Experimental mineralization of crustacean eggs leads to surprising tissue conservation : new implications for the fossilization of Precambrian-Cambrian embryos

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Introduction
Exceptional three-dimensional preservation of fossilized eggs and embryos from the Neoproterozoic, the Cambrian and the Ordovician provide a tantalizingly direct insight into embryology during the eve of animal radiation (Bengtson and Zhao, 1997;Li et al., 1998;Xiao et al., 1998Xiao et al., , 2000;;Chen et al., 2004;Steiner et al., 2004a,b;Donoghue and Dong, 2005;Donoghue et al., 2006;Liu et al., 2007;Dong et al., 2010).In particular, the terminal Neoproterozoic Doushantuo Formation algae and embryos occur in nearly rock-forming abundance, and second because radiometric ages of the Doushantuo Formation imply that the putative animal embryos predate the Precambrian-Cambrian boundary (542 Ma) for more than 20 million years.The latter finding in turn would rather confirm hypotheses based on molecular clocks indicating that the last common ancestor of Metazoa appeared in the Neoproterozoic between 676 and 766 Ma (Peterson et al., 2005(Peterson et al., , 2008)).Fossilized animal eggs and embryos, particularly of the Ediacaran Weng'an and Cambrian Kuanchuanpu biota (Fig. 1), are mainly preserved as diagenetically phosphatized replacements and encrustations in dolostone or limestone (Steiner et al., 2004a;Hubert et al., 2005) and perhaps exhibit the most remarkable variety of early biodiversity with extraordinary preservation of cell geometry and cytological structure.However, the classification and preservational context (taphonomy) of most of these fossils is still the subject of extensive research.To date for the Weng'an embryos, primarily the very earliest cleavage stages have been recovered, leading to the suggestion that the paleo-embryological record may have strong biases on developmental stages preserved (Dornbos et al., 2005;Raff et al., 2006).Furthermore, an alternative interpretation of these microfossils was introduced suggesting that some putative fossil animal eggs and embryos, such as Megasphaera inornata and Parapandorina, represent solitary and reductively dividing giant vacuolated sulphur bacteria (Bailey et al., 2007a).This intriguing reinterpretation and a recent publication describing helical spheroidal fossils as prehatchling forms (Xiao et al., 2007), has launched an "embryonic identity crisis" debate (Donoghue, 2007;Gostling et al., 2007).Additional data are therefore highly desirable to constrain the proposed interpretations (cf.Hagadorn et al., 2006;Bailey et al., 2007b;Chen et al., 2009).
Understanding the mechanism of decay and soft-tissue preservation through experimental taphonomic analyses, in which the conditions of decay and mineralization are explored experimentally (Briggs, 1995(Briggs, , 2003)), is crucial to interpreting the fossil record of early metazoan evolution.Previous experimental studies investigating the controls on the formation of authigenic minerals in association with decaying organic matter Introduction

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Full reported phosphatization of muscle tissue within weeks to few months, associated with a drop in the oxygen level and pH, after the introduction of an anaerobic bacterial community and/or sea floor sediment to shrimp carcasses (Briggs andKear, 1993a, 1994;Sagemann et al., 1999).The precipitation of either calcium phosphate or calcium carbonate is thereby related to local pH changes, with calcium phosphate being favored as pH decreases from neutral to acidic (Briggs and Wilby, 1996).Only a few studies have noted the experimental mineralization of invertebrate eggs.Experimental phosphatization of eggs was first reported from the modern shrimp Palaemon elegans, which was subjected to decay as a complete animal for 20 weeks (Briggs and Kear, 1993a).Later studies, which focused only on the eggs, demonstrated that it was possible to maintain the external shape of Homarus gammarus, Limulus polyphemus, and Nephrops norvegicus egg cases for at least a year, including surface mineralization mainly as calcium carbonate or as a combination of calcium carbonate and calcium phosphate (Martin et al., 2003(Martin et al., , 2005)).Laboratory decay experiments also achieved the replication of the outer morphology of H. gammarus eggs by coating the eggs with pre-existing clay minerals in the presence of metabolizing bacteria (Martin et al., 2004).None of these studies, however, considered the effects of inherent fragility on the internal morphology and histology of invertebrate eggs.In normal seawater, freshly killed embryos are generally prone to rapid decomposition within a few hours.Embryo preservation for a prolonged time prior to mineralization is thus rather unlikely.Recent studies thus focused on the factors, which affect the decay and preservation of the earliest stages on embryonic and larvae development (Gostling et al., 2008(Gostling et al., , 2009;;Raff et al., 2006Raff et al., , 2008)).Four stages of decay can be distinguished (Gostling et al., 2008): (a) The onset of autolysis, (b) some shrinkage within the fertilization envelope, enzymatic breakdown of the cyctoplasm and coalescence of sub-cellular lipids, often associated with the degradation of cell structures through the activity of endogenous and exogenous microbes (cf.Raff et al., 2006), (c) continued degradation of cell structures and breakdown of the fertilization envelope, (d) generation of amorphous organic matter due to the total breakdown of subcellular material.Embryo fossilization can Introduction

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Full therefore be attributed to three processes (Raff et al., 2008): (a) The rapid blockage of autolysis within an anaerobic environment, (b) bacterial invasion and consumption of the embryo, forming a replica that retains cell organization and morphology, and (c) bacterial development of microenvironments inducing changes in embryo chemistry and mineralization potential.
Here, we report a succession of simplified taphonomic experiments on eggs of the parthenogenetic crayfish Procambarus fallax f. virginalis (Martin et al., 2010), in order to elucidate alternative scenarios for embryo fossilization.We investigated the conditions that promote both the mineralization of invertebrate eggs as well as decay and preservation of organic tissue over a prolonged period of time under non-marine laboratory conditions.Our results show that experimental mineralization of invertebrate eggs can produce different qualities of preservation, which were similar in appearance to what has been documented from the fossil record.

Sample material
Eggs of the parthenogenetic crayfish Procambarus fallax f. virginalis (Fig. 2; Martin et al., 2010) were taken from living animals that were kept in culture at the Department of Comparative Zoology, Humboldt-University Berlin.Its capability, high fertility, fast growth, unisexuality and isogenic progeny (Scholtz et al., 2003;Martin et al., 2007) have made this species useful for physiological, ecological, evolutionary and genetic studies.Crayfish eggs, as those of decapod crustaceans in general, are considered as being encased by an inner vitelline membrane and a chorion (Winnicki et al., 2004) although the exact sequence and number of layers and their origin is debated (Cheung, 1966).The eggs are rich in yolk and reach diameters up to approximately 1000 µm.Ten defined stages from zygote to hatching (Alwes and Scholtz, 2006) were determined for most of the specimens used in the experiments.Introduction

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Experimental design
Taphonomy experiments (E1 to E7, Table 1) were conducted under different redox and pH conditions.In all experiments, crustacean eggs were embedded in a gel-like medium and transferred to an exsiccator at room temperature and atmospheric pressure.The embedding medium was prepared using analytically-grade synthetic calcite (CaCO 3 ), phosphoric acid (H 3 PO 4 , 85 %), distilled water and powdered activated carbon.According to Eq. (1) brushite crystals (CaHPO 4 • 2H 2 O) formed in the gel, which was verified by X-ray powder diffraction (Table 2).
Prior to the experiments, the eggs were stored in tapwater-filled glass vials at 9±1 • C.
Experimental series E1 and E2 were conducted to explore the effect of differences in the composition of the embedding medium on embryo preservation and mineralization.Phosphate was supplied in the form of phosphoric acid (H 3 PO 4 ), and brushite.Experiment E1 took place under normal air, whereas experiment E2 proceeded under an N 2 -atmosphere.Experimental series E3 to E7 were performed to study the temporal progress of preservation and mineralization.For this purpose, one to four embryos were removed from the experiment every week.After removal of the sampled specimens the exsiccator was refilled with nitrogen.The duration of these experiments was generally between seven and 44 days.The latter experiments were designed applying the excess CaCO 3 medium under the N 2 -atmosphere.In order to reduce oxidation processes, and thus the decay process of the embryo tissue, powdered activated carbon was added to the sample surfaces in experimental series E3.To explore whether and how initial heating might stabilize the eggs and prevent them from collapsing during fossilization, crustacean embryos were initially heated in distilled water at 80 ± 1 Full

Sample preparation
For scanning electron microscopy analyses, the treated crayfish eggs were first dehydrated in ethanol.Then they were either dried at the critical point (Balzer) and mounted on stubs with adhesive tape or prepared in histological blocs.They were infiltrated overnight in a basic solution of 100 ml Technovit ® 7100 and a hardener and subsequently filled in histo-moulds.Histoblocs were then prepared with Technovit ® 3040 and coated with silver and carbon.
For histological sections, the ethanol-dehydrated samples of experimental series E7 were infiltrated with a 1 : 1 mixture of araldite epoxy resin and epoxy propane overnight and subsequently transformed to solely araldite for one hour.Samples were then transferred to an araldite-loaded embedding mould and stationed till polymerisation at 70 • C overnight.Histological sections were trimmed applying a rotation microscope (HM 350, Micron), cut into semi-thin sections (2 µm) and transferred to slides.Histological sections of experimental series E7 were stained with toluidin blue (0.5 %) solution.

Analytical techniques
The mineralogical phase composition of the embedding medium was determined with a Philips PW1729 a primary monochromator unit and a 7 • -position detector (Gottschalk et al., 1998) at the GeoForschungsZentrum Potsdam, operated at 40 kV/40 mA.Measurements were conducted with a K α1 -beam between 5-125 • (2θ).The step size was 0.1 • and the measurement time was 5-20 s.Samples were ground in an agate mortar and mixed with Elmers white glue, superimposed on a plastic film, which was transferred to a transmission-sampling holder.Mineralized eggs were directly glued on the plastic film.Structural parameters were refined using the software package GSAS for Rietveld analyses.
Calcium and phosphorus content of fresh crayfish eggs were determined on a Varian (Vista-MPX) inductively coupled plasma-atomic emission spectrometer (ICP-AES) at the GeoForschungsZentrum Potsdam.Eggs were taken from mother animals during cell devision stage 5-6 (after Alwes and Scholtz, 2006).They were oven-dried at 105 • C for 24 h.Dried embryos were put in Savillex ® beakers which were filled with 10 ml of quartz-distilled ultra pure water, for five hours at room temperature.After rinsing of the embryos two times with ultra pure water, they were dried on a hot plate at 120 • C overnight for dry weight determination.About 2 ml of 7 N HNO 3 was added to the dry mass, which was then vaporized on a hot plate at 160 • C overnight.Since parts of the dried residues were not completely dissolvable, the 7 N HNO 3 step was repeated three times.For chemical analysis, another 2 ml of 1 N HNO 3 was finally added to the yellowish solution.
A Zeiss stereoscope (Stemi SV11) equipped with a Zeiss digital camera (AxioCam HRc) was used to examine the surfaces of the samples.In order to map the topography and determine the chemical composition of the samples, eggs were investigated using scanning electron microscopy (Hitachi S-2700, equipped with a tungsten cathode, operated at 20 kV/500 nA) at ZELMI, Technical University Berlin.Element determination was done applying an EDX system.The detection limit for measured elements was 0.1 weight-%.Introduction

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Results
We both determined and localized the initial calcium and phosphorus content of fresh crayfish eggs (Table 3).The dry mass contained 120 ppm Ca and 1.3 wt.-% P, comparable to concentrations described for other invertebrate eggs (Martin et al., 2005).Scanning electron microscopic (SEM) mapping of P of an untreated egg in crosssection furthermore revealed an almost uniform distribution of P, with higher initial P concentrations only in the germ area (Fig. 2).
Treatment with excess phosphoric acid (H 3 PO 4 ; Table 1) preserved fresh crayfish eggs neither in an oxygenated (air) nor in a reduced atmosphere (N 2 ).A few minutes after embedding the dark brown to black embryos became red colored, and tiny blebs occurred on the egg's envelope.After two weeks, the eggs decomposed and orange lipid droplets leached into the embedding medium.
Fresh crayfish eggs exposed to excess CaCO 3 (experiment E1b, E2b, E3 and E4; Table 1) in an oxygenated or a reduced atmosphere, in contrast were preserved over several weeks.The crayfish eggs, however, only partially retained their original spherical shape (Fig. 1).Collapse structures of the outer membrane were observed at least on one side.Commonly, polygonal ridges formed which we interpret as shrinkage patterns, resulting from atrophy of the internal body.Subtle mineralization of fine-grained flaky crystals (size range 10 µm), mostly along these polygonal structures, was evident after six days.The addition of activated carbon (experiment E3; Table 1) did not affect the tendency to mineralization.After two weeks, crystal envelopes covered the complete egg surface.In places, dense crystal accumulations up to 150 µm in diameter formed (Fig. 1).The SEM X-ray mappings highlight the post-experimental distribution of P and Ca with clear enrichment of these elements in surface crystallites (Fig. 3).
Crayfish eggs that were heated prior to embedding (experiments E6 and E7; Table 1), exhibit complete preservation of the spherical morphology and complete mineralization of the outer membrane after one week (Fig. 1).After two weeks, the crystal layer had accumulated to a thickness of approximately 100 µm.Continuous mineralization Introduction

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Full occurred as fine-grained botryoidal or kidney-shaped crystallites (Fig. 4).X-ray maps show that these are composed of P-and Ca-bearing phases (Fig. 3).
Stained histological sections showed that the embryonic tissues of the embryo, i.e. various cell types and components, e.g. the cell nuclei, the segmented caudal papillae, and muscle cells were exceptionally well preserved after two and three weeks, respectively (Fig. 5).The endochorional envelope was not preserved.A fine-grained crystal rind completely covered the outer surface of the embryo replacing the endochorional envelope.
X-ray diffraction and Rietveld refinement on the embedding medium and on surface crystallites of the mineralized layer of crayfish eggs of experimental series E7 confirmed the occurrence of brushite and calcite in approximately equal proportions (Table 2).However, the refinement of XRD data obtained from sample material of experiment E7.3 proved to be difficult, because the Durbin Watson factor (DW) and χ 2 (Table 2) were unsatisfactory and the background values of the X-ray spectra were elevated.
The ambiguous results of the Rietveld refinement might also reflect the low crystallinity of the sampled material (see Briggs and Kear, 1993b).

Mineralization
Our experiments show that the outer surface of crayfish eggs can be mineralized within one or two weeks under laboratory conditions in a manner that retains morphological detail similar to that preserved in fossil metazoan eggs (cf.Fig. 1).This finding holds true for all experiments applying the excess CaCO 3 medium and anoxic conditions.The crayfish specimens in our experiments became mineralized by a combination of fine-grained brushite and calcite, and showed no explicit signs of mineralized bacteria.
The ratio of calcium phosphate to CaCO 3 of the crystallites was measured to be 55 : 45 and 62 : 38 (in wt.-%), respectively (Table 2).Introduction

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Full Preferred calcium phosphate precipitation over calcium carbonate was previously reported for laboratory-mineralized body tissue of decapod crustaceans (Briggs andKear, 1993a, 1994;Sagemann et al., 1999) as well as for fossil specimens (Bengtson and Zhao, 1997;Steiner et al., 2004a).Our phosphate to carbonate ratios and the ratios of laboratory-mineralized body tissue of decapod crustaceans are, however, higher than the ratio obtained for laboratory-mineralized eggs of decapod crustaceans.Initially, laboratory mineralization of metazoan eggs had been thought to be closely associated with closed-system conditions and the presence of the decaying mother animal carrying the egg mass (Briggs and Kear, 1993a).In this case, decomposition of Palaemon elegans occurred over a period of up to 25 weeks and the organism itself acted as the source of phosphate.Mineralization of the body tissue and the eggs occurred within a few weeks, either as pure calcium carbonate or a mix of calcium carbonate and phosphate.The eggs were, however, less mineralized than the other tissue and with a much lower proportion of phosphate-to-carbonate (Briggs and Kear, 1993a).Whether changes in pH, the concentration of P, or a combination of both caused the observed differences in Palaemon elegans, however, remained unclear.Partial mineralization was also described for another range of decay experiments on invertebrate eggs of the European and Norwegian lobsters, Homarus gammarus and Nephrops norvegicus (Martin et al., 2005), in which experimental mineralization of invertebrate eggs has been closely associated with the presence of bacteria generating anoxic conditions and reduced pH.Mineralization of eggs occurred most commonly as calcium carbonate, or as a combination of calcium carbonate and calcium phosphate.The only study reporting complete mineralization of the egg surface was performed on H. gammarus, where a quantity of eggs equivalent to 520 mg dry mass was decomposed in artificial seawater (ASW) mixed with sediment over one month under anoxic conditions (Martin et al., 2003).In this experiment, the eggs were encrusted on the outside of the preserved egg envelope with a 50 µm thick layer of mostly amorphous calcium carbonate and mineralized bacteria, with the latter result providing further evidence that bacteria may promote mineralization.Replicate experiments, with the same Introduction

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Full or modified parameters (e.g.egg mass, volume of ASW, various additives), however, did not show complete surface mineralization with reasons for the observed variability in mineralization between treatments being unidentified.
A lack of mineralization and rapid decomposition of crayfish eggs was only observed in treatments with excess phosphoric acid, independent of the redox conditions.In this case crayfish eggs rapidly degrade through the process of autolysis, an internal cell destruction process caused by endogenous enzymes resulting in the coalescence of pigments and internal lipids or proteins, and finally the breakup of the endochorional envelope.

Preservation of organic tissue
Histological sections of treated crayfish eggs showed exceptional preservation of the embryonic tissue during the experiment.Our study is to our knowledge, the first investigation of artificially fossilized embryos at the tissue and cellular level, and is in general line with results of recent taphonomic experiments demonstrating that the morphology of metazoan embryos could be well preserved during prolonged periods for mineralization to occur (Raff et al., 2006;Gostling et al., 2008).These taphonomic experiments particularly showed that reducing conditions are a key factor preventing autolysis and precluding the activity of aerobic microbes.
Even though the embryonic tissue is well preserved, SEM images and histological sections clearly indicated that the endochorional envelope of crayfish eggs showed degradation.A thin sheat of fine-grained authigenic minerals replaces the endochorional envelope.This indicates that rapid decomposition of the endochorional envelope is attended by the rapid growth of authigenic minerals.Further evidence for this "replacement hypothesis" is provided by completely mineralized crayfish eggs showing different degrees of preservation, such as polygonal shrinkage patterns and collapse Introduction

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Full fossil record (cf.Fig. 1).Several studies on fossil metazoan eggs and embryos have shown that their organic envelopes are not originally preserved, but that their original composition and structure is altered diagenetically by phosphatic replacements or encrustations of the envelope (Steiner et al., 2004a).Particularly, the detailed petrographic and geochemical studies on some organic-walled microfossils of the Doushantuo fossil Lagerst ätte suggested that phosphatization was an early diagenetic process in shallow burial depth which occurred after rapid burial of the microorganisms and prior to the total decay of the organic matter and intergranular compaction (Hubert et al., 2005).All findings thus suggest that the mechanisms of decay and mineral replacement were likely similar in both this study and the fossil material.
Mineralization did not occur in the interior of the eggs even when the embryonic tissue had been exceptionally preserved, resembling results of similar experiments using eggs of Homarus gammarus (Martin et al., 2003).There are at least two possible explanations for this phenomenon: the newly formed mineral layer may either have acted as a barrier to microbial degradation of the internal cellular material or the dense structure of the yolk slowed or prevented the migration of substances necessary for mineralization.This "hollow egg" phenomenon is also known from Ediacaran and Cambrian fossil eggs (Steiner et al., 2004b).

Preservation of the spherical egg-morphology
Our experiments show that initial pre-heating of the crayfish eggs can stabilize their spherical morphology (Figs. 1 and 4).This is achieved both through pre-preservation of the yolk and the embryonic tissues and through prevention of internal decay, creating a stable template for mineralization.This result stands in marked contrast to the observations of all non-heated experiments, in which complete mineralization occurred but the original spherical shape was only partly retained.We therefore hypothesize that a moderate supply of heat, possibly through the proximity of a hydrothermal vent system, might assist the preservation and fossilization of yolk-bearing eggs and embryos, larvae and small animals.This is not a prerequisite for preservation, because 12064 Introduction

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Full spherically-preserved eggs and embryos are rare in the fossil record, whereas most show collapsed structures (Fig. 1).If eggs and embryos are preserved at all in Cambrian rock sequences only 9.5 % of them at best indicate a pristine external preservation without collapse structures.Several geochemical studies have demonstrated widespread hydrothermal venting on the Yangtze Platform in Early Cambrian times (Steiner, 2001).We therefore suggest that their emergence not only constitute a unique ecological niche for metazoan evolution, but also a potential environment for enhanced preservation and fossilization.

Conclusions
Our laboratory experiments show that complete surface mineralization of crayfish eggs, which were exposed to excess CaCO 3 , may occur under anoxic conditions after one or two weeks, producing different grades of preservation in a manner similar to those described from the fossil record.While we found exceptional preservation of embryonic tissue at the cellular and morphological level after the time of the experiment, the endochorional envelope was not preserved, but was replaced by a thin rind of fine-grained phosphate and carbonate minerals.This indicates a close link between rapid anaerobic decay of the endochorional envelope and mineral authigenesis, possibly microbially mediated.Pre-heating of the crayfish eggs stabilized the egg's spherical shape without changing the environment so that endochorional decay and mineralization could occur.Our simplified experiments provide further insights on the processes and conditions that might lead to exceptional egg and embryo fossilization, notably those at the Precambrian-Cambrian transition.Introduction

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Full  Full  Full  Full 1 , N. Hu 1 , M. Steiner 2 , G. Scholtz 3 , and G. Franz 1 Introduction (635 to 551 Ma; Condon et al., 2005) of South China has attracted considerable attention, first because fossilized Figures Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | X-ray diffractometer at the Department of Mineralogy and Petrology of the Technical University Berlin.The device is equipped with a PW1050 goniometer, a Cu-tube with a Ni-filter, a secondary monochromator unit, and a scintillation counter (LiF doped with Tl), operated at 40 kV/20 mA.Measurements were conducted with the mean K α -beam ([K α1 + K α2 ]/2) at 1.5419 Å between 5-80 • (2θ).Measurement time per scan (step size 0.02 • ) was 2 s.Samples were ground in an agate mortar by adding acetone and spread on a silica-sampling holder.Data were evaluated with the software packages X'Pert High Score and Origin.The mineralogical phase composition of samples of experimental series E7 was analyzed with a fully-automated STOE STADI P X-ray powder diffractometer, equipped with Introduction Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | C. Wolff (HU Berlin) are kindly acknowledged for providing XRD analyses and crayfish eggs, Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Fig. 2 .
Fig. 2. (A)Fresh egg of the crayfish Procambarus fallax f. virginalis.Black arrow points to the translucent endochorion covering the yolk and the embryonic tissue.(B) SEM X-ray mapping of phosphorus of a sliced untreated crayfish egg illustrating the original P distribution.An almost uniform distribution of P is documented for the yolk, whereas the germ area shows elevated P content (Scale bar: A = 500 µm; B = 400 µm).

Fig. 4 .
Fig. 4. SEM illustrations of pre-heated crayfish eggs incubated under the same experimental conditions conditions.(A) After 1 week.(B) After 3 weeks.Complete preservation of the spherical morphology is evident and continuous mineralization occurs as fine-grained botryoidal or kidney-shaped crystallites (Scale bar: 400 µm).

Fig. 5 .
Fig. 5. Preservation of embryonic tissue.Histological sections of mineralized preheated crayfish eggs.(A, B) After two weeks.(C, D) After three weeks.Various cell types and components (e.g., cell nuclei of the segmented caudal papillae) are exceptionally well preserved with finegrained authigenic brushite and calcite crystals (arrows) completely replacing the degraded endochorional envelope (Scale bars: A and C = 500 µm; B and D = 50 µm).

Table 1 .
Taphonomy experiments performed and corresponding conditions and duration.

Table 2 .
Phase fractions, lattice constants and statistical parameters of XRD-Rietveld refinement of experimental series E7.

Table 3 .
Comparison of initial phosphorus and calcium concentrations, occurrence of mineralization and embryo preservation in this and previous studies.