The occurrence of dinosaur pathologic eggs in the Late Cretaceous of Europe is well known, but their origin remains unclear. Here we expose the results of a detailed sampling of the conspicuous fossil record of Late Cretaceous titanosaurian eggs (oogenus Megaloolithius) from several southwestern Europe basins. After examining more than 450 samples, we observed a remarkable and statistically supported occurrence of multiple pathologic eggs in a relatively short stratigraphic range at the end of the early Maastrichtian, circa 71-70 Ma. All pathologic specimens exhibit multi-layered eggshell condition, a characteristic related to dystocia, or egg retention within the female uterus for an abnormal prolonged period of time. After exploring various scenarios, the occurrence of pathologic eggs is strongly correlated with an intense dinosaur faunal replacement that occurred during the early Maastrichtian in the Ibero-Armorican Island. Given that inter-species competiveness is proved to produce major affects in ecological communities, our results suggest that pathologies in the eggs of European titanosaurians could be a consequence of an increase in reproductive stress triggered by direct ecological competition between different dinosaurs. Thus, the present study provides a new perspective of how dinosaurs might have been affected by ecological/environmental disturbance.
To understand how species are affected by environmental disturbance is a crucial topic in both modern ecology and palaeoecology. Several studies demonstrate that environmental/ecological alterations force animal communities to restructure resource partitioning1,2,3,4,5. However, this reorganization is just one of the many consequences affecting the community when experiencing a new ecologic stability. Environmental changes and biotic disturbances can also drive ecological communities into stressed conditions, producing several negative effects upon organisms6. For instance, under unfavourable conditions oviparous amniotes may suffer an increase in reproductive stress that can be physically expressed in the malfunction of the reproductive system7,8,9,10.
The fossil record offers several examples of pathologic oological remains, especially in dinosaurs9,10,11,12,13,14, but very few convincing hypotheses are proposed to explain their origin. In this regard, the Upper Cretaceous formations of southwestern Europe offer an exceptional continuous record of pathologic megaloolithid egg14,15,16,17 (but see18; Fig. 1, Supplementary Information), an egg type attributed to titanosaurian sauropods19,20,21. This framework provides a unique opportunity to explore how dinosaur faunas could physiologically have responded to ecological/environmental perturbations.
Historically, the presence of dinosaur pathologic eggs has been linked with climatic changes22,23,24,25, or considered as one of the causes of the demise of dinosaurs15. Nevertheless, the former hypothesis has never been explored in detail, and the latter one has been widely rejected9,13,16,17,26,27. Here, we explore several scenarios to address the possible trigger event that favoured the overproduction of pathologic eggs in titanosaurians from Europe. In order to solve this question, the stratigraphic distribution of the pathologic eggs is firstly evaluated from a statistic perspective. Given that isotopic signature of eggshells can be used as a proxy for inferring change in both environmental and biological condition15, stable isotopes (δ13C and δ18O) of both normal and pathologic eggshells are measured and compared. Finally, these data are evaluated under different ecologic scenarios that occurred at the end of the Cretaceous (Maastrichtian time span) in southwestern Europe. As a result, we provide substantial evidence that abnormal eggs were the consequence of an intense ecological perturbation.
Stratigraphic distribution and statistical significance of pathologic eggs
In southwestern Europe, titanosaurian eggs occur in non-marine deposits ranging from the upper Campanian to the uppermost Maastrichtian28,29,30,31,32,33. After sampling more than 90 consecutive stratigraphic egg-levels in 22 continuous and composite stratigraphic sections of the southern Pyrenees (Catalonia), we documented the occurrence of several in situ abnormal eggs in 14 egg-horizons (see Fig. 1; see also Supplementary Information).
Taking the fossil egg record as a whole, pathologic eggshells are very scarce at the region (0.5–2.5%)16, but most of them occur in a relatively short stratigraphic range in the lower Maastrichtian; thereby representing 9.3% of the fossil egg record in that interval (Fig. 1B; see also Supplementary Table S1). According to poultry industry data, under controlled non-stressed conditions, pathologic eggs barely represent the 2% of the total production34,35. These values are much lower than that of the studied region, where pathologic remains represent about 9.3% of the record.
All the pathologic eggs and eggshells recovered from southwestern Europe can be ascribed to the egg type Megaloolithidae, and more precisely to Megaloolithus siruguei and Megaloolithus mamillare egg-types. It is worthy comment that pathologic remains attributed to M. siruguei appear located in the uppermost section of its stratigraphic range, whiles those of M. mamillare occur in the lower part of its respective stratigraphic interval (Figs 1B and 4; see also Supplementary Information Table S1).
In France, the La Bégude site is considered the lowest stratigraphic locality yielding pathologic eggshell, which is dated as early Maastrichtian in age (within the lowermost part of the C31r magnetochron30,36). On the other hand, the Vitrolles-La Plain site is the stratigraphically uppermost pathologic-egg-bearing locality in the region, and dated as early late Maastrichtian (near the C31r-C31n reversal37). The lowest pathologic remains in the southern Pyrenees are found in the Mas de Pinyes-1 site, which falls in the lower part of the C31r, and therefore dated as early Maastrichtian29,33. On the other hand, the uppermost pathologic oological remains in the region come from the Els Nerets locality, falling in the middle part of the chron C31r33 and considered late early Maastrichtian in age.
We found highly statistical significant (x2 = 4.37 × 10−17; df = 6; p < 0.05; see Supplementary Information) the occurrence of pathologic remains in the lower Maastrichtian. A non-parametric confidence interval correction38 was also applied to establish the most parsimonious time-range for the pathologic remains. By taking the European record as a whole, the confidence interval factor was −0.019 (n = 7; p < 0.05), and the confidence time-interval expands from ~71.3 Ma to 70.1 Ma (Fig. 1B).
Structure and stable isotope signal of abnormal titanosaurian eggshells
Most of analysed pathologic specimens come from egg debris (see Supplementary Information Table S2), which have been interpreted as disaggregated, or partially preserved but in situ eggs. A single complete egg exhibiting pathologic features was recovered from the Coll de Nargó nesting-area (Lleida province, Catalonia). The egg (MCD-5413) is ellipsoidal in shape, being the three semi-principal axes about X = 10.5 cm, Y = 8 cm, and Z = 5 cm, and its external surface is covered of irregular nodes. The ellipsoidal morphology of the egg is the result of tectonic compression linked to the Pyrenees formation rather than pathologic, given that all non-pathologic dinosaur eggs from Coll de Nargó exhibit similar morphology28.
Non-pathologic M. siruguei specimens are distinctively covered with rounded nodes of 0.5 to 1.23 mm in diameter17,29, while most of the pathologic eggshells exhibit aberrant ornamentation of irregular, enlarged (0.7 to 2.4 mm in diameter) and coalescent nodes (Fig. 2A). All the analysed pathologic specimens attributed to M. siruguei combine both superimposed eggshell layers and additional nucleation centres (Fig. 2B–E). A great disparity between the thicknesses of superimposed eggshell layers is observed (Supplementary Table S2), a condition that cannot be attributed to diagenetic alteration as occurs in other dinosaur egg-sites18,39. In some specimens the lower eggshell is well developed, showing a normal structure, while the upper one is thinner and shows more irregular shell units, as in some pathologic Argentinean sauropod eggs13. Extra nucleation centres occur at different points within pathologic eggshells, but mainly located at the basis of the overlaying eggshell, or near the boundary between superimposed eggshells. The boundary between the lower and the overlaying eggshell varies from well defined to irregular with vacuities frequently occupied by blocky sparry calcite and micrite cement. Because of that, no evidence of eggshell membrane is observed neither at the base of overlaying eggshells nor in the lower one.
The outer ornamentation of both normal and pathologic M. mamillare eggshells are nearly equal, being the size of nodes of 1 and 1.2 mm in diameter, respectively. The 5.35-mm-thick M. mamillare pathologic specimen from the Els Nerets locality (IPS-59132) is especially noteworthy because it consists of five superimposed eggshells layers (Fig. 2F). The normal lower eggshell layer is 1.35 mm in thickness, and covered by well-developed tubercular ornamentation. The overlaying abnormal eggshells are consecutively thinner than the previous ones (Supplementary Table S2), and calcite deposition follows the contour of the underlying eggshell, displaying smooth basal contacts (Fig. 2G,H). These structural features are equal to the Type III pathology described in Argentinean sauropod eggs13. The same pathological patterns are observed in other samples from the Els Nerets site (IPS-100376, IPS-100377), although they only show three superimposed eggshell layers. Similarly to pathologic M. siruguei, extra nuclear centres occur near the boundary of subsequent eggshell layers, but are especially abundant in the uppermost eggshell layer (Fig. 2G,H).
It is noteworthy that the occurrences of extra-spherulites have been used to infer pathologic conditions in dinosaur eggs8,9, but a recent study39 shows that those features occurring within primary shell units are crystallographic defects of taphonomic origin. However, this is not the case of the specimens presented herein, where extra nucleation centres and extra-spherulites mainly appears located between consecutive shell unit layers, thus reinforcing the suspicious of their pathologic origin.
Isotopic signature of eggshells can provide significant information about the physiological conditions in which they were formed. Thus, if mechanisms causing the formation of pathologic eggshells were linked to some kind of physiological disorders, these anomalies should be reflected in a distinctive isotopic signature. According to our results, pathologic eggshells exhibit minor variations in δ13C and δ18O values, with δ13C values ranging between −9.3‰ and −13.85‰, and δ18O values between −0.80‰ and −5.11‰ (Supplementary Table S3). When data is represented as a δ13C/δ18O plot, all pathologic eggshells fall into the cluster of normal eggshells (Fig. 3).
Various authors8,26,40,41,42 have indicated that multi-layered eggshells or additional deposition of calcite in the shell of extant reptiles result from prolonged periods of egg retention within the female uterus, a phenomena known as dystocia. This retention of the egg by the female can be caused by reproductive stress induced by environmental, physiological, or ecological factors. The relatively short stratigraphic range of pathologic eggshell occurrences in the southwestern European basins suggest that they were produced as a consequence of a very particular event that mainly affected titanosaurian sauropods. In order to elucidate which factors could force titanosaurian females to retain their eggs, three scenarios are explored.
Hypothesis 1, pathologic eggs result from an increase of reproductive stress by environmental changes
The hypothesis postulates that pathologies in titanosaurian eggs resulted from climatic perturbations that occurred at the end of the Cretaceous, as some authors already suggested more than 30 years ago15,22,23,24,25. According to this hypothesis climatic/environmental changes would have caused changes in their reproductive biology resulting in the production of abnormal eggs.
From a biological point of view, it is well known that certain physiologic and metabolic processes of reptiles are strongly temperature-dependent. Humidity and photoperiods are also important factors regulating the hormonal process involved in the reproductive cycle43. In extant reptiles, optimal environmental factors are essential for the reproductive health and immune system functioning. When environmental conditions exceed beyond the threshold of tolerance, many reptiles are obligated to suspend the development of the egg during the pre-ovipositional process44,45. As a consequence, several disorders may occur in both embryo and the egg.
Palaeoclimatic conditions of terrestrial environments can be inferred on the basis of the isotopic signal of pedogenic carbonates46,47. If drastic environmental shifts were the trigger event that led of the overproduction of pathologic eggs, these changes should be reflected as isotopic variations throughout stratigraphic sections48,49. According to the most recent isotopic data carried out in both northern Spain50 and southern France48, no significant climatic changes can be detected during the latest Cretaceous (Campanian-Maastrichtian) in southwestern Europe, at least in continental environments. Moreover, these studies point towards stable climatic conditions with low variation of the atmospheric temperature during the Maastrichtian, which agree with the global temperature fluctuation during that period51.
At the stratigraphic interval where pathologic eggshells occur (Fig. 4), no significant shifts in the δ18O or δ13C values are observed, and minor variations can be attributed to slight local fluctuations48,50. In the light of these data, there is not substantial evidence suggesting drastic climatic changes during the Maastrichtian in southwestern Europe, and hence it is no possible to establish any correlation between environmental perturbations and the occurrence of pathologic eggs.
Hypothesis 2, pathologic eggs result from an increase of reproductive stress by changes in the dietary behaviour
Ewert and collaborators26 considered that possible changes in the feeding sources of sauropods would have produced physiological alterations affecting their reproductive cycle. Here we consider changes in diet such as those related with the resource quality or feeding behaviour, regardless of whether the later are linked or not with a major climatic change.
It is well known in the poultry industry that nutrition has a capital importance in the final egg quality and changes in the diet or a poor feeding source, especially those with inadequate proportion of calcium and vitamins, may have dramatic effect upon final egg structure52. Chemistry of drinking water (e.g. electrolyte imbalance or saline water) also might influence on the final egg quality53,54. For instance, a diet low in amino acid or poor in selenium may limit the egg production, whereas calcium-limited nutrition may prolong the laying period and causing binding eggs55.
In archosaurs, both crocodiles and birds, the calcareous layer of the egg is deposited in a separate uterine region of the reproductive tract56, resulting in a sequential shelling of the eggs; a condition also proved in theropod dinosaurs57. However, while in hens eggshell formation occurs just few hours before the egg laying, in alligators and crocodiles the egg takes longer than 24 hours58. In any case, given that calcium deposition occurs in a relatively short time, stable isotopic composition of the eggshell may reflect the nature of the last meals taken by the producer59. Thus, changes in the isotopic composition of the eggshell may reflect changes in feeding sources or dietary behaviour49.
The analysis of pathologic titanosaurian eggshells shows isotopic δ13Cegg and δ18Oegg values ranging from −9.3‰ to −13.85‰ and from −0.8‰ to −5.11‰, respectively. These values are similar to those reported in non-pathologic megaloolithid eggshells from the Maastrichtian of Iberia, southern France, Romania and India (Fig. 3). The isotopic signal of δ13Cegg in dinosaur eggshells is determined by the δ13Cdiet value of the diet. Independently from the species, the eggshell is enriched in δ13Cegg about 16‰ relative to the ingested food60,61, and therefore, in herbivores, it may help in assessing the ingested vegetation. By considering this value of metabolic fractioning, the average δ13Cdiet of the ingested vegetation by titanosaurians producing pathologic eggs fall between −25.3‰ and −29.85‰. By assuming similar fractioning rates for non-pathologic eggs coming from the same area60, the δ13Cdiet of the ingested food is between −27.36‰ and −29.99‰. In both cases, values suggest an herbivorous diet based in C3 plants (average isotopic value of δ13C around −26‰, contra δ13C of −13‰ in C4 plants62) with very similar isotopic composition. Thus, it can be concluded that both normal and pathologic eggs were formed on the basis of similar feeding source.
The isotopic signal of δ18Oegg from eggshell informs on the nature of drinking water48,63, though the interpretation of its values is strongly subjected to the thermophysiology of the laying-taxon15,63. By assuming a virtual homeothermic condition for sauropods64,65,66, the δ18O values of their eggs can be directly related to the chemistry of the water they ingested. By considering the δ18Oegg values of studied pathologic eggshells, and using the equation correlating the δ18O of the eggshell and the δ18O of drinking water67, the resulting δ18Owater values of the ingested water for the analysed samples range between −2.71‰ and −9.55‰. These values are slightly lower compared to the average δ18Owater values inferred from non-pathologic eggshells reported from nearby areas of the southern Pyrenees (δ18Owater between −2.3‰ and −4.3‰), but similar to those observed in the Sallent site50 and the Provence area15,48. Because no differences in the δ18Owater values are recognized, we can conclude that both dinosaurs producing normal and pathologic eggshells probably drank water from similar sources. Finally, when the δ13Cegg and δ18Oegg values of normal and abnormal eggshells are plotted in a time-log chart, we observe that these are nearly invariable throughout the Maastrichtian section (Fig. 4), supporting the idea that there was not a significant change in the dietary behaviours, food type or quality of feeding resources of titanosaurian sauropods along the Maastrichtian.
Hypothesis 3, pathologic eggs result from an increase of reproductive stress by ecological competition
The hypothesis explores the possibility that ecological competition among dinosaurs could produce reproductive stress and consequent dystocia. In extant reptiles, an increase of the inter- or intraspecific competition for resources or nesting area, such as in a faunal replacement scenario, may cause a strong reproductive stress, favouring the malfunction of the reproductive system and the production of abnormal eggs68,69,70.
From a physiologic perspective, it is demonstrated that stress, whatever its origin, alters the normal hormonal activity of the hypothalamus-pituitary-adrenocortical (HPA) axis in amniotes. Particularly noticeable are the modification on the baseline levels of the hypothalamic luteinizing hormone-releasing hormone (LHRH), the pituitaric follicle-stimulating hormone (FSH), the luteinizing hormone (LH) and prolactine, being all of them involved in the ovulation process68,69,70. In addition, this unbalanced hormonal cascade affects the levels of oestrogens participating in the metabolic regulation of the calcium for the formation of the eggshell68,69,70. The combination of both anomalous hormonal functions and inappropriate calcium deposition; whether due to excess or deficiency, may favour the occurrence of dystocia and the production of abnormal eggshells. Because titanosaurian sauropods could exhibit reptilian reproductive physiological traits13, similar physiological responses are expected in a similar scenario.
In southwestern Europe, a dinosaur faunal replacement is well documented at the end of the Cretaceous71. According to this major faunal change, the upper Campanian-lower Maastrichtian plant-eating dinosaurs, characterized by rhabdodontid ornithopods, nodosaurid ankylosaurs and titanosaurian sauropod taxa, were replaced by numerous new hadrosauroids and titanosaurian taxa. New taxa reached the Ibero-Armorican domain at some time around the early Maastrichtian-late Maastrichtian boundary. Furthermore, fossil evidence suggest that both associations coexisted for some time71. This faunal change can be also recognized on the basis of dinosaur ootaxa (Fig. 4). In such ecological scenario, it has been stated that the changing dinosaur community was subjected to a high ecologic stress71. Interestingly, the occurrence of abnormalities in titanosaurian eggs is stratigraphically correlated with that major turnover event (Fig. 4).
Because this turnover clearly affected multiple taxa, it seems plausible to suggest that during the period of cohabitation “old” and “new” dinosaur faunas could compete for similar, if not the same, ecologic resources in overlapped environments. For instance, titanosauran sauropods showed a general affinity for nesting in ancient soils developed upon floodplains48,72,73,74,75; and hadrosauroids also had an environmental preference for fluvial settings76,77. In southwestern Europe the environmental distribution of herbivorous dinosaurs concurs with this pattern, with nesting grounds of sauropods and habitat of hadrosauroids being reported primarily in fluvial settings78,79,80. This concurrence in certain environments could certainly have produced a significant increase of the ecological competition among different dinosaur groups for habitat preference and uses.
It still remains unclear the particular role that each group of dinosaurs played during this faunal replacement, but apparently the ecological perturbation would have distinctly affected upon each taxonomic group since titanosaurian sauropods are the only clade that suffered reproductive stress, while other taxa like rhabdodontids and nodosaurids become extinct in this process71.
We compiled data from the megaloolithid egg localities reported in various basins of southwestern Europe, with special attention on the description and chronostratigraphic occurrence of pathological eggshells. In order to summarize the temporal occurrence of megaloolithid eggs in the entire region, we selected data from long stratigraphic successions of Provence area17,30,48, northern Pyrenees30,32, and southern Pyrenees29,31,33,50,81, which range from the upper Campanian to the uppermost Maastrichtian (Fig. 1, Supplementary Information Fig. S1). The stratigraphic position of ten egg levels yielding pathologic eggshells in France16,17,30,82 has been correlated with the most recent magnetostratigraphica data32,37,48 (Supplementary Information Fig. S2 and Table S1).
We sampled more than 90 consecutive stratigraphic egg-levels in 22 continuous and composite stratigraphic sections, of about 100 to 300 m thick each, distributed along the southern Pyrenees. For each egg-horizon, 50 eggshell samples were randomly collected from diverse both normal and abnormal in situ eggs or egg debris, taking only one sample per egg. As a result, we recovered 23 abnormal specimens in 14 egg-horizons along the southern Pyrenees (Fig. S1). A chronostratigraphic framework of the distribution of the Late Cretaceous dinosaur eggs from southern France (northern Pyrenees and Provence) was build up gathering information from the literature (see Supplementary Information for further details) and by first hand observations. As a result, 15 stratigraphic sections including 91 egg-horizons were correlated along this region (Fig. S2), but only 10 horizons have yield pathologic remains.
Eggshells preparation follows Val’s methodology83. Samples were preliminarily analysed using Leica® M60 binocular. Some eggshells were prepared as standard petrographic thin sections (30 µm), while others were examined and photographed using the environmental SEM Quanta 200 FEI, XTE 325/D8395 of the Scientific-technical Services of the University of Barcelona. Five pathologic eggshells (IPS-59122, IPS-59123, IPS-59124, IPS-59125, IPS-59127) were prepared for geochemical analysis using mass spectrometry. The isotopic analyses of dinosaur eggshells were performed at the Faculty of Geology of the University of Barcelona. The isotopic results are reported in per mil (‰) notation relative to the PeeDee Belemnite (VPDB) standard. The measured precision was over 0.1‰ for both the carbon and oxygen isotope compositions.
A chi-square test was performed in order to evaluate if there is, or not, a random stratigraphic distribution of pathologic dinosaur eggshells in southwestern Europe, assuming similar potential of fossilization for normal and pathologic eggshells, since both egg types were laid and incubated in the same fashion28. The chronostratigraphic log was divided in seven time-bins, from the upper Campanian to the late Maastrichtian (from 73 Ms to 66 Ma; Fig. 1), each one of 1 Ma (see Supplementary Information).
Bender, E. A., Case, T. J. & Gilpin, M. E. Perturbation experiments in community ecology: Theory and practice. Ecology 65, 1–13 (1984).
Schulze, E. & Mooney, H.A. Biodiversity and ecosystem function. (Srpimger-Verlag, 1993).
McCann, K. S. The diversity-stability debating. Nature 405, 228–233 (2000).
Cardinale, B. J., Nelson, K. & Palmer, M. A. Linking species diversity to the functioning of ecosystems: on the importance of environmental context. Oikos 91, 175–183 (2000).
Webb, A. E. & Leighton, L. R. Exploring the ecological dynamics of extinction in Quantifying the evolution of early life. (eds Laflamme, M., Schiffbauer, J. D. & Dornbos, S. Q.) 36, 185–220 (Topics in Geology, 2011).
Cohen, D. The evolutionary ecology of species diversity in stressed and extreme environments inOrigins: genesis, evolution and biodiversity of life. (ed. Seckbach, J.) 503–514 (Klu. Ac. Pub., 2003).
Hughes, B. O., Gilbert, A. B. & Brown, M. F. Categorisation and causes of abnormal eggshells: relationship with stress. Bri. Poul. Science 27, 325–337 (1986).
Hirsch, K. F. Pathological amniote eggshell- fossil and modern in Mesozoic Vertebrate Life (eds Tanke, D. H. & Carpenter, K.) 378–392 (Indi. Un. Press, 2001).
Jackson, F. D. & Schmitt, J. G. Recognition of vertebrate eggs abnormalities in the Upper Cretaceous fossil record. Cret. Res. 29, 27–39 (2008).
Jackson, F. D. & Varricchio, D. J. Ovum in ovo in Coturnix japonica eggs (Japanese quail): implications for dinosaur reproductive anatomy. J. Vert. Pal. 23, 699–702 (2003).
Zelenitsky, D. K. & Hills, L. V. Normal and pathological eggshells of Spheroolithus albertensis, oosp. nov. from the Oldman Formation (Judith River Group, late Campanian) southern Alberta. J. Vert. Pal. 17, 167–171 (1997).
Zhao, Z.-K. et al. A possible causal relationship between extinction of dinosaurs and K/T iridium enrichment in the Nanxiang Basin, South China: evidence from dinosaur eggshells. Palaeoge. Palaeocli. Palaeoeco. 178, 1–17 (2001).
Jackson, F. D. et al. Abnormal, multilayered titanosaur (Dinosauria: Sauropoda) eggs from in situ clutches at the Auca Mahuevo locality, Neuquen Province, Argentina. J. Vert. Pal. 24, 913–922 (2004).
Bravo, A. M. et al. Un nuevo yacimiento de cáscaras de huevo de dinosaurio de la provincial de Burgos, España (Maastrichtiense) in Actas de las III Jornadas Internacionales sobre Paleontología de Dinosaurios y su Entorno (ed. Colectivo Arqueológico y Paleontológico de Salas) 223–234 (Salas de los Infantes, 2006).
Erben, H. K., Hoefs, J. & Wedepohl, K. H. Paleobiologic and isotopic studies of eggshells from a declining dinosaur species. Paleobiology 5, 380–414 (1979).
Kérourio, P. La distribution des “Coquilles d’oeufs de Dinosauriens multistratifies” dans le Maestrichtien continental du Sud de la France. Geobios 14, 533–536 (1981).
Vianey-Liaud, M., Mallan, P., Buscail, O. & Montgelard, G. Review of French dinosaur eggshells: morphology, structure, mineral and organic composition in Dinosaur Eggs and Babies (ed. Carpenter, K., Hirsch, K. F. & Horner, J. R.) 151–183 (Camb. Uni. Press., 1994).
Grellet-Tinner, G., Corsetti, F. & Buscalioni, A. D. The importance of microscopic examinations of eggshells: Discrimination of bioalteration and diagenetic overprints from biological features. J. Iberian Geo. 36, 181–192 (2010).
Chiappe, L. M. et al. Sauropod dinosaur embryos from the Late Cretaceous of Patagonia. Nature 396, 258–261 (1998).
Wilson, J. A., Mohabey, D. M., Peters, S. E. & Head, J. J. Predation upon hatchling dinosaurs by a new snake from the Late Cretaceous of India. PLoS Biology 8, 3e1000322, https://doi.org/10.1371/journal.pbio.1000322 (2010).
Grellet-Tinner, G. et al. Description of the first lithostrotian titanosaur embryo in ovo with neutron characterization and implications for lithostrotian Aptian migration and dispersion. Gondwana Research 20, 621–629 (2011).
Dughi, R. & Sirugue, F. Observations sur les oeufs de dinosaurs du basin d’Aix- en-Provence: les oeufs ia coquille bistratifiees. Comp. Ren. Acade. Scien 246, 2271 (1958).
Dughi, R. & Sirugue, F. L’extinction des dinosaurs à la lumière des gisements d’oeufs du Crétacé terminal du Sud de la France, principalment dans le Bassin d’Aix-en-Provence. Paléobi. Cont 7, 1–39 (1976).
Erben, K. H. Ultrastrukturen und Mineralisation rezenter und fossiler Eischalen bei Vögeln und Reptilien. Biomineralisation. 1, 2–34 (1970).
Thaler, L. Les oeufs des dinosaurs du Midi de la France livrent le secret de leur extinction. La Nature 2, 41–48 (1965).
Ewert, M. A., Firth, S. J. & Nelson, C. E. Normal and multiple eggshells in batagurine turtles and their implications for dinosaurs and other reptiles. Can. Jour. Zoo. 62, 1834–1841 (1984).
Penner, M. M. The problem of Dinosaur extinction. Contribution of the study of terminal Cretaceous eggshells from Southeast France. Geobios. 18, 665–670 (1985).
Vila, B. et al. 3-D modelling of megaloolithid clutches: insights about nest construction and dinosaur behaviour. PLoS ONE 5, 5e10362, https://doi.org/10.1371/journal.pone.0010362 (2010).
Sellés, A. G. et al. Dinosaur eggs in the Upper Cretaceous of the Coll de Nargó area, Lleida Province, south-central Pyrenees, Spain: Oodiversity, biostratigraphy and their implications. Cret. Res. 40, 10–20 (2013).
García, G. & Vianey-Liaud, M. Dinosaur eggshells as biochronological markers in Upper Cretaceous continental deposits. Palaeogeo. Palaeocli. Palaeoeco. 169, 153–164 (2001).
Sellés, A. G. & Vila, B. Re-evaluation of the age of some dinosaur localities from the southern Pyrenees by means of megaloolithid oospecies. J. Iberian Geo. 41, 125–139 (2014).
Fondevilla, V. et al. Magnetostratigraphy of the Maastrichtian continental record in the Upper Aude Valley (northern Pyrenees, France): Placing age constraints on the succession of dinosaur-bearing sites. Cret. Res. 57, 457–472 (2015).
Fondevilla, V., Dinarès-Turell, J. & Oms, O. The chronostratigraphic framework of the South-Pyrenean Maastrichtian succession reappraised: Implications for basin development and end-Cretaceous dinosaur faunal turnover. Sedi. Geo. 337, 55–68 (2016).
Coutts, J. A. & Wilson, G. C. Optimum Egg Quality - A practical approach. Sheffield (5M Publishing, 2007).
Catania, S. et al. Treatment of eggshell abnormalities and reduced egg production caused by Mycoplasma synoviae infection. Avian Dis. 54, 961–964 (2010).
Westphal, M. & Durand, J. P. Magnéto-stratigraphie des séries continentales fiuviolacustres du Crétacé supérieur dans le synclinal de l’Arc (region d’Aix-en-Provence, France). Bull. Soc. Geol. France 8, 609–621 (1990).
Tabuce, R. et al. New eutherian mammals from the Late Cretaceous of Aix-en-Provence Basin, south-eastern France. Zoo. J. Linnean Soc. 169, 653–672 (2013).
Hammer, Ø. & Harper, D. Quantitative biostratigraphy in Paleontological data analysis. (eds Hammer, Ø. & Harper, D.) 279–316 (Blackwell Publishing, 2006).
Moreno-Azanza, M. et al. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells. PLoS ONE 11, e0153026, https://doi.org/10.1371/journal.pone.0153026 (2016).
Ferguson, M. W. J. Reproductive biology and embryology of the crocodilians in Biology of the Reptilia Volume 14. (eds Billet, F. & Gans, C.) 329–491 (Wiley and Sons, 1985).
Solomon, S. E. Egg and eggshell quality. (Manson Publishing, 1997).
Isles, T. E. The socio-sexual behaviour of extant archosaurs: implications for understanding dinosaur behaviour. Histo. Bio. 21, 139–214 (2010).
Norris, D. O. & López, K. H. Hormones and reproduction of vertebrates Volume 3 (Elsevier Inc., 2011).
López-Alcaide, S. & Macip-Ríos, R. Effects of climate change in amphibians and reptiles, Biodiversity Loss in A Changing Planet (eds Grillo, O.) 163–184 (In Tech, 2011).
Rafferty, A. R. & Reina, R. D. Arrested embryonic development: a review of strategies to delay hatching in egg-laying reptiles. Proc Biol. Sci. 279, 2299–2308 (2012).
Hays, P. D. & Grossman, E. L. Oxygen isotopes in meteoric calcite cements as indicators of continental paleoclimate. Geology 19, 441–444 (1991).
Dworkin, S. I., Nordt, L. & Atchley, S. Determining terrestrial paleotemperature using the oxygen isotopic composition of pedogenic carbonate. Earth Plan. Sci. Lett. 237, 56–68 (2005).
Cojan, I., Renard, M. & Emmanuel, L. Palaeoenvironmental reconstruction of dinosaur nesting sites based on a geochemical approach to eggshells and associated palaeosols (Maastrichtian, Provence Basin, France). Palaeoge. Palaeocli. Palaeoeco. 191, 111–138 (2003).
Ghosh, P. et al. C13-O18 bonds in carbonate minerals. A new kind of paleothermometer. Geoch. Cos. Acta. 70, 1439–1456 (2006).
Riera, V. et al. Dinosaur eggshell isotope geochemistry as tools of paleoenvironmental reconstruction for the upper Cretaceous from the Tremp Formation (Southern Pyrenees). Sedimentary Geology 294, 356–370 (2013).
Linnert, C. et al. Evidence for global cooling in the Late Cretaceous. Nat. Commun. 5, 4194, https://doi.org/10.1038/ncomms5194 (2014).
Robert, R. J. & Ball, M. J. Egg Shell Quality Problem: Causes and Solutions. (University of New England, 1998).
Senkoylu, N., Akyurek, H., Ersin Samli, H. E. & Agma, A. Assessment the impacts of dietary electrolyte balance levels on laying performance of commercial white layers. Pak. Jour. Nutrition 4, 423–427 (2005).
Gesen, S. S., Eren, M. & Denzi, G. The effect of different dietary electrolyte balances on eggshell quality in laying hens. Revue Méd. Vét. 156, 491–497 (2005).
Allen, M. E. & Ullery, D. E. Relationships among nutrition and Reproduction and relevance for wild animals. Zoo Biology 23, 475–487 (2004).
Palmer, B. D. & Guillette, L. L., Jr. Allogator proved evidences for the evolution of an archosaurian model of oviparity. Biol. Repr. 46, 39–47 (1992).
Sato, T. et al. A pair of shelled eggs inside a female dinosaur. Science 308, 375 (2005).
Packard, M. J. & DeMarco, V. G. Eggshell structure and formation in eggs of oviparous reptiles in Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles. (ed. Deeming, D. C. & Ferguson, M. W. J.) 53–70 (Camb. Uni. Press., 2004).
Koch, P.L., Fogel, M.L. & Tuross, N. Tracing the diets of fossil animals using stable isotopes in Stable Isotopes in Ecology and Environmental Science. (eds Lajtha, K. & Michener, R. H.) 63–92 (Blackwell Scientific Publications, 1994).
Von Schirnding, Y., Merwe, N. J. & Van Der Vogel, J. C. Influence of diet and age on carbon isotope ratios in ostrich eggshells. Archaeometry 24, 3–20 (1982).
Schaffner, F. C. & Swart, P. K. Influence of diet and environmental water on the carbon and oxygen isotopic signatures of seabird eggshell carbonate. Bull. Mar. Sci. 48, 23–38 (1991).
O’Leary, M. H. Carbon isotopes in photosynthesis. BioSciences 38, 328–336 (1988).
Folinsbee, R. E., Fritz, P., Krouse, H. R. & Robblee, A. R. Carbon-13 and oxygen-18 in dinosaur, crocodile and bird eggshells indicate environmental conditions. Science 168, 1353–1355 (1970).
Seymour, R. S. Maximal aerobic and anaerobic power generation in large crocodiles verus mammals: Implications for dinosaur giganthothermy. PLoS ONE 8, 7e69361, https://doi.org/10.1371/journal.pone.0069361 (2013).
Werner, J. & Griebeler, E. M. Allometries of maximum growth rate versus body mass at maximum growth indicate that non-avian dinosaurs had growth rates typical of fast growing ectothermic sauropsids. PLoS ONE 9, e88834, https://doi.org/10.1371/journal.pone.0088834 (2014).
Grady, J. M. et al. Evidence for mesothermy in dinosaurs. Science 344, 1268–1272 (2014).
Johnson, B. L., Fogel, M. L. & Miller, G. H. Stable isotopes in modern ostrich eggshells: A calibration for paleoenvironmental applications in semi-arid regions of southernAfrica. Geoch. Cos. Acta 62, 2451–2461 (1998).
Greenberg, N. & Wingfield, J. C. Stress and Reproduction: Reciprocal relationships in Hormones and reproduction in fishes, amphibians, and reptile (eds. Norris, D. O. & Jones, R. E.) 461–503 (Plenum Press, 1987).
Moorea, I. T. & Jessop, T. S. Stress, reproduction, and adrenocortical modulation in amphibians and reptiles. Hormones and Behavior 43, 39–47 (2003).
Radder, R. S. et al. Reproductive modes in lizards: measuring fitness consequences of the duration of uterine retention of eggs. Func. Eco. 22, 332–339 (2008).
Vila, B., Sellés, A. G. & Brusatte, S. L. Diversity and faunal changes in the latest Cretaceous dinosaur communities of southwesternEurope. Cret. Res 57, 552–564 (2016).
Butler, R. J. & Barrett, P. M. Palaeoenvironmental controls on the distribution of Cretaceous herbivorous dinosaurs. Naturwissenschaften. 95, 1027–1032 (2008).
Garrido, A. C. Paleoenvironment of the Auca Mahuevo and Los Barreales sauropod nesting-sites (Late Cretaceous, Neuquén province, Argentina). Ameghiniana 47, 99–106 (2010).
Mannion, P. D. & Upchurch, P. A quantitative analysis of environmental associations in sauropod dinosaurs. Paleobiology. 36, 253–282 (2010).
Jackson, F. D., Schmitt, J. G. & Oser, S. E. Influence of vertisol development on sauropod egg thaphonomy and distribution at the Auca Mahuevo locality, Patagonia, Argentina. Palaeoge. Palaeocli. Palaeoeco. 386, 300–307 (2013).
Nadon, G. C. The association of anastomosed fluvial deposits and dinosaur tracks, eggs and nests: implications for the interpretation of floodplain environments and a possible survival strategy for ornithopods. Palaios 8, 31–44 (1993).
Lyson, T. R. & Longrich, N. R. Spatial niche partitioning in dinosaurs from the latest Cretaceous (Maastrichtian) of North America. Proc. Roy. Soc. B 278, 1158–1164 (2011).
Prieto-Márquez, A., Dalla Vecchia, M. F., Galobart, À. & Gaete, R. Diversity, relationships, and biogeography of the lambeosaurine dinosaurs from the European archipelago, with description of the new aralosaurin Canardia garonnensis. PLoS ONE 8, 7e69835, https://doi.org/10.1371/journal.pone.0069835 (2013).
Vila, B. et al. The latest succession of dinosaur tracksites in Europe: hadrosaur ichnology, track production and paleoenvironments. PLoS ONE 8, e72579, https://doi.org/10.1371/journal.pone.0072579 (2013).
Dalla Vecchia, F. M. et al. The hadrosauroid record in the Maastrichtian of the eastern Tremp Syncline (northern Spain) in Hadrosaurs: Proceedings of the international hadrosaur symposium at the Royal Tyrrell Museum (eds. Eberth, D. & Evans, D. C.) 298–312 (Ind. Uni. Press, 2014).
Vila, B. et al. The chronology of dinosaur oospecies in southernEurope: refinements from the Maastrichtian succession of the Eastern Pyrenees. Cret. Res 32, 378–386 (2011).
García, G. Les coquilles d’œufs de dinosaures du Crétacé supérieur du Sud de la France: Diversité, paléobiologie, biochronologie et paléoenvironements. D. Phil. Thesis, Université de Montpellier II, Sciences et Techniques du Languedoc. (1998).
Val, S., García, R. & López, D. Preliminary results on the chemical preparation of dinosaur eggshells. J. Pal. Tech. 13, 29–37 (2014).
Riera, V., Oms, O., Gaete, R. & Galobart, À. The end-Cretaceous dinosaur succession inEurope: The Tremp Basin record (Spain). Palaeoge. Palaeocli. Palaeoeco 283, 160–171 (2009).
Sarkar, A., Bhattacharya, S. K. & Mohabey, D. M. Stable-isotope analyses of dinosaur eggshells. Paleoenvironmental implications. Geology 19, 1068–1071 (1991).
Bojar, A.-V., Csiki, Z. & Grigorescu, D. Stable isotope distribution in Maastrichtian vertebrates and paleosols from the Hateg Basin, South Carpathians. Palaeoge. Palaeocli. Palaeoeco. 293, 329–342 (2010).
This research paper is a contribution to the CERCA Program (Generalitat de Catalunya), the project CGL2016-73230-P, subsidised by the Ministerio de Economí y Competitividad of Spain, and the Project 2014/100927 funded by the Government of Catalonia (Generalitat de Catalunya). We thank Lucas E. Fiorelli for his comments in a very early version of the manuscript.
The authors declare that they have no competing interests.
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Sellés, A.G., Vila, B. & Galobart, À. Evidence of Reproductive Stress in Titanosaurian Sauropods Triggered by an Increase in Ecological Competition. Sci Rep 7, 13827 (2017). https://doi.org/10.1038/s41598-017-14255-6