The bone microstructure of polar “hypsilophodontid” dinosaurs from Victoria, Australia

High-latitude (i.e., “polar”) Mesozoic fauna endured months of twilight and relatively low mean annual temperatures. Yet non-avian dinosaurs flourished in this taxing environment. Fossils of basal ornithopod dinosaurs (“hypsilophodontids”) are common in the Early Cretaceous high-latitude sediments of Victoria, Australia, and four taxa have been described; although their ontogenetic histories are largely unexplored. In the present study, eighteen tibiae and femora were utilized in the first multi-specimen ontogenetic histological analysis of Australian polar hypsilophodontids. The sample consists of eleven individuals from the Flat Rocks locality (Late Valanginian or Barremian), and five from the Dinosaur Cove locality (Albian). In both groups, growth was most rapid during the first three years, and skeletal maturity occurred between five and seven years. There is a weak asymptotic trend in a plot of growth mark count versus femur length, with considerable individual variation. Histology suggests two genera are present within the Dinosaur Cove sample, but bone microstructure alone could not distinguish genera within the Flat Rocks sample, or across the two geologically separate (~ 26 Ma) localities. Additional histologic sampling, combined with morphological analyses, may facilitate further differentiation between ontogenetic, individual, and species variation.


Supplementary Text
Detailed histological descriptions of hypsilophodontid femora and tibiae from the collections of Museum Victoria (NMV), Victoria, Australia.
Flat Rocks Locality: NMV P208495 (left femur, Supplementary Figure 1): Compact coarse cancellous tissue is located on the posteromedial side and separated from the medullary cavity by a lamellar endosteal layer. This lamellar endosteal layer is only present on the medial side of the femur. Radial channels pierce through the lamellar endosteal layer. Primary tissue throughout the cortex varies between poorly organized parallelfibred to parallel-fibred, with small longitudinal primary osteons. Osteocyte lacunae are dense and plump within the cortex but are flattened in the outermost cortex near the periosteal surface. Two LAGs are visible. The first (innermost) LAG is partially destroyed by medullary expansion, while the second LAG is within the outermost cortex. Because the periosteal surface is mechanically eroded in places, the perimeter of the second LAG is not fully traceable.
NMV P199058 (left femur, Supplementary Figure 2): Preservation of bone microstructure is poor, possibly due to microbial invasion. On the posteromedial side adjacent to the medullary cavity this is an area of compact coarse cancellous tissue. A lamellar endosteal layer surrounds the medullary cavity and is thickest on the posteromedial side. The endosteal layers is pierced by numerous radial channels. There are three LAGs. Prior to the first LAG, and for a short distance after it, tissue is poorly organized parallelfibred. Osteocyte lacunae in this area are dense and plump. Between the first and second LAG the tissue becomes mostly parallel-fibred and osteocyte lacunae are flattened. Vascular canal orientation is primarily longitudinal but more laminar in places. The innermost LAG, which is followed by an annulus, is truncated on the lateral side due to medullary expansion. NMV P221151 (left femur, Supplementary Figure 3): Tissue preservation is poor and there is scattered pyrite invasion. Compact coarse cancellous bone is present on the posterior side of the medullary cavity. This area is separated from the medullary cavity by a thin lamellar endosteal layer. Cortical primary tissue is poorly organized parallel-fibred with longitudinal primary osteons. Osteocyte lacunae within the inner and mid-cortex are dense and rounded to somewhat flattened. There are five LAGs within the cortex, three of which are truncated by medullary drift. The outermost cortex is lamellar with dense, flattened osteocyte lacunae. Vascularity is sparse and consists of anastomosing simple vascular canals. The transition to primarily lamellar avascular bone at the periosteal surface after the fifth LAG may indicate the beginnings of an EFS, but it is thin.
NMV P180892 (left femur, Supplementary Figure 4): The innermost cortex on all but the medial side consists of compact coarse cancellous bone and scattered secondary osteons. The innermost cortex on the medial side is primary periosteal tissue, which was in the process of being resorbed due to medullary drift. On the lateral side, bone tissue is remodelled to the mid-cortex. The lamellar endosteal layer encircling the medullary cavity is thickest on the anterolateral side. Here, radial channels are present within the thick (1.5 mm) endosteal layer and open into the medullary cavity. Primary tissue within the cortex is poorly organized parallel-fibred with longitudinal simple vascular canals. Seven LAGs are within the cortex, and an eighth is within 50 microns of the periosteal surface. No EFS is visible, suggesting this individual was not skeletally mature even with a femur circumference of 10.08 cm, considerably larger than other samples.
NMV P150054 (right femur, Supplementary Figure 5): The cortex is crushed on the lateral side and fragments fill the medullary cavity. A small patch of compact coarse cancellous bone is visible on the medial side. Large secondary osteons are visible on the medial side in two groups, one in the inner cortex, and the other in the outer cortex. Otherwise the cortex is primary bone. The innermost primary cortical tissue is a combination of fibro-lamellar and poorly organized parallel-fibred, with small or incipient longitudinal primary osteons. Osteocyte lacunae density is high in the inner and mid-cortex but is lower near the periosteal surface. The cortex becomes more strongly parallel-fibred in the outer cortex. Within the cortex are three CGMs. The first two are LAGs, while the third is an annulus.
NMV P210062 (right tibia, Supplementary Figure 6): The primary cortical tissue varies between reticular and longitudinal fibro-lamellar, and poorly organized parallel-fibred. There is an area of compact coarse cancellous bone on the lateral side bordering the medullary cavity. A lamellar endosteal layer is located on the anterior surface bordering the medullary cavity. Secondary osteons are dense and confined to the lateral side from the endosteal to periosteal surface, forming the anterolateral plug of the tibial border 4 . There is a LAG present within the cortex but partially obliterated on the medial side by medullary expansion, and laterally by secondary osteons. Opaque areas of the slide are due to pyrite.
NMV P208204 (right tibia, Supplementary Figure 7): The innermost cortex of the anterior and posterior sides is fibro-lamellar, but otherwise cortex is parallel-fibred. Vascularity is longitudinal to anastomosing simple vascular canals and small primary osteons. Two LAGs are visible within the cortex. Osteocyte lacunae density is high prior to the first LAG but is less dense thereafter. Osteocyte lacunae density decreases from inner to outer cortex. The LAGs often appear as tightly stacked clusters of two or three lines, but because the lines merge and split when traced around the circumference, each grouping is considered to represent a single annual event. There is an eighth LAG very close to the periosteal surface in a region of decreased vascularity, suggesting a thin EFS is present.

Dinosaur Cove Locality:
NMV P186326 (left femur; Supplementary Figure 12): Compact coarse cancellous bone comprises the innermost cortex on the posteromedial and posterolateral sides. The entire medullary cavity is surrounded by an endosteal layer up to 440 m thick posteriorly. Frequent radial channels are found through the thick endosteal layer. Bone fibre orientation throughout the transverse section is poorly organized parallelfibred. Vascularity consists of anastomosing simple canals in the inner and mid-cortex, and is longitudinal in the outer cortex. Osteocyte lacunae density is high. There are three LAGs within the cortex. NMV P186334 (right tibia; Supplementary Figure 13): The medullary cavity is lined with a lamellar endosteal layer. Radial channels are within the endosteal layer between the primary tissue and the medullary cavity. The innermost primary cortex is fibro-lamellar with small longitudinal primary osteons and anastomosing simple primary vascular canals, and therefore resembles the incipient fibro-lamellar bone 1,5 observed in some modern alligators. Osteocyte lacunae are frequent in the innermost cortex but sparse by mid cortex. From middle to outer cortex, tissue is parallel-fibred, and is lamellar in the outermost cortex. There is a band of colour change in the outermost cortex, but appears to be due to mineral staining rather than a transition to EFS, as tissue organization and degree of vascularity remains the same when examined using polarized light. There are four LAGs within the cortex, the innermost of which is only visible on the anterolateral side due to medullary cavity enlargement. This tibia has a very pronounced anterior-lateral border. The inner to outer cortex of the anterior border is largely secondary.
NMV P228360 (right tibia; Supplementary Figure 14): Compact coarse cancellous bone is concentrated on the lateral side of the innermost cortex, and separated from the medullary cavity by a lamellar endosteal layer. The primary tissue of the innermost cortex varies between fibro-lamellar and very poorly organized parallel-fibred tissue, with longitudinal primary osteons. The cortex is parallel-fibred from middle to outer cortex with longitudinal primary osteons and anastomosing simple vascular canals. Osteocyte lacunae density is high, and lacunae become more flattened in the mid and outer cortex. There are seven annuli within the cortex. Osteocyte lacunae density is greatly decreased in the zone between the 5th and 6th annulus.  Figure S1.             The innermost cortex is fibro-lamellar and the medullary cavity is lined by a lamellar endosteal layer, which has within it radial channels. Arrow points to the innermost of four LAGs present within the cortex. This LAG is partially destroyed by medullary cavity enlargement. Circularly polarized light. EL = lamellar endosteal layer. c) The middle and outer cortex is primarily parallel-fibred and contains three LAGS (arrows). Plane polarized light. d) There is an abrupt band of colour change (arrow) in the outermost cortex, apparently due to mineral staining rather than a transition to EFS, as the tissue organization and vascular canal density remains constant across the boundary. Full wave plate.