Mandible histology in Metoposaurus krasiejowensis (Temnospondyli, Stereospondyli) from the Upper Triassic of Poland

Material

The material examined comes from an abandoned claypit near the village of Krasiejów in the Opole voivodship (Upper Silesia, southwest Poland). Geologically, this region is located along the south-easterly edge of the Fore-Sudetic Homocline and sedimentary strata at Krasiejów are of Late Triassic-Norian age, based on stratigraphical data (Racki & Szulc, 2014; Szulc, Racki & Jewuła, 2015a; Szulc et al., 2015b; Szulc, Racki & Bodzioch, 2017; Jewuła et al., 2019) or Carnian age, according to biochronological studies (Dzik & Sulej, 2007, 2016; Lucas, Spielmann & Hunt, 2007; Lucas, 2015).

Two mandibles of Metoposaurus krasiejowensis have been analysed histologically; both specimens are stored in the collections of the University of Opole, Institute of Biology, Laboratory of Palaeobiology (abbreviation: UOPB). UOPB 01145 is a complete (38 cm in length), well-preserved left ramus (Fig. 1), UOPB 01027 a near-complete (34 cm in length) right hemimandible (Fig. 2).

Methods

In total, 50 transverse thin sections of the mandibles of Metoposaurus krasiejowensis have been studied. UOPB 01145 was sectioned in its entirety, at distances of less than 10 millimetres. In this way, 45 samples were prepared from a single specimen (Fig. 1). UOPB 01027 was sectioned in five specific areas of the mandible (i.e., symphyseal region, medial and postglenoid area) in order to compare the histological variability between the two specimens.

Thin sections were prepared at the Institute of Geology, Adam Mickiewicz University (Poznań, Poland). Following standard petrographical procedures (Lamm, 2013), thin sections were ground and polished to a thickness of 60–80 µm using wet SiC grinding powders (SiC 600, 800). Microscopic observations were conducted using a LEICA DMLP light microscope in plane and cross-polarised light and with a gypsum filter, and were supplemented with a scanning electron microscope (SEM) Hitachi S-3700N and binocular microscopes Olympus SZH10 and SZ61.

The histological nomenclature used in the present study follows Francillon-Vieillot et al. (1990), Witzmann (2009) and Lamm (2013). Histological characteristics of the mandible of Metoposaurus were assessed and compared to results of previous work on the cranial bone histology of this taxon (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). Preliminary interpretations of biomechanical functions for bones that were examined histologically are those of Konietzko-Meier et al. (2018). In view of the large number of thin sections and high histovariability along the mandible of Metoposaurus krasiejowensis, it would be difficult to describe the histology of each bone individually. Instead, their histology and microstructure are briefly presented in Table 1. Thus, in the present study, we provide a description of the histological variability along the entire mandible arch.

A number of other problematic issues need to be addressed in order to understand correctly the histological characterisation of some bone components, i.e., external and internal cortices. The mandible of Metoposaurus is a tube-like structure, in which several bones (dentary, splenial, postsplenial and angular) possess variable shapes along their length. In many places they become U-shaped in cross section; thus, recognition of the position of external and internal cortices may be difficult. In the present study, it is assumed that term ‘external cortex’ refers to parallel-fibred bone which occurs in the uppermost and ornamented parts of the bones on the labial side and similarly in the uppermost and unornamented parts of the same bones on the lingual side. Conversely, ‘internal cortex’ refers to parallel-fibred bone which comprises the opposite and unornamented margins of bones, both on the labial and lingual side. To avoid confusion, we use the term ‘growth marks’ in relation to lines of arrested growth (LAGs), resting lines, zones and annuli (Francillon-Vieillot et al., 1990). A line of arrested growth (LAG) usually is interpreted as an annual episode of a growth cessation linked, for instance, with unfavorable external conditions (Francillon-Vieillot et al., 1990). Resting lines are also expressions of growth stops, that could occur several times during a year. This is a well-known phenomenon in long bones of Metoposaurus, in which numerous resting lines could be observed in a single annulus (Konietzko-Meier & Sander, 2013). Alternations of thick zones and thinner annuli illustrate periods of faster or slower growth. Moreover, two types of collagen fibres are described in the present work: Sharpey’s fibres, i.e., structures which indicate the point of soft tissue attachment to the bone (muscles, tendons or skin), and interwoven structural fibres (ISF), which indicate the metaplastic origin of dermal bones (Scheyer & Sander, 2004; Scheyer & Sánchez-Villagra, 2007).

For the most representative cross sections used herein (i.e., numbers 4, 14, 22, 29, 36, 39, 41 and 43), bone porosity was calculated using the pixel-counting software ‘bw-counter’ developed by Peter Göddertz at the Institute of Geoscience of the University of Bonn (© Peter Göddertz, IGPB). All cross sections are considered to represent a single sample record, without distinction of individual bones. Only the articular, visible in the two final sections, was invariably calculated separately because of the different origin and structure.

Histology of dermal bones

The external cortex consists usually of compact parallel-fibred bone with a thickness of 2 millimetres (Figs. 3A–3B). In some areas, Interwoven Structural Fibres (ISF) are visible in the external cortex (Fig. 3C). Growth marks appear as thick and well-vascularised zones, separated by thinner and avascular annuli (Figs. 3D–3F), in both the sculptural ridges (labial side) and unornamented parts of the external cortex (lingual side), especially in the dentary and the angular. Typical lines of arrested growth (LAG) do not occur. Sharpey’s fibres are numerous, well-mineralised and densely packed along the entire length of the mandible and are present in all dermal bones. However, they are the most common on the labial side of the mandible, mainly in the ornamented parts of the dentary, angular and surangular (Figs. 3G–3H). In these areas Sharpey’s fibres are very long and penetrate into the deeper parts of the external cortex (Figs. 3I–3K). In bones on the lingual side of the mandible, especially in the precoronoid, intercoronoid, coronoid and postsplenial, Sharpey’s fibres are shorter and thinner.

The external cortex is poorly vascularised. A high number of vascular canals and small primary osteons occur only in thick zones, mainly in the dentary and splenial. On the labial side, sculptural ridges are almost avascular in the postsplenial and angular, and alternations with an earlier generation of ridges are present (Figs. 4A–4C). Osteocyte lacunae with branched canaliculi occur numerously along the external cortex in all bones examined (Fig. 4D).

The external cortex clearly transits into the extensive middle region which covers the larger parts of every dermal bone in cross section (Figs. 4E–4F). The vascular network in the middle region varies from poorly developed to well developed. Small secondary osteons and primary vascular canals occur numerously next to the border between the middle region and the external cortex (Figs. 4G–4I). In many areas, dense clusters of small osteons create the beginning of Haversian tissue. These features are clearly seen, mainly in the posterior part of the dentary (Fig. 4G), the anterior areas of the angular and occasionally in the splenial and the postsplenial (Figs. 4H–4I). The deeper parts of the middle region are occupied by larger secondary osteons (50–200 µm in diameter) which create dense clusters mainly in the posterior parts of the postsplenial, angular and surangular (Figs. 5A–5B). The middle region is highly remodelled. Very large and irregularly shaped erosion cavities occur here. The degree of porosity may drastically change even along a single bone. For instance, in the angular, the anterior part is relatively thin and of low porosity, whereas in its distal parts it becomes thicker and also highly porous (Figs. 5C–5E). A similar pattern is present in the prearticular (Figs. 5F–5G) and surangular (Figs. 5H–5I). Posteriorly, the middle part of these bones is almost absent, and erosion cavities could exceed 3 millimetres in length.

The internal cortex consists of parallel-fibred bone and its thickness usually reaches around one millimetre (Fig. 5J). Vascularisation is moderate: longitudinal primary vascular canals are very numerous (Fig. 5K). Small secondary osteons are less common and filled with a thin layer of lamellar bone. Sharpey’s fibres are long, dense and packed in bundles; they occur along the subsurface and in the deeper part of the internal cortex, especially in the dentary and angular (Fig. 5L). Osteocyte lacunae are numerous as well. Growth marks are not visible.

Histology of the articular

The articular consists of extensive spongy bone, which is surrounded by a thin cortex (Figs. 6A–6B). Poorly preserved and avascular cortex consists of parallel-fibred bone. Growth marks and Sharpey’s fibres are not visible. The middle region consists of extensive spongy bone with large cavities between trabeculae (Figs. 6C–6D). Within the bony trabeculae, numerous and elongated osteocyte lacunae are present and remains of calcified cartilage are visible (Figs. 6E–6F).

Histological variability of the mandible

In the lower jaw of Metoposaurus krasiejowensis, the bone microanatomy and histological framework are highly variable. Symphyseal and anterior parts of the mandible represent a compact bony structure (calculated porosity below 20%—Table 1; Fig. 7) being a conglomerate of bones of low porosity, i.e., in the dentary, splenial, precoronoid and the anterior part of intercoronoid and postsplenial. On the labial side, the dentary, precoronoid and splenial possess a well-developed vascular network (Figs. 3D–3F and 4D) and abundant clusters of Sharpey’s fibres (Figs. 3G–3K). In the median part, the mandible is formed as a tube-like structure with a large Meckelian canal (sectioning sample no. 17–32 of UOPB 01145). Bone thickness varies across this part of the mandible. Dentary thickness varies between 1 and 5 millimetres, as in the coronoid, whereas in the postsplenial and angular it varies between 2 millimetres and almost 10 millimetres (Table 1). The porosity of these bones is low to moderate (between 20% and 30%, Table 1; Fig. 7). However, the most widely variable factor is the degree of vascularisation. Dense clusters of large, secondary osteons are visible in the thickest parts of the postsplenial and angular (Figs. 5A–5B). Distinct variations in bone thickness and compactness start in the area of the glenoid foramen (sectioning sample no. 33–45 of UOPB 01145). The average thickness of the surangular and angular is around 4 millimetres. However, in the upper part of the surangular and the posterior area of the angular, the thickness of these bones can reach even up to 10 millimetres. The prearticular thickness varies from 2 to 8 millimetres. The porous nature of all of these bones becomes very marked in contrast to the anterior and median areas of the mandible. The extensively remodelled middle region consists of large and irregular erosion cavities which can reach around 3 millimetres in length. The microanatomical variability could be easily followed, for instance in the angular (Figs. 5C–5E), prearticular (Figs. 5F–5G) and surangular (Figs. 5H–5I). These bones are thinner but more compacted in their anterior parts, while posteriorly they become much thicker and more porous. The high porosity makes documentation of histological characteristics difficult in the glenoid and postglenoid area.

Microstructure of the mandible bones

The overall thickness of bone walls increases along the mandible from the symphyseal to postglenoid area (Table 1). The symphyseal part is a tube-like structure, relatively massively built with only a few, small cavities. The calculated porosity in this area equals about 19% (Fig. 7). The highest compactness is observed in slide 14, where it reaches (89%). Posteriorly, the diameter of the entire mandible ramus increases rapidly, with a size increase of the inner cavities and a decrease of bone thickness. Simultaneously, bone porosity growths, attaining 42% in slide 43 (Fig. 7). The articular shows a different structure, with a very loose framework, the bone porosity reaching up to 51% in slide 41 and over 73% in slide 43.

Skeletochronology

In contrast to long bones in which growth mark structure could be easily determined and followed (Konietzko-Meier & Klein, 2013; Konietzko-Meier & Sander, 2013; Teschner, Sander & Konietzko-Meier, 2018; Teschner et al., 2020), dermal bones are not a good source of skeletochronological data (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). These structures appear in the skull of Metoposaurus only as resting lines and alternations of thick zones and annuli. Typical lines of arrested growth (LAGs) could not be found (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). Similar skeletochronological patterns occur along the mandibles of Metoposaurus. Lines of arrested growth are not visible. However, very common is the alternation of thick zones, separated by thinner and avascular annuli (Figs. 3D–3F). The presence of this alternation is not constant, but yet can clearly be followed along the dentary and angular. Highly vascularised zones are associated with more rapid growth of an organism which can be linked to favourable environmental conditions with high water levels and increased food availability (Konietzko-Meier & Sander, 2013). Avascular annuli are related to less beneficial seasons when life activity and body growth were slower (Konietzko-Meier & Sander, 2013). Due to the lack of continuity of growth marks along the mandible bones and high bone remodelling, it is difficult to assess the individual age of specimens studied; the same holds true for the skull (Gruntmejer, Konietzko-Meier & Bodzioch, 2016).

Ossification processes of the mandible

Dermal bones develop directly from the soft tissue, i.e., the deep parts of the dermis without periost, osteoblasts and osteoid, via metaplastic ossification (Francillon-Vieillot et al., 1990; Vickaryous & Hall, 2008). In temnospondyls, dermal bones form the skull, mandible and pectoral girdle (two clavicles and an interclavicle), the external surfaces of which are ornamented by grooves, ridges and polygonal pits (e.g., Von Meyer, 1858; Fraas, 1889; Fritsch, 1889; Zittel, 1911; Witzmann, 2009; Witzmann et al., 2010; de Buffrénil et al., 2016). In cross section, the dermal bone shows a diploë structure. Their external surface creates a compact layer of an intramembranous component which appears as parallel-fibred bone, lamellar bone or interwoven structural fibres (Scheyer & Sander, 2004; Scheyer & Sánchez-Villagra, 2007). The external cortex gradually passes into an extensive and highly porous middle region, usually with numerous clusters of secondary osteons (Witzmann, 2009). The internal cortex also consists of an intramembranous, lower part of the bone. In mandibles of Metoposaurus (specimens UOPB 01145 and UOPB 01027), dermal bones also represent a diploë combination in cross section. External and internal cortices usually consist of a compact layer of parallel-fibred bone (Figs. 3A–3C and 5J). Its metaplastic origin additionally confirms the presence of interwoven structural fibres (ISF) in the subsurface part of the cortex and along sutural margins of the surangular and prearticular (Gruntmejer et al., 2019a). The middle region is highly remodelled and forms the largest portion of the bone (Figs. 5C–5I).

Endochondral bones ossify throughout a cartilaginous predecessor and consist of a trabecular region, surrounded usually by a thin layer of cortex. In the skull of Metoposaurus, the quadrate and exoccipital are endochondral bones (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). In the case of the mandible, the articular does not constitute a dermal bone in origin, this having formed of a thin layer of avascular cortex and a very extensive, trabecular region (Figs. 6A–6D). Remnants of calcified cartilage show an endochondral development of articular bone (Figs. 6E–6F).

Sharpey’s fibres

Dense clusters of two types of Sharpey’s fibres have been recognised in long bones of Metoposaurus (Konietzko-Meier & Sander, 2013). Thick and long fibres are indicative of points of attachment of strong skeletal muscles and tendons, whereas shorter ones reflect areas in which the periosteum is connected to the bone. A similar distribution of Sharpey’s fibres can be observed within the skull of Metoposaurus (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). Short fibres are rare, but they do appear along the length of the skull and may indicate a delicate, soft-tissue attachment. More numerous and thick Sharpey’s fibres occur in the posterior part of the skull in the tabular and occipital condyle. The presence of well-mineralised and dense bundles in these areas suggests a strong skeletal muscle attachment of the skull to the vertebral column (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). Moreover, numerous clusters of short Sharpey’s fibres occur at sutural edges between adjacent bones (Gruntmejer et al., 2019b). Their presence along the lateral edges of dermal bones refers to the occurrence of collagen fibres within the sutural morphospace which bridged adjacent bones and increased the connections between them (Rafferty & Herring, 1999).

In mandibles of Metoposaurus (specimens UOPB 01145 and UOPB 01027), both types of Sharpey’s fibres are present as well. However, long and short fibres occur together along the whole length of the mandible on the labial side (Figs. 3G–3K). Their presence was noted in sculptural ridges and the unornamented parts of bones where longer Sharpey’s fibres penetrate into the deeper parts of the external cortex. Thinner fibres are present along the lingual side of the mandible and in the internal cortex of several bones (Fig. 5L). Dense clusters of two types of Sharpey’s fibres along the labial part of the mandible suggests abundant attachment of skeletal muscles, tendons and ligaments on this side of the lower jaw. In contrast, the occurrence of shorter fibres along the lingual part of the mandible may relate to delicate and soft-tissue attachment which filled the inner surface of the mouth. Similar to the case of the skull, short and numerous Sharpey’s fibres were also noted along the sutural edges between adjacent bones of the mandible (Gruntmejer et al., 2019a). Moreover, their orientation is an important indicator for deduction of stress distribution during feeding activity (Rafferty & Herring, 1999; Herring & Teng, 2000; Jasinoski et al., 2010). Sutural morphology and crucial orientation of Sharpey’s fibres in the anterior part of the dentary and at the sutural contacts between the articular, surangular and prearticular suggest tensile stress in these areas. The tensile loading regime in the symphyseal and postglenoid region could be an adaptation to open the jaw very wide during predation (Gruntmejer et al., 2019a).

Biomechanical condition of dermal bones in the mandible

Histological studies of dermal skull bones in Metoposaurus krasiejowensis have revealed that parameters such as bone thickness and porosity are the most variable along the skull and play an important role in its biomechanics (Konietzko-Meier et al., 2018). Bone thickness and compactness are genuine responses to stress distribution, i.e., bones lose strength and stiffness with increasing porosity; however, this can be partly compensated by an increase of structural thickness (Witzmann, 2009; Rinehart & Lucas, 2013). In addition to this, the histological properties are not related only to the bone, but strictly depend on the particular plane of sectioning (Gruntmejer, Konietzko-Meier & Bodzioch, 2016; Konietzko-Meier et al., 2018). In the skull of Metoposaurus krasiejowensis bones along the sagittal axis, such as the jugal and postorbital, sectioned along the sutural contact, represent the same histological framework and very good biomechanical properties (high thickness and low porosity) for stress resistance. Another example is the squamosal, sectioned in its anterior part and next to the otic notch, which showed different histological levels. Bones situated on the palatal side (e.g., vomer and pterygoid) display weaker biomechanical resistance, characterised by a low bone thickness and extreme porosity (Gruntmejer, Konietzko-Meier & Bodzioch, 2016). This suggests that the histological framework is not specifically limited to bone, but does reveal specific areas of the skull which probably were involved during feeding (Konietzko-Meier et al., 2018).

The wide range in microstructure of the mandible bones of Metoposaurus krasiejowensis may also provide preliminary data on its biomechanics. Similar to the skull, the microstructure of the mandible is not limited to a single bone either, but refers to a specific area of the mandible. The angular, sectioned in different areas along its length, shows a widely varying histological framework, from low thickness and high compactness to great thickness and an extremely high degree of porosity (Figs. 5C–5E). A similar microstructural pattern could be observed in the prearticular (Figs. 5F–5G). Moreover, some bones, such as the angular and surangular, sectioned along the sutural contact, represent the same histological framework (i.e., degree of bone thickness and compactness) which varies in other parts of these bones (Figs. 5H–5I).

Based on such high histovariability along the mandible of Metoposaurus, a preliminary interpretation of its biomechanics can be put forward. The symphyseal and other anterior parts of the mandible are characterised by low bone porosity which creates a thick conglomerate without any large cavity (Meckelian canal) between them (Fig. 7). Such microstructure may represent good biomechanical resistance of the anteriormost area of the mandible. A stable histological framework, accompanied by sutures that are related mostly to tensile loading (Gruntmejer et al., 2019a), suggests that the symphyseal part of the mandible was well adapted to resist stress created during feeding. The most stable structure is seen in slide 14, with the lowest porosity of the dentary, splenial and postsplenial and a complex structure without large inner cavity. This makes this part of the mandible a key point with the highest loading. In midline areas, bone thickness is low, reaching around 2–5 millimetres, but it increases to even 1 cm in sculptured regions. However, the compactness of these bones is lower than in the anterior part, thus biomechanical resistance of the central parts of the mandible can be assessed as moderate. A distinct increase of bone porosity begins in the glenoid area, whereas in postglenoid parts, all bones become extremely porous (up to 42% in slide 43) at thicknesses varying between 5 millimetres and 10 millimetres. However, the high level of porosity suggests the biomechanical parameters of these bones to have been moderate to low. Based on such varying microstructure among dermal bones, the mandible in general appears to be not such a strong structure. However, seemingly weak bones (caused by low compactness) could be strengthened by connections of strong skeletal muscles and other soft tissues. Dense clusters of thick and long Sharpey’s fibres support this assumption. Moreover, five types of sutures adapted to resist different stresses (tension vs. compression) have been noted along the mandible (Gruntmejer et al., 2019a). One of the biomechanical roles of sutures is to minimalise loading regimes acting within the skull or on the mandible during feeding. Thus, the complexity of sutural morphology and the abundance of Sharpey’s fibres along the mandible may compensate for the relatively weak microstructure of dermal bones of the mandible. These preliminary interpretations of mandible biomechanics should be worked out further and supplemented by additional studies, e.g., by computational modelling using finite element analysis. Based on previous works which focused on comparisons between FEA and histological results (Konietzko-Meier et al., 2018; Gruntmejer et al., 2019b), we know neither of these methods is flawless. For obtaining the most reliable results it is necessary to conduct investigations using histology as well as FEA in parallel.