Assessment of hydrothermal alteration on micro-and nanostructures of biocarbonates : quantitative statistical grain-area analysis of diagenetic overprint

The assessment of diagenetic overprint on microstructural and geochemical data gained from fossil archives is of fundamental importance for understanding palaeoenvironments. A correct reconstruction of past environmental dynamics is only possible when pristine skeletons are unequivocally distinguished from altered skeletal elements. Our previous studies (Casella et al., 2017) have shown that replacement of biogenic carbonate by inorganic calcite occurs via an interface coupled dissolution–reprecipitation mechanism. Furthermore, for a comprehensive assessment of alteration, structural changes have 25 to be assessed on the nanoscale as well, which documents the replacement of pristine nanoparticulate calcite by diagenetic nanorhombohedral calcite (Casella et al., 2018a, b). In the present contribution we investigated six different modern biogenic carbonate microstructures for their behaviour under hydrothermal alteration in order to assess their potential to withstand diagenetic overprint and to test the integrity of their preservation in the fossil record. For each microstructure (a) the evolution of biogenic aragonite and calcite replacement by 30 inorganic calcite was examined, (b) distinct carbonate mineral formation steps on the micrometre scale were highlighted, (c) microstructural changes at different stages of alteration were explored, and (d) statistical analysis of differences in basic mineral unit dimensions in pristine and altered skeletons was performed. The latter analysis enables an unequivocal determination of the degree of diagenetic overprint and discloses information especially about low degrees of hydrothermal alteration. 35 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-249 Manuscript under review for journal Biogeosciences Discussion started: 10 July 2018 c © Author(s) 2018. CC BY 4.0 License.


Introduction
Biomineralised hard parts composed of calcium carbonate form the basis of studies of past climate dynamics and environmental change.However, the greatest challenge that all biological archives face lies in 66 their capacity to retain original signatures, as alteration ofthem starts immediately upon death ofthe organism.
67 Biopolymers decay, and inorganic minerals precipitate within as well as at the outer surfaces ofthe hard tissue 68 (e.g., Patterson and Walter, 1994, Ku et aI., 1999, Brand et aI., 2004, Zazzo et aI., 2004).70 studies addressing the evolution of parameters whieh influence diagenetic alteration,are discussed in only a 71 qualitative manner (Brand andVeizer, 1980, 1981;Swart, 2015).In particular, deciphering the sequence of __ major element data.The shells of Mytilus edulis and Haliotis ovina consist of two layers with distinct microstructures.In Haliotis ovina the two layers are composed of aragonite, whereas the shell of Mytilus edulis consists of an outer calcite and inner aragonite layers.
To reliably identify low to moderate degrees of diagenetic overprint, we investigated the behaviour of biocarbonate skeletal microstructure during hydrothermal overprinting.We conducted laboratory-based hydrothermal alteration experiments for time spans between] and 35 days, at an alteration temperature of 175°C and in the presence of Mg-rich fluid.We investigated the skeletons of two modem bivalves (Arctica islandica and Mytilus edulis), one modern stony coral (Porites sp.) and one modem gastropod (Haliotis ovina).With this selection of hard tissue we are able to investigate the influence, during alteration, of variations in mineral surface area, control by primary (inherent) and secondary (induced) porosity, the effect of biopolymer fabric and pattern of distribution within the skeleton, and the role of~he size, form, and mode III of organization ofth~iC mineral~it)
2 Materials and Methods

Test materials
Shens of the modem bivalve Arctica islandica were collected from Loch Etive, Scotland, UK.The shells are 8-10 cm in size and represent adult specimens.Pristine specimens of the scleractinian coral Porites sp. were collected at Moorea, French Polynesia (Rashid et aI., 2014).Live specimens ofthe gastropod Haliotis ovina were collected from the reef flat of Heron Island, Queensland, Australia.All shell pieces used in this study were taken from the shell of one adult specimen with dimensions of approx.8 x 6.5 cm.Shells of the 2.0Yie' : modern common blue mussel, Mytilus edulis, were collected from 5-7 m depth in the sUbtida~ofMenai Strait Wales, UK.Shell sizes varied from 5 to 6 em and represent adult animals.

Selective etching of organic matrix 131
In order to image the organic matrix in modern (reference) and hydrothermally altered shell samples as well as the mineral reference (inorganic aragonite), shells or mineral pieces were mounted on 3 mm thick 133 cylindrical aluminium rods using super glue.The samples were first cut using a Leica Ultracut ultramicrotome with glass knifes to obtain plane surfaces.The cut pieces were then polished with a diamond knife by stepwise , 135 removal ofmaterial in a series of20 sections with successively decreasing thicknesses (90 nm, 70 nm, 40 nm, 20 nm, 10 nm and 5 nm, each step was repeated 15 times) as reported in Fabritius et a1.(2005).The polished samples were etched for 180 seconds using 0.1 M HEPES (pH = 6.5) containing 2.5 % glutaraldehyde as a 138 fixation solution.The etching procedure was followed by dehydration in 100 % isopropanol three times for 10 minutes each, before specimens were critical point-dried.The dried samples were rotary coated with 3 run 140 platinum and imaged using a Hitachi S5200 Field Emission-Scanning Electron Microscope (FE-SEM) at 4 141 kV.
142 143 2.2.2 Microstructure and texture For FE-SEM and Electron Backscatter Diffraction (EBSD) analyse~ x 5 mm thick pieces were cut 145 out of the shell and embedded in epoxy resin.The surface of the embedded samples was subjected to several sequential mechanical grinding and polishing steps down to a grain size of 1 ,.un.The final step was etch polishing with colloidal alumina (particle size --0.06JlITI) in a vibratory polisher.Samples were coated with 4-6 nm of carbon for EBSD analysis, and with 15 nm for SEM visualisation.EBSD measurements were carried out on a Hitachi SU5000 field emission SEM, equipped with an Oxford EBSD detector.The SEM was operated at 20 kV and measurements were indexed with the CHANNEL 5 HKL software (Schmidt and 151 Olesen, 1989;Randle and Engler, 2000).Information obtained from EBSD measurements is presented as band contrast images, and as colour-coded crystal orientation maps with corresponding pole figures.
---The EBSD band contrast represents the signal strength of the EBSD-Kikuchi diffraction pattern and is displayed as a grey-scale component of EBSD scanning maps.The strength of the EBSD signal is high when a crystal is detected (bright), whereas it is weak or absent when a polymer such as organic matter is scanned (darklblack).

Alteration experiments
Laboratory-based hydrothermal ,alteration experinlents mimicked burial diagenetic conditions.In all experiments pieces ofshells or skeletons up to 2 em x 1 em of modem A. islandica, modem M. edu/is, modem Porites sp., and modem H. oVirza were placed inside a PTFE vessel together with 10 mL of simulated burial 176 fluid (100 mM NaCI + 10 mM MgCIz aqueous solution) and sealed with a PTFE lid.Each PTFE vessel was @ 177 placed in a stainless steel autoclave, sealed and kept in the oven at a temperature of 175°C for different 178 periods of time ranging between 1 and 35 days.After the selected time period, the autoclave was removed from the oven, cooled down to room temperature and opened.Recovered solid material was dried at room ] temperature and prepared for XRD, EBSO and EOX measurements.

Microstructure and texture ofhydrothermally altered bivalve, gastropod and coral skeletons
The shells and skeletal elements of modem Arctica isJandica, Porites sp., HaJiotis ovina and MytiJus edulis were subjected to laboratory-based hydrothermal alteration.Experiments were carried out at 175°C in the presence of a Mg-rich fluid simulating burial water.Experiment durations varied between 1 and 35 days (Fig. A3).
The amount of newly-formed calcite was determined by Rietveld analysis of XRD data (Fig. A4).
Diagrams of calcite content versus experimental time (Fig. 2) demonstrate the difference in replacement kinetics between biogenic calcium carbonates and inorganic calcite and highlight the profound influence of the biogenic microstructure on carbonate replacement reactions.In hydrothermally altered aragonitic Arctica is/andica shells new calcite fonnation starts after 4 days of alteration and progresses constantly.After 7 days of alteration most shell aragonite was replaced by calcite (Figs.2A, A4A and Casella et at.2017).In contrast, the hard tissue of Porites sp. and of Ha/iotis ovina respond differently to alteration.Replacement of their 230 biogenic aragonite by newly-formed calcite is significantly slower in them compared to that in the shell of 231 Arctica islandica, such that after 35 days of alteration only 20 to 30% of biogenic aragonite is replaced by ---S ty e5 of Yt1vr-pho )~! ?! 5 Mvr-pro!o,a{ ~ :! f/-,'( ,,> the amount of newly funned caldte(1) calcite~~~2C04B, A4D).For all invost;gated~~, is ~o~ a continuous functi::~ fV1 € t\'1', 0 11 u..~;;-;-(,\~ r ); Q-hu .e,I) i F;j-:) A, material is hit by the electron beam, the backscattered signal is high and light grey colours form the image.
When an organic component is scanned, the backscattered diffraction signal is absent, and the band contrast measurement image is black.Carbonate mineral co-orientation strengths are given as MUD values (reference).These are derived from pole density distributions and are quoted for each EBSD scan.Figs. 3 to 5a M+,i'35 5 and A5 to A8 show the differences in microstructure and texture between pristine~and the most advanced stage of alteration carried out in this study (35 days, at 175°C in a Mg-rich fluid).At these conditions aragonite prisms in the shell of modem Arctica islandica (Fig. A5A) are quickly and almost completely replaced by inorganic calcite (Fig. A5B).In the modem shell, aragonite prisms are surrounded by a thin network of organic fibrils.These are easily destroyed with hydrothermal alteration, and space is created for fluid percolation and a pervasive and quickly progressing replacement of the biogenic aragonite by inorganic calcite.Calcite nucleation and growth in Arctica islandica shells start after a dormant period of about 4 days with a lower degree of crystallographic co-orientation.With progressively longer alteration large an@;:.'~ ~~ere is a resemblance intb~~i mineral unit morphology, size,"existence of primary porosi~d If"! fabric of occluded biopolymers between the prismatic shell part of Haliotis ovina and that of Arctica ,-Hou.k,/Q,'.) islandic~khe kinetics of carbonate phase replacement is distinct for the two microstructures (Figs.2A, 2C). /the..
While inlArctica islandica shelJ1.yplacementbetween carbonate phases is rapid and extensive, in the prismatic -IJI t-I-.o-tshell layer of Haliotis ovina it is slow and patchy.In Haliotis ovina we find prismatic shell areas wkieh are completely replaced by calcite, while in ther shell region some aragonite is still preserved and frames the newly-formed calcite grains (Fig. AI0B).In addition, the difference between pristine and altered prismatic

The dynamic evolution of hydrothermal alteration
Major changes to the microstructure that develop with different alteration times are depicted in Figs.
A9 to A1I.For all investigated skeletons one of the first steps in the alteration process is an increase in basic mineral unit dimension relative to that present in the pristine skeleton.In the Porites sp.coral skeleton, individual spherulites grow together (white stars in Fig. A9B, A9C) and form large and compact entities.
Even though the alteration fluid accessed the skeleton from all sides, calcite formation in Porites sp.starts within the skeleton and proceeds outward toward the outer perimeter of the hard tissue (Fig. A90).An increase in mineral grain size with progressive alteration can also be observed for both microstructures that constitute the shell of Haliotis ovina (Figs.Al 0) and that ofMytilus edulis (Figs.A I I).As the organic sheaths around the basic mineral units decompose, space becomes available for new mineral fonnation.Aragonite prisms, calcite fibres, and nacreous tablets increase in size until they abut each other.In particular, the nacreous microstructure, irrespective of its specific arrangement into columns or sheets, and the calcite fibres form compact entities in response to alte~tion.In addition to an increase in fibre dimension, Mytilus edulis calcite fibre morphology becomes highly distorted with progressively longer alteration.Even though the prisms of the prismatic shell layer in Haliotis ovina also amalgamated, due to their slightly rounded and irregular morpholo~oids get entrapped.The resulting structure becomes more .compactthan the pristine nacre, but not as compact as,.p~bk if smaller pores had formed within the tablets.

/'..
A further characteristic caused by hydrothennal alteration is the significant rise in porosity within individual basic mineral units (Fig. 6).Even though the latter grow together at their perimeters (Fig. 7) a multitude of nanopores develop within them, due to the decomposition of biopolymer fibrils, which were present in the pristine hard tissue (e.g., Griesshaber et aI., 2013, Casella et aI., 20 18a, 20 18b).In contrast, as Fig. 8 shows, the inorganic calcite that fonns from the altered biogenic aragonite is almost devoid of pores.
The patches of pores that are visible within the calcite (white arrows in Fig. 8) are all residues of the incorporated altered biogenic prismatic aragonite.Our results indicate that major features of the mesoscale original microstructure are retained even at advanced stages of alteration (Fig Our results highlight that among all investigated microstructures, the nacreous microstructures were most resistant to hydrothennal alteration for 35 days at 175°C in a Mg-rich fluid, irrespective of tablet thickness or their mode of assembly (columns or sheets).We observed that replacement of biogenic nacreous aragonite by inorganic calcite takes place in stages with vl!-rious microstructural and chemical intennediates.
These are described in detail for Haliotis ovina nacre, as illustrated in Figs.9-11 and A 13-A15.Alteration of bivalve and gastropod nacre starts with the decomposition oforganic biopolymers, which is followed by tablet amalgamation and the generation of increased porosity within the tablets.Ongoing alteration destroys the tablet assembly (blue stars in Figs.9A, 9B) up to the complete obliteration of the nacreous structure (yellow stars in Figs.9A, 9B, lOA, lOB).However, as the phase map in Fig. 9E shows, at that stage of overprint, a phase replacement of biogenic aragonite by inorganic calcite has not yet occurred.Thus, when altered, the microstructure is destroyed first; replacement ofone carbonate ph~e br the Ikother occurs subsequently (Fig.   Despite the ~ne carbonate phase into another, the newly formed calcite retained much of the f\ original mesoscale morphology of the basic mineral units inherited from the pristine biogenic skeleton.

Discussion
f hcrr Biomineralised tissue provides the bulk offossil material ~ is used for geochemical analysis.As +haf all fossil archives are overprinted to some degree, it is of major importance to identify those wftieh are subject to minor and moderate degree~ ~ overy>rint, as (I) these are the materials t.hat ~til1 contain mostly primary Ide~il+-iu,J,OI'\ cha (JPY!fe.~ information, an<itt(2) the deteetioR",of extensive overprint does not pose a p,obleml\as that microstructure is either highly distorted or completely destroyed.The latter two characteristics are easily identified, while, in contrast, microstructures with a low to moderate degree of overprint are difficult to recognize and to detect.the reaction is negative.A further source of porosity development during mineral replacement relates to the difference in solubility between the primary and secondary phases (Pollock et at.2011).Porosity is generated when the primary phase is more soluble than the secondary phas~ a small amount ofthe latter precipitates -after dissolution of the former.In the case of carbonates, even though the solubility of biogenic aragonite is 394 higher than the solubility of inorganic calcite, the solubility difference is not large enough to compensate the 395 positive volume change in the dissolution-recrystallization reaction.A positive molar volume change of only 396 8.12 % is associated with the replacement of aragonite by calcite (Perdikouri et aI., 2011, 2013).
These authors immersed inorganic aragonite in pure water and in solutions that contained calcium and carbonate, with the solutions being saturated with respect to calcite and undersaturated with respect to aragonite.In experiments that were carried out with water, a replacement was not observable, even after an I entire month, unless the solution temperature was equal or higher than 180 °e.Even at elevated temperatures there was only a narrow rim ofaragonite replaced by some calcite overgrowth.The newly formed calcite was (lncl devoid of pores,/fence there was no communication between the bulk aqueous phase and the phases at the reaction front,1hus,the overgrowth sealed the aragonite and prevented progressive replacement.However, byusing aqueous solutions containing calcium and carbonate Perdicouri et al. (20 II) obtained different results.
When the composition ofthe so ion was stochiometric, comparable results were obtained to the experiment vNre.. _._.-_..----' , ' .. " .. _." .with water: little replacemen W86 observed nd the formation ofa non-porous calcite overgrowti In contrast, ----•./?f. 0+ -: in the presence of a non-stochiometric solutioI'(, the amount of calcite overgrowth was still very small, {h'P ~a high degree of replacement was achievedtM effect tIM wa~ by the absence " of calcium in the solution.Thus, the experiments of Perdicouri et al. (20 II) demonstrate the importance of porosity and porosity generation for the progress of dissolution-precipitation reactions and allude to at least one fundamental difference between biologic and non-biologic hard materials.In the absence of primary \ .jt 1-'5 :) c£enl porosity and/or secondary porosity that should have been generated at early stages of alteration, &e ~o the r positive molar volume change involved in the aragonite by calcite replacemenj}'he only porosity that might be generated in inorganic systems will arise from the minor difference in solubility between aragonite and calcite.As the solubility products of the two main carbonate phases are similar, little porosity formation takes place, and, consequently, the replacement of inorganic aragonite by inorganic calcite occurs at a slow rate than and is significantly less pervasive AA _ in the case of biogenic aragonite.

!\.
Biological hard tissues are hierarchically organized and are composite materials where at all scale levels there is an interlinkage of biopolymers with minerals.The degradation of these biopolymers, being occluded within and between the basic mineral units of the hard tissuftP,rovides the necessary network of Moreov'C.r;V interconnected porosity (Figs. 6, 7, 8, A9, AIO, AI7).gllln mere, the porosity network not only facilitates

2 The effect of microstructure on alteration
A still unsolved problem in palaeoenvironment reconstruction is the assessment of the extent of @ diagenetic overprint that compromises the fidelity of geochemical proxies.One strategy is to use numerical (I) approaches for the quantification of the extent of diageneti~ ~lteration that are based on the comparison of fAJ A element to Ca ratios and associated partition coefficients and the comparison between isotope compositions /\ ofthe pore fluid and the precipitate (Regenberg et al. 2007 and references therein).In a previous study (Casella 45] et aI., 2017), we reported experimental data for Arctica islandica shell material for the replacement reaction of biogenic aragonite by inorganic calcite.In the present study, we extend our previous work with the investigation of additional (main Iy aragonitic) carbonate skeletons, and thus other mineral fabrics.One of the major aims of this study is the reliable identification of the first stages of alteration and the attempt to qualitatively assess diagenetic alteration based on microstructural reorganisation.For these targets, we apply statistical grain area evaluation and develop this approach as a qualitative tool for the detection of moderate diagenetic overprint.

I"
A. is/andica, M. edu/is calcite and Porites sp.aragonite, clearly an indication of fractal distribution ~ the microstructures of these skeletons.The least difference in grain area change between pristine and most altered states was observed for A. is/andica aragonite (Fig. 12A), while the most significant difference occurs for M edu/is fibrous calcite (Fig. 12E).For Porites sp.acicular aragonite and H ovina prismatic and nacreous aragonite, we find a ~Lt t th perceivable, .~Ismall difference in grain-area size betweeryr?rstineand the most altered states.
For M. edu/is nacre the majority of grain area data overlap~for this microstructurVas well~~me large grains formed in the altered shell ~(Fig.A 16).
As described in the results section, subsequent to the destruction of organic sheaths, membranes and fibrils, the amalgamation of basic mineral units is the next and =-: rastic step in the overprint process.

\7
Inorganic mineral precipitation starts in cavities between the basic mineral units and in voids within them VOid fll';Y1c/ (e.g., Figs. 7, AI7; Casella et aI., 2018a, 2018b).It is important to note that thisrbCcurs prior lo clrbonate ~ phase replacement, and thus, prior to abiogenic calcite formation.With EBSD we not only measure patterns ofcrystal orientation but determine the mineralogical phases ofthe hard tissue.At this early stage ofalteration crystallites that are deposited between the basic mineral units retain the phase of the host crystal and often even the crystallographic infonnation of th~ mineral in the pristine skeleton.Thus, in aragonitic biogenic microstructures, inorganic aragonite will precipitate, while in calcitic biogenic microstructures inorganic calcite will form.Syntactic nucleation of a secondary phase that has the same mineralogical nature as the l~ifJ, primary Pha 4 is prompted by the reduction of the energy barrier associated JIt heterogeneous nucleation in ~~r;:it& homogenous nucleation from a bulk aqueous solution.This barrier is further reduced as a result ~ red\Aet"py) '~n of a perfect match between the crystal lattice of the original and secondary phase.This~nergy barrier f,~explains the preference of inorganic aragonite formation on biogenic aragonite at the first stages of the alteration process, rather than the more stable inorganic calcite.
Due to its composite nature, biogenic aragonite is more soluble than inorganic aragonite and even more soluble than inorganic calcite.Thus, an aqueous solution in equilibrium with biogenic aragonite is supersaturated with respect to bo~inorganic aragonite and inorganic calcite.This supersaturation is higher with respect to calcite, an~ calcite nucleation on aragonite can be epitactic, the much better matc~ OtCr'OS$ 497~the interface makes it more likely that nucleation and growth of inorganic aragonite occurs on --biogenic aragonite.Hence, even though calcite is the more stable phase at Earth'{surface pressure and Accordingly, aspect ratios of the basic mineral units change as their original morphologies ~ hec [) Me distorted (Figs. 7, AS, A I7(f!Jd compaction ofthe hard tissue is the result (e.g., nacre tablets).However, even though already altered, t this early sta e of alteration he gross microstructure of the shell or skeleton is not modified to a large degre~We observe that alteration occurs in two stages: (1) Related to the original J carbonate phase of the hard tissuri'vergrowth and nucleation of abiogenic aragonite or abiogenic calcite in voids and pores, without,1major destruction of the original microstructure, and, (2) phase replacement, new formation with distortion of the original microstructure up to its complete destruction.These processes fo...es involve the constant rearrangement of ~, which in this case is driven by the free energy reduction associated with the increase in the volume/surface ratio of the basic mineral units.
We observed the above described features for all investigated microstructures (Figs.128 to 12F) except for the prismatic aragonitic microstructure of the shell of the bivalve A. islandica (Fig. 12A).islandica between the pristine and the most altered states.

Aragonitic prisms in
In contrast, M edulis calcite shows the most significant difference in grain area between the pristine and the most overprinted states (Figs.12E).When altered, the morphology of calcite fibres was distorted (Fig. A8A); fibre ama'igamation was substantial and led to the formation of large and highly irregularly-shaped mineral units (Fig. ASH).In the pristine state, calcite co-orientation strength is high in M edulis,_ a ---single-crystal-like distribution of c-and a*-axes is present (Figs.6 and 7 in Schmahl et aI., 2012).Hence, many neighbouring calcite fibres are highly co-aligned, a circumstance that favours the amalgamation of similarly oriented fibres (Fig. A8S).The nacreous shell layer in M edulis was little affected by alteration reOvdt'/y (Fig. 12F, Fig. A16A, AI6B), even though nacre tablet amalgamation was ~pefceivable.The nacreous shell part grows into a compact entity and becomes sealed and protected against fluid infiltration.This explains the observation of remnants of nacreous shell areas surrounded by calcite (Brand, 1994) as well as the increased prevalence of the nacreous shell layer of M. edulis relative to calcitic shell layers in seashore sediments.
[n H ovineacreous tablets are assembled in columns, and tablet dimensions are smaller than those ~sent in M edu/is.As for b0ttrM edulis and H avina, nacreous tablets are encased by organic sheaths.
(ompared to M edulis nacre, nacre in H. ovina has a larger organic-mineral interface and mineral surface area -per volume fraction ofshell.Nacreous tablet amalgamation and compaction ofthe nacreous shell layer occur! in the shell of H. ovina as well.In contrast to M. edulis, H ovina nacre exhibits a distinct increase in grain size in the altered hard tissue.Due to the larger interface and surface area in H ovina nacre alteration fluids infiltrate the shell more profusely, and dissolutionlrecrystallisation occurs to a higher extent.Hence, overprint becomes more significant and evident.The same argument holds for prismatic aragonite found in H. ovina (Fig. 12C) and acicular aragonite in Porites sp. (Fig. 12B), wherwor to replacement of biogenic aragonite by inorganic calcite, basic mineral units increase in size in the altered skeleton.It is important to note that this size increase is accompanied in H. ovina and Porites sp. by partial closure of the porosity, and the newly 556 formed calcite is completely devoid of pores (Figs. 8, 10, 11, ASS, AIOB).The partial closure of pores 557 explains the low degrees ofreplacement that is reached by these hard tissues even after long alteration periods.~1 he-,/ fa \Ale,) tho,t . .A 624 However, the greatest challenge that all biological proxies face lies in their capacity to retain their pristine record.The main conclusions are: I. Alteration of biogenic aragonite to inorganic calcite is fastest in hard tissues that contain primary porosity and are composed of irregularly shaped basic mineral units embedded in a network of biopolymer fibrils.The latter are easily destroyed anJProvid ,togefflei-wit~~.imary!_~~amPle space for extensive fluid infiltration into and percolation through the hard tissue.This mode of overprint is observed for the prismatic shell layer of the gastropod Haliotis ovina and for the shell of the bivalve Arctica islandica.Overprinting of these hard tissues is fast and completed with the formation of irregularly shaped and randomly oriented calcite units.2. The slowest alteration kinetics can be observed when biogenic nacreous aragonite is replaced by -r'he inorganic calcite, irrespective of the mode of assembly otTrtacre tablets.Alteration _ proceeds in -four subsequent stages: (a) decomposition ofbiopolymers and formation of secondary porosity, (b) lateral and longitudinal amalgamation of nacre tablets, (C)'t' the alteration front ,€~~Of a compact zone within the hard tissue where the original microstructure is entirely erased~theoriginal bioaragonite phase fstill retained, and (d) replacement by inorganic calcite.3. The acicular microstructure of the stony coral Porites sp. is highly resistant to alteration.With alteratio~aragonite needles fuse and form a compact aragonitic fabric, still retaining some R~F\ClC€\"I1QV)t 01 morphological aspects of the pristine microstructure.~Ologicai aragonite" \inorganic calcite b ~1 ~starts within the coral skeleton at the centers ofcalcification and proceeds ~om the latter 4.
the replacement by newly formed calcite.demonstrates an increase in grain area within the altered hard tissues relative to that in the pristine skeleton.Hence, even though at the very early stages of alteration the original phase is retained, ',,~ nqr-a.;1-\1:-h lo.r qe lr101r overprinf starts with the formation of overgrowths.This lis most pronounced in the calcitic shell layer f\ of Mytilus edulis and is least for the grains that constitute the shell of Arctica islandica.Thus, in the rdl«ble \VldtcOotl>r ~ case of aragon itic tissu~e survival of biological aragonite cannot be used as a distil1Ct jAElisaiep for pristine elemental and isotope signals.Statistical evaluation of grain area (basic mineral unit) values is a promising new tool for the estimation of the degree of diagenetic overprinting.j

Arctica islandica
Poritessp.• Figure 5: Colour-coded EBSD orientation maps with corresponding pole figures depict differences in microstructure and texture between pristine (A, B) and hydrothermally altered (C, D) Mytilus edu/is shells.Alteration lasted for 35 days and was carried out at 175°C in a Mg-rich fluid.The EBSD colour code used is shown in (B); crystal co-orientation strengths, expressed with MUD values are given on each EBSD map.Hydrothermal alteration induces a significant change in pristi~e Mytilus edu/is calcite fibres (compare maps (A) and (C)).The strength of calcite co-orientation decreas~~m an MUD on 81 in the pristine (A) to a MUD of79 in the altered shell(C), respectively.In the overprinted sample{ m'orphology ofcalcite fibres is highly distorted due to profound fibre amalgamation.In contrast, nacre in Mytilus edulis was little affected by the applied hydrothermal alteration conditions (D); a slight decrease in MUD and sporadic tablet amalgamation can be observe~erwisWletmorphology is not distorted.

7
IV\~e"t • lSHCo-orientation statistics are derived from pole figures obtained by EBSD scans and are given by the MUD (multiple ofunifonn (random) distribution) value.The MUD value measures crystal co-orientation (texture sharpness) in the scanned area, where a high MUD value indicates high crystal co-orientation, and a low MUD value reflects a low to random co-orientation.
Fig.1B).These grow radially outward from an organic template present at aragonite nucleation sites: the 203 centres of calcification (white dots in Fig.IBand Griesshaber et aI., 2017).As skeletal growth proceeds, aragonite crystallites increase in size, and form thin fibres that are bundled into loosely co-oriented units
r') I)f ~().\d te ; 9).During alteration in a Mg-rich fluid, a Mg-rich rim twas always present at the phase replacement front, between the newly formed calcite and the highly overprinted nacreous aragonite (white arrows in Figs.9A, 9D, Fig. A14, white arrows in Fig. AI5A).Based on Mg-contents, in addition to ther'final' calcite, two high .. s~p~r~.1 e_ _.-339 Mg-calcite phases can be ~istinguished (Figs. 10, II, A 15), which ~the 'final' calcite (calcIte HOYI/., 340 with a low Mg-contents) ~ the overprinted aragonite that was not yet replaced by calcite (Figs.II, AI5).-"The last step in the replacement of biogenic nacreous aragonite by inorganic calcite is the fonnation of low 342 Mg calcite, the 'final' calcite, which in the final stage of alteration constitutes the 6rin~ard tissue.
Y1S'To r VIi err I Dh ihot Accordingly, important questions whieh arise in this context are: What are the intermediate steps ofalteration A and diagenetic overprint?What is destroyed first, the original skeletal.microstructureor the original vJhDJ ho.p~en? to ".-r)'(,I};;'7I1y mineralogical Phase)and~le geochemical informationrstored by the biogenic archive?In general, what determines the preservation potential of a fossil archive?4.1.The process of overprintingDiagenetic overprinting of biogenic carbonates encompasses morphological and chemical changes that take place during post-mortem alteration.Fluids act as catalysts for the alteration reactions at fluid-rock contacts and allow the overprint reactions to proceed at a rapid rate(Brand, 1994).This response is in contrast to solid-state alteration in dry systems, where overprint kinetics are much slower.Brown et al. (1962) have shown that replacement of aragonite by calcite at Earth surface pressure and temperature conditions is 10 orders of magnitude faster in the presence of water compared to dry conditions.Accordingly, with the death of the organism and burial in sediments biomineralised hard tissues become subject to diagenetic overprint, to solvent mediated phase replacement (Cardew and Davey, 1985), and the coupled dissolution ofthe original /\ material and the precipitation of a new product(s) (Putnis, 2002, 2009).It has been shown for non-biologic systems that coupled dissolution-precipitation is highly influenced by the availability of interfaces, the reactivity of the involved surfaces, and the extent and topological characteristics of the original and newly formed porosity (Putnis, 2002, 2009, Ruiz-Agudo et aI., 2014, 371 Arvidson and Morse 2014).It is demonstrated for rocks and minerals that a coupling ofthe two (sub)reactions takes place when the rate of dissolution of the original phase and the rate of crystallisation of the product is almost equal.This has the effect that coupled dissolution-reprecipitation of mineral replacement proceeds with preservation ofthe external shape of the primary mineral, and leads to formation of pseudomorphs (Xia et aI., 2009; Quian et at, 2010).If the coupling between dissolution and recrystallisation is balanced, delicate microtextural features are well Mpreserved, such as twin boundaries (Xia et aI., 2009b) or exsolution lamellae (Altree-Williams et aI., 2015).It has been further demonstrated for non-biological materials that microstructural elements such as grain boundaries, are of key importance for the overprinting process.At the first stages ofalteration, these are pathways for fluid infiltration and percolation through the material and ensure a pervasive replacement ofthe original mineral (Jonas et al. 2014, Eschmann et al. 2014).In non-biologic systems, mass transfer along grain boundaries is an order of magnitude faster than through the porosity that is generated as a result ofthe mineral replacement reaction itself (Eschmann et al. 2014, Jonas et al. 2015).Even though, in non-biologic systems an interconnected pore system is also developed with progressive alteration (Putnis, 2002, 2009; Pollok et aI., 2011; Ruiz-Agudo et aI., 2014; Altree-Williams et aI., 2015).In fact, the formation of porosity is a requirement for the progress of the replacement reaction ~as it is the pore system that allows for the --, continuous communication between the bulk aqueous phase and the primary and secondary phases at the reaction front (Putnis, 2002, 2009, Etschmann et a1.2014).Pore formation also takes place as a direct consequence of the mineral replacement process,M-in cases when the molar volume change involved in --~-

~
)pMf..-;; alteration, it drives and accelerates iJrJasjiallow, for pervasive circulation of the alteration fluids within the v skeleton.Our results show~ tha~r biological carbonate tissuQJe presence of primary (inherent) and secondary (induced) porosi~ogether with the characteristics ofthe porosity netwo~tennines the kinetics and extent of the alteration.Furthermore, the transient character of porosity additionally influences mineral replacement reactions,4part from porosity generation, porosity closure and porosity coarsening on biological -ma~ are ~id~pr;;:I ph~ These modifY the geometry of the porosity network, increase its tortuosity,r;e~~~: its permeabili~therebY ~ffe~"mass transfer at the interface between the bulk solution and the original mineral phase and hinder(\&hysicochemical re-equilibration.APorosity characteristics are different for the different microstructures investigated in this study (Fig.1).Primary'porosities are present in the shell of Arctica islandica and in the prismatic shell layer of Haliotis Afthou.ahovina.1t is iMportant til Rite that the skeleton ofthe coral Porites sp. is compac,loIe" e ,e~ the coral skeleton has a particularly high surface areC!) the skeleton consists ofvarious combinations of vertical and transverse elements, with most ofthese being developed as thin lamellae.Basic mineral units that comprise these skeletal elements consist of irregularly organized clusters of closely packed aragonitic needles.The centres of calcification are the primary pores in the skeleton of Porites sp.• Vowever, these are in general not interconnected, and thus, do not facilitate transfer of solutes to and away from the reaction front to a large extent.Stacks of calcite fibres in Mytilus edulis and the nacreous tablet arrangements in Mytilus edulis and Haliotis ovina are the most compact microstructures investigated in this study.These materials lack primary -The 5~ -2\\ S (kCe.. 44] porosities.NontOheJess, whenNlltered, the extent of alteration-induced secondary porosity is high in the nacreous tablets, as the occluded intra-tablet membranes and inter-tablet fibrils ~ decomposeNand create space for fluid circulation.

n~
Figures12 and Al6show relative frequency and grain area (fbasic mineral unit in the case of +h biological hard tissues) diagrams foP1p~stine and the most altered (alteration for 35 days, at 175°C, in Mg rich fluid) skeleton equivalents for six microstructures.Grain area data are obtained fromEBSD .5"<:f.secf-t :y"\ 46\ measurements ~ for the definition of a grain in carbonate biological .hardtissue);;; 81:&,t85, 2.2.3:Grain area evaluation for the determination of alteration).A grain is defined tA~ug¥misorientation ~ relative 0.+ I) r, (~'/'\ qi e..to neighbouring grainsf'that i~ larger than a critical value, the critical misorientation value.Griesshaber et al.(2013) determined empirically that a critical misorientation value of2°best suits the microstructure ofmodem carbonate biological hard tissues to differentiate between individual basic mineral units (e.gi!)ibres, tablets, prisms, columns).Thus, we adopt a critical misorientation value of2° to define a grain.~~djacent grains are recognized as two individual grains when one unit is tilted relative to the adjacent unit by more than 2°.L I.+-The compilation in Fig.12clearly demonstrates the influence of the biogenic microstructure I!' o.Vi lIt tv " .~1)f 469A withstan~alteration.The relation,\~lIog (frequency) versus log (grain area) is linear for

~
temperature conditions, free energies and solubilities of the two carbonate phases are close enough that the lower energy barrier associated with epitactic nucleation kinetically favours the fonnation of new aragonite on the surface ofthe pre-existing aragonite (Fernandez Diaz et al.~009~oncal-Herrero et al.~201'~and 502 Cuesta Mayorga et aI.9018».Thi~observedin nature.Hover et aI.(2001) report early diagenetic overprint of Foraminifera and green algae skeletal hard tissues and demonstrate that the overprint 504 mechanism is the coupled process of dissolution and precipitation.The authors find thin overgrowths on.the mineral units of the original hard tissues and show that the precipitated material is largely similar in 506 composition and structure to that of the host crystallites.
A. islandica shell are sn1all and are embedded in a network ofbiopolymer fibrils (Casella et aI., 2017).The thin fibrils are easily destroyed when altered and leave behind a network of voids and cavitie~ch facilitate fluid infiltration and permeation through the shell.The large number ofsmall basic mineral units gives rise to exceedingly large surface areas where the fluid can get into contact with the mineral.tL--: once Carbonate phase alteration kinetics in A. islandica shell is sluggish at first, However, \l6Aenj1the nucleation -barrier is overcome and the alteration process is starte~ proceeds very rapidly (Figs.2A, A4A and Casella et aI., 2017).Thus, overgrowth of inorganic aragonite in voids and basic mineral unit amalgamation might well be masked by the almost instantaneous replacement of biogenic aragonite by inorganic calcite in the microstructure of A. islandica shells.The high volume of interconnected porosity in A. islandica ~ wh_' :-/ ttct'iJ~--, .1":----'"fY)or e-O V fr" explainsM~ alteration becomes ~ after only a short time in contact with diagenetic tluids.~etopological ' ) I characteristics of porosity facilitatej the coupling between the rate of aragonite dissolution and calcite crystallisation.Thitt9 tum, explains the little difference in mineral grain area found in the hard tissue of A.

558
Our study clearly shows th~fthe investigated aragonite microstructure~ nacreous tablets are the 559 most resistant to replacement by calcite, irrespective of the assembly pattern of the tablets in columns or 560 sheaths.Porosity closure and basic mineral unit (nacre tablet~ amalgamatio e bt..tt Yf(.ten+;on original microstructure, ~with the f~iAm8Rt of the original phase (Figs.9A, AI7A, AI7B).Hence, 562 even though nacreous aragonite is still preserved as aragonite, it is an overprinted aragonite that, most 563 probably, holds little of t h ~or geochemical signature.With increasing alteration, the ?@ 20 --'remoulded' aragonite finally becomes replaced by non-biologic calcite.In general, ~in our alteration experiments the microstructural signature is lost first, prior to a complete loss of the original phas1j) c,f, I; Y) e.while ~-;-~~~ in~~atio~~:~~n~.~!~~~i~~ § When alteration takes place in a Mg-rich fluid, @ ti&3 v«lijft~latlat the original material-product interface, in addition to the 'final' non-biogenic, low-Mg calcite. ..~_.+------------~o other calcite phases are presen These can be distinguished by their Mg-content (Figs. 9A, 11).We ~d\A~-Th clearly see an evolution in fluid composition with hydrothermal alteration~Jan evolution in catio"!~nion exchange between the alteration fluid, the overprinted origin~d the newly-formed carbonate products.4. 4 Implications for preservation of carbonate skeletons in the fossil record Several studies have shown that in modem cold and warm water environments aragonite dissolution takes place at burial diagenesis (e.g., Cherns et al. 2008 and references therein).It has been further ct~e.demonstrated th~ Palaeozoic marine faunawaxa with calcitic skeletons prevail, this being an indication of the preferential loss of aragonitic shells and skeletons, due to dissolution during diagenetic overprint (e.g., Wright et al. 2003, James et al. 2005).In addition to preferential carbonate phase preservation, experimental studies document that the microstructure of the biogenic skeleton influences fossil preservation (e.g., Harper 1998, 2000; Kidwell 2005), leading to a possibly distorted notion of paleoecological and evolutionary patterns.Accordingly, laboratory-based hydrothermal alteration experiments accounting for microstructural as well as mineral phase variability offer important insights into the fate of carbonate hard tissues during a) .,..---------_._--~shallow burial arly dissolutio~'and b) surviving dissolution and preservation in the fossil record.Do we see resemblances between the microstructural, chemical outcome of our alteration results and microstructural and geochemical features of fossilized hard tissues?It is remarkable~tha~en though our experiments lasted only 35 days, were carried out at~ingle w~Vt temperatur~nd'Performed in the presence of only one type of alteration flui~ere is much overlap between pr-odl-lC1:9 ~thDSf.-'\U our experimentaLresattsI\and pf carbonates that underwent diagenesis.Several decades ago Friedman (1964) and Land (1967) reported Qn the early diagenesis of skeletal carbonates and carbonate sediments exposed to iZ i'r1d•,c~ . . .ti VI! th~t marine ~aters, the b~ological.carbonates retained their original mineralogical and textural characteristi~s.
death of the organism, diagenetic overprinting starts immediately during which the original, replaced by inorganic features.We investigated the behaviour of six biogenic carbonat] ~ to ~~ samples and their associated microstructures at different degrees of hydrothermal alteration in order to ~~ evaluate their capacity to withstand alteration and thereby estimate their ability to be preserved in the fossil ~ i 5. ~;(t;r~tion in a fluid enriched in Mg, aq,igh-Mg ~;~~eloPs between the altered, compact ara&onite /1'Y) thE cQ\c; te -: and the newly formed calcite.With the progressive decrease ofMg~oncentrationlWe can clearly trace the chemical evolution of the alteration fluid at the biogenic aragonite to calcite interface.6. Statistical evaluation of differences in grain area size of pristine and altered skeletal equivalentsJ1.

Figure 1 :
Figure 1: SEM micrographs showing the characteristic microstructures ofskeletons ofthe modem specimens of(A) the bivalve Arctica isJandica, (B) the scleraetinian coral Porites sp., (C, D) the gastropod HaJiotis ovina and (E, F) the bivalve MytiJus eduJis.The shell ofArctica isJandica consists ofan assemblage of irregularly shaped and sized aragonitic basic mineral units, prisms, (white stars in (A» which are embedded in a network of biopolymer fibrils (this study and Casella et al., 2017).The acicular aragonitic skeleton of the modem coral Porites sp.(white star in (B» is composed of differently sized spherulites consisting of fibrils and needles.These grow outward from an organic template that lines the mineral nucleation sites, the centres of calcification (white dots in (B».The shell ofthe gastropod HaJiotis ovina and the bivalve MytiJus eduJis comprisehvo distinct carbonate layers.The shell of HaJiotis ovina consists of irregularly shaped and sized prisms (yellow stats in (C» next to a nacreous shell layer with nacre tablets assembled as columns (white star in (D».The outer shell layer in MytiJus eduJis is formed by stacks ofcalcite fibres (yellow star in (E», while the inner shell layer is nacreous with nacre tablets arranged in a 'brick wall fashion' (white star in (F».

Figure 3 :
Figure 3: EBSD colour-coded orientation and phase maps with corresponding pole figures which depict the

Figure 9 :C
Figure 9: Microstructural and chemical stages in the replacement process of biogenic nacreous aragonite by inorganic calcite.Haliotis ovina shell material was subjected to hydrothermal alteration for 35 days, at 175°C in a Mg-enriched hydrothermal fluid.(A) SEM image depicting the replacement front/zone between nacreous aragonite and the newly , formed calcite.Blue stars in (A): nacre tablets forming columns; some traces of the original microstructure can be still ( t'/I arl1.a0 tl ; it;observed.Yellow stars in (A): a formerly nacreous shell layer, but, at this stage ofalteration, the nacreous microstructure/\7 ./