THE ANATOMICAL RECORD 294:980–986 (2011) Vertebral Anomaly in Fossil Sea Cows (Mammalia, Sirenia) MANJA VOSS,1* PATRICK ASBACH,2 AND ANDRÉ HILGER3 Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Berlin, Germany 2 Institut für Radiologie, Charité – Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany 3 Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institut für Angewandte Materialforschung, Berlin, Germany 1 ABSTRACT Four incompletely preserved caudal vertebrae lacking the neural arches of two fossil sirenian individuals of Halitherium schinzii (Oligocene) from the Rhine area in Germany and northern Belgium reveal osteological alterations. The caudal vertebrae possess a transverse process with growth retardation. This asymmetry indicates that the affected transverse processes are less developed than their counterparts and, consequently, deviate from the norm. Computed tomography (CT) scans reveal osteosclerotic patterns, a morphological feature that characterizes sea cows and supports the nonpathological state of the vertebrae. Additionally, no indications of vertebral fractures or any other occurrences due to external factors are present. This is the oldest documentation of such an anomaly in any sirenian and is interpreted here as hypoplasia, the underdevelopment of an organ or parts of it that might cause a funcC 2011 Wiley-Liss, Inc. tional deﬁciency. Anat Rec, 294:980–986, 2011. V Key words: Oligocene; Germany; Belgium; caudal vertebrae; transverse process; asymmetry; hypoplasia Sirenia, or sea cows, are a group of aquatic mammals that have a fossil record extending from the early Eocene (50 Ma) to the present. Sirenians possess relatively large, stout but streamlined bodies, paddlelike ﬂippers, no hind legs, and a powerful horizontal tail ﬂuke. They are unique among living marine mammals in having an herbivorous diet and several morphological features distinguishing them from all other mammals, such as pachyostosis and osteosclerosis (Domning and de Buffrenil, 1991; Domning, 1994). The development of pachyostosis and osteosclerosis, or pachyosteosclerosis, is indicated by greatly thickened bones and the partial or complete loss of the marrow cavity and the spongiosa in the bones of the sirenian skeleton (Kaiser, 1974). Pachyosteosclerosis, in connection with the achievement of equilibrium in the aqueous medium, produces a weight increase corresponding to the environmental requirements for Sirenia (Kaiser, 1974; Kleinschmidt, 1982). According to Sickenberg (1934, p. 243), pachyosteosclerosis reaches a high degree in the vertebral column, especially in the spinous and transverse processes. The C 2011 WILEY-LISS, INC. V vertebral bone histology is dense in the vertebral arches and processes; greater parts of cancellous bone are rare and restricted to the centra. Sickenberg (1934) already noted the occurrence of left–right asymmetries in vertebrae of the fossil sea cow Halitherium schinzii from the Oligocene (30 Ma) of Belgium, but only referred to alterations of the spinous process and the centrum. Those observations of vertebral asymmetries are supplemented in this study by presenting four caudal vertebrae of two fossil sirenian individuals from the Oligocene of Germany and Belgium. These vertebrae possess an *Correspondence to: Manja Voss, Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Invalidenstraße 43, 10115, Berlin, Germany. Fax: þ49-30-20938868. E-mail: firstname.lastname@example.org Received 7 November 2010; Accepted 18 March 2011 DOI 10.1002/ar.21397 Published online 28 April 2011 in Wiley Online Library (wileyonlinelibrary.com). 981 VERTEBRAL ANOMALY IN FOSSIL SEA COWS underdeveloped transverse process causing a left–right asymmetry. The normally developed transverse processes are important as attachment areas for ligaments and muscles of the back (Nickel et al., 1984). They thus contribute to static and dynamic stability during the extension and ﬂexion of the vertebral column. Therefore, abnormalities of the development of the transverse processes have clinical relevance. One of the most important diseases that affect the attachment modes is enthesitis, a common ﬁnding in spondyloarthritis, which indicates an inﬂammatory disease of the musculoskeletal system (Rothschild and Martin, 1993, 2006; Hermann et al., 2005). Pathologic rotation of the transverse processes on the basis of blockage (blocking) of the spine can result in irritation or even compression of the spinal nerves, causing various neurologic symptoms (Biedermann and Sacher, 2002). Congenital defects (malformations) of the transverse processes are also known and can affect these either directly or indirectly (Wolfers and Hoeffken, 1974). Direct effects on the transverse processes include hypoplasia, the retarded development of an organ or parts of it caused by either a genetic defect or an ontogenetic alteration, and aplasia, which is the total absence of an organ. Assimilation malformations, for example the lumbosacral junction, which can be associated with pseudoarticulation of the transverse process and the sacrum, induce indirect effects. The observation of asymmetrical transverse processes in the fossil sea cow specimens presented here is interpreted as hypoplasia and documented for the ﬁrst time in this group of animals. In this study, we aim at clarifying and elucidating the development of this vertebral anomaly. For this purpose, we externally describe the affected vertebrae and provide Computed tomography (CT) scans for the internal investigations of the bones. This study may contribute to the knowledge of skeletal alterations in sea cows. MATERIALS AND METHODS Four caudal vertebrae of the extinct sirenian Halitherium schinzii were analyzed. H. schinzii was a dugongid sea cow of up to 3 m in body length living in the coastal areas of the early Oligocene (30 Ma) Sea of Europe, especially of Germany (e.g. Lepsius, 1882; Böhme, 2001; Voss, 2008) and Belgium (Sickenberg, 1934). One specimen is represented by a single vertebra (P 2068/5) of H. schinzii, which is deposited in the National Museum of Victoria (MV) in Melbourne, Australia. Vertebra P 2068/5 is assumed to be from the lower Miocene (20 Ma) of the Rhine area in western Germany, but may be most probably from the sandy facies of the Ratingen Formation (Rupelian, lower Oligocene), because no lower Miocene deposit is known in this area of the Rhine Valley. The other three vertebrae (Plt.M.137.1–3) belong to an individual of H. schinzii (Plt.M.137) from the Rupelian (lower Oligocene, 30 Ma) of Steendorp, southwest of Antwerp (Belgium), which is stored in the Institut Royal des Sciences Naturelles de Belgique (IRSNB) in Brussels. The specimen Plt.M.137 is a partial skeleton consisting of fragmentarily preserved cranial and postcranial elements including the frontal, maxilla, scapula, humerus, radius, and ulna of the right side; the humerus and ulna of the left side; the second cervical vertebra; 10 thoracics; 13 caudals; and 5 ribs and 4 chevron bones. CT was applied to investigate the internal structure of the H. schinzii vertebrae. Two objects of about 10 cm in diameter were scanned, the vertebrae Plt.M.137.2 and 3 of the Belgian specimen (Fig. 4). The CT scans were performed at the tomography station CONRAD (Cold Neutron Radiography) in the Helmholtz-Zentrum Berlin für Materialien und Energie (Hilger et al., 2006). At the end of a curved neutron guide, a pinhole aperture of 2 cm was installed. The sample position and the detector system were located 5 m behind the aperture. A combination of a 200-lm-thick lithium(6)ﬂuoride scintillator and a CCD-camera (Andor DW436N-BV) with a 50 mm lens system was used as a detector. For each scan, 400 projections were measured with an exposure time of 20 sec. The software Octopus was used for the reconstruction of the datasets. Subsequently, multiplanar visualization was obtained in the transverse, sagittal, and frontal planes with the VGStudioMax1.2 software (Volume Graphics GmbH, Heidelberg, Germany). RESULTS The vertebrae reported in this study are incompletely preserved, all missing the neural arches. Vertebrae Plt.M.137.1 and 2 also lack their right transverse processes (Fig. 1A and B). The vertebrae are identiﬁed as caudal vertebrae based on the presence of the characteristic and more or less prominent and paired anterior and posterior demifacets for the chevrons on the ventral side of each centrum. The centra are wider than high, having a slightly hexagonal outline. The cranial and caudal epiphyses are ﬂat to slightly concave. Even though the centra Plt.M.137.1–3 are disarticulated, their size might indicate their arrangement in a craniocaudal series (Fig. 1). The centra have a maximum width of 72 mm in Plt.M.137.1 and 73 mm in Plt.M.137.2 and 3, and a dorsoventral height of 44 mm in Plt.M.137.1 and about 52 mm in Plt.M.137.2 and 3. Vertebrae Plt.M.137.1–3 are from the middle part of the caudal vertebral column, as indicated by the single almost completely preserved right transverse process in Plt.M.137.3 (Fig. 2), which is more or less horizontally directed and only slightly caudally inclined. Vertebra P 2068/5 is one of the more posterior caudals within the vertebral column; because of its strongly caudally inclined left transverse process (Fig. 3), it is evidently from the peduncular region of the tail. Its centrum measures only 40 mm in width and about 33 mm in dorsoventral height. The transverse processes form lateral extensions of the centrum as is typical for caudal vertebrae. However, the left ones of caudal Plt.M.137.1–3 (Fig. 1) and the right one of P 2068/5 (Fig. 3) are exceptional. These transverse processes are present, but vestigial. This is best observable in the dorsal view of Plt.M.137.3 (Fig. 2A) and P 2068/5 (Fig. 3A), because these are the only vertebrae having a completely preserved counterpart and, therefore, provide the best contrast. The affected transverse processes have the form of a stump and, in contrast to their normal counterparts, do not extend to their full length laterally, but end in a slight caudal tip. They are miniature editions of transverse processes in normal condition without any indication of a pathological pattern or an association with other malformations and, therefore, reveal a 982 VOSS ET AL. Fig. 1. Caudal vertebrae Plt.M.137.1–3 of Halitherium schinzii with vestigially developed left transverse processes in dorsal views. c, vertebral centrum; fs, fracture surface of broken neural arch; tp, transverse process; and vtp, vestigial transverse process. hypoplasia, an anomaly or alteration of the vertebrae for which the reason is unknown. However, the diagnosis of associated malformations is limited because of the lack of preservation in the specimens of H. schinzii. Even though the right transverse processes of Plt.M.137.1 and 2 are broken (Fig. 1A and B), it can be assumed that they reached their normal extent during life, because their bases are distinctly longer in anteroposterior direction than their vestigial counterparts. Fractures or abrasion that could have caused the asymmetry of the transverse processes can be excluded, because the surfaces of the vertebrae are smooth. Generally, fractures can be easily identiﬁed by a sharply deﬁned area, which is missing at the hypoplastic transverse processes. The uniform shape of the affected transverse processes is another argument against external factors that may have caused the anomaly reported here. Their shape is similar not only among Plt.M.137.1–3 from one individual (Fig. 1), but also corresponds to P 2068/5 from another specimen (Fig. 3). Fractures along the bases of the neural arches of each vertebra (Figs. 1, 2A, and 3A) and along the right transverse processes of Plt.M.137.1 and 2 reveal the dense, osteosclerotic histology peculiar to sirenian bones. Neutron scans of Plt.M.137.2 and 3 (Fig. 4) conﬁrm the typical compact bone histology, therefore, supporting a nonpathological condition of the vertebrae. A distinction between compact and cancellous parts of the bone cannot be inferred from the CT scans. It is assumed that the dense bone situation prevents the vertebrae to be completely penetrated by the neutron scans, which indicates that the cancellous bone has been almost completely displaced (compare Sickenberg, 1934). Consequently, possible alterations in detail remain unrevealed. DISCUSSION The left transverse processes of the caudals Plt.M.137.1–3 (Figs. 1 and 2) and the right one of P 2068/5 (Fig. 3) deviate from the norm in showing less development than their counterparts. In contrast to aplasia (resulting in the total absence of an organ), the transverse processes are still present and cause an observable left–right asymmetry of the vertebrae, indicating a hypoplastic pattern. As stated above, hypoplasia of the transverse processes is a congenital defect (Wolfers and Hoeffken, 1974), which might be developmental and/or genetically induced. This is supported by Rathke (1952), who states that a constant type of alteration and its restriction to a certain vertebral region argue for a congenital defect. In the studies of Tanaka and Uhthoff (1981a,b) and Erol et al. (2002), congenital malformations of the vertebral body were classiﬁed into two categories. Type 1 includes failure of formation and Type 2 is a developmental failure of segmentation. Failure of formation implies total or partial defects of the vertebral body, and also speciﬁc abnormalities in the shape of the vertebral body. Failure of segmentation signiﬁes unsegmented vertebrae due to abnormalities of the intervertebral disc. Tanaka and Uhthoff (1981b) emphasize that this concept is accepted for all congenital vertebral malformations and, additionally, suggest a subdivision into defect and error. ‘‘Defect of formation’’ implies absence VERTEBRAL ANOMALY IN FOSSIL SEA COWS 983 Fig. 2. Caudal vertebra Plt.M.137.3 of Halitherium schinzii. A: Dorsal view; B: posterior view; and C: ventral view. c, vertebral centrum; d, demifacet for chevron bones; fs, fracture surface of broken neural arch; tp, transverse process; and vtp, vestigial transverse process. of the vertebral body or part of it and ‘‘defect of segmentation’’, total or partial absence of the intervertebral disc. ‘‘Error’’ signiﬁes some other malformation. A third category refers to a mixed type and can be any combination of the above anomalies. Ghebranious et al. (2007) enumerate failures of formation as butterﬂy vertebrae, hypoplasia, and hemivertebrae. According to the classiﬁcations of Tanaka and Uhthoff (1981a,b) and Ghebranious et al. (2007), the less developed transverse processes in this study can be explained by error of formation leading to hypoplastic transverse processes. The left–right asymmetry in the vertebrae caused by this failure of formation is conﬁrmed by Erol et al. (2002) stating that this may occur on the right or the left side of the body. Even though congenital malforma- tions are mostly referred to in the literature as affecting the vertebral body alone, Tanaka and Uhthoff (1981b) point out that the extent of the defect can involve different parts of the centrum to different degrees, as is observable in the specimens presented here. The vestigial transverse processes support their cartilaginous anlagen and even that of their bony cores. However, according to the comments of Rathke (1952) and Matsuura et al. (1998) on incompletely developed organs, the growth impulse of the transverse processes must have been disturbed during ontogeny, causing the development of only one normal transverse process. Following Tanaka and Uhthoff (1981a,b) and Erol et al. (2002), all types of congenital malformations result from disruption of normal vertebral development. 984 VOSS ET AL. Fig. 3. Caudal vertebra P 2068/5 of Halitherium schinzii with right vestigially developed transverse process. A: Dorsal view and B: ventral view. c, vertebral centrum; d, demifacet for chevron bones; fs, fracture surface of broken neural arch; tp, transverse process; and vtp, vestigial transverse process. The vertebrae of the spine are formed during somitogenesis and even a slight disruption of this process, as has been done in animal models, results in congenital vertebral defects (Erol et al., 2002). Erol et al. (2002) hypothesized that the close interaction of genes and environment produces the normal spine and the environmental factors affect the delivery of the genetic instructions during development. According to Erol et al. (2002) and Maisenbacher et al. (2005), the interaction of genes and environment is disrupted in embryonic somite formation leading to deformities. Tanaka and Uhthoff (1981b) even state that vertebral malformations may have greater environmental than genetic components to their origin. Concerning the genetic cause of vertebral malformations, developmental studies in animal models have identiﬁed many genes regulating somite formation and segmentation (Erol et al., 2002). One family of these somite genes is that in the ‘‘notch’’ pathway. The cycling of these genes regulates the periodic activation of the notch signaling pathway, which would be required for the somite segmentation process. A possible candidate gene for the congenital vertebral malformations is the protein-coding WNT3A gene, which has recently been identiﬁed as a negative regulator of notch signaling and somitogenesis (Ghebranious et al., 2007). Mutations in this gene are known to cause caudal vertebrae malfor- mations in mice. Some abnormalities of genes involved in mouse somitogenesis have been found to cause spinal deformities even in humans (Erol et al., 2002). Following Maisenbacher et al. (2005), genetic disruption is supposed to produce multiple severe defects. However, this is not the case in the vertebrae of this study. Environmental inﬂuences are more probably expected to induce single or localized axial defects (Maisenbacher et al., 2005). In this study, hypoplasia is only observed in the transverse processes, resulting in a localized defect of the vertebrae, which is most probably related to environmental factors. However, hypoplastic transverse processes are reported from neither extant nor fossil sea cows to date. Therefore, the environmental factors in the animal’s lifetime cannot be completely established. However, the developmental toxicity of the environmental factors, such as increased body temperature, carbon monoxide, and chemical reactions, and their undesirable effects on the development of the organism during the prenatal and postnatal period have been investigated experimentally in animal models (Edwards, 1986; Erol et al., 2002). Transient exposure to toxic substances such as carbon monoxide during the fetal period has been shown to induce hypoxia, causing congenital vertebral anomalies in mice (Erol et al., 2002). A possible environmental setting that could be associated with the development of hypoplastic transverse VERTEBRAL ANOMALY IN FOSSIL SEA COWS 985 Fig. 4. Neutron scans of vertebrae Plt.M.137.2 (A–C) and 3 (D–E) showing osteosclerosis characteristic of Sirenia (not to scale). A: Oblique anterodorsal view; B: oblique posterodorsal view; C: posterior view; and D and E: dorsal view. processes in the sea cow specimens of this study is naturally occurring harmful algal or red tide blooms. Recent studies point out that marine mammals including manatees are commonly susceptible during moderate and severe red tides and that they are affected by toxins produced by red tide dinoﬂagellates (Kimm-Brinson and Ramsdell, 2001; Flewelling et al., 2005; Walsh et al., 2005). However, skeletal malformations in manatees that might have resulted from red tides are not reported to date. This might be because toxication of sea cows resulting from intense algae blooms is a fast process indicating a short-term effect not leading to skeletal alterations, but to the animal’s death. This is supported by Flewelling et al. (2005), who showed that sea grass, the main food of Florida manatees (Trichechus manatus latirostris), has acted as the primary source of toxin during recent deaths of manatees, because of its ability to accumulate high concentrations of red tide toxins. The possibility of long-term effects such as skeletal malformations was apparently not investigated here. However, algal blooms are natural events in the habitat of manatees. Therefore, it cannot be excluded that extant sea cows are morphologically affected by algal blooms if they encounter low concentrations of red tide toxins over a long time. This hypothesis is supported by Kimm-Brinson and Ramsdell (2001), who describe adverse developmental effects of red tide toxins in embryos of Medaka ﬁsh (Oryzias latipes) in the form of morphologic abnormalities, such as lateral curvature of the spinal column. In view of the similar developmental processes found in higher and lower vertebrates, KimmBrinson and Ramsdell (2001) conﬁrm that developmen- tal toxicity and abnormalities potentially occur among different phylogenetic classes as a result of cumulative exposure to red tide events. The form of hypoplastic anomaly reported here is occasionally noted on X-rays of the human vertebral column (P. Asbach, pers. obser.) and the German shepherd dog (Julier-Franz, 2006). However, both observations are supposed to have probably no predisposition for clinical signiﬁcance. In humans, for example, asymmetries that refer to the transverse processes are used as landmarks during radiographical investigations (P. Asbach, pers. obser.). According to Rathke (1952), congenital anomalies especially in the centra cause changes in the stasis of the vertebral column. Junghans (1933, 1937) stated that congenital defects of the vertebral column may result in a compensatory growth of other vertebrae, particularly of the adjacent ones, maintaining an equilibrium. However, this cannot be conﬁrmed in this study because of the disarticulated preservation of the specimens. Although an impairment of caudal mobility during the lifetime of these specimens of H. schinzii cannot be excluded, because of the direct relationship of the transverse processes with the muscular system, it is assumed that the impact of the anomaly was to a minor degree. According to Biedermann and Sacher (2002), only an association of hypoplastic transverse processes with other malformations of the spine implies a relative clinical importance of such ﬁndings, which, however, is not the case in the specimens described here. In conclusion, this is the ﬁrst record of hypoplasia in vertebrae of extinct as well as extant sea cows. The 986 VOSS ET AL. hypoplastic transverse processes of H. schinzii reﬂect an anomaly caused by a deformity of one lateral anlage. Their development can potentially be explained by red tide toxin exposure affecting the delivery of the genetic instructions during ontogeny. However, further investigations on extant sea cows are required to verify this hypothesis. ACKNOWLEDGMENTS The authors thank Etienne Steurbaut, IRSNB, Brussels, for permission to borrow the vertebrae of H. schinzii, and Aneliese Folie for facilitating this loan. Eric Fitzgerald provided access to the specimen housed in the Museum Victoria Melbourne. We thank Jan MüllerEdzards for preparing the line drawings of the vertebrae. Daryl Domning and an anonymous referee provided helpful comments to improve this article. This research was funded by the German Research Foundation (DFG). LITERATURE CITED Biedermann H, Sacher R. 2002. 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