Citation:

Marzola, M., Russo J., & Mateus O. (2015).  Identification and comparison of modern and fossil crocodilian eggs and eggshell structures. Historical Biology. 27(1), 115-133.

Abstract:

Eggshells from the three extant crocodilian species Crocodylus mindorensis (Philippine Crocodile), Paleosuchus palpebrosus (Cuvier's Smooth-fronted Caiman or Musky Caiman) and Alligator mississippiensis (American Alligator or Common Alligator) were prepared for thin section and scanning electron microscope analyses and are described in order to improve the knowledge on crocodilian eggs anatomy and microstructure, and to find new apomorphies that can be used for identification. Both extant and fossil crocodilian eggs present an ornamentation that vary as anastomo-, ramo- or the here newly described rugosocavate type. The angusticaniculate pore system is a shared character for Crocodylomorpha eggshells and some dinosaurian and avian groups. Previously reported signs of incubated crocodilian eggs were found also on our only fertilised and hatched egg. Paleosuchus palpebrosus presents unique organization and morphology of the three eggshell layers, with a relatively thin middle layer characterised by dense and compact tabular microstructure.

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Identification and comparison of modern and fossil crocodilian eggs and eggshell structures.

Abstract. Eggshells from the three extant crocodilian species Crocodylus mindorensis (Philippine Crocodile), Paleosuchus palpebrosus (Cuvier’s Smooth-fronted Caiman or Musky Caiman) and Alligator mississippiensis (American Alligator or Common Alligator) were prepared for thin section and SEM analyses and are described in order to improve the knowledge on crocodilian eggs anatomy and microstructure, and to find new apomorphies that can be used for identification. Both extant and fossil crocodilian eggs present an ornamentation that vary as anastomo-, ramo- or the here newly described rugosocavate type. The angusticaniculate pore system is a shared character for Crocodylomorpha eggshells and some dinosaurian and avian groups. Previously reported signs of incubated crocodilian eggs were found also on our only fertilized and hatched egg. P. palpebrosus presents unique organization and morphology of the three eggshell layers, with a relatively thin middle layer characterized by dense and compact tabular microstructure.

Keywords: Extant and fossil crocodyliform eggshells, Crocodylus mindorensis, Paleosuchus palpebrosus, Alligator mississippiensis, eggshell structures, rugosocavate pore canals type

Introduction

Numerous references have been focused on crocodile reproduction, however very little is known about extant crocodilian eggs and eggshells morphological structure: Ferguson (1982), Grine and Kitching (1987) and Deeming and Ferguson (1990) report of Alligator mississippiensis (Daudin 1802);  Zhao and Huang 1986 report of Alligator sinensis Fauvel 1879, Schlëich and Kästle (1988) and Fernández et al. (2013) report of Caiman latirostris (Daudin 1802), Grine and Kitching (1987) report both of Crocodylus niloticus Laurenti 1768 and Crocodylus porosus Schneider 1807. An example of a rare comparison between eggs of two extant crocodiles, respectively Caiman latirostris and Caiman yacare (Daudin 1802), has been previously made by Paz et al. (1995).

Our samples belong to three extant crocodilian species, from to the infraclass Archosauromorpha and the suborder Crocodyliformes: Crocodylus mindorensis Schmidt 1935, Paleosuchus palpebrosus (Cuvier, 1807), and A. mississippiensis. The Philippine crocodile (C. mindorensis) is a relatively small freshwater crocodile endemic to the Philippines. Both male and female individuals reach their sexual maturity when about 1.5 m long and 15 kg in weight, with the longest individual ever reported of 3.02 m in length; females are slightly smaller than males (Hall 1898; van Weerd 2010). Van Weerd (2010) reports that the number of laid eggs by a female in two different localities in the wild is 20.1 and 26.0, while for two different localities in captivity is respectively 15.7 and 25.6, with an incubation time of 65 to 78 days in the wild and 77 to 85 in captivity. The Cuvier’s smooth-fronted caiman or musky caiman (P. palpebrosus) is an endemic to South America crocodile, the smallest of all living crocodilians; males can reach a length of 1.5 m while females are slightly smaller, reaching 1.2 m.; an adult typically weighs around 6 to 7 kg (Magnusson 1992). Around 10 to 19 eggs are laid, usually white, oblong, weighing between 61 to 70 grams and that hatch after about 90 days (Magnusson and Campos 2010; Medem 1971). The American alligator or common Alligator (A. mississippiensis) is the largest of the two extant species in the genus Alligator and it is endemic to the southeastern United States. Grown-up males can reach about 5 m in length, females 3 m, with the largest individuals up to 450 kg in weight. Nesting and egg-laying is initiated during the early part of the warm, wet summers (Ross and Ernst 1994; Elsey & Woodward 2010). Females construct a mound nest of vegetation and lay 30 to 50 eggs. Incubation takes 63 to 84 days, depending on temperature (Lang and Andrews 1994).

This study aims (1) to describe the morphology, the micro- and the ultrastructure of the eggshells from three extant crocodilian species; (2) to improve the general view over crocodilian eggshells, by comparing our samples with the known extant and fossil crocodilian eggshells; (3) to point out new apomorphies for crocodilian eggs that may help eggshell identification and further cladistic analyses. One other output is an overview of egg characteristics within the framework of the general consensus phylogeny of  Amniotes.

State of art about egg characters and eggshell ultrastructure

In extant crocodile eggs, the shell units start to outgrow from the uppermost fibers of the shell membrane (Mikhailov 1997). The entire eggshell is characterized by a tabular ultrastructure forming regular striations (Mikhailov 1997); the presence of this characteristic laminated tabular structure in the middle layer is given by most authors as the diagnostic condition for crocodylomorph eggshell, both extant and fossil (Hirsch 1985; Schlëich and Kästle 1988; Mikhailov 1997; Jackson & Varricchio 2010). No organic core is present at the base of the inner layer (IL); instead, an aggregation of calcite plates that serve as the nucleation center is evident (Mikhailov 1997; Carpenter 1999; Rogers 2001), as well as basal plate groups and basal knobs characterizing all the inner surface described in Hirsch (1985). On the contrary, Moreno–Azanza et al. (2013, p. 4) state about nucleation centers that ‘No crystallographic features can be identified either in SEM or in petrographic photographs, suggesting that the nucleation centres are poorly crystallised aggregates of calcite micro grains and organic matter’, suggesting the possibility of organic matter in the nucleation centers of crocodylomorph eggs. Moreover, the IL presents a series of ‘wedges’ (sensu Mikhailov 1997, p. 15, Fig. 5), large radiating subunits characteristic for the entire shell unit, distinguishable in observation with crossed nicols, under polarized light (Moreno–Azanza et al. 2013).

Generally, both extant and fossil crocodilian eggs are associated with a crocodiloid morphotype. The microstructure of this morphotype is characterized by discrete, large and rough shell units having a truncated cone shape. The ultrastructure is made of trapezoid-shaped units wider at the top (outer surface) than the bottom (inner surface), with a bulbous base, exhibiting a rosette-like structure in the inner eggshell surface. The shell units are built up by large and rough wedges with irregular boundaries; no fan-line pattern can be seen on radial sections (Mikhailov 1997; Carpenter 1999).

The state of the art on crocodilian eggs is given in Moreno–Azanza et al. (2013): ‘The micro– and ultrastructure of extant and fossil crocodilian eggshells remains controversial. Ferguson (1982) describes five distinct layers in the A. mississippiensis eggshell, four of which – the mammillary layer, the organic layer, the honeycomb layer and the outer, densely calcified layer – correspond to the calcified portion of the eggshell, or true eggshell. [...] Following Hirsch (1985), Mikhailov (1991, 1997) establishes the crocodyloid basic type and the corresponding crocodyloid morphotype as single-layered eggshell with ‘rough’ shell units. This approach is followed by Kohring and Hirsch (1996) in erecting the Krokolithidae oofamily. [...] More recently Jin et al. (2010) confirmed Ferguson’s observation that crocodilian eggshell is composed of several structural layers. [...] The presence of three structural layers is patent in Krokolithes wilsoni and in the eggshells of extant Crocodylus porosus and Crocodylus niloticus’.

The ultrastructures of few extant crocodilians have been so far described: A. mississippiensis and C. niloticus are described in Grine and Kitching (1987) as similar to one another. The eggs of these two species are described as made by an innermost layer consisting of mammillary processes densely packed. The mammillary crystals radiate outwards from a basal center and become gradually extinguished by tabular crystal lamellae. A second upper layer (‘palisade layer’) is then described, made of tabular aggregates with the lamellae disposed parallel to the outer surface of the egg. A. sinensis is described in Zhao and Huang (1986) as made by three differentiate layers: a mammillae layer, where the tips of each mammilla is a spherulitic aggregate of aragonite crystals radiating from the mammilla core, a cone layer and a columnar layer, characterized by small erosion pits on incubated eggs. C. latirostris is described in Fernández et al. (2013) as made up of one single calcareous ultrastructural layer characterized by ‘units formed from the irregular radial growth of tabular wedge-like crystals with a basal plate group (rosette); the organic core is absent’.

        Incubation has an influence on the eggshell morphology and preservation (Oliveira et al. 2011). An extrinsic degradation, characterized by many erosion pits (‘craters’ in previous literature) and stepped concentric erosion rings around the pore openings, has been reported for incubated eggs of A. mississippiensis (Ferguson 1981a; 1982; Hirsch 1985; Deeming and Ferguson 1989, Wink et al. 1990a), of A. sinensis (Zhao and Huang 1986; Wink and Elsey 1994) and of C. niloticus (Grine and Kitching 1987). Also, it has been previously documented on A. mississippiensis that the initial porosity of unfertilized eggshell is related to the density of mammillae on the inner surface of the shell and that incubation destroys the original relationship between pores and mammillae (Wink et al. 1990b), as well as that in A. mississippiensis the eggshell degrades progressively, losing thickness, because of the acidic metabolites of the microorganisms involved in the nest fermentation (Ferguson 1981b).

Materials and methods

A total of three eggs by three different species of extant crocodiles have been analyzed, so one eggshell per species. The eggshells were provided by Rene Hedegaard (Krokodille Zoo, Denmark) and Jesper Mìlan (Geomuseum Faxe, Denmark). The eggshell thin section slides are stored at FCT-UNL with the repository numbers FCT-UNL 707, 708 and 709 respectively. C. mindorensis and A. mississippiensis eggs were unfertilized, thus complete, while P. palpebrosus egg was fertilized and hatched. All the main parameters for the eggshells are reported in Tab. 1.

From each eggshell, selected samples were prepared for 30 μm thin sections using epoxy resin EpoThin 5 (resin) and 1.95 (hardener). Fragments imaged using a JEOL JSM T330A scanning electron microscope (SEM) at the Universidade Nova Lisboa (FCT-UNL) were previously treated with 10% formic acid for 30 seconds to dissolve the eggshell membrane, as well as those for observation and imaging under petrographic microscope. Polar and equatorial axis measures were taken with a caliper from the entire eggshell when possible; pores were counted from direct observation of the samples with a petrographic microscope and opening diameters were measured from the external surface using macro photographs; eggshell and structural layers thicknesses were measured from the thin sections. During the eggshell description, we standardized the orientation of the samples with the external (outer) surface to the top, and the internal (inner) surface to the bottom.

The following acronyms have been used: EA = equatorial axis of the egg (shortest); EI, elongation index (ratio PA/EA); IL, inner layer; IS, inner surface; ML, middle layer; n, number of measurements; OL, outer layer; OS, outer surface; PA, polar axis of the egg (longest); SD, standard deviation; V, egg volume.

Institutional abbreviations: FCT-UNL, Faculdade de Ciências e Tecnologia da Universidade de Lisboa (Portugal).

Density values in Tab. 2 were measured using different sets of data for masses and volumes. Due to this, they are merely indicative.

Main characters and structures in crocodilian eggs

[Tab. 1 near here]

[Tab. 2 near here]

[Tab. 3 near here]

1. Egg shape, dimensions and eggshell thickness

        Modern and fossil crocodilian eggs are generally ellipsoid and our two complete samples (C. mindorensis and A. mississippiensis) confer with this shape. Both poles are equal in width, so the eggs are symmetrical to the equatorial plane; thus, the eggs are not oval and  have dimensions and thickness which differ substantially from species to species. All the relative dimensions of our samples are presented in Tab. 1.

In a generic view, modern crocodilian eggs have a PA (polar or longest axis of the egg) between 58 mm and 102 mm in length, an EA (equatorial or shortest axis of the egg) between 34 mm and 63.5 mm, with an EI (elongation index) between 1.43 and 2. The eggshell thickness varies from 0.30 mm to 0.85 mm. The mass value ranges between 48.2 g in A. sinensis to 161.4 g in Gavialis gangeticus (Gmelin 1789) (see data in Thorbjarnarson 1996); volumes are variable in a range between 41.1 cm3 in A. sinensis and 189.2 cm3 in Tomistoma schlegelii (Müller 1838). Finally, modern crocodilian eggs have a very stable density, between 0.94 g/cm3 in T. schlegelii and 1.22 g/cm3 in Osteolaemus tetraspis Cope 1861 (Tab. 2). Fossil crocodilian eggs seem to have smaller dimensions than modern ones, with a PA between 70 mm and 30 mm and an EA between 16 mm and 54 mm; however, the EI does not differ so much, being included between 1.19 and 2.11, as well as the thickness, which goes between 0.15 mm and 0.76 mm (Tab. 3).

On Alligatoridea eggs the dimensions range goes from 62mm x 39 mm in P. palpebrosus (Medem 1971) to 76 mm x 42 mm in A. mississippiensis (Hirsch and Koring 1992) – 71.5 mm x 44.8 mm in our sample – with an EI between 1.43 in C. latirostris and 1.79 in P. palpebrosus (Medem 1971, Panadès I Blas and Patnaik 2009). The eggshell thickness ranges from 0.41 mm in P. palpebrosus (our sample) to 0.85 mm in C. latirostris (Schlëich and Kästle 1988); Fernández et al. 2013 gave a range of thickness for C. latirostris between 0.36 mm and 0.72 mm, calculating the thickness respectively without and with superficial ornaments. In Crocodylidae family the range goes from 58 mm x 40 mm in Crocodylus johnstoni Krefft 1873 (Hirsch and Kohring 1992) to 101.6 x 63.5 in T. schlegelii (Butler 1905), with our sample from C. mindorensis of 69.3 mm x 37.3 mm with an EI between 1.44 in C. johnstoni and 1.86 in C. mindorensis (Hirsch and Kohring 1992, our sample). The thickness ranges from 0.4 mm in Crocodylus acutus (Cuvier 1807) and C. johnstoni (Hirsch and Kohring 1992, Panadès I Blas and Patnaik 2009) and 0.60 mm in C. porosus (Hirsch and Kohring 1992), with our sample value from C. mindorensis of 0.43 mm. In Gavialidae family, the only extant species G. gangeticus presents dimensions equal to 82 mm x 56 mm, an EI equal to 1.46 and a thickness between 0.30 mm to 0.59 mm (Panadès I Blas and Patnaik 2009).

2. External surface

The classification for the external surface ornamentation proposed in Carpenter (1999) for dinosaurian eggs is not commonly used in the extant literature describing modern and fossil crocodilian eggs. The crocodilian eggs present some ornamentations, but those do not fit with the types already described.

The studied samples present an external hard and crystallized shell and an internal thin layer, the egg membrane. The color of the external eggshell surface is whitish in all our three samples; the thin egg membrane presents a leather-like aspect. In C. mindorensis, the external surface (Fig. 01a-b) presents an ornamentation characterized by an irregularly rugose surface scattered by subcircular pits, that not always correspond to pore openings: this kind of ornamentation seems unique in its kind and does not resemble any of the known and described type in Carpenter (1999); thus here propose the rugosocavate as a new type of external surface ornamentation for crocodilian eggs. In P. palpebrosus, the fragments bear bumps and nodes, more compact than in C. mindorensis, but somewhat resembling the surface of a golf ball (Fig. 02a). We interpret this ornamentation as a rugosocavate type as well, although differs from C. mindorensis for the denser and less irregular shape of the pits. The degradation of the outer surface is due to microbiological processes during the incubation and is characterized by many erosion pits and stepped concentric erosion rings around the pore openings (Fig. 02a, c and e). In A. mississippiensis, the external surface presents an anastomotuberculate-like ornamentation along the equatorial region with curly, ramified, bulbous and polar elongated ridges (Fig. 03a). On the contrary, the polar regions are smooth with some sporadic bulbs.

[Fig. 01, Fig. 02, Fig. 03 near here]

In modern crocodilian eggs, the texture and the ornamentation are smooth to rough, depending on the species and, in case of incubated eggs, on the grade of the degradation undergone during incubation (Schmidt and Schönwetter 1943, Ferguson 1982, 1985). The C. latirostris egg in Fernández et al. (2013, Fig. 1C) seems to have an ornamentation characterized by pronounced isolated bumps (‘towers of ornamentation’) and deep craters or pits of erosion. In Paz et al. (1995), both eggs from C. latirostris and C. yacare present an external surface made by a layer of craters and corresponding columnar structures formed by deposits of calcite crystals and with an anastomosed appearance.

Fossil crocodilian eggs usually present a smooth external surface due to the weathering and dissolution processes (Hirsch and Kohring 1992, Antunes et al. 1998, Novas et al. 2009). However, there are some fossil crocodilian eggs still presenting a slightly undulated external surface with few depressions and small pits: the sample from the Eocene of the Bridge Formation (Hirsch and Kohring  1992, Fig. 2C, p. 61) resemble the ramotuberculate ornamentation described in Carpenter (1999), with irregular chains of nodes splitting and joining other nodes spreading all over the surface. The sample from the Upper Miocene of Chinji Beds of Pakistan in Panadès I Blas and Patnaik (2009, Fig. 3, p. 3) presents ‘cracks, smooth and patchy surfaces, and craters containing pores’, characterizing an ornamentation that resemble the rugosocavate type described for C. mindorensis, with a golf ball-like general aspect made of rugose surface pitted by subcircular depressions.

3. Pores

In modern and fossil crocodilian eggs, pores always form between shell units and extent from the external surface through the calcified layers to the inner surface to end between the eggshell unit cones usually straight and with a simple shape, other times with an inclined angle, irregular shapes and pore openings (Hirsch 1985, Wink et al. 1990a, Wink and Elsey 1994, Antunes et al. 1998, Panadès I Blas and Patnaik 2009).

The C. mindorensis has an angusticanaliculate pore canals system (sensu Carpenter 1999, p. 141 (Fig. 01g-h). Pores mean diameter is 101 µm (n = 20, SD = 44 μm). The distribution of pores is uneven: the average density is 21 pores per cm2, however, in the polar regions the value decreases to 10 pores per cm2. Mean individual pore area is 0.009 mm2 (n = 20, SD = 0.008 mm2) and the relative pore area is 0.19% (Tab. 3). On the outer surface, pores present sub-circular openings (Fig. 01a), while on the inner surface openings have triangular, trapezoidal or irregular shape (Fig. 01h, 04).

P. palpebrosus presents an angusticanaliculate pore system. Pores have a diameter of 115 µm (n = 20, SD = 25 μm) and the mean density is 22 pores per cm2. Pores mean area is 0.01 mm2 (n = 20, SD = 0.005 mm2) and the relative pore area is 0.22%. Pore openings are circular to subcircular in shape both on the outer and the inner surface (Fig. 02, 05).

In A. mississippiensis pores distribution is much more uneven than in C. mindorensis and P. palpebrosus, without any relevant change between polar and equatorial regions porosity. The pore system is angusticanaliculate and pore openings are subcircular both on the outer surface and in the inner surface (Fig. 03a, b, d and 06). Pores average diameter is 129 µm (n = 20, SD = 42 μm) and the mean density is 5 pores per cm2. Pores mean area is 0.015 mm2 (n = 20, SD = 0.009) and relative mean area is 0.08%.

[Fig. 04, Fig. 05, Fig. 06 near here]

4. Eggshell sections

In C. mindorensis, the discrete shell units have a trapezoidal shape (Fig. 07a), wider at the top (external surface), with a width to height ratio of 0.58 for the single unit and a nucleation center and basal knobs at the bottom of each (Fig. 01d, f, Fig. 04 and 7b). The entire eggshell presents three distinct structural layers (Fig. 08): (1) a dark IL, consisting of nucleation centers characterizing the entire inner surface; (2) a pale ML, with noticeable linear brown growth lines and (3) an OL, darker than the ML probably for the higher presence of organic material. The typical crocodilian tabular ultrastructure is less visible on the thin section sample and can be only seen on SEM images, especially on the upper part of the OL. The growth lines are faint at the basal part of the ML and get more pronounced in outward direction, as well as there is not a clear distinction between the IN and the ML. The IL, ML and OL to total eggshell thickness ratios are, respectively, 18%, 55% and 27% calibrated to 100% of the eggshell total thickness (Fig. 07a). Visible with crossed nicols, single extinction wedges can be distinguished, with the tip at the base of the ML and the base at the upper part of the OL; the wedges present irregular shape and the typical crocodilian blocky extinction with an upside down triangular shape (Fig. 08).

[Fig. 07, Fig. 08, Fig. 09 near here]

In P. palpebrosus, the discrete shell units have a trapezoidal shape, wider at the top, with a height to width ratio of 0.65 for the single unit and a nucleation center at the bottom of each (Fig. b, d, f and 09a). The entire IL observed under transmitted light on a petrographic microscope presents pinholes in the middle of each nucleation center (Fig. 02d), which are not or rarely visible under reflected light or SEM (Fig. 02b, 05). These pinholes are similar to those presented in Garcia et al. (2008, Plate 1c) for Megapodius nicobariensis Blyth 1846, the Nicobar scrubfowl, and interpreted as marks of a possible reabsorption of calcite by the growing embryo or by weathering. The structure of this eggshell seems unique among all those described and known so far. Three different layers can be distinguished, like in the previous sample, but their organization is different than any other eggshell we observed: at the base of the shell units there are nucleation centers made by an aggregation of calcite plates; all the inner layer (IL) of the eggshell is characterized by the presence of these nucleation centers. Above this level, there is a middle, thin, dark, irregular layer (ML), probably an aggregation of fibers. The fibrous nature of the IL seems to be a unique feature of P. palpebrosus, when compared to the other samples and also to the so far described extant crocodilian eggshells (Fig. 09a). On SEM observation (Fig. 10), above the basal layer, a clear horizontal tabular ultrastructure can be observed for about a fourth of the entire eggshell thickness. No evidences of vertical lamination and fibers are present. Above this layer there is a thick outer layer (OL; approximately half of the entire eggshell thickness) characterized by a faint horizontal lamination, growth layering, and a more evident vertical lamination, corresponding to a fibrous fabric disposed perpendicularly to the eggshell surface, not radially like the tabular ultrastructure. Layer to entire eggshell thickness ratios are 32%, 11% and 57%, respectively for the IL, ML and OL, calibrated to 100% of the eggshell total thickness (Fig. 10). In crossed nicols observation, single wedges can be distinguished by the triangular shape (large side up), with the tip endorsed on the upper part of the middle layer and the base at the upper part of the OL; the wedges present regular shape and the typical blocky extinction (Fig. 09b).

[Fig. 10 near here]

        In A. mississippiensis, the discrete shell units are wedged shaped, widening to the outer surface, with a width to height ratio of 0.42 and a nucleation center at the bottom of each. Three different layers can be distinguished (Fig. 11a), organized in (1) a IL made of tightly packaged nucleation centers and basal knobs (Fig. 03c, e), which are approximately one third is size than those observed in the previous two samples (see Fig. 01d and f, 02b and d and 03c and e). Both a ML and an OL characterized by growth structures, a compact tabular ultrastructure and an evident fibrous vertical fabric, perpendicular to the eggshell surface (Fig. 12). The growth structures are more evident in the ML, while the fabric made by fibers are better defined on the OL. The IL, ML and OL to total eggshell thickness ratios are, respectively, 20%, 45% and 35% calibrated to 100% of the eggshell total thickness (Fig. 12). With crossed nicols, a blocky extinction can be noticed, shaped by irregular single extinction wedges, with an upside down triangular shape protracting from the upper part of the IL to the external surface (Fig. 11b).

[Fig. 11, Fig. 12, Fig. 13 near here]

Discussion

Among our samples, the external surfaces present two different kind of ornamentation: A. mississippiensis presents an anastomotuberculate type of ornamentation, while both C. mindorensis and P. palpebrosus present a new identified type ornamentation, here called rugosocavate (Fig. 01a, b and 02a), characterized by an irregularly rugose surface scattered by subcircular pits. This ornamentation seems characteristic also for a fossil crocodilian sample described from the Upper Miocene of Chinji Beds of Pakistan (Panadès I Blas and Patnaik 2009, Fig. 3, p. 3). We exclude this pattern to be a simple product of the degradation throughout incubation, because it is present on two different modern crocodilian samples, one unfertilized and the other incubated and hatched, as well as on a fossil fertilized one. The dissolution pits and stepped concentric erosion rings around the pore openings, identified on our only hatched samples (P. palpebrosus) and previously documented for A. mississippiensis in Ferguson (1981, 1982) are a good evidence for distinguishing the incubated eggs from the unincubated ones.

Our samples show an angusticanaliculate type of pore system which is typically associated to crocodiloid eggshells (Ferguson 1982; Mikhailov 1991, 1997; Zelenitsky & Hirsch 1997; Carpenter 1999). Crocodiles, however, share this character with some groups of dinosaurs and birds: the angusticaniculate type is described (i) for the theropodian oofamilies Prismatoolithidae and Elongatoolithidae, including the oospecies Elongatoolithus andrewsi Zhao 1975, Macroelongatoolithus carlylei Jensen 1970, M. xixianensis Li et al. 1995, Macroolithus yaotunensis Zhao 1975, M. rugustus Young 1965, Prismatoolithus coloradensis Hirsch 1994, P. levis Zelenitsky and Hills 1996, P. jenseni Bray 1999, Pseudogeckoolithus Vianey–Liaud and Lopez–Martinez 1997, Spheruprismatoolithus condensus Bray 1999, Spongioolithus hirschi, (ii) for the ornithopodian oofamily Ovaloolithidae, including the oospecies Ovaloolithus tenuisus Bray 1999 and O utahensis Bray 1999, and (iii) for the ornitid oofamilies Laevisoolithidae, Oblongoolithidae, Medioolithidae, Struthiolithidae, and Ornitholithidae (see Antunes et al. 1998, Bray 1999, Garcia 2000, Zelenitsky et al. 2000, Deeming 2006, Ribeiro et al. 2013). A. mississippiensis presents a lower porosity (5 pores per cm2) than C. mindorensis and P. palpebrosus. This low porosity seems, however, synapomorphic for this genus, because A. sinensis presents a pore density between 3 and 6 pores/cm2 (see Wink and Elsey 1994). The number of pores seems to change, however, by many environmental factors: Wink et al. (1990b) reports 94 pores per cm2 per unincubated fertile eggs of wild alligators. Furthermore, Wink et al. (1990b) and Bryan (2005) registered very low porosity values for A. mississippiensis in a wide range of environmental conditions lending additional support to the value described for this study. The pore diameter of the three samples is between 100 μm and 130 μm. The relative mean pore area percentage on the entire eggshell area is very similar for P. palpebrosus and C. mindorensis, respectively 0.22% and 0.19%, and lower for A. mississippiensis, about 0.08% (Tab. 3).

While an eggshell thickness between 0.30 mm and 0.59 mm seems to be typical for the extant Crocodyliformes, it does not appear to be a distinctive and useful character to identify specific taxa within this suborder. Our A. mississippiensis sample thickness is in agreement with the recorded range of this species, between 0.51 mm and 0.53 μm (Hirsch & Kohring 1992; Hirsch 1983), but is higher than a previous captive, fertile and unincubated sample described in Wink et al. (1990a) 0.43 ± 0.0236 mm.

Characteristic trapezoidal-wedge shaped shell units are clearly noticeable in our samples, with a width to height ratio between 0.42 and 0.65 (Tab. 3). While all the three inner layers are characterized by basal knobs with basal plate groups clearly distinguishable (Fig. 01d, f-h, Fig. 02b, d-f and 03c, e), the other two layers differ for the three samples. In C. mindorensis and A. mississippiensis can be recognized a thick ML, abundant in organic material and scarce in fibers, in contrast with an OL rich in fibers (so, darker on observation in thin section with normal light) and poorer in organic material. On the contrary, P. palpebrosus eggshell presents a characteristic and unique organization of the middle and outer layer among extant and fossil crocodiles described so far. The thin ML (middle layer) appears fully dark observed on thin section under direct light, while the OL (outer layer), relatively thick compared to the entire eggshell thickness, appears lighter in color. There is no direct observation of fibers in the ML, so the darker color of this layer could only depend on the dense microtabular horizontal lamination, in opposition to the sparse lamination present on the OL.

The absence of fibers and, subsequently, organic material in the ML is unique in P. palpebrosus and differs from the general microstructure of crocodylomorph eggs, which show ‘… an aggregate of prismatic calcite crystals that grow parallel to the shell surfaces, interwoven with protein fibers.’ (see Ferguson 1982). On thin section and SEM images observation (Fig. 09, 10), the ML is absent of all the characteristic that recall the presence of proteic fibers, evident in most of crocodylomorph eggs, as well as in our other two other samples (Fig. 07, 12).

On SEM observation, the three samples present a similar organization of the layers: the IL (inner layer) present the characteristic crocodilian basal knobs with basal plate groups; both the ML and the OL have the presence of a tabular horizontal ultrastructure, typical for crocodilian eggshells, denoting growth levels. Moreover, the OL presents a distinct vertical lamination, designating a fibrous fabric. In P. palpebrosus, the ML presents a denser horizontal lamination than the other two species, probably a unique characteristic of the eggshells of this species.

The crocodilian blocky extinction described in our samples is characterized by a V-shaped wedges that, on thin sections, appears like shaded triangular upside-down areas in the upper part of the eggshell. This particular extinction pattern is indicative of  an irregular distribution of the shell units, that superimpose one to another among the eggshell. On the contrary, the sweeping and columnar extinction pattern observed in dinosaurian eggs shows a more organized distribution of the shell units, packed one close to each other but with no superimposition (see i.e. Fang et al 2003; Ribeiro et al. 2013).

Fig. 13 compares the three types of eggshell here described.

[Fig. 13 near here]

Crocodilians exhibit a stable and well defined eggshell morphology, with only very slight variations at the structural level throughout the entire clade, as observed in this study. A cladogram summarizing the evolution of the egg in Amniotes was constructed in order to understand the relationships among the various oviparous groups and eggshell characteristics (Fig. 14). Packard et al. (1982, p. 142) recognize that ‘grouping of eggs on the basis of similarities in structure of eggshells is somewhat artificial’. Nonetheless, according to Carpenter (1999), Amniotes show a trend in hardening through further mineralization and an increase in the eggshell morphology complexity (see also Kohring 1995). Even though, there is a wide array of eggshell morphologies within some groups. By comparing our samples with other amniotic eggs, we are able to infer such a pattern. However, this analysis also reveals a complex evolution, with several groups developing a broad range of eggshells independently from each other (i.e. Chelonia, Lepidosauria) (Packard 1977; Packard et al. 1982; Packard and Seymour 1997; Stewart 1997; Carpenter 1999; Krachtovíl and Frynta 2006; Unwin and Deeming 2008). The primitive condition seems to be a leathery or parchment-like, flexible, most likely thin proteic membrane enveloping the egg (Grine and Kitching, 1987; Kohring 1995; Packard and Seymour 1997; Stewart 1997; Carpenter 1999; Oftedal 2002); nowadays, this condition can be observed in Monotremata (Grine and Kitching, 1987; Packard 1994; Packard and Seymour 1997; Stewart 1997; Oftedal 2002; Krachtovíl and Frynta 2006). A mineralized eggshell is considered a synapomorphy of Sauropsida (‘Reptilia’ in Packard 1994). It is plausible to assume that the leathery and semi-rigid eggshells in Chelonia, Lepidosauria and Pterosauria were either a retained primitive condition, as in the very primitive tuatara, or a secondary loss during the evolution of the group, as assumed for some more derived turtles or squamatans (Packard et al. 1977; Packard and Packard 1980; Packard et al. 1982; Kohring 1995; Stewart 1997; Carpenter 1999). In archosaurs, Pterosauria are the only group with a major change in eggshell morphology, characterized by a very thin, low mineralized, leathery eggshell but even so with some low degree of variation throughout the clade (Unwin and Deeming 2008). Crocodilians and dinosaurs (including birds) have very mineralized, rigid eggshells, although the Dinosauria show a greater variability in the eggshell structure, an organic core and higher porosity (i.e., Grine and Kitching, 1987; Antunes et al. 1998; Ribeiro et al. 2013).

[Fig. 14 near here]

Comparisons to fossils

Numerous fossil crocodylomorph eggs were collected and described. The oldest known are from the Late Jurassic of Lourinhã Formation in Portugal, which is known for the dinosaur fauna, including eggs and embryos (Araújo et al. 2013; Castanhinha et al. 2009; Mateus et al. 1998). The eggs bearing horizon are Upper Kimmeridgian/ Lower Tithonian. The fossil eggs putatively assigned to Crocodylomorpha from the same Formation were found in Paimogo, Peralta, Casal da Rola, and Cambelas. The origin of true crocodilians (members of the clade Crocodylia) occurred in the Late Cretaceous, so our Jurassic sample are not only the oldest known so far, but the best record for eggs of non-crocodilian crocodylomorphs. The eggs of Paimogo were the only subject of more detailed description by Antunes et al. (1998). These eggs measure 70 mm x 40 mm in dimensions (EI = 1.75) and 0.20 mm to 0.35 mm in thickness (Antunes et al. 1998). From the Cretaceous period there are several crocodilian eggs finds, the most of which are preserved only as fragments; from the complete known eggs, dimensions varies from 30 mm x 16 mm from the Upper Cretaceous of Bolivia to 65 mm x 36 mm from the Upper Cretaceous of Brazil (Adamantina Formation), with an EI between 1.14 and 1.88. The thickness goes from 0.15 mm of the Adamantina Formation specimen in Brazil to 0.75 mm of some fragments from the Upper Cretaceous of Spain (Hirsch and Kohring 1992, Rogers 2001, Ribeiro et al. 2006, Novas et al. 2009, Panadès I Blas and Patnaik 2009, Oliveira et al. 2011, Moreno–Azanza et al. 2013). Crocodilian eggs are known from the Cenozoic as well, with dimensions ranging from 35 mm x 30 mm (Eocene, Germany) and 64 mm x 54 mm (Pliocene, Upper Siwaliks, India), with an EI included between 1 and 2.11. The thickness is included between 0.15 mm of some Miocene fragments from the Chinji Beds of Pakistan and 0.76 mm from an Eocene sample from the Bridger Formation of the USA (Hirsch 1985, Hirsch and Kohring 1992, Patnaik and Schleich 1993, Kohring and Hirsch 1996).

All crocodylomorph eggs are ellipsoid in shape. Ovality, i.e. difference in pole curvature, seems to appear by the first time in coelurosaurian dinosaurs and being retained in birds until nowadays.

In general, the extinct crocodylomorph eggs are the same structure and shape than extant crocodile eggs, and synapomorphies for eggs of Crocodylia are also valid for the broader clade Crocodylomorpha. The main observed difference, however, is the smaller values of average eggshell thickness in non-crocodilian crocodylomorph.

Conclusions

We can conclude that: 1) anastomotuberculate, ramotuberculate and rugosocavate ornamentation types seem to be the most typical among extant and fossil crocodilian eggs (Crocodylomorpha clade); 2) angusticaniculate pore system is homoplasic for Crocodyliformes clade and some groups of dinosaurs and birds. Pore openings are circular in shape on the outer surface and sub-circular to irregular on the inner surface, having a relatively small diameter, between 100 μm and 130 μm and their relative percentage on the eggshell total area is low (0.08 to 0.22% per cm2); 3) Paleosuchus palpebrosus presents an autapomorphic eggshell ultrastructure, with a relatively thin middle layer, with a dense and compact tabular microstructure, and a thicker upper layer constituting more than half of the total eggshell thickness; and 4) dissolution pits and stepped concentric erosion rings around the pore openings are a constant characteristic of crocodylomorph incubated eggs only.

Acknowledgments. We thank the technicians of the GEAL – Museu da Lourinhã and the Faculdade de Ciências e Tecnologia da Universidade de Lisboa (Portugal) where this research was conducted. We want to thank Rene Hedegaard (Krokodile Zoo, Denmark) and Jesper Mílan (Geomuseum Faxe, Denmark) for kindly providing the extant crocodilian eggs. Our thanks also go to Carlos Galhano and Nuno Leal (FCT-UNL, Portugal) by making the SEM sessions possible, to Joaquim Simão (FCT-UNL) for allowing us the use of the petrographic microscope, and to Simão Mateus (GEAL – Museu da Lourinhã) for the illustrations. We are grateful to two former DinoEggs fellows and colleagues of our: Vasco Ribeiro, for the given support during the preparation of samples and SEM sessions, and Femke Holwerda (Bayerische Staatssammlung fur Paläontologie und Geologie/LMU, Germany), for preliminary sample selection and preparation. Finally, we thank Daniel Barta and an anonymous referee for the revision of the manuscript.

This research is part of Project Dinoeggs – dinosaur eggs and embryos in Portugal: paleobiological implications and paleoenvironmental settings (PTDC/BIA-EVF/113222/2009) funded by the Fundacão para a Ciências e Tecnologia (FCT), Portugal.

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Figure captions

Fig. 01 – Crocodylus mindorensis eggshell FCT -UNL 707. (a) Macro of the outer surface, ornamentation and pores (arrows). (b) Close-up image of the external surfacing showing the rugosocavate ornamentation. (c-f) Sample of FCT -UNL 707 observed under petrographic microscope; (c) outer surface under reflected light; (d) inner surface under reflected light showing nucleation centers; (e) outer surface under transmitted light; (f) inner surface under transmitted light showing nucleation centers. (g-h) Inset of respectively (d) and (f) showing nucleation centers; white holes in (h) are pore openings on the inner surface, right in-between the nucleation centers.

Fig. 02 – Paleosuchus palpebrosus eggshell FCT-UNL 708. (a) Macro of the outer surface showing pores (arrows) and the rugosocavate ornamentation; concentric erosion pits due to the incubation process are noticeable associated to pores. (b-e) Sample of FCT -UNL 708 observed under petrographic microscope; (b) inner surface under reflected light showing nucleation centers; (c) outer surface under reflected light; (d) inner surface under transmitted light showing nucleation centers; (e) outer surface under transmitted light. (f) Inset of (d) with clear pinholes in the center of each nucleation center.

Fig. 03 – Alligator mississippiensis eggshell FCT-UNL 709. (a) Macro of the outer surface of showing pores (arrows) and an anastomotuberculate ornamentation type. (b-e) Sample of FCT -UNL 709 observed under petrographic microscope; (b) outer surface under reflected light; (c) inner surface under reflected light showing nucleation centers; (d) outer surface under transmitted light; (e) inner surface under transmitted light showing nucleation centers.

Fig. 04 – Crocodylus mindorensis eggshell. SEM image of the inner surface of FCT-UNL 707 showing pores (arrow) and the packing of the basal knobs (BK) of the inner layer.

Fig. 05 – Paleosuchus palpebrosus eggshell. SEM image of the inner surface of FCT-UNL 708 showing pores (arrows) and an inset of a nucleation center with a pinhole.

Fig. 06 – Alligator mississippiensis eggshell. SEM image of a pore (arrow) on the outer surface of FCT-UNL 709.

Fig. 07 – Crocodylus mindorensis eggshell. (a) SEM image of an eggshell fragment in radial section of FCT-UNL 707. (b) Detail of (a) showing calcite plates on a nucleation center. IL = inner layer; ML = middle layer; NC = nucleation center; OL = outer layer; OS = outer surface.

Fig. 08 – Crocodylus mindorensis eggshell. Crossed nicols image with pore section of FCT-UNL 707.

Fig. 09 – Paleosuchus palpebrosus eggshell. Thin section under polarized light (a) and under crossed nicols (b) of FCT-UNL 708.

Fig. 10 – Paleosuchus palpebrosus eggshell. SEM image of an eggshell fragment in radial section of FCT-UNL 708. IL = inner layer; ML = middle layer; OL = outer layer; OS = outer surface.

Fig. 11 – Alligator mississippiensis eggshell. Thin section under normal light (a) and crossed nicols (b) of FCT-UNL 709.

Fig. 12 – Alligator mississippiensis eggshell. SEM image of an eggshell fragment in radial section of FCT-UNL 709. IL = inner layer; IS = inner surface; ML = middle layer; OL = outer layer.

Fig. 13 – Schematic 3D view of our three eggshell samples. Artwork by Simão Mateus.

Fig. 14 – Egg characteristics within the framework of the general consensus phylogeny of  Amniotes (see i.e. Benton 2005; Brusatte et al. 2011; Nesbitt 2011 for major clades relationships). Egg data after Packard et al. 1977, Packard and Packard 1980, Kohring 1995, Packard and Seymour 1997, Stewart 1997, Carpenter 1999, and Unwin and Deeming 2008.

Table captions

Tab. 1 – Main parameters of our three eggshell samples.

Tab. 2 – Egg mass, volume and density of modern crocodilian eggs. Mass values are taken by Thorbjarnarson 1996 (Table 1, p. 11). Volumes were obtained using PA and EA values in Tab. 3; when more than one couple of values per species was given, we calculated the mean value. For Tomistoma schlegelii we decided to use the value given Mathew et al. (2011), considered more accurate. All the data are merely indicative, because mass and volume values were taken from different sets of eggshells.

Tab. 3 – Egg size, elongation index and eggshell thickness of modern and fossil crocodilian eggs.