Semi thin sections through the mantle edge of <i>Neotrigonia</i> and details of the periostracal groove.

<p>A, C. <i>Neotrigonia gemma</i>. B, D. <i>Neotrigonia margaritacea</i>. Cr, forming crystals; DL, dark layer; IMF, inner mantle fold; MMF, middle mantle fold; OMF, outer mantle fold; Pa, pallial tentacle; PG, periostracal groove; Po, periostracum; TL, translucent layer.</p

Living specimen of <i>Neotrigonia lamarckii</i>, with the extended foot.

<p>Living specimen of <i>Neotrigonia lamarckii</i>, with the extended foot.</p

Shell microstructures of <i>Neotrigonia</i>.

<p>A. Vertical fracture through the prismatic and nacre layers of <i>N. lamarckii</i>. B. Fracture through the shell of <i>N. lamarckii</i>, showing the different shell layers. C. Detail of the upper part of the prismatic layer of <i>N. lamarckii</i>. The individual prisms are separated by thick periostracal membranes. D, E. Views of the external shell surface of <i>N. lamarckii</i>. The periostracal surface is ornated with nanometric pimples, which continue on top of the bosses. The bosses appear in high relief due to retraction of the periostracum upon dehydration. F. View of the free periostracum of <i>N. gemma</i> showing the regular distribution of bosses. G. Detail of the free periostracum of <i>N. gemma</i>. Etching of bosses reveals that they are composed of crystalline sectors. H. Surface of the periostracum of <i>N. margaritacea</i>, with cropping out bosses. Its position in J is indicated. I. Detail of the free periostracum of <i>N. gemma</i>. Intensive etching reveals lineations within bosses. J. Surface of the periostracum of <i>N. margaritacea</i>. Note the existence of bosses within the periostracal flap. K. Surface of the shell of <i>N. lamarckii</i>. The outlines of the prisms underlying the periostracum are clearly noticeable. Note the strict correspondence between each prism and its associated boss. L. Similar situation close to a rib of <i>N. lamarckii</i>. There appear small-sized additional prisms, which do not bear a boss. M. Fracture through the shell of <i>N. margaritacea</i>. The fractured prisms show a typical fibrous aspect. N. Fracture through the columnar nacre of <i>N. margaritacea</i>, close to the external surface. O. Fracture through the sheet nacre of <i>N. margaritacea</i>, at the mid shell thickness. B, boss; GD, growth direction; N, nacre; Pr, prismatic layer; Po, periostracum.</p

Schematic diagram depicting the initiation and early growth of bosses within the periostracal groove.

<p>Bosses inititate at subcellular positions which are equally spaced comarginally along the outer mantle fold. Growth proceeds as the periostracum slides between the mantle folds by secretions of the outer mantle fold. Below each boss there is a reticulum formed by extensions of the underlying microvilli, which also migrates across the cells in coincidence with the boss. The time of initiation alternates between rows, with the formation of bosses labelled 1 and 2 in the left diagram and of boss 3 in the right diagram. B, boss; Emv, reticulate extensions of the microvilli; MMF, middle mantle fold; Mv, microvilli; OMF, outer mantle fold; Po, periostracum.</p

Crystal evolution along the periostracum and shell margin of <i>Neotrigonia gemma</i>.

<p>A. Semi-thin section of the decalcified periostracum and shell margin. B. Enlarged view of the initial part, to show the relative thicknesses of the dark and translucent layers. The translucent layer has detached from the dark layer at the initialmost portion. Arrows point to positions where crystals have been dissolved. C–F. TEM views of forming crystals at progressively older positions. Along the age series the crystals increase in height. The growth surface stays always at the boundary between the dark and translucent layers. Note relative thickness of the translucent layer in C and remains of translucent layer between crystals in F. Cr, decalcified crystals; DI, dark inclusions; DL, dark layer; TL, translucent layer.</p

Crystallization within the periostracal groove of <i>Neotrigonia margaritacea</i>.

<p>A. General view of the periostracal groove area, showing the forming periostracum and incipient aragonite crystals. B. Forming crystals at about half a whorl after the initiation of the periostracal groove. Its position in A is indicated. C, D. Forming crystals at about three quarters of a whorl. The periostracal cover is much thinner than the surrounding periostracum. A thin translucent layer is visible below the dark layer. E. Detail of the reticulate extensions of the microvilli developed below the forming crystal shown in D (indicated). F. Crystal at two and a quarter whorls along the periostracal groove. There are thick dark and translucent layers. BC, basal cell; Cr, crystal; DL, dark layer; Ep, Extrapallial space; Gr, electron dense granules; Emv, reticulate extensions of the microvilli; Mv, microvilli; Po, periostracum; TL, translucent layer.</p

Semi-thin section of the periostracal groove area of <i>Neotrigonia margaritacea</i> stained with different toluidine Blue (TB) solutions.

<p>A. Greenish metachromasia of the epithelial glands of the outer mantle fold after staining with with 1% aqueous TB and sodium tetraborate (1%). B. Brown-greenish metachromasia with 1% aqueous TB and acetic acid (0.2%). C. Brown-greenish metachromasia with 0.5% aqueous TB and acetic acid (0.2%). D. Brown-greenish metachromasia obtained with 0.2% aqueous TB and acetic acid (0.2%). MMF, middle mantle fold; P, periostracum; PG, periostracal groove; OMF, outer mantle fold.</p

SEM views of the mantle margin of <i>Neotrigonia margaritacea</i> and its associated tentacles.

<p>A. Mantle around the exhalant siphon. B. Mantle around the inhalant siphon. C, D. Ventral mantle margin. The periostracum can be seen emerging behind the middle mantle fold in C, whereas it is absent in D, thus leaving the internal surface of the outer mantle fold and the periostracal groove exposed. IMF, inner mantle fold; MMF, middle mantle fold; OMF, outer mantle fold; PG, periostracal groove; Po, periostracum; Tl, tentacle.</p

SEM views of the periostracum and shell margin of <i>Neotrigonia gemma</i>.

<p>A. General view of the shell margin. The periostracum has adhered to the internal shell surface. B. Initial part of the periostracum. In this portion, the bosses increase in diameter in the growth direction. C. Detail of the initial bosses, some of which clearly retain the initial periostracal cover (inset). D. Aspect of the forming crystals half way to the shell margin showing that the initial bosses had grown into truncated conical crystals. E. Aspect of the crystals close to the shell margin. The position in F is indicated. F. General aspect of the folded shell margin. The position in H is indicated. Along the fold the conical crystals shown in E transform into short prisms separated by periostracal membranes. G. Detail of the interior surface of the fold. The bases of the crystals have expanded to a maximum and are about to impinge with each other. H. A general view the shell margin, which includes the completion of the prismatic layer and the transition to nacre. I. Detail of the transition prisms-nacre. The prisms are separated by gaps, which are clogged with crystalline material upon the onset of nacre secretion. The position in H is indicated. GD, growth direction; N, nacre; Po, periostracum.</p

Crystallography of the prismatic units forming the outer layer of <i>Neotrigonia</i>.

<p>A, B. Image quality and phase map, respectively, of a section through the outer prismatic layer of <i>N. margaritacea</i>. The blue color in E indicates that all the mapped material is aragonite, including part of the boss of the left prism. C, D, E. Surface views of bosses of <i>N. gemma</i>. The orientations of the lineations revealed upon natural etching have been indicated with arrows in all cases; the peripheral {110} faces, as well as the approximate boundaries between crystals (broken lines) have been indicated in D and E. F, G. Image quality and cleaned up orientation maps, respectively, of another section through the outer prismatic layer and nacre of <i>N. margaritacea</i>. H. 001 and 100 pole figures of the framed area in G, and color key (bottom) for the orientation map of G. The 001 pole distribution implies that the <i>c</i>-axes of the fibres are mostly parallel to the axes of the prisms, while the 100 pole distribution shows no preferential alignment either within or between prisms. I. Secondary electron image of a polished section through the outer prismatic layer of <i>N. margaritacea</i>. The diffraction contrast reveals the difference in texture between the upper part of the prisms (that enveloped by organic periostracal membranes) and the rest of the prisms. The approximate boundary has been delineated in the left prism. B, boss; Po, periostracum; Pr, prism.</p