Shell and long-bone histology, skeletochronology, and lifestyle of Araripemys barretoi (Testudines: Pleurodira), a side-necked turtle of the Lower Cretaceous from Brazil

Abstract

In this study we provide a comprehensive investigation of the microanatomical and microstructural aspects of the carapace and limb bones of the Early Cretaceous side-necked turtle, Araripemys barretoi, from the Araripe Basin, Brazil. Inter-elemental histovariability reveals different secondary remodelling of the skeletal elements within the same individual. The vascularisation is scarce and mainly longitudinal, also it ceases towards the bone surface, forming an avascular parallel-fibred bone with closely spaced LAGs. These traits indicate a late ontogenetic stage and a slow growth rate for one of the two A. barretoi specimens. The high cortical thickness of the costal plate suggests an increase of the shell stiffness. The elevated relative bone wall thickness of the ulna compared to other limb bones indicates a case of local pachyosteosclerosis, possibly to improve body stability in the aquatic environment.

Key words
Araripe Basin; bone histology; Early Cretaceous; shell histology; turtle

INTRODUCTION

The turtle shell is formed by the carapace (dorsal portion) and the plastron (ventral portion) connected by lateral bridges, which develop the physical protection of inner soft tissues (Gilbert et al. 2001). In most turtles, keratin scutes overlay the carapace and plastron, which increase the shell’s stiffness (Wyneken 2001). Furthermore, shell bones store water and fat besides acting as a pH buffer (Gilbert et al. 2001) and also play a structural role in other behaviours such as support the muscles attached to produce force during locomotion and ventilatory movements during breathing (Landberg 2003, Lyson et al. 2016). Scheyer & Sander (2007) and Jannello et al. (2020) attributed a correlation between shell microanatomy and lifestyle, establishing histological categories to determine crucial features in shell microstructure of Testudines with terrestrial, aquatic non-marine or marine habits. In the last few years, several studies demonstrated that lifestyle influences the shell compactness of these amniotes. For instance, terrestrial turtles exhibit higher compactness indices (Scheyer et al. 2015) than freshwater and marine turtles (Scheyer et al. 2014, Jannello et al. 2016).

The ontogenetic stage of the individuals can be identified through osteohistological studies in vertebrates. Limb bone microstructures have been established as a useful proxy for assessing skeletochronology among recent sea turtle species (e.g. Snover & Hohn 2004, Snover et al. 2011), and in terrestrial and freshwater Testudines (e.g. Chinsamy & Valenzuela 2008, Botha & Botha 2019). These investigations validate the annual periodicity of rapid and slow bone apposition, and periods of complete cessation of bone deposition in this group of vertebrates.

Among fossil turtles, the Araripemydidae is an extinct pleurodiran clade known mainly from deposits of the Santana Group in the Araripe Basin (Meylan 1996, Oliveira & Kellner 2017, Limaverde et al. 2020). Araripemys is one of these taxa and is considered an aquatic turtle living during the Early Cretaceous environment of Gondwana (Oliveira & Romano 2007). The taxon is the only turtle recorded in two distinct lithostratigraphic units of the Santana group: Crato and Romualdo formations (Oliveira et al. 2011, Carvalho & Barreto 2015, Oliveira & Kellner 2017), showing a higher tolerance for living in environments with different salinity levels (Oliveira & Kellner 2017). The Santana Group yielded a broad Testudines fauna from the Early Cretaceous, represented by five monotypic genera to date. Except for Santanachelys, all of them belong to Pleurodira (Romano et al. 2013). This diversity of body shape and environmental tolerance of pleurodirans allows for an ideal setting for studies of an osteohistological nature. Herein we present results of histological and microanatomical analyses of two costal plates, one neural plate, one peripheral plate, both humeri, one ulna, one radius and metatarsals of two specimens of Araripemys, from two geological units of the Santana Group, and compare them with other pleurodiran turtles, to test a correlation between its shell microstructure and lifestyle. In addition, their ontogenetic stage was estimated, using standard skeletochronology methods.

Institutional abbreviations

MPSC, Museu de Paleontologia Plácido Cidade Nuvens; UFRPE, Universidade Federal Rural de Pernambuco.

GEOLOGICAL SETTING

The Santana Group of the Araripe Basin is subdivided from base to top into four formations: Barbalha (formerly Rio da Batateira Formation), Crato, Ipubi and Romualdo (Assine et al. 2014). The timing for this Group varies and some studies indicate an Aptian-Albian stage (Benigno et al. 2021). Contrastingly, Arai & Assine (2020) suggested an Aptian age attributed by the presence of the guide palynomorph, Sergipea variverrucata in all formations of Santana Group.

The Crato Formation mainly consists of micritic laminated grey and light-beige limestones with halite pseudomorphs. The laminated limestones are interbedded with a series of claystones, siltstones and sandstones and the presence of halite pseudomorphs in some levels indicates deposition under fluctuating salinity levels (Neumann et al. 2003, Martill et al. 2007). Except for the report of the presence of marine foraminifera by Arai M. (unpublished data), the carbonate section is attributed to transgressive-regressive events associated with the expansion and contraction of a lacustrine system (Neumann V.H., unpublished data).

The Romualdo Formation represents a transgressive-regressive complete cycle (Assine et al. 2014). This formation is composed of a wide range of lithologies, including stratified conglomerates, fine to medium grained sandstones, interbedded shales, marls and limestones, and coquinas, which includes the highly fossiliferous carbonate concretion-bearing unit (Valença et al. 2003, Kellner et al. 2013, Assine et al. 2014, Almeida-Lima et al. 2020, Melo et al. 2020). Despite there being a consensus on its predominantly marine origin, several palaeoenvironment proposals have been made for this sequence. It has been previously interpreted as an estuarine environment (Silva Santos & Valença 1968), as a gulf connected with an open sea (Valença et al. 2003) or even as a non-marine deposit (Martill 2007). Despite this, a recently integrated study of foraminifera, ostracods and other microfossil data, from the Romualdo Formation, indicates a restricted environment after a brief connection with the sea, since some of these representatives are generally less abundant in open-ocean environments (Melo et al. 2020). While the Santana Group was recently considered to be Aptian in age (Arai & Assine 2020), the presence of certain planktonic foraminifera (Microhedbergellamini globularis Zone) point to an upper Aptian age for the Romualdo Formation, refining previous information (Melo et al. 2020).

MATERIALS AND METHODS

Material

The studied sample includes a fourth and a fifth costal plate, a fifth neural plate, a peripheral plate, metapodials (metatarsals), stylopodials (humeri) and zeugopodials (ulna and radius) corresponding to two individuals (Fig. 1). The first individual UFRPE 5302 includes a partially preserved carapace and the second specimen MPSC R 010 includes the axial and appendicular skeleton associated with a complete shell. Thin sections used in the current study are housed in the histological collection at the Department of Biology of the Universidade Federal Rural de Pernambuco. The specimens were referred to Araripemys barretoi Price, 1973 according to diagnostic characters: very flat and sculptured carapace in which first costals reach shell margin between nuchal and first peripherals; the carapace forms fontanelles between the costal and the peripheral plates; the epiplastra are long and narrow with long medial contact to the hyoplastra; the reduced plastron lacks mesoplastra and gular scutes; the entoplastron has an inverted V shape; the epiplastra present a J shape and form a point anteriorly and three midplastral fontanelles; hyoplastra and hypoplastra are nearly rectangular with broadly interdigitating sutures on the midline; and the xiphiplastra have deeply interdigitating sutures with the hypoplastra (Price 1973, Meylan 1996, Oliveira & Kellner 2005).

Figure 1
(Colour online) Selected bone elements of Araripemys barretoi specimens from Santana Group of the Araripe Basin before sectioning. A, Carapace (UFRPE 5302). B and C, Shell (MPSC R 010). D, Humerus associated with the ulna and radius (MPSC R 010). E, Distal portion of the humerus (MPSC R 010). F, Ulna and radius (MPSC R 010). G, Left pes (MPSC R 010). The dashed lines indicate the histological section planes. Scale bars represent 0.5 cm (e and g); and 1 cm (a, b, c, d and f).

Sampling methods

For histological analysis the specimens were hand-measured and photographed as per protocols proposed by Lamm (2013). Samples were removed from each bone element to prepare the histological slides. Thin sections were then prepared using standard fossil histology techniques (Chinsamy & Raath 1992, Scheyer et al. 2007) at the Laboratório de Paleobiologia e Microestruturas (LAPAMI) of the Universidade Federal de Pernambuco (CAV/UFPE) and at the Laboratório de Paleontologia & Sistemática of the Universidade Federal Rural de Pernambuco (LAPASI/UFRPE). The specimens were embedded in clear epoxy resin Resapol T-208, catalysed with Butanox M50 and cut with a diamond-tipped blade mounted on a saw. The mounting side of the sections was wet-ground using a metallographic polishing machine (Aropol-E, Arotec Ltda) with Arotec abrasive sandpapers of increasing grit size (60/P60, 120/P120, 320/P400, 1200/P2500) until a 60 μm thick section be reached.

Histological analyses

Samples were observed using two petrographic polarising microscopes under normal and polarised light with lambda waveplate. Images were obtained using an AxioCam digital sight camera (Zeiss Inc., Barcelona, Spain) mounted to an Axio Imager.M2 (Zeiss Inc. Barcelona, Spain) and an Olympus BX51 (Olympus Corporation, Tokyo, Japan), mounted to an Olympus DP26 (Olympus Corporation, Tokyo, Japan). The images were taken at the Laboratório de Gemologia (UFPE) and at the Laboratório Integrado de Tecnologia em Petróleo, Gás e Biocombustíveis (LITPEG).

Bone compactness analysis was done on all bone elements. The photographs of the cross-sections were transformed into binary images, using Adobe Photoshop® CS6. This method marks the bone tissue in black and vascular spaces (medullary cavity, vascular canals and resorption cavities) in white. The binary images (Supplementary Material – Figure S1) were quantitatively analysed with the software Bone Profiler, Windows-based version 4.5.8 (Girondot & Laurin 2003) to infer bone compactness of the bone sections. The bone wall thickness measurements were made using Fiji (ImageJ) Software (Schindelin et al. 2012) and the relative bone wall thickness (RBT), that is, the mean thickness of the cortical bone wall divided by the mean diameter of the total bone (Botha & Chinsamy 2004, Bhat et al. 2019), was calculated manually. For the shell bones, the terminology “external cortex” is adopted to the areas towards external surface of the bone, whereas “internal cortex” corresponds to the visceral surface of the bone.

RESULTS

Histological description

UFRPE 5302

Costal IV. Both external and internal cortices show similar histology (Fig. 2a-e). Primary bone mainly consists of lamellar bone tissue grading to parallel-fibred bone tissue. Lamellar bone tissue shows several osteocyte lacunae with a flatter and elongated appearance following a parallel arrangement towards the surface than the parallel-fibred bone tissue. The latter is composed of intrinsic fibres oriented parallel to the surface interrupted by thick and dark growth marks with a reduced amount of osteocyte lacunae interpreted as annuli (Fig. 2c–e). Simple vascular canals occur in the external and internal cortices and also infilling the inner region (Fig. 2c).

Figure 2
(Colour online) Histological section of costal IV plate of Araripemys barretoi (UFRPE 5302) from Crato Formation, Lower Cretaceous. A, Microanatomical overview of the perpendicular section of the costal IV plate. B, Detail of the composition of the external cortex. C, Detail of the inner region with reduced vascular spaces. D–E, Cortical bone of the costal composed of parallel-fibred bone tissue interrupted by annuli. Images: Normal transmitted light. Abbreviations: Blue arrowhead, Annulus; ECO, External cortex; ICO, Internal cortex; IR, Inner region; LVC, Longitudinal vascular canal; and PFB, Parallel-fibred bone tissue. Scale bars represent 1 mm (a); 200 µm (b and d); and 5 µm (c and e).

Neural V. The external cortex exhibits parallel-fibred bone tissue. The bone matrix shows an enormous amount of osteocyte lacunae following the orientation of the fibres (Fig. 3c). These lacunae have an elongated aspect in the parallel-fibred matrix and a globular appearance in the woven matrix. Some anastomosed vascular canals opening up to the bone surface and connect with ornamental pits. The inner region presents few and short resorption cavities, and secondary osteons feature the remodelling bone tissue. Some portions present primary bone represented by primary osteons (Fig. 3c and 3d). In its lateral part, the remodelling processes expand into the external cortex. The internal cortex consists of regions of woven and parallel-fibred bone tissues (Fig. 3b and 3d). The vascularisation pattern is formed by primary osteons, anastomosed and longitudinal simple vascular canals.

Figure 3
(Colour online) Histological section of neural V plate of Araripemys barretoi (UFRPE 5302). A, Microanatomical overview of the perpendicular section of the neural V plate. B, The external cortex is composed of woven bone tissue. C, Detail of the external cortex and inner region of the neural plate. D, Remodelling process occurs at the interior region. Images: Normal transmitted light. Abbreviations: ECO, External cortex; ICO, Internal cortex; LB, Lamellar bone tissue; PFB, Parallel-fibred bone tissue; PO, Primary osteon; SO, Secondary osteon; and WB, Woven bone tissue. Scale bars represent 100 µm (a); and 20 µm (b–d).

MPSC R 010

Costal V. The external cortex is broader and more vascularised than the internal cortex (Fig. 4a, Supplementary Material – Figure S1). The woven bone tissue has dispersed osteocyte lacunae. The vascular meshwork has a reticular and longitudinal pattern (Fig. 4e). The inner region is occupied by cancellous bone represented by short resorption cavities and secondary osteons. In the central core of the costal plate (in parallel section) occurs the largest circular cavity (Fig. 4b). The internal cortex consists of parallel-fibred bone tissue interrupted by growth marks (Fig. 4c and 4g). The vascularisation pattern is composed of scarce primary vascular canals (Fig. 4d).

Figure 4
(Colour online) Histological section of costal V plate of Araripemys barretoi (MPSC R 010) from Romualdo Formation, Lower Cretaceous. A, Microanatomical overview of the parallel section of the costal V plate. B, Centre of embryonic periosteal collar of the rib at the internal region. C and H, internal cortex composed of parallel-fibred bone tissue. D, Longitudinal vascular canals at the internal cortex. E, Anastomosed vascular canals are predominant at the external cortex. F, Microanatomical overview of the perpendicular section of the costal V plate. G, Growth marks are visible in the internal cortex. Images: Normal transmitted light. Abbreviations: AVC, Anastomosed vascular canal; CEPCR, Centre of embryonic periosteal collar of the rib; ECO, External cortex; ICO, Internal cortex; LVC, Longitudinal vascular canal; PFB, Parallel-fibred bone tissue; and White arrowhead, Line of arrested growth. Scale bars represent 300 µm (a); 100 µm (f) and 20 µm (b–e; g and h).

In the perpendicular section, the costal plate presents as a massive element formed by parallel-fibred bone tissue (Fig. 4h). In this bone tissue, the osteocyte lacunae show a flattened aspect following the orientation of the fibres. The diploë structure is only observed in the most medial part of the plate (Fig. 4f).

Peripheral. This plate presents a diploë structure (Fig. 5a). Both cortices are composed of parallel-fibred bone tissue and they have a similar thickness (Fig. 5c). Osteocyte lacunae show a flattened appearance following the orientation of the fibres. The external cortex is vascularised by longitudinal and reticular oriented vascular canals opening up to the external bone surface (Fig. 5b and 5e). The cancellous bone layer is composed of trabeculae and inter-trabecular spaces (Fig. 5d). The transition region between the internal cortex and the cancellous bone is marked by a resorption line (Fig. 5c). The internal cortex has its vascular system composed of longitudinal vascular canals.

Figure 5
(Colour online) Histological section of peripheral plate of Araripemys barretoi (MPSC R 010). A, Microanatomical overview of the transverse section of the peripheral plate. B, External cortex with vascular canals opening up to the surface of the bone. C, Limit between the cancellous bone and internal cortex marked by a resorption line. D, Trabeculae forming the cancellous bone layer. E, Longitudinal vascular canals dominate in the external cortex. Images: Normal transmitted light. Abbreviations: AVC, Anastomosed vascular canal; Black arrowhead, Resorption line; CB, Cancellous bone; ECO, External cortex; ICO, Internal cortex; ITS, Inter-trabecular space; LVC, Longitudinal vascular canal; PFB, Parallel-fibred bone tissue; RC, Resorption cavity; and Tr, Trabecula. Scale bars represent 500 µm (a); and 20 µm (b–e).

Humeri. The general epiphyseal and metaphyseal microanatomy of the thin sections are similar (Figs. 6a–e and 7a–e). Both cross-sections show a thin region of cortical bone, which surrounds a wide medullary region infilled by cancellous bone (Figs. 6a and 7a). Primary parallel-fibred bone tissues are intercepted by cyclical growth marks (Figs. 6c and 7c). The vascular network is composed of primary longitudinal vascular canals (Figs. 6b and 7d). The medullary region is composed of secondary trabeculae. The walls of the trabeculae are formed by secondary lamellar bone tissue (Figs. 6d–e; 7b and 7e) besides the inter-trabecular spaces are infilled with sedimentary matrix.

Figure 6
(Colour online) Histological section of the humeral epiphysis of Araripemys barretoi (MPSC R 010). A, Overall view of an epiphyseal cross-section of the humerus. B, Cancellous bone is dominated by slender bone trabeculae and some secondary osteons. C, Close-up of B showing parallel-fibred cortex interrupted by LAGs. D, Detail of the transition between cortical and cancellous bone demarcated by a thin resorption line. E, Details of the thin lamellar trabeculae of the cancellous bone. Images: Normal transmitted light (a, b, c and e); and Cross-polarised light with lambda waveplate (d). Abbreviations: Black arrowhead, Resorption line; ITS, Inter-trabecular space; LB, Lamellar bone; SO, Secondary osteon; Tr, Trabecula; and White arrowhead, Line of arrested growth. Scale bars represent 1 mm (a); 100 µm (b and e); and 50 µm (c and d).

Figure 7
(Colour online) Histological section of the humeral metaphysis of Araripemys barretoi (MPSC R 010). A, Overall view of a metaphyseal cross-section of the humerus. B, Note the presence of Sharpey’s fibres that extend perpendicularly to the bone margins. C, LAGs interrupting parallel-fibred cortical bone. D, Poor vascularised parallel-fibred bone tissue of the periosteal cortex and highly vascularised inner cortex of primary osteons and primary vascular canals. E, Secondary bone remodelling in the perimedullary region. Images: Normal transmitted light. Abbreviations: ITS, Inter-trabecular space; LVC, Longitudinal vascular canal; PO, Primary osteon; PFB, Parallel-fibred bone tissue; RC, Resorption cavity; Shf, Sharpey’s fibres; SO, Secondary osteon; White arrowhead, Line of arrested growth. Scale bars represent 1 mm (a); 100 µm (b and d); and 50 µm (c and e).

Ulna. The primary cortex is comprised of parallel-fibred bone with twelve lines of arrested growth (LAGs) (Fig. 8b–c). Cracks have run along some of the LAGs. Primary vascular canals with a longitudinal orientation form the vascularisation pattern. Osteocyte lacunae have a globular or flattened shape; they form rows following the fibre orientation. Secondary osteons are restricted to the innermost cortex (Fig. 8d). The medullary cavity presents an elongated narrow shape towards the periosteal surface (Fig. 8a).

Figure 8
(Colour online) Histological section of the ulna of Araripemys barretoi (MPSC R 010). A, Overall view of the cross-section of the ulna. B and C, Parallel-fibred cortex interrupted by cyclical growth marks. D, Inner cortex remodelled by secondary osteons and resorption cavities. Images: Normal transmitted light. Abbreviations: Blue arrowhead, Annulus; LVC, Longitudinal vascular canal; MC, Medullary cavity; PO, Primary osteon; RC, Resorption cavity; SO, Secondary osteon; and White arrowhead, Line of arrested growth. Scale bars represent 500 µm (a); and 5 µm (b–d).

Radius. The primary cortex is composed of parallel-fibred bone tissue interrupted by eleven LAGs and one annulus (Fig. 9b–c). As observed in the ulna, cracks have run along at least two of the LAGs (Fig. 9a). Primary osteons and primary longitudinally-oriented vascular canals constitute the vascularisation pattern (Fig. 9d). Osteocyte lacunae have a globular or flattened aspect dispersed throughout the bone matrix.

Figure 9
(Colour online) Histological section of the radius of Araripemys barretoi (MPSC R 010). A, Overall view of the cross-section of the radius. B, Cortical bone interrupted by cyclical growth marks. C, Detail of B showing parallel-fibred primary cortex. D, The inner and middle cortex contains several growth marks. Images: Normal transmitted light. Abbreviations: Blue arrowhead, Annulus; LVC, Longitudinal vascular canal; MC, Medullary cavity; PFB, Parallel-fibred bone tissue; PO, Primary osteon; and White arrowhead, Line of arrested growth. Scale bars represent 500 µm (a); 200 µm (b); and 5 µm (c and d).

Metapodials. The overall bone microstructure of the metatarsals consists of primary cortices formed by avascular parallel-fibred bone tissue (Fig. 10a). Metatarsals I and IV exhibit the narrowest and the widest cortices, respectively (Fig. 10b and 10e). Trabecular bone and secondary osteons constitute the medullary regions (Fig. 10c). The medullary cavity of metatarsal III is surrounded by convoluted compacted coarse cancellous bone (CCCB), which is separated from the periosteal cortex by a prominent resorption line (Fig. 10d).

Figure 10
(Colour online) Histological section of left metatarsals of Araripemys barretoi (MPSC R 010). A, Overall view of cross-sections of the metatarsals. B, Detail of metatarsal I exhibiting thin cortical bone and trabecular bone filling the medullary region. C, Detail of metatarsal II showing the remodelling process in the perimedullary region. D, Detail of metatarsal III showing compacted coarse cancellous bone tissue in the innermost cortex. E, Detail of metatarsal IV displaying parallel-fibred bone tissue in the cortical region. Images: Normal transmitted light. Abbreviations: Black arrowhead, Resorption line; CCCB, Compacted coarse cancellous bone; ITS, Inter-trabecular space; PFB, Parallel-fibred bone tissue; SO, Secondary osteon; and Tr, Trabecula. Scale bars represent 500 µm (a); and 50 µm (b–d).

DISCUSSION

Highly compacted carapace assessment

The most unexpected result of this study was the great difference between osteohistological pattern and palaeoenvironmental data. We used the categories suggested by Jannello et al. (2020) based on Scheyer & Sander (2007) to predict the lifestyle of Araripemys. The costal and neural plates present the terrestrial histological type composed of highly compact cortices (Table I) with similar thickness and small vascular canals. In general, the cancellous bone layer is sharply reduced composed by short trabeculae. This shell histology disagrees with the overall lacustrine and marine environment suggested by the sediments where Araripemys comes from (Oliveira et al. 2011, Carvalho & Barreto 2015, Oliveira & Kellner 2017). A similar case occurred with the pan-chelid Rionegrochelys caldieroi de la Fuente, Maniel and Jannello, 2017 (de la Fuente et al. 2017). This semiaquatic–aquatic species according to its fluvial depositional environment, disagrees with the terrestrial feature revealed by microanatomical features (de la Fuente et al. 2017). In this case, the authors suggested a semiaquatic habit with a tendency to forage in terrestrial environments. Another example is the coastal marine bothremydid, Cearachelys placidoi, which also presents a highly compacted plastron (Sena et al. 2021). Thus, the shell bone microstructure may incorrectly predict the palaeoenvironment in which these taxa lived. It is important to record that the costal IV plate has a higher density of osteocyte lacunae which suggests a higher metabolic rate than the appendicular bones sampled.

Table I
Estimated bone compactness and relative bone wall thickness of studied bone elements of Araripemys barretoi.

Bone element	Min (SE)	Max (SE)	S: slope (SE)	P: transition (SE)	Bone compactness	Relative bone wall thickness (%)
Costal V plate UFRPE 5302 (Perpendicular section)	91 (n.a)	91 (n.a)	5 (n.a)	50 (n.a)	0.999	–
Costal V plate UFRPE 5302 (Parallel section)	91 (n.a)	91 (n.a)	5 (n.a)	50 (n.a)	0.910	–
Neural V plate UFRPE 5302	91 (n.a)	91 (n.a)	5 (n.a)	50 (n.a)	0.951	–
Peripheral plate UFRPE 5302	91 (n.a)	91 (n.a)	5 (n.a)	50 (n.a)	0.834	–
Humeral epiphysis MPSC R 010	-4.860085 (0.0052233)	1122.01 (1.204783)	15.49973 (0.0017688)	83.44743 (0.0100576)	0.511	8.4
Humeral metaphysis MPSC R 010	-4.867664 (0.0025356)	3735.726 (1.945482)	10.70954 (0.0008052)	70.37807 (0.0057536)	0.682	7.82
Ulna MPSC R 010	0.9604937 (0.0001777)	-20.59331 (295.3171)	4.301269 (0)	97.16246 (0)	0.96	42.98
Radius MPSC R 010	-0.784116 (0.0004734)	551.3261 (0.2280387)	5.822927 (0.0017136)	34.38055 (0.0118601)	0.891	17.6
Metatarsal I MPSC R 010	0.2458498 (0)	800.1459 (0)	4.549157(0.0021647)	33.26039 (0.0197977)	0.859	–
Metatarsal II MPSC R 010	-5.601472 (0.0019807)	449.0123 (0.1584178)	8.71785 (0.0008965)	37.76353 (0.0042705)	0.722	–
Metatarsal III MPSC R 010	-2.110333 (0)	576.8223 (0)	6.235533 (0.0007363)	33.55287 (0.0045778)	0.795	–
Metatarsal IV MPSC R 010	0.530049 (0)	272.9994 (0)	5.981462 (0.0056199)	39.07308 (0.0416116)	0.971	–

• SE, standard errors.

The absence of LAGs in the UFRPE 5302 costal plate may suggests that annual environmental changes such as seasonal fluctuations in climate, salinity variation and food availability in the Crato palaeolake were not significant to cease the costal growth. Otherwise, this feature may correspond to the early ontogenetic stage for the specimen (Cerda et al. 2014). The possibility of bone resorption erasing the LAGs (Wang et al. 2019) is unlikely because there is no presence of resorption activity such as in Stupendemys geographicus Wood, 1976 (Scheyer & Sánchez-Villagra 2007). The presence of annuli in the costal plate might indicate periods of slow growth during unfavorable seasons (Francillon-Vieillot et al. 1990).

The flat external morphology of the carapace plates is reflected in their micromorphology. Most of the carapace is composed of high compacted costal and neural plates. However, the peripheral plate exhibits a cancellous layer more developed that suggests the highest capacity for acting as a water and waste reservoir within the carapacial plates. The thicker cortices and absence of the diploë structure might be interpreted as serving for the reduction of manoeuvering during swimming, since the presence of the diploë structure of the carapace offers high stiffness and decreasing the weight required for swift swimming (Balani et al. 2011). According to Clarac et al. (2020) massive shells facilitate the turtle’s efforts to dive and remain at the bottom of the lake or river. Araripemys barretoi was adapted to various salinities of the environmental medium. For example, this species habited the Crato palaeolake, which is characterised as an environment of high salinity and high evaporation rates associated with long dry periods (Martill et al. 2007). The exposition to this euryhaline environment might be compensating by short-term dives, also because of its highly compacted carapace plates; Araripemys probably had a low capacity of bone acidosis buffering. Furthermore, a massive carapace contributes to increased shell resistance to compensate for its weakness caused by several fontanelles of the Araripemys shell.

Based on a comparison with other fossil and extant turtles, the costal V plate presents an enlarged circular space in the inner region (Fig. 4b). This vascular space was found in Condorchelys antiqua and Heckerochelys romani being interpreted as the centre of embryonic periosteal collar of the rib early in costal growth (Scheyer et al. 2014, Cerda et al. 2016, Vieira et al. 2016).

Growth rate, relative bone wall thickness and osseous specialization in limb bones

The cortices of all the limb bones examined exhibit a similar microstructure, suggesting the same growth pattern, represented by parallel-fibred bone tissue with temporary cessation of growth during annual cycles. Humeri show a reduced cortical bone and its medullary regions contain a loose spongiosa like the freshwater cryptodire Annemys sp. (Danilov et al. 2018). However, it is not as pronounced as in marine turtles, Chelonia mydas (Linnaeus, 1758) (Laurin et al. 2011) and Dermochelys coriacea (Vandelli, 1761) (Houssaye 2013). Araripemys shares with the tortoises Chelonoidis carbonaria (Spix, 1824) and Homopus femoralis Boulenger, 1888, highly compacted zeugopodials (Laurin et al. 2011). In this way, high compactness in the ulna and radius might be related to the flat-shelled constraints in A. barretoi. Our findings corroborate an earlier study which indicated that turtles with different lifestyles can have similar high compactness indices for the long bones (Kriloff et al. 2008).

Within the limb bones, the coarse cancellous bone (CCCB) only occurs in the perimedullary region of the metatarsal III. This secondary reconstruction decreases the size of the medullary cavity by endosteal compactness of spongy bone (Enlow 1962). The CCCB deposition increases through ontogeny as observed in the stylopodials of the tortoise Chersina angulata (Bhat et al. 2019). In long bones with active growth, the metaphyseal region consists of spongy bone (Woodward et al. 2018). During ontogeny the bone shaft elongates, the diaphysis incorporates metaphyseal region and the trabeculae are subsequently infilled with primary bone to form CCCB (Enlow 1963). Given that herein the remodelled CCCB only occurs in one growing metapodial and it is likely related to the metaphyseal position of the section of the third metatarsal.

Inter-elemental variation in the relative bone wall thickness (RBT) is recorded in Table I. The ulna has the highest RBT (42%), suggesting a higher rate of appositional growth relative to the other limb bones and represents a case of pachyosteosclerosis, i.e., the development of hyperplasic periosteal cortical and an increase in inner bone compactness (see Buffrénil et al. 2008). The humeri and radius show the lowest values (<20%). It is evident from the RBT indices that the appositional growth of these long bones varies within the same individual. The low humeral RBTs suggest that these bones could not be used for digging (Ray & Chinsamy 2004). In a fossorial lifestyle the forelimbs are mainly used for digging, as a consequence the cortical bone becomes thicker increasing its RBT (Botha & Chinsamy 2004, Ray & Chinsamy 2004). The low RBT indices presented here in the forelimbs, therefore point to a non-fossorial behaviour for Araripemys barretoi.

In the MPSC R 010 ulna, pachyosteosclerosis indicates a local increase in bone density that is associated with a highly compacted carapace and improves the body trim and stability of Araripemys barretoi (Taylor 2000). However, the pachyosteosclerotic condition does not necessarily indicate a fully aquatic lifestyle for Testudines and their relatives. Amphibious and terrestrial turtles also sometimes show a high compactness of the long bones (Laurin et al. 2011, Schoch et al. 2019).

Skeletochronology in long bones: ontogeny and sexual maturity

Among the osteohistological features that reflect ontogeny presented by MPSC R 010 are the high number of LAGs, and the thin zones deposited towards the periosteal margin. Similar features have already been recorded in samples of the podocnemidid Podocnemis expansa (Schweigger, 1812) (Chinsamy & Valenzuela 2008) and the chelid Yaminuechelys maior (Staesche, 1929) (Pereyra et al. 2020). The counting of LAGs could be a good method for estimating the age of an individual. As the number increases, specimens reach older ontogenetic stages, allowing us to infer the actual age of the animal (Snover & Hohn 2004). Among the studied elements the ulna preserves the greatest amount of growth marks. We attribute thus a late ontogenetic stage for the MPSC R 010 and an age of 12 years old at death is estimated for this specimen. Supplemental LAGs were not considered in our analysis, these lines are less distinct and do not appear around the entire circumference of the bone section. Their presence is related to temporary stressful environmental conditions (Snover & Hohn 2004), an environmental condition that fits the proposal for the Romualdo Formation (Almeida-Lima et al. 2020, Melo et al. 2020).

The ulna of MPSC R 010 shows a change in its growth rate between the fourth and tenth years of life, featured by a decrease and a consecutive increase in growth rate. This change in growth dynamic was considered by Pereyra et al. (2020) to be probably related to the attainment of sexual maturity in Hydromedusa tectifera Cope, 1869. However, the authors indicated that the subsequent increase in growth rate may be caused by other factor instead the sexual maturation. Assuming that sexual maturity was reached between 4–10 years of age by MPSC R 010 it is similar to males of Australian chelids which reached their maturity at the same age range (Spencer 2002). Different species of the chelid Hydromedusa show distinct age range when obtaining their sexual maturity such as 8–12 years old (in Hydromedusa tectifera; Pereyra et al. 2020) or 11–16 years of age (in Hydromedusa maximilliani (Mikan, 1825); Martins & Souza 2008). The podocnemidid Podocnemis erythrocephala (Spix, 1824) attained its sexual maturity at nine years of life in both genders (Bernhard, unpublished data). Carapace length is another criterion that can accompany the analysis of long bones. Hofmeyr & Fritz (2018) observed that the pelomedusid Pelomedusa galeata (Schoepff, 1792) achieves its sexual maturity when the shell reaches a range from 110 to 160 mm in length. Following this trend the straight carapace length of MPSC R 010 (205 mm) is higher than the length considered for other mature turtles, corroborating the skeletochronological analysis of the ulna. Comparisons with other pelomedusoids and chelids indicate that MPSC R 010 reaches its sexual maturity between 4–10 years old. Moreover, it was at least 12 years old (based on the ulna which recorded the largest number of LAGs) before its death as evidenced by the presence of twelve LAGs. Finally, there is no evidence of an External Fundamental System (EFS), i.e., a rapid and successive deposition of growth marks at the periphery of the cortex, neither in the costal plate nor in the long bones sampled. The absence of this microstructural feature suggests that both individuals did not reach their skeletal maturity yet (Botha & Botha 2019).

CONCLUSIONS

Araripemys barretoi exhibits a terrestrial histological type according to Jannello et al. (2020) contrasting with the lacustrine and marine deposits where the species has been found. This histological pattern is probably more related to the functional role increasing shell resistance to compensate its weakness caused by several fontanelles.

Considering the histological features Araripemys was a slow swimmer and a skilled diver, which could be submerged for short-terms. Additionally, thick and compact cortices increase the carapace’s rigidity to prevent biomechanical damage. This discrepancy could be explained by an aquatic species with two extreme specializations in microanatomy of different parts of the skeleton (namely, in shell bones and in long bones; a view accepted herein) already reported in aquatic turtles (Skutschas et al. 2017). The growth strategy of A. barretoi suggests a moderate to slow growth rate shown by the parallel-fibred and lamellar bone tissues. As reported on other extinct and extant Testudines, A. barretoi attained its sexual maturity before reaching skeletal maturity, between the fourth and tenth years of age.

ACKNOWLEDGMENTS

We would like to express our gratitude to Dr. Diogenes de Almeida Campos who dedicated his life to the protection of Araripe fossils, inspiring our careers. We thank Professors Sandra Barreto, Lauro Santos and Virginio Neumann, who generously permitted analyses in the Laboratório de Gemologia (LABGEM/UFPE) and Laboratório Integrado de Tecnologia em Petróleo, Gás e Biocombustíveis (LITPEG/UFPE). We are also indebted to Pavel Skutschas and three anonymous reviewers for their helpful comments on the earlier version of this manuscript. Technical support for this research was provided by Charlisson Silva who performed UFRPE 5302 cross-section, Rafael Cavalcanti for previous chemical preparation of the specimens and William Alexandre for technical assistance helping us with equipment setup and procedures. This manuscript presents partial results of the doctoral thesis of the MVAS. This project was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq no. 311715/2017-6 and 314222/2020-0 to JMS), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP #BMD-0124-00302.01.01/19 to RAMB and #BP3-013900202.0100/18 to AAFS) and the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (finance code 001 CAPES #88887.162865/2018-00 to RAMB).

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SUPPLEMENTARY MATERIAL

Figure S1. Schematic black (bone) and white (vascular spaces and medullary cavities) drawings of the extinct Araripemys barretoi prepared for use with Bone Profiler. a, Perpendicular section of costal V plate (MPSC R 010). b, Parallel section of costal V plate (MPSC R 010). c, Perpendicular section of the neural V plate (UFRPE 5302). d, Transversal section of a peripheral plate (MPSC R 010). e, Transversal section of humeral epiphysis (MPSC R 010). f, Transversal section of ulna (MPSC R 010). g, Transversal section of humeral metaphysis (MPSC R 010). h, Transversal section of radius (MPSC R 010). i-l, Transversal sections of metapodials (MPSC R 010), metatarsal I (i); metatarsal II (j); metatarsal III (k); and metatarsal IV (l).

Publication Dates

• Publication in this collection
  02 Aug 2021
• Date of issue
  2021

History

• Received
  6 Oct 2020
• Accepted
  12 Mar 2021