A Comparative Analysis of the Postcranial Skeleton of Fossorial And

JOURNAL OF MORPHOLOGY 274:845–858 (2013)

A Comparative Analysis of the Post-Cranial Skeleton of Fossorial and Non-Fossorial Gymnophthalmid Lizards

Juliana G. Roscito* and Miguel T. Rodrigues

Departamento de Zoologia, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brasil

ABSTRACT Squamates are found in a wide range of is characterized by the presence of well-developed habitats and show a corresponding diversity of morpholo- limbs that act as the main propelling elements for gies that can often be correlated with locomotor mode. the body. However, in the snake-like morphotype, The evolution of a snake-like body form, frequently associ- usually seen in burrowing species and characterized ated with fossoriality, from a typical lacertiform morphol- by reduced limbs often in association with an elon- ogy involves changes in the morphology of vertebrae, girdles, and limbs; the changes are mainly manifested by the gated body, locomotion is primarily the result of reduction or loss of limbs and body elongation. In this undulating movements generated by the axial skele- study, we describe the axial and appendicular skeletons of ton (Wiens et al., 2006; Shine and Wall, 2008). six closely related gymnophthalmid species. Three of them Fossoriality and the associated morphological show a lizard-like morphology, with a four-digit forelimb changes for burrowing have evolved independently and a ﬁve-digit hindlimb, and the other three show a several times within squamates (lizards, ‘‘snakes’’ snake-like morphology associated with a burrowing habit, and ‘‘amphisbaenians’’; Wiens et al., 2006; Brand- with reduced limbs and a longer body in comparison to ley et al., 2008), and different degrees of body elon- the former three species. We show that vertebral morphol- gation and limb/girdle reduction can be observed ogy is similar among the six species, with the differences both in distantly-related groups as well as within being accounted for by an increase in the number of vertebrae and by the structural reduction of girdles and limbs the same genus (Lee, 1998; Wiens and Slingluff, in the snake-like species. Skeletal morphology provides 2001; Shapiro, 2002; Skinner et al., 2008). valuable information on locomotion type, physiology, diet, The South American Gymnophthalmidae (Estes and other biological features. The burrowing morphology et al., 1988) is an example of a clade where a usually involves accentuated reduction of girdle and limb snake-like morphology, i.e., an elongated body and elements, reﬂecting an undulating type of locomotion in reduced or absent limbs have evolved independ- which the limbs play little or no role in propelling the ently in a few lineages (Pellegrino et al., 2001; body; in contrast, well-developed limbs and girdles indi- Castoe et al., 2004). One such lineage is the Gym- cate a greater reliance on the limbs for body propulsion. nophthalmini, a monophyletic group comprised of Limb reduction is frequent among vertebrates, but many nine genera, Tretioscincus, Micrablepharus, Gym- different phenotypes are found in species exhibiting some kind of reduction, indicating that different mechanisms nophthalmus, Procellosaurinus, Vanzosaura, Psi- and evolutionary pressures may be involved in generating lophthalmus, Nothobachia, Scriptosaura, and the diverse morphologies. J. Morphol. 274:845–858, Calyptommatus (Pellegrino et al., 2001; Castoe et 2013. Ó 2013 Wiley Periodicals, Inc. al., 2004; Rodrigues and dos Santos, 2008). The

KEY WORDS: Gymnophthalmidae; limb morphology; Additional Supporting Information may be found in the online limb reduction; fossoriality version of this article.

Contract grant sponsor: Fundaçaõ de Amparo a` Pesquisa do INTRODUCTION Estado de Saõ Paulo (FAPESP); Contract grant sponsor: Conselho Nacional de Desenvolvimento Tecnolo´gico (CNPq). The morphology of the axial and appendicular skeletons is quite variable among reptilian saurop- JGR analyzed the data, conceptualized and drafted the manuscript; sids, and variation in form can often be correlated MTR participated in the coordination of the study and revised it with lifestyle (Losos, 1992; Gans and Fusari, 1994; critically giving important intellectual contributions. Losos et al., 1994, 1998; Benesch and Withers, *Correspondence to: Juliana G. Roscito; Departamento de Zoo- 2002). Of especial interest is locomotor mode, which logia, Instituto de Biocieˆncias, Universidade de Saõ Paulo, Rua do is the result of coordinated actions of both the axial Mataõ, Saõ Paulo, SP, Brasil. E-mail: [email protected] and appendicular systems (Renous et al., 1998), and is ultimately related to the morphology of such sys- Received 20 October 2012; Revised 17 December 2012; tems. The association between morphology and loco- Accepted 18 January 2013 motion is easily perceived when comparing lizard- Published online 18 March 2013 in like and snake-like species: the former morphotype, Wiley Online Library (wileyonlinelibrary.com) usually associated with a ground-dwelling habitat, DOI 10.1002/jmor.20139

Ó 2013 WILEY PERIODICALS, INC. 846 J.G. ROSCITO AND M.T. RODRIGUES

Fig. 1. Gymnophthalmini species analyzed in this study. A. Procellosaurinus tetradactylus. B. Vanzosaura rubricauda. C. Psilophthalmus paeminosus. D. Nothobachia ablephara. E. Scrip- tosaura catimbau. F. Calyptommatus nicterus. former six genera have a lizard-like body form MATERIAL AND METHODS with well-developed limbs, although minor limb Our description is based on adult specimens from the scien- reductions can be observed in each genera: in Tre- tific collection of Museu de Zoologia da Universidade de Saõ tioscincus, the two phalanges of the first digit are Paulo (MZUSP). We have analyzed two specimens of Psiloph- reduced in size, in Gymnophthalmus and Micrable- thalmus paeminosus Rodrigues, 1991, six specimens of Procello- pharus the distal-most phalanx from the first digit saurinus tetradactylus Rodrigues, 1991, nine specimens of Van- zosaura rubricauda (Boulenger, 1902), six specimens of Notho- is lost, and in Procellosaurinus, Vanzosaura, and bachia ablephara Rodrigues, 1984, two specimens of Psilophthalmus both phalanges of the first digit Scriptosaura catimbau Rodrigues & dos Santos, 2008, and eight are lost. The latter three genera have a snake-like specimens of Calyptommatus nicterus Rodrigues, 1991; refer- body form, with an elongated body and largely ence numbers and sampling sites for each specimen are sum- marized in the Supporting Information. reduced limbs: Nothobachia has a one-digit fore- Specific measurements of all specimens were taken with a limb and a two-digit hindlimb, and Scriptosaura digital caliper to the nearest 0.01 mm: snout-vent length (SVL), and Calyptommatus have no external forelimbs measured from the back of the occipital bone to the posterior and a one-digit hindlimb. end of the pelvic girdle (at the level of the acetabulum); trunk Despite being always recovered as monophyletic, length between pectoral and pelvic limbs, measured from the anterior end of the pectoral girdle to the posterior end of the relationships between Gymnophthalmini genera pelvic girdle; and tail length, measured, in specimens with are still a matter of debate: the phylogenetic hy- intact tails, from the posterior end of the pelvic girdle to the pothesis proposed by Pellegrino et al. (2001) groups end of the tail. the snake-like genera (Calyptommatus and Notho- All specimens were cleared and double stained for cartilage and bone following the methods from Potthoff (1983); Taylor bachia) together, while that proposed by Castoe and van Dyke (1985); Song and Parenti (1995); Springer and et al. (2004) places Calyptommatus as sister group Johnson (2000), and examined with an Olympus SZX12 stereo- to Psilophthalmus, and both to Nothobachia. Scrip- microscope (Olympus, Tokyo). Photographs were taken using an tosaura was only recently described (Rodrigues and Olympus digital camera (DP72) attached to the scope and sche- dos Santos, 2008) and therefore, has not been matic drawings were made using the digital images as shapes. The anatomical terminology follows Romer (1956), Hoffstetter included in any phylogenetic studies so far. and Gasc (1969), Russel and Bauer (2008), and Jerez and Tara- Here, we present a comparative description of zona (2009). the vertebral and appendicular skeletons of six gymnophthalmini species (Procellosaurinus tetradactylus, Vanzosaura rubricauda, Psilophthalmus RESULTS paeminosus, Nothobachia ablephara, Scriptosaura Vertebral Skeleton catimbau, and Calyptommatus nicterus; Fig. 1), and discuss the morphological adaptations shown The relative proportion of trunk and tail lengths by both lizard-like and snake-like species that may differs in lizard-like and snake-like species. In Psi- be related to each species’ lifestyle. lophthalmus paeminosus, Procellosaurinus tetra-

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 847 between these two morphotypes; therefore, the following description applies to all species and differences, when present, are emphasized. The atlas is the first trunk vertebra. It is composed of a small ventral intercentrum and a pair of neural arches that are not fused dorsally (Fig. 2A). Its centrum is incorporated into the centrum of the axis as the odontoid process (Fig. 2A), and does not contact the atlas’s neural arches; the lateral extremities of the intercentrum contact the antero-ventral surface of the odontoid process posteriorly. The vertebral column connects to the occipital condyle in the skull through the intercentrum and the base of the neural arches of the axis. The axis is the stoutest vertebra in all of the presacral region, with wide neural arches (Fig. 2B) and a well-developed neural spine; a post-zygapophysis, located at the posterior margin of the vertebra, articulates with the anteriorly located pre-zygaphophysis of the subsequent vertebra. The intercentrum is also the largest of the series (Fig. 2B). A transverse process is present but does not bear any ribs. The following presacral vertebra are similar to each other: all are procoelous, with the centrum showing a concave anterior surface and a convex posterior one, and with posteriorly directed neural spines showing a progressive reduction in size the more posterior is the vertebra. A series of ventrally located intercentra are associated with the more anterior vertebrae (Fig. 2B). The small Y-shaped intercentrum of the atlas Fig. 2. Anterior vertebrae of Calyptommatus nicterus. A. is located ventrally to the odontoid process but Atlas and axis in dorsal view; anterior to the right. B. Five not fused to it, while the following intercentra anteriormost vertebrae in lateral view; anterior to the right. Scale bars for A 5 0.5 mm; B 5 1.0 mm. ic, intercentrum; na, are fused to the anteroventral surface of the re- neural arch; ns, neural spine; od.p, odontoid process; p-z, post- spective vertebral centrum and show a progres- zygaphophysis; pr-z, pre-zygaphophysis; rb, rib; tr.p, transverse sive reduction in size, with the most posterior one process; zgs-zgn, zygosphene-zygantrum articulation. being very small. The morphology of the intercentra is similar in all species, but the number of intercentra differs: the lizard-like species show dactylus, and Vanzosaura rubricauda, the tail is seven intercentra while among the snake-like longer than the trunk, and tail size represents 60– ones the number is variable, with eight in N. 65% of the SVL (mean SVL of 2.8 cm, 2.4 cm, and ablephara, six in S. catimbau, and seven to nine 3 cm for the three species, respectively). The tail of in C. nicterus. Nothobachia ablephara and Scriptosaura catimbau The lateral articulation between successive ver- is slightly longer than the trunk, corresponding to tebrae is formed by the paired pre- and post-zyg- approximately 55 and 53% of the SVL (mean SVL apophyses, located at the posterior and anterior of 6.3 cm and 6.2 cm). Calyptommatus nicterus is margins of each neural arch, respectively (Fig. the only species in which the trunk is longer than 2B). The flat surface of the pre-zygapophysis faces the tail, with tail length corresponding to 43% of downward, and that of the post-zygapophysis faces the SVL (mean SVL of 61 cm). upward. The articulation between vertebrae is fur- The number of presacral vertebrae differs ther strengthened by paired zygosphenes, located between lizard-like and snake-like species, being in between and slightly dorsal to the pre-zyg- higher in the latter: N. ablephara, S. catimbau, apophysis, projecting from the anterior margin of and C. nicterus have 42, 40, and 42–48 presacral the neural arch, and fitting in a zygantrum at the vertebrae, respectively, while P. paeminosus, P. tet- posterior margin of the anterior vertebra. radactylus, and V. rubricauda have 30, 29, and 28 Ribs are present from the fourth vertebra and presacral vertebrae, respectively. Despite the dif- articulate with the transverse processes (Fig. 2B). ference in vertebral number, the morphology of Sternal ribs connected to the sternum articulate individual vertebrae does not differ greatly with the distal extremities of the vertebral ribs

Journal of Morphology 848 J.G. ROSCITO AND M.T. RODRIGUES

Fig. 3. Trunk ribs and associated structures. A. Psilophthalmus paeminosus in ventrolateral view; anterior to the right. B. Van- zosaura rubricauda in ventral view; anterior to the right. C. Nothobachia ablephara in ventral view; anterior to the right. D and E. Scriptosaura catimbau in ventral and lateral views, respectively; anterior to the top. Scale bars for A–D 5 1.0 mm; E 5 0.5 mm. is-rb, inscriptional rib; p-xph, post-xiphisternum; p-xph.rd, post-xiphisternal rod; rb, rib; st, sternum; st.rb, sternal rib; xph, xiphisternum; xph-rb, xiphisternal rib.

(Fig. 3A–E); the first sternal rib is associated with those of the other species: the proximal end of each the rib of the eighth vertebra in C. nicterus and S. rib is bifurcated into anterior and posterior rami, catimbau, and with the rib of the ninth vertebra in and the connection to the vertebral ribs is estab- the other species. V. rubricauda and P. te tr ad ac ty - lished by the posterior ramus; the anterior one lus have three pairs of sternal ribs (Fig. 3B); P. pae- ends freely. The medial end of each inscriptional rib minosus (Fig. 3A) and C. nicterus have two, and N. do not fuse to their pair at the midline, but connect, ablephara and S. catimbau have only one pair (Fig. instead, to the lateral end of a horizontal rod of cal- 3C–E). Xiphisternal ribs connected to the xiphister- cified cartilage (Fig. 3D,E). num and also associated with vertebral ribs are The sacral region is invariably composed of two found posterior to the sternum; two pairs of xiphis- sacral vertebrae strongly attached to each other ternal ribs are present in the lizard-like species through a stout neural arch from the anterior ver- (Fig. 3A,B), while C. nicterus and N. ablephara tebra that embraces that from the posterior one, have only one pair (Fig. 3C); no such ribs are pres- and through the stout transverse processes fused ent in S. catimbau (Fig. 3D). The post-xiphister- to each other (Fig. 4A,B); a small proximal fora- num, present in all species analyzed, is formed by men persists between these processes (Fig. 4B). the fusion of pairs of inscriptional ribs at the mid- The first four or five anterior-most caudal verte- line; in the snake-like species the fused inscrip- brae resemble presacral and sacral vertebrae, but tional ribs forms well-developed X-shaped post- the neural spine is shorter and wider anteroposter- xiphisternal plates, but in the lizard-like species iorly; the height of these processes decreases poste- these ribs may or may not be fused. P. tetradactylus riorly. Ventral chevrons are present from the and P. paeminosus bears one or two pairs of inscrip- boundary between the second and third vertebrae; tional ribs, in V. rubricauda the number is variable, all chevrons are Y-shaped, with the paired dorsal and N. ablephara and C. nicterus show seven to rami located in between successive vertebral cen- nine plates (Fig. 3C). The inscriptional ribs of S. tra (not illustrated). The chevrons decrease pro- catimbau differ in morphology when compared to gressively in size posteriorly.

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 849

Fig. 4. Sacral and caudal vertebrae. A. Scriptosaura catimbau in lateral view; anterior to the left. B. Procellosaurinus tetradactylus in dorsal view; anterior to the left. C. Scriptosaura catimbau in dorsal view; anterior to the left. Scale bars for A–B 5 0.5 mm; C 5 1.0 mm. cd.v, caudal vertebra; il, ilium; tr.p, transverse process; sc.v, sacral vertebra.

Autotomic sutures are present from the most an- sternum. The scapulocoracoid has three large fo- terior caudal vertebrae. The first autotomic suture ramina (anterior and posterior coracoid, and the line is seen in the fourth vertebra in C. nicterus scapulocoracoid), and a small coracoid foramen and S. catimbau, and in the fifth vertebra in P. (Fig. 5A); the glenoid fossa, with which the head of paeminosus, P. tetradactylus, N. ablephara, and V. the humerus articulates, is well marked and rubricauda (although in some V. rubricauda speci- located posteriorly. The epicoracoid forms the mens the sutures were present from the sixth ver- medial and anterior margins of the scapulocora- tebra on). The autotomic vertebrae are longer than coid (Fig. 5A). The suprascapula (Fig. 5A) is a other vertebrae and show two pairs of transverse large plate that connects medially to the scapular processes, the anterior one being very short and portion of the scapulocoracoid and projects latero- the posterior one being longer and stouter (Fig. dorsally. The sternum is longer than wide, with a 4C), similar to those of the anteriormost, non- U-shaped anterior margin and a large and central autotomic, caudal vertebrae. The line of suture is heart-shaped foramen (Fig. 5A). The rod-like and located in between these processes and extends for elongated xiphisternum is located posterior to the the entire length of the neural arch. The seg- sternum. mented musculature is organized so as to involve The well-developed forelimb of the lizard-like the posterior portion of one vertebra and the ante- species is formed by the humerus in the first seg- rior portion of the next vertebra adjacent to it, ment of the limb, radius and ulna in the interme- with each muscular package fitting in between diate segment, and carpals, metacarpals, and pha- successive suture lines (data not shown). langes in the distal segment (Fig. 5B). The proximal end of the humerus fits into the glenoid fossa of the scapulocoracoid and the distal end receives Pectoral Girdle and Forelimb the head of the radius and ulna, which articulate All skeletal elements that form the pectoral gir- with the radial and ulnar condyles of the humerus, dle of modern lizards are present in the species an- respectively; the radius is the outer and slender alyzed here, including the snake-like, limb- element and the ulna is more central with respect reduced, burrowers. The clavicle and interclavicle to the limb axis and stouter. are the only dermal elements found in the pectoral The carpal region (Fig. 5C,D) is composed of girdle; the scapulocoracoid is the only endochon- nine small bones distributed in two rows: in the dral element, and the epicoracoid, suprascapula, proximal row, the radiale and ulnare support the sternum, xiphisternum, post-xiphisternum and radius and ulna, respectively, and a small centrale associated ribs are formed by calcified cartilage. is found in between them (Fig. 5C). A pisiform, All limb elements are formed by endochondral located ventral to the ulnare, contacts the ulna, bone. and a large palmar rests ventral to the ulnare, The morphology of the pectoral girdle is similar centrale, and radiale (Fig. 5D). The distal row is in the lizard-like species. The stout and curved formed by the distal carpals (dc) II, III, IV, and V, rod-like clavicle (Fig. 5A), extends horizontally, articulating with digits II to V, respectively (Fig. with its medial end approaching its pair at the 5C,D). Digits are formed by long metacarpals and midline and its lateral end fitting into a recess at phalanges, except for digit I, which is formed by a the anterior margin of the suprascapula. The mid- reduced metacarpal and no phalanges; the phalan- ventral interclavicle is cruciform (Fig. 5A), with its geal formula of the forelimb is 0:3:4:5:3 (Fig. 5B). anterior process fitting in between the clavicles, Both the pectoral girdle and forelimb of snake- the lateral processes superposed to the scapulo- like species (Fig. 6) are greatly reduced, with some coracoids, and the posterior one superposed to the elements being reduced in size, lost, fused, or

Journal of Morphology 850 J.G. ROSCITO AND M.T. RODRIGUES

Fig. 5. Pectoral girdle and forelimb of the lacertiform species, represented by Psilophthalmus paeminosus. A and B. Pectoral girdle (A) and forelimb (B) in ventrolateral views; anterior to the right in both images. C and D. Detail of the carpal region seen in dorsal (C) and ventral (D) views; the drawings are schematic representations of each photograph (anterior to the right). Distal carpals are shaded. Scale bars for A and B 5 1.0 mm; C 5 0.5 mm; D 5 0.25 mm. acf, anterior coracoid foramen; ce, centrale; cf, coracoid foramen; cl, clavicle; epc, epicoracoid; hu, humerus; icl, interclavicle; mc, metacarpal; pa, palmar; pcf, posterior coracoid foramen; psi, pisiform; ra, radius; rd, radiale; sc, scapulocoracoid; scf, scapulocoracoid foramen; spc, suprascapula; st, sternum; st.rb, sternal rib; ul, ulna; un, ulnare. having lost part of their characteristic anatomical (Fig. 6D). The interclavicle of C. nicterus is more features. The degree of reduction is greater in S. reduced than that of the other two snake-like spe- catimbau and C. nicterus, while in N. ablephara cies, having slender but longer lateral processes, a the skeletal elements are more developed relative very short posterior process that does not project to the condition seen in the former species. from the body of the interclavicle, and no anterior The clavicle of N. ablephara is long, stout, and process (Fig. 6G,H). slightly sigmoidal in shape (Fig. 6A,B); its medial The scapulocoracoid of N. ablephara is similar to end is wider and contacts the anterior process of that of the lizard-like species, with distinct foram- the interclavicle, and the distal end is slender and ina (anterior coracoid, posterior coracoid and scap- fits into a small recess in the suprascapula (Fig. ulocoracoid fenestrae, and coracoid foramen) and a 6B). The clavicles of C. nicterus are similar to defined glenoid fossa (Fig. 6A,B). The scapulocora- those of N. ablephara, but are slender and farther coid of S. catimbau and C. nicterus, on the other apart from each other at midline; also, their curva- hand, is very reduced and simplified in morphol- ture is much less accentuated than that of the for- ogy, with no foramina (Fig. 6D,I; a few specimens mer species. The clavicle of S. catimbau is com- of C. nicterus show, however, a slight hook-shaped pletely different from that previously described for recess that resembles an incomplete foramen). The the other species, being arched anteriorly (Fig. 6D) glenoid fossa is recognized by a thickened region with a stouter medial end and a slender distal end at the posterior margin of the scapulocoracoid. The that fits into the recess in the suprascapula. epicoracoid is distinct in all three species at the The interclavicle in N. ablephara is cruciform medial margin of the scapulocoracoid (Fig. 6A). and has a long posterior process, but, differently The suprascapula in these species is a wide plate, from those of the lizard-like species, the anterior being wider in N. ablephara (Fig. 6B,D,H). and lateral processes are short and broad (Fig. The sterna of N. ablephara and C. nicterus are 6A). The interclavicle of S. catimbau is Y-shaped, similar in shape, although the sternum of C. nicte- with lateral and posterior processes of the same rus is somewhat wider than that of N. ablephara length and width; an anterior process is absent (Fig. 6A,G). The anterior margin has a U-shaped

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 851

Fig. 6. Pectoral girdle and forelimb of the serpentiform species Nothobachia ablephara (A–C), Scriptosaura catimbau (D–F), and Calyptommatus nicterus (G–I). A. Pectoral girdle in ventral view; anterior to the left. B. Pectoral girdle in lateral view, with detail to the suprascapula and lateral ends of the scapulocoracoid and clavicle; anterior to the top. C. Forelimb in dorsal view; anterior to the top. D and E. Pectoral girdle in ventrolateral and lateral views, respectively; anterior to the top (C) and to the left (D). F. Detail of the forelimb; anterior to the top. G. Pectoral girdle in ventral view; anterior to the left. H. Pectoral girdle in ventrolateral view, with detail to the suprascapula and lateral ends of the scapulocoracoid and clavicle; anterior to the top. I. Detail of the forelimb; anterior to the top left. Scale bars for A, B, D, F, and H–I 5 0.5 mm; B 5 0.25 mm; E and G 5 1.0 mm. acf, anterior coracoid foramen; cf, coracoid foramen; cl, clavicle; dc, distal carpal; epc, epicoracoid; hu, humerus; icl, interclavicle; mc, metacarpal; pcf, posterior coracoid foramen; ph, phalanges; ra, radius; rd, radiale; sc, scapulocoracoid; scf, scapulocoracoid foramen; spc, suprascapula; st, sternum; ul, ulna; un, ulnare; z.el, zeugopodial element; xph, xiphisternum. depression (that is more evident in N. ablephara) is wider, the central foramen is absent, and one and a central foramen. One pair of sternal ribs pair of sternal ribs connects to the sternum. A connects to the lateral margins of the sternal plate xiphisternum is absent. The post-xiphisternum is in N. ablephara, while two pairs connect to the composed of a series of 10 horizontal bars con- sternum in C. nicterus. A Y-shaped xiphisternum nected to the medial end of the inscriptional ribs is connected to the posterior margin of the ster- (Fig. 3D,E). num and one pair of xiphisternal ribs attach to The reduced forelimb of N. ablephara is formed each of its posterior ends both in N. ablephara and by a single digit (Fig. 6C). The short humerus is in C. nicterus. The post-xiphisternum in both spe- anchored proximally to the glenoid fossa and its cies is formed by a series of X-shaped plates, each distal end has two distinct condyles, the ulnar and united to one pair of inscriptional ribs. radial, that receive the head of the ulna and ra- The morphology of the sternum and post-xiphis- dius, respectively. Both ulna and radius are sup- ternum of S. catimbau differs from that of the ported distally by the carpals ulnare and radiale, other snake-like species (Fig. 6D,E). The sternum respectively. The palmar is located ventrally to

Journal of Morphology 852 J.G. ROSCITO AND M.T. RODRIGUES

Fig. 7. Pelvic girdle and hindlimb of the lacertiform species Procellosaurinus tetradactylus (A–B), Vanzosaura rubricauda (C), and Psilophthalmus paeminosus (D). A. Pelvic girdle in ventral view; anterior to the left. B. Detail of pelvic girdle showing the pectinal process; anterior to the left. C and D. Tarsal region in dorsal (C) and ventral (D) views, respectively; the drawings are schematic representations of each photograph (anterior to the right). Distal tarsals are shaded. Scale bars for A–D 5 0.5 mm. ac, astragalus-calcaneum; is, ischium; dt, distal tarsal; epi, epiischium; hpi, hipoischium; mt, metatarsal; obf, obturator foramen; pb, pubis; pc.p, pectinal process. both ulnare and radiale, and a flattened small cave acetabulum to which the femur is anchored. bone that most likely corresponds to distal carpal The pubes and ischia are joined to their pair at IV is observed distal to both; a metacarpal follows the midline forming a puboischiadic plate (Fig. this element, and on each side of it are two un- 7A,B). A well-developed pectinal process and an identified elements that may correspond to the dis- obturator foramen are present in the pubis; the tal carpals III and V or to the metacarpals III and ischium shows a developed posterior projection V, assuming that the only digit is digit IV. The (Fig. 7A,B). The ilium is wide and contacts distally digit is composed of one phalanx after the metacar- the transverse process of the sacral vertebrae, pal, and the ungual phalanx. establishing a connection between the pelvic girdle Although there is no external forelimb in S. cati- and the vertebral column. mbau and C. nicterus, a vestigial limb is retained The epipubis, epiischium, and hipoischium, next to the pectoral girdle (Fig. 6F,I). In S. cati- formed by calcified cartilage, are located medially mbau, the limb is formed by two minute bones with respect to the puboischiadic plate (Fig. 7A; (Fig. 6F), the proximal one lying next to the region epipubis not shown). The small epipubis is the an- of the glenoid fossa and called here a humerus, terior-most element, located anterior to the pubis, and a single, distal and even smaller one that is and the elongated epiischium and hipoischium are assumed to have originated from the second seg- located anterior and posterior to the ischium, ment of the limb (the zeugopodium); the identity of respectively; the hipoischium, slightly Y-shaped, is this element as either the radius or ulna, or a the longest. fusion of both, was not determined. The forelimb The hindlimb is formed by a long femur in the of C. nicterus is represented only by a short hu- first segment of the limb, and by the tibia and fib- merus (Fig. 6I). ula in the second segment. In the tarsal region (Fig. 7C,D), the large astragalus-calcaneum sup- ports the distal ends of the fibula and tibia. The Pelvic Girdle and Posterior Limb large fourth distal tarsal fits into a ventral concav- Both pelvic girdle and posterior limb (Fig. 7) are ity of the astragalus-calcaneum and contacts the very similar in the three lizard-like species. The metatarsals III, IV, and V. The small third distal pelvic girdle is composed of paired pubes, ischia tarsal is anchored between the fourth distal tarsal and ilia; their extremities meet to form the con- and the metatarsals II and III; in V. rubricauda

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 853

Fig. 8. Pelvic girdle and hindlimb of the serpentiform species Nothobachia ablephara (A–C), Scriptosaura catimbau (D–F), and Calyptommatus nicterus (G–H). A. Pelvic girdle in ventral view; anterior to the top. B and C. Tarsal region in dorsal (B) and ventral (C) views; anterior to the top and to the left, respectively. D–E. Pelvic girdle in ventral (D) and lateral (E) views; anterior to the top and to the left, respectively. F. Detail of the hindlimb; anterior to the top. G. Pelvic girdle in ventral view; anterior to the top. H. Detail of the hindlimb; anterior to the left. The unidentified element next to the metatarsal is marked with a question mark. Scale bars for A, D–E, and H 5 0.5 mm; B, C, and F 5 0.25 mm; G 5 1.0 mm. ac, astragalus-calcaneum; dg, digit; dt, distal tarsal; epi, epiischium; epp, epipubis; fe, femur; fi; fibula; hpi, hipoischium; il, ilium; is, ischium; mt, metatarsal; pb, pubis; pc.p, pectinal process; ph, phalanges; ti, tibia.

and P. paeminosus specimens examined, the third developed in N. ablephara (Fig. 8A), but is absent in distal tarsal is fused to metatarsal III, while in P. S. catimbau and C. nicterus (Fig. 8D,G); the obtura- tetradactylus this element is free. tor foramen is present in the three species. The The metatarsals from digits I to IV are long, ischium and ilium of N. ablephara are longer than slender, and phalanx-like. The fifth metatarsal is those of the other two snake-like species. The halves small and hooked-shaped (Fig. 7D), anchoring to of the puboischiatic plates are not united at midline the astragalus-calcaneum laterally, and articulat- in both S. catimbau and C. nicterus, being much far- ing to the first phalanx of the fifth digit. The pha- ther apart in the former (Fig. 8A,D,G). langeal formula is 2:3:4:5:4. The epipubis, epiischium and hipoischium (similar The pelvic girdle of the snake-like species (Fig. to those described for the lizard-like species) are pres- 8A,D,E,G) is morphologically simpler than that of ent in N. ablephara (Fig. 8A). In C. nicterus and S. the lizard-like ones, and, among the snake-like spe- catimbau only a hipoischium is present, being rod- cies, those of S. catimbau and C. nicterus are more like in C. nicterus, and Y-shaped in S. catimbau,with reduced. The pectinal process of the pubis is well- long anterior and posterior processes (Fig. 8D,G).

Journal of Morphology 854 J.G. ROSCITO AND M.T. RODRIGUES The hindlimb of N. ablephara is composed of two zygantrum articulation (as do some teiids; Veron- digits (Fig. 8B,C), while the hindlimb of C. nicterus ese and Krause, 1997), indicating that this feature and S. catimbau is very short and has only one is not exclusive to burrowing species. digit (Fig. 8E,F,H). The femur, tibia, and fibula are The minor differences observed in the axial skel- morphologically similar in the three species, but eton of the species analyzed, i.e., which rib con- the tibia and fibula are much longer in N. able- nects to the sternum, xiphisternum, and post- phara. xiphisternum (in the fossorial species); the pres- The tarsal region of N. ablephara (Fig. 8B,C) ence or absence of ribs associated with the last comprises the astragalus-calcaneum supporting presacral vertebra; and which caudal vertebra is the tibia and fibula, the flattened fourth distal tar- the first autotomic vertebra; most likely reflect sal fitting into a depression at the distal margin of individual or species-specific variations rather the astragalus-calcaneum, and the reduced third than functional adaptations to any particular type distal tarsal contacting the lateral surface of meta- of locomotion. tarsal III; a small bone (Fig. 8B, shown by a ques- The most significant difference between the tion mark), ventral to the proximal end of metatar- axial skeletons of the lizard-like and snake-like sal IV, may represent the fifth metatarsal, gymnophthalmids is the greater number of presac- although it is not hook-shaped as is that of the liz- ral vertebrae in the latter. The number of presac- ard-like species. In S. catimbau, only a small as- ral vertebrae in squamate reptiles is highly vari- tragalus-calcaneum is observed (Fig. 8F), and in C. able, and can range from 14 up to over 300 (Berg- nicterus the small astragalus-calcaneum is accom- mann and Irschick, 2011). Despite this variation, panied by a greatly reduced bone distal to it (Fig. the estimated modal number of trunk vertebrae 8H), probably representing the fourth distal tarsal. for lizards is 26 vertebrae, which is quite similar The phalangeal formula of N. ablephara is to the ancestral state for squamates of 25 trunk 0:0:2:4:0; the single digit of C. nicterus is formed vertebrae (based on Sphenodon punctatus; Berg- by a metatarsal and two phalanges (Fig. 8H), and mann and Irschick, 2011). The number of presac- the single digit of S. catimbau is formed by a ral vertebrae of the lizard-like gymnophthalmids metatarsal and one phalanx (Fig. 8F; the ungual analyzed here and of other closely-related species phalanx was not observed). of the Gymnophthalminae subgroup is slightly higher than the estimated modal number; however, other gymnophthalmids, such as some spe- DISCUSSION cies from the Cercosaurinae subgroup, and some The evolution of a snake-like body from a lizard- teiids (the sister group to Gymnophthalmidae), like one has occurred repeatedly among squamates have around 25–26 trunk vertebrae (Veronese and (Wiens et al., 2006). With respect to the axial and Krause, 1997, for teiids; Grizante, 2009, for gym- appendicular skeletons, such evolutionary transi- nophthalmids), which is equivalent to the modal/ tion involves modifications in vertebral number, ancestral number estimated by Bergmann and girdle and limb sizes and morphological complexity Irschick (2011). (i.e., loss of characteristic anatomical features of On the other hand, the snake-like species ana- particular bones), and in the number of digits and lyzed, as well as other elongated gymnophthalmids phalanges (Stokely, 1947; Gans, 1975; Greer, such as Bachia and Heterodactylus, have a greater 1991); these modifications are clearly illustrated in number of presacral vertebrae and, consequently, the evolutionary history of some gymnophthalmid a longer body when compared to the lizard-like lineages. species. The relationship between a greater num- The morphology of the vertebrae of the gym- ber of vertebrae and a longer body has been dem- nophthalmids analyzed here is quite similar, de- onstrated previously (Camp, 1923; Stokely, 1947; spite the differences both in trunk length and in Greer, 1987; Wiens and Slingluff, 2001; Bergmann the relative role of the axial skeleton for locomo- and Irschick, 2011). tion in lizard-like species in contrast to the snake- Previous works (Wiens and Slingluff, 2001; like ones that rely on undulating movements of Wiens et al., 2006) have documented two patterns the trunk. The presence of the zygosphene-zygan- of body elongation in snake-like reptiles, charac- trum articulation between successive vertebrae terizing two distinct ecomorphs: one in which elon- strengthens the column and restricts vertical flex- gation occurs via tail lengthening, and another in ion (although a limited degree of body torsion is which elongation occurs via trunk lengthening. possible in ‘‘snakes’’ with this complex type of These ecomorphs also differ in habitat use: the for- articulation; Moon, 1999); this type of articulation mer is represented by surface dwelling species is, thus, believed to be generally found in burrow- (‘‘long-tailed surface-dwellers’’), such as Ophiodes, ing animals that move through undulating move- the cordylids, and the gerrhosaurids, and the lat- ments of the body (Romer, 1956; Mosauer, 1932; ter is represented by burrowing species (‘‘short- Gans, 1974). However, both the lizard-like and tailed burrowers’’), such as Anniella, ‘‘amphisbae- snake-like gymnophthalmids show a zygosphene- nians’’, ‘‘snakes’’ (although ‘‘snakes’’ have experi-

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 855 enced a shift in habitat; Wiens and Slingluff, the burrowing species is the result of vertical 2001), and many scincids (see Wiens et al., 2006, movements of the ventre (belly), as was observed for more details). The snake-like gymnophthalmids for Uma notata inornata (Pough, 1969b). Further Calyptommatus nicterus, Nothobachia ablephara, anatomical and physiological studies need to be and Scriptosaura catimbau are all burrowers and carried out in order to determine the mechanisms fall within the second ecomorph. of lung ventilation in the burrowing gymnophthal- Reductions of limb and girdle elements can be mids, and would certainly help to access the func- manifested by the simplification of the anatomy of tional role of the post-xiphisternum, if any, in the a particular cartilage or bone, or by fusion and/or respiratory physiology of Scriptosaura, Calyptom- loss of elements, as noted by previous works (for matus, and Nothobachia. example Stokely, 1947; Greer, 1991; Jerez and Tar- Reduction in the pelvic girdle is evidenced by azona, 2009). The pectoral and pelvic girdles of the the smaller size and by the absence of some mor- snake-like species studied here, especially those of phological features of the girdle bones of snake- S. catimbau and C. nicterus, are greatly reduced like species when compared to those of the lizard- in comparison to those of the lizard-like ones and like. The absence of a pectinal process in S. cati- of N. ablephara. The dermal interclavicle, for mbau and C. nicterus, a condition also observed in example, has well-developed anterior, posterior, Chalcides striatus, Ophiodes striatus, and Anguis and lateral processes in the lizard-like representa- fragilis (Stokely, 1947), and the loss of the pubic tives, while in the snake-like species this element and ischiadic symphysis and the consequent sepa- is reduced. Reduction of the interclavicle however, ration of the puboischiadic halves at the midline follows distinct patterns: in S. catimbau and C. (also observed by Stokely, 1947), are examples of nicterus, reduction takes place in the anteroposte- the simplification of morphology. rior axis, with the posterior process being greatly Limb reduction can follow two distinct patterns shortened and the anterior one being absent, while among squamate lizards, one in which reduction is in N. ablephara (as well as in some Bachia species; more pronounced in the forelimb than in the hind- Presch, 1980; Jerez and Tarazona, 2009) reduction limb (such as is observed in some pygopodids and takes place in the horizontal axis, with the lateral some scincids, in the anguid Ophisaurus, and in processes being shorter than the anterior and pos- ‘‘snakes’’; Brandley et al., 2008), and other in terior ones. which reduction of the hindlimb is more pro- The presence of a xiphisternum fused to the nounced than that of the forelimb (such as is sternum is defined by Estes et al. (1988) as a syna- observed in ‘‘amphisbaenians’’; Brandley et al., pomorphy for the Gymnophthalmidae, and despite 2008). Most Gymnophthalmidae species, including the slight differences in morphology among the Nothobachia, Scriptosaura, Calyptommatus, gymnophthalmini species analyzed here, this ele- Bachia psamophila, and the lizard-like Psiloph- ment is present in all but S. catimbau. thalmus, Procellosaurinus, and Vanzosaura, are The series of cartilaginous plates that constitute examples of the first pattern, while other Bachia the post-xiphisternum (following Etheridge, 1965) species exemplify the second pattern (Rodrigues, are formed by the midline fusion of the inscrip- 1991c; Rodrigues et al., 2007). tional ribs to its pair during embryonic develop- Vanzosaura rubricauda, Procellosaurinus paemi- ment (Roscito and Rodrigues, 2012a). The mor- nosus, and Psilophthalmus tetradactylus show a phology of the post-xiphisternal plates differs reduction in the first digit of the forelimb, which greatly among the snake-like species: in N. able- has lost the phalanges and is represented only by phara and C. nicterus, the post-xiphisternum is the reduced first metacarpal. In contrast, the formed by X-shaped plates formed by the midline degree of reduction in Nothobachia ablephara, fusion of the inscriptional ribs; however, the post- Scriptosaura catimbau, and Calyptommatus nicte- xiphisternum of S. catimbau is composed of simple rus is much more pronounced, with the limbs ei- horizontal bars that are distinct from the inscrip- ther formed by one to two digits, or completely tional ribs. absent externally. It is possible that a developed post-xiphisternum The forelimbs of S. catimbau and C. nicterus are connected to the vertebral ribs, and the associated formed by internal bone vestiges (a single element musculature, helps in stabilizing the body wall in C. nicterus and two in S. catimbau). Analysis of and also the vertebral ribs themselves, that most the embryonic development of Calyptommatus likely support the body against vertical pressure sinebrachiatus (Roscito and Rodrigues, 2012b) from the substrate when the animal is burrowed. showed that a forelimb bud forms early but its de- This configuration imposes constraints on the re- velopment is arrested after a few days; however, a spiratory movements by restraining the lateral cartilage condensation develops during this short movement of the body wall (expansion and con- period of time, and ossifies later in development. traction of the rib cage; Pough, 1969a), since the Considering that the formation of limb skeletal ribs are attached to single ventral plates. Thus, it elements occurs in a proximo-distal sequence is reasonable to assume that lung ventilation in (Saunders, 1948), it is reasonable to identify this

Journal of Morphology 856 J.G. ROSCITO AND M.T. RODRIGUES vestigial element as the humerus. In addition, it The astragalus-calcaneum of S. catimbau and C. has been shown that interruptions in limb develop- nicterus is very reduced and has lost the charac- ment in progressively increased time points result teristic morphology seen in pentadactylous limbs in the formation of a greater number of skeletal of most reptile species (Romer, 1956) and of the liz- elements (see Saunders, 1948 for the initial experi- ard-like closely-related species, such as V. rubri- ments in chicken embryos, and more recent cauda, for example. Also, the small element reviews in Towers and Tickle, 2009 and Zeller located distally to the astragalus-calcaneum and et al., 2009, for example); thus, the later the trun- laterally to the single metatarsal of C. nicterus cation event, the more complete is the limb could not be identified by our analysis because it formed. In this context, the presence of a second shows no obvious connection to neither of the for- skeletal element in the forelimb of S. catimbau mer elements nor to the single digit; here, we distal to the humerus rudiment might indicate hypothesize that it can represent either a distal that its forelimb bud develops for a longer period tarsal or an extremely reduced metatarsal. of time than that of C. sinebrachiatus. This longer The configuration of the fore and hindlimbs of period of development implies an extended action N. ablephara, S. catimbau, and C. nicterus are of the signaling pathways involved in limb devel- similar to those observed in some species of the ge- opment, that would be sufficient to pattern a sec- nus Lerista (see Greer, 1990, for the phalangeal ond mesenchyme condensation corresponding to formulae). Digit number in both limbs of N. able- the intermediate segment of the limb, the zeugo- phara resemble those from the scincids Lerista pod. punctatovittata and Neoseps (0:0:0:2:0 and The hindlimb of N. ablephara is composed of 0:0:2:4:0 for the fore and hindlimb, respectively; digits III, with two phalanges, and IV, with four Greer, 1990, 1991), although the identity of the phalanges. The identity of digit IV can be deter- single digit in the forelimb of N. ablephara is still mined by its positional relationships to the uncertain. Also, digit configurations of the fore and neighboring elements, especially its connection to hindlimbs of N. ablephara match with specific distal tarsal IV; the digit anterior to it is, thus, limb morphologies in the morphocline observed in identified as digit III. A reduced element ventral Lerista species (Greer, 1990). The same is true for to distal tarsal IV could correspond to the fifth the limbs of S. catimbau and C. nicterus (except metatarsal, although it does not show the char- for the forelimb of S. catimbau, which shows no acteristic lepidosaurian hook-shaped morphology correspondence to any of the limb morphologies in (Gauthier et al., 1988). Truncated development the Lerista morphocline). All of these limb-reduced could explain the lack of anatomical correspon- species are sand burrowers and face similar ecolog- dence to the typical lepidosaurian morphology ical pressures, suggesting that such morphologies (Rieppel, 1992), however, contrary to this hypoth- have some sort of functional importance in these esis, a fifth metatarsal with a typical hooked sandy habitats (Gans, 1975; Greer, 1990; Wiens morphology was observed in limb-reduced species et al., 2006; Kohlsdorf et al., 2010). of the lizard genus Hemiergis that show no pha- It is possible to correlate the degree of develop- langes in digit V (Shapiro, 2002). ment of the appendicular skeletons of the gym- The hindlimbs of S. catimbau and C. nicterus nophthalmini described to each species’ locomotion and forelimb of N. ablephara are composed of a sin- and lifestyle: the well-developed limbs and girdles gle digit that could not be identified based solely on of the lizard-like species (P. paeminosus, P. tetra- this anatomical analysis due to the loss of morpho- dactylus, and V. rubricauda) act as the propulsive logical information of the limb bones and loss or elements for locomotion, while in the head-first fusion of neighboring elements. The morphogenetic burrowing snake-like species the primary mode of model for limb development proposed by Shubin locomotion is by undulating movements of the and Alberch (1986) states that limb structures are body, with the reduced limb playing either a minor formed proximo-distally from sequential events of or no role at all in locomotion and in burrowing. branching and segmentation following an axis that Nothobachia ablephara, for example, uses the runs through the humerus/femur, ulna/fibula, and limbs during slow walking in association with then the fourth digit (for anurans and tetrapods); a undulating movements of the trunk, but holds the direct consequence of this mode of development is limbs against the body at faster paces and moves that any interruptions in limb development would only through undulating movements (as does the result in reduction taking place in the opposite scincid Chalcides; Renous et al., 1998); on the sequence of development following the axes of other hand, S. catimbau and C. nicterus move only branching and segmentation (Oster et al., 1988), through body undulation. Thus, the organization with digit IV being lost last (Rieppel, 1992). Con- of the axial and appendicular skeletons of these sidering these hypotheses, then it is reasonable to three snake-like species may reflect a greater flexi- identify the single digit of the hindlimb of C. sine- bility of the selective pressures acting upon the brachiatus and S. catimbau and that of the fore- maintenance of the developmental pathways limb of N. ablephara as digit IV. involved in limb development because of the

Journal of Morphology POST-CRANIAL SKELETON OF GYMNOPHTHALMIDS 857 greater reliance of the axial skeleton in locomo- ACKNOWLEDGMENTS tion. The authors would like to thank Hussam Zaher Previous works have suggested an association and Carolina Castro-Mello for access to specimens between a burrowing habit and a snake-like mor- from Museu de Zoologia da Universidade de Saõ phology, accompanied with an elongated body and Paulo (MZUSP). They would also like to thank the reduced or absent limbs (for example Gans, 1975; two anonymous reviewers for their important com- Lee, 1998). Also, Gans (1975) proposed that body ments, which helped in improving the manuscript. elongation and an associated decrease in body diameter would be the initial morphological adaptation that would confer an advantage for allowing the animal to burrow or move through loose sub- LITERATURE CITED strate. Elongation would then be followed by the Barbadillo LJ, Barahona F, Sanchez-Herraiz J. 1995. Sexual dif- reduction first in size and then in loss of phalanges ferences in caudal morphology and its relation to tail autot- and/or loss of the limbs, since the presence of omy in lacertid lizards. J Zool 236:83–93. limbs projecting from the body would represent an Benesch AR, Withers PC. 2002. Burrowing performance and increase in body diameter, and would, thus, be a the role of limb reduction in Lerista (Scincidae, Lacertilia). Senck leth 82:107–114. disadvantage for locomotion through crevices. Bergmann PJ, Irschick DJ. 2011. Vertebral evolution and the However, at least for anguids there was no clear diversification of squamate reptiles. Evolution 66:1044–1058. association between a burrowing lifestyle and a Brandley MC, Huelsenbeck JP, Wiens JJ. 2008. Rates and pat- snake-like morphology, nor is there an evolution- terns in the evolution of snake-like body form in Squamate reptiles: Evidence for repeated re-evolution of lost digits and ary sequence that starts with body elongation, fol- long-term persistence of intermediate body forms. Evolution lowed by reduction in limb size, and then by digit 62:1–23. loss (Wiens and Slingluff, 2001). Nonetheless, the Boulenger GA. 1902. Descriptions of new fishes and reptiles dis- evolution of an elongated body and reduced limbs covered by Dr. F. Silvestri in South America. Ann Mag Nat is frequent among squamates, and estimates of the Hist Ser 9:284–288. Camp CL. 1923. Classification of the lizards. Bull Am Mus Nat number of times limb reduction has occurred vary Hist 48:289–481. according to taxon sampling (Skinner et al., 2008). Castoe TA, Doan TM, Parkinson CL. 2004. 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