The nature of aspidin and the evolutionary origin of bone

Abstract

Bone is the key innovation underpinning the evolution of the vertebrate skeleton, yet its origin is mired by debate over interpretation of the most primitive bone-like tissue, aspidin. This has variously been interpreted as cellular bone, acellular bone, dentine or an intermediate of dentine and bone. The crux of the controversy is the nature of unmineralized spaces pervading the aspidin matrix, which have alternatively been interpreted as having housed cells, cell processes or Sharpey's fibres. Discriminating between these hypotheses has been hindered by the limits of traditional histological methods. Here, we use synchrotron X-ray tomographic microscopy to reveal the nature of aspidin. We show that the spaces exhibit a linear morphology incompatible with interpretations that they represent voids left by cells or cell processes. Instead, these spaces represent intrinsic collagen fibre bundles that form a scaffold about which mineral was deposited. Aspidin is thus acellular dermal bone. We reject hypotheses that it is a type of dentine, cellular bone or transitional tissue. Our study suggests that the full repertoire of skeletal tissue types was established before the divergence of the earliest known skeletonizing vertebrates, indicating that the corresponding cell types evolved rapidly following the divergence of cyclostomes and gnathostomes.

Figure 1. Hypothesis of vertebrate relations based on Keating & Donoghue.

Heterostracans, together with the Ordovician aged Arandaspids and Astraspis comprise the clade Pteraspidomorpha, interpreted as the sister group to all other ostracoderms + crown gnathostomes (i.e. all other skeletonising vertebrates). Based on our histological analyses we inferred character evolution using parsimony. Black triangles represent character gain and white triangles represent character loss. Cartilage (1), dermal bone (2), dentine (3), enameloid (4), perichondral bone (5), endochondral bone (6), enamel (7), cell spaces in dermal bone (*).

Figure 2. Morphology and histology of the heterostracan dermal skeleton.

Gross external morphology of the dermal skeleton of Errivaspis waynensis (NHMUK P19789) from the Lochkovian of Herefordshire, UK (a); etched SEM section of Loricopteraspis dairydinglensis (NHMUK P75400) showing the 4-layered construction of the dermal skeleton. Aspidin spaces and Sharpey’s Fibre spaces are preserved as high relief pyrite diagenetic infill. Sharpey’s Fibre spaces pervading L3 can be distinguished from aspidin spaces in L2 by both their size and configuration (b); sectioned srXTM virtual model of the dermal skeleton ofTesseraspis tesselata (NHMUK P73617). The vasculature network is shown to comprise a series cancellae interlinked by reticular canals (c); SrXTM horizontal virtual thin section through aspidin trabeculae ofTesseraspis tesselata (NHMUK P73618). Aspidin spaces are preserved as diagenetic pyrite infill with high X-ray attenuation. Spaces are organised orthogonal to the trabecular lamellae, or else tangled at trabecular intersections (d); SEM detail of a cancellar chamber of Loricopteraspis dairydinglensis (NHMUK P73622), showing a centripetal fabric of coarse spicules, interpreted as mineralised (crystal) fibre bundles (e); SrXTM horizontal virtual thin section through aspidin trabeculae of Tesseraspis tesselata (NHMUK P73618). Aspidin spaces are preserved as diagenetic pyrite infill with high X-ray attenuation. Spaces are organised orthogonal to the trabecular lamellae, or else tangled at trabecular intersections (e). r.c., reticular canal; o., odontode; v.n., vascular network; ca, cancellae; a.s., aspidin space; o.a.s., orthogonal aspidin space; t.a.s., tangled aspidin space; s.f., Sharpey’s Fibre space; sup, superficial layer; L1, layer 1; L2, layer 2; L3, layer 3. Relative scale bar equals 21mm in (a), 183 μm in (b), 165 μm in (c), 83 μm in (d). and 49 μm in (e).

Figure 3. Histology of aspidin in phylogenetically disparate heterostracan taxa.

SEM etched section of a trabecular wall of Lepidaspis serrata NRM-PAL C.5940 showing bipartite construction (a); LM thin section of a junction of trabecular walls of Phialaspis symondsi NHMUK P73619 (b); etched SEM section through a polygonal cancellar chamber of Corvaspis kingi NHMUK P73616 (c); BSE SEM vertical section through a polygonal cancellar chamber of Corvaspis kingi NHMUK P73613 showing both the aspidin spaces and rystal fibre bundles (d); horizontal SrXTM virtual thin section through trabecular walls of Tesseraspis tesselata NHMUK P73617 (e); SEM BSE section of a trabecular junction of Amphiaspis sp. GIT 313-32 (f); SrXTM tomographic slice through a vertical trabecular of Anglaspis macculloughi NHMUK P73620 (g); SrXTM horizontal virtual thin section through a polygonal cancellar chamber of Pteraspis sp. NRM-PAL C.5945 (h); isosurface model of the same specimen showing the circular fabric of fibres enveloping the cancellae (i); vertical trabecular wall of Poraspis sp. NHMUK P17957 (j); junction of trabecular walls of Loricopteraspis dairydinglensis NHMUK P73623 (k). sup, superficial layer; L1, layer 1; L2, layer 2; L3, layer 3; ca, cancellae; o.a.s., orthogonal aspidin space; t.a.s., tangled aspidin space; c.f.b., crystal fibre bundles; cor., homogenous core of bipartite aspidin trabeculae. Relative scale bar equals 30 μm in (a), 48 μm in (b), 79 μm in (c), 30 μm in (d), 88 μm in (e), 180 μm in (f), 41 μm in (g), 100 μm in (h), 91 μm in (i), 40 μm in (j) and 52 μm in (k).

Figure 4. SrXTM virtual segmentation of aspidin spaces in Loricopteraspis dairydinglensis (NHMUK P75401) (a-c) and Tesseraspis tesselata (NHMUK P73617) (d, e).

Detail of orthogonal aspidin spaces pervading the trabecular walls dividing polygonal cancellae. These spaces exhibit linear morphology without ramifications, precluding interpretation that these are cell or cell process spaces (a); lateral (b), and transverse (c) views of the organisation of aspidin spaces radiating about a polygonal cancellar vacuity; Horizontal section of the middle layer of Tesseraspis tesselata showing orthogonal aspidin spaces within the trabeculae and tangled aspidin spaces at the intersection between trabeculae. Several orthogonal and tangled spaces have been segmented individually (highlighted in gold) to illustrate their linear, non-branching morphology (d), Detail of previous panel (e). Relative scale bar equals 55 μm in (a), 189 μm in (b) 160 μm in (c), 92 μm in (d) and 50 μm in (e).