Dinosaur paleohistology: review, trends and new avenues of investigation

Abstract

In the mid-19th century, the discovery that bone microstructure in fossils could be preserved with fidelity provided a new avenue for understanding the evolution, function, and physiology of long extinct organisms. This resulted in the establishment of paleohistology as a subdiscipline of vertebrate paleontology, which has contributed greatly to our current understanding of dinosaurs as living organisms. Dinosaurs are part of a larger group of reptiles, the Archosauria, of which there are only two surviving lineages, crocodilians and birds. The goal of this review is to document progress in the field of archosaur paleohistology, focusing in particular on the Dinosauria. We briefly review the "growth age" of dinosaur histology, which has encompassed new and varied directions since its emergence in the 1950s, resulting in a shift in the scientific perception of non-avian dinosaurs from "sluggish" reptiles to fast-growing animals with relatively high metabolic rates. However, fundamental changes in growth occurred within the sister clade Aves, and we discuss this major evolutionary transition as elucidated by histology. We then review recent innovations in the field, demonstrating how paleohistology has changed and expanded to address a diversity of non-growth related questions. For example, dinosaur skull histology has elucidated the formation of curious cranial tissues (e.g., "metaplastic" tissues), and helped to clarify the evolution and function of oral adaptations, such as the dental batteries of duck-billed dinosaurs. Lastly, we discuss the development of novel techniques with which to investigate not only the skeletal tissues of dinosaurs, but also less-studied soft-tissues, through molecular paleontology and paleohistochemistry-recently developed branches of paleohistology-and the future potential of these methods to further explore fossilized tissues. We suggest that the combination of histological and molecular methods holds great potential for examining the preserved tissues of dinosaurs, basal birds, and their extant relatives. This review demonstrates the importance of traditional bone paleohistology, but also highlights the need for innovation and new analytical directions to improve and broaden the utility of paleohistology, in the pursuit of more diverse, highly specific, and sensitive methods with which to further investigate important paleontological questions.

Keywords: Birds; Dinosaurs; Mineralized tissues; Molecular paleontology; New trends; Paleohistochemistry; Soft-tissues; Standard paleohistology.

Figure 1. Schematic representation of the cortex of a long bone seen in cross-section.

The degree of vascularization, the orientation of vascular canals (i.e., longitudinal, laminar, radial, or reticular orientations), and the degree of organization of the collagenous matrix, e.g., woven, lamellar (or parallel-fibered, not shown here) are a direct reflection of the local bone growth rate. When growth slows down and/or stops completely, which typically occurs annually, it is marked by a line of arrested growth (LAG) or other types of growth marks. Note that the schematized structures may not all necessarily be found together within a single section. From Huttenlocker, Woodward & Hall (2013); reproduced with permission; © 2013 by the Regents of the University of California. Published by the University of California Press.

Figure 2. Petrographic ground section of the ulna of Iteravis huchzermeyeri IVPP V18958.

Its histology shows a fairly vascularized tissue and an internal circumferential layer (ICL), the latter being found in extant birds with nearly complete skeletal growth. This microstructure and the absence of an annual growth mark suggests that this specimen was most likely less than a year-old and had not fully reached skeletal maturity. This represents the growth pattern typically found in most living birds, and is unlike that of more basal birds or non-avian dinosaurs who took multiple years to reach full skeletal maturity. Modified from O’Connor et al. (2015).

Figure 3. Petrographic ground section of a femur fragment of Tyrannosaurus rex (MOR 1125), showing cortical bone (cb) and medullary bone (mb).

The cb shows many secondary osteons interspersed with increasingly large erosion rooms (green arrows). Red arrows show distinct boundary between cb and mb. Avian mb is found in reproductive female birds and is used as a calcium reservoir during eggshell formation. Since a similar tissue was found in this non-avian theropod dinosaur, it illustrated similarities between birds and dinosaurs at the microscopic scale and suggested that MOR 1125 was a gravid, female T. rex. This tissue has since been used to infer sexual maturity in other fossil archosaurs. Modified from Schweitzer et al. (2016).

Figure 5. Petrographic ground sections in the maxilla of the duck-billed dinosaur Hypacrosaurus stebingeri (MOR 548, a nestling) showing the first evidence of “avian” secondary cartilage (SC) in a non-avian dinosaur.

(A) A nodule of SC (black arrows) is found nearing the more internal maxillary bone. (B) A close-up shows hypertrophic chondrocyte lacunae, typical of calcified cartilage. This nodule is found between the maxilla and the coronoid process of the dentary. Although not preserved here, some hyaline cartilage was certainly present as well, in continuity with the calcified cartilage. The cartilage may have facilitated the “rubbing” of the maxilla and coronoid process of the dentary during mastication, as cartilage has shock-absorbing, cushioning properties. It also suggests that some movement was possible at this joint (although the joint structure, or the exact amount of movement, is not clear at this point). Secondary cartilage is also found on the skull bones of living birds, and its discovery in an ornithischian dinosaur suggested that birds inherited this tissue from their non-avian dinosaur ancestors. Modified from Bailleul, Hall & Horner (2012).

Figure 4. Petrographic ground sections of the occipital condyles of two young Triceratops, MOR 1110 (A–C) and MOR 8657 (B–D).

(A) The first occipital condyle is still unfused (composed of a basioccipital (bo) and two exocipitals (exo) on each side, but only the right one is shown in (A). Magnification of the red square in (A) is shown in (C) and shows the condyle is composed of a highly vascularized, cancellous bone. Calcified cartilage islands can be seen within bony trabeculae (blue arrows). (B) The older occipital condyle (B) is fully fused. Magnification of the red square in (B) is shown in (D). It has a much less vascularized, and more compact bone (D). Sectioning the same cranial element in multiple ontogenetic stages can reveal unknown aspects of dinosaurian cranial growth. Modified from Bailleul & Horner (2016). ©John Wiley and Sons.

Figure 6. Ground section of a mineralized tendon from M. extensor carpi radialis of Bubo virginianus (Great Horned Owl), showing that it does not present the microstructure typically expected for bone.

The tissue is made of small collagen fiber bundles and fascicles (yellow arrows), separated from each other by arc-shaped spaces (white arrows). Red arrows are pointing at an irregular border between two types of tissues and probable unmineralized fiber fasciles. No regular bone cell lacunae (with an elongated morphology and canaliculi) can be seen anywhere in these sections, and this reflects a unique mode of skeletal tissue formation, different from that of typical bone. Many dinosaurian and archosaurian tissues have been found with a similar microstructure. Modified from Bailleul & Horner (2016).

Figure 7. Petrographic ground section of an isolated maxilla of a Hypacrosaurus embryo (MOR 559).

(A) Schematic representation of the skull of a Hypacrosaurus embryo with orientation of the cut on the maxilla. (B) Whole-view image of a transverse section through the maxillary battery near the occlusal surface. (C) A close-up image in the same transverse section shows different dental tissues (alveolar bone, ab; enamel, en; dentine; de; cellular cementun, cc). (D) Higher magnification shows that teeth contacted each other via soft tissues (i.e., periodontal ligament), as reflected by Sharpey’s fibers (Sf) within the cellular cementum and the space between the teeth filled with minerals and sediment. Therefore, in addition to possessing a ligamentous attachment with alveolar bone, hadrosaurs had a unique tooth-to-tooth fibrous attachments in which each individual tooth within the battery was suspended to its neighbors through soft tissue connections. Other abbreviations: ac, acellular cementum; rl, resorption line; Modified from Leblanc et al. (2016).

Figure 8. Ostrich (A) and dinosaur (B–C) cellular response to the DNA intercalating dye propidium iodine (PI).

Extant ostrich osteocytes (A), isolated osteocytes from the extinct theropod T. rex (B), and osteocytes from the hadrosaur B. canadensis (C) show identical response to PI (red arrows, consistent with the location of cell nuclei). This strongly suggests a compound chemically consistent with DNA, can survive tens of millions of years. PI requires double-stranded DNA to react, and only stains the nucleus of dead cells; these data support the presence of a compound with these characteristics. The data are not consistent with binding that occurs in bacteria, which are orders of magnitude smaller. However, although this binding pattern is consistent with that seen in extant samples, only sequence data can fully confirm the origin of this material (see Schweitzer et al., 2013 for additional data). Images are at the same scale.

Figure 9. Paraffin thin-sections paired with alcian blue histochemical staining of demineralized cortical bone (A) and medullary bone (B) of T. rex (MOR 1125).

This stain capitalizes on the differential presence of sulfated glycosaminoglycans found in cortical bone (CB) vs medullary bone (MB), with low amounts in the former (with a faint staining), and a higher amount in the latter (with a more intense staining). The same differential staining pattern is observed in these two tissues in extant birds; which provided additional histochemical similarities between the MB in T. rex and the gender-specific, reproductive MB found in extant birds. This new method (paleohistochemistry) can be applied to other fossilized tissues including soft-tissues, and if combined with other microscopic observations and/or techniques, has the potential to revolutionize paleohistology. Modified from Schweitzer et al. (2016).