Intervertebral and Epiphyseal Fusion in the Postnatal Ontogeny of Cetaceans and Terrestrial Mammals

Home , Epiphysis, Protocetidae, Rodhocetus

J Mammal Evol DOI 10.1007/s10914-014-9256-7

ORIGINAL PAPER

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 patterns 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 single fossil sacrum has been found, and the Vertebrae were identified using the descriptions for ontogenetic age of these fossil cetaceans is unknown. Given A. natans (Thewissen et al. 1996; Madar et al. 2002)and the slow pace of sacral centrum fusion after birth, low sample K. minimus (Thewissen and Bajpai 2009). J Mammal Evol

Fusion data for M. musculus were collected from the eruption sequences and wear pattern (Getty 1975; Hillson cleared and stained ontogenetic mouse series. This technique 1986). provided clear visualization of the epiphyseal fusion pattern. Fusion data in fossils were determined from inspection of the external surface of each vertebra. The inability to inspect the Modern Cetacean Fetuses internal morphology of these vertebrae introduces a level of variation that is not present in the other methods and is a likely Stenella attenuata specimens were acquired from the Los source of noise in our fossil data. Angeles Natural History Museum (LACM), and pertain to Carnegie Stage 21–23 (Thewissen and Heyning 2007). The Modern Cetacean Osteological Samples pelvic regions of S. attenuata LACM 94310 and B. mysticetus 2000B3F fetuses were dissected to identify the location of the We recorded vertebral counts, intervertebral and epiphyseal pudendal nerve before clearing-and-staining. LACM 94310 is fusion patterns in B. mysticetus and D. leucas,duringnative 185 mm in total length (TL) and is from C21-22. The Alaskan subsistence hunts in Barrow and Point Lay, Alaska. B. mysticetus fetus 2000B3F is 403 mm in TL and from These samples were collected under permit NOAA-NMFS C23. These specimens, along with LACM 94382 (TL= 814-1899-01 with permission from the Alaska Eskimo 225 mm), were cleared and stained to investigate the presence Whaling Commission and local subsistence hunters. Sagittal of morphological traces of sacrum development. samples of vertebral centra, intervertebral discs, growth plates, and epiphyses were cut using manual and power saws. Some Eocene Cetaceans samples were skeletonized by cold water maceration. Balena mysticetus were aged using criteria based on baleen length and Two of the most basal cetaceans (Geisler and Uhen 2005; body length (Lubetkin et al. 2012), but D. leucas could only Uhen et al. 2011) were chosen for study because nearly be relatively aged by comparing degrees of fusion and assum- complete vertebral columns are known for both A. natans ing that more epiphyseal fusion is present in older individuals. (Ambulocetidae, HGSP 18507; Thewissen et al. 1996; For D. leucas, a large number of partial vertebral columns Madar et al. 2002)andK. minimus (Remingtonocetidae, were available. Balena mysticetus and D. leucas specimens IITR-SB 2647; Bajpai and Thewissen 2000; Thewissen and provided data on epiphyseal fusion. Cervical fusion was stud- Bajpai 2009). ied in B. mysticetus only, because D. leucas do not fuse their cervical vertebrae. Computed Tomography Ontogenetic Series of Mus Musculus Computed tomography (CT) scans of modern and fossil ceta- We collected an ontogenetic series of M. musculus from birth cean specimens were completed at Loyola University Medical to 60 days postnatal every ten days, plus individuals over Center in Maywood, IL, using a Siemens Somatom Sensation one year old. Specimens were cleared and stained to show CT scanner that produced sequential slices at 0.6 mm thick- only bone (Alizarin Red) and cartilage (Alcian Blue) struc- ness and at NEOMED, Rootstown, OH, on a Norland Stratec tures, and all other tissues were chemically cleared, using a XCT Bone Densitometer μCTscanner yielding 0.25 mm thick protocol adapted from Wassersug (1976). This collection of- slices. Sequential CT images were studied using Image J fered details on the timing of bony epiphyseal and sacral (Rasband 2011). fusion in a terrestrial mammal.

Artiodactyl Osteological Collection Institutional Abbreviations We used S. scrofa as a second modern comparative sample to investigate epiphyseal and sacral fusion, because terrestrial CM, Carnegie Museum of Natural History; FMNH, Field artiodactyls are the closest mammalian relative of cetaceans Museum of Natural History; HGSP, Howard University- (Thewissen and Madar 1999; Geisler et al. 2007; Thewissen Geological Survey of Pakistan, Quetta, Pakistan; IITR-SB, et al. 2007; Spaulding et al. 2009). Osteological specimens Indian Institute of Technology, Roorkee, India- Sunil Bajpai were studied at the Field Museum of Natural History in collection; LACM, Los Angeles Natural History Museum, Chicago, IL, and the Carnegie Museum of Natural History specimens deposited at Northeast Ohio Medical University in Pittsburgh, PA. The S. scrofa studied were wild-caught or (NEOMED); LUMC, Loyola University Medical Center; domesticated individuals for which no exact ages were avail- NOAA-NMFS, National Oceanic and Atmospheric able.Instead,agesoftheS. scrofa were estimated from tooth Administration- National Marine Fisheries Service. J Mammal Evol

Results (Fig. 1v). The posterior lumbar epiphyses are the last to fuse (Fig. 1vi). Complete epiphyseal fusion is present Epiphyseal Fusion by 60 days postnatal (Fig. 1vii, viii), well after sexual maturity. The ontogenetic M. musculus series shows that, for In S. scrofa, epiphyseal fusion starts after the erup- individual vertebrae, the cranial epiphysis always fuses tion of the third molar (M3), when the animal is ap- to the centrum before the caudal epiphysis (Fig. 1). proximately 18–20 months old (Getty 1975), and the Epiphyseal fusion starts around ten days postnatal pattern is similar to that of M. musculus in that the (Fig. 1ii) in two regions: the neck and sacrum. Fusion cranial epiphysis fuses to the centrum before the caudal progresses caudally from the cervical and sacral regions epiphysis (Fig. 2). Similar to M. musculus, epiphyseal as ontogeny progresses (Fig. 1ii–vi). Epiphyses of the fusion progresses caudally through the vertebral column, seven cervical vertebrae and four sacral vertebrae are beginning in two different regions (Fig. 2i–v). In both completely fused at 20 days (Fig. 1iii). At that age, species, the anterior center of fusion is in the cervical cranial epiphyses of the thoracic vertebrae are complete- region. However, the posterior ossification center in ly fused, but the caudal epiphyses are not, and both S. scrofa is located in the anterior caudal vertebrae, epiphyses of the lumbar and anterior caudal vertebrae rather than in the sacrum as in M. musculus. are partially fused. Epiphyseal fusion progresses to the Epiphyseal fusion progresses cranially and caudally anterior thoracic and caudal vertebrae by 30 days from this posterior ossification center (Fig. 2v,vi). postnatal (Fig. 1iv). At 40 days postnatal, the cervical, The anterior thoracic vertebral epiphyses fuse last anterior thoracic, sacral, and anterior caudal vertebrae (Fig. 2viii, ix). are completely fused, as are the cranial epiphyses of the Epiphyseal fusion in B. mysticetus and D. leucas starts thoracics and most of the posterior caudal vertebrae from the cervical and the caudal regions; fusion continues

Fig. 1 Mus musculus postnatal fusion maps. i, 1 day (M81); ii, 10 day direction of epiphyseal fusion. C2, second cervical vertebra; T1,first (M54); iii, 20 day (M82); iv, 30 day (M78); v, 40 day (M143); vi, 50 day thoracic vertebra; L1, first lumbar vertebra; S1, first sacral vertebra; (M83); vii, 60 day (M84); viii, 230 day (M85). Blocks with two colors Ca1, first caudal vertebra. Light gray, no epiphyseal fusion; dark gray, show different amounts of fusion within a single vertebra. Arrows show partial epiphyseal fusion; black, complete fusion J Mammal Evol

Fig. 2 Sus scrofa postnatal fusion maps. i, FMNH 49844; ii, CM 59541; direction of epiphyseal fusion. Light gray, no epiphyseal fusion; dark iii, FMNH 42440; iv, FMNH 42439; v, CM 16; vi, CM 30427; vii, gray, partial epiphyseal fusion; black, complete fusion; white, missing FMNH 97884; viii, FMNH 92908; ix, FMNH 92907. Arrows show vertebra towards mid-column from both these end regions (Fig. 3). The the sacrum where fusion is completed before other areas anterior edge of the caudal ossification center in B. mysticetus (Fig. 4). A cervical center of fusion occurs in is located at vertebra 36 (post-cervical 29 or post-thoracic 15) K. minimus, whereas the A. natans specimen appears in one specimen (Fig. 3i) and four vertebral levels caudal to to be younger and lack this center (or possibly cervical that in another specimen (Fig. 3iv). Epiphyses of anterior vertebrae are too incompletely preserved to detect it). In thoracic vertebrae are starting to fuse by five to ten years old both fossil cetaceans, mid-caudal vertebrae have firmly in B. mysticetus (Fig. 3ii). Cervical epiphyseal fusion is not fused epiphyses and there is the suggestion that fusion complete in a one year old B. mysticetus (Fig. 3i) but is here does not start at the sacral center, but instead that complete in the 40 year old B. mysticetus. The thoracic and it commences at a separate, mid-caudal center (Fig. 4). lumbar vertebral epiphyses remain unfused through 20 years of age (Fig. 3iv). Delphinapterus leucas demonstrate the same Sacral Centrum Fusion and Sacral Homology epiphyseal fusion pattern as B. mysticetus (Fig. 3v–xiii). Posterior thoracics are the last vertebrae to fuse in D. leucas. Intervertebral sacral joints fuse in a cranial to caudal direction The posterior caudal vertebrae are not preserved in these wild in M. musculus. Intervertebral fusion causes sacral discs to caught specimens and therefore fusion pattern is unknown. decrease in width and eventually be replaced by bone As in modern mammals, including cetaceans, in gen- (Fig. 5i–iii). At 20 days postnatal all intervertebral discs eral, cranial epiphyses fuse before caudal epiphyses, and are intact, and the nucleus pulposus is present in each in both A. natans and K. minimus thereisanareanear sacral joint (Fig. 5i). By 40 days postnatal, the S1/S2 J Mammal Evol

Fig. 3 Balaena mysticetus and Delphinapterus leucas fusion maps. i–iv, epiphyseal fusion; ages are unknown as they were hunted, wild-caught B. mysticetus specimens: i, 1 year (09B11); ii, 5 years (11B8); iii, 5– individuals. Arrows show direction of epiphyseal fusion. Light gray,no 10 years (09B5); iv, 20 years (11B9). The ages of these individuals are epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete determined based on methods described by Lubetkin et al. (2012). v–xiii, fusion; white, missing data D. leucas specimens. Individual belugas are ranked according to

and S2/S3 intervertebral discs are ossified (Fig. 5ii), and In S. scrofa, the S1/S2 joint is the first to fuse and fusion the S3/S4 joint is maintained with the nucleus pulposus progresses caudally to the last sacral joint (S3/S4), similar to still intact (Fig. 5ii). At 60 days all three sacral joints M. musculus (Fig. 5iv–vi). At six to eight months of age, are fused (Fig. 5iii). Residual growth plates are present sacral intervertebral fusion is present in the S1/S2 and S2/S3 as thin strips of cartilage (Fig. 5ii, iii). This indicates joints, but not in the S3/S4 joint (Fig. 5iv). By 18–20 months, that full sacral fusion in M. musculus occurs around or the sacral centra are fusing (Fig. 5v), although at this age the just after sexual maturity. growth plates are still present. This implies that sacral J Mammal Evol

Fig. 4 Ambulocetus natans and Kutchicetus minimus fusion maps. i, A. natans (HGSP 18507). ii, K. minimus (IITR-SB 2647). Specimen numbers are listed for each vertebra. Light gray, no epiphyseal fusion; dark gray, partial epiphyseal fusion; black, complete fusion; white, missing data intervertebral fusion occurs before epiphyseal fusion in sacral homology. Dissection of the pudendal nerve of S. scrofa.InS. scrofa older than 20 months all sacral joints S. attenuata (Fig. 6i, ii) shows that it receives ventral rami are fused (Fig. 5vi). from three spinal nerves. The first root of the pudendal nerve Lacking intervertebral fusion in the sacrum of cetaceans exits the intervertebral foramen between the 10th and 11th makes homologizing these vertebrae difficult. We evaluated post-thoracic vertebrae (Fig. 6i, ii). These nerves join and Slijper’s(1936) pudendal nerve location as a criterion for descend through the abdominal wall to the genital region

Fig. 5 Terrestrial mammal sacra. i–iii, Cleared and stained mice 49844); v, 18–20 months (FMNH 42439); vi, greater than18–20 months (M. musculus) sacra, ventral view. i, 20 days; ii, 40 days; iii, 60 days (CM 30427) postnatal. S1 S2 S3 S4, sacral vertebrae 1–4; gp,growth postnatal. iv–vi, Skeletonized S. scrofa sacra. iv, 6–8months(FMNH plate; np, nucleus pulposus; tp, transverse process; uf,unfused J Mammal Evol

Fig. 6 Pudendal nerve of modern cetaceans. i, S. attenuata fetus, of the penis; ep, epididymus; pe,penis;pn, pudendal nerve; ri, stage C21/22 (LACM 94268); ii, black and white line drawing of right innominate; S1/Pt10, first sacral vertebra according to Slijper S. attenuata pudendal nerve; iii, B. mysticetus fetus, stage C23 or post-thoracic vertebra 10; sp, spinous process; te, testis; ti, (2000B3F); iv, black and white line drawing of B. mysticetus tibia; tp, transverse process; vr, ventral ramus pudendal nerve. cc, crus of corpus cavernosum; dn, dorsal nerve where they innervate the external anal sphincter and genitals (Fig. 7ii). The larger spinous process ossifications cor- (Fig. 6i, ii). This fetus (LACM 94310) and several respond to the location of the sacrum as identified by others were subsequently cleared and stained (Fig. 7i), the pudendal nerve dissection in both individuals and show that centers of ossification are present in most (Fig. 7). of the cartilaginous vertebral centra and vertebral arches Dissection of the B. mysticetus fetus (2000B3F) showed (Fig. 7). that the first root for the pudendal nerve passes through LACM 94310 has a total of 79 vertebrae, seven the intervertebral foramen between the 7th and 8th post- cervical, 12 rib-bearing thoracics, and 56 post-thoracics thoracic vertebrae (Fig. 6iii, iv). This fetus has a total of (Fig. 7i). Sixty-nine of these vertebrae are partly ossi- 55 vertebrae. Laminae and pedicles of the vertebrae are fied, and the last ten caudal vertebrae are cartilaginous. ossified for most of the column, but larger ossifications As is normal among mammals, independent ossification are present at post-thoracic vertebrae 3–7(Fig.7iii). These cores occur in the centrum, vertebral arch, and spinous larger ossifications are more anteriorly located within the process. Unusually though, the pedicle ossifications are vertebral column than those in S. attenuata, and are ante- larger in post-thoracic vertebrae 13–17 (Fig. 7i), with rior to the exit location of the pudendal nerve, which is those of post-thoracic vertebra 13 and 17 smaller than also unlike that in S. attenuata. those of 14–16. This pattern is similar to that in an CT scans of the four vertebrae A. natans sacrum older S. attenuata fetus (LACM 94382, Fig. 7ii). This (HGSP 18507.834) indicate complete fusion of the S1/S2 fetus also has 79 total vertebrae, but the larger sacral and S2/S3 joints (Fig. 8i, ii). The S3/S4 joint is fused at ossifications are present in post-thoracic vertebrae 15–18 the periphery of the joint, but an oval-shaped radiolucent J Mammal Evol

Fig. 7 Cleared and stained modern fetal cetaceans. i, S. attenuata LACM 94310; ii, S. attenuata LACM 94382; iii B. mysticetus 2000B3F. The black ovals surround the larger vertebral ossifications that correspond to the location of the sacrum as identified by pudendal nerve dissection in S. attenuata (i, ii). In B. mysticetus (iii) the larger vertebral ossifications are located anterior to the sacrum as identified through pudendal nerve location

space is visible in the joint on the CT scan (Fig. 8i,ii). Our postnatal series of B. mysticetus skeletal specimens shows This may be where the nucleus pulposus once resided. intervertebral fusion in a 1 year old individual (dark gray in There is also a thin radio-opaque line visible on the CT Fig. 9i). Bony intervertebral fusion (black) is present at the scans between S3 and S4 extending from the edges of this center of the cervical vertebrae and progresses peripherally oval space (Fig. 8i). This white line is a narrow extension (Fig. 9i–iv). By the age of 20, most cervical vertebrae have of the air-filled space between S3 and S4 that was infil- fused, with the exception of the most caudal cervical joints trated by mineral. (Fig. 9v, vi). By 40 years of age, all seven cervical vertebrae The four vertebrae of the sacrum (IITR-SB 2647.13) of and all growth plates are completely fused and the individual K. minimus were incompletely fused and broken after death, centra cannot be recognized (Fig. 9vii). resulting in individual sacral vertebrae that are still distinct on the CT scans (Fig. 8iii). Thewissen and Bajpai (2009)proposed that this specimen of K. minimus was immature (Fig. 8iii, iv); sacral centrum fusion pattern remains unknown. Discussion

Epiphyseal Fusion Cervical Intervertebral Fusion In M. musculus, epiphyseal fusion starts in cervical and sacral In B. mysticetus, the seven cervical vertebrae fuse together and vertebrae and simultaneously progresses from those two re- are remodeled to form a solid, immobile bony block (Fig. 9). gions caudally (Fig. 1). These two fusion centers are present in J Mammal Evol

Fig. 8 Archaic cetacean sacra. i, ii, CTscans of A. natans sacrum (HGSP 18507); i, horizontal; ii, sagittal; iii–iv, K. minimus sacrum (IITR-SB 2647); iii, ventral photograph, scale is 1 cm; iv, coronal CT scan. gp,growthplate;sp, air-filled space; uf, unfused

S. scrofa too, but epiphyseal fusion progresses cranially in Sacral Centrum Fusion and Sacral Homology addition to caudally from the sacral region (Fig. 2). This cranially directed fusion is absent in M. musculus, or, possibly, Developmental findings can help in establishing sacral ho- greatly delayed. The last vertebrae to fuse their epiphyses are mology. The sacrum is formed by two independent develop- the lumbar vertebrae in M. musculus, and the anterior thoracic mental processes. The first is the regionalization of the vertebrae in S. scrofa (Figs. 1 and 2). paraxial mesoderm into sections that will eventually be neck, In B. mysticetus, epiphyseal fusion is greatly delayed to thorax, lumbus, and sacrum. Well after regionalization is the point where, around the age of 20, only cervical verte- finalized, somites are formed by a clock-and-wave function brae and some anterior thoracic vertebrae have fused epiph- that cuts the paraxial mesoderm into somites (Pourquié 2007). yses (Fig. 3). Epiphyseal fusion therefore occurs well after Narita and Kuratani (2005) found remarkable stability in the sexual maturity (Chittleborough 1955). Like in presence of approximately 26 pre-sacral vertebrae across a B. mysticetus, epiphyseal fusion in D. leucas starts in two large diversity of vertebrates and considered this developmen- regions: it progresses caudally from the cervical vertebrae tally constrained. Minor heterochrony in the axial expression and cranially from the caudal region (Fig. 4), a pattern of Hox genes can lead to shifts in the location of the sacrum consistent with other cetacean species (Balaenoptera (Gérard et al. 1997;Zákányetal.1997). The regulatory physalus, Wheeler 1930;Ohsumietal.1958; factors controlling Hox expression are highly conserved Balaenoptera acutorostrata, Kato 1988; Stenella across species, although some variation in this expression coeruleoalba, Ito and Miyazaki 1990;Galatius2010; may occur in baleen whales (Shashikant et al. 1998). Neophocaena phocaenoides, Yoshida et al. 1994; Signaling related to the formation of regional territories Balaenoptera borealis,BestandLockyer2002; Phocoena in the axial skeleton occurs before the formation of so- phocoena, Galatius and Kinze 2003). Within a single ver- mites, but overall, the pattern is constrained, allowing for tebra, dolphins fuse the cranial epiphyses first followed by only minor heterochronic variations. This should be taken the caudal epiphyses (Ito and Miyazaki 1990) and our into account when evaluating criteria for homologizing results confirm this occurs in B. mysticetus and D. leucas. sacral vertebrae of modern cetaceans where there is no This epiphyseal fusion pattern is also present in terrestrial fused sacrum. mammals. Based on our limited evidence of two Eocene Data from terrestrial artiodactyls (including basal taxa such fossil vertebral columns, it is difficult to make generaliza- as Messelobunodon) and fossil cetaceans (Rodhocetus, tions, but the data appear consistent with the epiphyseal Maiacetus) are consistent with the view that pre-sacral verte- fusion pattern of modern cetaceans. bral numbers only allow minor variations: pre-sacral vertebrae J Mammal Evol

Fig. 9 Balaena mysticetus cervical specimen photographs and cervical (09B9); v, 20 years old (10B15); vi, 20 years old (11B9); vii, 40 years old fusion maps. On the left, photographs of skeletonized bowhead cervical (11B3). The vertebrae are black boxes separated by the cartilaginous samples, sagittal view. Scale bar: 1 cm for i–vi. Scale bar: 5 cm for vii. On intervertebral disc (i–vi). Growth plates are depicted as zigzag lines. the right, corresponding fusion maps for each specimen. i, 1 year old C1–C7, cervical vertebrae 1–7 (09B11); ii, 2 years old (10B16); iii, 5 years old (11B8); iv, 5–10 years old in these taxa vary between 26 and 28 (Table 1; Lessertisseur cetaceans, where most homology criteria show vastly different and Saban 1967). However, this developmental constraint was numbers of pre-sacral vertebrae (Table 1). released at least once early in the cetacean lineage, as even the A common practice among cetologists is to use the location Eocene cetacean A. natans has 31 pre-sacral vertebrae, and it of the first hemal arch to indicate the first caudal vertebrae in is likely that such constraints are released in modern modern cetaceans. In modern terrestrial artiodactyls and J Mammal Evol

Ta b l e 1 Interspecies comparison of vertebral column morphological landmarks

# of presacral vertebrae, as 1st hemal arch (Ca1) 1st root of pudendal 1st vertebral pedicle with Fetal hind limb bud based on fused sacrum nerve (S1) fetal sacral ossification location

Mus musculus (Mouse) 27 31 25 27 15–20 shifts to 25–30 Artiodactyla Sus scrofa (Pig) 26–28 x 30–31 x 18 Messelobunodon 26 x x x x Modern Cetacea Balaena mysticetus x332733x (Bowhead) Delphinapterus leucas x2922xx (Beluga) Stenella attenuata x46303345 (Dolphin) Eocene Cetacea Ambulocetus natans 31 x x x x Rodhocetus kasrani 26 31 x x x Maiacetus inuus 26 31 x x x

Eocene cetaceans, hemal arches indeed occur at the caudal assumptions about the location of their hind limb bud can end of the first caudal vertebra (Zhou et al. 1992; Buchholtz be assumed to be stable. 2007), although this is by no means a common feature of The vertebral pedicle in some modern cetaceans (Table 1) mammals, e.g., dogs lack hemal arches on the anterior caudal shows an area of early ossification that could indicate the vertebrae (Nickel et al. 1954; Evans 1993). If hemal arch presence of sacral vertebrae. Indeed, the first sacral vertebra location is an indicator of sacral position (by way of identify- in M. musculus fetuses at E18.5 also indicates stronger ossi- ing the first caudal vertebra) in cetaceans, the number of pre- fication in the pedicle (McIntyre et al. 2007). Moreover, the sacral vertebrae varies much more than in other mammal identical location of this area of ossification in two distantly orders (Table 1; Narita and Kuratani 2005; Buchholtz 2001, related modern cetaceans (B. mysticetus and S. attenuata, 2007; Buchholtz et al. 2005). In late fetal S. scrofa, the first Table 1) could be taken to suggest that some developmental and second caudal vertebra lack hemal arches, while they are signaling of sacral formation has been retained, but that their present in more posterior vertebrae (personal observation). initiation has been shifted to a more caudal somite position. Slijper (1936) suggested that the location of the first Study of terrestrial artiodactyls fetuses could be used to test spinal root of the pudendal nerve can be used as a marker the hypothesis that the first signs of ossification in the pedicle for the first sacral vertebra (S1). This feature cannot be indicate the location of the sacral region. checked in fossils, but comparison of three modern cetacean species indicates that this criterion correlates very Sacral Fusion poorly with the presence of the first hemal arch (Table 1). This holds true in M. musculus as well (Table 1), suggest- Figure 10 summarizes sacrum evolution in cetaceans based on ing the location of the first spinal root of the pudendal a phylogenetic framework presented by Geisler et al. (2007), nerve appears to be variable among mammals. The position Geisler and Theodor (2009), and Spaulding et al. (2009). of the hind limb bud would appear to be a robust way to Archaic artiodactyls, including the sister group to cetaceans determine the homology of sacral vertebrae in cetaceans, (Thewissen et al. 2007; Geisler et al. 2007), had three or four because the hind limb is initiated early in embryology by vertebrae in their sacrum. Only a few fossil sacra are available axial signaling, and is initially intact in cetaceans for dichobunid artiodactyls (Rose 1985;Thewissenand (Thewissen et al. 2009). Interestingly, the position of the Hussain 1990), raoellid artiodactyls, and pakicetid cetaceans hind limb bud in S. attenuata matches that of the first (Cooper et al. 2012); all have three fused sacral vertebrae. hemal arch, consistent with views by different authors, that Since full sacral fusion occurs late in postnatal ontogeny, it is these indicate sacrum position. However, there are no data possible that some of these are young animals and not all on the location of the hind limb bud with regard to the sacral vertebrae had fused yet. Usually, in Eocene cetaceans, somites in any other cetacean. In M. musculus embryos, the there are four firmly fused vertebrae with a well-developed hind limb bud shifts position greatly during ontogeny ilio-sacral joint on S1. The only specimen of A. natans has a (Table 1). More data on cetaceans are needed before four-vertebra sacrum (Fig. 8), even though the number of J Mammal Evol

Fig. 10 Cetacean phylogeny of sacral fusion. Notice the evolutionary loss of sacral intervertebral fusion through cetacean evolution. The loss of vertebral articulation with the pelvis can also be traced evolutionarily. ?- questionable articulation and fusion unfused vertebrae suggests that is not skeletally mature (Gingerich et al. 2001)andTakracetus simus (Gingerich et al. (Figs. 4 and 8). 2001) retain a fused joint between S1 and S2, but the latter has In cetaceans, the loss of fusion in the sacrum and the loss of lost any ilio-sacral joint contact (Fig. 10). ilio-sacral contact occur within the family Protocetidae (Fig. 10). The protocetid Rodhocetus was described as having Cervical Intervertebral Fusion an ilio-sacral contact, but having unfused sacral vertebrae (Gingerich et al. 1994). It is possible that, with age, some of In B. mysticetus, cervical intervertebral fusion starts in the the sacral centra of Rodhocetus would fuse. Qaisracetus arifi center of each of the intervertebral discs and progresses J Mammal Evol

Fig. 11 Phylogeny of cervical fusion related as to neck length. 0 fused Slijper 1936; Haldiman and Tarpley 1993;Buchholtz2001, 2007, 2010; cervical vertebrae, white, 2–4 fused cervical vertebrae, grey; 6–7fused Buchholtz et al. 2005), Remingtonocetus (Bebej et al. 2012), Dorudon cervical vertebrae, black. Phylogeny based on Messenger and McGuire (Uhen 2004) (1998) strict consensus phylogeny, cervical fusion counts (Wheeler 1930; J Mammal Evol peripherally: the first region to ossify is the nucleus pulposus is consistent with previous authors who studied the cetacean (Fig. 9). Cervical intervertebral fusion in B. mysticetus is vertebral column (Slijper 1936; Buchholtz 2001, 2010). gradual with complete fusion occurring after 20 years of age (Fig. 9). This fusion pattern is different from the intervertebral Acknowledgments We would like to thank the Department of Wildlife fusion pattern in the sacrum of M. musculus and S. scrofa, Management, North Slope Borough; the Alaska Eskimo Whaling Com- which starts in the periphery of the disc (annulus fibrosus) and mission, local subsistence hunters; Dr. Terrence Demos, Department of progresses towards the nucleus pulposus (Fig. 5). This central Radiology, LUMC, for his generosity with scanning fossil cetaceans; Dr. fusion in B. mysticetus cervical region causes ossification in Sirpa Nummela, University of Helsinki; Dr. Lisa Noelle Cooper, NEOMED; Dr. John F. Moran, LUMC; Dr. Larry Heaney and John the intervertebral discs and also in the vertebral growth plates Mead, FMNH and Sue McLaren, CM for their help and access to (Fig. 9). With larger sample sizes it could be possible to use museum collections; and Dr. Emily Buchholtz for her correspondence the ossification pattern of the neck to determine the age of during this project. B. mysticetus, a subject of interest in managing these popula- tions (Lubetkin et al. 2012). Fusion of cervical vertebrae limits neck mobility and full fusion makes movement impossible. Neck length and cervical References vertebrae width also limit neck mobility (Buchholtz 1998; Bebej 2011; Bebej et al. 2012). Figure 11 summarizes neck Bajpai S, Thewissen JGM (2000) A new, diminutive Eocene whale from length and cervical fusion in cetaceans in a phylogenetic Kachchh (Gujarat, India) and its implications for locomotor evolution of cetaceans. Curr Sci India 79:1478–1482 context. This figure shows that cervical fusion was lost or Bebej RM (2011) Functional morphology of the vertebral column in acquired independently a number of times, and that there is a Remingtonocetus (Mammalia, Cetacea) and the evolution of aquatic broad, but imperfect relation between neck shortening and locomotion in early archaeocetes. PhD Dissertation, University of cervical fusion. Michigan Bebej RM, ul-Haq M, Zalmout IS, Gingerich PD (2012) Morphology and Movements of the neck are partly determined by the function of the vertebral column in Remingtonocetus domandaensis vestibulocollic reflex (Sipla and Spoor 2008), and this reflex (Mammalia, Cetacea) from the middle Eocene Domanda Formation must be lessened or absent in animals lacking a mobile neck of Pakistan. J Mammal Evol 19:77–104 (Spoor et al. 2002). Cetacean semicircular canals are unusu- Best PB, Lockyer CH (2002) Reproduction, growth, and migrations of sei whales Balaenoptera borealis off the west coast of South Africa. S ally small for mammals (Spoor et al. 2002; Spoor and Afr J Marine Sci 24:111–133 Thewissen 2008; Kandel and Hullar 2010). Among cetaceans, Bruggeman BJ, Maier JA, Mohiuddin YS, Powers R, Lo Y, Guimarães- B. mysticetus have some of the largest semicircular canals Camboa N, Evans SM, Arfe BD (2012) Avian intervertebral disc (Spoor and Thewissen 2008). Given that cervical vertebrae arises from rostral sclerotome and lacks a nucleus pulposus: implications for evolution of the vertebrate disc. Dev Dynam 241:567– fuse later in life, it is possible that the vestibulocollic reflex is 683 operational, to some extent, in young B. mysticetus. Buchholtz EA (1998) Implications of vertebral morphology for locomotor evolution in early Cetacea. In: Thewissen JGM (ed) The Emergence of Whales, Evolutionary Patterns in the Origin of Cetacea. Plenum Press, New York, pp 325–351 Conclusion Buchholtz EA (2001) Vertebral osteology and swimming style in living and fossil whales (Order: Cetacea). J Zool 253:175–190 We studied three aspects of bony fusion in the vertebral Buchholtz EA (2007) Modular evolution of the cetacean vertebral col- columns of pre- and postnatal cetaceans in order to understand umn. Evol Dev 9:278–289 Buchholtz EA (2010) Vertebral and rib anatomy in Caperea marginata: the dramatic changes that the cetacean vertebral column implications for evolutionary patterning of the mammalian vertebral underwent as cetaceans became an obligate aquatic taxa. In column. Mar Mammal Sci 27:382–397 some respects, cetaceans retained terrestrial mammal patterns. Buchholtz EA, Booth AC, Webbnik KE (2007) Vertebral anatomy in the For instance, epiphyseal fusion patterns that start from two Florida manatee, Trichechus manatus latirostris: a developmental – areas, the neck and the sacro-caudal regions, are similar in and evolutionary analysis. Anat Rec 290:624 637 Buchholtz EA, Wolkovich EM, Cleary RJ (2005) Vertebral osteology cetaceans and terrestrial mammals. The evolution of some of and complexity in Lagenorhynchus acutus (Delphinidae) with the vertebral fusion patterns was gradual, exemplified by the comparison to other delphinoid genera. Mar Mammal Sci 21: loss of fused vertebrae in the sacrum. In other aspects, highly 411–428 derived morphologies evolved multiple times in the cetacean Chittleborough RG (1955) Puberty, physical maturity, and relative growth of the female humpback whale, Megaptera nodosa order, as demonstrated by the pattern of cervical vertebrae (Bonnaterre) on the Western Australian coast. Aust J Mar Fresh fusion in B. mysticetus. Taken as a whole, we view the Res 6:315–327 cetacean vertebral column as more plastic than that of other Cooper LN, Thewissen JGM, Bajpai S, Tiwari BN (2012) Postcranial mammals. Embryological study can elucidate developmental morphology and locomotion of the Eocene raoellid Indohyus (Artiodactyla: Mammalia). Historical Biol 24:279–310 constraints that may have been released in the evolution of the Dawson AB (1925) The age order of epiphyseal union in the long bones cetacean vertebral column (Thewissen et al. 2012). This view of the albino rat. Anat Rec 31:1–18 J Mammal Evol

Eschricht DF, Reinhardt J, Lilljeborg W (1866) Recent Memoirs on the Lubetkin SC, Zeh JE, George JC (2012) Statistical modeling of baleen Cetacea. Flower WH (ed), Published for the Ray Society by Robert and body length at age in bowhead whales (Balaena mysticetus). Hardwicke, London Can J Zool 90:915–931 Evans HE (1993) Miller’s Anatomy of the Dog. Third Ed. WB Saunders Madar SI, Thewissen JGM, Hussain ST (2002) Additional holotype Company, Philadelphia remains of Ambulocetus natans (Cetacea, Ambulocetidae) and their Fish FE (1996) Transitions from drag-based to lift-based propulsion in implications for locomotion in early whales. J Vertebr Paleontol 22: mammalian aquatic swimming. Am Zool 36:628–641 405–422 Fish FE, Peacock JE, Rohr JJ (2000) Stabilization mechanism in swim- McIntyre DC, Rakshit S, Yallowitz AR, Loken L, Jeannotte L, Capecchi ming odontocetes cetaceans by phased movements. Mar Mammal MR, Wellik DM (2007) Hox patterning of the vertebrate rib cage. Sci 19:515–528 Development 134:2981–2989 Fraas E (1904) Neue Zeuglodonten aus dem unteren Mitteleocän vom Mead JG, Potter CW (1990) Natural history of the bottlenose dolphins Mokattam bei Cairo. Geol Palaeontol Abh 6:199–220 along the Central Atlantic Coast of the United States. In: Galatius A (2010) Paedomorphosis in two small species of toothed Leatherwood S, Reeves RR (eds) The Bottlenose Dolphin. whales (Odontoceti): how and why? Zool J Linn Soc 99:278–295 Academic Press, San Diego, pp 31–43 Galatius A, Kinze CC (2003) Ankylosis patterns in the postcranial Messenger SL, McGuire JA (1998) Morphology, molecules, and the skeleton and hyoid bones of the harbour porpoise (Phocoena phylogenetics of cetaceans. Syst Biol 47:90–124 phocoena) in the Baltic and North Sea. Can J Zool 81:1851–1861 Moore JC (1968) Relationships among the living genera of beaked Gegenbaur C, Bell FJ (1878) Elements of Comparative Anatomy. whales with classifications, diagnoses, and keys. Fieldiana: Zool Macmillan and Co, London 53:209–298 Geisler JH, Theodor JM (2009) Hippopotamus and whale phylogeny. Munro ND, Bar-Oz G, Stutz AJ (2009) Aging mountain gazelle (Gazella Nature 458:E1–E4 gazelle): refining method of tooth eruption and wear and bone Geisler JH, Theodor JM, Uhen MD, Foss SE (2007). Phylogenetic fusion. J Archaeol Sci 36:752–763 relationships of cetaceans to terrestrial artiodactyls. In: Prothero Narita Y, Kuratani S (2005) Evolution of the vertebral formulae in DR, Foss SE (eds) The Evolution of Artiodactyls. The Johns mammals: a perspective on developmental constraints. J Exp Zool Hopkins University Press, Baltimore, pp 19–31 Part A 304B:91–106. Geisler JH, Uhen MD (2005) Phylogenetic relationships of extinct Nickel R, Schummer A, Seiferle E (1954) Lehrbuch der Anatomie der cetartiodactyls: results of simultaneous analyses of molecular, Haustiere, Vol. I. Bewegungsapparat. Parey P, Berlin, Hamburg, morphological, and stratigraphic data. J Mammal Evol 12: 503 pp 145–160 Ohsumi SK, Nishiwaki M, Hibiya T (1958) Growth of fin whale in the Gérard M, Zákány J, Duboule D (1997) Interspecies exchange of a Hoxd Northern Pacific. The Whales Research Institute, No. 13, Japan enhancer in vivo induces premature transcription and anterior shift of Pourquié O (2007) Building the spine: the vertebrate segmentation clock. the sacrum. Dev Biol 190:32–40 Cold Sh Q B 72:445–449 Getty R (1975) Sisson and Grossman’s The Anatomy of the Domestic Purdue JR (1983) Epiphyseal closure in white-tailed deer. J Wildlife Animals. Fifth Ed. WB Saunders Company, Philadelphia Manage 47:1207–1213 Gingerich PD, Raza SM, Arif M, Anwar M, Zhou X (1994) New whale Rasband WS (2011) Image J. U. S. National Institutes of Health, from the Eocene of Pakistan and the origin of cetacean swimming. Bethesda, Maryland, http://imagej.nih.gov/ij/ Nature 368:844–847 Roach HI, Mehta G, Oreffo ROC, Clarke NMP, Cooper C (2003) Gingerich PD, Ul-Haq, Khan I, Zalmount IS (2001) Eocene stratigraphy Temporal analysis of rat growth plate: cessation of growth with and archaeocete whales (Mammalia, Cetacea) of Drug Lahar in the age despite presence of a physis. J Histochem Cytochem 51:373– Eastern Sulaiman Range, Balochistan (Pakistan). Contrib Mus 383 Paleolotol Univ Mich 30:269–319 Rose KD (1985) Comparative osteology of North American dichobunid Graf W, de Waele C, Vidal PP (1995) Functional anatomy of the head- artiodactyls. J Paleontol 59:1203–1226 neck movement system of quadrupedal and bipedal mammals. J Shashikant CS, Kim CB, Borbély MA, Wang WCH (1998) Comparative Anat 186:55–74 studies on mammalian Hoxc8 early enhancer sequence reveal a Haldiman JT, Tarpley RJ (1993) Anatomy and physiology. In: Burns JJ, baleen whale-specific deletion of a cis acting element. Proc Natl Montague JJ, Cowles CJ (eds) The Bowhead Whale. The Society of Acad Sci USA 95:15446–15451 Marine Mammology Press, Lawrence, Kansas, pp 71–156 Sipla J, Spoor F (2008) The physics and physiology of balance. In: Hillson S (1986) Cambridge Manuals in Archaeology- Teeth. Cambridge Thewissen JGM, Nummela S (eds) Sensory Evolution on the University Press, London Threshold, Adaptations in Secondarily Aquatic Vertebrates. Hristova GI, Jarzem P, Ouellet JA, Roughley PJ, Epure LM, Antoniou J, University of California Press, Berkeley, pp 227–232 Mwale F (2011) Calcification in human intervertebral disc degener- Slijper EJ (1936) Die Cetaceen. A. Asher & Co, Amsterdam ation and scoliosis. J Orthop Res 29:1888–1895 Spaulding M, O’Leary MA, Gatesy J (2009) Relationships of Cetacea Ito H, Miyazaki N (1990) Skeletal development of the striped dolphin (Artiodactyla) among mammals: increased taxon sampling alters (Stenella coeruleoalba) in Japanese waters. Mammal Study 14:79–96 interpretations of key fossils and character evolution. PLoS ONE Kandel BM, Hullar TE (2010) The relationship of head movements to 4:1–14 semicircular canal size in cetaceans. J Exp Biol 213:1175–1181 Spoor F, Bajpai S, Hussain ST, Kumar K, Thewissen JGM (2002). Kato H (1988) Ossification pattern of the vertebral epiphyses in the Vestibular evidence for the evolution of aquatic behavior in early southern minke whale. Sci Rep Whales Res Inst 39:11–19 cetaceans. Nature 417:163–166 Kellogg R (1936) A Review of the Archaeoceti. Carnegie Institution of Spoor F, Thewissen JGM (2008) Balance: comparative and functional Washington, Washington anatomy in aquatic mammals. In: Thewissen JGM, Nummela S Kemper CM, Leppard P (1999) Estimating body length of pygmy right (eds) Sensory Evolution on the Threshold, Adaptations in whales (Caperea marginata) from measurements of the skeleton and Secondarily Aquatic Vertebrates. University of California Press, baleen. Mar Mammal Sci 15:683–700 Berkeley, pp 257–284 Lessertisseur J, Saban R (1967) Squelette axial. In: Grassé P-P (ed) Traité de Thewissen JGM, Bajpai S (2009) New skeletal material of Andrewsiphius Zoologie, Anatomie, Systematique, Biologie, Vol. 16.1 Mammifères, and Kutchicetus, two Eocene cetaceans from India. J Paleontol 83: Téguments, Squelette. Masson et Cie, Paris, pp 584–708 635–663 J Mammal Evol

Thewissen JGM, Cooper LN, Behringer RR (2012) Developmental biol- Uhen MD (1999) New protocetid (Mammalia, Cetacea) from the late ogy enriches paleontology. J Vertebr Paleontol 32:1223–1234 middle Eocene Cook Mountain Formation of Louisiana. J Vertebr Thewissen JGM, Cooper LN, Clementz MT, Bajpai S, Tiwari BN (2007) Paleontol 18:664–668 Whales originated from aquatic artiodactyls in the Eocene epoch of Uhen MD (2004) Form, function, and anatomy of Dorudon atrox India. Nature 450:1190–1194 (Mammalia, Cetacea): an archaeocete from the middle to late Thewissen JGM, Cooper LN, George JC, Bajpai S (2009) From land to Eocene of Egypt. Univ Michigan Pap Paleontol 34 water: the origin of whales, dolphins and porpoises. Evolution: Uhen MD, Pyenson ND, Devris TJ, Urbina M, Renne PR (2011) New Education and Outreach 2 272–288 middle Eocene whales from the Pisco Basin of Peru. J Vertebr Thewissen JGM, Heyning J (2007) Embryogenesis and development in Paleontol 85:955–969. Stenella attenuata and other cetaceans. In: Jamieson BGM (ed) Wassersug RA (1976) A procedure for differential staining of cartilage and Reproductive Biology and Phylogeny of Cetacea: Whales, bone in whole formalin fixed vertebrates. Stain Technol 51:131–134 Dolphins, and Porpoises. Science Publishers, New Hampshire, pp Wheeler JFG (1930) The Age of Fin Whales at Physical Maturity with a 307–329 Note on Multiple Ovulatons. Cambridge University Press, Cambridge Thewissen JGM, Hussain ST (1990) Postcranial osteology of the most Yoshida H, Shirakihara M, Takemura A, Shirokihara K (1994) primitive artiodactyls Diacodexis pakistanensis (Dichobunidae). Development, sexual dimorphism, and individual variation in the Anat Histol Embryol19:37–48 skeleton of the finless porpoise, Neophocaena phocaenoides,inthe Thewissen JGM, Madar SI (1999) Ankle morphology of the earliest coastal waters of western Kyushu, Japan. Mar Mammal Sci 10:266– cetaceans and its implications for the phylogenetic relations among 282 ungulates. Syst Biol 48:21–30 Zákány J, Gérard M, Favier B, Duboule D (1997) Deletion of a HoxD Thewissen JGM, Madar SI, Hussain ST (1996) Ambulocetus natans,an enhancer induces transcriptional heterochrony leading to transposi- Eocene cetacean (Mammalia) from Pakistan. Vol. 191 Courier tion of the sacrum. EMBO J 16: 4393–4402 Forschungsinstitut Senckenberg Zhou X, Sanders WJ, Gingerich PD (1992) Functional and behavorial Thewissen JGM, Williams EM, Roe LJ, Hussain ST (2001) Skeletons of implications of vertebral structure in Pachyaena ossifraga terrestrial cetaceans and the relationship of whales to artiodactyls. (Mammalia, Mesonychia). Contrib Mus Paleolotol Univ Mich 28: Nature 413:277–281 289–319