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The mineralized skeletons of echinoderms are characterized by their complex, open-cell porous microstructure (also known as stereom), which exhibits vast variations in pore sizes, branch morphology, and three-dimensional (3D) organization patterns among different species. Quantitative description and analysis of these cellular structures in 3D are needed in order to understand their mechanical properties and underlying design strategies. In this paper series, we present a framework for analyzing such structures based on high-resolution 3D tomography data and utilize this framework to investigate the structural designs of stereom by using the spines from the sea urchin Heterocentrotus mamillatus as a model system. The first paper here reports the proposed cellular network analysis framework, which consists of five major steps: synchrotron-based tomography and hierarchical convolutional neural network-based reconstruction, machine learning-based segmentation, cellular network registration, feature extraction, and data representation and analysis. This framework enables the characterization of the porous stereom structures at the individual node and branch level (~10 µm), the local cellular level (~100 µm), and the global network level (~1 mm). We define and quantify multiple structural descriptors at each level, such as node connectivity, branch length and orientation, branch profile, ring structure, etc., which allows us to investigate the cellular network construction of H. mamillatus spines quantitatively. The methodology reported here could be tailored to analyze other natural or engineering open-cell porous materials for a comprehensive multiscale network representation and mechanical analysis. Statement of Significance The mechanical robustness of the biomineralized porous structures in sea urchin spines has long been recognized. However, quantitative cellular network representation and analysis of this class of natural cellular solids are still limited in the literature. This constrains our capability to fully understand the mechanical properties and design strategies in sea urchin spines and other similar echinoderms’ porous skeletal structures. Combining high-resolution tomography and computer vision-based analysis, this work presents a multiscale 3D network analysis framework, which allows for extraction, registration, and quantification of sea urchin spines’ complex porous structure from the individual branch and node level to the global network level. This 3D structural analysis is relevant to a diversity of research fields, such as biomineralization, skeletal biology, biomimetics, material science, etc.

Holocrinid crinoids (Holocrinus) are among the oldest, if not the oldest, post-Paleozoic crinoids, which are widely considered the stem-group isocrinids for the post-Paleozoic monophyletic subclass Articulata. Owing to their phylogenetic and stratigraphic position, the morphology and paleobiology of these crinoids have received considerable attention. Although the holocrinid stalk is similar to that of fossil and living isocrinids, it lacks flat and rigid synostosial articulations, which are sites specialized for autotomy. Holocrinids were thus long considered sessile, without the ability to re-attach. However, taphonomic data, such as specific pattern of preferred disarticulation at the distal facet of nodals, indicated that Holocrinus was likely capable of stalk autotomy and relocation. This trait, that is considered an anti-predatory adaptation against benthic predation, has recently served as example of fundamental change in benthic marine communities, such as the Mesozoic marine revolution (MMR). Here I describe stereom microstructure of the stalk of Early-Middle Triassic Holocrinus. The holocrinid stalk is mainly constructed of straight stereom passageways passing through the columnals, that are diagnostic of long through-going ligaments. At the distal symplexial nodal facet, however, fine and dense stereom, in which pores are irregular in outline and show no alignment, is observed. The latter stereom type strongly resembles synostosial stereom in living isocrinids, that is associated with short ligaments, specifically designed for autotomy. These findings thus strongly support stalk shedding abilities in holocrinids. These results emphasize that microstructural observations, coupled with taphonomic data, may provide more insights into functional inferences than macromorphological data alone.

Uintacrinoids (Uintacrinoidea) are among the most bizarre of all the crinoids. These stratigraphically important Late Cretaceous crinoids are characterized by an atypical large stalkless globular cup without cirriferous centrodorsal and extremely long arms. Owing to this unusual morphology, the mode of life of these crinoids has confused palaeontologists for more than a century. While uintacrinoids have been commonly regarded as pelagic forms, either swimming actively or floating passively by means of a putative buoyancy mechanism, some recent authors have favoured a benthic mode of life. However, evidence supporting a benthic lifestyle hypothesis is still limited. Here we describe the stereom microstructure of Marsupites and Uintacrinus. In Marsupites, thecal plates are typically comprised of coarse to medium, clearly structural, galleried stereom, which alternates with zones of fine and deeply penetrating galleried stereom oriented perpendicular to the plate margins. The latter stereom type corresponds to the external ribbed ornament of the theca, and is indicative of collagenous sutural fibers for binding adjacent plates. In Uintacrinus, thecal plates are almost entirely composed of fine galleried stereom diagnostic for collagenous sutural fibers. In both taxa, a perforate stereom layer, that is known to increase plate strengthening and resistance to abrasion in some Recent echinoderms, is developed most externally, and also produces a characteristic interior growth banding pattern. No skeletal adaptations for maintaining buoyancy, such as skeletal lightening and hollow architecture, that are known to occur in nektonic and pseudoplanktonic crinoids, are observed. Instead, the stereom organization in uintacrinoids closely resembles those observed in Recent benthic crinoids. Our findings thus strongly support a benthic mode of life of uintacrinoids. Their widespread distribution within a short time interval can be best explained by having a very long-term planktonic larval stage as previously hypothesized by some authors.

One of the most profound environmental changes thought to be reflected in chemical composition of numerous geological archives is Mg/Ca ratio of the seawater, which has varied dramatically throughout the Phanerozoic. Echinoderms that today typically form high magnesium calcite skeletons are increasingly being utilized as a proxy for interpreting secular changes in seawater chemistry. However, accurate characterization of the diagenetic changes of their metastable high magnesium calcite skeletons is a prerequisite for assessing their original, major-element geochemical composition. Here we expand the existing models of diagenesis of echinoderm skeleton by integration of various analytical methods that up to now rarely have been used to assess the diagenetic changes of fossil echinoderms. We validated the preservation of a suite of differently preserved echinoderm ossicles, mostly crinoids, ranging in age from the Cambrian through Recent. In 13 of 99 fossil echinoderm ossicles we found well-preserved porous microstructure (stereom), non-luminescent behaviour or blotchy dark color in cathodoluminescence, and distinct nanostructural features (layered and nanocomposite structure). Moreover, in representatives of such preserved samples, distribution of sulphates associated with organic matter is identical to those in Recent echinoderms. Only such ossicles, despite of local micrometer-scale diagenetic changes, were herein considered well-preserved, retaining their original major-element skeletal composition. By contrast, majority of samples show transformation to the stable low magnesium calcite that leads to obliteration of the primary geochemical and micro/nanostructural features and is accompanied with increase in cathodoluminescence emission intensity. Using only well-preserved fossil echinoderm samples, we found purely random variation in Mg/Ca in echinoderm skeletons through the observed time series; any periodicities in echinoderm skeletal Mg/Ca ratio which might be related to the secular transitions in calcite and aragonite seas were not confirmed. These findings suggest that, in contrast to some groups of organisms with relatively weak control over their biomineralization (such as algae, sponges, and bryozoans), in which polymorph mineralogies consistently changed according to the seawater type, application of fossil echinoderms, in particular crinoids, to seawater Mg/Ca reconstructions is unreliable. These data emphasize a key-role of physiological factors (the so-called vital effects) in echinoderm biomineralization.

Anthropogenic CO2 – driven ocean acidification (OA) is causing a decrease in seawater pH and the saturation state of calcium carbonate minerals, compromising the ability of calcifying species to produce and maintain their skeletons. Sea urchins are ecologically important calcifying species and we investigated the impacts of long-term (9 month) exposure to near-future OA (Ambient – pHNBS 8.01; OA – pHNBS 7.6) on the skeleton microstructure of Heliocidaris erythrogramma using scanning electron microscopy (SEM), micro-computed tomography (μCT) and nanoindentation. SEM revealed that the youngest plates (apical plates) which had likely grown in experimental conditions had larger pores in the OA group (pore surface area ~ 72% larger) compared with those of urchins maintained in ambient pH. High-resolution, μCT 3-D reconstructions of the apical plates revealed that the experimental OA treatment urchins had a ~14% greater porosity and ~17% less biomineral, suggesting an inability to finely regulate skeletogenesis. The mid-test ambital plates established prior to this study did not show any OA associated change in porosity. Nanoindentation of the apical plates indicated that OA reduced skeletal hardness and elasticity. Stereom pore size is a key trait of the sea urchin endoskeleton and increased porosity in H. erythrogramma is likely to impact its biological functions as well as its biomechanical capacity to defend against predation and physical disturbances.

The chemical composition and microstructure of the calcite cuticles of eleven species of phacopine trilobites have been investigated by electron beam imaging, diffraction, and microanalysis, and results reveal that the lenses of their schizochroal eyes differed significantly in chemical composition from the rest of the cuticle in vivo. Apart from the eye lenses, most cuticles are inferred to have escaped extensive recrystallisation because their constituent crystals are sub-micrometre in size and have a preferred orientation that is consistent between species. Their current compositions of ~ 1.4 to 2.4 mol% MgCO3 are likely to be close to original values, although as they commonly luminesce and contain detectable manganese and iron, some diagenetic alteration has taken place. The associated lenses have a microstructure that is suitable for focusing light, yet are optically turbid owing to the presence within calcite of micropores and crystals of microdolomite, apatite, celestite and pyrite. The microdolomite indicates that lenses recrystallised from an original high-Mg calcite composition and this is supported by the presence of nanometre-scale modulated microstructures in both the calcite and dolomite. These lenses currently contain ~ 1 to 6 mol% MgCO3, and by comparison with the proportion of magnesium lost from echinoderm stereom in the same thin sections, may have contained ~ 7.5 mol% MgCO3 in vivo. In some samples, more extensive diagenetic alteration is evidenced by recrystallisation of the cuticle including lenses to coarse equant calcite or enrichment of the cuticle, but not necessarily the lenses, in magnesium accompanying replacement by a Mg–Fe phyllosilicate. The phacopine trilobites had to modify partition coefficients for magnesium considerably in order to grow lenses with contrasting compositions to the rest of their cuticles, and such a strong vital effect on biomineralisation suggests that incorporation of magnesium was essential for functioning of their calcite optical systems.

In various mineralizing marine organisms, calcite or aragonite crystals form through the initial deposition of amorphous calcium carbonate (ACC) phases with different hydration levels. Using X-ray PhotoEmission Electron spectroMicroscopy (X-PEEM), ACCs with varied spectroscopic signatures were previously identified. In particular, ACC type I and II were recognized in embryonic sea urchin spicules. ACC type I was assigned to hydrated ACC based on spectral similarity with synthetic hydrated ACC. However, the identity of ACC type II has never been unequivocally determined experimentally. In the present study we show that synthetic anhydrous ACC and ACC type II identified here in sea urchin spines, have similar Ca L2,3-edge spectra. Moreover, using X-PEEM chemical mapping, we revealed the presence of ACC-H2O and anhydrous ACC in growing stereom and septa regions of sea urchin spines, supporting their role as precursor phases in both structures. However, the distribution and the abundance of the two ACC phases differ substantially between the two growing structures, suggesting a variation in the crystal growth mechanism; in particular, ACC dehydration, in the two-step reaction ACC-H2O → ACC → calcite, presents different kinetics, which are proposed to be controlled biologically. Keywords: Sea urchin spine regeneration, Anhydrous amorphous calcium carbonate, Hydrated amorphous calcium carbonate, Ca L2,3-edge spectra, PhotoEmission Electron spectroMicroscopy

This paper reports results of a novel approach, X-ray microCT, for quantifying stereom structures applied to ossicles of the sea urchin Lytechinus variegatus. MicroCT, a high resolution variant of medical CT (computed tomography), allows noninvasive mapping of microstructure in 3-D with spatial resolution approaching that of optical microscopy. An intact pyramid (two demipyramids, tooth epiphyses, and one tooth) was reconstructed with 17 microm isotropic voxels (volume elements); two individual demipyramids and a pair of epiphyses were studied with 9-13 microm isotropic voxels. The cross-sectional maps of a linear attenuation coefficient produced by the reconstruction algorithm showed that the structure of the ossicles was quite heterogeneous on the scale of tens to hundreds of micrometers. Variations in magnesium content and in minor elemental constitutents could not account for the observed heterogeneities. Spatial resolution was insufficient to resolve the individual elements of the stereom, but the observed values of the linear attenuation coefficient (for the 26 keV effective X-ray energy, a maximum of 7.4 cm(-1) and a minimum of approximately 2 cm(-1) away from obvious voids) could be interpreted in terms of fractions of voxels occupied by mineral (high magnesium calcite). The average volume fraction of mineral determined for a transverse slice of the demipyramid near where it joins an epiphysis was 0.46; for a slice 3.3 mm adoral it was 0.70. Local volume fractions of mineral approached 1, and, away from resolvable voids, considerable portions of the demipyramids had volume fractions of calcite at or below approximately 0.33. MicroCT imaging of a demipyramid before and after infiltration with a high absorptivity fluid (sodium polytungstate) confirmed the determination of the volume fractions of minerals.

Interambularcral plates of echinoid Heterocentrotus trigonarius, composed of Mg calcite 1 (≈14 mol% MgCO3), were heated in three timed series of experiments at 300°C. Dried plate fragments and fragments with added water were heated separately in pressurized bombs. X-ray powder diffractometry, unit cell dimensions, and phase compositions are used to monitor reaction progress. After 10 h heating in the bombs dolomite (43.5 mol% MgCO3) and Mg calcite appear (4–7 mol% MgCO3); by 20 h all Mg calcite 1 is consumed, and at 120 h dolomite composition has evolved to ≈47 mol% MgCO3 and calcite to ≈2 mol% MgCO3. Whole plates heated at 300°C in an open muffle furnace develop dolomite (≈42 mol% MgCO3) and Mg calcite 2 (≈6 mol% MgCO3) after 10 h and remain compositionally invariant throughout subsequent heating to 620 h. Limited skeletal water catalyzes the early reaction but escapes from the open furnace and consequently reaction ceases after ≈10 h. The experimentally produced dolomite has relative Mg-Ca ordering of 75% to 79%. The stabilization of echinoid Mg calcite by heating at 300°C to a mixture of dolomite and calcite occurs through a dissolution/precipitation reaction. The alteration fabric produced within the stereom consists of irregularly shaped, branched dolomite crystals > 5 μm homoaxially set in a calcite 2 (bomb) or Mg calcite 2 (furnace) matrix. Round and tubular pores 1 to 5 μm are randomly distributed throughout this fabric. The stereom pore system remains intact during furnace heating but is destroyed during heating in bombs. The texture of experimentally stabilized echinoid skeletons is different from that of fossil echinoderms that are composed of microrhomic dolomite homoaxially set in a single calcite crystal.

The similar proximal and distal attachments to the stereom of primary spine ligament in the echinoid Eucidaris tribuloides are described, from thin sections and SEM studies on frozen and fractured spine articulations and ligaments from decalcified material. The orthogonal structure of the general stereom is modified on the attachment zones where bundles of collagen cylinders enter approximately hexagonally arranged channels. Straps of collagen extend in parallel series between adjacent bundles via regularly placed ports and collagen loops rather than non-striated 'tendons' pass over skeletal trabeculae. The regular pattern of collagen straps is most evident on the proximal and distal attachment zones. Mechanical features of the non-adhesive mode of attachment are considered, together with similarities and differences between insertion of muscle cells and mutable collagenous tissue (ligament) in echinoderms.