Wilkie IC .

	Fig 1. A specimen of O. nigra photographed in situ in Loch Leven, Scotland.(Photograph: James Lynott)
	Fig 2. Experiment on individual spine joints.(A) Diagrammatic side-view of the apparatus (not to scale). ch, chain; cl, clamp; ho, hook; il, isotonic lever; lo, load; pl, Plasticineâ„¢; pp, plastic platform; sp, arm spine. (B) Representative recording of one test: after application of the load (arrow), the preparation was left undisturbed for 3.5 min then stimulated with seawater containing 100 mmol l-1 K+ ions (K), which was followed by an increase in the extension rate and rupture of the spine joint (asterisk). Horizontal scalebar: 1 min; vertical scalebar: 1 mm.
	Fig 3. Main anatomical components of the arm spine joint.Diagrammatic representation of part of a transverse section of the arm of O. nigra showing one arm spine joint. This is not to scale and is a simplified and idealised depiction of anatomical relationships, since in a real animal not all the components would be included in the same plane of section. ep, epidermis; ict, interarticular connective tissue; im, intervertebral muscle; jln, juxtaligamental node; jlp, juxtaligamental cell processes; ln, lateral arm plate nerve; lp, lateral arm plate; lpl, ligament connecting adjacent lateral arm plates; m, spine muscle; s, spine; sl, spine ligament; sn, spine nerve; t, tubercle; te, tendons.
	Fig 4. Main anatomical components of the arm spine joint.Transverse histological section showing actual anatomical relationships. ab, aboral (dorsal) side; ep, epidermis; im, intervertebral muscle; jln1, juxtaligamental node associated with the ligament of the featured joint; jln2, juxtaligamental node associated with the ligament of the adjacent joint; jlp, juxtaligamental cell processes; lp, lateral arm plate; lpl, ligament connecting adjacent lateral arm plates; m, spine muscle; or, oral (ventral) side; s, spine; sl1, ligament of featured joint; sl2, ligament of adjacent joint; sn, spine nerve (sectioned obliquely); t, tubercle; te, tendon. Scalebar: 0.1 mm.
	Fig 5. Scanning electron micrographs of the skeletal components of the arm spine joint.(A) Lateral view of three segments of an incompletely digested arm. The asterisk marks the joint shown at higher magnification in B. Scalebar: 0.5 mm. (B) More magnified view of the disarticulated joint marked by an asterisk in A. Scalebar: 0.1 mm. (C) Isolated lateral arm plate. View of proximal side showing tubercles. Scalebar: 0.25 mm. (D) Lateral view of a tubercle. The line below the oral lobe shows the approximate position of the gap in the insertion area of the ligament associated with the illustrated tubercle. Scalebar: 0.1 mm. (E) Distal view of a tubercle. Scalebar: 50 μm. (F) Adradial end of a detached spine: oblique view of aboral and articular surfaces. Scalebar: 0.1 mm. (G) Articular surface of a spine. Scalebar: 50 μm. (H) Adradial end of a detached spine: oblique view of oro-proximal and articular surfaces. The asterisks mark the proximal (left) and distal (right) imperforate regions of the proximal condyle. Scalebar: 0.1 mm. a, abrasions; ab, aboral (dorsal) side; al, aboral lobe of tubercle; c, opening of nerve canal; dc, distal condyle; di, distal (nearer arm-tip) end; f, muscle fossa; fs, fascicular stereom; gs, galleried stereom; la, ligament insertion area; lp, lateral arm plate; ol, oral lobe of tubercle; op, oral arm plate; or, oral (ventral) side; pc, proximal condyle; pr, proximal (nearer central disc) end; rc, reniform concavity; s, spine; t, tubercle; thin arrow, muscle insertion area; thick arrow, position of gap in ligament insertion area.
	Fig 6. Microstructure of soft tissue components of the joint.Light micrographs. (A, B) Circumarticular epidermis. Transverse sections stained with Bargmann’s chrome alum haematoxylin and phloxine (CAHP: connective tissue grey) showing epidermal constrictions (arrows). Scalebars: 40 μm (A) and 20 μm (B). (C, D) Spine joint, the spine being in the fully erect position. Two sections from the same series, stained with Milligan’s trichrome (MT: connective tissue blue). The plane of these sections is vertical and parallel to the longitudinal axis of the spine (which projects to the left); their location is shown by the dashed lines in E. The thin arrows indicate fibres connecting the interarticular connective tissue to the skeleton, and the thick arrow indicates the area of direct contact between the proximal condyle and oral lobe of the tubercle. Note that the spine ligament is not visible at the oral side of the joint in C, because the section plane passes through the ligament gap. Scalebars: 40 μm. (E-H) Spine joint. Transverse sections through the tubercle in plane perpendicular to the longitudinal axis of the erect spine. (E) Stained with MT. Plane of section shown in D. Note the gap in the oral side of the ligament (between c’ and d’). Scalebar: 40 μm. (F-H) Stained with CAHP. Three sections from the same series, the plane of section being progressively abradial (towards the spine and away from the lateral arm plate). (F) Plane of section is below the tubercle lobes. The muscle (thin arrow) occupies a wide fossa. The spine nerve (asterisk) has bifurcated into larger and smaller branches, which are still contiguous. The ligament contains sparse cell bodies, as revealed by their nuclei. Scalebar: 40 μm. (G) Plane of section is below the spine base, but the tubercle lobes are visible (al, ol). The arrow indicates the spine muscle. Scalebar: 40 μm. (H) Plane of section is at about same level as in E (see ee’ in D) and includes both tubercle and spine base. The arrow indicates the abradial edge of the spine muscle insertion area. Scalebar: 40 μm. Asterisk, spine nerve; ab, aboral side; al, aboral lobe of tubercle; bm, basement membrane; ca, cavity; cc’ and dd’, planes of sections shown in C and D respectively; cu, cuticle; dc, distal condyle of spine base; ee’, plane of section shown in E; ep, epidermis; ict, interarticular connective tissue; n, nucleus; ol, oral lobe of tubercle; pc, proximal condyle of spine base; pr, proximal side; s, spine; sb, spine base; sl, spine ligament; t, tubercle.
	Fig 7. Ultrastructure of the circumarticular epidermis.Transmission electron micrographs. (A) Epidermal constriction (between arrows). Scalebar: 2 μm. (B) Extreme epidermal constriction (between arrows). Scalebar: 2 μm. (C) More magnified view of epidermal constriction shown in B. The arrows indicate large hemidesmosome-like junctions between the lamina densa and the support cells. The asterisks mark small hemidesmosome-like junctions. Scalebar: 1 μm. (D) Large hemidesmosome-like junction (indicated by lower arrow in C). Scalebar: 0.2 μm. (E) Edge of lamina densa next to cavity between it and spine ligament, showing delaminated appearance. Scalebar: 0.2 μm. (F) Edge of spine ligament next to cavity. Scalebar: 0.5 μm. ca, cavity between epidermis and spine ligament; cc, possible coelomocyte; co, collagen fibrils; cu, cuticle; ld, lamina densa; ll, lamina lucida; mf, microfibrils; ph, possible phagocyte; se, secretory cell; su, support cell; tf, tonofibrils.
	Fig 8. Ultrastructure of the spine ligament and interarticular connective tissue.Transmission electron micrographs. (A-G) Spine ligament. (A) Low magnification view showing the main components. Scalebar: 2 μm. (B) Edge of a fibril bundle showing collagen fibrils and microfibrils. Scalebar: 0.2 μm. (C) Cell containing secondary lysosome-like vesicle (arrow). Scalebar: 1 μm. (D) Cell that appears to have phagocytosed collagen fibrils (arrows). Scalebar: 1 μm. (E) Cluster of cell processes. 1, type 1 DCV-containing process; 2, type 2 DCV-containing process; 3, process containing only electron-lucent vesicles. Scalebar: 1 μm. (F) Cluster of cell processes. Arrows indicate microtubules. Scalebar: 1 μm. (G) Cluster of cell processes, including an omega-profile suggestive of exocytosis (arrow and inset). Scalebars: 1 μm (main panel) and 0.2 μm (inset). (H) Interarticular connective tissue, which consists of loosely arranged collagen fibrils and microfibrillar meshworks. Scalebar: 0.2 μm. co, collagen fibrils; hvc, cell containing heterogeneous vesicles; mf, microfibrils; pr, cell process containing dense-core vesicles.
	Fig 9. Microstructure of the juxtaligamental node and muscle.Light micrographs. (A-F) Transverse sections. (A) Overview of the anatomical relationships between the juxtaligamental node, spine ligament and spine nerve. Stained with Milligan’s trichrome (MT). Scalebar: 40 μm. (B) Juxtaligamental node shown in A. 1, 2 and 3 are the cell types described in the main text. The arrow indicates cell processes extending between the spine nerve and the node. The asterisk marks the perineurium of the spine nerve. Stained with MT. Scalebar: 20 μm. (C) Juxtaligamental node, showing continuity of a branch of the spine nerve (arrow) with the neuropil-like region. Stained with MT. Scalebar: 20 μm. (D) Juxtaligamental node, showing presence of cell type 1 processes (arrow) in the neuropil-like region and in the cluster extending to the spine ligament. Stained with Bargmann’s chrome haematoxylin and phloxine (CAHP). Scalebar: 20 μm. (E) Remainder of the same juxtaligamental node shown in D. Stained with CAHP. Scalebar: 20 μm. (F) Spine muscle: insertion into spine base at top left and insertions into lateral arm plate at bottom right. Stained with CAHP. Scalebar: 40 μm. (G-J) Horizontal sections showing the anatomical relationships between the spine muscle (arrows), lateral arm plate nerve and juxtaligamental node. The plane of the sections and orientation of the micrographs are the same as in Fig 6F–6H. Also as in Fig 6F–6H, the plane of section is progressively abradial (towards spine base). Stained with CAHP. (G) The muscle consists of two widely separated myocyte bundles (arrows). Scalebar: 40 μm. (H) The adradial edge of the juxtaligamental node is visible between the two myocyte bundles. Scalebar: 40 μm. (I) The main body of the juxtaligamental node is closely adjacent to both myocyte bundles and the spine nerve has branched from the lateral arm plate nerve. Scalebar: 40 μm. (J) The two myocyte bundles have merged to form a single muscle (arrow). Scalebar: 40 μm. ab, aboral side; cp, capsular layer; jln, juxtaligamental node; jlp, juxtaligamental cell processes; ln, lateral arm plate nerve; m, muscle; np, neuropil-like region; pr, proximal side; sl, spine ligament; sn, spine nerve; te, tendon connecting muscle to spine base.
	Fig 10. Ultrastructure of the juxtaligamental node.Transmission electron micrographs. (A) Type 1 cell body. Scalebar: 2 μm. (B) Type 2 cell body. Scalebar: 2 μm. (C) Type 3 (agranular) cell body. Scalebar: 2 μm. (D) Edge of a node, showing the capsular layer and extensions of capsular cells (arrows) separating adjacent DCV-containing cells (1, 2). The asterisk marks a capsular cell vacuole containing possible cell debris. Scalebar: 2 μm. (E) Capsular cell extension (arrow) between type 1 and 2 DCV-containing cells (1, 2). The asterisk marks a Golgi complex. Scalebar: 0.5 μm. (F) Cell undergoing degradation in the capsular layer. Scalebar: 2 μm. (G) Capsular cell with large vacuoles and basement membrane. Scalebar: 1 μm. (H) Same capsular cell as shown in G, with cilium, lysosome-like vesicles and Golgi complex (asterisk). Scalebar: 2 μm. (I) Cilium of cell shown in H. Scalebar: 0.5 μm. (J) Neuropil-like region of a juxtaligamental node, which includes profiles containing small spherical vesicles (arrows). Scalebar: 1 μm. (K) Neuropil-like region, which is separated from the spine muscle by a thin cellular partition (arrow). Scalebar: 1 μm. (L, M) Cell processes in the neuropil-like region, which contain two types of small vesicles and are adjacent to DCV-containing cell processes. That in M includes a synapse-like junction (arrow). Scalebars: 0.5 μm. bm, basement membrane; ci, cilium; cp, capsular layer; ly, lysosome-like vesicle; m, spine muscle; va, vacuole.
	Fig 11. Microstructure of the spine muscle tendons.Light micrographs of transverse sections. (A) Tendon connecting the muscle to the proximal condyle of the spine base. Stained with CAHP. Scalebar: 40 μm. (B) Tendons (arrow) connecting the muscle to stereom bars (asterisks) of the lateral arm plate. Stained with MT. Scalebar: 20 μm. al, aboral lobe; ep, epidermis; jln, juxtaligamental node; jlp, juxtaligamental cell processes; ln, lateral arm plate nerve; m, muscle; ol, oral lobe; pc, proximal condyle; sl, spine ligament; t, tubercle; te, tendon.
	Fig 12. Ultrastructure of the spine muscle and tendons.Transmission electron micrographs. (A) Longitudinal section of two myocytes, showing oblique striations. Scalebar: 1 μm. (B) Longitudinal section of a myocyte. Arrows indicate Z-bodies. Scalebar: 0.5 μm. (C) Desmosome-like junctions (arrow) between two myocytes. Scalebar: 0.2 μm. (D) Myocyte extensions (thin arrow) are in contact with a cell process containing small vesicles (thick arrow). Scalebar: 2 μm. (E, F) Tendon connecting the muscle to the spine base: region adjacent to the muscle. (E) The tendon has a dense delimiting layer (arrow), which is connected to the myocytes at hemidesmosome-like junctions. Scalebar: 0.5 μm. (F) The spine base tendon includes prominent fibre-like aggregations of microfibrils. Scalebar: 0.5 μm. (G, H) Tendon connecting the muscle to the lateral arm plate: tendon loop adjacent to a stereom bar (see arrow in Fig 11B). (G) The tendon is closely adpressed against the extracellular “matrix coat” (arrow) that envelopes the calcite stereom (see [46]). Scalebar: 0.2 μm. (H) Point at which the tendon diverges from the stereom bar. Arrow indicates the “matrix coat”. Scalebar: 0.5 μm. hd, hemidesmosome-like junction; st, calcite stereom (decalcified); te, tendon.
	Fig 13. Arm self-wiping behaviour of O. nigra.Drawings (not to scale) illustrating the response of arms to particles settling on their aboral surface. (A-F) A complete sequence of bolus formation and transfer to the mouth. (G) The dragging action of the arm-loop inclines spines distally and aboro-distally. b, bolus; es, fully erect spines; is, inclined spines.
	Fig 14. Results of the spine tagging experiment.Each dotted horizontal line represents one animal and the symbols indicate the day on which tagged spines were detached.
	Fig 15. Diagrams illustrating aboral inclination of a spine.(A) Spine fully erect. Accurate representation of the articular surfaces based on a transverse histological section. No ligament is present at the oral side of the joint, because the plane of the section passes through the gap in oral region of the ligament. The arrow indicates the apposition where interarticular connective tissue is lacking and the spine base and tubercle are in direct contact. (B, C) Two stages in the aboral inclination of the spine, assuming minimal separation between the articular surfaces. The aboral portion of the spine ligament is compressed; the articular surfaces do not maintain a close fit; and the muscle is stretched. al, aboral lobe of tubercle; ict, interarticular connective tissue; m, muscle; ol, oral lobe of tubercle; pc, proximal condyle of spine base; sl, aboral portion of spine ligament; t, tubercle.

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