Multi-scale mineralogical characterization of the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea)

Journal of Structural Biology 176 (2011) 315–329

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Multi-scale mineralogical characterization of the hypercalcified sponge Petrobiona massiliana (Calcarea, Calcaronea) Melany Gilis a,b,⇑, Olivier Grauby c, Philippe Willenz a,b, Philippe Dubois b, Laurent Legras d, Vasile Heresanu c, Alain Baronnet c a

Department of Invertebrates, Royal Belgian Institute of Natural Sciences, B-1000 Brussels, Belgium Laboratoire de Biologie marine, Université Libre de Bruxelles, B-1050 Brussels, Belgium Université Paul Cézanne & Centre Interdisciplinaire de Nanosciences de Marseille, Campus de Luminy 1328, France d Electricité de France, R&D Division, Material and Mechanics of Components Department, Les Renardières, 77818 Moret-sur Loing Cedex, France b c

a r t i c l e

i n f o

Article history: Received 23 June 2011 Received in revised form 12 August 2011 Accepted 13 August 2011 Available online 23 August 2011 Keywords: Biomineralization Coralline sponge Calcification Amorphous Calcium carbonate

a b s t r a c t The massive basal skeleton of a few remnant living hypercalcified sponges rediscovered since the 1960s are valuable representatives of ancient calcium carbonate biomineralization mechanisms in basal Metazoa. A multi-scale mineralogical characterization of the easily accessible Mediterranean living hypercalcified sponge belonging to Calcarea, Petrobiona massiliana (Vacelet and Lévi, 1958), was conducted. Oriented observations in light and electron microscopy of mature and growing areas of the Mg-calcite basal skeleton were combined in order to describe all structural levels from the submicronic to the macroscopic scale. The smallest units produced are ca. 50–100 nm grains that are in a mushy amorphous state before their crystallization. Selected area electron diffraction (SAED) further demonstrated that submicronic grains are assembled into crystallographically coherent clusters or fibers, the latter are even laterally associated into single-crystal bundles. A model of crystallization propagation through amorphous submicronic granular units is proposed to explain the formation of coherent micron-scale structural units. Finally, XRD and EELS analyses highlighted, respectively, inter-individual variation of skeletal Mg contents and heterogeneous spatial distribution of Ca ions in skeletal fibers. All mineralogical features presented here cannot be explained by classical inorganic crystallization principles in super-saturated solutions, but rather underlined a highly biologically regulated formation of the basal skeleton. This study extending recent observations on corals, mollusk and echinoderms confirms that occurrence of submicronic granular units and a possible transient amorphous precursor phase in calcium carbonate skeletons is a common biomineralization strategy already selected by basal metazoans. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Porifera were among the first metazoans engaged in the formation of calcium carbonate biominerals. Indeed, hypercalcified sponges, able to produce an aragonitic or calcitic basal skeleton in addition to their spicules, were the most important reef builders in late Paleozoic and early Mesozoic (Reitner and Gautret, 1996; Vacelet, 1983). The phylogenetic relationship of these organisms or even their poriferans affiliation was subject to a long history of varied interpretations (Vacelet, 1979; Wood and Reitner, 1986). However, since the late 1960s, scuba diving and submersible explorations allowed the fortuitous findings of about 20 living hypercalcified species (Hartman, 1969, 1979; Hartman and Goreau, 1970, 1975, 1976; Vacelet, 1964; Vacelet and Lévi, 1958; Willenz and Pomponi, 1996). First regarded as a new class of Porifera ⇑ Corresponding author at: Laboratoire de Biologie Marine, Université Libre de Bruxelles, CP 160/15 50, Avenue F.D. Roosevelt, B-1050 Bruxelles, Belgium. E-mail address: [email protected] (M. Gilis). 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.08.008

(‘‘Sclerosponges’’), today their polyphyletic relationship to different orders of Demospongiae as well as to a few species belonging to Calcarea is obvious (Reitner, 1992; Vacelet, 1977, 1979, 1985; Wood and Reitner, 1986). Most of these remnant ‘‘coralline sponges’’ are distributed in deep or cryptic habitats like bathyal cliffs, sublittoral dark caves and coral reef tunnels (see Vacelet et al. (2010) for review). Living hypercalcified sponges rapidly appeared as valuable organisms either to understand sponge phylogeny by relating them to fossil records (e.g. Basile et al., 1984; Cuif, 1979; Cuif et al., 1979; Cuif and Gautret, 1991; Finks, 2010; Gautret, 1986; Reitner, 1992; Vacelet, 1979, 1985; Wendt, 1979), as potential recorders of past environmental conditions (Benavides and Druffel, 1986; Böhm et al., 2000; Fallon et al., 2005; Hermans et al., 2010; Rosenheim et al., 2004, 2005, 2009; Swart et al., 1998) or even as living illustration of early biomineralization mechanisms in metazoans (e.g. Bergbauer et al., 1996; Cuif et al., 1990; Gautret et al., 1996; Jackson et al., 2010; Jones, 1979; Lange et al., 2001; Reitner, 1989; Reitner and Gautret, 1996; Reitner et al., 1997, 2001;

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Willenz and Hartman, 1985, 1999; Wood, 1991;Wörheide, 1998; Wörheide et al., 1996). In the present work, we investigated the Mg-calcite basal skeleton of a hypercalcified sponge belonging to Calcarea, Petrobiona massiliana Vacelet and Lévi, 1958 (Calcaronea, Baeriida, Petrobionidae). This species, living at shallow depths in dark sublittoral caves in the Mediterranean Sea, has been chosen as a suitable model to provide insights into old mechanisms of biomineralization in Metazoa. Furthermore, the high magnesium content in this biogenic calcite is a still unresolved biomineral feature that stimulates much interest in this field (e.g. Loste et al., 2003; Politi et al., 2010; Raz et al., 2000; Wang et al., 2009). The basal skeleton of P. massiliana has already been described externally after soft tissue digestion or internally in thin sections and fracture in light (LM) and scanning electron microscopy (SEM) (Cuif and Gautret, 1991; Gautret, 1986; Hermans et al., 2010; Reitner, 1989; Stolarski and Mazur, 2005; Vacelet, 1964, 1991; Wendt, 1979) and in atomic force microscopy (AFM, Stolarski and Mazur, 2005). Most authors agreed on the external morphology to be formed by the superposition of spiny elongated or irregular layers called ‘‘sclerodermites’’. However, the internal organization, mostly the microstructure, has received different denominations, from ‘‘spherulitic’’ (Stolarski and Mazur, 2005; Vacelet, 1964; Wendt, 1979) to ‘‘pennular’’ (Cuif and Gautret, 1991) or ‘‘composite’’ (Cuif et al., 1979; Gautret, 1986). Stolarski and Mazur (2005) revealed a granular morphology at the submicronic scale in a partially etched section of the basal skeleton of P. massiliana. While this observation still needs to be demonstrated in the intact skeleton, it already contrasts with previous fibrillar descriptions (e.g. Gautret, 1986) and reflects the complexity of the structural organization of the basal skeleton in P. massiliana. In all these works, the skeleton has been described according to differently oriented sections or fractures and, for some of them, after partial decalcification (‘‘etching’’), leading to various or even misleading microstructural interpretations. The complexity to generalize the internal organization of this basal skeleton might be linked to its multidirectional and spatially discontinuous growth highlighted by in situ calcein staining (Hermans et al., 2010). In order to obtain a coherent description of the internal organization, the morphology observed in LM or SEM has to be systematically related to the orientation of the sections or fractures. Furthermore, ‘‘etching’’ is a relevant tool to reveal traces of successive micro to nano-scaled growth steps or intraskeletal organic matrix. However, as it does not really illustrate structural units in a native state, it should not be used alone in micro or nano-structural characterization but rather as a complementary observation. Another way to recognize the organization of structural units in highly compact skeleton is to investigate intact but growing areas of skeletons in addition to mature ones. Finally, as indicated by previous investigators, mechanisms of formation of this highly complex basal skeleton are still poorly understood (Vacelet, 1991; Vacelet et al., 2010). In order to recognize all structural levels of the basal skeleton in P. massiliana, we investigated mature and growing areas from macroscopic to submicronic scales in LM, SEM and transmission electron microscopy (TEM). In addition, selected area electron diffraction (SAED) and energy electron loss spectroscopy (EELS) gave further insight into the structure of this magnesium calcite skeleton.

2. Material and methods 2.1. Material Specimens of P. massiliana were collected in early May 2008 and September 2009 in an underwater cave at 14 m in the Calanque of La Vesse, Marseille (France). Sponges were fixed immediately

after collection either in absolute ethanol or in a solution of 4% glutaraldehyde buffered with sodium cacodylate, sodium chloride and sucrose (0.2 M, pH 7.4, 1700 mOsM). The latter were rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide in 0.2 M sodium cacodylate and 0.3 M NaCl (pH 7.4, 1040 mOsM), rinsed again in cacodylate buffer and then dehydrated in a graded ethanol series and stored in ethanol 95% at 4 °C before further treatment. For light microscopy (LM) and transmission electron microscopy (TEM) observations, samples were embedded in a low viscosity epoxy resin (Spurr Low Viscosity Embedding Media, Polysciences, Inc.). For scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD), some sponges were exposed to a 10% sodium hypochlorite solution for short times in order to digest superficial soft tissue. Calcareous spicules included in the soft tissue were then isolated by multiple gentle centrifugations from the remaining digesting solution progressively replaced by absolute ethanol. The tissuecleaned skeletons were dehydrated in a graded ethanol series and stored in absolute ethanol until further treatments for SEM and XRD analyses. Other skeletons subjected to internal morphological observations in SEM were stored in absolute ethanol without soft tissue digestion. 2.2. Methods 2.2.1. Light microscopy (LM) Thick sections (1–2 mm) of skeletons embedded in a low viscosity epoxy resin (Spurr Low Viscosity Embedding Media, Polysciences, Inc.) were obtained with a low speed diamond saw (Bennet Labcut 1010) prior to grinding and polishing using diamond disks. Photomicrographs were taken in Differential Interference Contrast (DIC) with a Nikon Coolpix camera mounted on a Nikon Optiphot 2 microscope. 2.2.2. Scanning electron microscopy (SEM) Intact and gently crushed calcareous spicules extracted from tissues were air dried, mounted on aluminum stubs, coated with a 15–20 nm thick carbon layer and observed with a SEM JEOL JSM-6320F. External and internal morphology of the basal skeleton were, respectively, observed after soft tissue digestion or cryofracture in liquid nitrogen using a razor blade. In addition, partial decalcification (i.e. ‘‘etching’’) was used on longitudinal sections of resin-embedded specimen to complete microstructural observations (0.2 M trichloroacetic acid, 2 min, followed by dehydration in graded ethanol series). Samples, stored in absolute ethanol, were dried at 50 °C in an oven or by the critical point method using carbon dioxide (Critical Point Dryer Balzers CPD 030), mounted on aluminum stubs, sputter-coated with gold (Sputter coater Balzers SCD 50) and observed with a Philips/FEI XL30 ESEM TMP at 30 kV. 2.2.3. Transmission electron microscopy (TEM) Thick sections (1–2 mm) of mature areas of basal skeletons embedded in epoxy resin obtained with a low speed diamond saw were ground until a section of about 30 lm thick was obtained. Buehler diamond/water polycrystalline suspension 3 and 1 lm and then colloidal silica suspension were used for grinding. Resulting thin sections were glued on single-hole Cu grids and then Ar+ ion-milled with a precision ion polishing system (PIPS) GATAN in order to obtain a hole in the skeleton that was surrounded by an electron-transparent thin wedge suitable for TEM examinations. The procedure comprised 7 h of thinning at 5.2 keV accelerating voltage of Ar+ ions under grazing incidence and finished by 45 min at 2.8 keV. Grids were coated by 15 nm-thick amorphous carbon and observed on a JEOL 3010 TEM at 300 kV for TEM images recording and selected area electron diffraction (SAED). Furthermore, finely crushed cleaned-spicules were mounted on holey-carbon grids for TEM. For both preparations, a low dose illumination

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during tuning of the objective focus and astigmatism correction was used to reduce damages of biogenic calcite. SAED patterns were obtained by using a set of apertures, selecting skeleton areas with homogenous absorption/diffraction contrasts. Moreover, ions polished sections have been investigated by energy-filtering transmission electron microscopy (EFTEM) on a FEG STEM TECNAI G2 20F fitted with a GIF system (GATAN) for electron energy loss spectroscopy (EELS) analyses and with an EDAX X-rays detector for energy dispersive X-ray spectroscopy (EDX). EDX elemental analyses were first carried out in a selected area of the mature basal skeleton in order to verify the presence of targeted elements (i.e. Ca, Mg). The area was examined by EELS to confirm EDX results and then by EFTEM at 200 keV. After eliminating electrons that had undergone inelastic interactions, zero loss filtered images provided a general view of the ultrastructural organization of the skeleton with a higher contrast than non-filtered images. Mappings of calcium and magnesium were carried out by the ratio method using two windows with a width of 5 eV (respectively before and after the considered edge). The ratio imaging method consists in mapping the ratio post-/pre-edge intensities acquired with a slit of the same width before and after the considered edge. 2.2.4. X-ray diffraction (XRD) Powder diffraction measurements were realized using an INEL diffractometer equipped with a 120° curved position sensitive detector (CPS-120), in transmission mode, at 45 kV and 20 mA. The radiation length corresponded to Cu Ka (i.e. 1,5418 Å). Samples from the superficial crests and from the center of two soft tissuecleaned basal skeletons were powdered with a mortar and pestle and measured separately in thin-walled (0.5 and 0.7 mm) capillary tubes for X-ray diffraction. The basal skeletons were cleaned and air-dried before the preparation of the powder. In order to have a better accuracy of the collected data, pure quartz was used as

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internal standard. Magnesium concentrations were estimated from the shift in the diffraction peak d104 according to the calibration curve of Goldsmith and Graf (1958). Furthermore, the relative percentage of aragonite and calcite phases occurring in skeletons was established from a calibration curve (IAr/ICal = k (MAr/MCal)). The ‘‘k’’ factor was obtained by measuring the peak intensity of powders obtained by mixing pure aragonite and calcite in proportions 20/ 80, 50/50 and 80/20. 3. Results 3.1. Spicules The spicular skeleton of P. massiliana included four types of calcareous spicules: sagittal triactines (Fig. 1a), tuning fork triactines (Fig. 1b), cruciform tetractines (Fig. 1c) and microdiactines (Fig. 1d). Crushed spicules showed conchoidal fracture patterns (Fig. 1e). However, higher magnification observation in SEM revealed a finely submicronic granular structure rather than a smooth typical crystal surface (Fig. 1f). In TEM, the granular morphology was more obvious with submicronic domains less than 50 nm in size (Fig. 1g). The electron diffraction pattern demonstrated the monocrystallinity of the selected set of grains as shown by the SAED pattern taken along [0 0 1] calcite (Fig. 1h). 3.2. Basal skeleton 3.2.1. External morphology P. massiliana can display various morphotypes according to the habitats and/or the location in the underwater cave (Vacelet, 1964; Vacelet et al., 2002; Manconi et al., 2009). The population from the cave of La Vesse, from which sponges have been collected for this study, presented different shapes from plate-encrusted or

Fig.1. Calcareous spicular skeleton of Petrobiona massiliana. Spicules types (SEM): (a) sagittal triactine, (b) tuning fork triactine, (c) cruciform tetractine, (d) microdiactine. Interne spicular structure: (e) conchoidal fracture patterns and (f) finely submicronic granular structure in a crushed spicule (SEM). (g) TEM bright-field image at the edge of a crushed spicule showing submicronic and rounded domains of less than 50 nm in size. (h) SAED pattern showing single-crystal behavior in the c-axis orientation of calcite.

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Fig.2. Macrostructure of the basal skeleton of Petrobiona massiliana (SEM). (a) Cleaned basal skeleton of a subspherical specimen with serpulid tubes at the bottom. (b) View of spiky crests delineating depressions between them. (c) Transverse section in the basal skeleton showing alternation of crests and depressions and narrow canals opening at the bottom of the latter. (d) Schematic representation of the macrostructure of the basal skeleton. C, canal; Cr, crest; D, depression; ST, serpulid tubes.

subspherical to finger-like shapes with sometimes multiple lobes. The subspherical type was the most commonly observed one. For each morphotype, the surface of the massive basal skeleton was composed of large spiky crests, up to 1 mm high, delineating depressions between them (Fig. 2a–d) in which lay the soft tissue. At the bottom of the depressions opened narrow canals extending into the massive part of the basal skeleton. The surface of the latter displayed spikes, 50–100 lm long, principally in the lower portion of the crests and at the bottom of the depressions (Fig. 3a and b). Furthermore, numerous minute spines, less than 5 lm long, conferred a rough aspect to the surface (Fig. 3c–e). The significance of those tiny protuberances will be discussed further. Elongate and sinuous layers, 20–70 lm width, described as sclerodermites by Vacelet (1964, 1990), extended laterally along the crests (Fig. 3a–c). They presented a thinner highly sinuous extremity at the bottom of the crest. At the surface of these sclerodermites, minute spines were single cone-shaped and often cooriented (Fig. 3c). The extremities of crests terminated in globulous finger-like structures (Fig. 3a and b). Wider flake-shape sclerodermites coated the bottom of depressions resembling lava flows (Fig. 3d) with superficial minute spines less co-oriented than those on elongate sclerodermites and often presenting growth in multiple directions (Fig. 3e). Finally, those superficial layers also extended on serpulid tubes occurring on the basal surface of the skeleton (Fig. 3f). 3.2.2. Internal morphology In transverse cryofracture, a crest of the basal skeleton appeared as a superposition of elongate sclerodermites covering an initial deposit (Fig. 4a and b). Each of these sinuous sclerodermites, including the initial deposit, presented a radiating microstructure when seen in transverse section (Fig. 4c and d). The fibrous composition of this microstructure was highlighted by TEM observation (Fig. 4e). Fibrous structures, 50–100 nm in width and 300 nm to 10 lm long, were closely packed in a fibroradiate organization. The latter, frequently partially tilted upward the crest, was

repeated along the longitudinal axis of the sinuous layer (Fig. 4c). Accordingly, the longitudinal section of the apical portion of the layer gave rise to a pennular arrangement (Fig. 4f–h). In ontogenetically younger superficial elongate sclerodermites, the fibrous texture could be more easily recognized than in highly compact mature layers. The transverse cryofracture of Fig. 5a and b represents a superposition of two layers recently deposited on the crest. Moreover, as observed at higher magnification, the microstructure is actually composed of granular fibers of different lengths laterally associated (Fig. 5b and c). In deeper and thus older parts of the same growing sclerodermite, fibers were more closely packed or even fused together (Fig. 5d). According to some fracture orientations, they appeared somewhat forming stacked planes (Fig. 5d). However, in the most superficial portion of the sclerodermite (i.e. ontogenetically younger), individual or clusters of submicronic grains from 50 to 100 nm large, have been observed instead of fibers (Fig. 5e). Such individual grains have also been observed between bundles of fibers from deeper parts of sclerodermites and, in this case, might represent a way to fill up spaces produced by the fibro-radiate organization. Interestingly, flake-shape sclerodermites covering the bottom of depressions were not organized as fibers but as layers of the same closely packed submicronic grains (Fig. 6a and b). Sharp spikes rose up from the basal skeleton (Figs. 3a and 7a). Their main surface presented a coat with morphological patterns similar to spiny superficial sclerodermites observed on the entire skeleton, although seemingly lightly etched on the Fig. 7a. In longitudinal polished sections (LM, Fig. 7b) as well as in transverse cryofractures (SEM, Fig. 7c), these spikes exhibited, respectively, a light translucent and compact core surrounded by a radiating fibrous ring typical to the already observed basal skeleton microstructure except for their tip which was always externally smooth and internally compact. Minute superficial spines appeared co-oriented with underlying fibers of the elongate sclerodermite (Fig. 8a). Cryofracture revealed sometimes a central long filament, deeply anchored in the

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Fig.3. External macro- and microstructure of the basal skeleton of Petrobiona massiliana (SEM). (a and b) Crest viewed from above with globular tips, lateral sinuous elongate sclerodermites, spikes, and canals at the bottom of the depression. (c) Lateral sinuous elongate sclerodermites along a crest with co-oriented superficial minute spines. (d) Flake-shape sclerodermites covering the bottom of a depression, with less co-oriented minute spines. (e) Close-up view of minute spines on a sclerodermite in a depression. (f) Sclerodermites colonizing (arrows) the surface of a serpulid tube. C, canals; D, depression; FS, flake-shape sclerodermites; GT, globulous tip; SES, sinuous elongate sclerodermites; Sp, spines; ST, serpulid tube.

sclerodermites, around which small granular fibers radiated (Fig. 8b and c). The latter organization was separated from the perpendicularly oriented fibroradiate microstructure of the rest of the basal skeleton by other filaments or even a sheath (Fig. 8b). This peculiar organization was not observed below minute spines of flake-shape superficial sclerodermites lying in the bottom of depressions. Finally, the four types of spicules have been observed partially (Fig. 9a) or entirely (Fig. 9b) entrapped in the massive basal skeleton. However, they did not appear to undergo diagenetic changes. They maintained their compact monocrystalline structure while the growth of surrounding fibers from the basal skeleton seemed to be blocked at their sharp contact (Fig. 9b and c). 3.2.3. Diffraction patterns 3.2.3.1. Electronic diffraction (SAED). Identical diffraction patterns were always obtained in different areas of the same fiber (Fig. 10a), indicating crystallographic coherency along granular fibers composing the fibroradiate microstructure. Furthermore, crystallographic coherency has been also revealed in a selection of adjacent fibers clustered in bundle (Fig. 10b). The fibers elongate occasionally but not always along the c-axis of calcite. The monocrystallinity was expected, as continuous Bragg fringes

were crossing over the entire bundle when tilting the sample. The fibroradiate organization of contiguous fibers (ca. 50– 100 nm width) was also shown in TEM (Fig. 10c). The diffraction patterns display only approximate single-crystal features, in the form of slight rotations of crystallographic positions between adjacent fibers (Fig. 10d). Additionally, between fibers or bundles of fibers, thin layers of material were observed that might correspond to some organic substance as it was rapidly altered under the beam (Figs. 10e and 11a and c). Furthermore, Moiré fringes sometimes appeared between adjacent fibers (Fig. 10e), confirming slight misorientations of the crystal lattices between adjacent fibers. In transverse sections, fibers displayed a granular morphology that might however also correspond to the section of individual grains observed in flake-shape sclerodermites or between fibers of elongate sclerodermites (Fig. 11a and c). A crystallographic coherency was still present, although less strict (Fig. 11b). Intriguingly, two deformation patterns occurred between contiguous fibers (or grains) leading either to curved or plane surfaces at their contact (Fig. 11c). This pattern occurred when a mushy roundshape structure gets in touch with, respectively, another soft or hard round structure.

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Fig.4. Internal microstructure of the mature basal skeleton of Petrobiona massiliana. (a) SEM image of a crest in transverse fracture showing superposition of elongate sclerodermites around an initial deposit. (b) Schematic drawing of (a). (c) Sketch of an elongate sclerodermite with partially tilted fibroradiate microstructure repeated along the growth axis. (d) Transverse fibroradiate organization in a superficial elongate sclerodermite growing on a crest. (e) Bright-field TEM micrograph with diffraction contrasts highlighting juxtaposed fibers of the fibroradiate microstructure. Pennular organization of elongate sclerodermites in longitudinal section: (f) TCA etched section (SEM), (g) Schema of the pennular organization in longitudinal section, (h) ground section (LM). Cr, crest; GA, growth axis of the sclerodermite; ID, initial deposit; SES, sinuous elongate sclerodermite.

3.2.3.2. X-ray diffraction. In order to check whether calcite was the only crystal phase present and to compare shifts of the d104 peak induced by incorporated magnesium ions in different skeletal parts, X-ray diffraction analyses of powders from the center and from apical crests of two cleaned basal skeletons were carried out. The X-ray diffraction analyses from the center of the skeleton and crests of both specimens showed a decrease of d104 values of 0.022 Å (±0.001) to 0.031 Å (±0.001) in comparison to the pure inorganic calcite value (3.035 Å) indicating the presence of magnesium ions in the lattice of the skeletal calcite in concentration, respectively, from 6.8 to 9.5 mol% MgCO3, according to the Goldsmith and Graf’s calibration curve (1958). The same shift was recorded from powders of the center and apical crests of both cleaned skeletons, while it was different between both individuals. Finally, only the center of the first specimen presented 3.85% (±0.85) of aragonite in addition to the Mg-calcite.

The zero loss-filtered image (EFTEM, Fig. 12a) provided a general view of some longitudinally sectioned contiguous fibers. EDX elemental (Fig. 12b) as well as EELS analysis both confirmed the presence of Ca and Mg in the selected area. Mappings of calcium and magnesium, carried out by the ratio method using two windows 5 eV wide, are shown in Fig. 12c and e. Higher calcium content (with higher contrast) was shown as lighter central or lateral bands along most fibers (Fig. 12c), revealing a heterogeneous spatial distribution of calcium (Fig. 12d). A high Ca-content was particularly clear in fiber 1 as demonstrated by an enhanced Ca signal in its selected central band (Fig. 12d). Magnesium was globally more homogenously distributed than calcium in mapping. Nevertheless, in the lower part of fiber 1, a lower longitudinal distribution of magnesium signal (Fig. 11e) was even inversely correlated to higher Ca concentrations, as highlighted when comparing corresponding signals in the same selected area (see quadrants) (Fig. 12d and f).

3.2.4. Chemical analysis The granular submicrostructure of the basal skeleton led us to verify whether some variation of Mg and Ca distribution occurred at this scale. Electron energy loss spectroscopy (EELS) was used to provide, at the same time, high energy and scale resolution in the mapping of those elements in ultra-thin sections of the basal skeleton produced by precise ions polishing system (PIPS).

4. Discussion The macromorphology of the basal skeleton of P. massiliana is defined by a massive basal part covered by crests alternating with depressions. In the bottom of the latter, narrow canals open and extend deeply into the massive part. These openings are filled by a peculiar portion of soft tissue, ‘‘trabecular tracts’’, containing

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Fig.5. Internal submicro- to microstructure of growing elongate sclerodermites of Petrobiona massiliana (SEM). (a) Transverse fracture of two superposed elongate sclerodermites growing laterally on the crest. (b) Enlargement of (a) illustrating the superposition of the elongate growing sclerodermites and (c) their granular fibers composition. (d) Lateral association of ontogenetically older granular fibers. (e) Closely packed submicronic grains (ca. 50–100 nm large) in the superficial part of a growing elongate sclerodermite (i.e. ontogenetically younger). Cr, crest; SES1, sinuous elongate layer 1; SES2, sinuous elongate layer 2.

Fig.6. Internal submicro- to microstructure of growing flake-shape sclerodermites at the bottom of depressions of the basal skeleton of Petrobiona massiliana (SEM). (a) Fracture in a sclerodermite highlighting a granular microstructure. (b) Enlargement of closely packed submicronic grains (ca. 50–100 nm large). MS, minute spine.

storage cells (Vacelet, 1964, 1990). While crests could be considered as an active growth area of superposed elongate sclerodermites, mineralization seemed to be very slow or even absent in these narrow canals as they are still observed empty in deep older parts of the massive skeleton. Therefore, superficial crests could be regarded as preferred sites of mineralization. Vacelet (1964) suggested that the absence of the potentially mineralizing basopinacoderm in canals hollowed out in the skeleton, could explain that skeletal growth does not occur in such portions. Nevertheless, this assumption has to be checked by an investigation of real cellular mechanisms implicated in the biomineralization of P. massiliana. Sinuous elongate layers cover the surface of crests and flakeshape layers the bottom of depressions until the beginning of narrow canals. In his first study of P. massiliana, Vacelet (1964) observed the former as spherulitic bodies regularly assembled on crests while deformed and more stratified in depressions. Later, he defined them as ‘‘sclerodermites’’ (Vacelet, 1991). The external stratification of these elongate sclerodermites as well as their superposition featured in transverse fracture of crests obviously demonstrate that the latter are formed by successive addition of layers. Furthermore, their highly sinuous shape may indicate their biologically controlled formation by mobile cells. The formation of the basal skeleton by addition of elongate or flake-shape newly formed sclerodermites, would explain the spatially discontinuous calcein labeling observed by Hermans et al. (2010) in a growth rate investigation of P. massiliana. Calcein lines corresponded in size

and shape to the external boundaries of sclerodermites, curved or flatter if revealed, respectively, on crests or in skeletal depressions (Fig. 3. in Hermans et al., 2010). At the microstructural scale, sclerodermites are organized differently, depending on their location. The wider flake-shape sclerodermites observed at the bottom of depressions do not show a fibrillar organization but instead a compact granular microstructure. The elongate sinuous sclerodermites could be schematized as semi-cylindrical shapes presenting a fibroradiate microstructure in transverse section, with their fibers radiating obliquely from the longitudinal axis. The microstructural fibrous organization of sclerodermites in P. massiliana strongly reminds the fibrous assemblage found in coral skeletons (Cuif and Dauphin, 2005a,b; Stolarski, 2003). In the center of crests or at the very top of smaller ones, the first deposited sclerodermite could be further characterized as a whole cylinder with fibers radiating all around a central longitudinal axis. Therefore, as this first sclerodermite deposited during crest formation (‘‘initial deposit’’) displayed a similar fibrous microstructure than other sclerodermites, they could not be functionally compared with early mineralizing zones found in coral skeletons (e.g. Cuif and Dauphin, 2005a), which structurally differ from the surrounding fibrous microstructure zones. When observed in transverse section, the first sclerodermite displayed a spherulitic-like structure, although fibers are radiating from a central linear core rather than from a spherical one, as also pointed out by Gautret (1986). These fibroradiate features led some authors to

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Fig.7. Spikes rising up from the basal skeleton of Petrobiona massiliana. (a) External morphology with main surface similar to the basal skeleton and smooth tips (SEM). Internal morphology exhibiting, respectively, (b) a light translucent (LM) or (c) compact core (SEM) surrounded by a radiating fibrous ring. Co, core; Mi, microdiactine; Ri, ring; SmT, smooth tip.

Fig.8. Co-oriented minutes spines on elongate sclerodermites of Petrobiona massiliana (SEM). (a) Fracture at superficial minute spines (arrows) level indicating their coorientation with underlying fibers of the elongate sclerodermite. (b and c) Minute spines composed of a central filament, anchored in the sclerodermite, surrounded by radiating small granular fibers. CF, central filament; MS, minute spines; RF, radiating fibers; SES, sinuous elongate layer.

Fig.9. Incorporation of spicules in the basal skeleton of Petrobiona massiliana (SEM). (a) Partially entrapped microdiactine in a sclerodermite. (b and c) Entrapped spicule in the mature basal skeleton without diagenetic changes (SEM, TEM). BS, basal skeleton; Sp, spicule.

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Fig.10. TEM and SAED of submicronic structural units of the basal skeleton of Petrobiona massiliana in longitudinal sections. (a) TEM micrograph of a fiber in longitudinal section with similar SAED patterns from both extremities demonstrating the single-crystal behavior of the fiber. (b) TEM micrograph of fibers laterally assembled into monocrystalline (SAED) bundles. (c) TEM micrograph illustrating fibroradiate organization of adjacent fibers differently oriented (various diffraction contrasts) or laterally assembled (same diffraction contrasts). (d) SAED pattern from the selection in (c) showing approximate single-crystal feature marked by spotty arcuate reflections, i.e. slight rotations of crystallographic positions between selected contiguous fibers. (e) TEM micrograph showing Moiré fringes at fibers boundaries due to overlap of misaligned crystal lattices and rapidly altered thin material between contiguous fibers. Bu, bundle; F, fiber; RAM, rapidly altered organic material.

conclude that the microstructure of P. massiliana was spherulitic (Stolarski and Mazur, 2005; Vacelet, 1964; Wendt, 1979). Moreover, the pennular-shape or feather-like microstructure described by Cuif et al. (1979), Gautret (1986) and Cuif and Gautret (1991) corresponds to longitudinal sections of semi-cylindrical sclerodermites. Furthermore, the microstructure appeared even more complex, as some authors have considered the basal skeleton of P. massiliana as a composite organization with spherulitic, irregular or pennular microstructures (Cuif et al., 1979; Gautret, 1986). By systematically linking our structural observations with the orientation of sections or fractures, we can state that all previous descriptions actually correspond to different views of a same microstructure in an elongate sclerodermite. Numerous spikes project from the basal skeleton surface principally in lower portions of crests and at the bottom of depressions. In longitudinal or transverse sections, they exhibit a light translucent (LM) or compact (SEM) core surrounded by a radiating fibrous ring typical of the microstructure observed in elongate sclerodermites. The translucent or compact feature of the core was very sim-

ilar to the internal appearance of calcareous spicules isolated from the soft tissue. Moreover, the regular length of spikes (about 100 lm long) and their persistently smooth edge relate to rays of sagittal triactines, the most abundant spicules. Therefore, we suggest that spikes are projecting rays of spicules entrapped in the basal skeleton. While their main surface was progressively coated by the typical fibrous organization of superficially formed sclerodermites, their tip, emerging in the soft tissue, may have been kept smooth as it was separated from the calcifying process. This spicular origin of spikes on the skeleton of P. massiliana had been proposed by Reitner (1989) while this interpretation was not supported by other authors (Vacelet, 1991; Hermans et al., 2010). However, Reitner (1989) claimed that the fibrous ring around the compact core of the spike was a diagenetic change of the spicule occurring in a hypothetic ’’extrapinacodermal mucus’’. Minute crystal-shaped spines abundantly covered the surface of the basal skeleton. In elongate sclerodermites, they were more co-oriented with each other with their growth axis following underlying fibers of the sclerodermite. This contrasts with minute

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Fig.11. TEM and SAED of submicronic structural units of the basal skeleton of Petrobiona massiliana in transverse sections. (a) TEM micrograph of transversally sectioned fibers or grains. (b) SAED pattern from the selection in (a) showing single-crystal behavior of a few transversally sectioned fibers or grains. (c) TEM micrograph illustrating deformation contacts between contiguous fibers or grains. (Arrows: curved surface. Arrowheads: plane surface). Bu, bundle; RAM, rapidly altered organic material.

spines observed on flake-shape sclerodermites located in depressions, which exhibited more irregular orientation. This could be linked to the absence of fibrillar radiating organization of these sclerodermites. In some fractures, oriented minute spines found on elongate sclerodermites seemed to be composed of a central long filament, deeply anchored within the sclerodermite, surrounded by small radiating granular fibers. However, this unexplained peculiar organization was not found below minute spines of flake-shape superficial sclerodermites lying at the bottom of depressions. According to Reitner (1989), minute spines represent prismatic crystals generated by epitactic overgrowth of ‘‘single crystallites’’ with a typical rhombohedral crystal shape. However, the continuity of minute spines with underlying fibers of the sclerodermites makes this epitactic interpretation unconvincing. Nevertheless, some of them (as Plate 6, Fig. 3 in Reitner, 1989 and Fig 6a in the present study) could also be dissolution– recrystallization events induced by washing or fixation treatments, or by uncontrolled contact with seawater even during the life of the sponge. In order to identify micronic to submicronic growth units of the basal skeleton we investigated sclerodermites in younger stages of formation rather than in partially decalcified samples. In growing superficial elongate sclerodermites, fiber composition and arrangement were more easily recognizable than in the more compact mature ones. An ontogenetic process was revealed, extending from superficial individual submicronic grains (less than 100 nm large) to radiating granular fibers of various lengths more or less laterally assembled, reaching finally bundles of fused fibers in the deepest part of the sclerodermites. Therefore, the initial formation of fibers and their subsequent lateral association are likely to be two distinct ontogenetic stages occurring during formation of these sclerodermites. Furthermore, the same submicronic grains (50– 100 nm) were found in fractures of flake-shape sclerodermites growing at the bottom of depressions. These grains are not organized into fibers but are closely packed together and delineate bands probably corresponding to different growth periods that suggests some parallel with the ‘‘two step mode of growth’’ found in scleractinian coral skeletons formation (Cuif and Dauphin, 2005b). Therefore, we suggest that submicronic grains represent

the smallest mineralogical units that are later assembled in fibers in elongate sclerodermites and closely packed with each other in flake-shape sclerodermites. The granular submicronic morphology of the basal skeleton has been previously suggested in P. massiliana by atomic force microscopy (Stolarski and Mazur, 2005), although it was shown only in partially decalcified sections of the skeleton. The occurrence of these grains (50–100 nm) in the intact but ontogenetically nonmature basal skeleton allows us to confirm their function as a first step of biomineralization in this sponge. Furthermore, in fractured spicules of P. massiliana, observed in SEM and TEM, a finely submicronic granular structure (<100 nm) rather than a smooth typical crystal surface could be recognized as well after a slight etching. Similar submicronic grains found in calcitic spicules of two other Calcarea, Pericharax heteroraphis (ca. 10–50 nm; Sethmann et al., 2006; Sethmann and Wörheide, 2008) and Leuconia johnstoni (ca. 60–120 nm; Kopp et al., 2011) support this observation. The production of skeleton by assemblage of submicronic grains might be a universal biomineralization feature, since it has been described, in the last few years, in other biogenic calcium carbonate: coral skeletons (Isa, 1986; Cuif and Dauphin, 2005a,b; Dauphin et al., 2008; Cuif et al., 2008; Przeniosło et al., 2008; Sethmann et al., 2007; Stolarski, 2003; Stolarski and Mazur, 2005), mollusk shells (Baronnet et al., 2008; Cuif et al., 2008; Dauphin, 2003; Okumura et al., 2010; Rousseau et al., 2005) and echinoderm skeletons (Robach et al., 2005; Stolarski et al., 2009). Transverse section of contiguous fibers (or cluster of grains) showed in TEM either curved or plane surfaces at the contact of each other. This relevant feature suggests that grains (or fibers) had a mushy consistence before their crystallization. A curved surface would occur when a mushy round-shape structure (submicronic grain or part of a fiber) comes in contact with a hard one. A plane surface would arise when two adjacent mushy grains/fibers are tightly squeezed up against each other. This suggested the occurrence of a mushy stage during calcium carbonate biomineral formation, before crystallization of the smallest mineralogical structures. An appealing correlation could be made between this soft stage and the occurrence of an amorphous phase, since it is currently being suggested that some stable crystalline biogenic cal-

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Fig.12. Calcium and magnesium distribution in the basal skeleton of Petrobiona massiliana at the submicronic scale. (a) Zero loss-filtered image (EFTEM) of four longitudinally sectioned contiguous fibers (F1–F4). (b) EDX elemental analysis confirming the presence of Ca, Mg and S in the selected area. (c) Filtered image on calcium edge corresponding to Fig. 11a using the ratio method (slit width of 5 eV). Linear higher Ca-content appeared in each fiber as longitudinal central or lateral pale bands (arrows). (d) Profile of the integrated intensity of calcium signal extracted from the quadrant selection in the Fig. 11c demonstrating heterogeneous spatial distribution of calcium. The vertical unit «e » in Fig. 12d and f corresponds to the variation of the post-/pre-edge intensities ratio. (e) Filtered image on magnesium edge corresponding to Fig. 11a. showing a more homogenous distribution than for calcium using the ratio method (slit width of 5 eV). (f) Profile of the integrated intensity of magnesium signal extracted from the quadrant selection in the Fig.11e. In fiber 1, a lower Mg-content in the sub-quadrant selection (f) is correlated with higher Ca signal (Fig. 11d) as indicated by arrowheads.

cite or aragonite develop by a non-classical mineralization pathway involving an amorphous calcium carbonate precursor phase, the so-called transient ‘‘ACC’’ (Addadi et al., 2003, 2008; Beniash et al., 1997; Levi-Kalisman et al., 2002; Loste et al., 2003; Raz et al., 2000, 2003; Wang et al., 2009; Weiner et al., 2003, 2005; Weiner, 2008). Amorphous calcium carbonate as permanently stabilized or as precursor transient phase, is a biomineralization strategy being selected by widely separated organisms in the animal kingdom: sponges (Aizenberg et al., 1995, 1996, 2003) corals (Meibom et al., 2004), ascidians (Aizenberg et al., 2002), mollusks (Baronnet et al., 2008; Cuif et al., 2008; Marxen et al., 2003; Weiss et al., 2002) crustaceans (Dillaman et al., 2005; Raz et al., 2002) and echinoderms (Beniash et al., 1997; Killian et al., 2009; Ma et al.,

2007; Politi et al., 2004, 2006, 2008; Raz et al., 2003). However, amorphous calcium carbonate in biominerals is often described as a solid phase. Therefore, the occurrence of an initial disordered mushy phase in submicronic grains before their crystallization in the basal skeleton of P. massiliana, might suggest that another ions-rich phase with soft consistency may exist either before or instead of a solid ACC phase. In spite of their submicronic granular composition, we found that each fiber from the fibroradiate microstructure of elongate sclerodermites behaved as single-crystal, since similar SAED patterns obtained from extremities of fibers revealed crystallographic coherency. Moreover, morphological observations in SEM have pointed out that grains or fibers are, in a further step, arranged

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in clusters or bundles. SAED studies revealed that they are crystallographically coherent units as well. These data lead us to propose the model depicted in Fig. 13. In a first step, submicronic mushy amorphous grains, probably limited by an organic envelope, would coalesce in an organized way forming a fiber (Fig. 13a) in elongate sclerodermites or granular clusters (Fig. 13b) in flake-shape sclerodermites. An unknown nucleation signal would then or in the same time initiate crystallization that would propagate from one grain to another in the fiber or in the cluster, resulting in the progressive transformation of the mushy amorphous phase into longrange ordered single-crystal (Fig. 13, dotted arrows). Similarly, crystallization would propagate even across adjacent fibers through lateral association leading to co-oriented fibrillar bundles (Fig. 13c). Deformation features between adjacent grains or fibers would occur before or during crystallization propagation (Fig 13c and d). In the former case, the deformation would correspond to a compression caused by the addition of new structures against still mushy or already crystallized ones. In the second case, the crystallization event would make the grains or fibers hard which might induce the deformation of adjacent mushy structures. It is highly probable that grains and fibers are enveloped in an organic material, since thin and rapidly altered amorphous material was observed in TEM leading to gaps a few nanometers large between grains, fibers or bundles. This organic envelope would limit crystallization propagation between structural units and therefore preserve their amorphous state. Robach et al. (2005) identified occluded macromolecules in similar spherical cavities between nanodomains of sea urchin tooth calcite. Furthermore Sethmann et al. (2006), Sethmann and Wörheide (2008) and later Kopp et al. (2011) showed the presence of an intercalated organic matrix between submicronic granular units composing calcareous sponge spicules. The requirement of membrane compartmentalization of a

precursor amorphous phase for its transient stabilization has been purposed for example in the case of sea urchin larvae spicules (Addadi et al., 2003; Beniash et al., 1999). On the contrary, rupturing the organic envelope in our model would offer a route of propagation from already crystallized grains (Fig. 13c) or fibers (Fig. 13d) toward still amorphous neighboring ones. The occurrence of organic material in the basal skeleton of P. massiliana is under investigation. Such biomineralization mechanism involving crystallization propagation through submicronic amorphous granular units supports previous similar models for the formation of scleractinian coral fibers (Przeniosło et al., 2008), calcitic prisms of mollusk shells (Baronnet et al., 2008; Cuif et al., 2008), sea urchin larval spicules (Politi et al., 2008) and sea urchin tooth (Killian et al., 2009). Both latter authors further suggested that transformation of ACC nanoparticles into long-range coherent calcitic crystal in, respectively, the sea urchin spicule and tooth is a solid-state transformation. It would occur through a secondary nucleation during which each already-crystallized nanograin acts as a nucleation template for neighboring still amorphous ones (Killian et al., 2009; Politi et al., 2008). The propagation of crystal orientation through these nanodomains has been explained by occurrence of connecting bridges (Killian et al., 2009; Przeniosło et al., 2008; Rousseau et al., 2005) probably formed on an organic template. The basal skeleton in P. massiliana is composed of magnesium calcite as previously established by X-ray diffraction analysis (Vacelet, 1964; Wendt, 1979), energy dispersive spectroscopy (EDX, Reitner, 1989) and inductively coupled plasma atomic emission spectroscopy (ICP-AES, Hermans et al., 2010). However, we exceptionally found at the center of one sample 3.85% of aragonite. This small amount of aragonite could be either attributed to a naturally induced diagenesis event or to boring activities of clionid sponges

Fig.13. Model of submicronic mushy amorphous grains assemblage, idealized from observations of the basal skeleton of Petrobiona massiliana. (a) Formation of single-crystal fiber of elongate sclerodermites by linear fusion of submicronic amorphous grains and crystallization propagation. (b) Formation of granular single-crystal cluster in a flakelike sclerodermite by random fusion of submicronic amorphous grains and crystallization propagation through fused grains. (c) Crystallization propagation between contiguous fibers leading to co-oriented fibrillar bundles through connecting bridges (disruption of the organic envelope?). (d) Curved or plane contact deformations occurring, respectively, between a grain or fiber (1) against a still mushy amorphous one (2) or between two contiguous still mushy amorphous grains or fibers (2 and 3).

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or to a loss of polymorphism control by the organism during biomineralization. Reitner (1989) and Hermans et al. (2010) found higher Mg concentrations in the basal skeleton of P. massiliana, respectively, by EDX (2.86–14.00 mol% MgCO3) and by ICP-AES (11.477 ± 0907 mol% MgCO3) analyses than we have estimated through our XRD analysis (i.e. 6.8–9.5 mol% MgCO3). This discrepancy could be explained either by a high interindividual variation or by occurrence of Mg ions outside of the calcite lattice, detected by EDX and ICP-AES analysis but not by XRD. Such additional Mg might then be associated to intra-skeletal organic macromolecules, as suggested for Mg-calcite sponge spicules (Sethmann et al., 2006; Sethmann and Wörheide, 2008). Nevertheless, Politi et al. (2010) support in their recent study on Mg role in ACC stabilization, that most Mg ions are in lattice positions. On the two samples investigated here by XRD, an important variation of Mg concentration between both samples was shown whereas no intra-individual difference between bulk powder from crest and center of the basal skeleton appeared. The latter observation contrasts with large range spatial variation of Mg content obtained by different EDX point analysis in a unique specimen by Reitner (1989). Nevertheless, this spatial variation might be hidden in our XRD results on bulks (crests or center) instead of micrometric resolved points. However, at the submicronic range, we highlighted slight spatial variation of Mg by EFTEM images (EELS) of skeletal fibers in P. massiliana. According to some sections, higher Mg content was negatively correlated to Ca distribution. In the last decade, it became well admitted that most biologically controlled biomineralizations do not follow rules of classical inorganic mineralization from a super-saturated solution (Addadi et al., 2006; Weiner et al., 2005; Weiner, 2008), allowing organisms to produce higher magnesium calcite even in ambient conditions. Production of biogenic calcite through a partially disordered transient amorphous phase is most likely one of the biomineralization pathway that could explain higher magnesium to be incorporated in the calcite lattice (e.g. Hermans et al., 2011; Loste et al., 2003; Raz et al., 2000; Wang et al., 2009). If such transient ACC phase occurred during the basal skeleton formation in P. massiliana, it might therefore explain its high-magnesium calcite composition. EFTEM images highlighted a spatially heterogeneous distribution of Ca and Mg in the calcitic fibers of P. massiliana. While this pattern may probably result from their non-classical mineralization through assemblage of amorphous submicronic grains, we could not explain it entirely without further investigation. Nevertheless, we assume that a nano-scale fibrillar organic matrix might be involved as a linear framework to organize amorphous grains into a fiber. The occurrence of organic macromolecules in this linear transient amorphous assemblage would influence ion organization in their vicinity leading to spatially unusual patterns distribution of Ca and maybe Mg along the fiber. Ca/Mg spatial segregation has been observed in synthetically produced round-shape minerals through transient ACC phase in presence of acidic organic additives (Raz et al., 2003). Organic matrix, often containing highly acidic proteoglycans and glycoproteins, is now acknowledged to play a key role in the regulation of nucleation and subsequent growth in very diverse biomineralization models (see Arias and Fernández (2008) for a review). This multi-scale mineralogical characterization of the hypercalcified sponge P. massiliana has finally brought forward a series of structural features in the basal skeleton that could not be explained by classical inorganic mineralization rules but rather underlined a highly biologically regulated biomineralization. These more ‘‘biological’’ patterns include: highly sinuous-shaped superficial layers, granular morphology instead of polyhedral calcitic crystals, formation in early ontogenesis of individual submicronic grains in a mushy state (ACC?), intercalation of an organic material

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between micron to submicron scale structural units, inter-individual variation of skeletal Mg contents and heterogeneous spatial distribution of Ca ions in fibers. Furthermore, we promote a model of crystallization propagation through amorphous submicronic units to explain the formation of coherent micron-scale structural units. Our observations on this very basal metazoan organism stress further that occurrence of submicronic granular units and a transient amorphous precursor phase are ancient and likely universal strategies in calcium carbonate skeleton biomineralization. The biological machinery (calcifying cells and organic matrix) regulating the formation of structural units revealed in the present study is currently under investigation in order to acquire a broader understanding of initial steps and of the biological control over biomineralization in this hypercalcified sponge.

Acknowledgments We thank V. Studak, J. Blanquart, D. Plunet, J.M. Roux and Ph. Guimier from ‘‘Au Delà Plongée’’ (La Vesse) for their scuba diving support, J. Cillis and L. Despontin (RBINS) for technical support. We also thank Claude Henry for accessing the microscopy facilities of the Centre Interdisciplinaire de Nanosciences de Marseille (CINaM). This work was partly funded by the CALMARS II project (No. NR SD/CS/02A-) from the Belgian Federal Science Policy and by FRFC contract (No. 2-4532.07-). Ph. Dubois is a Senior Research Associate of the Fund for Scientific Research of Belgium (F.R.S.FNRS). M. Gilis benefits a FRIA PhD Grant conceded by the FNRS (Belgium).

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