Russian Journal of Herpetology ol. 1, No. 1, 1994, pp. 30 36 ON UNIQUE FORMS OF ANOMALOUS SACRAL STRUCTURE IN TAILLESS AMPHIBIANS E. E. Kovalenko 1 and S. E. Danilevskaya 1 Submitted September 11, 1993. The unique structural anomalies of spinal column caused by unilateral development of pelvis are described in an adult specimen of Xenopus laevis and 2 larvae of Rana temporaria. In all the specimens the unilateral development of sacrum was observed. The described anomalies support the hypothesis of deforming influence of developing ilium on posteromost presacral segments as well as some assumptions on the factors determining the morphogenetic process in anurans. Key words: Amphibia, Anura, sacral structure, variations, unique forms. ariations of the structure of urostyle-sacral bones of Anura were the subject of many studies, because the origin of this group is traditionally linked with evolution of locomotor system (hind legs, pelvic girdle, sacroiliac articulation). arious normal forms of the structure of this part of skeleton were described. Characters important for diagnostics and classification were analyzed (Lynch, 1973; Trueb, 1973; Böhme, 1977; Duellman and Trueb, 1986; etc.). The dependence between the structure of sacroiliac articulation and functional capabilities of constructions was studied in detail (Emerson, 1979, 1982). Morphogenetic mechanisms ensuring the formation of normal or anomalous forms of this part were investigated (Kovalenko and Anisimova, 1987; Kovalenko, 1992). Many papers containing data on individual variation of sacrum in various species of Anura were published (see Kovalenko, 1992). Known data concerning variability of urostylesacral part of skeleton in Anura were generalized and preliminary analyzed (Kovalenko, 1992) using a great amount of data on the subject obtained in our own studies as well as in literature. On structural anomalies only, published data on more than 40 existing and extinct species were compiled. The total number of specimens studied by various authors for individual variability of that part of skeleton exceeds 10,000 (summarizing the total number of samples 1 St. Petersburg State University, Universitetskaya nab. 7 9, St. Petersburg 199084, Russia. cited in papers whenever such information is given; see Kovalenko, 1992). The forms of sacral structure are shown to be different in the number of vertebrae forming the articulation with pelvis, in presence or absence of fusions between the elements of axial skeleton, in the symmetrical or asymmetrical arrangement of sacral diapophyses, and the shape of these latter. The expanded (sacral) diapophyses can be found on sacral vertebrae normally devoid of them and lie anterior or posterior to ordinary ones. All the possible combinations of these features were compiled. Analysis of their frequencies in interspecific and individual variability was performed (see Kovalenko, 1992). In the above-mentioned paper in the analysis of possible constructions of sacrum did not consider we structures where sacral diapophyses are missing on one or both sides of all the vertebrae, because these constructions presuppose complete or unilateral absence of pelvis. The last circumstance was assumed to be hardly probable or even impossible under the natural development without any experimental surgical operation. Indeed, such constructions were never found until the present among a huge number of studied specimens of various species. However, soon after the paper was accepted for publication we obtained material showing that the emergence of such constructions is possible. The description of these unique forms of sacral structure is the subject of the present paper. 1994 Folium Publishing Company
On Unique Forms of Anomalous Sacral Structure in Tailless Amphibians 31 MATERIAL AND METHODS The structure of axial skeleton and pelvis was studied in 3 specimens of Xenopus laevis and Rana temporaria. One of the hind legs was absent in two specimens and was rudimentary in the third one. One skeleton belonged to a mature (approximate age 2 years) Xenopus laevis, reared in laboratory (conditions unknown); natural death. Material was provided by. K. Uteshev (Institute of Biophysics, Pushchino) in 1991. Two skeletons belonged to specimens of Rana temporaria prepared just after the end of their metamorphosis (75 and 85 days after hatching); reared in laboratory; natural death during the first day of terrestrial life. Reared in 1990 by S. E. Danilevskaya from the frog roe of two clumps collected in a natural pond near St. Petersburg. The total number of larvae examined to detect structural anomalies of the axial skeleton was 194. The above described anomalies were found only in two specimens. In other larvae only insignificant morphological deviations from the normal structure or anomalies common to Ranidae (e.g., fusion of two anteromost vertebrae or posterior ones) were found. The material was fixed in formalin (solution 1:9) and totally stained by alizarin and alcian blue. Soft tissues were clarified in glycerol (Borkhvardt and Karavayeva, 1982). RESULTS Specimen 1 (No. A1410 CACZ 2 ). A larva of Rana temporaria (85 days after hatching) the left hind leg of which is rudimentary. The right hind leg and the right half of pelvic girdle are normally developed. The right process of the ilium forms an articulation with the presacral vertebra. The transversal process of the vertebra is extended in a way that normally occurs at the end of metamorphosis. The left hind leg is considerably shortened and curved, its musculature is weakly developed. The left process of the ilium is shorter by half than the right one and does not reach the axial skeleton (therefore, it is not seen on the total preparation and in Fig. 1); the left sacral diapophyses is absent. The larva is curved to the right; the scoliosis begins from the segment (Fig. 1a, b). 2 CACZ) the Collection of Anomalies, Department of ertebrate Zoology, St. Petersburg State University. a I cna II I II stp cna Fig. 1. The axial skeleton of Rana temporaria after the end of metamorphosis (specimen 1; No. A1410 CACZ). a) From above; b ) from below. Only the right process of ilium is seen; the left one is rudimentary and lies significantly more ventrally than spinal column; it is not depicted in Fig. 1a. I X) Numbers of vertebrae; cna) caudal neural arches; il) ilium; stp ) transversal process of sacrum. The axial skeleton has 9 body somites on the right side and 8 ones on the left; the -th left neural arch is morphologically similar to normal caudal one and dorsally fused with other caudal elements (Fig. 1a). The right half of the last presacral semivertebra has a typical sacral structure. Taking into account the fusion of the corresponding left element with caudal ones, assumption can be made, that in further development both somites would be fused and become a part of the urostyle; the spinal column would be formed by 8 presacral vertebrae and urostyle with a sacral diapophyses on the left side. In other respects, the spinal column is absolutely normal except for a few details (dorsal parts of to II neural arches are somewhat thickened, the left transversal process of the vertebra is absent). Specimen 2 (No. A1411 CACZ). A larva of Rana temporaria (75 days after hatching) without left hind leg. The right leg is situated medially at the boundary between the sacrum and the caudal part of spinal column. Multiple structural anomalies are found in most of the organs of this specimen (Fig. 2). The larva is significantly curved. Its spinal column is characterized by scoliosis (the curvature is pointed to the right) and torsion to the left. The left foreleg resembles a short stump with humerus as the only skeletal element; the left scapula is rudimentary. The cranium is considerably asymmetric (Fig. 2a): the right ear capsule and eyeball are significantly il b
32 E. E. Kovalenko and S. E. Danilevskaya a c ec ec I II I X I I II stp II hpc il Fig. 2. The axial skeleton of Rana temporaria after the end of metamorphosis (specimen 2; No. A 1411 CACZ). a) From above, b) from below; c) lower part from the left. The spinal column was twirled to the left (torsion); it was straightened to make all its elements visible on the same plane. The process of ilium is not depicted in Figs. 2a and 2c. I + II) Fusion of the I and the II vertebrae; ec) ear capsule; hpc) hypochordal cartilage; sb) base of skull. For other designations see Fig. 1. I II b sb larger than the left ones; the left cranial elements (i.e., nostril, orbit, parachordal, maxillary elements) are displaced somewhat caudally compared to the right ones; the mandible is shortened, left mandibular and maxillary elements are deformed. The left hind leg and left half of the pelvis are completely absent. The right hind leg is developed almost normally; the right pelvic bones form a somewhat flattened disc. The process of right ilium is normally developed, though its anterior end is more than normally protruding anterodorsally to the sacral diapophyses (Fig. 2b). Therefore, a protrusion increasing the external asymmetry of the larva is seen on the right side of the sacral part of its body. Left myomeres are completely undeveloped throughout the body from the ear capsules to the caudal end. The spinal column is formed by 9 complete vertebral primordia in its right half, by 8 bases and 6 ascending parts of neural arches in the left one (Fig. 2c). The right elements of neural arches arc considerably longer than the left ones and join together to the left of the medial axis (Fig. 2a). This disproportion increases caudally. Neurocentrums are absent. Intervertebral tissues arc, probably, undeveloped on the left (Fig. 2b, c), because the typical spaces between the bases of neural arches arc absent on that side (undifferentiated intervertebral mesenchyme cannot be stained in whole mounts). Intervertebral folds of perichordal sheath are absent on the left as well (Kovalenko, 1986). The right half of the spinal column: ascending part of the I neural arch is scarcely developed and not connected with its dorsal part; the latter is completely fused above the neurotubule with the II neural arch, only in the medial part of this arch its duality is visible. other vertebral segments including the caudal neural arches are developed quite normally. The left half of the spinal column: the I neural arch consists only of a base and a very short ascending part, the distal end of which is fused with the base of skull (Fig. 2b, c); other parts of the arch are completely absent; the ascending part of the II neural arch is not fully developed (it is narrow, the transversal process and the upper part are absent; the transversal processes are rudimentary in and segments; in other segments (from the ) neural arches are of abnormal shape and consist only of ascending parts; in the II segment, the last presacral one, only the base is present; the neural arch is morphologically similar to an ordinary first caudal one and is fused with
On Unique Forms of Anomalous Sacral Structure in Tailless Amphibians 33 a b I aa I II II zj u X X u u Fig. 3. The spinal column of Xenopus laevis (specimen 3; No. Ax50 CACZ). a) From above; b) from below. The caudal part of urostyle is shown on the right. aa) Auxiliary articulation formed by the II vertebra and sacral process; u ) urostyle; zj ) joint formed by the zygapophyses of II and vertebrae. For other designations see Figs. 1, 2. subsequent arches; the number of caudal primordia is more than normal due to 5 or 6 accessory small cartilaginous elements lying posteriorly to the ordinary ones (Fig. 2c); sacral diapophyses is absent. A disparity between the arrangement of right elements and that of left ones was observed [the increasing asymmetry syndrome, see Kovalenko (1983, 1992)]; the left elements of the and subsequent segments are caudally displaced. Therefore, the dorsal parts of and segments are arranged in staggered rows (two right neural arches opposite a left one), the number of the left presacral primordia is less by one primordium than of the right ones (Fig. 2a). Bases of the left arches are not continued laterally to chord and closer adjoin each other so that separate elements are almost indiscernible. Probably all the bases of arches on the left side would be fused during the further development. The hypochordal element has an aberrant clublike shape (Fig. 2c). Specimen 3 (No. Ax50 CACZ). An adult specimen (age no less than 2 years) of Xenopus laevis without the left hind leg and left half of pelvis. The right hind leg and right half of pelvic girdle are normally developed. The process of the right ilium together with the vertebra forms a sacral diapophyses of a peculiar shape. The left sacral process is absent (Fig. 3). The spinal column is characterized by left-side scoliosis with an apex near the I vertebra. Most of neurocenters, neural arches, zygapophyses and transversal processes have abnormal shape; the right parts of the to neural arches are extremely deformed. Their transversal processes seem to be turned so that the sacral wings press them to vertebrae, zygapophyses are turned laterally. The right parts of the II and neural arches form an additional articulation; in front of the ordinary articulation between two zygapophyses an additional one is formed by the base of the II transversal process and the anterior margin of the sacral wing (Fig. 3b ). The adjoining skeletal elements of additional articulation have smooth glenoid surface and resemble an ordinary zygapophyseal articulation. The glenoid elements between all the neurocenters are somewhat deformed. Three anteromost vertebrae are fused. Among these, the I and II vertebrae are completely fused so
34 E. E. Kovalenko and S. E. Danilevskaya that the border separating them is scarcely discernible and seen only on the dorsal side; the vertebra was probably, fused with the II one later in onthogenesis, and the dorsal parts of their neural arches are fused only near the zygapophyses. Intervertebral foramina are found between all the vertebral segments. The vertebra is completely fused with the urostyle similarly to the normal structure, and has a wide sacral diapophyses including the transversal process of the I caudal neural arch. The last presacral vertebra (the ) has a small left transversal process similar to the processes of other presacral vertebrae. The urostyle has a rudimentary left transversal process closely adjoining the vertebra but not articulating with it. DISCUSSION Ridewood (1897) held that a vertebra lying immediately near the sidebone becomes the sacral one. At present this opinion is beyond any doubt Firstly the correlation was supported, though not undoubtedly, by Hodler (1949, 1949a) who carried out experiments with amputation and transplantation of primordia of pelvis and legs. However, the essence of morphogenetic role of ilium remained unknown for a long time. Later, a special morphological study (Kovalenko and Anisimova, 1987) revealed that the process of iliac element influences the formation of transversal process in the last presacral segment. The earlier in onthogenesis the chondrification of ilium take place, the more this influence become apparent. The pelvic process has mechanical influence upon myomeres and the septae separating them: it widens the space in which the formation of transversal process occurs, and therefore more mesenchyme cells are attracted. It favors the formation of a transversal process larger than these of other vertebrae. This conclusion was confirmed by the data on normal morphogenesis of urostyle-sacral bones in a lot of anurans, by the comparison between the supposed and the observed nature of variations in this part of skeleton (Kovalenko, 1992). The most complicated problem consisted in verification of the assumption of the physical influence of the iliac process on anteromost presacral segments causing the deformation of these latter. Strictly speaking, only experimental studies could support this assumption. If the ilium presses the segments with certain force, a deformation of spinal column could be anticipated when one of the ilia is absent, and this force is not balanced on another side. In amphibians with iliac element, where chondrification takes place long before metamorphosis, this element is anterior to the last presacral segment (see Kovalenko and Anisimova, 1987; Kovalenko, 1992), and asymmetrical deformation of the presacral neural arches could be expected. Unfortunately, the experiments with amputation of pelvis and legs are too gross to study the subtle and multifactorial morphogenetic processes. Hodler (1949a) acknowledged that his experiments are only able to show that the development of pelvis and of sacrum are correlated. Not all of these tests supported the assumption of the influence of the iliac process, and some even disproved it. The given examples of anomalous structure represent a natural experiment, the display of formation of spinal column in unique conditions. These specimens not only confirmed the assumption of the pelvic influence on the formation of sacral diapophyses, but also showed that developing ilium is a peculiar disturbing factor of morphogenesis that changes the conditions of formation by means of mechanical influence. It is supported also by the observed scoliotic changes (Figs. 1 3)and the deformation of presacral neural arches on the side of a developing process of ilium (Fig. 3). Certainly, the assumption could be made that the abnormal development of pelvis and that of spinal column are mediated, for example, by disturbed segmentation. This idea seems to be suggested by the structure of the above described larva (specimen 2, Fig. 2). However, if the lack of development in a pelvis is caused by disturbed segmentation, then scoliosis and deformations of neural arches would occur at the side where pelvis is undeveloped. Specimen 1 and 3 show the opposite. The structure of neural arches (Fig. 3) shows that the influence deforming the spinal column arose rather late at the final stage of morphogenesis after the formation of principal structural elements. Therefore, the structure of larva 2 shows, that initially disturbed segmentation could not be the disturbing factor of morphogenesis. Specimen 2 is such an unique one that a morphologist studying morphogenetic principles could consider it a perfect illustration of a lot of propositions on the development of spinal column in anurans. This concerns in particular the hypothesis on the morphogenetic significance of myomeres, septae and the reliable connection of the latter with chord and covering tissues (Borkhvardt and Kovalenko, 1985, 1986). Indeed, the shape and dimensions of myomeres are
On Unique Forms of Anomalous Sacral Structure in Tailless Amphibians 35 among the factors determining the morphogenesis of vertebrae. Neural arches developing without restriction of space by myomeres lose their characteristic features and form almost shapeless primordia (Fig. 2). The number of caudal neural arches was assumed to be limited by too large caudal myomeres (Kovalenko, 1985) closely enveloping the chord and leaving no room for aggregating mesenchyme cells. This assumption is confirmed by the presence of 5 or 6 additional caudal primordia when this factor is absent. Morphogenetic data confirm the assumption that the contact of septae with chord and their tension caused by myomeres are the obligatory conditions of the formation of intervertebral chordal plicae (Kovalenko, 1986), where condylar primordia develop. Reduction of myomeres disturbs these conditions. As a result, the chordal plicae are absent, and the bases of neural arches fuse (Fig. 2). We suppose that the presence of septae separating skeletogenous mesenchyme is the most important morphogenetic factor determining the discrete structure of vertebral primordia (Borkhvardt, 1982, 1990; Kovalenko, 1985, 1992). Despite the absence of left myomeres in specimen 2, it is clear, that the discrete mesenchymatous primordia were formed and the septae were present at that stage of onthogenesis. We could assume that the myomeres were reduced after the formation of mesenchymatous primordia. However, the alternative interpretation must be taken into account as well: either the discrete vertebral primordia are not determined by the primary segmentation (which is contrary to many facts of anomalous morphogenesis), or the septae develop irrespectively of the development of muscular tissue. Unfortunately, it cannot be verified on wholly stained mounts. We listed here only the most obvious conclusions which can be made basing on the preliminary analysis of the unusual structures. However, they demonstrate the value of morphological anomalies compared with normal structures for the study of general morphogenetic principles. Thus, multiple anomalies in the development of cranium, eyes, spinal column, legs and their girdles in specimen 2 suggest an idea, that unilateral absence of sacrum is not directly caused by rudimentary state of pelvic process, but there is a common cause at the bottom of all these defects. The known course of normal morphogenesis makes it possible to draw a conclusion that abnormal development of the skeleton in these specimens began at the initial stages of its formation and was caused by disturbed larval segmentation. Analysis of different forms of structure in connection with the disturbance of segmentation at early stages (Kovalenko, 1983, 1992) shows that neither asymmetry of axial skeleton or skull, nor torsion of the spinal column, nor the rudimentary state of legs correlate with rudimentary state of sacrum, if processes of ilium are normally developed. Therefore, the given interpretation of cause and effect relationship seems to be the most reasonable at present. The analysis of morphologic anomalies makes it possible to reconstruct the morphogenetic relationships more precisely. Therefore, more substantiated conclusions can be made on the possible ways of evolution as well as tin diagnostic and taxonomic value of some characters. Acknowledgments. We are sincerely grateful to. K. Uteshev for lending us a specimen of Xenopus laevis. The work was supported by the Russian Fund for Fundamental Investigations (grant No. 93-04-6661). REFERENCES Böhme G. (1977), Zur Bestimmung guartarer Anuren Europus an Hand von Skelettelementen, Wiss. Zeit. Humboldt Univ. Berlin, 3, 283 300. Borkhvardt. G. (1982), On Morphogenesis and Evolution of Axial Skeletal Structures ( the Skeletal Segment Theory ), Leningrad [in Russian]. Borkhvardt. G. (1990), Axial complex of vertebrates in onthogenesis and phylogenesis, in: Organization, Integration, and Regulation in Biological Systems. Trudy Biol. Inst. Leningr. Univ., 41, 120 138 [in Russian]. Borkhvardt. G. and Karavayeva K. Yu. (1982), Methods of cleaning and staining of small vertebrates skeletons, Zool. Zh., 61(1), 120 121 [in Russian]. Borkhvardt. G. and Kovalenko E. E. (1985), On significance of mechanical interactions for the development of myomeres and axial skeleton in Anamnia, est. Leningr. Univ., 3(1),3 10[inRussian]. Borkhvardt. G. and Kovalenko E. E. (1986), Septae in the embryos and larvae of Anamnia, Dokl. Akad. Nauk SSSR, 287(3), 764 768 [in Russian]. Duellman W. E. and Trueb L. (1986), Biology of Amphibians, New York. Emerson Sh. B. (1979), The ilio-sacral articulation in frog: form and function, Biol. J. Linn. Soc., 2, 155 168. Emerson Sh. B. (1982), Frog, postcranial morphology: identification of functional complex, Copeia, 3, 603 613. Hodler F. (1949), Zur Etwicklung der Anurenwirbelsaule, Rev. Suisse Zool., 56(2), 327 330.
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