THE ANATOMICAL RECORD 290:900–912 (2007) Developmental Basis of Limb Homology in Lizards MARISSA FABREZI,1* VIRGINIA ABDALA,2 1 AND MARÍA INÉS MARTÍNEZ OLIVER 1 Instituto de Bio y Geociencias-Museo de Ciencias Naturales, Universidad Nacional de Salta, Salta, Argentina 2 Instituto de Herpetologı́a-Fundación Miguel Lillo, San Miguel de Tucumán, Argentina ABSTRACT Shubin and Alberch (Evol Biol 1986;20:319–387) proposed a scheme of tetrapod limb development based on cartilage morphogenesis that provides the arguments to interpret the homologies of skeletal elements and sets the basis to explain limb specialization through later developmental modiﬁcation. Morphogenetic evidence emerged from the study of some reptiles, but the availability of data for lizards is limited. Here, the study of adult skeletal variation in 41 lizard taxa and ontogeny in species of Liolaemus and Tupinambis attempts to ﬁll in this gap and provides supporting evidence for the Shubin-Alberch scheme. Six questions are explored. Is there an intermedium in the carpus? Are there two centralia in the carpus? Is there homology among proximal tarsalia of reptiles? Does digit V belong to the digital arch? Is the pisiform an element of the autopodium plan? And should the ossiﬁcation processes be similar to cartilage morphogenesis? We found the following answers. Some taxa exhibit an ossiﬁed element that could represent an intermedium. There is one centrale in the carpus. Development of proximal tarsalia seems to be equivalent with that observed among reptiles. Digit V could arise from the digital arch. Pisiform does not arise as part of the limb plan. And different patterns of ossiﬁcation occur following a single and conservative cartilaginous conﬁguration. Lizard limb development shows an early pattern common to other reptiles with clear primary axis and digital arch. The pattern then becomes lizard-speciﬁc with specialization involving some reduction in prechondrogenic elements. Anat Rec 290:900–912, 2007. Ó 2007 Wiley-Liss, Inc. Key words: limb; morphogenesis; lizards; homology Shubin and Alberch (1986) reviewed the literature on the development and evolution of the tetrapod limb and proposed a formalism to describe limb skeleton based on the spatial connectivities among cartilaginous condensations during earlier stages of limb morphogenesis (Fig. 1). This proposal allowed comparisons among tetrapod taxa and provided a conceptual basis to interpret the homologies of the skeletal components of the tetrapod limbs. From this approach, several paleontological, embryological, and developmental genetic studies have contributed with important data to explain the evolution and diversiﬁcation of vertebrate locomotor appendages and have established the homologies of their components. The interspeciﬁc variation of limb skeleton in lizards has been explored by different authors with different emphasis (e.g., Wellborn, 1933; Gasc, 1963; RenousÓ 2007 WILEY-LISS, INC. Lécuru, 1973; Mathur and Goel, 1976; Rieppel, 1992a, 1992b, 1992c, 1993a; Maisano, 2001, 2002a, 2002b). Nevertheless, data on early limb development in lizards Grant sponsor: Agencia Nacional de Promoción Cientı́ﬁca y Tecnológica; Grant number: PICT/2002 12418; Grant sponsor: Consejo Nacional de Investigaciones Cientı́ﬁcas y Técnicas; Grant number: PIP 2829. *Correspondence to: Marissa Fabrezi, Instituto de Bio y Geociencias-Museo de Ciencias Naturales, Universidad Nacional de Salta, Mendoza 2, 4400-Salta, Argentina. Fax: 54-387-4255455. E-mail: firstname.lastname@example.org Received 24 May 2006; Accepted 23 February 2007 DOI 10.1002/ar.20522 Published online 5 April 2007 in Wiley InterScience (www. interscience.wiley.com). LIMB DEVELOPMENT IN LIZARDS Fig. 1. Pattern of cartilages morphogenesis in tetrapod limbs modiﬁed from Shubin and Alberch (1986: Fig. 20). Limb development is characterized by differentiation of de novo condensation (humerus/ femur), which bifurcates to give rise to the ulna/ﬁbula and radius/tibia cartilages. Bold lines depict connectivities and gray ﬁgures denote the postaxial side of limb where most differentiation events take place. Condensation, bifurcation, and segmentation of the ulna/ﬁbula produce the formation of cartilages of the primary axis in a proximodistal sequence (black bold line), the digital arch in a posteroanterior sequence (black medium bold line), and the intermedium and centralia in proximomedial direction (gray bold line). Differentiation of the primary axis involves the origin of digit IV, from which the digital arch arises. Digit V could represent a de novo condensation in this model. The intermedium is segmented from the end of ulna/ﬁbula and bifurcates in centrale series. Radiale/tibiale originates from the distal end of radius/tibia in the preaxial side of the limb (black thin line). cd1–5/ td1–5, distal carpalia 1–5/distal tarsalia 1–5; crd/ctd, centrale radiale distale/centrale tibiale distale; crp/ctp, centrale radiale proximale/centrale tibiale proximale; cud/cfd, centrale ulnare distale/centrale ﬁbulare distale; cup/cfp, centrale ulnare proximale/centrale ﬁbulare proximale; I, intermedium; I–V, metarcarpalia I–V/metatarsalia I–V; r/t, radiale/ tibiale; Ra/Ti, radius/tibia; u/f, ulnare/ﬁbulare; Ul/Fi, ulna/ﬁbula (Shubin and Alberch, 1986). are scarce and limited to Agama (Holmgren, 1933), Calotes (Mathur and Goel, 1976), Chamaleo (Rieppel, 1993a), Hemiergis (Shapiro, 2002), and Tarentola and Chalcides (Goldschmidt, 1943). At present, lizards are the only tetrapod group in which the interpretation of limb morphology does not have an embryological basis (for urodeles, see Blanco and Alberch, 1992; Vorobyeva and Hinchliffe, 1996; Hinchliffe and Vorobyeva, 1999; for anurans, see Fabrezi and Alberch, 1996; Fabrezi and Barg, 2001; for turtles, 901 see Burke and Alberch, 1985; Sheil, 2003, 2005; for crocodiles, see Müller and Alberch, 1990; for birds, see Hinchliffe and Hecht, 1984; Burke and Feduccia, 1997; Feduccia et al., 2005; for mammals, see Prochel et al., 2003, 2004; Sanchez-Villagra and Döttling, 2003). This problem arises from the difﬁculty to get complete embryonic series of lizard development. There are six questions regarding the interpretation of the skeletal elements that compose lizard limbs that need developmental data in order to be answered. One, is there an intermedium in the lizards carpus? The intermedium, both as embryonic and as ossiﬁed element, is free in turtles (Burke and Alberch, 1985; Sheil, 2003, 2005). An embryonic condensation representing the intermedium fuses with the radiale condensation in crocodiles and birds (Hinchliffe and Hecht, 1984; Müller and Alberch, 1990; Burke and Feduccia, 1997). In lizards, some authors have identiﬁed an ossiﬁed element as an intermedium in the carpus of some Anguidae (Renous-Lécuru, 1973), Iguanidae (Avery and Tanner, 1964), Lacertidae (Rieppel, 1992b; Maisano, 2001), Teiidae (Fischer and Tanner, 1979), Varanidae (Rieppel, 1992c), and Xantusiidae (Maisano, 2002b). Others (Mathur and Goel, 1976) have described this structure as a transient cartilage that disappears during the embryonic development of the lacertid Calotes versicolor. To answer this question, it is necessary to review developmental and interspeciﬁc variation in order to identify the mentioned intermedium as an embryonic cartilage and as an ossiﬁed element. Two, are there two centralia in the lizard carpus? The intermedium bifurcates to form condensations of the centralia (Fig. 1). The chelonian carpus has three centralia (Burke and Alberch, 1985; Sheil, 2003, 2005). The carpus of crocodiles presents a single centrale that forms distal to the fused radiale-intermedium after distal carpalia are fully formed (Müller and Alberch, 1990), and no centralia differentiate in the wing of birds (Hinchliffe and Hecht, 1984; Müller, 1991). Some authors have pointed out two elements as centralia in lizards: one in a median position named lateral centrale, and the second one proximal to metacarpale I, named medial centrale (Maisano, 2002a, 2002b). For other authors (e.g., Renous-Lécuru, 1973), the medial centrale is the distal carpale 1. Data on morphogenesis of primary cartilages could reveal the identity of this condensation as a second centrale or as part of the digital arch. Three, is there homology among the lizard proximal tarsalia, and the same cartilages in turtles and crocodiles? The tarsus of reptiles has two proximal tarsalia representing the ossiﬁed calcaneum (ﬁbulare) and astragalus that may fuse into a large proximal tarsale. The number and identity of the primary cartilages forming the astragalus have been discussed in several contexts (Holmgren, 1933; Mathur and Goel, 1976; Rieppel, 1993b). Embryological studies showed that the astragalus is formed by the intermedium and centralia in turtles (Burke and Alberch, 1985; Sheil, 2005) and the intermedium and a single centrale in crocodiles (Müller and Alberch, 1990). In lizards, the proximal cartilage that is positioned directly anterior to the ﬁbulare was described as the astragalus (Rieppel, 1992a; Shapiro, 2002) and its identity was discussed in at least two studies (Holmgren, 1933; Mathur and Goel, 1976). Revision of early stages of hindlimb development in lizards is 902 FABREZI ET AL. necessary to update the discussion on the homologies of proximal tarsalia among reptiles. Four, does digit V belong to the embryonic series of primary cartilages of the digital arch? Shubin and Alberch (1986) suggested digit V is formed as a condensation de novo, independently of the digital arch, and therefore it would not be equivalent to other digits. Patterns of connectivity between digit V and cartilages of the primary axis and digital arch were described for anurans (Fabrezi and Barg, 2001) but has not been studied yet in lizards. Five, is the pisiform an element of the autopodium plan? Shubin and Alberch (1986) described the pisiform through ontogeny as a de novo condensation in the reptilian carpus; other authors recognized it as a sesamoid (Haines, 1969). It is necessary to review and discuss the pisiform as an element in the limb plan. Six, should the patterns of ossiﬁcation be similar to cartilage morphogenesis? Embryological evidence provides strong arguments to identify limb skeletal elements and interpret their homologies (Feduccia et al., 2005). However, some authors have criticized the morphogenetic plan of tetrapod limbs (Fig. 1), claiming that the ossiﬁcation sequence in lizards does not show the same pattern as the differentiation of primary cartilages; and so, the identity of ossiﬁed elements could not be the same as that of primary cartilages (Maisano, 2002a, 2002b). This issue could be solved by the analysis of the differences between cartilage morphogenesis and ossiﬁcation. Here we present a study of lizard limbs in adult specimens belonging to 41 taxa (see Appendix) and ontogenetic series of Liolaemus multicolor, L. quilmes, L. ruibali, L. zullyi, and Tupinambis merianae in order to describe the morphogenesis of primary cartilages as well as set the embryological basis for interpreting the adult ossiﬁed elements; address differences between morphogenesis and ossiﬁcation processes affecting the limb morphologies; and establish interspeciﬁc variation in carpus and tarsus as well as discuss the constraints. We propose a scheme to interpret the lizard limb elements with an embryological basis. The description of skeletogenic patterns of a selection of lizards in the present study attempts to ﬁll in the gap of limited evidence for the group, providing useful arguments for the Shubin and Alberch (1986) scheme, and to serve for future comparative analyses of limb homologies in lizards and tetrapods. MATERIALS AND METHODS In this study, different analyses were performed: description of primary cartilage development in embryonic series; study of ossiﬁcation pattern in ontogenetic series; and examination of interspeciﬁc variation of carpalia and tarsalia in a sample of 41 species belonging to 12 lizard families. All observations were made in clearing and double staining with Alcian Blue and Alizarin Red whole mounts obtained following the procedure described in Wassersug (1976), and histological serial sections of 7 mm thick, stained with hematoxyilin-eosin (Martoja and Martoja, 1970). Descriptions, illustrations, and photographs were made with a stereo dissection microscope Nikon SMZ1000 and light microscope Leica DM equipped with digital camera and camera lucida. Fig. 2. Dorsal view of forelimb bud in Liolaemus quilmes embryo at early paddle-shaped stage (stage 30). A: The primary axis and digital arch display proximodistal and posteroanterior morphogenesis (bold lines). This image agrees with Shubin and Alberch’s interpretation (1986). B: The same view in detail showing radius, ulna, ulnare, distal carpale 4, and metacarpale IV. Differentiation of distal carpale 3 has begun. White arrows point out embryonic connectivities between ulna and ulnare, and ulnare and distal carpale 4. da, digital arch; pa, primary axis; pre, preaxial axis. Scale bar ¼ 0.2 mm. Primary cartilage development was examined in embryonic specimens staged following Dufaure and Hubert (1961). This table of development was chosen because stages can be identiﬁed on the basis of limb bud morphologies. The embryonic series were selected from museum collections, and studied specimens are Liolaemus multicolor (stages 32, 34, 36, and 40), Liolaemus quilmes (stages 30, 32, 34, 35, and 36), Liolaemus zullyi (stage LIMB DEVELOPMENT IN LIZARDS 903 Fig. 3. Earlier chondrogenic foci in an embryo of Tupinambis merianae at paddle-shaped stage of limb development (stage 33). A: Right forelimb where primary axis condensations, distal carpale 3, metacarpalia V–II, and centrale are differentiated. The centrale appears distal to the ulnare. B: Right hindlimb with cartilages of primary axis, distal tarsale 3, and metatarsalia V–II become evident. A diffuse condensation is present distal to the ﬁbula and tibia ends. This condensation is interpreted as the primary fusion of intermedium and centrale. Scale bar ¼ 0.2 mm. 39), and Tupinambis merianae (stages 33, 37, 40, 49, and 50). Embryos at limb paddle-shaped stages of Liolaemus spp. were removed from preserved gravid females. Left limbs of Liolaemus zullyi were chosen for histological sections and all specimens were cleared and double-stained. Patterns of ossiﬁcation were analyzed in whole mounts of cleared and double-stained specimens of ontogenetic series of Liolaemus multicolor and Liolaemus ruibali. Because the Alizarin Red S is a speciﬁc dye for the histochemical detection of calcium, ossiﬁcation sequence is inferred when positive Alizarine Red S coloration is observed. However, we must notice that bone development may start before calciﬁcation. Whole mounts of cleared and double-stained adult specimens of 41 taxa of Anguidae, Chamaleontidae, Gekkonidae, Gerrhosauridae, Gymnophthalmidae, Lacertidae, Leiosauridae, Liolaemidae, Polychrotidae, Scincidae, Teiidae, and Tropiduridae were studied in order to describe interespeciﬁc carpal and tarsal variation. Species, specimen numbers, and collection data are listed in the Appendix. All specimens are deposited in the herpetological collections of Instituto de Herpetologı́a, Fundación Miguel Lillo, Tucumán (Argentina); Museo de Ciencias Naturales, Universidad Nacional de Salta (Argentina); Museo de Zoologı́a, San Pablo (Brazil); and the private collection of Richard Thomas. in proximodistal direction (to elongate and give rise to phalanges) and postaxial-preaxial direction (to differentiate digits II and I; Figs. 3A and 4A and B). Thereafter, a medial condensation appears distal to the ulnare in Tupinambis merianae (Fig. 3A). Based on its position, this condensation is identiﬁed as the centrale; however, the absence of connectivity between this centrale cartilage and the ulnare was a reason to prefer its identiﬁcation as the intermedium (Mathur and Goel, 1976; Shapiro, 2002). Distal to the radius, a bilobed cartilage representing the radiale becomes evident and grows medially (Fig. 5A and B). Differentiation of digit II progresses and the cartilage of distal carpale 2 is better deﬁned. In Liolaemus spp., condensation of the centrale appears after the metacarpale I cartilage is fully formed (Fig. 5A and B). Segmentation of distal carpale 1 from distal carpale 2 was observed in embryos of Liolaemus spp. and Tupinambis merianae (Fig. 6). The origin of distal carpale 1 from distal carpale 2 conﬁrms the identity of this cartilage as a distal carpale and invalidates any argument to name it as a centrale or medial centrale (Gauthier et al., 1988; Carroll and Currie, 1991; Maisano, 2002a, 2002b). Patterns of connectivity among digital arch cartilages as depicted in Figure 20 (Fig. 1 here) of Shubin and Alberch (1986) have been found in limb buds of a malformed embryo of L. quilmes with asymmetrical limb development (Fig. 7A). RESULTS Primary Cartilages Morphogenesis Forelimbs. In lizards, primary axis and digital arch differentiation were observed in the embryo of Liolaemus quilmes, where connectivities among ulna and ulnare, ulnare and distal carpale 4 are still evident (Fig. 2). The primary axis and digital arch cartilages develop Hindlimbs. In the same species and at similar stages of development (Figs. 3B and 4C and D), the primary axis cartilages that are already differentiated correspond to the ﬁbula, ﬁbulare, distal tarsale 4, and metatarsale IV. At these early stages, there is a diffuse and heterogeneous condensation between ﬁbula and tibia and anterior to the ﬁbulare. In Liolaemus spp., this 904 FABREZI ET AL. Fig. 4. Left limb buds at paddle-shaped stage in Liolaemus multicolor embryos. A: Forelimb with primary axis cartilages (stage 32). B: The following stage (stage 34), forelimb where digital arch has started to develop. C: Hindlimb showing primary axis foci and a medial condensation distal to the tibia and ﬁbula ends (stage 32). D: The following stage (stage 34), hindlimb displaying digital arch differentiation and better development of the medial tarsal condensation. Scale bar ¼ 0.2 mm. condensation grows medially from the distal end of the tibia (Fig. 4B and C). This cartilaginous focus and the ﬁbulare will fuse to form the large proximal tarsale in later stages (Fig. 5C and D). Cartilages of metatarsalia and phalanges grow distally. Connections among metatarsale V and distal tarsalia 4–3, and ﬁbula and ﬁbulare were observed in a malformed embryo of L. quilmes (Fig. 7B). The complete set of primary cartilages of limbs differentiates simultaneously when the disappearance of interdigital tissue has ended and digits are free. Ossiﬁcation Patterns The skeleton of limbs ossiﬁes from preformed cartilages, a process called chondral ossiﬁcation. Chondral ossiﬁcation happens simultaneously with cartilage growth. However, growth of cartilage and ossiﬁcation are two different and independent processes that are controlled by intrinsic (genetic and programmed growth) and extrinsic (hormones, nutrition) factors (Hinchliffe and Johnson, 1983). There are four mechanisms of deposition and bone growth in cartilages (Hall, 2005): periosteal ossiﬁcation, endosteal ossiﬁcation, perichondral ossiﬁcation, and endochondral ossiﬁcation. Perichondral and endochondral ossiﬁcations are observed in reptiles and take place in mineralized cartilage or following removal of mineralized cartilage by cells brought into the marrow cavity by invading blood vessels (Fig. 8). In lizard limbs, perichondral ossiﬁcation precedes endochondral ossiﬁcation. Perichondral ossiﬁcation centers appear in diaphyses of long cartilages (stylopodium, zeugopodium, metapodium, and phalanges; Fig. 8A and B). Calciﬁcations, as is observed in whole mounts, suggest that they start to differentiate in a proximodistal direction after development of primary cartilages (Figs. 9A and 10A). Endochondral ossiﬁcation occurs in carpalia and tarsalia in LIMB DEVELOPMENT IN LIZARDS 905 Fig. 6. Dorsal view in right hand. The distal carpale 1 arising from distal carpale 2 in Tupinambis merianae at the end of paddle-shaped stage embryo (stage 37). Scale bar ¼ 0.5 mm. Fig. 5. Right limbs of Liolaemus quilmes embryos in advanced paddle-shaped stages with interdigital tissues in regression. A: Stage 35, the hand exhibits advanced differentiation of primary condensations of the digital arch. The radiale cartilage becomes evident distal to the radius. B: Stage 36, differentiation of the centrale has took place and the radiale condensation became more deﬁned distal to the radius. C: Stage 35, differentiation of primary cartilages in the tarsus has ended and development of phalanges is in progress. Cartilages of the ﬁbulare and intermedium-centrale are separated. D: Stage 36, fusion between ﬁbulare and intermedium-centrale has started in the tarsus. Scale bar ¼ 1 mm. later stages of embryonic development, but it increases in postnatal stages (Figs. 8C and F, 9B and C, and 10B and E). Most cartilages of carpalia/tarsalia show single calciﬁed centers but there are two centers in the proximal tarsale and radiale. Calciﬁcation centers in proximal tarsale differentiate at different times; the ﬁrst one to appear corresponds to the preaxial and medial portion, which forms the astragalus, followed by calciﬁcation of the postaxial portion (ﬁbulare) that develops into the calcaneum. Differentiation of two ossiﬁcations in the proximale tarsale is common to all lizards, with the exception of Chamaleo spp. (this paper and Rieppel, 1992a), in which there is only one ossiﬁcation center identiﬁed as astragalus (Rieppel, 1992a). In the radiale cartilage, we observed two calciﬁcation centers; the ﬁrst one to differentiate is anterior and the second one is medial (Fig. 9C). The anterior center of calciﬁcation was identiﬁed as the radiale ossiﬁcation center, and the posterior one as an accessory ossiﬁcation center, as was described in xantusiids and phrynosomatids (Maisano, 2002a, 2002b). Development of two ossiﬁcation centers in the radiale seems to be common to all lizards. Secondary centers of ossiﬁcation develop in epiphyses in advanced embryonic stages. During postnatal growth, they may fuse with diaphyses in adult stages when development rates become slow (Figs. 9D and 10E). Sesamoids are ectopic elements that appear related to the limb skeleton; they are formed by cartilaginous nodules that may ossify. Sesamoids originate in tendinous or ligamentous tissues, especially where tendons cross an articulation (Hall, 2005). Differentiation of sesamoids starts in embryonic stages after morphogenesis of primary cartilages. The ﬁrst sesamoid to appear is the pisiform (Figs. 8C and 10A–D). It locates ventrally to the articulation between ulna and ulnare. Other sesamoids differentiate in a proximodistal direction but they vary among species (Fig. 9D). Ossiﬁcation of each sesamoid is endochondral and takes place during postnatal growth. Interspeciﬁc Variation of Carpal Elements in Adult Lizards Most lizard species with well-developed limbs show similar forelimbs with a carpus formed by two proximal elements, the ulnare and radiale; one medial element, the centrale; and ﬁve distal elements, distal carpalia 1– 5. Distal carpale 4 is always the largest of the distal series. The radiale, ulnare, and centrale articulate with distal carpalia. The pisiform is also present in the articulation between distal epiphysis of ulna and ulnare. Deviation from this pattern was observed in Chamaleo unicornis (Fig. 11A). In this species, the radiale and ulnare articulate with a single and large distal carpale, representing the fusion of the centrale with distal carpalia 4, 3, and 2 (Renous-Lécuru, 1973), placed medially and bearing ﬁve metacarpals; the centrale is absent. 906 FABREZI ET AL. Fig. 7. Pattern of cartilage connectivity in Liolaemus quilmes embryo at stage 32, in which condensation events in right limbs are more advanced than in left ones but segmentations are retained. A: Hand with embryonic connectivities between ulna and ulnare, ulnare and digital arch, distal carpalia 5 and 4, distal carpalia 4 and 3, distal carpale 2 and digit I. This image shows the origin of digit I (distal car- pale 1 and metacarpale I) from distal carpale 2, and the relationship between digit V with the digital arch. B: Foot displaying connectivities between metatarsale V and distal tarsalia 4–3, and ﬁbula and ﬁbulare. This image exhibits connectivity of metatarsale V with digital arch. Curved arrows indicate embryonic connectivities. Scale bar ¼ 0.2 mm. Fig. 8. Light microscopic cross-sections of Liolaemus zullyi limbs at the end of embryonic development (stage 39). A: Perichondral ossiﬁcation in proximal diaphyses of metatarsalia III, IV, and V. Scale bar ¼ 0.2 mm. B: Perichondral ossiﬁcation at level of expanded head of metatarsale V. Scale bar ¼ 0.2 mm. C: Pisiform, epiphysis of ulna, ulnare, and radiale. Hyperthrophic cells indicate the endochondral ossiﬁcation in the ulnare. Scale bar ¼ 0.2 mm. D: Endochondral ossiﬁcation in the ulnare. Scale bar ¼ 0.2 mm. E: Proximal tarsalia displaying astragalus and ﬁbulare ossiﬁcation centers. Scale bar ¼ 0.2 mm. F: Detail of cartilaginous hyperthrophic cells in the ﬁbulare. Scale bar ¼ 0.5 mm. coas, ossiﬁcation center of the astragalus; cof, ossiﬁcation center of the ﬁbulare; eu, distal epiphysis of the ulna; ps, pisiform. LIMB DEVELOPMENT IN LIZARDS Fig. 9. Sequence of ossiﬁcation in Liolaemus multicolor, dorsal view of left forelimbs. A: Advanced embryo at stage 40. Black arrows point out perichondral ossiﬁcations in radius, ulna, metacarpalia, and phalanges. B: Juvenile specimen. Black arrows show endochondral ossiﬁcation in ulnare, centrale, radiale (a medial center), and distal carpalia 1–5. White arrows depict centers in distal epiphysis of ulna, and proximal epiphyses of metacarpalia I–V. C: Juvenile specimen. Black arrows point out a second center of endochondral ossiﬁcation in the radiale, and ossiﬁcation in pisiform. Epiphyses ossiﬁcations are indicated with white arrows. D: Adult specimen. A fully ossiﬁed manus; no distinct ossiﬁcation centers are evident. Open arrow points out a suprarticular sesamoid. Scale bars ¼ 0.1 mm (A); 0.2 mm (B–D). The metacarpalia are short, have similar lengths, and articulate with the large distal carpale in two directions. Metacarpalia V and IV articulate with the distal carpale in postaxial position, and metacarpalia III, II, and I articulate with the distal carpale in preaxial position. Thus, digits form two divergent groups (zygodactylia). In some species of Scleroglossa (Panaspis breviceps and Gerrhosaurus nigrolineatus), we found a small ossiﬁed element between ulnare and radiale (Fig. 11B). A similar osseous element has been identiﬁed as the intermedium (Avery and Tanner, 1964; Renous-Lécuru, 1973; Fischer and Tanner, 1979; Rieppel, 1992b, 1992c; Maisano, 2001, 2002b). Interspeciﬁc Variation of Tarsal Elements in Adult Lizards The tarsus is formed by a large proximal tarsale and distal tarsalia 4 and 3. The proximal tarsale articulates proximally with the tibia and ﬁbula, and distally with distal tarsalia 4 and 3 and metatarsalia V, II, and I. The stereotypical hooked metatarsale V of lizards is short and has an expanded proximal head that articulates with proximal tarsale and distal tarsale 4. 907 Fig. 10. Sequence of ossiﬁcation in Liolaemus multicolor, dorsal view of left hindlimbs. A: Advanced embryo at stage 40. Perichondral ossiﬁcations in diaphyses of ﬁbula, tibia, metatarsalia I–V, and phalanges (black arrows). B: Posthatched specimen. Astragalus, calcaneum, distal tarsalia 4 and 3, and metatarsale V are the endochondral centers in the tarsus (black arrows). Distal epiphyses of metatarsalia are indicated with white arrows. C: Juvenile specimen. Distal epiphyses of the tibia and ﬁbula show ossiﬁcation centers; the same is observed in proximal epiphyses of metatarsalia (white arrows). D: Juvenile specimen. Sesamoids and accessory calciﬁcations are pointed with open arrows; sesamoid are present between distal tarsale 4 and metatarsale V, and a lateral plantar tubercle on the metatarsale V. Two ossiﬁed centers as epiphyses are identiﬁed in the proximal tarsale (white arrows). E: Fully ossiﬁed pes in adult specimen. Scale bar ¼ 0.2 mm. The tarsus of Chamaleo unicornis differs also from the tarsus of most lizards (Fig. 11C); the proximal tarsale is reduced in size, placed between the distal ends of the tibia and ﬁbula (which are convergent), and articulated with a single distal tarsale, representing an early fusion of distal tarsalia cartilages. Metatarsalia are short and form divergent groups. Metatarsalia V, IV, and III articulate with the distal tarsale and are oriented in postaxial direction. Metatarsale II articulates with the distal tarsale and metatarsale I articulates with the proximal tarsale, both oriented in preaxial direction. Another variation from the basic lizard pattern was observed in some Gekkonidae (Garthia gaudichaudii, Hemidactylus mabouia, and Phyllopezus pollicaris). In these species, there is a small bone proximal to the epiphyses of metatarsalia I and II, which articulates with proximal tarsale (Fig. 11D). This bone has been considered a preaxial distal tarsale 1 (Mathur and Goel, 1976; Khalil and Sabri, 1977; Mohammed, 1989) or the fusion of distal tarsale 2 and 1 (Holmgren, 1933). Embryological data to support its identity are absent. At present, we prefer to name it anterior distal tarsale. 908 FABREZI ET AL. Fig. 12. Skeletal elements of the left limbs of Ophiodes striatus. This species lack forelimbs. The primary axis elements are conserved in the vestigial hindlimbs that are represented by the ossiﬁcation of ﬁbula, calcaneum (ﬁbulare), and metatarsale IV. The astragalus ossiﬁcation is also retained. as, astragalus; ca, calcaneum. Scale bar ¼ 0.5 mm. Fig. 11. Carpal and tarsal elements in selected adult lizards. A: Dorsal view of carpus in Chamaleo unicornis. The radiale, ulnare, and pisiform lack ossiﬁcation. A single distal carpale, representing the fusion of distal carpalia, with an endochondral ossiﬁcation center bears ﬁve short metacarpalia. Cartilages are in gray. B: Dorsal view of carpus in Gerrhosaurus nigrolineatus. A small ossiﬁcation between ulna distal epiphysis and radiale is identiﬁed as the intermedium. C: Dorsal view of tarsus in Chamaleo unicornis. The proximal tarsale has only the astragalus ossiﬁcation and a single distal tarsale articulates with ﬁve short metatarsalia. D: Dorsal view of tarsus in Phyllopezus pollicaris. There is a preaxial distal tarsale between the tarsale proximale and metatarsalia II and I. cd, distal carpale; td, distal tarsale; tda, anterior distal tarsale; tp, proximal tarsale. Scale bars ¼ 1 mm (A–C); 1.5 mm (D). DISCUSSION Pattern of Primary Cartilages Morphogenesis The scheme proposed by Shubin and Alberch (1986) represents cartilage morphogenesis, which deﬁnes the tetrapod limb plan. Morphogenesis of cartilages is different from chondrogenesis. Morphogenesis of cartilage implies the generation and emergence of a variety of less easily deﬁned three-dimensional shapes of cartilaginous entities, whereas chondrogenesis involves cell differentiation (Thorogood, 1983). Shubin and Alberch (1986) described the limb skeleton based on spatial connections during early stages of limb morphogenesis, where skeletal condensations ﬁrst appear proximally and yield other condensations through segmentation and bifurcation events that occur predominantly along the posterior side of the limb (Fig. 1). The study of cartilage morphogenesis of limbs has conﬁrmed the existence of an early primary axis in Tetrapods (Mathur and Goel, 1976; Hinchliffe and Hecht, 1984; Burke and Alberch, 1985; Shubin and Alberch, 1986; Müller and Alberch, 1990; Fabrezi and Alberch, 1996; Burke and Feduccia, 1997; Fabrezi and Barg, 2001; Feduccia et al., 2005; Sheil, 2005). The early differentiation of a primary axis that invariably passes through digit IV in early limb development is a morphological tool that allows one to interpret the identity and homology of digits and speciﬁc elements of the autopodium, as well as identify and discuss the relative positions of structures and directions of growth. Primary axis development in lizard limbs as we show it (Fig. 2) has been described in other studies (Goldschmidt, 1943; Mathur and Goel, 1976; Shapiro, 2002) and is conserved in those taxa with strong limb reductions (Fig. 12). In forelimbs of crocodiles and birds, after differentiation of primary axis, a condensation continuous with the end of the ulna and the end of the radius develops and segments, giving rise to the intermedium, after fusing to the radiale (Müller and Alberch, 1990; Burke and Feduccia, 1997). In turtles, the intermedium is segmented from the distal end of the ulna as a single cartilage, from it centralia 4 and 3 originate, and the radiale is absent (Burke and Alberch, 1985; Sheil, 2003, 2005). In the lizard Calotes versicolor, the intermedium was described as a condensation of mesenchyme that may disappear by necrosis, and the radiale differentiates after disappearance of intermedium (Mathur and Goel, 1976). Our observations of the lizard carpus development failed to ﬁnd an embryonic intermedium and showed the origin of the radiale to be a condensation from the end of the radius. However, the origin of the intermedium should be studied in those taxa, e.g., some Scleroglossa such as Panaspis breviceps and Gerrhosaurus nigrolineatus, with adults having an ossiﬁed intermedium to elucidate its origin. In spite of embryological data showing the intermedium of lizards as being weak, the presence of a centrale suggests the possibility that the intermedium differentiates only as a mesenchyme condensation and may be LIMB DEVELOPMENT IN LIZARDS incorporated to the centrale in those taxa in which the intermedium is absent in adults. Differing from what is the case in turtles, the lizard carpus shares with crocodiles the development of a distinct and single radiale condensation. In amniote hindlimbs, the pattern of morphogensis of proximal tarsalia is complex. The ﬁbulare forms as part of the primary axis, the medial condensation anterior to the ﬁbulare in which the astragalus ossiﬁes is interpreted as the intermedium-centrale, and the tibiale is absent (Burke and Alberch, 1985; Müller and Alberch, 1990; Rieppel, 1993b; Sheil, 2005). In lizards, we observed both cartilages, and the anterior condensation seems to be formed by two foci without connectivity with the distal end of the tibia (Fig. 3B). In crocodiles, the cartilage of the intermedium-centrale differentiates as a single chondrogenic condensation with two foci; the proximal focus is the intermedium and the distal focus is the centrale (Müller and Alberch, 1990). Heterochrony produces the failure of the centrale to separate completely from the intermedium (Müller and Alberch, 1990). In turtles, the well-deﬁned and continuous condensations of the intermedium and proximal centrale appear in a proximodistal sequence and the centrale 4 differentiates as an independent element to be incorporated into the growing condensation of the intermedium-centrale (Burke and Alberch, 1985; Sheil, 2003, 2005). We propose that the medial condensation where the astragalus ossiﬁes in lizards represents the intermedium-centrale and is equivalent to that described in crocodiles sharing the heterochronic change, for which the event of segmentation of the centrale represents a primary modiﬁcation of the pattern (Müller, 1991). Therefore, it seems that in most reptiles, the astragalus forms by ossiﬁcation of the intermedium and central, whereas in turtles it is formed by the inclusion of additional centralia elements. Differentiation of digital arch cartilages in lizards with well-developed limbs shows a sequence and connectivities in agreement with Shubin and Alberch’s interpretation (1986). There are ﬁve digits and the carpus has ﬁve distal carpalia. Distal carpale 1 was named medial centrale by some authors (Gauthier et al., 1988; Carroll and Currie, 1991; Maisano, 2002a, 2002b), but our observations conﬁrm the identity of this element as part of the digital arch and distal carpal series (Figs. 6 and 7A). Shubin and Alberch (1986) recognized the digit posterior to the primary axis as digit V. The participation of digit V in the primary axis and digital arch has not been discussed and there are few references of the origin of digit V in the literature (Fabrezi and Barg, 2001). Shubin and Alberch (1986) mentioned the differentiation of digit V as a de novo condensation in turtles based on Burke and Alberch (1985). Fabrezi and Barg (2001) described distal carpale 5 and metacarpale V as included in the global morphogenetic events involved in the postaxial autopodium development showing patterns of connectivity between ulnare and the fused distal carpalia 5 and 4 in forelimbs of some species of Neobatrachia. In this study, we found the ﬁrst evidence that digit V develops as part of the digital arch and it is not a de novo condensation in reptiles (Fig. 7). However, this ﬁnding is limited to the right limbs of a single abnormal specimen, and more embryological studies are needed to conﬁrm digit V is part of the digital arch. 909 Digit V and the pisiform were described as de novo condensations by Shubin and Alberch (1986). Different from other primary cartilages of the limb, including those of digit V, the pisiform does not exhibit spatial connections with any condensation of the autopodium. Haines (1969) described it as a sesamoid, and as most sesamoids, it develops into a tendon and links muscle and bone. However, the pisiform is a highly conserved sesamoid associated with the reptilian carpus (Carroll and Currie, 1991). Ossiﬁcation Patterns of Lizard Limbs Morphogenesis of the cartilages of limbs seems to happen during paddle-shaped embryonic stages (this study; Mathur and Goel, 1976; Shapiro, 2002). Ossiﬁcation begins when primary cartilages are fully differentiated. Perichondral ossiﬁcation is characteristic of diaphyses, whereas endochondral ossiﬁcation occurs in carpal and tarsal elements, epiphyses, and sesamoids. Perichondral ossiﬁcations start to calcify before endochondral ossiﬁcations. What type of ossiﬁcation will ensue is related to the shape of the preformed cartilage; in anurans, perichondral calciﬁcation usually occurs in diaphyses, and because proximal tarsals are long, they calcify perichondrally before other tarsal and carpal cartilages. Perichondral calciﬁcation progresses in proximodistal sequence in diaphyses, whereas during advanced embryonic development, the ﬁrst ossiﬁcation centers appear in postaxial carpal and tarsal cartilages (Mathur and Goel, 1976; Rieppel, 1992b; 1993a; Shapiro, 2005). Ossiﬁcation of sesamoid and epiphyses seems to take place at postembryonic stages with variable patterns (Rieppel, 1992a, 1992b, 1993a; Maisano 2002a, 2002b; Shapiro, 2002). Limb development involves cartilage morphogenesis, cartilage growth, and ossiﬁcation. These are developmental processes that may happen simultaneously, but they can be regarded as independent and different (Hinchliffe and Johnson, 1983; Thorogood, 1983). Morphogenesis of cartilages deﬁnes a primary pattern that may be altered by heterochrony and a secondary pattern involving modiﬁcations to primary pattern (Müller, 1991). Secondary modiﬁcations are earlier than ossiﬁcation and thus ossiﬁcation takes place on preformed cartilages. At present, data on skeletal lizard limb development suggest that ossiﬁcation does not deviate from a conservative cartilaginous pattern. Ossiﬁcation sequences may exhibit great variation without consequences on the identity of the elements of the limb plan. We do not agree with Maisano (2002a, 2002b), who stated that the proximodistal gradient, primary axis, and digital arch are not appropriate bases for either predicting postnatal ossiﬁcation sequences or identifying ossiﬁed elements. Interespeciﬁc Variation in Lizard Carpus and Tarsus A similar variation of ossiﬁed carpus/tarsus elements to the one we found in our study is described in the literature. We summarized it as follows. There is a small osseous intermedium between the ulnare and radiale in Ameiva spp. and Cnemidophorus spp. (Fischer and Tanner, 1979), anguids (RenousLécuru, 1973), lacertids (Rieppel, 1992b; Maisano, 2001), Sauromalus, Ctenosaura, and Crotaphytus (Avery and 910 FABREZI ET AL. Fig. 13. Schematic representation of primary chondrogenic foci of lizard limbs. A: In the forelimb, the primary axis and digital arch represent the ﬁrst cartilages to differentiate. Digit V could arise from the digital arch. The radiale is segmented from the radius end. An intermedium condensation was absent in our observations in these embryonic series, but it is present in adults of some species. No connectivity between the only centrale of the carpus and primary axis cartilages has observed. The ossiﬁed carpus in most lizards with pentadactyl forelimbs exhibits this pattern of primary cartilages. Chamaleontidae have reductions that could represent an early modiﬁed pattern. B: In the hindlimb, the primary axis and digital arch represent the ﬁrst cartilages to differentiate. Digit V could have relationships with digital arch. Proximal tarsale is formed by the fusion of ﬁbulare and the condensation of intermedium-centrale. This last develops the ossiﬁcation center named astragalus. Distal tarsalia show a reduced pattern with only distal tarsale 4 and 3 differentiated. In some Gekkonidae, an anterior distal tarsale is present. The ossiﬁed tarsus in most lizards with pentadactyl hindlimb exhibits this pattern. Chamaleontidae have reductions that could represent a modiﬁed developmental pattern. Tanner, 1964), varanids (Rieppel, 1992c), and xantusiids (Maisano, 2002b). Distal carpale 1 is absent in the carpus of Callisaurus draconoides (Maisano, 2002a). The number of distal carpalia/tarsalia that corresponds to a pentadactyl pattern is reduced only in chamaleontids, representing a modiﬁed early limb pattern that includes absence of postaxial ossiﬁcation (ﬁbularecalcaneum) in the tarsus (Gasc, 1963; Renous-Lécuru, 1973; Rieppel, 1993a). The anterior distal tarsale, anterior to distal tarsale 3, is present in species of gekkonids and lacertids (Siebenrock, 1893; Wellborn, 1933; Stephenson, 1960; Mathur and Goel, 1976; Mohammed, 1988; Bauer, 1990). The elements of the primary axis are conserved (Fig. 12) in those lizard taxa with reduced or vestigial limbs (Shapiro, 2003). These data reveal that the lizard carpus and tarsus are conservative in their formation and have strong developmental constraints for which variation is limited. Observed variation, such as the failure of some primary cartilages to segment (e.g., absence of distal carpale 1, absence of the centrale) or the progress in segmentation of distal anterior tarsale, may be interpreted as primary modiﬁcations by heterochrony in respect to those patterns of limb morphogenesis of turtles and crocodiles (Müller, 1991). Variation related to absence of ossiﬁcation in primary cartilages does not imply a modiﬁed limb pattern, but rather a modiﬁcation of the sequence of ossiﬁcation. The identiﬁcation and determination of equivalent entities is the basis of any comparative analysis. Morphogenesis of cartilages underlies and determines the form and identity of a large part of the endoskeleton of vertebrates. The cartilaginous primordium provides the structural template for subsequent ossiﬁcation in the bones that arise by endochondral and perichondral ossiﬁcation (Thorogood, 1983). Shubin and Alberch (1986) described the cartilage morphogenesis of the tetrapod limb and proposed a scheme of spatial connections among primary cartilaginous condensations, providing a tool to interpret the skeletal elements of limbs. From this framework, we have analyzed some aspects of lizard limb development, and we found some evidence in agreement with data presented in primary literature to answer the following questions. One, is there an intermedium in the lizard carpus? Adults of some taxa present an ossiﬁed element placed between the ulnare and radiale that may be interpreted as the intermedium, but embryological evidence is weak and more taxonomically comprehensive examination of limb development at paddle-shaped embryonic stages is needed to conﬁrm its identity. Two, are there two centralia in the lizard carpus? No, the carpal previously identiﬁed as a medial centrale (Gauthier et al., 1988; Carroll and Currie, 1991; Maisano, 2002a, 2002b) is identiﬁed herein as distal carpale 1. There is a single centrale in the lizard carpus. LIMB DEVELOPMENT IN LIZARDS Three, is there homology among the lizard proximal tarsalia, and the same cartilages in turtles and crocodiles? Yes, lizards share with other reptiles the presence of a cartilaginous condensation of the ﬁbulare and an anterior condensation representing the intermediumcentrale. Four, does digit V belong to the embryonic series of primary cartilages of the digital arch? We present evidence of patterns of connectivity between digit V and digital arch in lizard’s fore- and hindlimbs. However, more embryological studies supporting digit V forms part of the primary axis are needed. Five, is the pisiform an element of the autopodium plan? No, the pisiform is one of the numerous sesamoids that develop without spatial connectivities with the primary cartilages of the limb. Six, should the patterns of ossiﬁcation be similar to cartilage morphogenesis? No, cartilage morphogenesis and ossiﬁcation are different. The pattern of morphogenesis of cartilages deﬁnes the cartilaginous primordia in which ossiﬁcations occur, but the ossiﬁcation sequences seem to have variable patterns. The later requires further examination. At present, the morphogenetic approach of Shubin and Alberch (1986) provides a satisfactory hypothesis to interpret the origin and identiﬁcation of the skeletal elements of lizard limbs. On this basis, we depicted a particular pattern of primary cartilage morphogenesis for fore- and hindlimbs in lizards, from which most variations known in ossiﬁed elements can be interpreted using developmental arguments (Fig. 13). Nevertheless, we emphasize that these conclusions need a more taxonomically comprehensive examination of lizard limb ontogeny. ACKNOWLEDGMENTS The authors are grateful to two anonymous reviewers for comments, criticisms, and suggestions made on the submitted draft of this article. They also thank Dr. Natalia von Ellenrieder for reading and improving the English. Specimens referred to in this study were kindly provided by Marta Cánepa and Sonia Ziert (Instituto de Herpetologı́a, Fundación Miguel Lillo, Tucumán, Argentina), Juan Daza (personal collection of Dr. Thomas), Paulo Vanzolini (Museo de Zoologı́a, San Pablo, Brazil), and the technical staff of Museo de Ciencias Naturales (Universidad Nacional de Salta, Argentina). 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Vorobyeva EI, Hinchliffe RJ. 1996. Developmental pattern and morphology of Salamandrella keyserlingii limbs (Amphibia, Hynobiidae) including some evolutionary aspects. Russ J Herpetol 3:68–81. Wassersug RJ. 1976. A procedure for differential staining of cartilage and bone in whole formalin ﬁxed vertebrates. Stain Technol 51:131–134. Wellborn V. 1933. Vergleichende osteologishe Untersuchungen an Geckoniden, Eublephariden und Uroplatiden. Sber Ges Nat Freunde Berlin 1–3:126–199. APPENDIX ABBREVIATIONS OF MUSEUMS FML: Instituto de Herpetologı́a, Fundación Miguel Lillo, Tucumán, Argentina. MCN: Museo de Ciencias Naturales, Universidad Nacional de Salta, Argentina. MZUSP: Museo de Zoologı́a, San Pablo, Brazil. RT: private collection of Richard Thomas. SPECIMENS EXAMINED Anguidae: Ophiodes sp. (FML 2000, one adult specimen); Ophiodes striatus (FML 2001, one adult specimen). Chamaleontidae: Chamaleo unicornis (MCN 2151, one adult specimen). Gekkonidae: Bogertia lutzae (MZUSP 54747, one adult specimen); Garthia gaudichaudii (MZUSP 45329, one adult specimen); Garthia penai (MZUSP 60938, one adult specimen); Hemidactylus mabouia (FML 2421, two adult specimens); Homonota borelli (FML 933, two adult specimens; FML 2409, one adult specimen; FML 2414, one adult specimen); Homonota darwini (FML 794, two adult specimens); Homonota fasciata (FML 062, one adult specimen; FML 1495, two adult specimens; FML 777, one adult specimen; FML 1751, one adult specimen; FML 2415, one adult specimen); Homonota underwoodi (FML 1310, one adult specimen; FML 1490, one adult specimen; FML 2031, two adult specimens); Homonota uruguayensis (FML 1746, two adult specimens); Homonota whitii (FML 3547, one adult specimen); Phyllodactylus gerrhopygus (FML 1563, two adult specimens); Phyllopezus pollicaris (FML 2813, two adult specimens); Thecadactylus rapicauda (MZUSP 11476, one adult specimen); Sphaerodactylus sp. (RT 13875-13848-1385513873-14137, ﬁve adult specimens); Vanzoia klugei (MZUSP 59135, two adult specimens). Gerrhosauridae: Gerrhosaurus nigrolineatus (MCN 2148, one adult specimen). Gymnophthalmidae: Pantodactylus schreibersii (FML 731, two adult specimens). Lacertidae: Podarcis sicula (FML 03714, one adult specimen); Poromera fordi (MCN 2149, one adult specimen). Leiosauridae: Leiosaurus paronae (FML 00035, one adult specimen); Leiosaurus catamarcensis (FML 00670, one adult specimen); Leiosaurus sp. (FML 7496, one adult specimen); Pristidactylus achalensis (FML 61816182, two adult specimens). Liolaemidae: Liolaemus bitaeniatus (FML 2441, one adult specimen); Liolaemus cuyanus (FML 1803, one adult specimen); Liolaemus dorbingy (FML 1757, one adult specimen); Liolaemus multicolor (FML s/n, seven juvenile specimens, between 5–7 days posthatched; MCN 2142, posthatched specimen 29.8 mm SVL; MCN 2143, juvenile specimen 42.4 mm SVL; MCN 2144, juvenile specimen, 40.8 mm SVL; MCN 2141, juvenile specimen 48.4 mm SVL; MCN 2140, adult specimen; MCN 2152, embryo at stage 32; MCN 2153, embryo at stage 34; MCN 2154, embryo at stage 3; MCN 2155, embryo at stage 40); Liolaemus cf quilmes (FML 1227, one adult specimen); Liolaemus quilmes (FML s/n, eight embryos at stages 30, 32, 34, 35, and 36; FML 1227, one adult specimen); Liolaemus ramirezae (FML 1228-2288, two adult specimens); Liolaemus ruibali (FML s/n, eight juvenile specimens, between 5 and 7 posthatched); Liolaemus scapularis (FML 1846, one adult specimen); Liolaemus zullyi (MCN 2139, embryo at stage 39). Polychrotidae: Polychrus acutirostris (FML 48156, one adult specimen). Scincidae: Mabuya dorsivittata (FML 0896-1979, two adult specimens); MCN 2150 Panaspis breviceps (FML 0896-1979, one adult specimen). Teiidae: Cnemidophorus longicauda (FML 1619, one adult specimen), Kentropix lagartija (FML 1186, two adult specimens), Kentropyx viridistriga (FML 1204, two adult specimens), Teius cyanogaster (FML 02941978, two adult specimens), Tupinambis merianae (FML s/n, two embryos at stages 49 and 50; FML s/n, three embryos at stages 33, 37, and 40); Tupinambis rufescens (FML 2559, one adult specimen). Tropiduridae: Tropidurus etheridgei (FML 1984–1986, two adult specimens); Tropidurus melanopleurus (FML 2055, one adult specimen).