Azzabaremys moragjonesi, from the Paleocene of Mali, is a member of Nigeremydini. This is a clade of bothremydid pleurodiran turtles that includes large putatively marine forms which inhabited the African Trans-Saharian Seaway from the Maastrichtian to the Paleocene. This work represents the first neuroanatomical reconstruction of a putative sea pleurodire. Some of the neuroanatomical modifications observed for Azzabaremys moragjonesi differ from those in the other lineages of Bothremydidae in which these structures have been documented, corresponding to freshwater instead of pelagic marine forms. In fact, the primitive condition for Pleurodira is the adaptation to freshwater environments, as is the case with all extant representatives, but also with most documented extinct forms. The neuroanatomy of Azzabaremys moragjonesi shows convergences with that of the members of Pan-Cryptodira with adaptations to marine environments (i.e., Chelonioidea, including Cheloniidae and Dermochelyidae), but also with other clades of marine turtles exclusive to the fossil record (i.e., several Mesozoic and Palaeogene clades with uncertain affiliations: Plesiochelyidae, Sandownidae, and Protostegidae). Thus, aspects such as the position of the geniculate ganglion into the canalis cavernosus, the absence of an anterior vidian canal piercing the pterygoid, and the possession of wide semicircular canals of the endosseous labyrinth, as well as the possible presence of enlarged lacrimal glands, are recognized here as convergent modifications developed in Cryptodira, extinct clades with uncertain affiliations, and Pleurodira in response to adaptation to marine environments.

Bothremydidae, convergent evolution, marine adaptations, neuroanatomy, Nigeremydini, Tran-Saharan Seaway

All extant pleurodires are freshwater turtles (TTWG 2021), this being the primitive and generalized condition for the members of this lineage (de Broin 1988; Gaffney et al. 2006). Despite being freshwater forms, some extant species of Chelidae can tolerate high levels of salinity (Frazier 1986; Bower et al. 2016), allowing them to move around coastal or brackish estuarine environments. However, some extinct pleurodiran species or even lineages have been associated with marine lifestyles, being generally interpreted as coastal forms (i.e., most Stereogenyini, some erymnochelyinid podocnemidids, and several lineages of Bothremydidae) (Gaffney et al. 2006, 2011; Ferreira et al. 2015; Pérez-García et al. 2017). These interpretations are generally based on the sedimentary environments in which they or their putative nesting areas have been found, but without robust arguments based on their anatomy (e.g., modifications in the limb, shell, palatal anatomy or cranial morphology, which could indicate greater hydrodynamism and mobility than in freshwater environments; and also adaptations to durophagous diets, very suitable for some marine ecosystems) (Lapparent and Werner 1998; Gaffney et al. 2006; Winkler and Sánchez-Villagra 2006; Weems and Knight 2013; Ferreira et al. 2015; Pérez-García 2016a; Moreno-Azanza et al. 2021; Hermanson et al. 2022).

Bothremydidae was a successful lineage of pleurodiran turtles that inhabited large areas of both Laurasian and Gondwanan territories from the Early Cretaceous to the Eocene at least (e.g., Lapparent de Broin 2000; Gaffney et al. 2006; Pérez-García 2018, 2023; Maniel et al. 2021). The adaptation to transitional or marine, mainly coastal, environments has been proposed for some lineages as Taphrosphyini and Nigeremydini, but also for some representatives of Bothremydini (Lapparent and Werner 1998; Gaffney et al. 2006; Pérez-García 2016a, 2023; Lapparent de Broin et al. 2021). In fact, the more extreme case is that of Nigeremydini, identified as a clade of large Maastrichtian to Paleocene bothremydids interpreted as well-adapted to a fully marine environment, that could inhabit both coastal and pelagic regions of the African Trans-Saharan Seaway (for a detailed list of the taxa included in this clade see Pérez-García 2023, and references therein).

Adaptation to coastal environments, and particularly to open marine environments, involves numerous anatomical modifications, especially considering the higher percentage of salt in these environments in relation to freshwater ones and, therefore, the different density values of those waters (Rhodin et al. 1981; Davenport et al. 1984; Wyneken 2001; Sánchez-Villagra et al. 2007; Scheyer et al. 2014). This can be observed in the skeleton of the pan-cryptodiran and other non-pleurodiran lineages of sea turtles, with several representatives forming part of the current biodiversity, and with an abundant and diverse fossil record, not only during the Cenozoic but, especially, at the Cretaceous (Hirayama 1995, 1997; Cadena et al. 2018). These adaptations include autopodial modifications into flippers (e.g., Hirayama 1995; Evers et al. 2019), humeral changes (e.g., Hirayama 1992; Krahl et al. 2019; Hermanson et al. 2024), or fontanelles in the shell (e.g., Lapparent de Broin et al. 2018; Gentry et al. 2018). The modifications not only affect the postcranial skeleton, but also the skull. Thus, marine cryptodires have developed lacrimal glands to remove excess salt from its organism with an osmoregulatory function (e.g., Schmidt-Nielsen and Fange 1958; Hudson and Lutz 1986; Marshall and Cooper 1988; Wyneken 2001), these structures having also been putatively inferred for some Mesozoic marine turtles, based on poor osseous evidence (Hirayama 1998; Anquetin et al. 2017). Some neuroanatomical elements are also modified in all these clades of marine turtles, as is the case of the nasal cavities with the presence of some diverticula to improve the underwater olfactory capacity (Parsons 1959; Schwenk 2008), the reduced size of the cavum tympani and the antrum postoticum of the middle ear (Foth et al. 2019), and the morphology of the semicircular canals of the inner ears to increase the diving and locomotion capabilities in pelagic environments (Neenan et al. 2017; Evers et al. 2019), although this last character is currently under debate (Evers et al. 2022).

The information on the neuroanatomy of extinct pleurodires was, until now, very limited. In the case of Bothremydidae, the first detailed neuroatomical description of a member of this lineage had been published in 2021, corresponding to the freshwater Bothremydini Tartaruscola teodorii Pérez-García 2016b, from the Ypresian of France (Martín-Jiménez and Pérez-García 2021). Scarce neuroanatomical information about this lineage had been previously documented, corresponding to the North American Upper Cretaceous freshwater representatives of Bothremydini Bothremys cooki (Leidy 1865), Chedighaii barberi (Schmidt 1940) and Chedighaii sp. (Gaffney and Zangerl 1968; Gaffney 1977). Some works about the neuroanatomy in Bothremydidae were subsequently published, corresponding to the complete reconstruction and description of some internal cranial cavities and canals of two members of Cearachelyini (Galianemys whitei Gaffney, Tong & Meylan, 2002 and Galianemys emringeri Gaffney, Tong & Meylan, 2002 from the Cenomanian of Morocco; Martín-Jiménez and Pérez-García 2022), and the description or figuration of some neuroanatomical elements of several taxa to study the endosseous labyrinth [based on the Moroccan Bothremydini Bothremys maghrebiana Gaffney, Tong & Meylan, 2006 from the Paleocene (Hermanson et al. 2020) and the Taphrosphyini Ummulisani rutgerensis Gaffney, Tong & Meylan, 2006 from the Eocene (Evers et al. 2022)], the canals of the facial nerves, and the circulatory system (based on Galianemys whitei and three Moroccan Paleocene and Eocene representatives of Taphrosphyini; Hermanson et al. 2020). The present study includes the first neuroanatomical study of a member of the clade of putative marine forms Nigeremydini, Azzabaremys moragjonesi Gaffney, Moody & Walker, 2001. The study is based on the analysis of the complete and well-preserved skull of the holotype and so far only known remains of the species. Therefore, we intend to investigate if neuroanatomical structures have been modified in relation to those of freshwater and even coastal representatives to live in those environments, as occurs with the lineages of marine Mesozoic turtles and cryptodiran sea turtles. Also, we explore if any of these modifications that were not previously studied for pleurodires are convergent with those of the other lineages of pelagic turtles.

The present neuroanatomical study is based on the holotype of Azzabaremys moragjonesi, corresponding to the skull NHMUK R16370, housed in the Natural History Museum (NHMUK, London, United Kingdom). The detailed anatomical description of this skull was performed in previous publications (Gaffney et al. 2001, 2006). The specimen was scanned at the Micro-CT Scanning service of the NHMUK. The scanner used for this analysis corresponds to a high resolution Nikon Metrology HMX ST 225, with a current of 150 µA, a voltage of 200 kV, and 3,600 projections over 360°. 2,545 images in .raw format with a voxel size of 84 µm were obtained. Due the large size of this set of files, the images were converted to .tiff format using the software Avizo 7.1 (VSG). The segmentation of the obtained CT images and the generation of 3D models of both the skulls and the inner structures were also carried out with the tools of the software Avizo 7.1. The files obtained were processed into three packages of 500 images and one more of 561, obtaining three-dimensional meshes of both the osseous and the neuroanatomical elements. The final models were obtained from the merge of the meshes generated from the four packages, using tools of the Geomagic Studio 2014.3.0 software. The 3D models were exported as .stl files. The inner cavities and canals linear and angular measurements were performed using the tools of Avizo 7.1. The volumetric measurements of the nasal and cranial cavities were obtained using the Geomagic Studio 2014.3.0 tools. The 3D models obtained have been compared with those of all other bothremydid taxa described or figured until now (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022); the marine pan-cryptodiran turtles in which neuroanatomical structures are known, corresponding to extant representatives of Chelonioidea, including the cheloniids Chelonia mydas (Linnaeus 1758), Lepidochelys olivacea (Eschscholtz 1829), Eretmochelys imbricata (Linnaeus 1766), and Caretta caretta (Linnaeus 1758) (Zangerl 1960; Neenan et al. 2017; Lautenschlager et al. 2018; Evers et al. 2019; Kondoh et al. 2019; Yamaguchi et al. 2020; Rollot et al. 2021; Ferreira et al. 2022; Yoshida et al. 2022), and the dermochelyid Dermochelys coriacea (Vandelli 1761) (Evers et al. 2019; Rollot et al. 2021); some extinct members of Chelonioidea, corresponding to the cheloniid Argillochelys cuneiceps (Owen 1849), from the Eocene of United Kingdom (Neenan et al. 2017; Evers et al. 2019), and the dermochelyid Corsochelys haliniches Zangerl 1960, from the Late Cretaceous of United States (Zangerl 1960); and several marine members of extinct turtle clades with uncertain affiliations, corresponding to the plesiochelyid Plesiochelys etalloni (Pictet and Humbert 1857), from the Jurassic of Switzerland (Paulina-Carabajal et al. 2013), the sandowniid Sandownia harrisi Meylan, Moody, Walker & Chapman, 2000, from the Early Cretaceous of the United Kingdom (Evers and Joyce 2020), and the protostegid Rhinochelys pulchriceps (Owen 1851), from the Cenomanian of the United Kingdom (Evers et al. 2019).

Testudines Batsch, 1788

Pleurodira Cope, 1864

Pelomedusoides Cope, 1868

Podocnemidoidea Cope, 1868

Bothremydidae Baur, 1891

Bothremydodda Gaffney, Tong & Meylan, 2006

Nigeremydini Lapparent de Broin and Prasad, 2020

Azzabaremys Gaffney, Moody & Walker, 2001

 Azzabaremys moragjonesi Gaffney, Moody & Walker, 2001

Figs 1, 2, 3, 4, 5

Type locality

North of In Fargas, Samit, eastern Mali.

Horizon and depositional environment

Shallow marine sediments of the Teberemt Formation, Paleocene (see Moody and Sutcliffe 1990, 1991, 1993).

Holotype

NHMUK R16370, corresponding to a complete skull without the mandible, being the only material currently known for this taxon (Figs 1, 2).

Figure 1. 

Skull, osseous, and neuroanatomical three-dimensional reconstruction of NHMUK R16370. A–C. Dorsal view of the skull of the holotype of Azzabaremys moragjonesi (Pleurodira, Nigeremydini), from the Paleocene of Mali; D–F. Anterior view; G–I. Left lateral view.

Figure 2. 

Skull, osseous, and neuroanatomical three-dimensional reconstruction of NHMUK R16370. A–C. Ventral view of the skull of the holotype of Azzabaremys moragjonesi (Pleurodira, Nigeremydini), from the Paleocene of Mali; D–F. Posterior view; G–I. Right lateral view.

Neuroanatomical description

The cranial inner cavities (i.e., endocranial, nasal, and labyrinths), the canals of the carotid and anterior maxillary arteries, the canalis and sulcus cavernosus, and the canals of most nerves that cross the bones were completely reconstructed for this skull (Figs 1C, D, F, 2C, D, F, 3). The canal of the abducens nerve (cranial nerve VI) cannot be observed due to the brightness of the sections in that area, and most of the canal of the vidian branch of the facial nerve (CN VII) is absent, because this nerve was not covered by bone. The anterior foramen for the exit of the glossopharyngeal nerve (CN IX) had not been observed.

Figure 3. 

Three-dimensional reconstruction of neuroanatomical structures, inner ears, and osseous elements of NHMUK R16370. A-–D. Neuroanatomical reconstructions of the holotype of Azzabaremys moragjonesi (Pleurodira, Nigeremydini), from the Paleocene of Mali, in dorsal (A), ventral (B), left lateral (C), and right lateral (D) views; E. Posterior right region of the skull, in posterior, showing the foramina related with neuroanatomical elements; F,H. Right inner ear in lateral (F) and dorsal (H) views; G, I. Left inner ear in lateral (G) and dorsal (I) views. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; cas, canalis alveolaris superior; cc, crus communis; cci, canalis caroticus internus; ccv, canalis cavernosus; cer, cerebral hemispheres; cio, canalis infraorbitalis; cst, canalis stapedio-temporalis; dp, dorsal protuberance; fja, foramen jugulare anterius; fjp, foramen jugulare posterius; fm, foramen magnum; fng, foramen nervi glossopharyngei; fnh, foramen nervi hypoglossi; fpcci, foramen posterius canalis carotici interni; fpo, fenestra postotica; hyo, hyomandibular branch of facial nerve; I, olfactory nerve; IX, glossopharyngeal nerve; lsc, lateral semicircular canal; nc, nasal cavity; npd, nasopharyngeal duct; ob, olfactory bulbs; pit, pituitary fossa; psc, posterior semicircular canal; scv, sulcus cavernosus; sot, septum orbitotemporale; V, trigeminal nerve; vesn, vestibulum nasi; VII, facial nerve; VIII, vestibulocochlear nerve; XII, hypoglossal nerve.

The dorsal surface of the endocranial cavity forms an angle of 133.8° between the forebrain and the hindbrain (Fig. 3C, D). The forebrain is slightly dorsally directed to reach the dorsal protuberance [dural peak sensu Evers et al. (2019) or cartilaginous rider sensu Werneburg et al. (2021)]. The olfactory bulbs are weakly developed (Fig. 3A). The cerebral hemispheres are weakly expanded laterally, its width corresponding to 23.8% of the total length of the endocranial cavity (Fig. 3A). The ratio between the width of hemispheres and that of the medulla oblongata is 1.32. The dural peak is a dorsomedial protuberance of the endocranial cavity (Fig. 3A, C, D), being bulbous and oval in dorsal view. This structure is relatively tall, being equivalent to 9.6% of maximum height of the endocranial cavity. The peak represents 12% of the total length of the endocranial cavity. The pituitary fossa is located ventral to the hemispheres (Fig. 3B). This cavity is small and oval, being wider than long. The dorsal surface of the medulla oblongata descends to reach the foramen magnum (Fig. 3C, D).

The sulcus that housed the olfactory nerve (CN I) is deep and wide. The olfactory nerve was long, equivalent to 44% of the endocranial cavity length (Fig. 3A). The nerve was posterodorsally directed and connected the nasal cavity with the olfactory bulbs region. The trigeminal nerve (CN V) exited from the endocranial cavity through the foramen nervi trigemini (Fig. 3C, D). This foramen is a big oval-shaped opening, ventral to the cerebral hemispheres. The nerve reached the canalis cavernosus laterally. The anterior region of the canalis cavernosus is not covered by bone at the level of the trigeminal foramen (Fig. 3A–C). Thus, it shows a gap between the canal and the sulcus cavernosus. Posteriorly, the canalis cavernosus contacts the canalis stapedio-temporalis, which is short and anterodorsally directed (Fig. 3A). The sulcus cavernosus continues anteriorly ventral to the endocranial cavity (Fig. 3A–C). The facial and the vestibulocochlear (CN VIII) nerves exited laterally to the endocranial cavity through two foramina located in the fossa acustico-facialis of the prootic (Fig. 3A, B, D). This fossa is located posterior to the trigeminal foramen. The facial nerve reached the geniculate ganglion into the canalis cavernosus. The vidian branch of the facial nerve exited ventrally through a short canalis pro ramo nervi vidiani, and posteriorly entered the canalis caroticus internus (Fig. 4B). The vidian nerve ran anteriorly without the presence of a canalis nervi vidiani and it anteriorly pierced the pterygoid by a small canal (Fig. 4C). The nerve continued across the sulcus palatinopterygoideus. The distal hyomandibular branch of the facial nerve exited laterally from the canalis cavernosus (Fig. 3B). The vestibulocochlear nerve entered the labyrinthic cavity to innervate the inner ear anteriorly (Fig. 3D). The vagus (CN X) and the spinal accessory (CN XI) nerves crossed the foramen jugulare anterius and reached the recessus scalae tympani (Fig. 3D). These nerves exited the skull posteriorly through the foramen jugulare posterius (Fig. 3E). The foramen jugulare posterius is an opening separated by bone from the fenestra postotica. The distal region of the glossopharyngeal nerve crossed the skull through a canal from the recessus scalae tympani (Fig. 3A–B). The canal splits and the nerve exited the skull by two foramina located in the opisthotic, between the foramen jugulare posterius and the fenestra postotica (Figs 3E, 5). The hypoglossal nerve (CN XII) exited the endocranial cavity through a single foramen with a canal that bifurcates (Fig. 3A, B). Two external foramina are observed for the right exoccipital but only a foramen is present in the left one (Fig. 3E).

The nasal cavity of Azzabaremys moragjonesi is relatively enlarged (Fig. 3A–D), being more than a half (54.2%) the total volume of the cranial cavities (i.e., including the endocranial and the nasal cavities). The nasal opening is wide (Fig. 1D–F) and the vestibulum of the nasal cavity is anteroposteriorly short (Fig. 3C, D). The posterodorsal olfactory region of the nasal cavity is slightly expanded dorsally. The nasopharyngeal ducts are wide and short. They are posteriorly directed in a straight trajectory (Fig. 3B).

The interorbital foramen is largely expanded in lateral view (Figs 1G–I, 2G–I). The septum orbitotemporale is present in Azzabaremys moragjonesi, as in the rest of pleurodiran turtles. Until recently, this structure was identified as exclusive to Pleurodira. However, recent studies have shown that it is more widespread than previously reported, being present in other groups such as Paracryptodira and Trionychidae (e.g., Evers et al. 2020, 2021, 2023). The septum has a large opening that widely connects the orbital and temporal fossae (Fig. 3E). The dorsal surface of the maxilla shows an anteroposteriorly directed sulcus crossing the ventral surface of the orbital fossa (Fig. 4D). This sulcus enters the maxilla ahead of the level of the anterior margin of the orbit.

The inner ear cavity presents relatively robust semicircular canals (Fig. 3F–I). They are slightly expanded dorsally. The anterior semicircular canal is the longest, displaying a wide anterior ampulla. The diameter of all semicircular canals is relatively wide (i.e., with a range between 2.53 and 3.37 mm, and a ratio between its maximum diameter and its length that varies from 0.37 to 0.63). The inner spaces formed by the semicircular canals and the vestibulum are oval, with a reduced diameter. The dorsal area of the anterior and posterior semicircular canals is at the same level than the dorsal surface of the crus communis, forming a horizontal surface (Fig. 3F, G). Anteroposteriorly, the crus communis is very long. The angle formed between the vertical semicircular canals is acute, being 77° for the left inner ear and 77.4° for the right one (Fig. 3H, I).

The carotid artery entered the skull through the foramen posterius canalis carotici interni, which is in the ventral surface of the pterygoid (Fig. 3E). The artery ran into the canalis caroticus internus reaching the posterior end of the pituitary fossa (Fig. 3B–D). The carotid canals form an angle of almost 89° (Fig. 3B). The lateral canal for the palatine artery is absent. The arteria inframaxillaris crossed the ventral region of the septum orbitotemporale and continued anteriorly through the canalis infraorbitalis, located into the maxilla (Fig. 3A-C). This canal bifurcates at the level of the nasopharyngeal duct. The anterior branch contained the nasal posterior artery, which irrigated the anteroventral region of the nasal cavity. The canalis alveolaris superior contained the alveolar superior artery, which also bifurcates, irrigating the lateral portion of the nasal cavity.

Most neuroanatomical structures observed in non-Nigeremydini bothremydids are very conservative, considering both the pattern of the nerves and vascular canals, and the configuration of the semicircular canals of the endosseous labyrinth (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022). The most characteristic neuroanatomical feature within Bothremydidae is the presence of a dorsal dural peak with a lateroventrally directed cartilaginous ridge (Fig. 3A, C, D) (Martín-Jiménez and Pérez-García 2021, 2022). This structure is very variable in other pleurodires, being absent or poorly dorsally expanded in Chelidae, Pelomedusidae, and Euraxemydidae (Martín-Jiménez and Pérez-García 2021, 2023a), or showing different morphologies into the clade Podocnemidoidae, becoming as developed as in the Bothremydidae in some taxa (Ferreira et al. 2018; Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2023b). The length of the dural peak relative to the length of the endocranial cavity in Bothremydidae varies between the longest structure of the Bothremydini Tartaruscola teodorii, Bothremys cooki, and Chedighaii barberi, and the Cearachelyini Galianemys whitei (representing between 17 to 20% of the endocranial cavity length); and the shortest dorsal protuberance in Galianemys emringeri (being less than 10% of it) (Martín-Jiménez and Pérez-García 2022). The value observed for Azzabaremys moragjonesi (i.e., corresponding to 12% of the endocranial cavity length) is close to the lesser value of the range previously documented for Bothremydidae. Two other neuroanatomical features are also identified in Azzabaremys moragjonesi as shared with all other members of Bothremydidae. The first corresponds to the value of the angle between the forebrain and the hindbrain, which reflects the cephalic flexure of the brain. This angle is lesser than 150° in Azzabaremys moragjonesi (Fig. 3C, D), as occurs in all podocnemidoids (i.e., Bothremydidae and Podocnemidoidae) (Martín-Jiménez and Pérez-García 2021, 2022, 2023b), but not in the other members of the Pleurodira (Martín-Jiménez and Pérez-García 2021, 2023a). The value range documented for Bothremydidae varies from about 120° for the Taphrosphyini Phosphatochelys tedfordi Gaffney and Tong 2003, from the Eocene of Morocco (based on the figure 15 of the supplementary file of Hermanson et al. 2020), to almost 150° for the Cearachelyini Galianemys emringeri (see Martín-Jiménez and Pérez-García 2022). Azzabaremys moragjonesi presents an angle close to that in the Bothremydini Bothremys cooki (i.e., 133°; see Martín-Jiménez and Pérez-García 2022). The second character is related to the angle formed by the carotid canals. In Bothremydidae, this angle shows a wide range, the more acute being those observed in the lineage of Cearachelyini (i.e., lesser than 80°; Martín-Jiménez and Pérez-García 2022), and the more obtuse angles corresponding to those in the representatives of Taphrosphyini (i.e., values greater than 140°; Hermanson et al. 2020). The angle measured for Azzabaremys moragjonesi (Fig. 3B) (i.e., near to 90°) is close to that observed for Cearachelyini. The angle of the carotid canals of Bothremydini, only documented in the species Tartaruscola teodorii (being recognized as 98° in the holotype and 108°, in the paratype of the species; Martín-Jiménez and Pérez-García 2021), is more obtuse than that in Azzabaremys moragjonesi. Within Pleurodira, the angle formed by the carotid canals in Azzabaremys moragjonesi is similar to that recognized for Pelomedusidae (Martín-Jiménez and Pérez-García 2021), being more acute than that of Chelidae, and more obtuse than in the non-bothremydid podocnemidoids (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2023b).

Several neuroanatomical elements of Azzabaremys moragjonesi display clear differences with those so far documented for all other pleurodiran taxa [extant chelids, pelomedusids, and podocnemidids (Lautenschlager et al. 2018; Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021); extinct euraxemydids (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2023a); the Brazilian Upper Cretaceous podocnemidoids Amabilis uchoensis Hermanson, Iori, Evers, Langer & Ferreira, 2020 (Hermanson et al. 2020) and Yuraramirim montealtensis Ferreira, Iori, Hermanson & Langer, 2018 (Ferreira et al. 2018); and the French Eocene podocnemidid Neochelys arenarum de Broin 1977 (Martín-Jiménez and Pérez-García 2023b)], including members of several lineages of Bothremydidae (Cearachelyini, Bothremydini, and Taphrosphyini; Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022; Evers et al. 2022). Previous works described one foramen located in the posterior opisthotic region of the skull of Azzabaremys moragjonesi (Gaffney et al. 2001, 2006), between the foramen jugulare posterius and the fenestra postotica, suggesting that it corresponded to the exit of the glossopharyngeal nerve. The three-dimensional reconstruction of a canal connecting the recessus scalae tympani with the posterior cranial region in the same skull of this taxon, and the identification of two foramina in the opisthotic for the exit of the glossopharyngeal nerve (Figs 3A, B, E, 5), being absent in all other studied bothremydids (Gaffney et al. 2006; Martín-Jiménez and Pérez-García 2021, 2022), confirms this character as exclusive of Azzabaremys moragjonesi. All extant and most extinct representatives of Pleurodira, including Bothremydidae, are freshwater forms, in which the nasal cavities are relatively reduced, its volume being less than 30% of the total volume of the endocast (Lautenschlager et al. 2018; Martín-Jiménez and Pérez-García 2021, 2022, 2023a, 2023b). By contrast, the nasal cavity of Azzabaremys moragjonesi is greatly expanded (Fig. 3A–D), its volume exceeding 50% of it. The morphology of the different regions of the nasal cavity (i.e., the diverticula) is conditioned by the layout and development of the cartilage and connective tissue (Kondoh et al. 2019; Yamaguchi et al. 2020). Therefore, the shape of the functional area of the nasal cavity of Azzabaremys moragjonesi remains unknown and it is not possible to compare it with that of extant forms of turtles in which it has been documented (Parsons 1959; Schwenk 2008).

Most of the neuroanatomical characters which differentiate Azzabaremys moragjonesi from the other members of Bothremydidae so far analyzed are shared with those of all members of the clades of marine turtles Chelonioidea (i.e., Cheloniidae and Dermochelyidae), Plesiochelyidae, Sandownidae, and Protostegidae in which these features are known. Thus, the olfactory nerve is very long in Azzabaremys moragjonesi, its length corresponding to 44% of the total length of the endocranial cavity (Fig. 3A, C, D), being less than 30% in all other bothremydids (a character so far documented for members of Cearachelyini and Bothremydini; Martín-Jiménez and Pérez-García 2021, 2022). The olfactory nerve in pan-cryptodiran and marine extinct clades with uncertain affiliations is relatively long (more than 30% measured for the endocasts of Chelonia mydas and Caretta caretta based on the plate 30 of Zangerl 1960; almost 40% measured for Plesiochelys etalloni based in the fig. 2 of Paulina-Carabajal et al. 2013; 36% measured for Rhinochelys pulchriceps based on the fig. 3 of Evers et al. 2019), exceeding the values documented for the freshwater forms of Pleurodira (Martín-Jiménez and Pérez-García 2021) and non-marine cryptodiran representatives (ranging between almost 15% and 26% for freshwater forms of different lineages as Trionychidae, Chelydridae, Platysternidae, Emydidae, and Geoemydidae, measured from Paulina-Carabajal et al. 2017, Lautenschlager et al. 2018, and Evers et al. 2019; and being relatively shorter in terrestrial cryptodires, Paulina-Carabajal et al. 2017, Lautenschlager et al. 2018).

Despite the morphology of the inner ear of the members of Testudines being relatively conservative (Paulina-Carabajal et al. 2013; Ferreira et al. 2022; Evers et al. 2022), the endosseous labyrinth of the marine and terrestrial turtles presents thicker and shorter semicircular canals than in freshwater representatives of both Cryptodira and Pleurodira (Neenan et al. 2017; Paulina-Carabajal et al. 2017; Lautenschlager et al. 2018; Evers et al. 2019; Menon et al. 2024), including all so far documented members of Bothremydidae (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022; Evers et al. 2022). However, the endosseous labyrinth of the aquatic taxa (both marine and freshwater forms) differs from that of the tortoises considering the angle formed between the anterior and the posterior semicircular canals in dorsal view, being more acute in aquatic turtles than in terrestrial forms (Paulina-Carabajal et al. 2017; Lautenschlager et al. 2018). The angle formed by the semicircular canals in terrestrial turtles belonging to Cryptodira is variable, showing a relatively wide range, from acute angles (Evers et al. 2022; Pérez-García et al. 2022; Evers and Al Iawati 2024) to angles close to those in terrestrial forms of stem-Testudines (Paulina-Carabajal et al. 2017). This could be due more to plesiomorphic causes than to the ecology of each group. The relatively wide semicircular canals of Azzabaremys moragjonesi, being anteroposteriorly short (Fig. 3F–I), differ notably from those of the other bothremydids in which this is known (i.e., the Cearachelyini Galianemys whitei and Galianemys emringeri, the Bothremydini Tartaruscola teodorii and Bothremys maghrebiana, and the Taphrosphyini Ummulisani rutgerensis), with elongated and narrow canals (Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022; Evers et al. 2022). The semicircular canals of Azzabaremys moragjonesi present a similar thickness to that observed for the extant dermochelyid Dermochelys coriacea (Evers et al. 2019), being relatively thicker than in the representatives of Cheloniidae (Neenan et al. 2017; Lautenschlager et al. 2018), Protostegidae (Evers et al. 2019), Sandownidae (Evers and Joyce 2020), Plesiochelyidae (Paulina-Carabajal et al. 2013), and other putative marine bothremydids as Phosphatochelys tedfordi or Ummulisani rutgerensis (Evers et al. 2022; Hermanson et al. 2022). However, a relation between a relatively wide thickness of the semicircular canals with a well-developed perilymphatic system has been proposed as associated with pelagic deep divers (Neenan et al. 2017; Evers et al. 2019), so that, following this hypothesis, Azzabaremys moragjonesi can be considered as a species well adapted to a pelagic way of life. Nevertheless, the presence of wide canals in tortoises included within Cryptodira (i.e., Testudinidae), suggests that this character is not exclusive to forms adapted to marine environments.

The facial nerve of the representatives of Podocnemidoidea, including all members of Bothremydidae in which it had been identified [i.e., the Cearachelyini Galianemys emringeri and Galianemys whitei (Martín-Jiménez and Pérez-García 2022), the Bothremydini Tartaruscola teodorii (Martín-Jiménez and Pérez-García 2021), and the Taphrosphyini Ummulisani rutgerensis (see supplementary file in Hermanson et al. 2020)] splits in the geniculate ganglion, which is located in the prootic, between the medial foramen of the facial nerve and the canalis cavernosus (Rollot et al. 2021; Martín-Jiménez and Pérez-García 2021). However, the geniculate ganglion of Azzabaremys moragjonesi is located in the canalis cavernosus, as occurs in most members of Cryptodira (both aquatic and terrestrial forms) and in all representatives of the extinct Sandownidae and Protostegidae (Evers et al. 2019; Evers and Joyce 2020; Rollot et al. 2021). Contrary to the condition observed in Azzabaremys moragjonesi, the geniculate ganglion is located in the prootic in the pleurodiran euraxemydids, sahonachelyids, and podocnemidoids (including both Podocnemididae and Bothremydidae); the freshwater group of cryptodiran turtles Carettochelyidae, and the marine plesiochelyids (see Appendix S.1 in Evers and Benson 2019; supplementary in Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022, 2023a, 2023b; Rollot et al. 2021; Joyce et al. 2021). The geniculate ganglion is located close to the canalis caroticus internus in the chelids, pelomedusids, and dermochelyids (only documented for the extant form, i.e., Dermochelys coriacea) (Martín-Jiménez and Pérez-García 2021; Rollot et al. 2021). Nevertheless, the vidian nerve in the members of Pleurodira (including the taxa of Bothremydidae in which it had been documented, i.e., representatives of Cearachelyini, Bothremydini, and Taphrosphyini; see Hermanson et al. 2020; Martín-Jiménez and Pérez-García 2021, 2022) and the freshwater and terrestrial lineages of Cryptodira crosses the pterygoids anteriorly through the canalis nervi vidiani (Rollot et al. 2021). The condition observed here for the Nigeremydini Azzabaremys moragjonesi, in which the vidian canal of the pterygoid is absent, is shared with that observed for the extant sea turtles (both Cheloniidae and Dermochelyidae) (Rollot et al. 2021). However, in Azzabaremys moragjonesi a very short canal crosses the pterygoid to reach the sulcus palatinopterygoideus in this bothremydid (Fig. 4C), but the vidian nerve runs inside the sulcus cavernosus in these lineages of marine cryptodires (Albretch 1976; Rollot et al. 2021). The presence of a longer vidian canal was suggested for Protostegidae and Plesiochelyidae, which present foramina for the anterior exit of the vidian nerve (Hooks 1998; Anquetin et al. 2015; Püntener et al. 2017). The anterior trajectory of the vidian nerve remains unknown in Sandownidae (Evers and Joyce 2020).

Figure 4. 

Three-dimensional reconstruction cross section images of NHMUK R16370. A. Three-dimensional model of the skull of the holotype of Azzabaremys moragjonesi (Pleurodira, Nigeremydini), from the Paleocene of Mali, in left anterolateral view; B–D. Anteroposterior slices. Abbreviations: cci, canalis caroticus internus; ccr, cavum cranii; ccv, canalis cavernosus; cio, canalis infraorbitalis; cprnv, canalis pro ramo nervi vidiani; fnt, foramen nervi trigemini; mxs, maxillary sulcus; orb, orbit; pcvn, pterygoid canal for the vidian nerve.

Figure 5. 

Three-dimensional reconstruction cross section images of NHMUK R16370. A. Three-dimensional model of the skull of the holotype of Azzabaremys moragjonesi (Pleurodira, Nigeremydini), from the Paleocene of Mali in left lateral view; B–F. Dorsoventral (B) and anteroposterior (C–F) slices, showing the presence of a bifurcated canal and two foramina for the glossopharyngeal nerve. Abbreviations: ccr, cavum cranii; ccv, canalis cavernosus; cgn, canal of glossopharyngeal nerve; cnh, canalis nervi hypoglossi; fja, foramen jugulare anterius; fjp, foramen jugulare posterius; fm, foramen magnum; fnt, foramen nervi trigemini; fpo, fenestra postotica; hyo, hyomandibular branch of facial nerve; lfng, lateral foramen nervi glossopharyngei; lgc, lateral glossopharyngeal canal; mfng, medial foramen nervi glossopharyngei; mgc, medial glossopharyngeal canal; rst, recessus scalae tympani.

Extant marine reptiles have several types of specific cephalic glands to inhabit this environment, with different functions (Schmidt-Nielsen and Fange 1958). So, marine lizards present nasal glands located in the nasal cavity, and marine crocodiles and snakes have oral glands (Babonis and Brischoux 2012). The lacrimal or salt glands, observed in extant turtles, together with the harderian ones, are cephalic organs located into the orbit fossa (Schmidt-Nielsen and Fange 1958; Baccari et al. 1992; Yoshida et al. 2022). The harderian glands are located in the anteroventral region of the orbit and drain the eyeball (Cowan 1973). The lacrimal glands are enlarged organs located in the posterior region of the orbital fossa, and they have an osmoregulatory function (Schmidt-Nielsen and Fange 1958). The presence of salt glands has been inferred in several clades of extinct marine reptiles by the development of structures as, for example, a single foramen nariale obturatum in mesosaurs (Piñeiro et al. 2012), or lobulated rostral structures to the orbits in metriorhynchid crocodilomorphs (Fernández and Gasparini 2000, 2008; Gandola et al. 2006). However, the lacrimal glands in turtles are not directly reflected in the bones (Schmidt-Nielsen and Fange 1958; Wyneken 2001, Yoshida et al. 2022). In extinct turtles the presence of the salt glands is inferred by the presence of a greatly enlarged foramen interorbital and a reduced processus inferior parietalis, as occurs in Chelonioidea, the Cretaceous clade Protostegidae, and the Jurassic Plesiochelyidae (Hirayama 1998; Anquetin et al. 2017). The presence of a posteroventral excavation in the orbital fossa has been identified as a synapomorphic character of Bothremydidae (Gaffney et al. 2006), being also observed in Stereogenyini turtles (Gaffney et al. 2011). This region coincides with the position of the harderian glands, and could be interpreted as the accommodation area of these structures in the bones (Ferreira et al. 2015). The presence of a posteriorly enlarged foramen interorbital with a narrow processus inferior parietalis (Figs 1G–H, 2G–H) observed in Azzabaremys moragjonesi, and also identified in marine plesiochelyids, protostegids, and cryptodiran turtles, and the bothremydid genus Taphrosphys (Hirayama 1998; Gaffney et al. 2006; Anquetin et al. 2017), allows to infer the presence of big salt glands housed in the posterior region of the orbit. In addition, the reduction of the septum orbitotemporale (Fig. 3E) is also observed in the bothremydids Taphrosphys spp. and Phosphatochelys tedfordi, which could be related to the posterior arrangement of the harderian glands. Furthermore, the presence of a sulcus in the ventral surface of the orbital fossa overpassing the anterior margin of the orbit (Fig. 4D), not identified in other members of Bothremydidae, could also suggest the presence of ducts associated to the cephalic glands (i.e., the lacrimal or the harderian glands).

The neuroanatomical differences observed here between Azzabaremys moragjonesi (the first representative of the lineage of Nigeremydini for which a neuroanatomical study has been carried out) and the members of other lineages of Bothremydidae (i.e., Cearachelyini, Bothremydini, and Taphrosphyni), shared with marine non-pleurodiran turtles, could be identified as ecological convergences for the adaptation of the representatives of Nigeremydini to pelagic environments. Thus, the shared combination of several neuroanatomical characters in Azzabaremys moragjonesi exclusively with sea turtles (both stem Testudines and members of the crown group, belonging to Cryptodira) (i.e., the absence of ossification in the anterior region of the canalis cavernosus, the lack of the vidian nerve canal, the presence of relatively wide semicircular canals of the endosseous labyrinth, and the expanded foramen interorbital and the reduced processus inferior parietalis for the potential possession of salt glands), allows the identification of this species of Pleurodira as a form well adapted to marine environments.

Bothremydidae is a very diverse lineage of pleurodiran turtles. Most of its representatives were forms adapted to freshwater ecosystems. By contrast, the members of Nigeremydini have been interpreted as putative marine coastal or pelagic forms, based on indirect evidence, although no adaptations to this mode of life were so far justified for this lineage. The three-dimensional reconstruction of the neuroanatomical structures of a member of Nigeremydini is performed here, based on the study of the holotype and only known specimen of Azzabaremys moragjonesi, from the Paleocene of Mali.

As occurs in the other representatives of Podocnemidoidea (i.e., Podocnemidoidae and Bothremydidae), but not in other lineages of Pleurodira, the endocranial cavity of Azzabaremys moragjonesi presents an angle between the forebrain and the hindbrain lesser than 150°, and a dorsally expanded protuberance at the posterior end of the cerebral hemispheres. The angle formed by the carotid canals ranges into the values measured for other bothremydid lineages, being more obtuse than in Cearachelyini and more acute than in Bothremydini and Taphrosphyini. Nevertheless, the skull of Azzabaremys moragjonesi differs from the other representative of Bothremydidae in the presence of a distal canal and two foramina for the glossopharyngeal nerve. In addition, the nasal cavity of Azzabaremys moragjonesi is greatly expanded in contrast with those of all other pleurodiran turtles in which this value had been documented, including the other bothremydids.

Most of the neuroanatomical elements that allow us to differentiate Nigeremydini from the other lineages of Bothremydidae are identified as vinculated to marine adaptations, being convergent with those observed for the different lineages of non-pleurodiran marine turtles. So, the olfactory nerve of all these forms is longer than that in freshwater and terrestrial forms. The semicircular canals of the endosseous labyrinth of Azzabaremys moragjonesi are relatively short and wide as occurs in Dermochelyidae, which are adapted to a pelagic locomotion and to a deep diving behavior, being wider than those of Cheloniidae, more littoral forms as the plesiochelyids or sandowniids, and the freshwater forms of Cryptodira and Pleurodira. The geniculate ganglion is located into the canalis cavernosus in Nigeremydini, as occurs in Cheloniidae, most of freshwater forms of Cryptodira, and the Cretaceous Protostegidae, differing from the condition in the pleurodiran Euraxemydidae, Sahonachelyidae, and Podocnemidoidea (i.e., Podocnemidoidae and the other members of Bothremydidae) , Carettochelyidae, and Plesiochelyidae, in which the ganglion is located in the prootic, between the medial foramen of the facial nerve and the canalis cavernosus. This geniculate ganglion also differs to that in pleurodiran Chelidae and Pelomedusidae, Dermochelyidae, and Sandownidae in which it is located close to the canalis caroticus internus. The vidian nerve of Azzabaremys moragjonesi runs anteriorly out of a bony canal, as occurs in Dermochelyidae and Cheloniidae. This condition differs from that observed in all other pleurodires (including the other lineages of Bothremydidae), all other cryptodiran clades (both freshwater forms and tortoises), and in the members of the extinct marine clades Plesiochelyidae and Protostegidae, in which the vidian nerve pierces the pterygoid through the canalis nervi vidiani. The presence of lacrimal glands, observed in extant forms of Chelonioidea, is inferred here for Azzabaremys moragjonesi by the possession of both an enlarged interorbital foramen (as also occurs in Protostegidae and Plesiochelyidae), and a sulcus located in the ventral surface of the orbit (a feature not shared with any of those clades). Therefore, Azzabaremys moragjonesi shows numerous neuroanatomical differences as compared to the other bothremydids, as well as with the other lineages of Pleurodira. Thus, Nigeremydini developed several convergences with non-pleurodiran marine turtles for the adaptation to pelagic environments. Therefore, the first neuroanatomical study of a marine pleurodiran turtle is performed here.

The holotype of Azzabaremys moragjonesi, NHMUK R16370, is housed in the Natural History Museum (London, United Kingdom). The obtained CT scan files and the 3D models of the skull and the neuroanatomical structures of Azzabaremys moragjonesi are available in MorphoSource: https://www.morphosource.org/concern/media/000677617 (November 4, 2024). Additional media related 3D meshes for neuroanatomical and skull reconstructions of both specimens are also available there (November 7, 2024).

Conceptualization M. Martín-Jiménez (MMJ), A. Pérez-García (APG); Data Curation MMJ; Formal Analysis MMJ; Funding Acquisition APG; Investigation MMJ, APG; Methodology MMJ; Project Administration APG; Resources APG; Supervision APG; Visualization MMJ; Writing – Original Draft Preparation MMJ, APG; Writing – Review & Editing MMJ, APG.

The authors would like to thank Mike Day and Sandra Chapman (NHMUK) for access to the specimen studied here, Brett Clark (NHMUK) for his job scanning it, Andrea Guerrero (UNED) for photographs of the specimen, and Alejandro Serrano (ICP) for the technical assistance. The authors would also like to thank the editor J. Müller and the reviewer S. Evers for their comments and suggestions for the improvement of this manuscript.