Inter-element variation in the bone histology of Anteosaurus (Dinocephalia, Anteosauridae) from the Tapinocephalus Assemblage Zone of the Karoo Basin of South Africa

Introduction

The anteosaurs, a monophyletic group of dinocephalians (Therapsida: Dinocephalia: Anteosauria), first appeared during the Middle Permian (Guadalupian) and formed a key component of the terrestrial tetrapod fauna (Rubidge, 1995; Rubidge & Sidor, 2001; Kammerer, 2011; Cisneros et al., 2012; Kemp, 1982, 2012; Smith, Rubidge & Walt, 2012). The family Anteosauridae comprises two major clades, Syodontinae (Australosyodon, Notosyodon and Syodon) and Anteosaurinae (Anteosaurus, Sinophoneus and Titanophoneus) with Russian taxa Archaeosyodon and Microsyodon representing the most basal anteosaurs (Kammerer, 2011). Liu (2013) revised the phylogeny of Anteosauridae using the modified character lists and data matrices of Kammerer (2011) and Cisneros et al. (2012) and recovered Sinophoneus as a basal anteosaur, falling outside the clade Anteosaurinae. Anteosaurs are known from Middle Permian rocks of both Laurasia (Efremov, 1954; Tchudinov, 1968; Ivachnenko, 1995; Li, Rubidge & Cheng, 1996; Cheng & Ji, 1996; Cheng & Li, 1997; Golubev, 2015) and Gondwana (Owen & Bain, 1845; Owen, 1879; Watson, 1921; Broom, 1929; Boonstra, 1954, 1969; Rubidge, 1994; Cisneros et al., 2012; Kruger, Rubidge & Abdala, 2018). There are more than eight valid genera of anteosaurs (King, 1988; Kammerer, 2011; Kemp, 2012; Cisneros et al., 2012; Liu, 2013) but only two, Australosyodon and Anteosaurus, are represented in the Beaufort Group of the Karoo Supergroup of South Africa (Boonstra, 1969; King, 1988; Rubidge, 1994; Kemp, 2012; Kruger, Rubidge & Abdala, 2018). They were the largest apex predators during the Middle Permian Period; however, by the end of the Tapinocephalus Assemblage Zone (Smith & Keyser, 1995; Kemp, 2012; Day & Rubidge, 2020; Day & Smith, 2020), they along with other dinocephalians completely disappear from the fossil record leaving no descendants (Boonstra, 1971; Kemp, 1982, 2005, 2012). Their extinction within the Karoo Basin is linked either to the increasing aridification or to the loss of their food sources (Bond et al., 2010; Rey et al., 2018; Day & Rubidge, 2021). Anteosaurs are characterized by large pachyostotic skulls, which range in length from 280 to 805 mm (Boonstra, 1954; Kemp, 1982; Kammerer, 2011; Kruger, Rubidge & Abdala, 2018). The members of the family Anteosauridae are diagnosed by combined synapomorphic features: anterodorsally canted premaxilla, convex ventral maxillary margin, ‘scroll’ vomers, quadrate rami of the pterygoid that bifurcate the anterior margin of the basisphenoid, a ridge on the jugal-lacrimal suture, and a ‘scoop’-shaped (strongly anteroventrally curved) postorbital bar (Kemp, 1982; Ivakhnenko, 2008; Kammerer, 2011).

Anteosaurs were fully terrestrial animals feeding on large tapinocephalid dinocephalians, bull-sized armored pareiasaurs and even scavenging on kills made by the lycosuchid and scylacosaurid therocephalians (Boonstra, 1955; Rubidge, 1995; Lee, 1997; Nicolas & Rubidge, 2010; Kammerer, 2011; Kemp, 2012; Canoville, Thomas & Chinsamy, 2014). They have also been considered riparian, obligate fish-eaters (Boonstra, 1955, 1962; Ivakhnenko, 2001, 2003, 2008), or even amphibious (Olson, 1962; King, 1988; Ivakhnenko, 2008). Kammerer (2011) supported a fully terrestrial lifestyle for anteosaurs as they possessed the heaviest skulls of all the carnivorous synapsids, and were equipped with large canines and incisors, that would have been effective for preying on large land animals (Sennikov, 1996; Van Valkenburgh & Jenkins, 2002). Canoville, Thomas & Chinsamy (2014) performed isotopic analysis of the teeth of dinocephalians and pointed out that most of the dinocephalians had lower oxygen isotope compositions than contemporaneous pareiasaurs and therocephalians. They further suggested that dinocephalians and pareiasaurs inhabited different ecological niches and that the herbivorous pareiasaurs may have shared a terrestrial habitat with carnivorous therocephalians. However, their isotopic differences also reflected higher water turnover rates for dinocephalians suggesting niche partitioning (Canoville, Thomas & Chinsamy, 2014). Later, Rey et al. (2020) supported a terrestrial adaptation for Anteosaurus even though their oxygen values point towards water dependency. Since their sample size was small, they were unable to confirm whether anteosaurs were riparian or more in-land dwellers. Recently Benoit et al. (2021) concluded that anteosaurs were agile predators, based on the enlarged fossa for the floccular lobe of the cerebellum and semicircular canals of the inner ear.

Bone microanatomical studies of tetrapods have demonstrated that bone architecture (microanatomy) and internal tissue structure (histology) varies according to lifestyle adaptations (Wall, 1983; Chinsamy, 1991; Ray, Chinsamy & Bandyopadhyay, 2005; Gray et al., 2007; Hayashi et al., 2013; Houssaye et al., 2016; Canoville, de Buffrénil & Laurin, 2016; Canoville & Chinsamy, 2017). Furthermore, bone histology of extant and extinct vertebrates provides direct assessment of life history and the biology of an animal (de Ricqlès, 1969, 1972; Francillon-Vieillot et al., 1990; Chinsamy, 1997; Horner, de Ricqlès & Padian, 1999, 2000; Chinsamy-Turan, 2005, 2012; Klein & Sander, 2008; Chinsamy et al., 2013, 2019, 2020; Woodward, Horner & Farlow, 2014; Woodward et al., 2015; Woodward et al., 2020; Huttenlocker & Botha-Brink, 2014; Botha-Brink, Soares & Martinelli, 2018; Cullen et al., 2020; Huttenlocker & Shelton, 2020; Botha, 2020). Several studies have examined the bone histology of the non-mammalian therapsids (see Chinsamy-Turan, 2012 and references therein), however, except for the early work of de Ricqlès (1972), dinocephalians have been relatively under-studied. Recent histology research on Titanosuchus led to the identification of osteomyelitis in a femur of Jonkeria parva (Shelton, Chinsamy & Rothschild, 2019). More recently, additional bone histology studies have been conducted on Jonkeria (Bhat, Shelton & Chinsamy, in press (a)) and have shown that these fast growing omnivorous animals were adapted for a semi-aquatic lifestyle, like modern graviportal Hippopotamus. Subsequently, a comprehensive histological study of various limb bones of several herbivorous dinocephalian taxa found richly vascularized fibrolamellar bone tissue, as well as extensively developed medullary spongiosa, and supported the semi-aquatic lifestyle hypothesis for herbivorous dinocephalian taxa Bhat, Shelton & Chinsamy, in press (b).

Since tetrapods display a wide range of histological characteristics (e.g., Horner, de Ricqlès & Padian, 1999, 2000; Ray & Chinsamy, 2004; Ray, Botha & Chinsamy, 2004; Chinsamy-Turan, 2005, 2012), as well as bone depositional rates (e.g., Amprino, 1947; de Margerie, Cubo & Castanet, 2002; de Margerie et al., 2004; Starck & Chinsamy, 2002), multi-element studies of individuals of a particular species provide a better assessment of their growth patterns, lifestyle habits and various aspects of their life history (Horner, de Ricqlès & Padian, 1999, 2000; Botha & Chinsamy, 2004, 2005; Chinsamy-Turan, 2005, 2012; Ray, Mukherjee & Bandyopadhyay, 2009; Woodward et al., 2015). This study represents the first histological and microanatomical study of multiple skeletal elements of two Anteosaurus taxa. Our study aims to assess the inter-elemental variation in their bone histology as well as to shed light on their lifestyle adaptations.

Materials & Methods

The specimens studied here were excavated from the Tapinocephalus Assemblage Zone of the South African Karoo Basin (Boonstra, 1969). For our analysis, several skeletal elements (femora, radius, ulnae, fibula, and ribs; Fig. 1) were examined to assess inter-elemental histological variability. These skeletal elements are positively identified at species or generic levels (Table 1; references therein). In the text, different elements of the same individual have the same specimen numbers but are differentiated by the suffixes of a/b/c. The skeletal elements with specimen number SAM-PK-12088 belong to Anteosaurus magnificus whereas the fibula (BP/1/5591b) and ribs (BP/1/5591c–d) belong to another Anteosaurus taxon. All specimens were obtained from Iziko South African Museums, Cape Town, and Evolutionary Studies Institute (formerly the Bernard Price Institute) at the University of the Witwatersrand, Johannesburg, South Africa. Permission to section the fossils was obtained from the South African Heritage Resources Agency (SAHRA; Permits 2076, 2131, and 3752–4658).

Transverse sections were prepared from midshaft levels wherever possible as these are the regions of the bone that undergo the least amount of secondary remodeling (Enlow, 1963; Chinsamy, 1995; Chinsamy-Turan, 2005) and limb bones were preferentially selected because they retain the best record of growth (Francillon-Vieillot et al., 1990; Chinsamy-Turan, 2005; Bhat, Chinsamy & Parkington, 2019). Both stylopodial (femur) and zeugopodial (radius, ulna; fibula) bones were sampled as they can show different ecological signals and respond differently to any change in the habitat (de Buffrénil & Schoevaert, 1989; de Margerie et al., 2005; Canoville & Laurin, 2010; Quemeneur, de Buffrénil & Laurin, 2013). We also sectioned non-weight bearing bones (ribs) to investigate whether they exhibit a better growth mark record in their proximal ends like those of sauropod dinosaurs (Stein & Sander, 2009; Waskow & Sander, 2014). The destructive nature of histological analyses and the scarcity of complete specimens prohibited the sectioning of a large number of bones; however, an optimal sample was obtained by selecting diagnostic though incomplete skeletal elements.

Thin sections of long bones were petrographically prepared using cutting and grinding techniques following Chinsamy & Raath (1992). Considering the technical challenge of sectioning large dinocephalian skeletal elements, limb bones were sampled by the hydraulic coring method using a drill with a one cm diamond encrusted coring bit or cut using a Dremel Precision Tool, following the standard procedures outlined in Stein & Sander (2009). Core drilling was preferentially performed in an area that would cause the least amount of damage to the anatomy of the element. Note that permission was granted to section skeletal elements only in clearly specified areas (e.g., ends of the bone/broken regions) and therefore this prevented us from sectioning both proximal and midshaft regions from the same element. After the cores were obtained, the holes were infilled with plaster to preserve the overall morphology of the bone. Depending on the fossil itself, each core was either embedded in an epoxy resin (EpoxAcast 690 and/or Struers Epofix; Chinsamy & Raath, 1992; Chinsamy-Turan, 2005) or subjected to direct cutting/slicing along the preferred direction. The coring, sectioning, embedding and thin sectioning, as well as the microscopy were performed in the thin sectioning laboratory of the Palaeobiology Research Group at the Department of Biological Sciences, University of Cape Town. The embedded bones were mounted on frosted glass slides and thin sectioned using a Struers Accutom-50 and ground and polished using carborundum (silicon carbide) discs of various grit sizes (400–1,200 μm). This was followed by a final polish on a lap wheel with a velvet cloth using aluminium oxide (Al₂O₃) solution. The final thickness of the section was between 45–50 microns; this proved to be optimum for our analyses. Bones were processed in serial sections in case of slide breakages or loss of slide. All prepared sections were studied and photographed using a digital compact camera Nikon DS-Fi1 mounted on Carl Zeiss Axio Lab A1 polarizing microscope. Histological nomenclature follows that of Francillon-Vieillot et al. (1990) and Chinsamy-Turan (2005, 2012).

Results

The femur (SAM-PK-12088a; Fig. 1A) belongs to A. magnificus (Table 1). The histology is well preserved even though the section is damaged by numerous cracks (Fig. 2A). The maximum diameter of the cross-section is approximately 73 mm. It has a distinct, outer cortex and medullary region partially infilled by trabeculae (Fig. 2A). The bone wall is thick, but the extensive resorption of vascular canals gives the inner cortex a spongy aspect. The overall cross-section is divided into three regions: inner spongy bone, a middle plexiform layer and an outer band of circumferential fibrolamellar bone. The medullary region is large, and the perimedullary region consists of unevenly distributed cancellous bone that grades into the more compacted mid-cortex (Fig. 2A). The predominant bone tissue of the outer cortex is highly vascularized uninterrupted fibrolamellar bone tissue (Figs. 2B–2F) with a woven matrix as defined by fiber orientation, globular and randomly distributed osteocyte lacunae (Figs. 2D–2F). Fibrolamellar bone deposition continues up to the peripheral margin of the bone without any decrease in vascularization or change in the tissue type (Fig. 2B). The primary osteons in the peripheral cortex have a circumferential orientation (Figs. 2B, 2D–2F). A change in vascularity to a more plexiform pattern with radial and circumferential channels is observed in the inner mid-cortex (Figs. 2A, 2C). Growth marks are not present in the compacta.

A transverse section of the A. magnificus radius (SAM-PK-12088b; Fig. 1B) displays an open medullary cavity with thick struts of trabeculae (Fig. 3A). Given that the section is from the epiphyseal region, the medullary cavity is quite irregular and resorptive (Fig. 3A). Like the femur SAM-PK-12088a (Fig. 2A), the outer cortex is comprised of highly vascularized fibrolamellar bone tissue (Figs. 3C–3D) with a woven matrix as shown by the orientation of the collagen fibers and the profuse, globular, haphazardly oriented osteocyte lacunae. The vascular channels within the outer cortex are mostly longitudinally orientated and arranged circumferentially (Figs. 3B–3C), and they are surrounded by osteonal deposits forming primary osteons (Fig. 3C). The size and density of vascular canals are mostly consistent throughout the compacta (Fig. 3B), although, secondary reconstruction is extensive in the perimedullary region and has resulted in numerous enlarged cavities (Fig. 3D). Many of these erosional spaces are secondarily infilled by centripetally deposited lamellar bone (Fig. 3E).

Two ulnae (SAM-PK-12088c–d; Figs. 1C–1D) of A. magnificus were sectioned at the distal epiphyseal (Figs. 4A–4C) and midshaft regions (Figs. 4D–4F). They exhibited similar histological features as both are composed of highly vascularized cortex with an inner medullary region infilled by a dense network of bony trabeculae. Near the distal epiphyseal end (Fig. 4A), the bone is highly remodeled with many secondarily enlarged erosional cavities showing centripetal deposits of lamellar bone tissue (Figs. 4B–4C). The cross-section of the ulna at midshaft region (Fig. 4D) shows a compact bone with a large, partially filled medullary region. The medullary region has numerous islands of bony trabeculae. Circumferentially arranged longitudinal vascular canals are abundant in the compacta (Figs. 4E–4F).

The fibula (BP/1/5591b; Fig. 1E) of another Anteosaurus taxon shows an inner medullary region infilled by a dense network of bony trabeculae (Fig. 5A) while the pore spaces are occupied by diagenetic minerals (Fig. 5A, 5F). The outer margin of the medullary cavity and the inner medullary region have islands of trabeculae with a woven matrix (Fig. 5F). Resorption is intense in the inner cortex resulting in large erosional cavities (Fig. 5A). The size of the eroded cavities decreases from the inner cortex to the peripheral cortex (Fig. 5A). Overall, the compact bone tissue is laminar fibrolamellar with woven matrix (Figs. 5D–5E). The vascularization pattern is laminar towards the inner cortex whereas subperiosteally there tends to be more radial anastomoses between the circumferentially organized vascular canals (Figs. 5B–5C).

Two ribs (BP/1/5591c–d; Figs. 1F–1G) of Anteosaurus from the same individual were sectioned; one rib (BP/1/5591c; Fig. 1F) was sectioned at the proximal end (Figs. 6A–6F) and the other rib (BP/1/5591d; Fig. 1G) at the midshaft region (Figs. 6G–6K). The cortical thickness and overall shape of the section varies in different regions of the rib shaft (Figs. 6A, 6G). Near the proximal end (Fig. 6A), the bone is highly remodeled with many secondarily enlarged erosional cavities (Fig. 6B). Narrow bands of lamellar bone tissue occur between open spaces in the inner cortex (Figs. 6B–6C) whereas subperiosteally fibrolamellar bone tissue forms a thin peripheral layer (Figs. 6D, 6F). Several closely spaced rest lines and a few primary osteons are present in the subperiosteal region (Fig. 6E). The cross-section of rib BP/1/5591d taken at the midshaft level (Fig. 6G) displays a thick outer cortex of highly vascularized fibrolamellar bone tissue. However, like the proximal section, the inner cortex is spongy comprising of bony trabeculae (Fig. 6G). The outer cortex has numerous longitudinal primary osteons arranged circumferentially within the fibrolamellar bone preserving dense randomly organized globular osteocyte lacunae (Figs. 6H–6K). Circumferentially oriented longitudinal canals are dominant in the compacta (Figs. 6H–6K). A few localized radial and reticular organized vascular canals are visible (Figs. 6H, 6K). A line of arrested growth (LAG) is present in the outer peripheral cortex (Figs. 6J–6K).

Discussion

Conclusions

Three stages of growth dynamics are deduced for the genus Anteosaurus: the first stage during early ontogeny is rapid, the second stage has periodic interruptions (LAGs) in the bone deposition, while the third stage shows a marked slowing down of bone deposition and overall growth as indicated by the presence of several peripheral rest lines. Most of the skeletal elements are characterized by thick bone walls, extensive secondary reconstruction and the complete infilling of the medullary cavity. The radius of Anteosaurus magnificus and previously studied femur of Anteosaurus sp. have open medullary cavities with struts of bony trabeculae. Based on the mixed histological features evident in the skeletal elements studied (i.e., complete infilling of medullary cavity in some elements and open medullae in others), and considering the previous oxygen isotopic studies (Canoville, Thomas & Chinsamy, 2014), we propose that Anteosaurus may have been primarily terrestrial but it may have occasionally occupied ephemeral ponds as compared to the contemporaneous fully terrestrial pareiasaurs.

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