Biomechanical evidence suggests extensive eggshell thinning during incubation in the Sanagasta titanosaur dinosaurs

Introduction

Recent studies have changed our perspective on titanosaur palaeobiology. These highly diversified dinosaurs were the largest terrestrial organisms that ever roamed the earth and, according to recent investigations, their thermophysiology was similar to that of large modern endotherms (Seymour et al., 2012; Seymour, 2013; Eagle et al., 2015). Titanosaur eggs were incubated in holes excavated in the soil or in mounds of soil and leaf litter, comparable to the nests of the modern megapodes (Grellet-Tinner & Fiorelli, 2010; Hechenleitner, Grellet-Tinner & Fiorelli, 2015) and their chicks had a rapid ontogenetic development (Werner & Griebeler, 2014; Curry Rogers et al., 2016). Perinatal embryos preserved in ovo also revealed that titanosaurs developed an “egg-tooth”-like structure (García, 2007) that could have served to break the shell during hatching. Such anatomical structure is present in all the archosaurs (from crocodilians to birds) and presently, is the only known to be specifically involved in the hatching process (Honza et al., 2001; García, 2007; Hieronymus & Witmer, 2010; Hermyt et al., 2017).

Titanosaurs laid amniotic eggs with a calcitic shell. This genetically and physiologically controlled, biomineralized hard layer that protects the developing embryo from damage (mechanical or chemical), dehydration and infection, is specifically adapted to particular nesting environments, hence functionally optimized for each species (Ferguson, 1981; Board, 1982). Titanosaur eggshells consist of monolayered calcium carbonate, growing from densely packed shell units of rhombohedric, acicular calcite crystals that radiate from nucleation centers located at the external surface of the membrana testacea (Grellet-Tinner, Chiappe & Coria, 2004). Although titanosaur eggshells typically are 1.35–2.0 mm thick, the exceptionally thick-shelled eggs of the Sanagasta nesting site, in La Rioja, Argentina, reach 7.9 mm (Grellet-Tinner & Fiorelli, 2010; Grellet-Tinner, Fiorelli & Salvador, 2012; Hechenleitner et al., 2016a) (Table 1).

At Sanagasta, more than 80 titanosaur egg clutches were found to be synchronous with a Cretaceous geothermal process (Grellet-Tinner & Fiorelli, 2010; Fiorelli et al., 2012). Although unique among non-avian dinosaurs, the evidence at hand suggests that several species of titanosaurs may have utilized geothermalism as a source of heat for egg incubation (Grellet-Tinner & Fiorelli, 2010; Hechenleitner, Grellet-Tinner & Fiorelli, 2015). Yet, nesting in active geothermal settings is still a strategy exploited by several modern vertebrates, chiefly iguanas, snakes, birds, and even deep-sea skates (Werner, 1983; Göth & Vogel, 1997; Guo et al., 2008; Salinas-de-León et al., 2018), because it ensures a nesting thermal stability. Such association between titanosaur nesting and palaeogeothermalism led to hypotheses that thickness of the Sanagasta eggshells was an adaptation to resist the extrinsic dissolution by pore fluids in a harsh nesting environment (Grellet-Tinner & Fiorelli, 2010; Grellet-Tinner, Fiorelli & Salvador, 2012). This hypothesis received additional paleobiological support from more recent studies on the striking thickness of these eggshells (Grellet-Tinner, Fiorelli & Salvador, 2012; Hechenleitner et al., 2016a). The new data confirmed that these titanosaur eggs were physiologically functional; that is, they would have allowed an appropriate gas exchange under burial conditions in the substrate, even when their shells were as thick as 7.9 mm. Moreover, calculations based on micro-CT data showed that the eggshells were also physiologically functional even when they thinned up to 80% or 1.5 mm (Hechenleitner et al., 2016a). This implies that the suggested external chemical erosion of the shell by hydrothermal fluids would not have compromised the incubation with respect to gas exchange. However, whether or not this dissolution of the shell was essential for the hatchability of the Sanagasta eggs (as well as other titanosaur eggs) is a hypothesis that has not yet been tested.

Therefore, the present investigation aims to test the mechanical strength of the Sanagasta eggs using finite element analyses (FEA) on models of titanosaur eggs from several nesting sites by evaluating the required force to break them from inside. Furthermore, it will shed light on the importance of the external dissolution of the shell by chemical leaching, and its paramount role for their hatchability and the survival of several titanosaur species.

Methods

Specimens and modeling

We analyzed data of Haţeg (Romania), Boseong (South Korea), Tama, Sanagasta, and Auca Mahuevo nesting sites (Argentina) (Table 1). Measurements of the eggs from Tama and Sanagasta were obtained from digital 3D reconstructions of specimens curated at the Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja (CRILAR-PV 530/1 and CRILAR-PV 400 SA-C6-e1, respectively). Egg models for other sites are based on personal observations (Haţeg and Auca Mahuevo) and literature (Boseong) (Hechenleitner, Grellet-Tinner & Fiorelli, 2015; Hechenleitner et al., 2016b). In addition, we included data from Hahn et al. (2017) for four kinds of living birds: quail, hen, goose, and ostrich (Table 1). A comparison of their size and shape is given in Fig. 1A.

Breaking force estimation

In all instances (birds and titanosaurs), we conducted exploratory analyses. Using internal forces of different magnitude we recorded the eggshell displacement at the load point (Figs. 2A–2J; Table 2). Based on these results, we estimated the inner load force required to obtain a displacement equal to the eggshell thickness in each case (Fig. 3; Table 2).

Table 2:

Summary of the breaking force tests for each egg model.

Model	T#	F [N]	D [mm]
Tama	T1	1	0.5910
	T2	2	0.7619
	T3	3	1.1230
	T4	4	1.4720
	T5	5	1.8120
	BP	4.077752	1.4950
Sanagasta (thick eggshell)	T1	5	0.2934
	T2	10	0.5857
	T3	50	2.8980
	T4	100	5.7960
	T5	150	8.7950
	BP	136.09665	7.9500
Sanagasta (thin eggshell)	T1	1	0.4560
	T2	2	0.8959
	T3	3	1.3210
	T4	4	1.7340
	BP	2.73	1.2000
Auca Mahuevo (egg levels 1–3)	T1	1	0.4935
	T2	2	0.9523
	T3	3	1.3830
	T4	4	1.7920
	BP	3.0424	1.3900
Toteşti	T1	1	0.2400
	T2	3	0.7200
	T3	5	1.1800
	T4	7	1.6300
	T5	10	2.2800
	BP	7.5836250	1.7500
Boseong	T1	1	0.1825
	T2	2	0.3639
	T3	3	0.5442
	T4	4	0.7234
	T5	10	1.7760
	BP	9.9240465	1.7650
Ostrich	T1	5	1.7410
	T2	10	3.3090
	T3	2.5	0.8963
	T4	7.5	2.5420
	BP	7.580065	2.5500
Goose	T1	0.5	0.3736
	T2	0.2	0.1517
	T3	1	0.7295
	T4	1.2	0.8675
	BP	0.919825	0.6700
Hen	T1	0.25	0.1575
	T2	0.5	0.3114
	T3	0.75	0.4621
	T4	1	0.6096
	BP	0.666208	0.4100
Quail	T1	0.05	0.1515
	T2	0.1	0.2946
	T3	0.15	0.4306
	T4	0.025	0.0769
	BP	0.07477	0.2200

DOI: 10.7717/peerj.4971/table-2

Note:

BP, break point estimated by regression; D, maximum displacement at the load point; F, inner load force; T#, test number.

Effect of the eggshell dissolution on the egg mechanical resistance

In order to evaluate the effect of the dissolution of the eggshell in the Sanagasta eggs, as was previously hypothesized (Grellet-Tinner & Fiorelli, 2010; Grellet-Tinner et al., 2012; Hechenleitner et al., 2016b), we generated and analyzed models with different shell thicknesses between 7.9 and 1.2 mm (the maximum and minimum thicknesses recorded at this site). Each of these models was evaluated with an internal load force of 5 N (Figs. 4A and 4B). This magnitude corresponds to the average of forces previously estimated for all the titanosaur eggs in our sample, excluding the estimation for maximum thickness of the Sanagasta eggs. Based on the data of maximum displacement at the load point (Table 3), we estimated the maximum shell thickness that can be broken applying 5 N.

Table 3:

Results of FEA on Sanagasta egg models with different eggshell thicknesses.

Eggshell thickness [mm]	Dissolution [mm]	Displacement at the load point [mm]
7.95	0	0.29
6.26	1.69	0.39
4.58	3.38	0.56
2.89	5.06	0.87
2.47	5.48	1.05
2.04	5.91	1.42
1.62	6.33	1.78
1.2	6.75	2.14

DOI: 10.7717/peerj.4971/table-3

Statistical analysis

We performed a multiple linear regression analysis to test the influence of the egg volume and shell thickness on the strength of the eggs (Fig. 5). To perform the statistical analysis we used the lm function from the package stats version 3.4.3 of the open source software R (R Development Core Team, 2017).

Two models were performed in order to evaluate the relationship between variables; one model with interaction of the variables volume and thickness and one without interaction. The Akaike information criterion (AIC) method was used to select the model that better fits to data. A residual vs leverage plot of the fittest model helped to identify extreme values within the data set.

Results

According to the present 3D reconstruction, the Sanagasta eggs were originally spherical (Fig. 1B). This is consistent and supports all previous publications on titanosaur eggs (Grellet-Tinner, Chiappe & Coria, 2004; Grellet-Tinner, Fiorelli & Salvador, 2012; Hechenleitner, Grellet-Tinner & Fiorelli, 2015). Furthermore, the present CT-scan-based analysis shows that previous studies overestimated the size of these eggs (Fig. 1B). After digitally rearranging the eggshell fragments, the external egg diameter decreased from ∼210 mm (∼4,850 cm³ in volume) to ∼180 mm (∼3,370 cm³). Such a reduction in volume involves much less internal space for nutrient storage and embryo development. In addition, the diameter of the embryonic chamber of the Sanagasta eggs only reaches 169.2 mm due to the considerable shell thickness of these eggs (Fig. 1B). Therefore, although the Sanagasta eggs are larger than those of Tama, a nesting site found less than 150 km away in the same stratigraphic unit (Hechenleitner et al., 2016b), both display an identical chamber space available for the developing embryo (Table 1).

The 3D FEA conducted here, which are the first of their kind, allowed estimations that an effort of 3.04–9.77 N could break most of the titanosaur egg samples, namely Tama, Toteşti, Boseong, and Auca Mahuevo (Figs. 2A–2F and 3). In contrast, the eggs of Sanagasta are 14–45 times stronger, requiring up to 136 N to break.

Porosity could affect the eggshell’s strength, although to date, there is no quantitative information in this regard (Hahn et al., 2017). Eggshell strength in modern birds has been correlated with several factors, e.g., calcium diet, shell microstructure, incubation period; however, shell thickness is the main factor affecting strength (Ar, Rahn & Paganelli, 1979). The statistical model corroborated that there is an important linear association between egg internal volume and shell thickness (F_(1,8) = 16.93, R² = 0.64, p = 3.40⁻⁴), although an over-dispersion of thickness values becomes evident as volume increases (Fig. 5A). From the two multiple linear regression models tested, the model that better explains the relationship between internal volume and eggshell thickness as independent variables, and the shell mechanical strength as response variable was the model without interaction (AIC = 14.13). The regression analysis showed a statistical association between eggshell thickness and the mechanical strength of the eggs (F_(2,7) = 107.1, R² = 0.96, p = 8.53⁻⁵; Fig. 5B), whereas there is not a direct association with egg internal volume (F_(2,7) = 107.1, R² = 0.96, p = 0.80; Fig. 5C). The residual vs leverage plot shows that the thick-shelled egg from Sanagasta and the quail egg represent outlier values, and according to the Cook’s distance, they are strong influential observations for the model (Fig. 5D).

Considering that the geological and palaeontological data, as well as the evidence from modern analogues, suggest that the eggshells of Sanagasta would have partially dissolved during incubation, we further tested the mechanical effect of their constant thinning (Figs. 4A and 4B; Table 3). Results indicate that the average estimate for the other titanosaur eggs (5 N), has little effect on the Sanagasta egg, when its shell is thick (Figs. 4A and 4B). However, as the thinning progresses, the shell strength drops abruptly. When thinning reaches ∼1.6 mm, the shell reaches its fracture threshold and, as previously speculated (Grellet-Tinner, Fiorelli & Salvador, 2012; Hechenleitner et al., 2016a), it breaks easily at and below this threshold (Figs. 4A and 4B).

Discussion

The concept that all of the eggs of titanosaurs are spherical is well established. However, several sites preserve deformed and/or incomplete eggs (Huh & Zelenitsky, 2002; Salgado et al., 2009; Jackson, Schmitt & Oser, 2013; Hechenleitner et al., 2016b), and there is little CT information available to reconstruct their original shape and volume. The CT scan of the specimen CRILAR-Pv 400 SA-C6-e1 confirmed that the Sanagasta eggs were spherical. A spherical shape in eggs is mechanically and physiologically optimal. It has a greater resistance to impacts and is the smallest surface with respect to any geometric figure of equal volume (Bain, 1992; Stoddard et al., 2017). As such it is advantageous in terms of strength, shell economy, and heat conservation (Kratochvil & Frynta, 2006; Stoddard et al., 2017).

Currently, there is strong evidence for titanosaurs’ precociality or hyperprecociality (Hechenleitner, Grellet-Tinner & Fiorelli, 2015; Curry Rogers et al., 2016). Precociality requires a relatively greater amount of available nutrients and therefore a larger egg size. Egg internal diameter constitutes a valuable proxy for the size of a fully developed embryo, so its precise measurement is important to figure out how big (and, eventually estimate, how strong) the embryo could have been. The new data shows that the Sanagasta and Tama eggs have nearly the same internal space for accommodating an embryo. This suggests that the hatchlings of Sanagasta could have been strong enough to pip through (at least) a 1.5 mm thick eggshell (Table 1). However, hatching through a 7.9 mm thick shell, more than three times thicker than other titanosaur eggs (depending on which species), seems unlikely.

The characteristics present in the archosaur eggshells result from a compromise between several factors (Board, 1982). They must be strong enough to prevent fracture, but sufficiently weak to allow hatching. This relationship is corroborated by the statistical analysis of the present data, which shows an association between the eggshell thickness and strength of the eggs (F_(2,7) = 107.1, R² = 0.96, p = 8.53⁻⁵; Fig. 5B). The titanosaur eggs show, in general, a good fit to the statistical model (Fig. 5C). However, the Sanagasta eggs with thick shell fall entirely outside these predictions. According to the FEA, they were 14–45 times stronger than any other titanosaur eggs that have nearly the same space for accommodating a late term embryo, such as those of Tama and Boseong. Thus, the Sanagasta embryos would have had to invest a considerable amount of energy to be able to hatch, if the eggs kept their thickness constant during the whole incubation.

Recapitulating on the adaptive advantage of such a thick shell for the Sanagasta specimens, two reasons that are not mutually exclusive can be considered: mechanical strength and resistance to chemical abrasion. Most titanosaurs laid biologically and mechanically viable eggs with thinner shells (e.g., Auca Mahuevo, Toteşti), which rarely exceed 2 mm, thus suggesting that strength was not a primary reason for developing thick eggshells. This shows that the excessive thickness of the Sanagasta shells would not respond to a mechanical need (e.g., withstand shock from outside).

However, keeping the shells thick during the whole incubation process could have had serious consequences for the Sanagasta titanosaurs. First, it would be detrimental for the development of the embryo because, as it grows, its needs change from preventing water loss to increasing gas exchange, due to the increase in energy consumption of a late embryo (a process documented among mound-nester archosaurs (Ferguson, 1981; Booth & Seymour, 1987; Hechenleitner et al., 2016a)). Second, a very thick eggshell might also represent a problem during hatching, as is suggested by the new results (Figs. 2A and 3). The case was pointed out by empirically studying Alligator mississippiensis, which bury their eggs in mounds of vegetation, in a way similar to that used by some titanosaurs and megapode birds (Hechenleitner, Grellet-Tinner & Fiorelli, 2015). Eggs incubated artificially (without natural substrate) develop normally, but then, the fully grown embryos are unable to break their shell (Ferguson, 1981). In nature, the dissolution of the Alligator mississippiensis eggshell is mediated by bacterial decomposition, which acidifies the nesting environment. Given the environmental similarities for ground-nesting, it is not surprising that the shells of several titanosaur nesting sites show evidence of extrinsic dissolution (Grellet-Tinner, Chiappe & Coria, 2004; Hechenleitner, Grellet-Tinner & Fiorelli, 2015). This type of dissolution should not be confused with the internal calcium absorption produced in the late stages of the embryogenesis, which is ubiquitous among archosaurs (Chien, Hincke & McKee, 2009). During ossification the calcium is removed from the shell, getting to reduce up to 20% of its thickness in precocial birds, such as the megapodes (Booth & Seymour, 1987). However, these high values are associated with very thin eggshells, in which the removal mostly affects the base of the structural units of calcite, in the innermost portion of the shell. Indeed, some internal dissolution in the Sanagasta eggshells was related with calcium resorption, but is negligible compared to the shell’s thickness (Grellet-Tinner, Fiorelli & Salvador, 2012).

The results of FEA conducted on models of Sanagasta eggs with different shell thicknesses, between the minimum and maximum shell thickness reported for this site, show that an effort similar to the one necessary to break other titanosaur eggs would have had very little effect on those of Sanagasta immediately after oviposition (Figs. 4A and 4B). However, when the thickness is reduced to less than 1.6 mm, the shell becomes as fragile as for other titanosaur eggs.

The nesting strategies of titanosaurs have been compared with those of modern megapodes (Kerourio, 1981; Cousin & Breton, 2000; Garcia et al., 2008; Grellet-Tinner & Fiorelli, 2010; Grellet-Tinner, Fiorelli & Salvador, 2012; Hechenleitner, Grellet-Tinner & Fiorelli, 2015; Grellet-Tinner, Lindsay & Thompson, 2017). To date, only a handful of dinosaur species are confirmed to exploit and have exploited the geothermalism as a source of heat for incubating their eggs (Jones & Birks, 1992; Grellet-Tinner & Fiorelli, 2010; Harris, Birks & Leaché, 2014; Hechenleitner, Grellet-Tinner & Fiorelli, 2015; Grellet-Tinner, Lindsay & Thompson, 2017). The eggshell structure of modern dinosaurs differ from those of their ancestors by having three to four structural layers that confer a greater strength for a thinner eggshell thickness (Grellet-Tinner, 2006), instead of one structural layer like the Sanagasta dinosaur eggs. Macrocephalon maleo and Megapodius pritchardii are two modern megapode species that resort or revert to geothermal incubation, although the former, in Sulawesi Island, have two populations that do not interbreed and respectively utilize black sand with solar radiation and geothermal heated sand. However, the latter do oviposit in sands heated by in geothermal activities and Megapodius pritchardii in the volcanic ashes of calderas. In both instances the megapode eggs are not in direct contact with geothermal fluids. Leipoa ocellata and Alectura lathami, two mound-builder megapodes that inhabit Australia, must also deal with the risks of external acidic erosion. In their mound-nests the activity of microorganisms that maintains a high incubation temperature (Seymour & Ackerman, 1980) also produces organic acids as a by-product (Grellet-Tinner, Lindsay & Thompson, 2017). The eggshells of both species have an accessory layer composed of nanospheres of calcium phosphate on their outer surface (Board, 1980). D’Alba et al. (2014) showed that this accessory layer has antimicrobial properties. In addition, the calcium phosphate of the nanospheres is, compared to the calcite present in the structural layers of the eggshell, a relatively insoluble salt (Board, 1980). For this reason it has been recently suggested that the accessory layer also constitutes a protective cover that prevents the external erosion of the shell (Grellet-Tinner, Lindsay & Thompson, 2017). In addition, the pronounced nodular surficial ornamentation of these eggs complements the calcium phosphate nanospheres against chemical erosion by limiting most of the external erosion of their eggshell to these nodes. Therefore, although a few species of modern megapodes may display a reversal that utilizes ground generated heat as a passive incubating energy, their incubating strategies differ from the Sanagasta dinosaurs, which eggs were in direct contact with acidic geothermal fluids (Grellet-Tinner & Fiorelli, 2010).

Conclusions

The FEA data suggest that hatching through a 7.9 mm thick shell was impossible for the embryos from Sanagasta. However, the analyses carried out on egg models with different shell thicknesses further suggest that thinning below 2 mm would have allowed these titanosaurs to hatch. With regard to the relationship between eggshell thickness and egg strength, the thick-shelled Sanagasta eggs are completely out of the prediction of the statistical model. In other words, the model shows that in terms of the strength/thickness ratio, the Sanagasta eggshells are disproportionately thick with respect to those recorded for birds and other titanosaurs. As the original thickness would have been a strong limitation for hatching, the present results are consistent with previous arguments of outer eggshell thinning in the Sanagasta nesting site (Grellet-Tinner & Fiorelli, 2010; Grellet-Tinner, Fiorelli & Salvador, 2012). Considering that titanosaur eggs were incubated in fairly acid nesting environments, such as mounds or dug-out holes as seen in the modern megapodes (Hechenleitner, Grellet-Tinner & Fiorelli, 2015), it is plausible that the force required for hatching would be even less than estimated. Regardless of the factors (intrinsic and/or extrinsic) involved in the wear of ∼80% of the eggshell, our results strongly suggest that external chemical dissolution, here complemented by the typical internal ontogenetic dissolution, throughout the incubation process would have been essential for allowing hatching of the titanosaurs that nested at Sanagasta.

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