The morphology and ultrastructure of the shells of two balanid species have been examined, paying special attention to the three types of boundaries between plates: (i) radii-parietes, (ii) alae-sheaths, and (iii) parietes-basal plate. At the carinal surfaces of the radii and at the rostral surfaces of the alae, there are series of crenulations with dendritic edges. The crenulations of the radius margins interlock with less prominent features of the opposing paries margins, whereas the surfaces of the longitudinal abutments opposing the ala margins are particularly smooth. The primary septa of the parietes also develop dendritic edges, which abut the internal surfaces of the primary tubes of the base plates. In all cases, there are chitino-proteinaceous organic membranes between the abutting structures. Our observations indicate that the very edges of the crenulations and the primary septa are permanently in contact with the organic membranes. We conclude that, when a new growth increment is going to be produced, the edges of both the crenulations and the primary septa pull the viscoelastic organic membranes locally, with the consequent formation of viscous fingers. For the abutting edges to grow, calcium carbonate must diffuse across the organic membranes, but it is not clear how growth of the organic membranes themselves is accomplished, in the absence of any cellular tissue., This research was funded by projects CGL2017-85118-P (A.G.C., C.S. and C.G.) and CGL2015-64683-P (A.B.R.N.) of the Spanish Ministerio de Economía, Industria y Competitividad, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada (A.G.C. and A.B.R.N.) and the Research Group RNM363 of the Junta de Andalucía (A.G.C.). N.A.L. acknowledges support from CONICYT-Chile through grant nos. FONDECYT 1140938, PCI REDES 170106 and PIA ANILLOS ACT172037, for international collaborative research with A.G.C. and A.B.R.N.

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules., A.G.C., A.G.-S. and F.V.-F. acknowledge funding provided by projects CGL2013-48247-P and CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovación. A.G.C. also acknowledges the Research Group RNM363 (Junta de Andalucía) and the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada for financial support.

An organic-rich columnar prismatic outer shell layer, which extends far beyond the underlying nacre, has allowed pterioid bivalves (the pearl oysters and their allies) to develop flexible valve margins, allowing a tight hermetic seal when shut. In some taxa, the microstructural arrangement is known to be asymmetrically developed between the two valves. The asymmetry was surveyed across 29 taxa of pterioids (including representatives of known genera) confirming that it is typically the right valve which has a greater expanse of prism-only shell (and less nacre) and showing that this portion of the right valve has more organic content (more than twice the value in some instances) than the equivalent in the left. A more detailed investigation of prismatic material in Pteria penguin comparing the right and left valves revealed that the right valve flange has a higher density of smaller prisms, each with its organic envelope, and not a greater thickness of the organic envelopes themselves. The flange is also thinner on the right valve and shown here to be very flexible when wet. This allows it to bend against the rigid left valve when the shell is closed. Comparison of this structural asymmetry in the pterioids with five outgroup taxa in the Ostreidae and Pinnidae suggests that clades with the asymmetry have been freed from the constraints of a flattened valve morphology and to develop inequivalved forms., Funding was provided by projects CGL2013-48247-P and CGL 2017-85118-P of the Spanish Ministerio de Ciencia e Innovación. A.G.C. also acknowledges the Research Group RNM363 (Junta de Andalucía) and the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada for financial support. We are grateful to Philippe Maestrati for access to the MNHN collections, Mercedes Checa for assistance with measurements and the constructive comments of two reviewers., Peer reviewed

In biomineralization, it is essential to know the microstructural and crystallographic organization of natural hard tissues. This knowledge is virtually absent in the case of barnacles. Here, we have examined the crystal morphology and orientation of the wall plates of the giant barnacle Austromegabalanus psittacus by means of optical and electron microscopy, and electron backscatter diffraction. The wall plates are made of calcite grains, which change in morphology from irregular to rhombohedral, except for the radii and alae, where fibrous calcite is produced. Both the grains and fibres arrange into bundles made of crystallographically co-oriented units, which grow onto each other epitaxially. We call these areas crystallographically coherent regions (CCRs). Each CCR elongates and disposes its c-axis perpendicularly or at a high angle to the growth surfaces, whereas the a-axes of adjacent CCRs differ in orientation. In the absence of obvious organic matrices, this pattern of organization is interpreted to be produced by purely crystallographic processes. In particular, due to crystal competition, CCRs orient their fastest growth axes perpendicular to the growth surface. Since each CCR is an aggregate of grains, the fastest growth axis is that along which crystals stack up more rapidly, that is, the crystallographic c-axis in granular calcite. In summary, the material forming the wall plates of the studied barnacles is under very little biological control and the main role of the mantle cells is to provide the construction materials to the growth front., This research was funded by projects CGL2017-85118-P (A.G.C., A.G.-S.) and CGL2015-64683-P (A.B.R.-N.) of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada (A.G.C., A.B.R.-N.) and the Research Group RNM363 of the Junta de Andalucía (A.G.C.). N.A.L., A.G.C. and A.B.R.-N. acknowledge support from CONICYT-Chile through grant nos. FONDECYT 1140938, PCI REDES 170106 and PIA ANILLOS ACT172037, for international collaborative research., Peer reviewed

Oyster shells are mainly composed of layers of foliated microstructure and lenses of chalk, a highly porous, apparently poorly organized and mechanically weak material. We performed a structural and crystallographic study of both materials, paying attention to the transitions between them. The morphology and crystallography of the laths comprising both microstructures are similar. The main differences were, in general, crystallographic orientation and texture. Whereas the foliated microstructure has a moderate sheet texture, with a defined 001 maximum, the chalk has a much weaker sheet texture, with a defined 011 maximum. This is striking because of the much more disorganized aspect of the chalk. We hypothesize that part of the unanticipated order is inherited from the foliated microstructure by means of, possibly, 011 8 twinning. Growth line distribution suggests that during chalk formation, the mantle separates from the previous shell several times faster than for the foliated material. A shortage of structural material causes the chalk to become highly porous and allows crystals to reorient at a high angle to the mantle surface, with which they continue to keep contact. In conclusion, both materials are structurally similar and the differences in orientation and aspect simply result from differences in growth conditions., Special thanks are given to Isabel Sánchez-Almazo (Univ. Granada) for her help with scanning microscopy and to Antonio Sánchez-Navas (Univ. Granada) for advice on the crystallography of calcite. Funding was provided by projects CGL2013-48247-P and CGL2017-85118-P (to A.G.C., E.M.H. and A.G.-S.) of the Spanish Ministerio de Economía, Industria y Competitividad. A.G.C. also acknowledges the Research Group RNM363 (Junta de Andalucía).

Molluscs are grand masters in the fabrication of shells, because these are composed of the largest variety of microstructures found among invertebrates. Molluscan microstructures are highly ordered aggregates of either calcite or aragonite crystals with varied morphologies and three-dimensional arrangements. Classically, every aspect of the fabrication of microstructural aggregates is attributed to the action of proteins. There was, however, only direct evidence that the mineral phase, and indirect evidence that nucleation and the crystal shape, are determined by the types of soluble proteins. Some authors imply that crystal competition may also play a role. In addition, the fabrication of intergranular organic matrices typical of some microstructures (nacre, columnar prismatic) cannot have a protein-based explanation. Over the last decade I and collaborators have been applying a holistic view, based on analyzing and interpreting the features of both the organic (mantle, extrapallial space, periostracum, organic matrices) and inorganic (crystallite morphology, arrangement, and crystallography) components of the biomineralization system. By interpreting them on biophysical principles, we have accumulated evidence that, in addition to the activity of proteins, other mechanisms contribute in an essential way to the organization of molluscan microstructures. In particular, we have identified processes such as: (1) crystal nucleation on preformed membranes, (2) nucleation and growth of crystals between and within self-organized membranes, (3) active subcellular processes of contact recognition and deposition. In summary, besides the activity of organic macromolecules, physical (crystal competition, self-organization) and/or biological (direct cellular activity) processes may operate in the fabrication of microstructures. The balance between the physical and biological determinants varies among microstructures, with some being based exclusively on either physical or biological processes, and others having a mixed nature. Other calcifying invertebrates (e.g., corals, cirripeds, serpulids) secrete microstructures that are very similar to inorganic crystal aggregates, and only some brachiopods and, to a lesser extent, bryozoans may have secretory abilities comparable to those of molluscs. Here I provide a new perspective, which may allow microstructures to be understood in terms of evolutionary constraints, to compare the secretional abilities among taxa, and even to evaluate the probability of mimicking microstructures for the production of functional synthetic materials., AC received funding from Project CGL2013-48247-P and CGL2017-85118-P of the Spanish Ministerio de Economía y Competitividad (MINECO) and the Fondo Europeo de Desarrollo Regional (FEDER), from the Research Group RNM363 (Consejería de Economía, Innovación, Ciencia y Empleo of the Junta de Andalucía) and from the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada.

Many biological structures use liquid crystals as self-organizing templates for their formation. We review and analyse evidence that the crossed-lamellar biomineral microstructure of mollusc shells may be formed from such a liquid-crystal precursor., We acknowledge funding from projects CGL2017-85118-P and FIS2016-77692-C2-2-P of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada and the Research Group RNM363 of the Junta de Andalucía.

Nacre, or mother of pearl, is a biomaterial with a layered structure. In a recent geological study, researchers found that the width of the nacre layers depends on the formation temperature, which is determined by the ocean water temperature. A linear dependence of layer width with respect to temperature is understandable within the transient liquid-crystalline nature of incipient nacre. Thus, developing nacre is a liquid-crystal thermometer recording its formation temperature. A more complete understanding of nacre formation is of interest not only for biology and geology, in terms of biomineralization and paleoclimatology, but also for materials science: for reproducing nacre or fabricating synthetic analogues and also potentially for developing new classes of layered materials with layer spacings tunable by pH and temperature., .H.E.C. and C.I.S.D. acknowledge the financial support of the Spanish MINCINN FIS2016-77692-C2-2-P, and A.G.C. acknowledges CGL2017-85118-P. J.H.E.C. would like to acknowledge the contribution of the COST Action Eutopia, CA17139.

The shells of the bivalves Glycymeris glycymeris and Glycymeris nummaria are widely used for environmental studies. They consist of aragonite and comprise four different microstructures and textures from outer to inner shell surfaces: crossed-lamellar, myostracal, complex crossed-lamellar and fibrous prismatic. We characterize with SEM, EBSD, laser-confocal microscopy and AFM imaging mineral unit size, morphology and orientation of crystallites in the different microstructural arrangements and at the transition from one microstructure to the other. We also characterize the microstructure and texture of adductor and pedal retractor myostraca and address structural characteristics at the transition from crossed-lamellar to myostracal assemblies. We find that the crossed-lamellar layer has a three-dimensional crystallographic orientational order. Each set of first-order lamellae consists of twinned aragonite; the two sets of first-order lamellae are misoriented to each other by about 30 to 40° while retaining an approximately parallel a-axis; they do not show any particular twin relationship. Myostracal aragonite grows homoepitactically onto the crossed-lamellar aragonite, but is clearly a separate microstructure, with its own crystallite size and morphology. Within adductor and pedal myostraca, prisms increase in size towards inner surfaces. In contrast to the other shell layers, the myostraca form through competitive growth. The complex crossed-lamellar aragonite initially inherits the three-dimensional texture of the crossed-lamellar microstructure, but with growth develops an axial texture, which is transmitted to the underlying fibrous prismatic microstructure. With this work we provide a modern, unaltered, reference for fossil Glycymeris shells to be used for detection of diagenetic overprint in fossil Glycymeris analogs., GC acknowledge financial support of the 2017 Italian Ministry PRIN RX9XXYProject “Biota resilience to global change: biomineralization of planktic and benthic calcifiers in the past, present and future” to E. Erba. AGC is funded by project CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada, and the Research Group RNM363 of the Junta de Andalucía (AGC).

Stingless bees of the genus Tetragonula construct a brood comb with a spiral or a target pattern architecture in three dimensions. Crystals possess these same patterns on the molecular scale. Here, we show that the same excitable-medium dynamics governs both crystal nucleation and growth and comb construction in Tetragonula, so that a minimal coupled-map lattice model based on crystal growth explains how these bees produce the structures seen in their bee combs., J.H.E.C. and C.I.S.D. acknowledge the financial support of theSpanish MINCINN FIS2016-77692-C2-2-P and A.G.C., CGL2017-85118-P.

The calcite grains forming the wall plates of the giant barnacle Austramegabalanus psittacus have a distinctive surface roughness made of variously sized crystalline nanoprotrusions covered by extremely thin amorphous pellicles. This biphase (crystalline-amorphous) structure also penetrates through the crystal’s interiors, forming a web-like structure. Nanoprotrusions very frequently elongate following directions related to the crystallographic structure of calcite, in particular, the <− 441> directions, which are the strongest periodic bond chains (PBCs) in calcite. We propose that the formation of elongated nanoprotrusions happens during the crystallization of calcite from a precursor amorphous calcium carbonate (ACC). This is because biomolecules integrated within the ACC are expelled from such PBCs due to the force of crystallization, with the consequent formation of uninterrupted crystalline nanorods. Expelled biomolecules accumulate in adjacent regions, thereby stabilizing small pellicle-like volumes of ACC. With growth, such pellicles become occluded within the crystal. In summary, the surface roughness of the biomineral surface reflects the complex shape of the crystallization front, and the biphase structure provides evidence for crystallization from an amorphous precursor. The surface roughness is generally explained as resulting from the attachment of ACC particles to the crystal surface, which later crystallised in concordance with the crystal lattice. If this was the case, the nanoprotrusions do not reflect the size and shape of any precursor particle. Accordingly, the particle attachment model for biomineral formation should seek new evidence., This research was funded by projects CGL2017-85118-P (AGC) and CGL2015-64683-P (ABRN) of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada (AGC, ABRN) and the Research Group RNM363 of the Junta de Andalucía (AGC). NAL, AGC, and ABRN acknowledge support from ANID-Chile through grants FONDECYT 1140938, PCI ANID REDES 170106, and PIA ANID ANILLOS ACT172037, for international collaborative research with AGC and ABRN. Images of Fig. 3a to c were obtained by Dr. Adrian Sandu (Thermo Scientific NanoPort Europe), Eindhoven, with the ultra-high-resolution SEM Verios. Prof. Elizabeth M. Harper (Dept. Earth Sciences, University of Cambridge, UK) assisted with the terminology.

Shells of calcifying foraminifera play a major role in marine biogeochemical cycles; fossil shells form important archives for paleoenvironment reconstruction. Despite their importance in many Earth science disciplines, there is still little consensus on foraminiferal shell mineralization. Geochemical, biochemical, and physiological studies showed that foraminiferal shell formation might take place through various and diverse mineralization mechanisms. In this study, we contribute to benthic foraminiferal shell calcification through deciphering crystallite organization within the shells. We base our conclusions on results gained from electron backscattered diffraction (EBSD) measurements and describe microstructure/texture characteristics within the laminated shell walls of the benthic, symbiontic foraminifera: Ammonia tepida, Amphistegina lobifera, Amphistegina lessonii. We highlight crystallite assembly patterns obtained on differently oriented cuts and discuss crystallite sizes, morphologies, interlinkages, orientations, and co-orientation strengths. We show that: (i) crystals within benthic foraminiferal shells are mesocrystals, (ii) have dendritic-fractal morphologies and (iii) interdigitate strongly. Based on crystal size, we (iv) differentiate between the two layers that comprise the shells and demonstrate that (v) crystals in the septa have different assemblies relative to those in the shell walls. We highlight that (vi) at junctions of different shell elements the axis of crystal orientation jumps abruptly such that their assembly in EBSD maps has a bimodal distribution. We prove (vii) extensive twin-formation within foraminiferal calcite; we demonstrate (viii) the presence of two twin modes: 60°/[0 0 1] and 77°/~[6 –6 1] and visualize their distributions within the shells. In a broader perspective, we draw conclusions on processes that lead to the observed microstructure/texture patterns., We thank Prof. A. Altenbach, Department of Earth and Environmental Sciences, LMU Munich, Germany and Prof. S. Redfern, Department of Earth Sciences University of Cambridge, UK for providing the foraminifera samples. We thank the staff of Central Facility for Electron Microscopy at Ulm University for their technical support at sample preparation for EBSD. This project was partly funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 643084 and supported by Deutsche Forschungsgemeinschaft, DFG Grant GR-1235/9-1. A.C. and I. S.-A. were funded by Project CGL2017-85118-P, Spanish Ministry of Education and Science.

The reinforcement function of shell ribs depends not only on their vaulted morphology but also on their microstructure. They are part of the outer layer which, in the case of the Pinna nobilis bivalve, is built from almost monocrystalline calcitic prisms, always oriented perpendicular to the growth surfaces. Originally, prisms and their c-axes follow the radii of rib curvature, becoming oblique to the shell thickness direction. Later, prisms bend to reach the nacre layer perpendicularly, but their c-axes retain the initial orientation. Calcite grains form nonrandom boundaries. Most often, three twin disorientations arise, with two of them observed for the first time. Nano-indentation and impact tests demonstrate that the oblique orientation of c-axes significantly improves the hardness and fracture toughness of prisms. Moreover, compression tests reveal that the rib area achieves a unique strength of 700 MPa. The detection of the specific microstructure formed to toughen the shell is novel., The work was supported by the Polish National Agency for Academic Exchange (grant PPI/APM/2018/1/00049/U/001) and the National Science Center (grant UMO-2018/29/B/ST8/02200). MS was supported by the European Union from the resources of the European Social Fund (Project No.WNDPOWR. 03.02.00-00-I043/16). AGC was funded by project CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovación.

The shell of the cephalopod Argonauta consists of two layers of fibers that elongate perpendicular to the shell surfaces. Fibers have a high-Mg calcitic core sheathed by thin organic membranes (>100 nm) and configurate a polygonal network in cross section. Their evolution has been studied by serial sectioning with electron microscopy-associated techniques. During growth, fibers with small cross-sectional areas shrink, whereas those with large sections widen. It is proposed that fibers evolve as an emulsion between the fluid precursors of both the mineral and organic phases. When polygons reach big cross-sectional areas, they become subdivided by new membranes. To explain both the continuation of the pattern and the subdivision process, the living cells from the mineralizing tissue must perform contact recognition of the previously formed pattern and subsequent secretion at sub-micron scale. Accordingly, the fabrication of the argonaut shell proceeds by physical self-organization together with direct cellular activity., We acknowledge TESCAN and Carl Zeiss Microscopy GmbH for the acquisition and early processing of the images by FIB-SEM. This research was funded by projects CGL2017-85118-P (A.G.C., C.G.), PID2020-
116660GB-I00 (A.G.C., C.G., A.B.R.-N.) (Spanish Ministerio de Ciencia e Innovación), and B-RNM-265-UGR18 (A.G.C., C.G., A.B.R.-N.) (Junta de Andalucía), the Unidad Científica de Excelencia UCE-PP2016-05
(A.G.C., C.G., A.B.R.-N.) (University of Granada), and the Research Group RNM363 (A.G.C., C.G.) (Junta de Andalucía).

Among bivalve muscles, the adductors are particularly important for animal survival because they control valve closure. Most studies have addressed the type and morphology of this muscle in bivalves but few have focused on the mechanism that anchors it to the shell myostracum layer. Moreover, the possible calcium transport mechanism through the adductor muscle cells to the myostracum shell layer, which is necessary for bivalve biomineralisation, has never been addressed. Our results indicate that the muscle cell-shell attachment is mediated by the outer mantle epithelial cell layer, here termed tendon cells. These cells are modified at the muscle scar zone by the presence of actin cytoskeletal bundles, which anchor cells to the extracellular matrix via focal adhesion (or focal contact) junctions at the basal side and to extrapallial matrix at the apical side, both rich in collagen. From apical focal adhesions, bundles of collagen-rich fibres cross the extrapallial space and penetrate the myostracum shell layer. The latter constitutes one of the strongest anchoring structures among invertebrates. Numerous vesicles protrude from the tendon cells into the extrapallial space. TEM-EDX analysis reveals the presence of Ca inside some of these vesicles both in tendon cells and in the extrapallial space. This suggests a potential mechanism for calcium transport from cells to the myostracum. Statement of Significance: The interfaces between bivalve shells and muscular attachments are unique and of special interest as adhesive functional biomaterials, being one of the strongest invertebrate anchoring structures. We present an updated ultrastructural model of the adductor muscle-shell attachment. Muscle cells connect with the shell through epithelial `tendon cells`, which have a cytoskeleton of actin microfilaments that connect to the extracellular matrix via focal adhesions. Collagen-rich fibres arise from apical focal adhesions, cross the nanometric extrapallial space and penetrate the myostracum where they form an organic network. Calcium is present inside vesicles that are released into the extrapallial space. The lack of direct cellular control on secretion restricts the myostracal microstructure to prismatic aragonitic similar to its inorganic counterpart., This research was funded by the project CGL2017-85118-P of the Spanish Ministry of Economy, Industry and Competitiveness. Castro-Claros (FPU18/00547) was supported by a pre-doctoral FPU grant (Ministry of Science, Innovation and Universities).

This work is a detailed study of the giant barnacle shell micro-nano-structure and the relationship between the organic matrix and the mineral. We have studied the composition of the organic matrix and how this interacts with the mineral part, controlling crystal orientation and modifying the nanostructure and crystal lattice. The incorporation of organic matter into the mineral produces a rough topography in the calcite crystals surface, reducing the size of coherent crystal domains and inducing an isotropic straining of the calcite crystal lattice (about 0.08% along the a- and c- axes). We show that insoluble organic matrixes are mainly chitin-rich membranes that can induce the nucleation of calcite crystals on their surface. Also, we have studied the effect of the soluble organic matter (composed of proteins and polysaccharides) on the calcium carbonate precipitation, inducing the formation of calcite, which is the only mineral phase present in the barnacle shell. It has an important effect on nucleation (reducing the number of crystals and promoting their aggregation). It also modifies the calcite morphology by the expression of prismatic crystals faces without interfering with epitaxial growth., We are grateful for financial support through grants CGL2015-64683-P, CGL2016-76723-P, and CGL2017-85118-P (Ministerio de Ciencia e Innovación, Spain), UCE-PP2016-05 (U. Granada) and RNM-179 group (Junta de Andalucía, Spain). A.B.R.-N., A.G.C., and N.A.L. acknowledge support from ANID PIA ANILLOS ACT172037 and PCI REDES 170106 (Chile).

Abstract: Planktonic gastropods of the suborder Euthecosomata (order Pteropoda) secrete a unique microstructure made of tightly interlocked, space-filling helically coiled aragonite fibers. This material has a high degree of ordering because all fibers are in phase at a given growth plane. The recognition of the helical nature from mere scanning electron micrographs is somewhat uncertain. In addition, three-dimensional (3D) reconstructions of individual helices are hindered by the small dimensions of fibers, and the virtual absence of intervening organic interphases. Here, we (1) reexamine the morphological characteristics of the fibers in fracture and find them compatible with a helical model, (2) provide direct evidence of the helical mode of coiling in the form of anaglyphs, and (3) develop a 3D model, which is fully consistent with the patterns observed in fractures and polished sections. Proposals based on motifs different from helices are not congruent with the observations. Graphical abstract: [Figure not available: see fulltext.] Impact statement: Some planktonic gastropods of the order Pteropoda secrete a unique microstructure that was originally described as made of tightly interlocked helically coiled continuous aragonite fibers. Despite this material having been described 50 years ago, there is still debate as to the helical design of such fibers. We provide additional unambiguous evidence that fibers describe true helical trajectories during growth. Establishing their true nature is essential in order to know the spectrum of calcified biomaterials produced by molluscs. This is also a prerequisite for future biomimetic studies aiming at producing bioinspired highly functional materials., Funding for open access charge: Universidad de Granada/CBUA. This research was funded by projects CGL2017-85118-P (A.G.C.), PID2020-116660 GB-I00 (A.G.C., K.B., A.B.R.-N.) (Spanish Ministerio de Ciencia e Innovación), and B-RNM-265-UGR18 (A.G.C., A.B.R.-N.) (Junta de Andalucía), KB PPI/APM/2018/1/00049/U/001 (A.C.G., K.B.) (Polish National Agency for Academic Exchange), the Unidad Científica de Excelencia UCE-PP2016-05 (A.G.C., A.B.R.-N.) (University of Granada), PCIN-2017–098 (C.I.S.-D) (Junta de Andalucía), and the Research Group RNM363 (A.G.C., C.P., J.H.E.C., C.I.S.D.) (Junta de Andalucía), and European COST Action CA17120 (C.I.S.-D.). C.P. acknowledges Juan de la Cierva-Formación postdoctoral contract (ref. FJC2018-035820-I) from the Spanish Ministry of Science. P.R.-S. was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 844345 [project EPIC] and the Malacological Society of London Travel Award.

The bryozoan Cass Stenolaemata is currently represented by the Order Cyclostomata. They produce skeletons made exclusively of calcite crystals assembled into different microstructures. Despite extensive previous research, no appropriate crystallographic techniques have been applied to decipher their crystallographic organization. We present an in-depth crystallographic study of the microstructures of three extant species (Fasciculipora ramosa, Hornera robusta, and Cinctipora elegans) using scanning electron microscopy and associated techniques (energy-dispersive spectroscopy and electron backscatter diffraction), together with atomic force microscopy and micro-computed tomography. We differentiate two microstructures, foliated and tabular calcite, and establish consistent models of their crystallography. Foliated calcite of F. ramosa and C. elegans consists of co-oriented laths arranged with their c-axes parallel to their elongation axis and to their main surfaces, and one a*-axis perpendicular to the latter (sheet texture). Tabular calcite of H. robusta consists of polygonal tablets with the c-axis as fiber axis (axial texture), perpendicular to the tablet surface. The foliated calcite of bryozoans is homeomorph to that of bivalves but has a significantly different crystallography. The tabular calcite of bryozoans lacks the spiral morphology of the tablet-shaped calcite of craniiformis brachiopods and has a different orientation of the c-axis with respect to the constituent tablets., Proyectos CGL2017-85118-P y PID2020116660GB-I00 (Ministerio de Ciencia e Innovación); Proyecto B-RNM-265UGR18 (Junta de Andalucía); Grupo de Investigación RNM363 (Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía); Open Access financiado por la Universidad de Granada y por el Consorcio de Bibliotecas Universitarias de Andalucía (CBUA), Peer reviewed

The external surface microornament of the glass scallops Catillopecten natalyae and malyutinae is made by calcitic spiny projections consisting of a stem that later divides into three equally spaced and inclined branches (here called aerials). C. natalyae contains larger and smaller aerials, whereas C. malyutinae only secreted aerials of the second type. A remarkable feature is that aerials within each type are fairly similar in size and shape and highly co-oriented, thus constituting a most sophisticated microornament. We demonstrate that aerials are single crystals whose morphology is strongly controlled by the crystallography, with the stem being parallel to the c-axis of calcite, and the branches extending along the edges of the {104} calcite rhombohedron. They grow epitaxially onto the foliated prisms of the outer shell layer. The co-orientation of the prisms explains that of the aerials. We have developed a model in which every aerial grows within a periostracal pouch. When this pouch reaches the growth margin, the mantle initiates the production of the aerial. Nevertheless, later growth of the aerial is remote, i.e. far from the contact with the mantle. We show how such an extremely sophisticated microornament has a morphology and co-orientation which are determined by crystal growth., We are grateful to John Taylor for drawing the aerials to our attention. GK is very grateful to A. Brandt, and M.V. Malyutina for invitation to join the deep-sea KuramBio and KuramBio II expeditions and to the scientific staff and ship crews of the expeditions. This research was funded by projects CGL2017-85118-P (AGC, CS, FMVF, CG, EMH) and PID2020-116660GB-I00 (AGC, CS, ABRN, FMVF, CG, EMH) of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada (AGC, ABRN), the Research Group RNM363 of the Junta de Andalucía (AGC, CG), and Grant No. 13.1902.21.001 (Agreement No. 075-15-2020-796) of the Ministry of Science and Higher Education of the Russian Federation (GK).

We analyzed, by optical and transmission electron microscopy, the morphology and function of the mantle edge, including the formation of the periostracum, of ten species of protobranchs. Five species from the order Nuculida, four species from the order Nuculanida and one species from the order Solemyida were studied. A second outer fold, which seems to function as a template for the internal marginal crenulations of the valves, is present in the crenulated species of Nucula. The minute non-crenulated Ennucula aegeensis shows the glandular basal cells displaced toward the periostracal groove, resembling a minute additional fold between the outer and middle folds. Intense secretion of glycocalyx, together with active uptake of particles, have been observed in the inner epithelium of the middle mantle fold and the whole epithelium of the inner mantle fold in all the studied species. Contrary to the rest of the bivalves, all the protobranchs analyzed have two basal cells involved in the formation of the external nanometric pellicle of the periostracum, a character that would support the monophyly of protobranchs. A three-layered pattern is the general rule for the periostracum in protobranchs, like for other bivalves. The presence of pouches of translucent layer inside the tanned dark layer under periostracal folds is characteristic of the species with a folded periostracum; its function is unclear but could give flexibility to the periostracum. The non-nacreous internal shell layer and the presence of translucent pouches under periostracal folds in Sarepta speciosa resemble those found in nuculanids. However, the free periostracum is rather similar to those of N. hanleyi and E. aegeensis, with a continuous vesicular layer. All the latter supports the inclusion of Sarepta in the order Nuculanida but could indicate either a basal lineage or that the translucent vesicular layer is an adaptive trait., This research was funded by the projects CGL2017-85118-P and PID2020-116660GB-I00 of the Spanish Ministry of Economy, Industry and Competitiveness . Funding for open access charge has been provided by the University of Málaga/CBUA .

The mechanical properties such as compressive strength and nanohardness were investigated for Pinctada margaritifera mollusk shells. The compressive strength was evaluated through a uniaxial static compression test performed along the load directions parallel and perpendicular to the shell axis, respectively, while the hardness and Young modulus were measured using nanoindentation. In order to observe the crack propagation, for the first time for such material, the in-situ X-ray microscopy (nano-XCT) imaging (together with 3D reconstruction based on the acquired images) during the indentation tests was performed. The results were compared with these obtained during the micro-indentation test done with the help of conventional Vickers indenter and subsequent scanning electron microscopy observations. The results revealed that the cracks formed during the indentation start to propagate in the calcite prism until they reach a ductile organic matrix where most of them are stopped. The obtained results confirm a strong anisotropy of both crack propagation and the mechanical strength caused by the formation of the prismatic structure in the outer layer of P. margaritifera shell., The research was co-financed by the European Union from the resources of the European Social Fund (Project No.WND-POWR.03.02.00-00-I043/16), the DAAD program. A.C. acknowledges project CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovación for funding.

Here, we provide the dataset associated with the research article ¿Orientation patterns of aragonitic crossed-lamellar, fibrous prismatic and myostracal microstructures of modern Glycymeris shells¿ [1]. Based on several tools (SEM, EBSD, laser confocal microscopy and FE-SEM) we present original data relative to the microstructure and texture of aragonite crystallites in all Glycymeris shell layers (crossed-lamellar, complex crossed-lamellar, fibrous prismatic and pedal retractor and adductor myostraca) and address texture characteristics at the transition from one layer to the other, identifying similarities and differences among the different layers. Shells were cut transversely, obliquely and longitudinally in order to obtain different orientated sections of the outer and inner layer and of the myostraca. The identification of major microstructural elements was provided by detailed SEM and laser confocal microscopy images. Microstructure and texture characterization was based on EBSD measurements presented as band contrast images and as color-coded crystal orientation maps with corresponding pole figures. Crystal co-orientation was measured with the MUD value. Finally, the distribution of the organic matrix occluded within the outer crossed-lamellar layer was revealed using FE-SEM. These data, besides providing a modern unaltered Glycymeris reference to detect diagenetic alteration in fossil analogs used for paleoenvironmental reconstructions, are useful to better comprehend the mechanisms of bivalve shell formation., GC acknowledges financial support of the 2017 Italian Ministry PRIN RX9XXY Project "Biota resilience to global change: biomineralization of planktic and benthic calcifiers in the past, present and future" to E. Erba. AGC is funded by project CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovación, the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada, and the Research Group RNM363 of the Junta de Andalucía.

Gymnolaemata bryozoans produce CaCO skeletons of either calcite, aragonite, or both. Despite extensive research, their crystallography and biomineralization patterns remain unclear. We present a detailed study of the microstructures, mineralogy, and crystallography of eight extant cheilostome species using scanning electron microscopy, electron backscatter diffraction, atomic force microscopy, and micro-computed tomography. We distinguished five basic microstructures, three calcitic (tabular, irregularly platy, and granular), and two aragonitic (granular-platy and fibrous). The calcitic microstructures consist of crystal aggregates that transition from tabular or irregularly platy to granular assemblies. Fibrous aragonite consists of fibers arranged into spherulites. In all cases, the crystallographic textures are axial, and stronger in aragonite than in calcite, with the c-axis as the fiber axis. We reconstruct the biomineralization sequence in the different species by considering the distribution and morphology of the growth fronts of crystals and the location of the secretory epithelium. In bimineralic species, calcite formation always predates aragonite formation. In interior compound walls, growth proceeds from the cuticle toward the zooecium interior. We conclude that, with the exception of tabular calcite, biomineralization is remote and occurs within a relatively wide extrapallial space, which is consistent with the inorganic-like appearance of the microstructures. This biomineralization mode is rare among invertebrates., Funding for open access publishing: Universidad de Granada/CBUA. CG and AGC were funded by projects CGL2017-85118-P, PID2020116660GB-I00 (Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033; \u201CFEDER Una manera de hacer Europa\u201D), and PCM 00092 (Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía), the Research Group RNM363 (latter institution), and the Unidad Cient\u00EDfica de Excelencia UCE-PP2016-05 (University of Granada). Funding for open access was provided by Universidad de Granada/Consorcio de Bibliotecas Universitarias de Andalucía (CBUA).

Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033; National projects CGL2017-85118-P and PID2020116660GBI00; “FEDER Una manera de hacer Europa”; PCM 00092 (Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía); Research Group RNM363 and the Unidad Científica de Excelencia UCE-PP2016-05 (University of Granada); Universidad de Granada/Consorcio de Bibliotecas Universitarias de Andalucia (CBUA), Peer reviewed