Skull of Dinilysia patagonica , MLP 79‐II‐27‐1. Scale bar equals 10 mm. ( A ) Dorsal view, ( B ) ventral view, ( C ) right lateral view and (D) left lateral view. Abbreviations: aa, anterior ampulla; av, aortic vessels; bo, basioccipital; bot, basioccipital tubers; cbl, cerebellum; ie, inner ear; lic, left inner carotid; oc, occipital condyle; p, parietal; pb, posterior brain; pg, pituitary gland; pr, prootic; pt, pterigoid; q, quadrate; ric, right inner carotid; so, supraoccipital; V, trigeminal nerves; VI, abducens nerves; vc, V f, foramen of the trigeminal nerves; VII f, foramen of the facial nerves, vidian canal; vv, vascular vessels.

In this paper, a natural cranial endocast of an extinct snake is described for first time. The material corresponds to a fragmentary skull of Dinilysia patagonica (MLP 79‐II‐27‐1) previously reported by Caldwell and Albino ( 2002 ) and Albino ( 2007 ). This skull includes an endocast preserved by sedimentary filling of the cranial cavity in which the posterior brain, the vessels, the cranial nerves, the inner ear and the semicircular canals are preserved (Figs. 1 and 2 ). Both the osteology and the soft tissue cast of this unpublished skull are also described herein. The results of this study are compared with the conclusions published by Yi ( 2013, 2015 ) and Yi and Norell ( 2015 ) in order to test the hypothesis about the burrowing origin of modern snakes.

Dinilysia patagonica is an Upper Cretaceous snake from Argentina, known for exceptionally well preserved cranial and postcranial remains recovered in northwest Patagonia (Smith‐Woodward, 1901 ; Estes et al., 1970 ; Hecht, 1982 ; Rage and Albino, 1989 ; Albino and Caldwell, 2003 ; Albino, 2007 ; Scanferla and Canale, 2007 ; Caldwell and Calvo, 2008 ; Filippi and Garrido, 2012 ; Zaher and Scanferla, 2012 ; Triviño and Albino, 2015 ). Taking into account the osteological anatomy, Dinilysia patagonica is considered basal in most ophidian cladograms (Caldwell, 1999 ; Rieppel and Zaher, 2000 ; Scanlon and Lee, 2000 ; Tchernov et al., 2000 ; Lee and Scanlon; 2002 ; Zaher and Scanferla, 2012 ; Hsiang et al., 2015 ); thus, relevant neuroanatomical data of this species could add substantial information about the early evolution of snakes.

Although for a long time Palaeoneurology has been based exclusively on natural or artificial endocasts (Jerison, 1973 ; Edinger, 1975 ; Hopson, 1979 ), the use of high resolution X‐ray computed tomography has increased the number of artificial endocranial casts of the nervous system in a wide range of extinct vertebrates, especially mammals and archosaurs (Witmer et al., 2008 and references cited there). Studies on Palaeoneurology in snakes were nonexistent (Hopson, 1979 ), but Yi ( 2013, 2015 ) and Yi and Norell ( 2015 ) recently analyzed some aspects of the inner ear anatomy in a variety of snakes through X‐ray computed tomography which builds three‐dimensional models that are virtual endocasts of the bony inner ear labyrinth. The materials analyzed by these authors included a skull of the extinct snake Dinilysia patagonica (MACN‐RN 1014).

MATERIALS AND METHODS

What exactly does a natural endocranial cast represent? The nervous system of reptiles is tubular, linear in organization, and has some degree of dorsoventral flexure along its length (Wyneken, 2007). The brain cavity is limited by a tubular cranium composed rostrally by the cartilaginous ethmoids, laterally by the bony otic series, ventrally by the basisphenoid and laterosphenoids, and caudally by the occipital series (Wyneken, 2007). The tubular cranium is covered by the supraoccipital, parietal, and frontal bones, and there is a subdural space (below the dura mater) and an epidural space (above the dura mater). An endocast is the sedimentary infill of a cavity that forms a three‐dimensional structure. Therefore, what it is known as a natural cranial endocast is the filling of the intracranial cavity that contains the brain with its cranial nerves, the meninges that cover and protect them, and the blood vessels. These casts may provide approximations of the brain morphology, with the possibility to see details of some superficial structures (Macrini et al., 2006).

In extant reptiles, the volume of the brain does not determine the topographic relationships between brain and skull (Starck, 1979). The size of the brain is determined by the body size whereas the volumes of individual cerebral segments are dependent on the development of sense organs (Starck, 1979). In most reptiles, the brain is smaller than the intracranial cavity. Starck (1979) reminds us that these relationships not only show group specific differences, but also ontogenetic and possibly sexual ones. Wyneken (2007) suggests that the nervous system of reptiles is relatively simple in anatomical structure yet allows greater functional diversity in species‐specific behaviors and adaptation to diverse niches.

According to Hopson (1979), the reptile brain does not fill the brain cavity and the extra neural elements occupy part of the intracranial space. This would means that the endocast shows the place previously occupied by the brain and intracranial space, providing only a superficial overview of the topography of the brain (Jerison, 1969; Hopson, 1979; Norman and Faiers, 1996; Larsson et al, 2000; Wyneken, 2007). Although several groups of reptiles present mostly ossified braincases, the brain still does not completly occupy the cranial cavity; thus endocasts would neither be representative of the brain topography in these cases. In this sense, a well‐developed subdural space is observed in marine turtles, Sphenodon and many lizards. Most Testudines and Crocodylia present a moderate subdural space. However, this space is very narrow in snakes and amphisbaenians (Starck, 1979). Because of this, Wyneken (2007) considers that endocasts of snakes would allow us to hope a good morphological copy of their brains. Recently, Olori (2010) studied a digitized endocast of an uropeltid and enunciated the hypothesis that the relationship between the cranium and the endocraneal cavity is similar to that of mammals, involving the possibility that endocraneal space is completely occupied by the brain. This condition could be a consequence of the high degree of fusion in the bones of the uropelid skull, thus determining a completely closed cranial cavity (Olori, 2010).

For this paper, several skulls of Dipsadidae (Colubroidea) were dissected to test the hypothesis of Olori (2010) in other extant snakes, focusing on the disposition of the brain and subdural space in the cranial cavity. These snakes were collected after they died flattened by cars on the road. The heads of some of these snakes were sectioned in sagital plane and others in longitudinal plane (Table 1), to observe the disposition of brain regions, the cranial space occupying these regions, and the arrangement of the veins and cranial nerves. As Figures 3 and 4 shows, the cranial bones surround the tissue of the nervous system very closely, leaving a very small space between the bone and the nervous tissue for the blood vessels to pass through. Thus, the brain fills around 90% of the endocranial cavity in snakes, leaving a very narrow intracavitary space.

Table 1. Extant dissected individuals for comparative purpose Specie Collection number Provenance Date of collection Colector Dissection mode Erythrolamprus poecilogyrus MLP –R 6462 Ruta provincial 11, La Plata‐ Magdalena 11/14/2015 Jorge Williams Section longitudinal to the main axis of the body Erythrolamprus poecilogyrus MLP –R 6463 Ruta provincial 11, La Plata‐ Magdalena 11/14/2015 Jorge Williams Section parallel to the main axis of the body Thamnodynastes strigatus MLP –R 6464 Ruta provincial 11, La Plata‐ Magdalena 11/14/2015 Jorge Williams Dorsal dissection and left lateral Lygophis anomalus MLP –R 6465 Ruta provincial 11, La Plata‐ Magdalena 11/20/2016 Laura Triviño Bone conservation, observation of cranial nerves Lygophis anomalus MLP –R 6466 Ruta provincial 11, La Plata‐ Magdalena 12/09/2016 Laura Triviño Bone conservation, observation of cranial nerves Philodryas patagoniensis MLP –R 6467 Ruta provincial 11, La Plata‐ Magdalena 11/20/2016 Laura Triviño Section longitudinal to the main axis of the body Philodryas patagoniensis MLP –R 6468 Ruta provincial 11, La Plata‐ Magdalena 11/20/2016 Laura Triviño Dorsal dissection and right lateral Xenodon dorbignyi MLP –R 6469 Ruta provincial 11, La Plata‐ Magdalena 12/09/2016 Laura Triviño Dorsal dissection and right lateral

Figure 3 Open in figure viewer PowerPoint Dissection of skull of Erythrolamprus poecilogyrus. Scale bar equals 10 mm. (A) Dorsal view, (B) left lateral view. Abbreviations: cbl, cerebellum; cer, cerebrum; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe.

Figure 4 Open in figure viewer PowerPoint Dissection of skull of Thamnodynastes strigatus. Scale bar equals 10 mm. (A) Dorsal view, (B) left lateral view. Abbreviations: b: bones; cbl, cerebellum; cer, cerebrum; i ear, inner ear; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe.

Fossil specimens examined. An exceptionally well‐preserved specimen of Dinilysia patagonica (MLP 79‐II‐27‐1) was used for the present study. The material was collected by Santiago Roth at the end of XIX century in Boca del Sapo, Neuquén city, at the confluence of the Neuquén and Limay Rivers, Neuquén province, Argentina. The specimen was recovered from rocks belonging to the Bajo de La Carpa Formation (Santonian), Río Colorado Subgroup, Neuquén Group. It is represented by the posterior portion of a well preserved skull including the corresponding fragment of a natural endocast exposed on the dorsal right side. The specimen was mechanically prepared through the extraction of the following bones to have better endocast exposure: parietal, prootic, a supraoccipital segment, and a portion of the right otooccipital. The extraction of these bones exposed the sedimentary fillings that occupied the place of the soft tissues of the brain, nerves, blood vessels, and inner ear, forming the first natural endocranial cast of a snake.

The described specimen was directly compared with the following specimens of Dinilysia patagonica: MLP 26–410 (holotype), MACN‐PV RN 1013, and MACN‐PV RN 1014.

Living specimens examined. The fossil specimen was also compared with skulls of the extant Boa constrictor occidentalis (UNMdP‐O 44), Salvator merianae (MLP‐R 5969, MLP‐R 6029), Broghammerus reticulatus (MLP‐R 6030), and Bothrops alternatus (MLP‐R 6031).

Moreover, for comparative studies, latex endocasts (CNP‐ME 146, CNP‐ME 147, CNP‐ME 148) were obtained from skulls of the extant snake species Boa constrictor occidentalis (UNMdP‐O 50, UNMdP‐O 47), and Hydrodynastes gigas (UNMdP‐O 54). Table 1 details the dissected skulls of Dipsadidae used in this study to see the relationships among the brain and the braincase.

The material was studied using a stereoscopic microscope and was photographed with digital cameras.

Abbreviations. CNP‐ME: Colección de moldes endocraneanos del Centro Nacional Patagónico (CENPAT), Puerto Madryn, Argentina; MACN‐PV RN: Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Sección Paleontología de Vertebrados, Colección Río Negro, CABA, Argentina; MLP: Museo de La Plata, División Paleontología de Vertebrados, La Plata, Argentina; MLP‐R: Museo de La Plata, sección Zoología de Vertebrados, División Herpetología, Reptiles, La Plata, Argentina; UNMdP‐O: Universidad Nacional de Mar del Plata, Colección Herpetológica, Sección Osteología, Mar del Plata, Argentina.

Systematic Paleontology

REPTILIA Linnaeus, 1758

SQUAMATA Oppel, 1811

SERPENTES Linnaeus, 1758

DINILYSIA Smith‐Woodward, 1901

DINILYSIA PATAGONICA Smith‐Woodward, 1901

Figures 1, 2, 5-9, and Figure 12

Figure 5 Open in figure viewer PowerPoint Blood vessels. Scale bar equals 10 mm. (A) posterior view of the venous vessels, (B) medial view of the venous vessels, (C) arterial vessels, (D) arterial vessels, (E) medial view of the venous vessels, (F) posterior view of the venous vessels. Abbreviations: c, capillaries; cc, common crus; cv, cerebral vein; lic, left inner carotid; lv, longitudinal vein; mcv, median cerebral vein; pg, pituitary gland; sv, venous sinuses; ric, right inner carotid.

Figure 6 Open in figure viewer PowerPoint Natural endocranial cast. Scale bar equals 10 mm. (A) right lateral view, (B) ventral view. Abbreviations: aa, anterior ampulla; cbl, cerebellum; lsc, lateral semicircular canal; pb, posterior brain; pg, pituitary gland; pt, pterigoid; q, quadrate; V, trigeminal nerves; VI, abducens nerves; VII h, hyomandibular facial nerves; VII p, palatine facial nerves; vb, vestibule; vc, vidian canal.

Figure 7 Open in figure viewer PowerPoint Diagram lateral view of natural endocranial cast. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; cbl, cerebellum; pb, posterior brain; pg, pituitary gland; pt, pterigoid; q, quadrate; V, trigeminal nerves; VII h, hyomandibular facial nerves; VII p, palatine facial nerves; vb, vestibule.

Figure 8 Open in figure viewer PowerPoint (A) Ventral view of the middle ear. (B) Inner ear. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; bo, basioccipital; cc, common crus; lsc, lateral semicircular canal; oc, occipital condole; psc, posterior semicircular canal; s, stapes; vb, vestibule.

Figure 9 Open in figure viewer PowerPoint Diagram Inner ear. Scale bar equals 10 mm. Abbreviations: aa, anterior ampulla; asc, anterior semicircular canal; cbl, cerebellum; cc, common crus; lsc, lateral semicircular canal; pb, posterior brain; psc, posterior semicircular canal; q, quadrate; vb, vestibule.

Figure 10 Open in figure viewer PowerPoint Latex endocast, Hydrodynastes gigas. Scale bar equals 10 mm. (A) Dorsal view, (B) ventral view. Abbreviations: cbl, cerebellum; cer, cerebrum; i ear, inner ear; spm, spinal medulla; olb, olfactory bulb; olt, olfactory tract; opl, optic lobe; opt ch, optic chiasma; pit gl, pituitary gland; II, optic nerves; V, trigeminal nerves; X, vagus nerves.

Referred specimen. MLP 79‐II‐27‐1 posterior portion of an articulated skull including the endocast.

Provenance. Boca del Sapo, Neuquén city Neuquén province, Argentina.

Horizon. Neuquén group, Río Colorado Subgroup, Bajo de la Carpa Formation (Santonian, upper Cretaceous).

Description

Braincase and Basicranium

Among elements of the braincase, the parietal, prootic, supraoccipital and otooccipital bones are well preserved on the left side (Figs. 1 and 2). The parietal bone forms the roof as well as the lateral walls of the braincase, and makes up the anterior limit of the trigeminal foramen. Furthermore, the parietal surrounds the dorsal part of the inner ear and posterior brain. On the left wall of the braincase, behind the parietal bone, the prootic forms the posterior margin of the trigeminal foramen. Posterior to the trigeminal foramen there is a small foramen for the facial nerve. Both foramina are separately found, each one having a sole opening where nerves pass through.

In the occipital region, the supraoccipital, otooccipital, and basioccipital bones form the dorsal and posterior portion of the braincase and basicranium. The supraoccipital is located dorsally and posteriorly to the parietal, united by a zig‐zag suture. This bone covers the posterior and dorsal extreme of the myelencephalon. The otooccipitals and the basioccipital constitute the ellipsoidal occipital condyle. A small part of the supraoccipital and otooccipitals forms the foramen magnum. In the otooccipital portion of the spinal canal, near the foramen magnum, there is a pair of foramina; the anterior is the exit of the vagus nerve and the posterior foramen is for the hypoglossal nerve. The metotic foramen opens externally, near the neck of the occipital condyle and over the otooccipitals. The glossopharyngeal, vagus and hyploglossal nerves, as well as the jugular vein, exit the cranial cavity through this foramen.

The basicranium is formed by the basioccipital and basiparasphenoid (Figs. 1 and 2). Ventrally, the basioccipital has a pair of basal tubercles anterior to the occipital condyle, which also appears in other specimens of Dinilysia. The floor of the basiparasphenoid is lost, thus, it is possible to see the elements that run within this bone. The cast shows the pituitary gland surrounded by a groove that corresponds to the Vidian Canal. The internal carotid and the dorsal branch of the facial nerve (palatine ramus or Vidian nerve) pass through the Vidian Canal. A portion of nerve VI and several blood vessels, which would have irrigated the base of the brain, are also recognized.

Natural Endocranial Cast The natural endocast preserved in the specimen MLP 79‐II‐27‐1 exhibits the posterior brain, that is, the hindbrain (cerebellum and medulla oblongata). The forebrain is exposed in ventral view; it is represented by the diencephalon with the ventrally extended pituitary gland. The brain is horizontal, without flexures between regions. The natural endocast also includes sedimentary fillings corresponding to some cranial nerves, the right ear (middle and inner), and the impressions related to venous and arterial craniocerebral circulatory elements (Figs. 1 and 2).

Brain In ventral view, the pituitary gland cast is observed as an expansion of the diencephalon. The position of this gland is posterior, next to the output of the trigeminal nerve (Figs. 1 and 2). Vessels related to the ventral circulatory system of the skull are immediately behind the gland. The Abducens nerve (VI) surrounds the vessels, and external to this nerve are the natural casts of the Vidian Canal system. The internal carotids, emerging from their respective Vidian Canals, enter at the most anterior part of the pituitary gland. The hindbrain is observed in dorsal view; it is formed by two smaller regions, the metencephalon and the myelencephalon. The structures observed in the hindbrain of the endocast are the following: cerebellum, cranial nerves V, VI and VII, and medulla oblongata. The cerebellum and cranial nerves V, VI, and VII are observed forming part of the metencephalon. Next to the inner ear and branching outwards from the lateral wall of this region are the trigeminal nerve (V) and the facial nerve (VII). The root of the trigeminal nerve is bigger than the facial nerve and is located in front of it. The abducens nerve (VI) is noted in the floor of the metencephalon, posterior to the exit of the trigeminal nerve and it runs near the pituitary gland, internal to the Vidian canal. The medulla oblongata is observed in the ventral face of the myelencephalon, where nerves IX, X and XII exit. The cast of the cerebellum is located in the medial region of the parietal, in the area where this bone begins to taper laterally. The cerebellum consists of a simple structure (corpus cerebelli) separated into two hemispheres by an anteroposterior groove where the dorsal longitudinal venous sinus that drains the brain would have run (Figs. 1, 2, and 5). The natural endocast only preserves the corresponding filling of the trigeminal (V), abducens (VI) and facial (VII) nerves. The trigeminal nerve (V) originates in the metencephalon and exits the braincase through a large and single foramen anterior to the inner ear (displayed on the left side) (Figs. 1D and 2). This nerve innervates the muscles of the jaw and the eye regions. The trigeminal ganglion corresponding to this nerve [=gasser (gasserian) or lunate] is located outside the braincase; it is a motor‐sensitive element which is divided into four branches (Figs. 6A and 7). The abducens nerves (VI) exit from the floor of the metencephalon, posterior to the trigeminal nerve (Figs. 2C and 6B) and runs parallel to the pituitary gland in the ventral face of the basicranium. The facial nerves (VII) originate on the lateral wall of the metencephalon, posterior to the root of the trigeminal nerve (Figs. 6A and 7). They have a dorsal branch (hyomandibular branch) that goes lateroventrally to the external edge of the endocast immediately before the inner ear and the ventral branch (palatine branch), which is directed towards the posterior foramen of the Vidian canal and enters the channel. Externally, in the prootic bone, it is possible to see a single foramen that is independent from the trigeminal recess.

Ear An area crossed by the massive columella (stapes) is located ventrolaterally, viewed from the occipital plane (Figs. 2E and 8A). The distal end of this element contacts the quadrate. Its major axis is directed towards the ventral part of the inner ear. The stapes is in contact with the inner ear through the footplate that rests in the oval window. The endocast of the right inner ear is preserved. It is a compact and massive structure located behind the base of the trigeminal nerve. The inner ear is predominantly represented by a large element in the center, the vestibule, which would have contained a large central mass, the statolith, when the animal was alive (Figs. 3, 4, 7, 8B, and 9). This structure is ellipsoid shaped (although in lateral view it is spherical) and is compact in appearance. It is directed towards the midplane of the skull, passing over the medulla oblongata and behind the small lobes of the cerebellum. The vestibule is surrounded by very thin and close semicircular canals: anterior, posterior and lateral. These delicate canals maintain their continuous diameter and are smoothly curved around the vestibule. The lateral canal is long and positioned in a horizontal plane close to the vestibule and the quadrate. Between the lateral semicircular canal and the vestibule there is no bone separates them and the two structures are very close to each other. Also, the lateral canal follows the contour of the vestibule. The anterior and posterior semicircular canals originate from the foremost and hindmost ends of the vestibule, respectively. They are directed vertically and laterally to the median plane of the skull. In dorsal view, these canals form a flattened cone with a hemispherical end at the base, around the vestibule. The anterior and posterior canals form the sides of the cone, whereas the lateral canal produces the contour of the hemisphere. The anterior ampulla is also preserved, connecting the anterior canal and the small fragment of the lateral canal. The ampulla is a compact structure with an anterior prominence; it is preserved at the front end of the vestibule, very close to the exit of the trigeminal nerve. At the junction between the anterior and posterior semicircular canals, a very small common crus is observed. The semicircular canals are connected together forming different angles: the angle is greater than 90° between the anterior and posterior canals, whereas it is close to 90° between the posterior and lateral canals, as well as between the anterior and lateral canals.