ArticlePublisher preview available

The first dinosaur egg was soft

July 2020
Nature 583(7816)

DOI:10.1038/s41586-020-2412-8

Projects: What is the material nature of modern and fossil biomineralized tissues, and how did they evolve?
The dinosaur-bird transition: can reproductive features explain why birds are the only dinosaurs to survive into the modern?

Authors:

Mark A Norell

American Museum of Natural History

Jasmina Wiemann

California Institute of Technology

Matteo Fabbri

Yale University

Yu Congyu 余琮煜

American Museum of Natural History

Show all 9 authorsHide

Calcified eggshells protect developing embryos against environmental stress and contribute to reproductive success¹. As modern crocodilians and birds lay hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs. Known dinosaur eggshells are characterized by an innermost membrane, an overlying protein matrix containing calcite, and an outermost waxy cuticle2,3,4,5,6,7. The calcitic eggshell consists of one or more ultrastructural layers that differ markedly among the three major dinosaur clades, as do the configurations of respiratory pores. So far, only hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered; the paucity of the fossil record and the lack of intermediate eggshell types challenge efforts to homologize eggshell structures across all dinosaurs8,9,10,11,12,13,14,15,16,17,18. Here we present mineralogical, organochemical and ultrastructural evidence for an originally non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of in situ Raman spectra obtained for a representative set of hard- and soft-shelled, fossil and extant diapsid eggshells clusters the originally organic but secondarily phosphatized Protoceratops and the organic Mussaurus eggshells with soft, non-biomineralized eggshells. Histology corroborates the organic composition of these soft-shelled dinosaur eggs, revealing a stratified arrangement resembling turtle soft eggshell. Through an ancestral-state reconstruction of composition and ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those from other diapsids, revealing that the first dinosaur egg was soft-shelled. The calcified, hard-shelled dinosaur egg evolved independently at least three times throughout the Mesozoic era, explaining the bias towards eggshells of derived dinosaurs in the fossil record.

| Photographs, histology and Raman spectroscopy of Protoceratops and Mussaurus soft eggshells. a, b, Clutch assigned to the basal ceratopsian Protoceratops (a) on the basis of embryonic remains (b). The white outlined area in a corresponds to b; the arrow in b indicates one of the white, egg-shaped halos surrounding the embryos. c, Egg assigned to the basal sauropodomorph Mussaurus. The arrow indicates the black egg-shaped halo. Scale bars, 50 mm (a), 10 mm (b, c). d, Schematic (top) of the microstructures observed in a representative Protoceratops section (bottom; one of six technical replicates), depicted in normal (bottom left) and cross-polarized (bottom right) light. The eggshell is multilayered and dark brown; birefringence is observed. The maximum eggshell thickness measures 0.3053 mm (0.3353 mm including the phosphatized layer). Raman point spectra (right) were acquired at the positions labelled with bold numbers, each spectrum with ten technical replicates, at three distinct eggshell positions (the same applies to e). PFPs are in brown, and epoxy and sediment are in grey. e, Schematic (top) of microstructures in the Mussaurus section (bottom) under normal (bottom left) and cross-polarized (bottom right) light. Multiple layers are evident within the eggshell, which lacks birefringence. The maximum eggshell thickness measures 0.1227 mm.

…

Hierarchical cluster analysis of biomineralization signatures preserved in eggshell proteins (extant samples) and their fossilization products (fossil samples) The topology represents a cluster analysis of n = 24 selected eggshell protein and PFP bands (Methods). Sampling of both biomineralized proteins (in situ analysis) from hard-shelled eggs and extracted, non-biomineralized membranes from soft and decalcified hard-shelled (Caiman, Alligator, Emys, Mesoclemmys, Phrynops and Gallus) eggs avoids phylogenetic attraction of the included fossil samples, and thereby allows eggshell clustering on the basis of the protein and PFP biomineralization signal. Two separate clusters of biomineralized and non-biomineralized eggshell proteins/PFPs are recovered. Pink nodes illustrate biomineralized egg proteins/PFPs, and blue nodes represent non-biomineralized eggshell proteins/PFPs. The egg icons illustrate whether samples represent originally hard or soft eggshell. One spectrum only was used for Mussaurus, as there is not much compositional variation across the eggshell (Fig. 1e), whereas all three eggshell spectra were sampled for Protoceratops, owing to the differences in composition across the egg section (Fig. 1d). Hard-shelled Alligator and turtle eggshells were excluded from this biomineralization analysis, as they do not produce any substantial organic signal with the spectroscopy protocol used (Supplementary Information and ref. ⁴⁴). Both Protoceratops and Mussaurus eggshells are nested within the cluster of originally non-biomineralized eggshell proteins/PFPs.

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Biomineralization and evolution of hard- and soft-shelled eggs a, Discriminant analysis of informative Raman bands (n = 14) in fossil eggshells (n = 20) and sediments (n = 9). The blue (eggs) and grey (sediment) vectors characterize how the sample groups diverge. b, Discriminant analysis of Raman bands (n = 12; spectra acquired with 10 technical replicates) of proteins (and PFPs) in soft-shelled (n = 13) and hard-shelled (n = 13) fossil and extant eggs. Turquoise and pink dots indicate soft-shelled and hard-shelled eggs, respectively, and the corresponding vectors characterize how samples diverge. Discriminant factors are listed for each cluster. Chelating ligands are present only in biomineralized eggshells, and are absent in soft eggs. c, Simplified phylogeny showing the evolution of eggshell in Archosauria (n = 18 taxa shown, based on n = 112 diapsid taxa). Mechanically soft eggshell is found to be ancestral for Archosauria, Ornithodira and Dinosauria. The schematics of eggshell structures distinguish membrane (blue) and crystalline (pink) layers. Coloured eggs mark the appearance of egg colour¹⁹.

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406 | Nature | Vol 583 | 16 July 2020

Article

The first dinosaur egg was soft

Mark A. Norell1 ✉, Jasmina Wiemann2 ✉, Matteo Fabbri2 ✉, Congyu Yu1, Claudia A. Marsicano3,

Anita Moore-Nall4, David J. Varricchio4, Diego Pol5 & Darla K. Zelenitsky6

Calcied eggshells protect developing embryos against environmental stress and

contribute to reproductive success1. As modern crocodilians and birds lay

hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs.

Known dinosaur eggshells are characterized by an innermost membrane, an overlying

protein matrix containing calcite, and an outermost waxy cuticle2–7. The calcitic

eggshell consists of one or more ultrastructural layers that dier markedly among the

three major dinosaur clades, as do the congurations of respiratory pores. So far, only

hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered;

the paucity of the fossil record and the lack of intermediate eggshell types challenge

eorts to homologize eggshell structures across all dinosaurs8–18. Here we present

mineralogical, organochemical and ultrastructural evidence for an originally

non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian

Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of

insitu Raman spectra obtained for a representative set of hard- and soft-shelled, fossil

and extant diapsid eggshells clusters the originally organic but secondarily

phosphatized Protoceratops and the organic Mussaurus eggshells with soft,

non-biomineralized eggshells. Histology corroborates the organic composition of

these soft-shelled dinosaur eggs, revealing a stratied arrangement resembling turtle

soft eggshell. Through an ancestral-state reconstruction of composition and

ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those

from other diapsids, revealing that the rst dinosaur egg was soft-shelled. The

calcied, hard-shelled dinosaur egg evolved independently at least three times

throughout the Mesozoic era, explaining the bias towards eggshells of derived

dinosaurs in the fossil record.

Hard-shelled eggs are an important character defining modern birds

and are thought to have had a key role in their survival through the

Cretaceous–Palaeogene extinction (approximately 66 million years

ago)

. The calcified avian eggshell stands in contrast to the primitive

amniote eggshell condition: early amniotes and more primitive tet-

rapods

2–7

laid soft eggshells. Extant archosaurs share assembly-line

oviducts

, corpus luteum morphology

and the embryonic resorption

of eggshell calcite—factors that would seem to suggest homology of

hard, calcitic eggshell among crocodilians and all dinosaurs, non-avian

and avian

10–14

. However, pterosaurs—the sister group to dinosauro-

morphs—laid soft eggs15–18.

Non-avian dinosaurs are thought to have shared with crocodil-

ians, extant birds and most turtles an innermost shell membrane6, a

biomineralized protein matrix and an outer cuticle6. Such architecture

is found in most previously described dinosaur eggs, regardless of

shape, size or colour

. Both the shell membrane and the biomineral-

ized protein matrix are arranged in multiple layers of varying internal

patterning. Calcitic dinosaur eggs

are generally considered hard

tissues, and their fossil record is patchy in terms of diversity and age21.

Only eggs of afewtaxa—such as ornithopods, sauropodomorphs,

titanosaurs and tetanurans—have been reliably identified

21–26

. The

vast majority of these eggs arefrom the Cretaceousperiod

21–28

. How-

ever, the diversity of dinosaur taxa from the Triassicperiod to the

Cretaceous suggests that the apparent biases in the egg fossil record

cannot be explained solely by preferential preservation of certain

nesting sites, as previously hypothesized. Even in highly fossiliferous

localities, such as the Mongolian Djadoktha26–29 and the Tugrugeen

Shireh site30, where eggs and embryonic remains are relatively com-

mon, eggshells attributable to more basal dinosaur taxa have not

been recovered.

Previous attempts to homologize archosaur eggshell ultrastructures

failed24,29 because of fundamental differences in the layer organiza-

tion

23–29,31

. Ornithopod eggshells

23–25,29

have one calcified spherulitic

layer. Basal sauropodomorph eggshell32–35 consists primarily of a

thick membrane covered by a thin, nondescript calcitic layer32–35, and

titanosaurid sauropod eggshells

22,36

possess a single, well-calcified

spherulitic layer on a thinner membrane22,36,37. The number of calcified

ultrastructural layers in theropod eggshells varies between one and

three22–24,26–29,31,38,39. Current hypotheses assume a single evolutionary

origin of the dinosaurian calcified egg11–13.

https://doi.org/10.1038/s41586-020-2412-8

Received: 19 March 2019

Accepted: 14 May 2020

Published online: 17 June 2020

Check for updates

1Division of Vertebrate Paleontology, American Museum of Natural History, New York, NY, USA. 2Department of Geology & Geophysics, Yale University, New Haven, CT, USA. 3Departamento de

Ciencias Geológicas, Universidad de Buenos Aires, Buenos Aires, Argentina. 4Department of Earth Sciences, Montana State University, Bozeman, MT, USA. 5CONICET, Museo Paleontológico

Egidio Feruglio, Trelew, Argentina. 6Department of Geoscience, University of Calgary, Calgary, Alberta, Canada. ✉e-mail: norell@amnh.org; jasmina.wiemann@yale.edu; matteo.fabbri@yale.edu

Content courtesy of Springer Nature, terms of use apply. Rights reserved

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Full-text available

Oct 2016

The reproductive biology of living birds differs dramatically from that of other extant vertebrates. Although some attributes of modern avian reproduction had their origin within theropod dinosaurs like oviraptors and troodontids, even the most derived non-avian theropods lack key features of modern birds. We review the current knowledge of reproduction in Mesozoic birds and 3 lines of evidence that contribute to our understanding of the evolution of the modern avian reproductive mode: (1) efforts to define the ancestral reproductive condition on the basis of extant birds, (2) the fossil record of non-avian theropod dinosaurs, and (3) the fossil record of reproduction in primitive Mesozoic birds (e.g., Enantiornithes). The fossil evidence from Mesozoic birds and non-avian theropods suggests that reproduction passed through 5 stages from basal theropods to neornithines: (1) pre-maniraptoran theropods, (2) oviraptor-grade maniraptorans, (3) troodontid-grade paravians, (4) Enantiornithes, and (5) basal Neornithes. Major changes occurred incrementally in egg size, shape, and microstructure; in nest form; in incubation method; and in parental care. Reproduction in troodontid theropods concurs with this clade representing the sister taxon to birds. Reproduction in enantiornithine birds included sequential ovulation from a single ovary and oviduct, eggs planted upright within sediments, and incubation by a combination of sediment and attendant adult or eggs fully buried with superprecocial young. Incubation modes of derived non-avian theropods and enantiornithines may have favored paternal care. Significant changes between enantiornithines and neornithines include an additional increase in relative egg size and sediment-free incubation. The latter permitted greater adult-egg contact and likely more efficient incubation. Associated changes also included improved egg shape, egg rotation, and chalazae-the albumin chords that suspend the yolk and facilitate proper embryonic development during rotation. Neornithes are the only Mesozoic clade of Dinosauria to nest completely free of sediment, and this may have played a crucial role in their surviving the K-Pg mass extinction event.

Dinosaur incubation periods directly determined from growth-line counts in embryonic teeth show reptilian-grade development

Article

Jan 2017

Birds stand out from other egg-laying amniotes by producing relatively small numbers of large eggs with very short incubation periods (average 11-85 d). This aspect promotes high survivorship by limiting exposure to predation and environmental perturbation, allows for larger more fit young, and facilitates rapid attainment of adult size. Birds are living dinosaurs; their rapid development has been considered to reflect the primitive dinosaurian condition. Here, nonavian dinosaurian incubation periods in both small and large ornithischian taxa are empirically determined through growth-line counts in embryonic teeth. Our results show unexpectedly slow incubation (2.8 and 5.8 mo) like those of outgroup reptiles. Developmental and physiological constraints would have rendered tooth formation and incubation inherently slow in other dinosaur lineages and basal birds. The capacity to determine incubation periods in extinct egg-laying amniotes has implications for dinosaurian embryology, life history strategies, and survivorship across the Cretaceous-Paleogene mass extinction event.

Re-evaluation of the eggshell structure of eggs containing dinosaur embryos from the Lower Jurassic of South Africa

Article

Jul 2002

Some of the oldest known dinosaur eggs with embryos were reported from Lower Jurassic sediments of South Africa's Elliot Formation. The osteological data from the embryos suggest that the eggs were laid by dinosaurs, and the presence of skeletal materials of the sauropodomorph Massospondylus carinatus at the egg-producing locality prompted the tentative hypothesis of a prosauropod parentage for these eggs. Original analysis of eggshell microstructure, however, suggested that they shared crocodilian characteristics. Re-evaluation of eggshell microstructure suggests that the 'crocodilian' characteristics of the eggshell are instead artefacts of diagenesis. The available data from the eggs and embryos suggest that the clutch is probably dinosaurian in origin, but no particular dinosaurian lineage can be identified as the egg-layer.