Cartilage in the Cloaca: Phallodeal Spicules in Caecilians (Amphibia: Gymnophiona)

Home , Cloaca, Scolecomorphidae, Scolecomorphus kirkii, Scolecomorphus vittatus

JOURNAL OF MORPHOLOGY 237:177–186 (1998)

MARVALEE H. WAKE Department of Integrative Biology and Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720–3140

ABSTRACT The presence of spicules (also termed spines, teeth, or denticles) on the intromittent organ, a character unique to male members of some species in the African caecilian family Scolecomorphidae, has long been known (Noble, ’31; Taylor, ’68; Wake,’72; Nussbaum,’85). However, their organization and structure has not been examined. Series of males of three species of Scolecomorphus were examined in order to determine the gross and histological organization of the spicules. Spicule morphology changes with ontogeny within species and differs grossly among species. The cellular organization of the spicules is remarkably similar among species, however. The spicules are composed of chondroid cartilage. The large discoid cells are arranged in ‘‘stacks’’ to form the projections, which emerge from an extensive base plate of the same cartilage. The spicules have a connective tissue sheath, which is not continuous with the cloacal epithelium. The sheath of the spicules may or may not be mineralized; the cartilage itself does not appear to be calciﬁed. Mineralization apparently is not correlated with age, reproductive status, or season. Cartilage in the cloaca of these few species poses a series of questions about the interactions of developmental, structural, functional, and phylogenetic properties in the evolution of the reproductive biology of caecilians. J. Morphol. 237:177–186, 1998. ௠ 1998 Wiley-Liss, Inc. KEY WORDS: Caecilians; Amphibia: Gymnophiona; cloacal structures

Copulation in apparently all caecilians is avoided postulating a morphological ‘‘lock- effected by the male everting the rear part of and-key’’ mechanism. his cloaca (the phallodeum) and inserting it As part of a study of the structure and into the vent of the female, thus providing biomechanics of phallodeal eversion in caecil- sperm transport. Phallodeal and cloacal ians, I have reexamined phallodeal morphol- structures in both males and females have ogy in many species. A particular morphol- been examined by Tonutti (’31, ’32, ’33), Tay- ogy unique to members of several species of lor (’68), and Wake (’72, ’77, ’81). Male phal- one family, the African Scolecomorphidae, is lodeal morphology is species-speciﬁc, charac- that of elaboration of the phallodeum to in- terized by arrangements of connective tissue clude spicules (occasionally termed denticles, spines, or teeth) that are arranged in ﬂanges, sacs, and other structural features. rows. Noble (’31) illustrated these in the Females of the respective species have a everted phallodeum of Scolecomorphus ulu- morphology that complements that of the guruensis; Taylor (’68, ’69) commented on males but which is simpler in form, suggest- them, as did Wake (’72). Nussbaum (’85) ing a functional association (see Wake, ’72, found the spicules to be mineralized in some for a comparison of the cloacas of males and species of Scolecomorphus. Because Nuss- females of several species). However, noth- baum had not found spicules in all species in ing is known of courtship in caecilians to date, at least as reported in the literature, though copulation has been observed in aquatic caecilians in captivity (Barrio, ’69; Contract grant sponsor: National Science Foundation. Murphy et al., ’77; Billo et al., ’85). Given *Correspondence to: M. H. Wake, Department of Integrative Biology, 3060 VLSB, University of California, Berkeley, CA 94720– this paucity of information, workers have 3140. E-mail: [email protected]

௠ 1998 WILEY-LISS, INC. 178 M.H. WAKE the family, he did not consider them diagnos- cised, cleared, and stained with alizarin tic, as Taylor had implied they were. Nuss- red-S to ascertain calcium-specific mineral- baum (’85) mentioned the presence of spicules ization. In addition, sectioned cloacas of fe- in S. uluguruensis and S. kirkii; Taylor (’68) male Scolecomorphus and those of males commented that S. vittatus has a ‘‘penis with and females of species representing genera very numerous spines.’’Taylor also implied that in five of the six families of caecilians were the spines are present in S. bornmuelleri (’68, available for reference. p. 632: Taylor transferred the species bornmuelleri from the genus Herpele to Scolecomorphus RESULTS on the basis of having ‘‘the four essential char- Gross morphology acters of Scolecomorphus,’’ which include the penis ‘‘very different, being provided with spiny When the cloaca is opened ventrally, spic- structures larger or smaller, few or very numer- ules of Scolecomorphus uluguruensis are vis- ous’’). The species was placed by Nussbaum in ible as two lateral rows of several spicules, a new genus of scolecomorphid, Crotapha- often, but not always, with a mediodorsal trema, which includes an additional species, row of 1–3 spicules. The spicules increased but Nussbaum does not report the presence of in size from anterior to posterior in all speci- spicules on the intromittent organ in C. born- mens, with the posterior-most spicules often muelleri (or C. lamottei), nor does Taylor refer bicuspid and four times the length of the to them in his account of the species. anterior-most (Fig. 1). Of 40 males of S. No attention had been given inter- and uluguruensis examined, seven lack spicules intra-specific variation in spicule number entirely. Five of the latter individuals were and form, nor to the internal structure of the immature (gonads differentiated, but not yet spicules. As part of on-going studies of caecil- functional); two, however, were mature males ian reproductive biology, including the mech- with spermatogenic testes (sensu Wake, ’68). anism of sperm transport via phallodeal in- In addition, one immature male (168 mm tromission, I systematically examined total length [TL]) had small, soft spicules in spicule and cloacal morphology in scoleco- reduced numbers (4 right, 1 medial, 5 left). morphids. The ontogeny and variation of There was no indication of any correlation of spicules, and their peculiar internal struc- numbers of spicules with size (body length) ture, are unique among amphibians and ap- and presumed age other than in the latter parently among all vertebrates. animal, which probably is commencing matu- ration. In fact, a smaller immature (151 mm MATERIALS AND METHODS TL) has well-developed spicules within the Males of Scolecomorphus uluguruensis numerical range for adults, but a large ma- (N ϭ 40), S. kirkii (N ϭ 7), and S. vittatus ture male (253 mm TL) has only the faintest (N ϭ 10) were examined. A longitudinal ven- indication of precursors of spicules (see be- tral incision was made just anterior to the low). For 31 mature males, the mean num- vent, the body wall musculature pinned ber of spicules in the right row was 10.42 aside, and the cloaca opened via a mid- (range ϭ 8–13.5 [the minor cusp on the ventral slit. The cloaca was pinned open, largest spicule was counted as half a spic- and spicules counted and internal gross mor- ule]; SD ϭ 1.18); left was 9.73 (range ϭ phology examined using a dissecting micro- 6–12.5; SD ϭ 1.51), and median 1.5 (range ϭ scope. Representatives from each species 0–3; SD ϭ 0.647). were drawn using a camera lucida for refer- Seven males of Scolecomorphus kirkii were ence; examples were photographed. Cloacae mature (252–300 mm TL), and all had spic- from two to five members of each species ules. The gross arrangement of the spicules were excised and prepared for microscopic is quite different from that in S. uluguruen- examination by embedding and sectioning sis. Numerous, very tiny spicules are ar- at 7–10 µm, mounting serially on slides, and rayed on four to eight raised ridges on each staining alternate slides with either hema- side of the opened cloaca (Fig. 2). The middle toxylin-eosin, Mallory’s azan, or picro-pon- ridges are the longest, the anterior and pos- ceau (Humason, ’79), the latter two stains terior of a set the shortest. Full counts can- especially useful for delineating connective not be made, for the numbers are more than tissue structure. Sections were photographed 20 on longer ridges, but the lateral- and using a Zeiss Auto-photomat (Zeiss; Jena, medial-most small spicules could not be visu- Germany). Cloacae of four S. uluguruensis, alized because they disappeared into the two S. vittatus, and two S. kirkii were ex- epithelium of the cloaca, and many spicules CLOACAL SPICULES IN CAECILIANS 179

Fig. 1. Spicules in the uneverted cloacas of Scoleco- uneverted cloaca as it lies in the coelomic cavity. Ante- morphus uluguruensis. Cloacas from adult male speci- rior is to the top. A illustrates mineralized spicules in mens with spermatogenic testes were excised and cleared the cloaca; in B, arrows indicate unmineralized spicules and stained with alizarin red-S, which is calcium- in the cloaca of another male. ms, mineralized spicules. specific. The cloacas were opened via a ventral slit and Bar ϭ 1.0 mm. pinned open, so the view is of the dorsal wall of the in each row, especially at the margins, do not tinuous, three-dimensional plate of tissue of have mineralized, stain-reactive tips, so they the same characteristics as the spicules. Nei- are not visible in whole-mount cleared and ther female Scolecomorphus nor males or stained preparations. females of any other species available for Of ten male Scolecomorphus vittatus ex- histologic examination have such a cartilagi- amined, seven were mature with spermato- nous structure, nor are there suggestions of genic testes. Three were immature and such structures in the literature; however, lacked spicules entirely. Of the mature ani- as part of the larger study, a more exhaus- mals, only two had distinct spicules; they tive examination is under way. Second, the were in the S. kirkii pattern of small spicules plate is composed of large (100–150 µm diam- on ridges. The remaining five appear to have eter), flat (5 µm deep), round cells with large incipient but poorly developed spicules. nuclei (10 µm diameter) that are arranged in a diversity of orientations, but which are Histology continuous. The spicules are projections, of- The general cellular structure of the spic- ten in linear stacks that curve, composed of ules in all three species examined is very those cells. Third, the cells strongly re- similar, though the visible gross form and semble large pre-cartilage cells that lack a some details of histology differ considerably. matrix; this is typical of vertebrate chon- Several features of spicule histology are note- droid cartilage (Studnicka, ’03; Schaffer, ’30; worthy. First, the spicules are not indepen- Moss and Moss-Salentijn, ’83; Benjamin, ’88, dent structures, but they project from a con- ’89ab, ’90). Fourth, the sheath over the emer- 180 M.H. WAKE with cells in different planes. Fibrous connective tissue (and possibly elastic fibers) di- vides components of the plate in association with the spicules (Fig. 3ABC). The fibrous

Fig. 2. Mineralized spicules in the cloaca of an adult male Scolecomorphus kirkii. The cloaca is treated as in Figure 1. Arrows indicate sets of rows of tiny spicules. Bar ϭ 1.0 mm. gent spicules, not the spicule core, is mineralized in some specimens, but not in others, of all three species. In most S. uluguruensis, the cartilage cells at the tips of the spicules, but not the entire spicule structure, is mineralized as well. The sheath is not cellular. Fifth, the spicules typically project through a thin layer of epithelium that lines the entire cloaca; the epithelium is not continuous over the spicules. This condition is most pronounced in S. uluguruensis, with its stout spicules; the epithelium does cover many of Fig. 3. Cellular structure of spicules and base plates the small lateral spicules in the other spe- in the cloacae. Parasagittal sections of spicules (A, B) cies examined. and base plate (C)inScolecomorphus uluguruensis; The continuous basal sheet, or plate, of anterior is to the left. Mallory’s azan stain. Red blood chondroid cartilage is organized similarly in cells from the dilated cloacal vascular sinuses are present in the sections. In A, the spicules are sectioned all three species, with some variation in tangentially to their tips; in B, the section shows the Scolecomorphus uluguruensis. The plate is spicules at full extent. The chondroid cartilage of the composed of linear arrays of the chondroid spicules and base plate is apparent. Note the extent of cartilage cells; the cells lie mostly in the the cloacal epithelium relative to the spicules. In C, the ‘‘stacks’’ of chondroid cartilage cells are illustrated; the same plane, except when they form the pedi- nuclei indicate the positions of the large, flat cells. A cels of the spicules. In S. uluguruensis, the cluster of nuclei lies in the circle. bp, base plate; e, plate is stouter and more three-dimensional, epithelium; s, spicules. Bar ϭ 100 µm for A–C. CLOACAL SPICULES IN CAECILIANS 181 connective tissue boundaries are stouter in S. uluguruensis than in the two other species. The spicules of Scolecomorphus uluguruensis are multicellular projections from the basal sheet (Fig. 3AB); they have broad bases (2–5 cells wide) and extend to a small apical point (cells approx. 50 µm ϫ 10 µm), giving the denticular shape observed grossly (Figs. 1, 3AB). The spicules in S. kirkii and S. vittatus are linear stacks of the large round cells that project from the sheet, and the stacks curve as they extend (Figs. 4A–C, 5AB), thus effecting the observed small but numerous denticles characteristic of the species. In S. kirkii, the cells at the bases of the spicules are approximately (the approxima- tion is meant to indicate some variation in cell sizes, but it is not significant) 100 µm ϫ 5 µm, and those at the tip are 50 µm ϫ 10 µm (Fig. 4A–C). These spicules are structurally more elongate and delicate in appearance than those of S. uluguruensis. Spicules of all three species have a thin connective tissue sheath that is mineralized (Figs. 3A,B, 4A–C, 5A,B) in most specimens. In most males of S. uluguruensis, the sheaths of all of the relatively stout spicules are mineralized; there are no instances of mineralization of some, but not all, spicule sheaths. However, par- tial mineralization characterizes spicule morphology in both S. kirkii and S. vittatus. More anterior and more medial spicules in the several transverse rows are mineralized, more posterior and more lateral ones are not. Also, in S. uluguruensis, the apical cartilage cells of each spicule are often mineralized as well, but the mineralization does not extend below the apical region; the mineralization observed in cleared and stained specimens is primarily in the sheath. Fig. 4. Cellular structure of spicules and base plate The vascularization of the cloaca is exten- in Scolecomorphus vittatus. Parasagittal sections; anterior is to the left. Mallory’s azan stain. In A, B, and C, sive. Thin, elongate sinuses lie beside the aggregations of spicules and their associations with the chondroid base plates in all three species base plate are illustrated. Compare with Figures 3 and examined. Only a few cloacae had the si- 5; the spicules and base plates of S. vittatus and S. kirkii are much more similar to each other in structure (smaller nuses dilated; they contained large numbers size and larger number of spicules; more intricate ar- of red blood cells (Fig. 5A; also note blood rangement of the cells and connective tissue of the base cells that were scattered from sinuses dur- plate; extent of mineralization) than either is to the structural pattern in S. uluguruensis. The nuclei of the ing sectioning in Fig. 3A,B). The presence of chondroid cartilage cells indicate the complexly stacked some erythrocytes in the sinuses, whether structure of the base plate and the intricate arrange- they included many cells or were relatively ments of the spicules. Mineralization is scant, but present at the tips of the spicules in the connective tissue evacuated, indicated the extent of the si- covering the cartilage. bp, base plate; e, epithelium; n, nuses in all specimens. It is not clear how nuclei; s, spicules; ss, spicule series. Bar ϭ 100 µm for large the sinuses would be when filled with A–C. blood (see discussion below). 182 M.H. WAKE

Fig. 5. Cellular structure of spicules and base plate ment of the cartilage of the spicules and base plate. in Scolecomorphus kirkii. Parasagittal sections; ante- Aggregations of small spicules and elongate, narrow rior is toward the top. In A, the section is lightly stained spicules from the cloaca of a single male are illustrated. with hematoxylin and eosin to emphasize cell structure bp, base plate; e, epithelium; s, spicules; ss, spicule and epithelium; in B, the section is stained with Mallo- series; v, venous sinus. Bar ϭ 100 µm for A and B. ry’s azan and emphasizes the nuclei and cellular arrange- CLOACAL SPICULES IN CAECILIANS 183 DISCUSSION scolecomorphid material does not allow infer- A number of aspects of the morphology of ences about the mechanism of mineraliza- cloacal spicules in Scolecomorphus are unex- tion of the cartilage or its sheath, so correla- pected, based on the limited conjectures in tion with Ali’s hypotheses is not possible at the literature and my own initially cursory present. observations. They affect considerations of Functional morphology development, structure, function, and evolution. The most obvious question of the functional morphology of spicules is the least answerable: why are spicules present at all, Structure and development and only in members of the genus Scoleco- Chondroid cartilage is found in a diversity morphus? The cloacas of members of the of vertebrate structures (Studnicka, ’03; genus have the same general configuration Schaffer, ’30; Moss and Moss-Salentijn, ’83; found in other caecilians, in that patterns of Benjamin, ’88, ’89ab, ’90). It is particularly connective tissue ridges and flanges, and of prevalent in fishes, including mouth parts of blind sacs and other features, appear to be lampreys, and many sites in teleosts, includ- species-specific. The distribution and num- ing barbels, beak, surrounding large blood bers of spicules on connective tissue ridges vessels, branchial arches, surrounding cra- in the three species examined is indeed con- nial nerves and/or the brainstem and spinal sistent within the species. I infer that the cord, labial folds (including suckers), oral presence of spicules is a derived state, an valves, tongue, nares, surrounding the olfac- autapomorphy for the genus. I assume that tory sacs, pharyngeal bones, pharyngeal mu- all species assigned to Scolecomorphus have cosa, beneath the skin, supporting the sen- spicules, and those assigned to Crotapha- sory epithelia of the statoacoustic organ, trema lack them; if members of Crotapha- scales, menisci, ligaments, articular carti- trema are found to have spicules when they lages, etc. (Benjamin, ’88, ’90). Chondroid are examined, spicules are then an autapo- cartilage also occurs in the Achilles tendons morphy for the family Scolecomorphidae. To of certain frogs, the auricular and epiglottal date, I have not been able to obtain speci- cartilages of some rodents and bats, and the mens of Crotaphatrema in order to ascertain articular surfaces of the limb bones of small presence or absence of spicules. The pres- reptiles, birds, and mammals (Moss and ence of spicules is a synapomorphy of the Moss-Salentijn, ’83). Moss (’61) reported that three species examined; the similar pattern chondroid cartilage in some fishes cytodiffer- and distribution found in S. kirkii and S. entiates directly to bone, and that it is in- vittatus is a synapomorphy that unites these volved in bone fracture healing in some fishes species as sister taxa (assuming both are (Moss, ’62). Beresford (’81) summarized work good species) and distinguishes them from on chondroid bone, metaplasia, and endo- their sister taxon, S. uluguruensis. The modi- chondral ossification in a diversity of verte- fication of connective tissue in these species brates, especially mammals; his discussion to form chondroid cartilage cannot be ex- points out that bone and cartilage can form plained. Chondroid cartilage is found in a in many soft tissue sites, both pathologically diversity of situations in vertebrates (see and evolutionarily. Chondroid cartilage also above), but not reported to date in the cloaca. characterizes certain invertebrate struc- An extensive review of the literature on de- tures, such as the branchial crown supports velopment of the bacculum and the os clitori- of sabellid worms, and occurs in molluscs dis of mammals (Williams-Ashman, ’90; Wil- and arthropods as well (Person, ’83). As Ben- liams-Ashman and Reddi, ’91) and on jamin (’90) notes, little work has been done mineralized structures in the hemipenes of on development and evolution of the many lizards (see Card and Kluge, ’95, for the most variations of chondroid cartilage. Associa- recent review) sheds no light on this questions of chondroid cartilage appear to fall tion, except to indicate the ability of connec- into three broad classes: (1) those with epi- tive tissue to develop and mineralize in a thelia, (2) those included in bones or liga- great many ways (see below). ments, and (3) those providing support or The function of spicules on the everted ‘‘cushioning.’’ The condition in Scolecomor- male phallodeum can only be conjectured at phus meets class 1, and perhaps the support best. Nothing is known of mate recognition function of class 3. Ali (’83) summarized or courtship in caecilians. Any suggestion mechanisms of calcification of cartilage. The that the spicule pattern might be involved in 184 M.H. WAKE mate recognition or a ‘‘lock-and-key’’ mecha- most male caecilians; (2) the situation in nism is speculative, but cannot be rejected. Epicrionops, members of the basal family Spicules in Scolecomorphus may be inter- Rhinatrematidae, in which large, paired, preted as an enhancement of the species- ovoid vascular sinuses lie beside the cloaca, specific patterns of cloacal connective tissue and each sinus has a large striated muscle morphology found in all caecilians, or, con- beside it whose contraction would exert force, versely, these patterns may have nothing to presumably propulsive, on the cloaca, with do with reproduction per se. However, the or without that of other muscular contrac- literature includes discussion pertinent to tions (Wake, personal observations); and (3) this issue. Arnold (’86ab) discusses the elabo- the Scolecomorphus morphology, in which rate species-specific structure of the hemi- vascular sinuses, muscular contractions, and penes of lacertid lizards in terms of its pos- the cartilaginous base plate are involved. sible evolution either as a physical isolating The plate is densely packed, but must be mechanism or as a consequence of genetic somewhat flexible, as it must be folded dur- pleiotropy, but points out that either hypoth- ing eversion of the intromittent organ. I esis would be difficult to test in lizards. hypothesize that hydrostatic (vascular si- Eberhard (’85) cogently presents the limited nuses and cloacal lumen) and muscular (cloa- evidence for lock-and-key and genitalic rec- cal and body wall) pressures build to a ognition, pleiotropism, and mechanical con- threshold, at which point the cartilaginous flict of interest, and the more extensive evi- plate buckles, folds, and rolls over itself un- dence against these hypotheses. He proposes til it is straight again, thus generating mo- that male genitalia evolve via sexual selec- mentum via energy storage that perhaps tion by female choice and develops the hy- facilitates eversion of the cloaca after the pothesis with a number of supporting ex- threshold is reached. Retraction of the phal- amples; he also cites apparent contradictions lodeum in caecilians apparently occurs as a consequence of contraction of the musculus to the hypothesis. Caecilians would be diffi- retractor cloacae, which originates on the cult, but not impossible, organisms in which body wall epithelium and inserts on the to test these hypotheses, given the paucity of cloaca. The cloacal retractor muscle of the information about their reproductive biol- species of Scolecomorphus examined is not ogy and the relative rarity of their breeding distinctly larger than that of other male in the laboratory. Further, fossorial animals caecilians, despite having to resist the stiff- of apparently low vagility, especially those ness of the cartilaginous base plate in order that are viviparous and presumably with to initiate retraction (the same principle of relatively long gestation periods, such as energy storage propelling the plate to Scolecomorphus, would require special provi- straighten after buckling would obtain as sion for sexual selection experiments. At this retraction occurs). Thus the caecilian intro- stage, we cannot yet assess the functional mittent organ is an unusual muscular hydro- morphology or the evolution of the genitalia stat, sensu Kier and Smith (’85), but it ap- of caecilians. pears to be one, nevertheless, and its The association of spicules with a continu- mechanism is currently under investigation ous base plate of unmineralized cartilage (Wake, unpublished data). poses another functional question: How is the cloaca everted, given the presence of a Mineralized cloacal structures relatively massive structure that presum- in other vertebrates ably reduces the flexibility of the cloaca? Skeletal support structures occur in the Tonutti (’31, ’32, ’33), Taylor (’68), and Wake intromittent organs of a diversity of verte- (’72) conjectured that the filling of cloacal brates. Those that are associated with intro- vascular sinuses, coupled with the contrac- mittent organs that are derived, at least in tions of body wall and certain cloacal muscu- part, from the cloaca include the os baculum lature, combined to eject the rear part of the and os clitoridis in a number of mammalian cloaca from the vent of the male. My prelimi- taxa (insectivores, rodents, carnivores, pinni- nary investigation of the morphological ba- peds, bats, and primates; Williams-Ashman sis of cloacal eversion in caecilians provides and Reddi, ’91) and mineralized structures evidence for three different biomechanical in the horns of the hemipenes of certain variations of cloacal eversion: (1) the vascu- lizards (see below). The development of the lar sinus-cloacal-body wall contraction sug- os baculum and the os clitoridis has been gested by the morphology of the cloaca of well studied; in some instances, bone arises CLOACAL SPICULES IN CAECILIANS 185 directly from central mesenchyme, in oth- initiated a comparative study of the cellular ers, there is a cartilage precursor, but it is structure of the mineralized elements of the characterized as hyaline (Williams-Ashman, hemipenis. Several aspects of the ontogeny ’90; Williams-Ashman and Reddi, ’91). (Note and adult structure of hemipenial mineral- that Nelsen [’53] characterizes the urogeni- ized elements warrant both further study, tal sinus as a cloacal derivative; it contrib- and comparison with the scolecomorphid con- utes to penial development, possibly to some dition: 1) the basic information on tissue- of the mesenchyme that later organizes into level structure is needed, for data to deter- internal penial structures.) Mammalian and mine whether the structures are fully bone, squamate intromittent organs are clearly or cartilage that is mineralized, or, if carti- nonhomologous in either developmental or laginous, the kind of cartilage is essential to phylogenetic terms. Further, the mineral- comparison; 2) the comment by Kluge (’82) ized structures are clearly bone or bone and in his summary of hemipenial structure that cartilage in mammals, and presumed to be the ‘‘bones’’ in some taxa protrude through bone in lizards. Mineralized cartilage or car- the covering epithelium; 3) Card and Kluge’s tilage sheaths, as in the species of Scoleco- (’95) mention of the ontogeny of one species morphus investigated, has not been re- of varanid showing that the hemipenial ported. ‘‘bones’’ were not yet mineralized at a year The structure of the hemipenes of squa- post-hatching—a situation not dissimilar to mates includes a contribution from the cloa- the caecilian situation. It would be interest- cal wall to each of the paired organs ing indeed if lizard hemipenes, as well as (Raynaud and Pieau, ’85). This situation is caecilian (scolecomorphid) intromittent or- clearly not homologous to the single intromit- gans, could be added to the long and diverse tent organ of caecilians, but the presence of list of taxa that have structures composed of mineralized structures merits further atten- chondroid cartilage; it would be especially unusual if chondroid cartilage in association tion. Only a few lizards, and no snakes, are with cloacal derivatives for intromission reported to have mineralized structures as- evolved independently in two effectively un- sociated with the hemipenes (e.g., some vara- related lineages of vertebrates. nids [Branch, ’82; Shea and Reddacliff, ’86; Werner, ’88; Bo¨hme, ’88, ’91]; all pygopodids ACKNOWLEDGMENTS and most gekkonids have cloacal bones, but I thank curators at the California Acad- they are not associated with the hemipenes emy of Sciences, the Field Museum of Natu- except in Aristelliger, which lacks cloacal ral History, the Museum of Vertebrate Zool- bones and sacs, but has one or two pairs of ogy at the University of California, Berkeley, bones within each hemipenis [Kluge, ’82]; and the Museum of Comparative Zoology, xantusiids have post-cloacal bones [Rieppel, Harvard University (which has relatively ’76; Kluge, ’82]; many lacertids have an ‘‘ar- large series of Scolecomorphus) for allowing mature’’ in their hemipenes, but it is dense examination of their specimens, and MCZ fibrous connective tissue that is apparently and CAS for permission to section and clear unmineralized [Arnold, ’83, ’86ab; Arnold and stain cloacas. Tina Rouse, Michelle Koo, confirmed this by histological examination]; and Reneé Dickie sectioned cloacas, and Tina certain teiids [Uzzell, ’69] and anguids [Cope, Rouse counted spicules. I thank Michael Ben- ’00] have mineralized external ornamenta- jamin for confirming that spicule tissue is tion on the ends of the hemipenes). Branch chondroid cartilage. This research was sup- (’82) commented that the element in some ported by the National Science Foundation. varanids appeared to be of ‘‘fibrous cartilage,’’ but that this had not been determined LITERATURE CITED by histological staining. In fact, all of the Ali, S.Y. (1983) Calcification of cartilage. In B. K. Hall work to date on mineralized structures in (ed): Cartilage, Vol. 1. New York: Academic Press, pp. lizard hemipenes is based on cleared-and- 343–378. alizarin-stained or x-rayed material, or ma- Arnold, E.N. (1983) Osteology, genitalia and the relationships of Acanthodactylus (Reptilia: Lacertidae). 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