastron, however, can only be resolved by future discoveries of taxa intermediate between www.nature.com/scientificreports www.nature.com/scientificreports/ Pappochelys (and Eorhynchochelys) and Odontochelys or by examination of the bone microstructure of the plastral elements in Odontochelys. Although occupying an intermediate position between Pappochelys and Odontochelys 2 , Eorhynchochelys does not add evidence here, because only few dislocated gastral ribs are visible between the dorsal ribs.
The crucial question is whether Pappochelys evolved these new shell features within the same functional context as Eunotosaurus, namely a fossorial lifestyle, or whether it was an aquatic animal that used its 'proto-shell' for protection against predators or as skeletal ballast to remain submerged.
Unlike Eunotosaurus, which is found in floodplain deposits together with other probably burrowing tetrapods 8 , Pappochelys was discovered in mudstones that formed in a small freshwater lake 47 , whereas the more derived Eorhynchochelys and Odontochelys occur in shallow marine strata 1,2 . What do these occurrences tell us about the setting in which the turtle shell evolved?
Recently, the analysis of skeletal features in Eunotosaurus indicated that this taxon shares various traits with fossorial amniotes 8 . It was argued that the broadened and imbricating dorsal ribs provided rigidity during digging/burrowing, as did the foreshortened trunk. Numerous features in the limbs fit this interpretation, such as the robust humerus and ulna, the short manus and pes with long and robust claws suited for powerful digging. For Eorhynchochelys an amphibious lifestyle in near shore-terrestrial habits, as well as digging activity based on robust limb morphology and enlarged terminal phalanges was also hypothesised 2 . This raises the question whether Pappochelys shows similar features, or which correlates can be found in that taxon regarding its lifestyle. The grade formed by Pappochelys, Eorhynchochelys and Odontochelys is especially interesting, because the evolution of the plastron has been considered to have taken place in the water 1,48 . Furthermore, consistent with Eunotosaurus, the short manus and pes in Pappochelys, Eorhynchochelys and Odontochelys are found also in fossorial taxa, and the long and robust unguals suggest a mode of life that involves digging 8 .
The occurrence of Pappochelys, Eorhynchochelys and Odontochelys in lake or shallow marine sediments does not imply a fully aquatic lifestyle. At Vellberg, the type locality of Pappochelys, terrestrial taxa were found in large numbers together with remains of aquatic taxa, indicating that land-dwelling forms were easily washed in, or terrestrial taxa were preserved during episodes of drought, for which sedimentological evidence has been presented 47 . Pappochelys is a rather common reptile in that lake deposit, but the skeletons are usually heavily affected by predation (most specimens forming regurgitates and coprolites of larger predators). Further evidence is provided by the autecology of Pappochelys: the skeleton of the holotype of P. rosinae contains bones of two tiny www.nature.com/scientificreports www.nature.com/scientificreports/ reptiles, which are juveniles of a small diapsid that is similar to the Early Triassic lepidosauromorph Sophineta, a terrestrial taxon 49 .
The available morphological and microanatomical evidence indicates that Pappochelys was not a fully or predominantly aquatic taxon. Instead, some features identified in Eunotosaurus as indicative of fossorial habits, such as the broadened ribs and robust claws, are also present in Pappochelys and Odontochelys. In addition, the microanatomical structure of the dorsal vertebra in Pappochelys points to a fossorial life style as well. The feature potentially indicating an amphibious lifestyle, the osteosclerosis present in all studied bones, but especially in the long bones, is equivocal, because it is also found in fossorially adapted terrestrial taxa 8,30 . The enlarged but hollow gastralia of Pappochelys, which very unusually exceed the length of the ribs, are likely to have formed a basket that gave additional rigidity to the trunk, which would have been important during digging. They might have compensated for the reduced role of the thoracic ribs, which were proportionately shorter than in most amniotes and could not strengthen the flanks.
Pappochelys may have preferred riparian habitats, as suggested by its abundance in the Vellberg lake deposit. It evidently fed on terrestrial tetrapods and fell prey to larger amphibious or aquatic predators. Based on the currently available data, the early phases in the evolution of the turtle shell took place in a terrestrial rather than aquatic setting, and the driving selective forces were likely not protection in an aquatic environment but rather functional demands to strengthen the trunk during digging.
Material. All material of Pappochelys rosinae included in this study (Table 1) was recovered from the type locality (Schumann quarry, Eschenau, Vellberg municipality, Baden-Württemberg, Germany) and type horizon (Lower Keuper [Erfurt Formation]; Middle Triassic: Ladinian: Longobardian) 3 . The specimens are housed in the Staatliches Museum für Naturkunde Stuttgart, Germany (SMNS). The sectioned bones are throughout highly diagnostic for Pappochelys and were mostly sampled from partial skeletons 12 .
Two humeri (SMNS 92084, SMNS 91013), two femora (SMNS 91013, 92085), and one dorsal vertebra (SMNS 96939) have been micro-CT-scanned. The quality of the scans varied due to different levels of infiltration by pyrite in the bones. Thin-sections of one femur (SMNS 91357), two dorsal ribs (SMNS 91115, SMNS 91968), and three gastralia (SMNS 91895, SMNS 91115a, b) were produced. The samples came from different individuals except for the rib and two gastralia that were taken from specimen SMNS 91115. Samples of long bones and ribs were taken at mid-shaft.

Methods
The thin-sections were produced following standard petrographic methods (Klein and Sander) 50 and then studied and photographed with a Leica ® DM 750 P compound polarizing microscope equipped with a digital Leica ® ICC50HD camera. Histological terminology follows Francillon-Vieillot et al. 51 . Some samples were micro-CT-scanned with a v|tome|xs by GE phoenix|x-ray at the Steinmann Institut für Geologie, Mineralogie und Paläontologie (StIPB) in Bonn (Germany). Image visualization was performed using VGStudio MAX 2.0 software (Volume Graphics GmbH) and Adobe Photoshop. Cross-sections of samples were transformed into black (bone) and white (cavities and vascular spaces) images to measure bone compactness with a custom-designed pixel-counting computer program developed by P. Göddertz (StIPB).

Data Availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information Files).