Journal of Vertebrate Paleontology 33(1):121–130, January 2013 © 2013 by the Society of Vertebrate Paleontology
ARTICLE
GROWTH MECHANISMS IN DINOSAUR EGGSHELLS: AN INSIGHT FROM ELECTRON BACKSCATTER DIFFRACTION ´ I. CANUDO1 MIGUEL MORENO-AZANZA,*,1 ELISABETTA MARIANI,2 BLANCA BAULUZ,3 and JOSE ´ Grupo Aragosaurus–IUCA, Area de Paleontolog´ıa, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain,
[email protected];
[email protected]; 2 Department of Earth and Ocean Sciences, University of Liverpool, 4 Brownlow Street, Liverpool L69 3GP, U.K.,
[email protected]; 3 ´ Area de Mineralog´ıa, Facultad de Ciencias, Universidad de Zaragoza. Pedro Cerbuna 12, 50009 Zaragoza, Spain,
[email protected]
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ABSTRACT—The direct relationship between eggshell structure and eggshell formation is well established for avian eggs, but has never been studied in depth in non-avian dinosaurs. Both biological and crystallographic mechanisms take part in eggshell formation, due to its dual mineralogical and proteinaceous nature, but the exact relative contributions of these processes are still poorly known. Competitive growth has been postulated to be the general mechanism leading to the characteristic columnar construction seen in dinosaur eggshell. Here we analyze the eggshell structure of both ornithopod and non-avian theropod ootaxa with orientation contrast imaging and electron backscatter diffraction and present the first misorientation angle boundary maps of fossil eggshell, in order to ascertain whether competitive growth can explain the development of the columnar structure in non-avian dinosaur eggshell. Our results show that both eggshell types can be constructed via competitive growth, and that small changes in organic core spacing and crystal grain size, which are organically controlled, may develop into major changes in general eggshell structure, which will determine the physical properties of the egg. However, interseed distance cannot be directly correlated with organic core spacing as the competitive growth model predicted.
INTRODUCTION Dinosaurs, including birds, are the most diverse group that uses amniotic hard-shelled eggs from the Triassic to the present (Carpenter, 1999). Whereas eggshell ultrastructure in birds is comparable across different species, the eggshells of non-avian dinosaurs display diverse internal crystal structure and, potentially, diverse crystal growth mechanisms. Dinosaur eggshell consists of organic matter, mainly proteins, and calcium carbonate (CaCO3 ) in the form of calcite crystals (Hirsch, 1994). These calcite crystals form the mineralized layer, which can be arranged in a single layer, as in the sauropod and ornithopod dinosaurs (Horner and Makela, 1979; Chiappe et al., 1998, 2001), or in multiple, distinct layers, as observed in theropods, including birds (Norell et al., 1995; Varricchio and Jackson, 2004; Grellet-Tinner and Makovicky, 2006), and possibly in neoceratopsians (Balanoff et al., 2008, but see comments on Jackson and Varricchio, 2010). In birds, eggshell growth mechanisms during egg nucleation and development have been studied extensively, especially in hens (e.g., Hernandez-Hernandez et al., 2008; Nys et al., 2010), due to the economic importance of these farm animals and the easy access to samples at different stages of their growth (Simons, 1971; Mikhailov, 1997a, and references within). By contrast, the processes involved in the formation of non-avian dinosaur eggshells are still poorly understood. Mechanisms of crystal nucleation and growth are crucial to the final structure and strength of the eggshell that protects the embryo. In birds, the mineralization of the eggshell starts with the secretion of the organic cores, which are distributed at quasiperiodic, discrete locations on the eggshell membrane (Wyburn et al., 1973; Fernandez et al., 1997). These are amorphous CaCO3 spherules that form on the inner membrane, and develop into small crystallites, or mammillae. The mammillae grow radially at *Corresponding
author.
first, competing for space, and then form elongated calcite crystals (palisades). Palisades form the greater part of the eggshell and extend to the outer surface (Grigor’ev, 1965; Garc´ıa-Ruiz and Rodriguez-Navarro, 1994; Checa et al., 2006; Chien et al., 2008; Nys et al., 2010; Freeman et al., 2010). Competitive crystal growth has been postulated as the mechanism responsible for eggshell formation in hens (Garc´ıa-Ruiz and Rodriguez-Navarro, 1994). In this model, growing crystals compete for space with their adjacent neighbors, and only crystals with their c-axis parallel to the direction of eggshell growth—i.e., orthogonal to the eggshell surface—‘survive’ competition and reach the top of the eggshell (Grigor’ev, 1965). In most crystalline aggregates, nucleation of a large number of small grains in arbitrary orientations is a prerequisite for competitive crystal growth (e.g., Epishin and Nolsen, 2006). This is not the case with eggshells, where nucleation at the organic cores occurs around a protein nucleus (i.e., the organic core), from which crystals fan out in radial orientations. Two different stages of competition take place. First, crystals compete with neighbors from the same organic core, resulting in the radial fanning geometry. Afterwards, surviving crystals finally reach their homologues from adjacent organic cores, and a new stage of competition starts. The relative importance of competition between crystals from the same organic core and competition between crystals from different organic cores remains uncertain (see Grigor’ev, 1965, for a detailed description of this process). Electron backscatter diffraction (EBSD) and orientation contrast imaging (OCI) techniques have only recently been applied successfully to paleontology to study different biomineralized structures, such as mollusk shells (e.g., Checa et al., 2006; Fryda et al., 2009) and recent eggshells (Dalbeck and Cusack, 2006). Only two EBSD studies of dinosaur eggshells have been conducted to date (Shaw, 2009; Grellet-Tinner et al., 2011), mainly focusing on ruling out a potential diagenetic overprint on eggshell structure.
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Differences in the ultrastructure of the eggshells of ornithopod and theropod (at least troodontid and avian) dinosaurs have been reported previously on the basis of their characteristics under the optical microscope and scanning electron microscopy (SEM) (see, e.g., Mikhailov, 1991, and references within). EBSD provides new information on the crystallography of the eggshell because, although relative misorientation between larger crystals and domain areas can be inferred qualitatively from crosspolarized microphotographs, undulating extinction areas can be resolved quantitatively and with higher resolution using EBSD in a SEM, particularly when analyzing small crystals (72% successful indexing in cf. Maisaura eggshell and >87% in the prismatoolithid resulting from full automation, allowing a minimum amount of data processing after acquisition. Noise reduction was applied: first wild spikes—one pixel misindexed grains—were removed. Then, extrapolation of zero solutions was carried out first for pixels with eight and seven neighbors with automatic iteration. Further extrapolation was then carried out using six neighbors twice without iteration. This resulted in a 5% improvement of the indexing in both samples. Additional
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MORENO-AZANZA ET AL.—DINOSAUR EGGSHELLS UNDER EBSD extrapolation was not carried out to avoid artefacts in grain size and shape (Prior et al., 2009). The prismatoolithid possesses a distinctive, highly packed compactituberculated ornamentation in the outer surface. This ornamentation makes the upper part of the thin sections irregular in topography, resulting in poor sample polishing and charging effects that interfere with the acquisition of EBSD data. Thus, the outer part of this eggshell type (∼150 µm) was not analyzed. To study the differences between the two eggshells, grain boundary maps, inverse pole figure (IPF) coloring maps, and pole figures were elaborated. Boundary maps for grains with relative misorientations of >20◦ , >10◦ , and >5◦ were obtained (Fig. 2). The IPF coloring maps (Fig. 3A, C) show absolute crystal orientation, with red representing crystals with their c-axis oriented orthogonal to the eggshell curvature and thus parallel to the eggshell direction of growth, and blue to green representing crystals with the c-axis orthogonal to the direction of eggshell growth. The pole figures (Fig. 3B, D) show crystal axis orientation plotted on a stereonet: pole figures represent orientation of the c-axis, whereas and pole figures show the corresponding orientation of the a- and b-axes of the crystals. All pole figures are equal-area projections of the upper hemispheres. Contours are calculated with a half width of 20◦ and a cluster size of 5◦ . In order to test if the hypothesis of competitive crystal growth applies to the samples analyzed in this study, the number of grains with a misorientation angle >20◦ , >10◦ , and >5◦ with all of their neighbors was subsequently counted along 30 equidistant line intercepts through the complete eggshell thickness. The rate at which the number of growing grains decreases with eggshell height was quantified using an empirical mathematical relationship. The data acquired were then compared with the computer simulation model proposed by Garc´ıa-Ruiz and RodriguezNavarro (1994). RESULTS Overall macroscopic differences between the two eggshells examined are patent: the two eggshells possess different ornamentation patterns—sagenotuberculate in cf. Maiasaura and compactituberculate in the prismatoolithid. The cf. Maiasaura eggshell presents growth lines that are subparallel to the outer surface in the upper part of the eggshell, which are absent in the prismatoolithid. Finally, cf. Maiasaura eggshell presents undulose extinction, whereas the indeterminate prismatoolithid shows discrete extinction of individual crystals. Nonetheless, the general crystallographic architecture of the eggshells is similar in both specimens. Two distinct crystallographic fabrics can be observed in both eggshells: in the inner part of the eggshell units, elongated calcite crystals radiate out from organic cores in the former inner organic membrane, and the crystallographic preferred orientation (CPO) is relatively weak, although not random. In the middle and outer parts of the eggshell units, crystals display a strong fabric, with their c-axes subparallel and contained within the shell cross-section—i.e., the data acquisition surface—(Figs. 1–3). Despite the comparable architecture of the eggshell, the orientation data show important differences. The cf. Maiasaura eggshell presents discrete spherulitic shell units consisting of aggregates of prismatic calcite crystals arranged in a fan-like structure, which radiate out from the inner membrane (Fig. 1A, B). Each of these aggregates has an internal radial ultrastructure consisting of elongated calcite grains and subgrains, which emphasize the general fanning aspect of the eggshell units (Fig. 1A, B). Adjacent units present interlocking crystals, which may increase the stiffness of the eggshell (Dalbeck and Cusack, 2006). In the prismatoolithid (Fig. 1C, D), two different layers can be observed, an inner layer with elongated crystals radiating from the organic cores, and a prismatic outer layer with large prismatic
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FIGURE 1. cf. Maiasaura eggshell, MPZ 2000/3563 (A, B); Prismatoolithidae indet., MPZ 2000/3565 (C, D). A–D, comparison between usual light microscope photographs (A, C) and orientation contrast (OC) images (B, D) of the two eggshells included in this study. Note highlighted crystallography in OC images. Misorientation between subgrains causes a grayscale zonation that highlights the ultrastructure of the eggshell. Dashed areas were not analyzed due to charging and consequent misindexing. Scale bars equal 200 µm. (Color figure available online.)
crystals, which grow normal or subnormal to the eggshell surface. These prismatic crystals present a tabular ultrastructure. These differences can be observed under the optical microscope, whereas details of the microstructure and some of the crystallographic features, such as the crystal size and shape-preferred orientation (SPO), and the ultrastructure at the subgrain level, are highlighted in the OCI (Fig. 1B, D). EBSD data show that in cf. Maiasaura the size of the grains varies between 20 and 200 µm, with elongated grains in the palisade part of the structure (Fig. 2A), whereas in the prismatoolithid the grain size varies between 10 and 150 µm (Fig. 2B). The transition between the radial and the parallel fabrics is rapid in the cf. Maiasaura eggshells, occurring within 150 µm from the organic membrane outwards, with a strong alignment of the caxis of the crystals parallel to the direction of eggshell growth (Fig. 3A, B). In contrast, the prismatoolithid presents a slow transition between the radial and growth-parallel c-axis fabrics, with elongated crystals progressively increasing their alignment as they reach the upper part of the eggshell (Fig. 3C, D). Importantly, the alignment is not as strong as in the cf. Maiasaura eggshell, as some dispersion of the c-axis around the direction of eggshell growth can be observed (Fig. 3C, D). The distribution of misorientation angles between neighboring grains is strongly skewed towards low-angle boundaries (20◦ boundaries. Note the near absence of low-angle boundaries in the prismatoolithidae, whereas these are most frequent in the cf. Maiasaura eggshell. Red arrow indicates direction of eggshell growth during egg formation. Scale bars equal 100 µm.
be applied more generally to dinosaur eggshells, we measured the number of >20◦ , >10◦ , and >5◦ grain boundaries relative to neighboring grains along 30 equally spaced lines perpendicular to the growth direction, spanning the whole thickness of each eggshell thin section analyzed. The number of surviving grains (n) on each line was counted and plotted as a percentage. This value was then represented graphically versus the eggshell thickness, h (percentage of the distance of each line from the eggshell substrate). In order to identify a systematic mechanism of crystal growth, a power-law function was fitted to the data for both the cf. Maisaura eggshell (Fig. 5A) and the prismatoolithid (Fig. 5B) because competitive growth is best described with these relations (Grigor’ev, 1965). Our observations were then compared with the numerical model proposed by Garc´ıa-Ruiz and RodriguezNavarro (1994). In both eggshells, the best fitting was found considering grains with a critical misorientation of >5◦ . In cf. Maiasaura there is a high variation in the quality of the fitting with grains of low-angle boundaries, which clearly do not follow a power law, whereas a mediocre fitting is achieved for high-angle-boundary grains. On the other hand, in the prismatoolithid there are no important differences in the quality of the fittings for the three critical misorientation angles measured. It is noticeable that in both eggshells there is a relative increase in the number of crystals in the upper half of the eggshell (at 700 µm in the cf. Maiasaura eggshell and at 400 µm in the prismatoolithid), followed by a new decay to the top of the eggshell. This point coincides with the end of the interlocking area in the cf. Maiasaura eggshell, whereas in the prismatoolithid there is no clear correlation with the eggshell microstructure. On the basis of the initial model of Garc´ıa-Ruiz and Rodriguez-Navarro (1994), we derived a new empirical relationship that could describe the data rigorously, accounting for the important influence of interseed distance (a) on the final eggshell architecture. In the case of eggshells, the interseed distance can a priori be related to the organic core distance, which is the distance between the nuclei of the crystal units. As noted above, competition does not only exist between crystals from different eggshell units, as the first stages of crystal growth occur within the spherulites. Thus, the relation between core distance and interseed distance cannot be established beforehand. The technique used to obtain a rigorous empirical equation is described below.
Firstly, n and h were plotted as natural logarithms, ln(n) versus ln(h) (Fig. 6A). This allowed us to fit linear relationships to our data and to those from Garc´ıa-Ruiz and Rodriguez Navarro (1994). As noted by these authors, for low values of a (a = 1, a = 10) the empirical data follow a power law. However, we have found that for higher values of a (a = 100), the data are best described by an exponential relationship (Fig. 6B). Bearing this in mind, we plotted our data in both a log-log graph and a semi-log graph. Unexpectedly, the sets of data for both cf. Maiasaura and the prismatoolithid. fit best to exponential relationships, despite the expected values of the interseed distance being low. The slopes (s) of such linear regressions become steeper with decreasing values of interseed distance, a, following the equations: y = 340.05x + 6.4739 for the power-law relationship and y = 11.049x + 5.8713 when exponential relationships are considered. This allowed us to calculate the values of a expected for the cf. Maiasaura eggshell (Fig. 6C) and the prismatoolithida (Fig. 6D). The cf. Maiasaura eggshell has higher calculated values of a: 10.95 if power laws are considered, 16.08 if exponential relations are taken into account. The prismatoolithid has values of 2.62 and 3.77, respectively. DISCUSSION In this study, EBSD mapping provided high-resolution, complete, and quantitative information on crystal orientation in the dinosaur eggshell structures analyzed. These data include complete relative and absolute orientations, misorientation angles and misorientation angle distribution, and crystal shapes and sizes. Such data sets are powerful tools that can be used to characterize and compare ornithopod and theropod eggshells, and can provide new characters that can be useful in phylogenetic analysis. A more detailed study of a wider sample of different dinosaur eggshells, including all oofamilies described to date, would be of high interest. The main differences between non-avian theropod and ornithopod eggshells emerge in the grain boundary maps, especially in the palisade layer (Fig. 2A, B). Even though both present a palisade layer composed of strongly oriented prismatic crystals, the cf. Maiasaura eggshell has a high number of intracrystal lowangle boundaries (10◦ , and >5◦ misorientation grain boundaries are considered for the cf. Maiasaura eggshell, MPZ 2000/3563 (A), and Prismatoolithid indet., MPZ 2000/3565 (B). Equations and R2 figures are shown for each case. A, the number of surviving grains is independent of the height in the eggshell where low-angle boundaries are considered, whereas there is a power-law relationship between them if higher-angle boundaries (>10◦ and >20◦ ) are considered. The first three data points represent the first 120 µm of the eggshell, comprising the lower part of the spherulites, where the crystals radiate downwards, so the fitting has only been performed for the remaining 27 data points, which cover the area of interest. B, the low number of low-angle boundaries means that, independently of which critical boundary misorientation is considered, the decay in the number of grains follows a power law. Note the relative increase in number of grains at 700 µm in A and at 400 µm in B.
The competitive growth model plays a role in several nonbiological processes, such as geode and speleothem formation (Grigor’ev, 1965), and has been observed in the laboratory with certain artificial minerals (Messier, 1990). These studies suggest that if several crystal seeds are left to grow with enough space in a suitable environment, they will grow competitively if no restrictions are applied. The early development of strong c-axis alignment in cf. Maiasaura eggshell and the high space between organic cores reduces competition between crystals from the same eggshell unit, as observed in other strongly aligned materials (Epishin and Noltze, 2006), and certainly minimizes the interaction between crystals across different eggshell units. In fact, this is limited to the uppermost part of the eggshell. As a consequence of this, the strength of the whole structure is significantly reduced. The benefits of an eggshell made by independent eggshell units, but significantly weaker, may be understood if egg
size and nesting strategies are examined, as suggested by Shaw (2009). Spheroolithid eggs are small to medium in size (7–10 cm sensu Carpenter, 1999) and are spherical in shape, which allows a better distribution of stress. Furthermore, the huge sizes that hadrosaur dinosaurs can reach suggest that incubation strategies do not include sitting on the eggs, but burial. In this context, weaker eggshells may be preferred because there are no significant stresses applied to the egg and this will allow the hatchlings to break the egg more easily. Conversely, prismatoolithid eggs are medium to large in size (ranging between 10 and 17 cm in their long axis) and have elongated to ovoid shape (Mikhailov, 1997b), which affects the stress distribution in the egg, with important differences in resistance between the equatorial part of the egg and the poles. In addition, nest sitting in theropods, which lay prismatoolithid eggs, has been
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FIGURE 6. A, log-log plot of number of surviving grains versus eggshell thickness for numerical model relations proposed by Garc´ıa-Ruiz and Rodriguez-Navarro (1994), for cf. Maiasaura eggshell, MPZ 2000/3563, and the Prismatoolithid indet., MPZ 2000/3565. B, semi-log plot of number of surviving grains versus eggshell thickness for numerical model relations proposed by Garc´ıa-Ruiz and Rodriguez-Navarro (1994) for cf. Maiasaura eggshell and the Prismatoolithid indet. Note that the computed values follow a power law for low values of interseed distance (a), whereas high values of a are best explained with an exponential relationship. Contrary to what is expected, an exponential fitting to the measured data results in a better matching, particularly for cf. Maiasaura, despite the low values of a of both eggshells. Relationship between the slopes of lines calculated in A and a. C and D, relationship between the slopes of lines calculated in A and B, respectively, and interseed distance (a). The value of a is approximately 4 times greater in cf. Maiasaura than in the prismatoolithid, in both cases. This value contrasts with the distance between organic cores, which is less than 3 times greater in cf. Maiasaura than in the Prismatoolithid indet.
reported (Varricchio et al., 1997), involving a large weight placed over the egg during incubation. In these circumstances, strong eggshells are required to prevent the eggs hatching too soon or been accidentally crushed. Proteins commonly found in avian eggs have been reported to control the preferred growth of crystals and crystal faces in avian eggshells, modifying the fabric and ultrastructure and the physical properties of the eggshell as a consequence (Nys et al.,
2001, 2004; Chien et al., 2008; Freeman et al., 2010). Evidence of molecular compounds similar to those that form the proteins in avian eggshell has been reported in pristine preserved dinosaur eggshells (Schweitzer el al., 2005). In modern birds, the quasiperiodic distribution of the organic cores is under strong proteinic, and therefore genetic, control (Wyburn et al., 1973; Fernandez et al., 1997). Our results show that organic core spacing controls the main architecture of the eggshell, from ultrastructure
MORENO-AZANZA ET AL.—DINOSAUR EGGSHELLS UNDER EBSD to microstructure, and can affect physical properties of the egg. Further research is needed to test if organic core spacing can be used as a key character in future phylogenetic analysis of eggshell structure.
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CONCLUSIONS EBSD and OCI have been used for the first time to understand the growth of the eggshell in non-avian theropod and ornithopod dinosaurs. EBSD and OCI are powerful tools for use in the description of fossil eggshells. Orientation contrast images provide good-quality qualitative data that help in the description and understanding of eggshell structures. EBSD is a source of quantitative data, including crystal shape, size, orientation, and relations between crystals, which can be useful in eggshell description and classification. If the phylogenetic signal is confirmed in the control of the crystallography and geometry of the eggshell, EBSD data will allow new characters to be included in the data matrix for cladistic analysis, such as the relative frequency of lowmisorientation-angle boundaries, crystal size, dispersion of the orientation of crystals, and relations between crystals from different shell units. In addition, this technique can be used to better understand previously used characters. The avian model of eggshell crystal growth fits theropod and ornithopod eggshell structure, but interseed distance cannot be directly correlated with organic core spacing as the model predicted. In addition, our results suggest that ornithischian dinosaurs, or at least hadrosaurs, have active biological control of the growth and formation of the eggshell, via a looser spacing of organic cores and a wider interseed distance. These features reduce competition between crystals and allow the eggshell units to grow discretely, likely making the egg more fragile than the theropod egg. Also, the lack of space for the crystals and the low-angle relationship between neighbors result in a high number of low-angle boundaries in the ornithopod eggshell, which are not present in the theropod eggshell, thus weakening the general structure even more. These features allow the eggshells to be broken by the hatchlings of hadrosaur dinosaurs, which seem not to sit on their eggs and do not need hard-shelled eggs. On the other hand, tighter organic core distribution may have helped the nonavian theropod dinosaurs to strengthen the eggshell, allowing different strategies of egg incubation.
ACKNOWLEDGMENTS This paper forms part of the projects CGL2010-16447 and CGL2009-07574, subsidized by the Ministry of Science and Innovation, the “Gobierno de Aragon” and European Social Fund ´ General de Patrimonio “Grupos consolidados”, and “Direccion Cultural”. M.M-.A. is supported by the Spanish Ministry of Science and Innovation, grant FPI BES-2008-005538. C. Shaw, A. Rodriguez-Navarro and the editor Randall Irmis revised the paper and greatly improved our work. M.M.-A. wants to thank O. Kalin for his inspiring comments and references in the ´ preliminary phase of this work. J. Gomez and F. Jackson made helpful comments that improved the paper in its final form. R. Glasgow edited the text in English. The cf. Maiasaura eggshells were donated by F. Sweeney.
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