High incidence of cervical ribs indicates vulnerable condition in Late Pleistocene woolly rhinoceroses

Introduction

The number of cervical vertebrae in mammals is remarkably constant at seven, in contrast to other tetrapods, where this number varies considerably (Leboucq, 1898; Schultz, 1961; Starck, 1979; Narita & Kuratani, 2005). The only exceptions are manatees (Trichechus, Sirenia) and extant sloths (Bradypus and Choloepus, Xenarthra). These latter taxa have an exceptional number of cervical vertebrae (Bateson, 1894; Starck, 1979; Varela-Lasheras et al., 2011). The extreme evolutionary conservation of the number of cervical vertebrae in mammals implies that there must be selection against intraspecific variation of this number. Yet intraspecific variation does occur. The most common variation is a partial or complete homeotic transformation of a cervical into a thoracic vertebra (involving a change in the activity of Hox genes) in the form of the presence of ribs on the seventh vertebra, so-called cervical ribs (Galis, 1999; Li & Shiota, 2000; Varela-Lasheras et al., 2011; Wéry et al., 2003). These cervical ribs are usually rudimentary and not fused to the sternum, but they may be fused to the first rib instead. In humans, remarkably strong selection against such cervical ribs was shown to exist (Galis, 1999; Galis et al., 2006; Furtado et al., 2011; Ten Broek et al., 2012). Approximately 90% of individuals with a cervical rib die before reaching reproductive age (Galis et al., 2006). The cervical rib itself is relatively harmless, but its presence is associated with multiple and major congenital abnormalities (Galis et al., 2006; Ten Broek et al., 2012). This is because its development is induced by a (genetic or environmental) disturbance of early embryogenesis (Li & Shiota, 2000; Wéry et al., 2003; Chernoff & Rogers, 2004; Galis et al., 2006). Usually, such a disturbance has multiple effects, due to the highly interactive nature of early embryogenesis. Hence, the strong selection against cervical ribs is indirect and due to the severity of the associated medical problems (Galis et al., 2006; Ten Broek et al., 2012). Indeed, we have found an association with abnormalities in other mammalian species as well (Varela-Lasheras et al., 2011). Even in the exceptional manatees and sloths, support was found for an association between abnormal numbers of cervical vertebrae and severe skeletal abnormalities, including fused vertebrae and ossification defects. The selection against these abnormalities is apparently sufficiently relaxed in these latter taxa to allow for the breaking of the constraint on changes of the number of cervical vertebrae (Varela-Lasheras et al., 2011).

An unusually high incidence of cervical ribs (33.3%) was found in the woolly mammoth Mammuthus primigenius (Blumenbach, 1799) from the southern North Sea (Reumer, Ten Broek & Galis, 2014). This was explained either as a direct result of a reduced gene pool due to population fragmentation towards its final extinction or as due to adverse conditions during early stages of pregnancy.

Our aim here is to test whether there are indications for similar conditions in another megaherbivore member of the mammoth steppe fauna: the woolly rhinoceros Coelodonta antiquitatis (Blumenbach, 1799). To this end, we investigated developmental abnormalities in the neck vertebrae, specifically the presence of cervical ribs. We checked the extensive collection of Late Pleistocene C. antiquitatis material in the Naturalis Biodiversity Centre (Leiden, the Netherlands) to estimate the incidence of cervical ribs. Additionally, we compared the resulting incidence of cervical ribs with those that we found in skeletons of all five extant African and Asian species of rhinoceros (Dicerorhinus sumatrensis, Ceratotherium simum, Rhinoceros unicornis, R. sondaicus, Diceros bicornis).

Methods

Cervico-thoracic transitional vertebrae

We analyzed the C7 vertebrae for the presence or absence of articulation facets of cervical ribs. We follow a conservative approach here by assuming that the ribs, if present, are cervical ribs and not rudimentary first ribs, because the incidence of cervicalized thoracic vertebrae in mammals is extremely low. In a meta-analysis of deceased human foetusses and infants, Galis et al. (2006) and Ten Broek et al. (2012) found that respectively 0.98% (9.8% erroneously in the former article) and 1.3% of all C/T transformations consisted of rudimentary first ribs (of which half unilateral) against ∼99% cases of cervical ribs. Bradley (1901) describes such a transitional T1 in a horse, and he as well remarks that the condition is extremely rare. The extra-ordinary low incidence of rudimentary first ribs does in our view not support such an interpretation for cervico-thoracic transitional vertebra in the woolly rhinoceros. In addition, a somewhat cervicalized first thoracic vertebra (albeit not involving a change in the ribs) with a cervical type of zygapophyses, or atypical cervico-thoracic junction, as found in the extant giraffe (Giraffa camelopardalis) coincides with homogenization of C3–C7 and an extremely long neck (Danowitz & Solounias, 2015). This condition is not present in the woolly rhino, where C3–C7 each have their own distinctive identity.

The presence of cervical ribs can be deduced from articulation facets on the anterior side of the centrum and the transverse processes of C7 (Fig. 1). To distinguish a transitional cervico-thoracic (C/T) vertebra with ribs from a first thoracic vertebra (T1), we scored 12 discrete characters diagnostic for either C7 or T1 (Figs. 1–3), amongst others shape parameters of the vertebra and size of the rib facets (Table 3), based on Borsuk-Białynicka (1973), Protero (2005), Diedrich (2006) and our own observations on 56 complete skeletons of extant rhinoceroses. When a vertebra had rib facets and six or more cervical or cervico-thoracic transitional characters, thus 50% cervical in morphology, we considered the vertebra to have a transitional C/T identity, whereas a vertebra with rib facets but fewer cervical or cervico-thoracic transitional characters, we considered it to be a regular T1. An additional diagnostic character considered here is that C/T vertebrae often display left–right asymmetry in the extent of the homeotic transformation, for instance differences in the size of the left and right ribs (Varela-Lasheras et al., 2011; Ten Broek et al., 2012).

Table 3:

List of diagnostic characters.

Features of cervical and thoracic vertebral elements used in this study to identify a vertebra as either C7, T1 or intermediate.

Feature		View	C7	T1
Centrum
a	Shape of anterior articular face	Rostral	Oval—higher than wide, with dorsal border convex	Heart-shaped—at least as wide as high, with approx. straight dorsal border
b	Shape of posterior end (adult)	Caudal	Dorsal width clearly exceeds ventral width, may have dimple midway dorsally, ventrally convex	Squarish, dorsal width approx equals ventral width, ventrally straight or tapering
	Shape of posterior end (juvenile)	Caudal	Wider than high (at least 69%), Ventrally rounded, Shallow dimple midway dorsally and indistinct trace of rib facets	Wider than high (less than 64%), ventrally tapering, sharp dimple midway dorsally and prominent impression of rib facets
c	Ventral keel	Ventral	Single or bilateral median tubercle, strongly developed	No tubercles, may be (very) weakly developed
d	Shape of anterior articular face	Ventral	Deep and rounded, A–P length approx. 40–50% of the width of centrum	Shallow and flat, A–P length approx. 25–35% of width of centrum
Vertebral foramen
e	Shape	Rostral	Wide (large), H/W ratio 75% or more	Narrow (small), H/W ratio 73% or smaller
Prezygapophyse
f	Left-right distance	Rostral	Large—shortest distance approx. 50% of total width at same level	Small—shortest distance approx. 35% of total width at same level
g	Position relative to transverse process	Dorsal	More anterior than transverse process	Overhangs transverse process
Postzygapophyse
h	Articular facet	Lateral	Visible for greatest part (latero-caudally directed)	At most minimally visible (caudally directed)
i	Articular facets	Caudal	Widely spaced (inner side lateral of neural spine)	Narrow spaced (inner side close to centre of neural spine)
Mammilary process, pedicle and transverse process
j	Outer arch formed by mammilary process, pedicle and transverse process	Rostral	Long—at least 85% of height of centrum	Short—at most 70% of height of centrum
Rib facets
k	At transverse process	Lateral	Absent	Present (large)
l	At centrum	Rostral	Absent	Present (large)

DOI: 10.7717/peerj.3684/table-3

Results

We found five transitional C/T vertebrae, 24 normal C7 vertebrae, and three C7 vertebrae with one or two transitional characters for C. antiquitatis (Figs. 4–6, Tables 1 and 4). The incidence of transitional C/T vertebrae (15.6%) is significantly higher than for the extant Rhinocerotidae where we found no transitional C/T vertebrae and 56 normal C7 vertebrae (p = 0.005) (Table 2). Judging from the rib facets, the cervical ribs were quite large and none of them was fused to the transverse process as is usually the case for small cervical ribs (Cave, 1975; Varela-Lasheras et al., 2011). Two of the five C/T vertebrae show small anterior rib facets relative to those of a T1 (RGM 93477, RGM146833) (Fig. 4), and three of the five C/T vertebrae displayed a conspicuous asymmetry in the size of the rib facets and other shape parameters (RGM 94549, RGM 445933, RGM 93790) (Fig. 5). The most prominently asymmetric specimen is RGM 94549, where the left anterior rib facet is considerably smaller and lies more dorsally than the one on the right and the transverse process and mammilary process are asymmetrically placed (Fig. 6).

Discussion

The incidence of transitional C/T vertebrae in our set of Late Pleistocene C. antiquitatis from the North Sea and the Netherlands is high (15.6%, n = 32) and significantly higher than that of extant rhinocerotids (0%, n = 56). In humans, the incidence in the general population is estimated to be between 0.5 and 1% and is only higher than 1% in diseased or isolated populations (Galis et al., 2006). A particularly high incidence has been found in children with cancer (15–25%; Schumacher, Mai & Gutjahr, 1992; Merks et al., 2005; Galis & Metz, 2003) and deceased fetuses (approximately 50%; Galis et al., 2006; Furtado et al., 2011; Ten Broek et al., 2012; Schut et al., 2016). Investigations of skeletons of other extant mammalian species found support for an association between cervical ribs (C/T vertebrae) and defects, such as fused vertebrae and ossification abnormalities (Varela-Lasheras et al., 2011; Ten Broek et al., 2012). This applies inter alia also to rudimentary first ribs, which were in all cases coupled to multiple major abnormalities (Galis et al., 2006). Late Pleistocene woolly mammoths from the North Sea display an unusually high incidence of cervical ribs, much higher than that of extant elephants (Reumer, Ten Broek & Galis, 2014). Interestingly, both woolly rhinos and mammoths have quite large cervical rib articulation facets, indicating cervical ribs that are substantially larger than usually found in humans and other extant mammals (see Cave, 1975; Bots et al., 2011; Varela-Lasheras et al., 2011; Ten Broek et al., 2012; Reumer, Ten Broek & Galis, 2014 for examples), though not as large as first ribs. Size of cervical ribs was found to be negatively correlated with fitness in transgenic mice (Jeannotte et al., 1993; see also Bots et al., 2011). Importantly, there are a few cases of woolly rhinos with developmental abnormalities. An isolated deciduous molar of a woolly rhino from Italy (Grotta di Fumane, Verona) retrieved from a Palaeolithic layer dated between 37,000 and 32,000 years ago (C¹⁴; Bartolomei et al., 1994) shows an enamel defect (hypoplastic form of amelogenesis imperfecta) (Billia & Graovac, 1998). Perhaps not coincidentally, the geological age coincides with the last occurrences of this species in the west (Stuart & Lister, 2012). In humans, such a structural enamel anomaly is hereditary (Stephanopoulos, Garefalaki & Lyroudia, 2005). Also, there are three reported cases of supernumerary teeth (hyperodontia) in C. antiquitatis (4th upper molar, 3rd lower molar in Garrutt, 1990; 3rd lower molar in Hillman-Smith et al., 1986). In humans, supernumerary teeth are commonly seen with several congenital genetic disorders (Subasioglu et al., 2015). One of the woolly rhino specimens reported by Garrutt (1990) also has an osteoma (a benign bone tumor) at the top of the occipital bone (Garrut, 1994), which might indicate a wider spectrum of abnormalities in this specimen.

The exceptionally high incidence of large cervical ribs in Late Pleistocene woolly rhinos and mammoths can be due to two factors. Firstly, it can be due to a high rate of inbreeding in declining populations potentially resulting in drift and preservation of less favorable genetic variants. A large frequency of cervical ribs (7.5%) in adults has been observed in an isolated human population in Sicily (Palma & Carini, 1990), in inbred pedigreed dogs (11.4% Breit & Kunzel, 1998) and in inbred minipigs (11%, Jorgensen, 1998). Generally, in inbred mammals there is an increased incidence of congenital anomalies (Cristescu et al., 2009; Räikkönen et al., 2013). Recent studies have shown that the genetic diversity was indeed extremely low in Late Pleistocene mammoths in Siberia (Miller et al., 2008; Nyström et al., 2012). In woolly rhinoceroses, unfortunately no research has been carried out on genetic diversification. An indirect indication that such a loss of genetic variation might have existed is that the range of the woolly rhinoceros gradually disintegrated into isolated spots before their extinction towards the end of the Late Pleistocene (Markova et al., 2013).

A second cause of the increased incidence of cervical ribs may be harsh conditions that impact early pregnancies, because diseases, famine, cold and other stressors can lead to disturbances of early development, that can result in the induction of cervical ribs (e.g., Sawin, 1937; Li & Shiota, 2000; Wéry et al., 2003; Chernoff & Rogers, 2004; Steigenga et al., 2006). Harsh conditions during the Late Pleistocene, a period of intense climatic fluctuations and ecosystem instability, are plausible (Brace et al., 2012). The extinction of the woolly mammoth and the woolly rhinoceros has been related to the Late Glacial interstadial warming event and increased precipitation, especially snowfall, together with a replacement of forbs (non-graminoid herbaceous vascular plants) and mosses—the typical diet of woolly mammoths and rhinos according to (Willerslev et al., 2014)—by less nutritious shrubs and trees and an increase in the amount of fibrous grasses with low nutritional value (Stuart & Lister, 2012). The high incidence of bone dystrophy and severe arthrosis found in young mammoths of Northern Eurasian Late Pleistocene populations are assumed to be caused by mineral deficiencies in pregnant females and developing calves, especially in the geologically youngest populations (Leshchinskiy, 2012; Leshchinskiy, 2015). Spondylosis (a collective term for degenerative conditions affecting the disks, vertebral bodies, and/or associated joints; Middleton & Fish, 2009) also occurs and may lead to vertebral fusions (Fig. 7). Segmentation defects, due to disturbances of the early embryonic segmentation process of the prevertebrae (somites) can also cause fusions of vertebrae (Fig. 8). Such congenital defects are commonly associated with cervical ribs (Varela-Lasheras et al., 2011; Ten Broek et al., 2012). A perhaps similar segmentation defect in a woolly rhinoceros may be provided by a pathological fusion of a second and third vertebra (Fig. 9). As in the case of the woolly mammoth (Fig. 8), these abnormalities were not necessarily fatal but persisted into adulthood. In general, studies of abnormalities in extinct megaherbivores are scarce, other than incidental observations of abnormalities (see also the above-mentioned amelogenesis imperfecta and supernumerary teeth in woolly rhinoceroses), hampering any statistical evaluation of their occurrence or their relation to other developmental anomalies. At the present stage of our knowledge, a combination of inbreeding and harsh conditions may be the most likely explanation for the extremely high incidence of cervical ribs in two Ice Age megaherbivores, the woolly mammoths and rhinoceroses.

The relatively high incidence of cervical ribs might present a transitional stage in an evolution towards a shorter neck in the woolly rhinoceros lineage. This is, however, in our opinion unlikely for two reasons. First, there does not seem to be a selective advantage to a shorter neck, because this species likely carried a fat hump above the shoulders as inferred from the presence of extremely long dorsal spines of the anterior thoracic vertebrae (Kahlke, 1999), which is mechanistically incompatible with a shorter neck. Second, the evolution towards fewer neck vertebrae in mammals is restricted to the sloth Choloepus and manatee Trichechus. Both are extremely slow moving species with an extremely low metabolism. Both taxa experienced, therefore, relaxed selection with adverse side-effects of a changed number of cervical vertebrae not negatively affecting their fitness (Varela-Lasheras et al., 2011). This is unlikely to have been the case for the woolly rhinoceros, a species that had to be relatively fast and agile to escape or fight predators like wolves and hyenas.

Our study found no cervical ribs in the investigated extant Asian and African rhinos. This might erroneously lead us to the conclusion that rhinoceros populations today are thriving and healthy. However, we have to keep in mind that all analysed specimens date back to times when populations were indeed almost certainly thriving. With a few exceptions, they were captured before the mid-20th century. Especially the past few decades saw very severe declines in their numbers, for example, greater than 80% in 60 years for the Sumatran rhinoceros (Van Strien et al., 2008), and a loss of 69% of the mitochondrial genetic variation in the black rhinoceros (Moodley et al., 2017). Our data are thus based on a situation that differs essentially from today. Furthermore, there are indications that the level of genetic variation in extant rhinos is still high because of the very rapid severe population decline. Such is the case in the greater one-horned rhinoceros of Chitwan National Park of Nepal, and is likely due to a compression of surviving populations into the park area, thereby concentrating genetic variation (Dinerstein, 2003). Monitoring of the skeletal health of rhinos, including the presence of cervical ribs, would improve our power to assess their genetic status on the long term and predict their chances of survival.

Conclusion

In conclusion, the high incidence and large size of the cervical ribs in the Late Pleistocene woolly rhinoceros of the North Sea indicates a strong vulnerability, given the association of cervical ribs with diseases and congenital abnormalities in mammals in general. If so, that vulnerable condition in woolly rhinoceroses and mammoths may well have contributed to their eventual extinction (Reumer, Ten Broek & Galis, 2014).