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 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 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-μm-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).
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 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.
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 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 malformations 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.
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 malformations 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 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, Kimm-Brinson and Ramsdell (2001) confirm that developmental 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 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.
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 Müller-Edzards 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).