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Home , Amphisbaenia, Amphisbaenidae, Anadia ocellata, Blanus, Colobodactylus dalcyanus, Colobodactylus taunayi

THE ANATOMICAL RECORD 297:482–495 (2014)

Are Hemipenial Spines Related to Limb Reduction? A Spiny Discussion Focused on Gymnophthalmid (: )

PEDRO M. SALES NUNES,* FELIPE F. CURCIO, JULIANA G. ROSCITO, AND MIGUEL T. RODRIGUES Departamento de Zoologia, Instituto de Biociencias,^ Universidade de Sao~ Paulo, Caixa Postal 11.461, CEP 05422-970, Sao~ Paulo, SP,

ABSTRACT Calcified spines in the hemipenial surface occur convergently in sev- eral gymnophthalmid and in advanced . Based on the pronounced degrees of limb reduction in these distantly related line- ages, such hemipenial structures were suggested to play a functional role in couple-anchoring during , partly assuming the function of the limbs during mating. Herein, we assessed the hemipenial morphology of virtually all the valid genera of the family Gymnophthalmidae to test for a phylogenetic correlation between limb reduction and the presence of calcified hemipenial spines. The occurrence of calcified structures was mapped on the two most comprehensive phylogenies of the family. We concluded that spiny hemipenes are by no means necessarily associated with reduction of limbs. Conversely, the presence of well-developed hemi- penial spines in specific limb-reduced taxa does not allow one to disregard the possibility that in some instances such structures might indeed be functionally associated with couple-anchoring, improving the success of mating. Anat Rec, 297:482–495, 2014. VC 2014 Wiley Periodicals, Inc.

Key words: lepidosaurs; hemipenis; ornamentation; limb reduction; copulation

INTRODUCTION Limb reduction, defined as the partial or complete phylogenetic loss of skeletal elements of the limbs rela- tive to a typical four-limbed, pentadactyl condition (Greer, 1991; Shapiro, 2002), is widespread in the evolu- Additional Supporting Information may be found in the online version of this article. tion of Tetrapoda. Different degrees of limb reduction ~ are particularly common in the order Squamata, with Grant sponsor: Fundac¸ao de Amparo a Pesquisa do Estado de Sao~ Paulo (FAPESP); Grant numbers: 2011/50146-6, 2012/ several independent events among its lineages (Greer, 00492-8, 2012/00547-7, 2012/01319-8; Grant sponsor: Conselho 1991; Wiens et al., 2006; Skinner, 2008). Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq). The most pronounced instances of fore and/or hind *Correspondence to: P. M. S. Nunes; Departamento de Zoo- limb reduction in squamates (complete limblessness or logia, Instituto de Biociencias,^ Universidade de Sao~ Paulo, partial reduction affecting functionality) occur in the Caixa Postal 11.461, CEP 05422-970, Sao~ Paulo, SP, Brazil. major Serpentes and , as well as in Fax: 155 11 3091 75 13. [email protected] the lizard families , Annielidae, , Received 25 February 2013; Accepted 23 May 2013. , Gymnophthalmidae, , and Scin- DOI 10.1002/ar.22876 cidae. Among lizards, the vast majority of reduction Published online 31 January 2014 in Wiley Online Library events occur in the Scincidae [16 to 20 events (Miralles (wileyonlinelibrary.com).

VC 2014 WILEY PERIODICALS, INC. ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 483 et al., 2012) or 27 events only among Lerista (Skinner of Anguis fragilis, an assumption likely based on the et al., 2008)]. Another group showing multiple events of descriptions of Cope (1896) and on an illustration by limb reduction is the Gymnophthalmidae. According to Bohme€ (1988), since they provide no evidences that such Pellegrino et al. (2001), at least five independent events structures are in fact calcified. The absence of any traces of limb reduction have occurred within the Gymnoph- of calcified structures in the hemipenis of other Angui- thalmidae: one in the subfamily Rachisaurinae (Rachi- dae (Thomas and Hedges, 1998; personal observation), saurus brachylepis); two in the subfamily even after staining tests with Alizarin Red solution casts Gymnophthalminae (tribes Gymnophthalmini and Het- further doubts upon their work and leaves the question erodactylini); and two in the subfamily Cercosaurinae open for further investigation. [genera (subfamily Epleopodinae, sensu The remarkable degrees of limb-reduction seen in Castoe et al., 2004) and (tribe Bachiini, sensu snakes and in some gymnophthalmids, combined with Castoe et al., 2004)] (see Pellegrino et al., 2001; Castoe the presence of calcified hemipenial spines in both et al., 2004; Rodrigues and Dos Santos, 2008; Rodrigues groups, may have led Presch (1978) to associate the com- et al., 2009 for systematic accounts). plexity in hemipenial ornamentation with dramatic Limb reduction is usually associated with body elon- instances of limb reduction and/or total limblessness. gation, resulting in a -like body form that is often This assumption is based on the premise that, in the related to , sand-dwelling, or grass-dwelling absence of well-developed limbs, other anatomical struc- habits (Caputo et al., 1995; Lee, 1998; Wiens and Sling- tures (i.e., hemipenial spines) could take on the role of luff, 2001). In addition, such snake-like morphology keeping male and female tightly attached while mating. implies in biomechanical and physiological adaptations However, with respect to gymnophthalmids, Presch’s of most body systems (Underwood, 1976; Caputo et al., (1978) observations were not supportive of such a 1995). However, there is only a handful of revisions straightforward rationale, since he reported virtually about modifications of the genital system and/or repro- nude hemipenes both in taxa with well-developed limbs ductive behavior of squamates and its correlation with (e.g., species of the genera , , Gym- limb reduction (e.g., Presch, 1978; Olsson and Madsen, nophthalmus, , and ), as well as in 1998; Sanchez-Mart ınez et al., 2007). two greatly limb-reduced species of the Bachia (B. The use of genital morphology as an indication of kin- intermedia and B. trisanale). Posterior studies (Myers ship (Dowling, 1957; Arnold, 1986b; Nunes et al., 2012) and Donnelly, 2001; Nunes, 2011) revealed the presence is mostly supported by evidence from sexual selection of calcified spicules embedded in the hemipenial flounces (Eberhard, 1985, 2010). In contrast, most functional of the fully limbed species , which were interpretations of hemipenial ornaments [e.g., mechani- unnoticed by Presch (1978), most likely due to prepara- cal stimulation (King et al., 2009), increase of copula tion artifacts. Thus, Presch’s (1978) observations sug- duration (King et al., 2009; Olsson and Madsen, 1998), gested an opposite pattern to the one predicted in his gaping of female (Pisani, 1976) and lifting of the first assumption, with limb-reduced taxa showing poorly anal plate (Pisani, 1976)] are widely speculative. Even ornamented organs, whereas conspicuous ornaments though, there seems to be a widespread assumption that were common among taxa with well-developed limbs. such structures must be directly involved in couple- Despite the inconclusiveness of Presch’s (1978) results, anchoring during the sexual act to ensure efficient the putative relationship of limblessness with complex sperm transfer (Pope, 1941; Edgren, 1953; Pisani, 1976; hemipenial ornamentation deserves further attention for Murphy and Baker, 1980). the following reasons: (i) Presch’s (1978) sampling was far Calcified hemipenial spines and spicules are appa- from comprehensive, including only one of the limb- rently restricted to some gymnophthalmid lizards (e.g., reduced gymnophthalmid lineages (i.e., the tribe Bachiini, Uzzel, 1965; Estes et al., 1988; Nunes, 2011; Nunes sensu Castoe et al., 2004); and (ii) the family Gymnoph- et al., 2012) and several representatives of a major line- thalmidae is especially informative for studies concerning age of snakes [Colubroides sensu Zaher et al. (2009)]. the “by-products” of limblessness because the several Calcified hemipenial structures have also been reported reduction events can be traced in the two recent phyloge- for other squamate groups, but the homology and gen- nies (Pellegrino et al., 2001; Castoe et al., 2004). There- eral structure of such elements are debatable. For exam- fore, if the patterns of hemipenial ornamentation are ple, Arnold (1973, 1986a) mentions a possible seasonal confronted with well-supported phylogenetic trees, one presence of minute spines in the , but this can directly assess the possible hierarchic relationship observation has received no further attention. In addi- between limb morphology and hemipenial ornamentation. tion, distinct calcified hemipenial structures have been In this study, we dedicate special attention to hemipe- reported in some (McDowell and Bogert, nial spines. A recent study on the hemipenial morphol- 1954; Ziegler et al., 2007; Koch et al., 2009; Welton ogy of the Gymnophthalmidae allowed the unequivocal et al., 2010), and (Kluge, detection of distinct patterns of calcified spines and spi- 1982; Rosler€ and Bohme,€ 2006) representing internal cules in the hemipenes of several genera of the family skeletal structures referred to as a hemibaculum,as (Nunes, 2011). Based on the considerable variation in well as apical horns acting as extensions of the retractor size and organization of such structures among the gym- muscles (Branch, 1982). Observations of hemipenial nophthalmid taxa presenting variable degrees of limb spines in Chamaeleonidae (Olsson and Madsen, 1998) reduction, we reassess the hypothesis of Presch (1978), are intriguing, considering that descriptions and illus- regarding the phylogenetic congruence between major trations of the hemipenis of several made by reductions in limb morphology, including complete loss Klaver and Bohme€ (1986) provide no indication of the of the limbs, and the presence of hemipenial spines, in presence of such structures. Finally, Olsson and Madsen light of the current phylogenetic hypothesis for the (1998), mention the presence of spines in the hemipenis family. 484 NUNES ET AL.

Fig. 1. (A) Sulcate, lateral, and asulcate views of the hemipenis of panamensis (KU 80584). Arrow 1 indicates a hook-shaped spine; arrow 2 indicates flounces bearing minute spicules (“comb-like flounces”). Scale bar 5 3 mm; (B) Live specimen of E. panamensis (Photo: Sara Ruane).

MATERIAL AND METHODS shape, and number of ornaments, represents important taxonomic information and will be addressed in more We examined the hemipenes of the vast majority of appropriate contexts of taxonomic and/or systematic valid genera of Gymnophthalmidae, including all taxa approaches. with limb reduction. Additionally, we also analyzed the hemipenes of some representatives of Serpentes and Amphisbaenia, as well as selected taxa of the lizard fam- RESULTS ilies Anguidae, Anniellidae, and Scincidae (see Support- Comments on the Hemipenial Morphology of ing Information Appendix I for a list of the specimens the Gymnophthalmidae examined). Our observations were complemented by an extensive survey of the literature and, in some cases, The results presented below represent our own obser- such data represented the sole information regarding vations combined with a collection of literature data the hemipenial morphology of taxa to which we had no regarding calcified structures (e.g., spines and spicules) access to (e.g., Dibamidae; see Darevskyi, 1992). We in gymnophthalmid lizards and in five other squamate could not obtain any information about the hemipenial groups with limb-reduced representatives (i.e., Ser- morphology of limb-reduced species of the families Pygo- pentes, Amphisbaenia and the lizard families Anguidae, podidae, , and of the genus Chamaesaura Anniellidae, Dibamidae, and, Scincidae). (family Cordylidae). We found no evidence of the presence of calcified hem- Hemipenial preparations follow a combination of the ipenial structures being exclusive to limb-reduced spe- techniques proposed in earlier studies (Manzani and cies among the 46 gymnophthalmid genera sampled (out Abe, 1988; Pesantes, 1994; Zaher, 1999; Zaher and Pru- of the 47 valid genera so far). Enlarged hemipenial body dente, 1999), added by a staining step (immersion of the spines occur both in genera with well-developed limbs everted organ in a 70% alcoholic solution of Red Alizarin (e.g., Echinosaura) and in limb-reduced taxa (e.g., Calyp- for 24 hr) used to enhance calcareous structures and tommatus); in contrast, poorly ornamented or even virtu- improve their detection (see Uzzell, 1973; Harvey and ally nude hemipenis occur in genera, such as Embert, 2008; Nunes et al., 2012). The retractor muscle Amapasaurus and , which have well-developed was manually separated and the everted organ filled limbs, as well as in some representatives of the long- with a mixture of stained petroleum jelly and melted tailed genus Bachia, which displays an extreme degree paraffin (liquefied). The hemipenes of currori of limb reduction. (Scincidae) and Anniella pulchra [Annielidae, sensu Fully ornamented hemipenis, with large and hook- Uetz (2013)] could not be manually everted; alterna- shaped body spines, as well as series of minute calcified tively, the inverted organs were longitudinally dissected spicules associated with hemipenial flounces, are present to allow access to their external surface, and stained in the genus Echinosaura (three species examined accordingly. The terminology of hemipenial structures herein) (Fig. 1; Uzzell, 1965) and in Teuchocercus keyi. follows Dowling and Savage (1960), Klaver and Bohme€ Judging from their shape and general organization, it is (1986), Savage (1997), and Myers and Donnelly (2001, possible that such structures are functionally associated 2008). with the reinforcement of couple-anchoring, independent Information regarding the presence/absence of calci- of the role of limbs during the sexual act. Similar exam- fied structures in the hemipenes of Gymnophthalmidae ples among gymnophthalmids are the genera Cerco- were mapped onto the phylogenies of Pellegrino et al. saura, , , Riama, and Proctoporus, (2001) and Castoe et al. (2004) with the aid the soft- as well as some species of (A. reticulata, A. ware Mesquite v. 2.75 (Maddison and Maddison, 2011). kocki) (C. modesta), and (L. scin- The analysis simply illustrates the presence or absence coides, L. southi and L. rugiceps). of calcified hemipenial spines in the distinct gymnoph- Conversely, in the monotypic taxon Rachisaurus bra- thalmid lineages. Nonetheless, one must emphasize chylepis (Rachisaurinae) as well as in all the representa- that the considerable variation reported regarding size, tives of the limb-reduced genus Bachia (Cercosaurinae: ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 485

Fig. 2. (A) Sulcate and asulcate views of the hemipenis of Bachia scaea (MZUSP 103414) Scale bar 5 1 mm; (B) Live specimen of B. scaea (Photo: Renato Gaiga).

Fig. 3. (A) Sulcate and asulcate views of the hemipenis of Rhachisaurus brachylepis (MZUSP 78569) Scale bar 5 1 mm; (B) Live specimen of R. brachylepis (Photo: Mauro Teixeira-Jr).

Bachiini; 12 species examined) present very poorly orna- Psilophthalmus, bear no hook-shaped hemipenial spines, mented hemipenes, lacking spines, spicules, and any but exhibit different ornamentation patterns such as vestiges of calcified structures (Figs. 2, 3), [see also finger-like and noncalcified papillae (e.g., Gymnophthal- Presch (1978), Rodrigues et al. (2008), and Teixeira Jr. mus vanzoi and G. pleei), flounces modified into calcified et al. (2013)]. In Bachia, nude hemipenial flounces occur laminae (e.g., speciosus, in B. cophias, B. flavescens, B. heteropa, and B. scole- agilis, T. bifasciatus, and T. oriximinensis), or completely coides, whereas the organs of B. bresslaui, B. dorbignyi, nude and roughly circular flounces (e.g., Micrablepharus B. oxyrhina, and B. scaea are completely unornamented. spp., Procellosaurinus spp., and rubricauda). The tribe Gymnophthalmini (subfamily Gymnophthal- The genera and both minae; Pellegrino et al., 2001; Castoe et al., 2004; allocated in the tribe Heterodactylini (following Pelle- Rodrigues and Dos Santos, 2008) includes genera with grino et al., 2001), together with the genus Caparaonia variable degrees of limb reduction. The fossorial and (Rodrigues et al., 2009), represent another limb-reduced remarkably limb-reduced taxa , Nothoba- radiation within Gymnophthalmidae. However, limb chia, and Scriptosaura, have enlarged and hook-shaped reduction in this group is not as remarkable as is in calcified hemipenial spines (Figs. 4, 5). Calcified spines some Gymnophthalmini and in the tribe Bachini, and are also present in the four-limbed genus Psilophthalmus, the Heterodactylini species show only minor reductions although not as developed as those of the previously men- in phalanx number and/or size. tioned Gymnophthalmini. The remaining gymnophthal- Among the Heterodactylini, the presence of calcified mini genera, with limb morphology similar to that of structures is subjected to intrageneric variation. Minute 486 NUNES ET AL.

Fig. 4. (A) Sulcate, lateral and asulcate views of the hemipenis of Calyptommatus leiolepis (MZUSP 71149). Arrow 1 indicates a hook-shaped spine. Scale bar 5 1mm;(B) Live specimen of C. leiolepis (Photo: Mauro Teixeira-Jr).

Fig. 5. (A) Sulcate, lateral, and asulcate views of the hemipenis of ablephara (MZUSP 70275). Arrow 1 indicates a hook-shaped basal spine. Scale bar 5 1mm;(B) Live specimen of N. able- phara (Photo: Mauro Teixeira-Jr). calcified spines are detected on the extremities of some according to Rodrigues et al. (2005)], and flounces of the hemipenial body of Heterodactylus imbri- cearensis (sister taxon of the composed by the two catus (Fig. 6A,B), whereas the hemipenis of H. lundi former genera) (Pellegrino et al., 2001; Castoe et al., 2004; (Fig. 6C,D) lack any traces of calcified structures. Simi- Rodrigues et al., 2005) exhibit stout and short limbs when larly, shows minute spines on compared to the remaining Ecpleopodini genera [i.e., the hemipenial flounces (Fig. 7A,B), while C. dalcyanus Arthrosaura, Ecpleopus, Leposoma, and probably Amapa- has a virtually nude hemipenis (Fig. 7C,D). We found no saurus, Kaieteurosaurus, Marinussaurus,andPantepui- calcified structures in Caparaonia itaiquara. saurus (Pellegrino et al., 2001; Kok, 2005, 2009; Peloso Calcified spicules on the hemipenial body flounces et al., 2011)]. Despite differences in size and position of the were detected in the five Iphisini genera (sensu Rodrigues hemipenial lobes, as well as in the general shape of the et al., 2009), which also present minor reductions of hemipenial body, the hemipenes of limbs, mainly represented by a decrease in phalanx size. (Fig. 8), nordestina (see Rodrigues et al., In these genera, the hemipenial spicules are organized in 2005) and Colobosauroides cearensis share the presence of rows along the flounces, in an arrangement referred to in small and isolated calcified spines at the margin of the the literature as “comb-like” flounces (Myers and Don- transversal body flounces. Dryadosaura nordestina also nelly, 2001; Rodrigues et al., 2005, 2007). presents series of mineralized comb-like spicules through- Among the tribe Ecpleopodini, Anotosaura spp., Dryad- out the body flounces, an unusual characteristic among osaura nordestina [sister taxa of Anotosaura vanzolinia, members of the tribe, which is only shared with three ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 487

Fig. 6. Sulcate, lateral, and asulcate views of the hemipenes of (A) Heterodactylus imbricatus (MD 3405) and (C) H. lundii (AMNH 131871). Scale bars 5 1 mm; Live specimens of (B) H. imbricatus (Photo: Renato Gaiga) and (D) H. lundii (Photo: Jose Cassimiro). species of Arthrosaura (A. reticulata, A. synaptolepis, and advanced and highly diverse clade Colubroides (sensu A. montigena). The recently rediscovered species Anoto- Zaher et al., 2009). Hemipenial spines of snakes are saura collaris differs from the congener A. vanzolinia by scattered throughout the hemipenial body and vary exhibiting the hemipenis ornamented with nude flounces greatly in size. Some authors suggest that such struc- lacking any vestige of calcified structures (see Rodrigues tures are functionally related to couple-anchoring, as et al., 2013). Conversely, the Ecpleopodini taxa with well- well as to the positioning of the hemipenis for sperm developed limbs Adercosaurus vixadnexus (sensu Myers transfer (Pope, 1941; Edgren, 1953; Pisani, 1976; Mur- and Donnelly, 2001), Amapasaurus , and phy and Baker, 1980). some species of the Leposoma parietale group (sensu Rui- The direct examination of several colubroid genera (e.g., bal, 1952; Rodrigues, 1997) exhibit poorly ornamented Apostolepis, Boiruna, Erythrolamprus, Helicops, Hydrops, hemipenes, with nude flounces sparcely distributed along Philodryas, Pseudoeryx, Sordellina, Xenodon—see Sup- the hemipenial body, whereas Ecpleopus gaudichaudii has porting Information Appendix I for a list of the species completely nude hemipenes (Uzzell, 1969; Myers et al., analyzed), combined with literature data (e.g., Mao et al., 2009; Nunes, 2011). In contrast, species of the Leposoma 1984; Zaher, 1999; Harvey and Embert, 2008; Silva and scincoides group and some species of Arthrosaura (A. guia- Rodrigues, 2008; Guo et al., 2009; Klaczko et al., 2010; nense and A. kocki) show isolated calcified spines on the Montingelli et al., 2011; Zaher et al., 2012) corroborates border of each hemipenial body flounce. the widespread occurrence of hemipenial spines in the lin- eage. In contrast, more basal radiations of the Serpentes, Comments on the Hemipenial Morphology of such as Cylindrophiidae (Stuebing, 1994), Leptotyphlopi- dae (Passos et al., 2005, 2006; Pinto and Curcio, 2011; Other Limb-Reduced Squamata Pinto and Fernandes, 2012), (Passos and Fer- Serpentes. Among Serpentes, the presence of calci- nandes, 2008), and (Stull, 1928; Gibson, fied spines and spicules are confirmed only in the 1970; Curcio et al., 2012) seem to lack calcified ornaments. 488 NUNES ET AL.

Fig. 7. Sulcate, lateral, and asulcate views of the hemipenes of (A) Colobodactylus taunayi (MZUSP 91447) and (C) C. dalcyanus (MZUSP 95600). Scale bars 5 1 mm. Live specimens of (B) C. taunayi (Photo: Renato Gaiga) and (D) C. dalcyanus (Photo: Pedro H. Bernardo).

Fig. 8. (A) Sulcate and asulcate views of the hemipenis of Anotosaura vanzolinia (MZUSP 95328) Scale bar 5 1 mm; (B) Live specimen of A. vanzolinia (Photo: Renato Gaiga). ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 489

Fig. 9. (A) Sulcate and asulcate views of the hemipenis of brasiliana (TM 180) Scale bar 5 1 mm; (B) Live specimen of A. brasiliana (Photo: Christine Strussmann).€

Hemipenial ornamentation in basal snake lineages is and Anguis fragilis) have been previously described or mostly represented by flounces, papillae, and/or calyces, illustrated (Bohme,€ 1988). Those of A. fragilis present as well as modified structures that may also aid in couple- spine-like structures, but it is not possible to recognize anchoring (e.g., the “heart-shaped” papillate structures the presence of calcified structures only by examining described in some tropidophiids; see Curcio et al., 2012). the illustrations provided in Bohme’s€ work (1988). The report of seven to nine spines on the asulcate face of The analyses of hemipenes of Ophiodes fragilis (a the hemipenes of reticulatus (see Dixon and Hen- limb-reduced anguid; Fig. 10) and Diploglossus fasciatus dricks, 1979) demonstrates that poorly studied snake taxa (an anguid lizard with no reduction of limbs; Fig. 11) (e.g., ) deserve further attention regarding confirmed the presence of pleated hemipenial flounces hemipenial morphology. (Cope, 1896; Thomas and Hedges, 1998; Balej and Jablonski, 2006) in both taxa, but no calcified structures Amphisbaenia. The general structure of amphis- were detected after Alizarin Red staining (not performed baenian hemipenes is much simpler than that of the vast in any of the previous studies). Therefore, the presumed majority of other squamates. The direct examination of presence of spines [Thomas and Hedges (1998); the hemipenis of Amphisbaena brasiliana (Fig. 9), A. “recurved osseous spines”, sensu Cope (1896); “spinous cuiabana, A. mertensi, and A. microcephala, coupled with hemipenis”, sensu McDowell and Bogert (1954)] was not sparse literature information (Rosenberg, 1967; Broadley supported by our data. et al., 1976; Gans, 1978; Bohme,€ 1989; Rosenberg et al., In Diploglossus, pleated flounces cover the entire 1991; Pinna et al., 2010; Pinna, 2012), brings no evidence hemipenis [Fig. 11; Thomas and Hedges (1998 – Fig. 4)], of calcified structures. Ornamentation of amphisbaenian whereas in Ophiodes fragilis they are restricted to lobes hemipenes are either entirely absent, resulting in com- and to the distal end of the hemipenial body (Fig. 10). pletely nude organs [e.g., Amphisbaena carli (Pinna Regardless of the absence of spines or spicules, the gen- et al., 2010), A. bolivica (Pinna, 2012) and canali- eral structure of the flounces is highly suggestive of a culatus (Bohme,€ 1989)], or is restricted to the presence of couple-anchoring role during mating. flounces on the body and/or lobes [e.g., A. caudalis (Thomas and Hedges, 2006), A. microcephala, A. tra- Annielidae. Literature data on annielid hemipenes chura (Pinna, 2012), galeata (Cope, 1896), are scarce: Coe and Kunkel (1904) illustrated the partly Trogonophis wiegmanni (Bohme,€ 1989)]. Some reports in everted organ of an embryo of Anniella pulchra, and Cope the literature point the presence of hemipenial lobes dis- (1896) provided a brief description of the hemipenis of an tinctly curved or perpendicular to the hemipenial body, unidentified Anniella specimen. We were unable to prop- such as that seen in A. brasiliana (Fig. 9; Pinna, 2012), erly prepare the hemipenis of the single specimen of A. A. cuiabana (Strussmann€ and Carvalho, 2001), A. inno- pulchra (MZUSP 2227) available for examination; alter- cens (Rosenberg et al., 1991), A. schmidti (Rosenberg, natively, we performed a longitudinal dissection of the 1967), and cinereus (Rosenberg et al., 1991). In organ that allowed us to examine its external surface. The other instances, there are reports of lateral papillae posi- Alizarin Red staining failed to show any particular calci- tioned as secondary lobes, as is the case for A. kingi fied structures in this specimen, suggesting that such ele- (Rosenberg, 1967; Vanzolini, 1999). Lamellate tips are ments must be absent. In addition, like Coe and Kunkel documented in some species of Amphisbaena (Rosenberg (1904), we were unable to detect vestiges of other orna- et al., 1991; Thomas and Hedges, 2006; Pinna, 2012), ments, such as the wrinkled body flounces described by pistillum (Broadley et al., 1976), and Monopel- Cope (1896). However, we regard our data as inconclusive tis sphenorhynchus (Rosenberg et al., 1991). due to the absence of a well-prepared hemipenis.

Anguidae. Hemipenes of some limb-reduced Dibamidae. The data on hemipenial morphology of anguids (e.g., spp., Ophiodes intermedius, dibamids rely strictly on a brief description of the 490 NUNES ET AL.

Fig. 10. (A) Sulcate and asulcate views of the hemipenis of Ophiodes fragilis (MZUSP 45812) Scale bar 5 3 mm; (B) Live specimen of O. fragilis (Photo: Renato Gaiga).

Fig. 11. (A) Sulcate and asulcate views of the hemipenis of Diploglossus fasciatus (MZUSP 89131) Scale bar 5 3 mm; (B) Live specimen of D. fasciatus (Photo: Marco Sena). hemipenis of the holotype of greeri (Darevsky, (Greer, 1979: Fig. 15), as well as minor additional infor- 1992); in addition, the organ is not fully everted and no mation on other Australian given in the same staining tests were performed for detecting calcareous paper. The illustration provided by Greer (1979) reveals structures. The illustrations of the anal region of D. greeri a completely nude organ with no evident ornaments in (Darevsky, 1992: Figs. 8, 11) suggest a very reduced organ the hemipenial body or lobes. We had access to a male (1.1 mm length and 0.4 mm width, according to the origi- specimen of the limbless Feylinia currori (MZUSP nal description), and the absence of ornaments is incon- 97826), but the small size of its hemipenis prevented clusive given that eversion is not complete. Hence, further proper preparation. Nonetheless, the dissection of the analyses are needed in order to access the diversity of organ and the staining test revealed no traces of calci- hemipenial morphology in dibamid lizards. fied structures, following the condition seen in limb- developed skinks for which hemipenial morphology is known (Cope, 1896; Greer, 1979; Linkem et al., 2011; C. Scincidae. There are virtually no descriptions of Linkem pers. comm.). hemipenes among the more than 15 remarkably limb- reduced genera (according to Miralles et al., 2012). DISCUSSION The single record on hemipenial morphology of a limb- reduced skink relies on a brief characterization and a Despite scattered evidence on anguid lizards (Bohme,€ single illustration of the hemipenis of Lerista terdigitata 1988) and scolecophidians (Dixon and Hendricks, 1979), ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 491

Fig. 12. The presence of calcified structures on the hemipenes mapped onto the topologies generated by (A) Pellegrino et al. (2001: Fig. 3) and (B) Castoe et al. (2004: Fig. 6). our results reinforce the assumption that calcified orna- associated with limb reduction, one should expect their ments are circumscribed to advance snakes (Colubroides) predominant occurrence in the limb-reduced squamate and gymnophthalmids. Moreover, the significant degree taxa. of limb reduction seen in both lineages is tempting to Observations from the present study, together with associating this feature with the presence of calcified most literature data, do not support such a relationship. spines as an apomorphic trait related to couple- Basal snake lineages, all known amphisbaenians, and anchoring, following Presch’s hypothesis (Presch, 1978). several limb-reduced lizards lack any traces of calcified However, our comprehensive sampling of the Gymnoph- spines or spicules. In contrast, extremely developed, thalmidae, combined with data for other squamate hook-shaped and calcified spines are present in gym- groups, suggests that such an association may be exceed- nophthalmid genera with well-developed limbs, such as ingly simplistic. Teuchocercus and Echinosaura (Fig. 1). In addition, Even in the absence of specific morpho-functional despite lacking spines, the pleated flounces of anguid liz- approaches, the general structure of calcified hemipe- ards are also suggestive of couple-anchoring and are nial ornaments is, in itself, highly suggestive of a present both in the limbed genus Diploglossus (Fig. 11) couple-anchoring role. Nonetheless, if the presence of and in the limb-reduced genus Ophiodes (Fig. 10), sug- calcified structures was in any way phylogenetically gesting that the development of couple-anchoring 492 NUNES ET AL. systems are by no means phylogenetically associated unique within the subfamily for presenting distinctly with particular events of reduction/loss of limbs. developed hook-shaped and calcified hemipenial spines. The presence of hemipenial spines was already consid- In contrast, the other genera included in Gymnophthal- ered synapomorphic for the Gymnophthalmidae (Estes minae exhibit reduced comb-like spicules in the hemi- et al., 1988), thus rendering the absence of calcified penes or organs completely lacking calcified structures. ornaments in the several taxa analyzed here as isolated The enlarged spines of the limb-reduced gymnophthal- instances of secondary losses. However, distinct and minis might represent an adaptation to limb-reduction more parsimonious optimizations are possible in light of and/or to psammophilic environments, considering that the two most recent and comprehensive phylogenies of all of these genera have sand-dwelling habits. Similar the family (Pellegrino et al., 2001; Castoe et al., 2004). hook-shaped spines on the basal region of the hemipe- The topologies of Pellegrino et al. (2001) and Castoe nial body are also found in other psammophilic squa- et al. (2004) are congruent regarding the placement of mates such as the snake species Apostolepis the subfamily Alopoglossinae as the sister-group to all longicaudata (Curcio et al., 2011). other gymnophthalmids. Since alopoglossine species Reports of the seasonal presence of micro- have well-developed limbs and no vestiges of calcified ornamentations such as minute spines, hooks or multi- hemipenial structures, Este’s hypothesis of the spines spined tubercles in hemipenial ridges of sexually active being present in the most recent common ancestor of the lacertids (Arnold, 1986b), a family with no limb-reduced Gymnophthalmidae should be set aside. taxa, support the assumption that these anchoring struc- Considering the topology obtained by Pellegrino et al. tures must have arisen independently multiple times (2001), the presence of calcified structures may repre- within Squamata. However, the presumed seasonal sent either a synapomorphy for a less inclusive clade development of calcified hemipenial structures deserves within the family (i.e., Gymnophthalmidae except Alopo- further investigation. glossinae) with independent reversions to the plesiomor- The results of this study do not refute the putative phic condition, or a homoplastic character acquired at functional role of calcified hemipenial ornaments for least twice within the family (Fig. 12A). Alternatively, couple-anchoring. Nonetheless, the wide occurrence of considering the topology presented by Castoe et al. these structures in several gymnophthalmids with well- (2004) implies in assuming the homoplastic appearance developed limbs, combined with their virtual absence in of calcified hemipenial structures in at least three amphisbaenians, several snake lineages and limb- clades, each containing one single reversal event (Fig. reduced gymnophthalmid genera demonstrates that 12B). Regardless of these scenarios, none of the topolo- such structures are not necessarily associated with par- gies support a direct phylogenetic association between ticular events of limb-reduction. the presence of calcified hemipenial structures and limb Hemipenial ornaments seem to be related to couple- reduction, because limb-reduced taxa (i) are nested anchoring in some limbless squamate lineages, but cer- within terminals represented by species with spiny hem- tainly do not represent the single mechanism that keeps ipenes and well-developed limbs (e.g., Heterodactylus male and female attached during mating. For example, imbricatus, and Colobodactylus taunayi); and (ii) may in many snakes and lizards the tail can aid in this pro- present no calcified hemipenial structures at all (e.g., cess (e.g., Tryron, 1979; Crews and Fitzgerald, 1980). Bachia spp, Rachisaurus brachylepis, Heterodactylus However, the poor knowledge of reproductive behavior of lundii, and ). squamates deter further functional inferences about the Within the Gymnophthalminae, interspecific variation role of limbs or hemipenial ornamentation during regarding the presence of calcified hemipenial spines is mating. seen in the genera Heterodactylus and Colobodactlylus. Calcified spines are present in the hemipenial body ACKNOWLEDGEMENTS flounces of H. imbricatus and C. taunayi, whereas H. lundii and C. dalcyanus lack such structures, presenting We are grateful to D. Frost and D. Kizirian (AMNH); K. no vestiges of calcium in the organs. Despite being con- de Queiroz, R. McDiarmid, R. Heyer and G. Zug sidered representatives of a limb-reduced lineage among (USNM); W. Duellman and L. Trueb (KU); M. Altamir- the Gymnophthalmidae (Pellegrino et al., 2001), these ano (DHMECN), P. Kok (IRSNB); J. H. C. Santa Gadea species show minor degrees of limb reduction (reductions (MHNSM); G. Rivas and T. Barros (MBLUZ); L. Coloma in size or loss of individual phalanges). These minor and O. Torres-Carvajal (QCAZ); A. L. Prudente and T. C. changes are unlikely to affect limb functionality, in con- Avila-Pires (MPEG); J. Hanken and J. Rosado (MCZ); E. trast to the snake-like species, which exhibit more dras- La Marca (ULABG); D. Borges-Nojosa (UFC); A. Resetar tic reductions. Thus, intrageneric variation regarding and K. Kelly (FMNH); T. Mott (UFAL); and H. Zaher the presence or absence of calcified spines in species and C. Castro-Mello (MZUSP) for providing access to with similar body architecture, like that observed in the specimens under their care. We are also grateful to M. genera Heterodactylus, Colobodactylus, and Leposoma, Sena, M. Teixeira-Jr, R. Gaiga, T. Mott, C. Strussmann,€ reinforces the lack of a relationship between hemipenis S. Ruane and J. Cassimiro for making available some of ornamentation and limb development, and suggests that the pictures of live specimens that illustrate this work, other historical factors might be more strongly associ- to Mary Andriani for revising the English writing and to ated with the evolution of hemipenial structure. P. Pinna for providing unpublished information on The subgroup of gymnophthalmini lizards formed by amphisbaenian hemipenial morphology. We would like to the limb-reduced genera Calyptommatus, Nothobachia, thank to S. J. Sanchez-Pacheco and to an anonymous and possibly Scriptosaura (Rodrigues and Dos Santos, reviewer for their important comments, which helped in 2008), and by the four-limbed Psilophthalmus (included improving the manuscript. Finally, we are specially in this group in the topology of Castoe et al., 2004), is grateful to Juan D. Daza and Scott Miller for the ARE HEMIPENIAL SPINES AND LIMB SIZE RELATED? 493 invitation to take part in this volume and for the contri- Eberhard WG. 2010. Evolution of genitalia: theories, evidence, and new directions. Genetica 138:5–18. butions during the revision process of the manuscript. Edgren RA. 1953. Copulatory adjustment in snakes and its evolu- The work was funded by Fundac¸ao~ de Amparo a Pes- tionary implications. Copeia 1953:162–164. quisa do Estado de Sao~ Paulo (FAPESP) and Conselho Estes R, Queiroz K, Gauthier J. 1988. Phylogenetic relationships Nacional de Desenvolvimento Cientıfico e Tecnologico within the Squamata. In: Estes R, Pregill G, editors. Phylogenetic (CNPq). relationships of the lizard families - Essays commemorating Charles L. Camp. Stanford: Stanford University Press. p 119–281. Gans C. 1978. The characteristics and affinities of the Amphisbae- LITERATURE CITED nia. Trans Zool Soc London 34:347–416. Gibson FW. 1970. The “quadrifurcate” hemipenis of . 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