Journal of Vertebrate Paleontology Three-Dimensional Pelvis and Limb

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Three-dimensional pelvis and limb anatomy of the Cenomanian hind- limbed snake Eupodophis descouensi (Squamata, Ophidia) revealed by synchrotron-radiation computed laminography Alexandra Houssayea; Feng Xub; Lukas Helfenb; Vivian De Buffrénila; Tilo Baumbachb; Paul Tafforeauc a UMR 7207 du CNRS, Département Histoire de la Terre, Muséum National d'Histoire Naturelle, Paris, France b Institute for Synchrotron Radiation, Ångströmquelle Karlsruhe, Karlsruhe Institute of Technology, Karlsruhe, Germany c European Synchrotron Radiation Facility, Grenoble Cedex, France

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To cite this Article Houssaye, Alexandra , Xu, Feng , Helfen, Lukas , De Buffrénil, Vivian , Baumbach, Tilo and Tafforeau, Paul(2011) 'Three-dimensional pelvis and limb anatomy of the Cenomanian hind-limbed snake Eupodophis descouensi (Squamata, Ophidia) revealed by synchrotron-radiation computed laminography', Journal of Vertebrate Paleontology, 31: 1, 2 — 7 To link to this Article: DOI: 10.1080/02724634.2011.539650 URL: http://dx.doi.org/10.1080/02724634.2011.539650

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THREE-DIMENSIONAL PELVIS AND LIMB ANATOMY OF THE CENOMANIAN HIND-LIMBED SNAKE EUPODOPHIS DESCOUENSI (SQUAMATA, OPHIDIA) REVEALED BY SYNCHROTRON-RADIATION COMPUTED LAMINOGRAPHY

ALEXANDRA HOUSSAYE,*,1 FENG XU,2 LUKAS HELFEN,2 VIVIAN DE BUFFRENIL,´ 1 TILO BAUMBACH,2 and PAUL TAFFOREAU3 1UMR 7207 du CNRS, Departement´ Histoire de la Terre, Museum´ National d’Histoire Naturelle, 57 rue Cuvier CP 38, 75005 Paris, France, [email protected]; [email protected]; 2Institute for Synchrotron Radiation, Angstr˚ omquelle¨ Karlsruhe, Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany, [email protected]; [email protected]; [email protected]; 3European Synchrotron Radiation Facility, BP220, 6 rue Jules Horowitz, 38043 Grenoble Cedex, France, [email protected]

ABSTRACT—Cretaceous marine hind-limbed snakes are considered to be key fossils for understanding the origin and evolution of snakes. In view of the rarity of such fossils, performing new analyses on described specimens using emerging, cutting- edge techniques should bring important new insights on these forms. We investigated the three-dimensional morphology and inner architecture of the pelvic girdle and hind-limb bones of the type specimen of Eupodophis descouensi Rage and Escuillie,´ 2000, one of the three taxa for which at least one hind-limb is known, using synchrotron-radiation computed laminography (SRCL), a recently developed non-destructive technique that overcomes some of the limitations of synchrotron microtomography for ﬂat, laterally extended objects. This experiment allowed a virtual exhumation of the second, hidden leg of the specimen. The morphology and proportions of the regressed pelvic and hind-limb bones of Eupodophis resemble those of the hind-limbed snakes Pachyrhachis and Haasiophis.AsinHaasiophis, four tarsals are observed in each limb, but there are no traces of metatarsals or phalanges. Moreover, despite the presence of osteosclerosis and pachyostosis in the vertebrae and the ribs of Eupodophis, the inner structure of its limb bones is devoid of these osseous specializations and displays a microanatomical organization similar to that of extant terrestrial lizards. This suggests that limb regression in Eupodophis was not due to a qualitative alteration of growth but, more likely, to a local decrease in growth rate or shortening of growth duration.

INTRODUCTION hind-limbed snakes may (or may not) represent the most primitive snakes, the study of their remains is of great signiﬁcance for

Downloaded At: 21:32 8 February 2011 During the Cenomanian (early Late Cretaceous), the tropi- the question of the origin of snakes. cal carbonate platforms of the northern and southern margins Despite the presence of pelvic girdle remains (lacking contact of the Mediterranean Tethys hosted the radiation of the marine with the vertebral column) and (reduced) femora in most extant hind-limbed snakes (Bardet et al., 2008). These ophidians have snake families (Rage and Escuillie,´ 2003), snakes are generally an elongated (0.5–1.5 m long), laterally compressed body, and considered to be limbless squamates. The presence of reduced (where known) a short, strongly laterally compressed tail. They but fully developed hind-limb bones in Eupodophis offers an op- all display pachyostotic vertebrae and ribs. But, above all, they portunity to further document the mechanisms responsible for are characterized by the possession of short, regressed hind-limb limb regression within Ophidia. bones of plesiomorphic morphology (Bardet et al., 2008). Among The aim of the present study was to document the three- hind-limbed snakes, only three specimens attributed to differ- dimensional (3D) morphology and inner structure of the pelvic ent genera (Eupodophis Rage and Escuillie,´ 2000, Haasiophis girdle and hind-limb bones of the type specimen of Eupodophis Tchernov et al., 2000, and Pachyrhachis Haas, 1979) have hind- descouensi, in order to better understand their mode of regres- limb elements preserved. The other genera (Mesophis Bolkay, sion. 1925, Pachyophis Nopcsa, 1923, and Simoliophis Sauvage, 1880) Institutional Abbreviations—ESRF, European Synchrotron are represented by specimens with no preserved (or exposed) Radiation Facility, Grenoble, France; KIT, Karlsruhe Institute limbs. They are nevertheless considered as possible hind- of Technology, Karlsruhe, Germany; WDC, Wyoming Dinosaur limbed snakes based on other morphological features (Rage and Center, Thermopolis, Wyoming, U.S.A. Escuillie,´ 2003). Anatomical Abbreviations—A, astragalus; Ca, calcaneum; dt, There is currently no consensus about the phylogenetic status distal tarsal; F, femur; Fi, ﬁbula; Il, ilium; Is, ischium; Pu, pubis; and position of hind-limbed snakes (Rage and Escuillie,´ 2003). T, tibia. Whereas some authors consider them to be the sister group (if monophyletic) or stem group (if not) of all other snakes (e.g., Lee and Caldwell, 1998; Lee et al., 1999; Caldwell, 2000; Lee et al., 2007; Lee, 2009), others place them within macrostom- MATERIALS AND METHODS atan snakes, a derived group of snakes (e.g., Zaher and Riep- Materials pel, 1999; Rieppel and Zaher, 2000; Zaher et al., 2009). Because The material investigated is the type specimen of Eupodophis descouensi Rage and Escuillie,´ 2000, discovered in the Ceno- *Corresponding author. manian of Al Nammoura, Lebanon, and recorded under the

2 HOUSSAYE ET AL.—EUPODOPHIS PELVIS AND LIMB ANATOMY 3

FIGURE 1. Eupodophis descouensi. A–B, WDC-C-L-N-0030A. A, ventral half of the specimen. The white rectangle indicates the position of the hidden limb. Scale bar equals 2 cm. B, detail of the visible (right) hind limb. Scale bar equals 1 mm. C–D, WDC-C-L-N-0030B. C, ischium. Scale bar equals 500 µm. D, visible (right) hind limb. Scale bar equals 1 mm.

catalog numbers WDC-C-L-N-0030A and WDC-C-L-N-0030B. also to some missing data. SRCL is thus characterized by spe- It consists of two calcareous plates (part and counterpart) on cific artifacts (loss of contrast and of resolution in the direction of which the two (ventral and dorsal) halves of the fossil specimen the rotation axis), but these nevertheless are less problematic for are preserved (Fig. 1). Whereas the right leg and associated is- data processing than those resulting from tomography on such ex- chium are exposed in this individual, the left half of the pelvic tended objects. As for microtomography, the use of a synchrotron girdle and the left hind-limb are not. source for laminography instead of a conventional one leads to In order to compare the inner structure of Eupodophis limb far better results, allowing higher spatial resolution, higher sig- bones with that of four-legged extant squamates, traditional X- nal-to-noise ratio, elimination of artifacts due to polychromatic- ray radiographs of various terrestrial lizards (Heloderma suspec- ity, and the possibility of using propagation-based phase contrast tum, Iguana iguana, Timon lepidus, Lacerta viridis, Tupinambis (Cloetens et al., 1996; Tafforeau et al., 2006; Helfen et al., 2009). teguixin, and numerous species of Varanus) have been examined. Experiments were performed at the ESRF on beamline ID19. Downloaded At: 21:32 8 February 2011 We used a double-crystal monochromator in Bragg geometry us- Methods ing a Si 111 reflection to obtain a monochromatic X-ray beam at an energy of 60 keV. The detector was a FReLoN (fast read- The uniqueness of the Eupodophis type specimen completely out low noise) charge-coupled device camera (Labiche et al., excluded the possibility of invasive investigations. The experi- 2007) imaging a scintillating Gadox powder screen via a magni- ment was carried out with the purpose of non-destructively imag- fying light–optical lens system and yielding an effective isotropic ing the three-dimensional outer and inner structures of particular pixel size of 30.3 µm. The sample-detector distance was set to 1 regions of interest. The shape and the X-ray absorption contrast m, leading to a weak edge enhancement due to phase contrast of the specimen being poorly suited for a conventional microto- (Helfen et al., 2009) in order to obtain a better visibility of speci- mographic investigation, we performed the experiment using the men structures (Tafforeau et al., 2006). The axis inclination angle ESRF synchrotron. Using a third generation synchrotron source ◦ was set to 30 with respect to the beam normal. For each scan, (see Kunz, 2001) instead of a conventional laboratory one to im- ◦ we acquired 900 projections over 360 specimen rotation with 0.7 age fossil specimens allows the collection of much better qual- s of exposure time. Data were reconstructed using a specific al- ity data (Tafforeau et al., 2006). In the present case, however, gorithm developed at KIT for laminographic reconstruction. Its microtomography was not suitable due to the shape of the speci- principles are explained in more detail in Helfen et al. (2006). men, composed of two broad and relatively thin calcareous plates Image segmentation and visualization were performed using the (ca. 30 × 30 × 3 cm), because such objects engender high het- software Amira, version 4.1.1. erogeneity in beam transmission during object rotation in a clas- sic computed tomography (CT) experiment, leading to complete absorption of the beam when the latter crosses the long axes of RESULTS the plate. In order to overcome that limitation and to achieve Pelvic Girdle data of sufficient quality for image segmentation, we employed synchrotron-radiation computed laminography (SRCL) (Helfen Three pelvic bones were successfully segmented on the left et al., 2005). For SRCL, the rotation axis is inclined at a known side of the specimen (Fig. 2A). As in the two other hind-limbed angle (typically between 15◦ and 45◦), instead of being perpendic- snakes, the bones are much reduced and nearly featureless. They ular to the incoming beam, as in tomography. As a consequence, were identified by comparisons with the general shape of the the beam is never transmitted in the directions where the speci- pelvic girdles of the hind-limbed snakes Pachyrhachis (Lee and men is extended. Such a geometry leads to a rather homogeneous Caldwell, 1998) and Haasiophis (Rieppel et al., 2003). Because integral absorption by the sample during its entire rotation, but these bones are disarticulated, alternative identifications are not 4 JOURNAL OF VERTEBRATE PALEONTOLOGY, VOL. 31, NO. 1, 2011

FIGURE 2. Eupodophis descouensi,WDC- C-L-N-0030B. A, 3D reconstruction of the left half of the pelvis in lateral view, with femur, as preserved; B, 3D reconstruction of the left half of the pelvis in lateral view, with femur ren- dered transparent to show supposed original positions of pelvic elements. Scale bar equals 500 µm.

impossible. The absence of sutures between these elements im- fibula. The astragalus is much bigger than the calcaneum, as in plies that the pelvic bones in Eupodophis were very loosely at- ‘aigialosaurs’ (Caldwell et al., 1995). The two bones are neither tached to each other, which could explain the displacement of sutured nor fused together (whereas they are coossified in some these bones from their original position (as reconstructed in Fig. ‘aigialosaurs’; Polcyn et al., 1999; Smith and Buchy, 2008), which 2B). might represent paedomorphosis. The two other bones are iden- The curved morphology of the ilium resembles that of Haasio- tified as the distal tarsals III (small) and IV (larger), because the phis. As it seems to narrow medially, the ilium is distinguished presence of only these two tarsals is a common feature of the from that of Pachyrhachis, which displays an expanded medial Pythonomorpha (Caldwell et al., 1995; Caldwell, 2006; Smith and end. There is some indication of two articular surfaces, for the is- Buchy, 2008). There is no trace of other distal tarsals and, con- chium and the pubis, on the ventral end of the ilium. However, trary to what is observed in Haasiophis (Rieppel et al., 2003) and because of a probable displacement of this bone, there is no in- suggested in Pachyrhachis (Lee and Caldwell, 1998), there are formation about a possible sacral contact. no traces of metatarsal bones. No phalanges could be observed The ischium is very similar to that of Pachyrhachis in its shape either. and proportions with respect to the other bones (about half the length of the ilium). Its middle region is subcircular in cross- section, whereas its extremities are more expanded and flattened. DISCUSSION A fossa on the expanded (proximal) portion of the ischium might A Remark about Limb Regression

Downloaded At: 21:32 8 February 2011 be part of the acetabulum. The pubis is rod-like and displays slightly expanded ends, of Limb reduction, a process characterized by a decrease in the which the larger is probably the medial. The morphology and size of the limb elements, poor development of osseous struc- the relative size of this bone are roughly consistent with those of tures (engendering featureless bones), and/or complete loss of the very poorly known pubes of Pachyrhachis (Lee and Caldwell, 1998) and Haasiophis (Rieppel et al., 2003).

Limb Anatomy The femur is straighter and more slender than that of other aquatic pythonomorphs, with moderately expanded ends. Con- trary to what is observed in Pachyrhachis (Lee and Caldwell, 1998), the posterior margin is nearly straight and the anterior one is concave. The adductor crest seems to be absent, as in Pachyrhachis. The distal margin seems to display distinct articular surfaces for the tibia and the fibula. Tibia and fibula are similar in length, as in Pachyrhachis,al- though the tibia is stouter as is common in reptiles (Romer, 1956). Whereas the anterior margin of the tibia appears straight, the posterior margin is concave. Conversely, the posterior margin of the fibula, which appears particularly bowed, is markedly con- vex and the anterior one concave. The spatium interosseum is spindle-shaped. SRCL revealed the second (right) leg of Eupodophis. In each limb, four tarsal bones are observable and display the same spatial organization (Fig. 1B, D; Fig. 3). They look subcircular in shape and quite featureless. On the left limb, the astragalus and FIGURE 3. Eupodophis descouensi, WDC-C-L-N-0030B. 2D section of the calcaneum are in contact with, respectively, the tibia and the the hidden (left) leg. Scale bar equals 500 µm. HOUSSAYE ET AL.—EUPODOPHIS PELVIS AND LIMB ANATOMY 5

FIGURE 4. Eupodophis descouensi,WDC-C- L-N-0030B. 3D reconstruction of the inner cav- ities of the femur, the tibia, and the ﬁbula of the hidden leg. Scale bar equals 500 µm.

some bones (Jerez and Tarazona, 2009), is encountered in nu- According to Caldwell (2003), a presacral vertebral number merous squamate taxa (Greer, 1991). There appears to be a cor- higher than 90, as occurs in Eupodophis, would be associated relation between decrease in relative limb length, digit loss, and with complete loss of the forelimbs and severe reduction of the body elongation (Raynaud, 1989; Greer, 1991; Cohn, 2001; Wiens hind limbs. Body elongation increases the efficiency of an undu- and Slingluff, 2001; Brandley et al., 2008), which probably reflects latory mode of propulsion. This mode of locomotion, for which an influence of shared functional and developmental constraints limbs can be an obstacle to efficiency, is considered as particu- on the evolution of body form in the adaptation to lateral undu- larly adapted to burrows or dense grass (Gans, 1975), but it also lation (Gans, 1975; Brandley et al., 2008). facilitates anguilliform swimming. Correlation of snake-like body The transition from lizard-like to snake-like body form is more form and a burrowing lifestyle has been shown by different stud- or less gradual (Wiens and Slingluff, 2001), and so does not in- ies (Gans, 1975; Rieppel, 1988; Caputo et al., 1995; Lee, 1998) volve sudden anatomical transformations, as shown by the nu- but was not significant in anguids (Wiens and Slingluff, 2001). merous species with intermediate degrees of limb loss and body Loss of limbs also reduces drag and hence the energetic cost of elongation (e.g., in scincids and gymnophthalmids). However, swimming (Bejder and Hall, 2002). Such a developmental pro- though the correlation between body elongation and limb loss cess would have thus increased performance in both burrowers

Downloaded At: 21:32 8 February 2011 is well established, no common developmental mechanism links and axial undulatory swimmers. these specializations (Sanger and Gibson-Brown, 2004). Limb reduction seems to be caused by Hox genes. They act through the Inner Structure of the Limb Bones loss of axial regionalisation via homeotic changes of genes in- volved in the thoracic or sacral regions. This mechanism can no- Comparison of the reconstructed microanatomical architec- tably explain why forelimb loss is not common in non-ophidian ture of Eupodophis limb bones with X-ray radiographs of the taxa that retain cervical vertebrae (Sanger and Gibson-Brown, same bones from extant four-legged lizard species reveals that 2004). It must be pointed out that it is suggested that differ- the femur, the tibia, and the ﬁbula of Eupodophis (Fig. 4) have ent developmental mechanisms might be responsible for forelimb the same microanatomical organization as those of extant terres- loss and hind-limb reduction in squamates (Sanger and Gibson- trial lizards. In particular, the well-developed medullary cavity Brown, 2004). As for loss of digits, it appears as a developmental and metaphyseal trabecular system of Eupodophis are indicative by-product of limb reduction. A reduction in the number of cells of a qualitatively normal pattern for the growth in length of the in the developing limb bud might engender this phenomenon bones (endochondral growth with no inhibition of bone resorp- (Raynaud, 1985; Wiens and Slingluff, 2001). It was previously tion). This suggests that limb regression in this taxon was not noted that a consistent relationship between reduction in abso- due to a qualitative alteration of growth but, more likely, to a lute limb size and loss of digits is likely to occur in anguids (Wiens global decrease in limb bones growth rate or to an ontogenetic and Slingluff, 2001), but the absence of distal elements in the shortening of local growth duration. Testing the latter hypothe- forelimb of only one specimen of the ophidiomorph Adriosaurus, ses would require the study of cyclical growth marks in virtual which is not the smallest specimen assigned to that genus (Palci sections of bone diaphyses (cf. Castanet, 1985). Further develop- and Caldwell, 2007), and the absence of metatarsal bones in ment of SRCL in a near future may allow such kind of investi- femora of Eupodophis that are of similar size to those of Haa- gations at a sub-micron scale in phase-contrast mode, which is siophis, seems to invalidate this relationship. Conversely, body already possible using phase contrast microtomography on teeth elongation results from heterochronic changes in the segmenta- (Tafforeau et al., 2006; Tafforeau and Smith, 2008). tion clock (Pourquie, 2001, 2003), i.e., from molecular mecha- Eupodophis characteristically displays osteosclerosis (increase nisms acting on the timing (rate or duration) of somitogenesis in in bone inner compactness) all along its axial skeleton (A.H., vertebrates, which increases either the total number of vertebrae pers. observ.) and pachyostosis (increase in cortical bone de- or their length, because somites are the precursors of vertebrae posits) in most of its presacral vertebrae and ribs (Rage and (Jouve et al., 2000). Escuillie,´ 2000). However, this study reveals that such osseous 6 JOURNAL OF VERTEBRATE PALEONTOLOGY, VOL. 31, NO. 1, 2011

specializations did not occur in the limb bones of Eupodophis the interpretation of the data, and to F. Goussard and R. Boistel (whereas the latter bones are affected in several ‘pachyostotic’ (MNHN) for their help during the experiments. We also thank taxa such as Claudiosaurus [Buffrenil´ and Mazin, 1989] and some the anonymous reviewers for useful comments that improved the Basilosauridae [Uhen, 2004] and Choristodera [Buffrenil´ et al., manuscript and S. Modesto for editorial work. 1990]). In aquatic squamates, pachyostosis is always found in association with osteosclerosis (Buffrenil´ et al., 2008; Houssaye LITERATURE CITED et al., 2008), whereas the reverse is not true (Houssaye, 2008). It Bardet, N., A. Houssaye, X. Pereda Suberbiola, and J.-C. Rage. 2008. has already been remarked that these two specializations are gen- The Cenomanian-Turonian (Late Cretaceous) radiation of marine erally restricted to particular bones or skeletal regions (Domning squamates (Reptilia): the role of the Mediterranean Tethys. Bulletin and Buffrenil,´ 1991), but the ultimate causes of these two special- de la Societ´ eG´ eologique´ de France 179:605–622. izations and the reasons for the diversity of their intra-skeletal Bejder, L., and B. K. Hall. 2002. Limbs in whales and limblessness in other localizations remain unclear. This study clearly shows that the vertebrates: mechanisms of evolutionary and developmental trans- limbs, like (most probably) the skull, were unaffected by these formation and loss. Evolution and Development 4:445–458. osteological specializations. It thus supports the hypothesis of Bolkay, S. J. 1925. Mesophis nopcsai n.g. n.sp. ein neues, schlangenahn-¨ a controlled—rather than diffuse—determinism for pachyostosis liches reptil aus der unteren Kreide (Neocom) von Bilek-Selista and osteosclerosis, probably relying on local factors, such as the (Ost-Hercegovina). Glasnik zemaljskog Muzeja u Bosni i Herce- govini 37:125–135. local differentiation, development, and activation of osteoblasts Brandley, M. C., J. P. Huelsenbeck, and J. J. Wiens. 2008. Rates and and osteoclasts. patterns in the evolution of snake-like body form in squamate rep- It is worth pointing out that secondary ossification centers can tiles: evidence for repeated re-evolution of lost digits and long- be observed in neither the proximal nor the distal epiphyses of term persistence of intermediate body forms. Evolution 62:2042– the long bones in Eupodophis. On the contrary, the trabecular 2064. network seems continuous from the metaphyses up to the epiphy- Buffrenil,´ V. de, and J. M. Mazin. 1989. Bone histology of Claudiosaurus seal surfaces. Secondary ossification centers occur in the appen- germaini (Reptilia, Claudiosauridae) and the problem of pachyosto- dicular bones of all limbed lepidosaurians. Conversely such ossi- sis in aquatic tetrapods. Historical Biology 2:311–322. fication centers are absent in the vertebral centra of both lizards Buffrenil,´ V. de, I. Ineich, and W. Bohme.¨ 2004. Comparative data on epiphyseal development in the family Varanidae. Journal of Her- and snakes (Haines, 1942, 1969). petology 37:328–335. The absence of secondary centres in the limb bones of Eu- Buffrenil,´ V. de, N. Bardet, X. Pereda Suberbiola, and B. Bouya. 2008. podophis might therefore either indicate (1) that such centers Specialization of bone structure in Pachyvaranus crassispondylus were absent in this taxon or (2) that they occurred, as in other Arambourg, 1952, an aquatic squamate from the Late Cretaceous lepidosaurs possessing limbs, but tended to fuse completely with of the southern Tethyan margin. Lethaia 41:59–69. the metaphyseal region by the end of growth. The latter process Buffrenil,´ V. de, A. de Ricqles,` D. Sigogneau-Russell, and E. Buffe- is common in small- to medium-sized lizard taxa (see Buffrenil´ taut. 1990. L’histologie osseuse des champsosaurides:´ donnees´ de- et al., 2004, for varanid lizards). Such fusion, in a normally devel- scriptives et interpretation´ fonctionnelle. Annales de Paleontologie´ oped limb bone, signifies that somatic growth stops, at least lo- 76:255–275. Caldwell, M. W. 2000. On the phylogenetic relationships of Pachyrhachis cally. If secondary ossification centers actually existed in the limb within snakes: a response to Zaher (1998). Journal of Vertebrate bones of Eupodophis, it could be possible that an early fusion of Paleontology 20:187–190. these centers with the metaphyses contributed, in synergy with a Caldwell, M. W. 2003. “Without a leg to stand on”: on the evolution global decrease in limb growth rate, to limb regression. and development of axial elongation and limblessness in tetrapods. Canadian Journal of Earth Sciences 40:573–588. Caldwell, M. W. 2006. A new species of Pontosaurus (Squamata,

Downloaded At: 21:32 8 February 2011 CONCLUSION Pythonomorpha) from the Upper Cretaceous of Lebanon and a phy- The use of synchrotron-radiation computed laminography logenetic analysis of Pythonomorpha. Memorie della SocietaItal-` (SRCL) enabled us to obtain three-dimensional data on the mor- iana di Scienze Naturali e del Museo Civico di Storia Naturale di phology of the regressed pelvic girdle and hind-limb bones of Eu- Milano 34(3):1–39. podophis descouensi, which were then compared with those of Caldwell, M. W., R. L. Carroll, and H. Kaiser. 1995. The pectoral girdle the hind-limbed snakes Pachyrhachis and Haasiophis.Theinter- and forelimb of Carsosaurus marchesetti (Aigialosauridae), with a nal structure of the limb elements was also revealed. It notably preliminary phylogenetic analysis of mosasauroids and varanoids. showed two interesting features of these bones under regression: Journal of Vertebrate Paleontology 15:516–531. Caputo, V., B. Lanza, and R. Palmieri. 1995. Body elongation and limb a microanatomical organization similar to that of extant lizards reduction in the genus Chalcides Laurenti 1768 (Squamata: Scinci- and the absence or complete fusion of secondary ossification cen- dae): a comparative study. Tropical Zoology 8:95–152. ters. Moreover, it also revealed the absence of osteosclerosis in Castanet, J. 1985. La squelettochronologie chez les Reptiles I. Resultats´ this part of the skeleton, which probably bears a functional signifi- experimentaux´ sur la signification des marques de croissance cance. The method used in this study could be used to investigate squelettiques chez les lezards´ et les tortues. Annales des Sciences the occurrence of limb bones in other fossil snakes assumed to Naturelles, Zoologie et Biologie Animale 7:23–40. have retained partial hind limbs. More generally, it demonstrates Cloetens, P., R. Barrett, J. Baruchel, J.-P. Guigay, and M. Schlenker. the potential of SRCL for detailed non-destructive studies of fos- 1996. Phase objects in synchrotron radiation hard X-ray imaging. sil specimens preserved on large flat slabs. This is all the more Journal of Physics D: Applied Physics 29:133–146. Cohn, M. J. 2001. Developmental mechanisms of vertebrate limb evolu- noteworthy and promising that this kind of preservation is fairly tion; pp. 47–62 in C. Wiley (ed.), The Molecular Basis of Skeletoge- common in the fossil record. nesis. Novartis Foundation Symposium 232, London. Domning, D. P., and V. de Buffrenil.´ 1991. Hydrostasis in the Sirenia: ACKNOWLEDGMENTS quantitative data and functional interpretations. Marine Mammal Science 7:331–368. We are very grateful to F. Escuillie´ for authorizing the loan Gans, C. 1975. Tetrapod limblessness: evolution and functional corollar- of the specimen, and to the ESRF for providing beamtime and ies. American Zoologist 15:455–467. support in the framework of the proposal EC-193. We would like Greer, A. E. 1991. Limb reduction in squamates: identification of the to thank M. Geze and J. Jovet for providing and accommodat- lineages and discussion of the trends. Journal of Herpetology ing the use of Amira software at CEMIM (Museum´ National 25:166–173. d’Histoire Naturelle [MNHN], Paris, France). We are also par- Haines, R. W. 1942. The evolution of epiphyses and of endochondral ticularly thankful to J.-C. Rage (MNHN) for useful comments on bone. Biological Reviews 17:267–292. HOUSSAYE ET AL.—EUPODOPHIS PELVIS AND LIMB ANATOMY 7

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