J Mammal Evol DOI 10.1007/s10914-014-9256-7 ORIGINAL PAPER Intervertebral and Epiphyseal Fusion in the Postnatal Ontogeny of Cetaceans and Terrestrial Mammals Meghan M. Moran & Sunil Bajpai & J. Craig George & Robert Suydam & Sharon Usip & J. G. M. Thewissen # Springer Science+Business Media New York 2014 Abstract In this paper we studied three related aspects of the Introduction ontogeny of the vertebral centrum of cetaceans and terrestrial mammals in an evolutionary context. We determined patterns The vertebral column provides support for the body and of ontogenetic fusion of the vertebral epiphyses in bowhead allows for flexibility and mobility (Gegenbaur and Bell whale (Balaena mysticetus) and beluga whale 1878;Hristovaetal.2011; Bruggeman et al. 2012). To (Delphinapterus leucas), comparing those to terrestrial mam- achieve this mobility, individual vertebrae articulate with each mals and Eocene cetaceans. We found that epiphyseal fusion other through cartilaginous intervertebral joints between the is initiated in the neck and the sacral region of terrestrial centra and synovial joints between the pre- and post- mammals, while in recent aquatic mammals epiphyseal fusion zygapophyses. The mobility of each vertebral joint varies is initiated in the neck and caudal regions, suggesting loco- greatly between species as well as along the vertebral column motor pattern and environment affect fusion pattern. We also within a single species. Vertebral column mobility greatly studied bony fusion of the sacrum and evaluated criteria used impacts locomotor style, whether the animal is terrestrial or to homologize cetacean vertebrae with the fused sacrum of aquatic. In aquatic Cetacea, buoyancy counteracts gravity, and terrestrial mammals. We found that the initial ossification of the tail is the main propulsive organ (Fish 1996;Fishetal. the vertebral pedicles in the fetus may be a reliable indicator of 2000). As a result, vertebral column design in cetaceans is sacral homology in modern cetaceans. Finally, we also studied very different from that of terrestrial mammals (Slijper 1936; fusion of the centra of cervical vertebrae in B. mysticetus and Buchholtz 1998, 2010). Since cetaceans are derived from found that it is not completed until after sexual maturity, and terrestrial mammals, evolutionary changes occurred in their after 20 years of age. vertebral column, and these changes are recorded in modern adult morphologies as well as ontogeny. In this paper we Keywords Intervertebral . Epiphyseal . Fusion . Cetacea . compare aspects of vertebral centrum joint ontogeny between Vertebra cetaceans and terrestrial mammals to understand the similar- ities among mammalian morphologies in the vertebral col- M. M. Moran (*) umn, regardless of habitat; and follow vertebral column mor- Department of Anatomy and Cell Biology, Rush Medical College, phologies as they change during cetacean evolution. 600 S. Paulina Street, Suite 506Ac/Fac, Chicago, IL 60612, USA Our first objective is to document ontogenetic epiphyseal e-mail: [email protected] fusion patterns in cetaceans and compare them to those of S. Bajpai some terrestrial mammals. Epiphyseal fusion of the vertebral Department of Earth Sciences, Indian Institute of Technology, centrum is epiphyseal (or growth) plate closure, an ossifica- Roorkee, Uttarakhand 247667, India tion event that results in cessation of longitudinal growth. J. C. George : R. Suydam Vertebral epiphyses fuse over an extended postnatal period, Department of Wildlife Management, North Slope Borough, and in some species, such as Balaena mysticetus,someepiph- P.O. Box 69, Barrow, AK 99723, USA yses fuse long after sexual maturity. Epiphyseal fusion pat- terns have been studied in terrestrial mammals (Dawson 1925; S. Usip : J. G. M. Thewissen Department of Anatomy and Neurobiology, Northeast Ohio Medical Purdue 1983; Roach et al. 2003; Munro et al. 2009), as well as University, 4209 State Route 44, Rootstown, OH 44272, USA some cetaceans (Moore 1968; Ito and Miyazaki 1990;Mead J Mammal Evol and Potter 1990; Yoshida et al. 1994;Galatius2010;Wheeler sizes complicate determination of the number of vertebrae that 1930;Kato1988, Kemper and Leppard 1999;Bestand eventually make up the sacrum in fossils. Lockyer 2002), but little comparative work has been done. Our third objective is to document the fusion of cervical Our second objective is to study the fusion of the sacral vertebrae in B. mysticetus. Unlike most mammals, vertebrae in ontogeny and evolution and the absence of this B. mysticetus and some other cetaceans undergo intervertebral process in modern cetaceans. The sacrum is the area of least fusion of their cervical vertebrae centra (Eschricht et al. 1866; mobility in most mammalian vertebral columns since it is the Wheeler 1930; Slijper 1936; Haldiman and Tarpley 1993; site where the weight-bearing hind limb is anchored to the Buchholtz 2001, 2007; Buchholtz et al. 2005, 2007). This axial skeleton. In terrestrial mammals, the process of sacral severely limits neck mobility in cetaceans, even though this fusion, i.e., the ossification of the intervertebral disc, occurs is the area of greatest vertebral mobility in most other mam- well after birth, affecting the stability of the pelvis in immature mals (Graf et al. 1995; Bebej 2011; Bebej et al. 2012). Just like animals. In modern cetaceans and sirenians, there is no weight sacral fusion in terrestrial mammals, cervical fusion is a pro- bearing hind limb and the sacral vertebrae remain unfused cess with a protracted ontogeny, but no age series of cetacean (Eschricht et al. 1866; Slijper 1936; Buchholtz 2001, 2007; necks with different levels of fusion have been described. Buchholtz et al. 2005). Because of this, it is difficult to distinguish cetacean sacral vertebrae from adjacent lumbar and anterior caudal vertebrae, causing different authors to Materials and Methods use different criteria to homologize cetacean vertebrae. There is no consensus on criteria to distinguish sacral This study used an ontogenetic series of 20 mice (Mus vertebrae in cetaceans and we compare different methods of musculus); skeletal samples of nine pigs (Sus scrofa); anatom- distinguishing and homologizing sacral vertebrae in terrestrial ical specimens of eight bowhead whales (Balaena mysticetus) mammals and cetaceans, and evaluate them against observa- and nine beluga whales (Delphinapterus leucas); two pan- tions on cetacean fetuses. Many marine mammal anatomists tropical spotted dolphin (Stenella attenuata) fetuses; one avoid the issue altogether and use the term lumbar vertebrae B. mysticetus fetus; and skeletons of the Eocene cetaceans for all vertebrae between the last rib-bearing vertebra and the Ambulocetus natans and Kutchicetus minimus. Techniques first hemal-arch-bearing vertebra (e.g., Buchholtz 2001, used include computed tomography imaging, whole mount 2010). Lumbar, in this definition, includes traditionally de- clearing and staining, anatomical dissections, and osteological fined lumbar vertebrae plus unfused sacral, and even anterior study. caudal vertebrae since the first hemal arch is often not present on the first caudal vertebra in animals that have a sacrum Epiphyseal Fusion (Eschricht et al. 1866;Slijper1936;Evans1993; Buchholtz 2010). Slijper (1936) proposed that the modern cetacean sa- Epiphyseal fusion within a single vertebra and across the crum could be recognized on the basis of the location of the vertebral column was illustrated in a diagram that we refer to pudendal nerve as it exits the intervertebral foramen; the first as a fusion map. Each vertebra is represented by a rectangle root of this nerve passes through the foramen between S1 and and the degree of epiphyseal fusion is indicated in shades of S2 (Slijper 1936). gray. To determine the epiphyseal fusion in the fossils, spec- Ontogenetic fusion of sacral vertebrae is of special interest imens were scored based on the fusion scar along the epiph- in fossil cetaceans. Cetaceans originated from terrestrial mam- yses. If an epiphysis was missing, the joint was scored as mals (Thewissen et al. 2007, 2009), and early cetaceans unfused (light gray). If a fusion scar was visible for part or the possessed fused sacra (Gingerich et al. 2001; Thewissen entire circumference of the centrum, it was scored as partially et al. 2001) and were able to locomote on land (Thewissen fused (dark gray). If no fusion scar was visible along any part et al. 1996, 2001; Madar et al. 2002). The origin of cetaceans of the epiphysis, the joint was scored as fused (black). Scoring is well documented by fossils and vertebral columns are is more difficult in fossil specimens than in our recent sam- known for many of these (Fraas 1904; Kellogg 1936; ples. In some fossils the entire circumference of a vertebra was Thewissen et al. 1996;Uhen1999; Gingerich et al. 2001; not visible because of damage to the specimen, or because it Madar et al. 2002; Uhen 2004; Thewissen and Bajpai 2009; was covered with sediment. In those cases, specimens were Bebej et al. 2012). For many Eocene fossil cetaceans, there is scored on the basis of the visible segment. If the vertebra was no ambiguity about the homology of the sacrum because not recovered, it was scored as missing data (white). This multiple vertebrae are fused together and this vertebral region procedure is likely to have introduced some noise in our data articulates with the illium. However, for some of these spec- that is not present in the scores of the modern animals. imens, only a