Beschreibung: Fossil carbonate skeletons of marine organisms are archives for understanding the development and evolution of palaeo-environments. However, the correct assessment of past environment dynamics is only possible when pristine skeletons and their biogenic characteristics are unequivocally distinguishable from diagenetically-alteredskeletal elements and non-biogenic features. In this study, we extend our work on diagenesis of biogenic aragonite (Casella et al. 2017) to the investigation of biogenic low-Mg calcite using brachiopod shells. We examined and compared microstructural characteristics inducedby laboratory-based alteration to structural features derived from diagenetic alteration in natural environments. We used four screening methods: cathodoluminescence (CL), cryogenic and conventional field emission-scanning electronmicroscopy (FE-SEM), atomic force microscopy (AFM) and electron backscatter diffraction (EBSD).We base our assessments of diagenetic alteration and overprint on measurements of, a) images of optical overprint signals, b) changes in calcite crystal orientation patterns, and c) crystal co-orientation statistics. According to the screening process, altered and overprinted samples define two groups. In Group 1 the entire shell is diagenetically overprinted, whereas in Group 2 the shell contains pristine as well as overprinted parts. In the case of Group 2 shells, alteration occurred either along the periphery of the shell including the primary layer or at the interior-facing surface of the fibrous/columnar layer. In addition, we observed an important mode of the overprinting process, namely the migration of diagenetic fluids through the endopunctae corroborated by mineral formation and overprinting in their immediate vicinity, while leaving shell parts between endopunctae in pristine condition. Luminescence (CL) and microstructural imaging (FE-SEM) screening give first-order observations of the degree of overprint as they cover macro-to micron scale alteration features. For a comprehensive assessment of diagenetic overprint these screening methods should be complemented by screening techniques such as EBSD and AFM. They visualise diagenetic changes at submicron and nanoscale levels depicting the replacement of pristine nanocomposite mesocrystal biocarbonate (NMB) by inorganic rhombohedral calcite (IRC). The integration of screening methods allows for the unequivocal identification of highly-detailed alteration features as well as an assessment of the degree of diagenetic alteration.

Beschreibung: To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles. At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, incross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference. FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of miner-alization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

Beschreibung: Fossil carbonate skeletons of marine organisms are archives for understanding the development and evolution of palaeo-environments. However, the correct assessment of past environment dynamics is only possible when pristine skeletons and their biogenic characteristics are unequivocally distinguishable from diagenetically-altered skeletal elements and non-biogenic features. In this study, we extend our work on diagenesis of biogenic aragonite (Casella et al. 2017) to the investigation of biogenic low-Mg calcite using brachiopod shells. We examined and compared microstructural characteristics induced by laboratory-based alteration to structural features derived from diagenetic alteration in natural environments. We used four screening methods: cathodoluminescence (CL), cryogenic and conventional field emission-scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) and electron backscatter diffraction (EBSD). We base our assessments of diagenetic alteration and overprint on measurements of, a) images of optical overprint signals, b) changes in calcite crystal orientation patterns, and c) crystal co-orientation statistics. According to the screening process, altered and overprinted samples define two groups. In Group 1 the entire shell is diagenetically overprinted, whereas in Group 2 the shell contains pristine as well as overprinted parts. In the case of Group 2 shells, alteration occurred either along the periphery of the shell including the primary layer or at the interior-facing surface of the fibrous/columnar layer. In addition, we observed an important mode of the overprinting process, namely the migration of diagenetic fluids through the endopunctae corroborated by mineral formation and overprinting in their immediate vicinity, while leaving shell parts between endopunctae in pristine condition. Luminescence (CL) and microstructural imaging (FE-SEM) screening give first-order observations of the degree of overprint as they cover macro-to micron scale alteration features. For a comprehensive assessment of diagenetic overprint these screening methods should be complemented by screening techniques such as EBSD and AFM. They visualise diagenetic changes at submicron and nanoscale levels depicting the replacement of pristine nanocomposite mesocrystal biocarbonate (NMB) by inorganic rhombohedral calcite (IRC). The integration of screening methods allows for the unequivocal identification of highly-detailed alteration features as well as an assessment of the degree of diagenetic alteration.

Beschreibung: Highlights • Cell-reorganization; commissure: muti-cell-layered, central shell: single-cell-layered. • Individual fibres are secreted by several cells at the same time. • Tight cooperation of cells for the coordinated secretion of organic membrane and mineral. • Lack of extrapallial space between OME cells and developing fibres. • Mineral transport to sites of mineralization occurs via ion transport through cell membrane. Abstract To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles. At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, in cross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference. FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of mineralization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

Beschreibung: The fibrous calcite layer of modern brachiopod shells is a hybrid composite material and forms a substantial part of the hard tissue. We investigated how cells of the outer mantle epithelium (OME) secrete calcite material and generate the characteristic fibre morphology and composite microstructure of the shell. We employed AFM, FE-SEM, and TEM imaging of embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze substituted samples. Calcite fibres are secreted by outer mantle epithelium (OME) cells. Biometric analysis of TEM micrographs indicates that about 50% of these cells are attached via hemidesmosomes to an extracellular organic membrane present at the proximal, convex surface of the fibres. At these sites, mineral secretion is not active. Instead, ion transport from OME cells to developing fibres occurs at regions of closest contact between cells and fibres, however only at sites where the extracellular membrane at the proximal fibre surface is not developed yet. Fibre formation requires the cooperation of several adjacent OME cells. It is a spatially and temporally changing process comprising of detachment of OME cells from the extracellular organic membrane, mineral secretion at detachment sites, termination of secretion with formation of the extracellular organic membrane, and attachment of cells via hemidesmosomes to this membrane.

Beschreibung: The present data in brief article provides additional data and information to our research article “Micro- and nanostructures reflect the degree of diagenetic alteration in modern and fossil brachiopod shell calcite: a multi-analytical screening approach (CL, FE-SEM, AFM, EBSD)” [1] (Casella et al.). We present fibre morphology, nano- and microstructure, as well as calcite crystal orientations and textures found in pristine, experimentally altered (hydrothermal and thermal), and diagenetically overprinted brachiopod shells. Combination of the screening tools AFM, FE-SEM, and EBSD allows to observe a significant change in microstructural and textural features with an increasing degree of laboratory-based and naturally occurring diagenetic alteration. Amalgamation of neighbouring fibres was observed on the micrometre scale level, whereas progressive decomposition of biopolymers in the shells and fusion of nanoparticulate calcite crystals was detected on the nanometre scale. The presented data in this article and the study described in [1] allows for qualitative information on the degree of diagenetic alteration of fossil archives used for palaeoclimate reconstruction.