Palaeontology A gravid fossil turtle from the early cretaceous reveals a different egg development strategy in contrast to extant marine turtles Journal: Palaeontology Manuscript ID PALA-10-18-4335-OA.R3 Manuscript Type: Original Article Date Submitted by the Author: n/a Complete List of Authors: Cadena, Edwin-Alberto; Universidad Del Rosario, Faculty of Natural Sciences and Mathematics Parra, Mary Luz; Centro de Investigaciones Paleontológicas, Parra-Ruge, Juan de Dios; Centro de Investigaciones Paleontológicas , boyaca; Cineca, boyaca Padilla, Santiago; Centro de Investigaciones Paleontológicas, Boyaca Key words: Fossil eggs, Protostegidae, Testudines, Villa de Leyva, gravid turtle Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online. SIappendixVideoS1HorizontalView.mov SIappendixVideoS2CoronalView.mov SIappendixVideoS3SagitalView.mov Palaeontology Page 1 of 29 Palaeontology 1 2 3 A GRAVID FOSSIL TURTLE FROM THE EARLY CRETACEOUS REVEALS A 4 DIFFERENT EGG DEVELOPMENT STRATEGY IN CONTRAST TO EXTANT 5 6 MARINE TURTLES 7 1,2 2 8 EDWIN-ALBERTO. CADENA , MARY L. PARRA-RUGE , JUAN DE D. PARRA- 9 RUGE2, and SANTIAGO PADILLA-BERNAL2 10 1Universidad del Rosario, Facultad de Ciencias Naturales y Matemáticas, Grupo de 11 Investigación en Paleontología Neotropical Tradicional y Molecular (PALEONEO), 12 Bogotá, Colombia; e-mail: [email protected] 13 2 14 Centro de Investigaciones Paleontológicas, Villa de Leyva, Colombia; e-mails: 15 [email protected], [email protected], [email protected] 16 17 Abstract: Extant sea turtles develop and lay pliable (flexible) eggs; however, it is 18 unknown whether they inherited this reproductive strategy from their closer fossil 19 relatives or if it represents an evolutionary novelty. Here, we describe the first 20 21 undisputable gravid marine fossil turtle ever found, from the early Cretaceous of 22 Colombia, belonging to Desmatochelys padillai Cadena and Parham, which constitutes a 23 representative of the Protostegidae. Using thin sectioning of one of the eggs, as well as 24 scanning electron microscopy coupled with elemental characterisation, 25 cathodoluminescence, and computer tomography, we established that Desmatochelys 26 padillai produced rigid eggs similar to those associated with some extant and fossil 27 freshwater and terrestrial turtles. At least 48 spherical eggs were preserved inside this 28 29 gravid turtle. We suggest that the development of rigid eggs in the extinct marine turtle 30 Desmatochelys padillai resulted as an adaptation for egg-embryo requirements dictated 31 by the physical attributes of the nesting site. 32 33 Key words: gravid turtle, fossil eggs, Protostegidae, Testudines, Villa de Leyva. 34 35 Turtles have been a successful group of vertebrates since at least the middle Triassic 36 37 (~240 Ma), adapted to almost all types of terrestrial, freshwater, and marine 38 environments. However, how the embryological development and reproduction of extant 39 linages of turtles differ from or resemble those of their ancestors remains a poorly 40 understood aspect of turtle evolution. Evidence of reproductive strategies in fossil turtles 41 is restricted to eggs, nests, embryos, and coupling pairs (Lawver & Jackson 2014); very 42 few of these findings correspond to gravid mothers. 43 44 At present, the fossil record of gravid turtles with their eggs preserved is restricted to 45 the Upper Cretaceous (Campanian) Adocus sp. (Cryptodira, Pan-Trionychia, Adocidae) 46 egg clutch and a gravid individual from the Oldman and Dinosaur Park formations in 47 Alberta, Canada preserving 26 eggs, only two of which are relatively uncrushed 48 (Zelenitsky et al. 2008), and a specimen of Basilemys variolosa (Cryptodira, 49 Nanhsiungchelyidae) from the Upper Cretaceous (late Campanian), found in the 50 Dinosaur Park Formation, Alberta, Canada, in which some eggs were found by accident 51 52 inside its carapace (Braman & Brinkman 2009). Both of these gravid fossil turtles 53 represent freshwater and/or terrestrial forms, and their egg morphologies are consistent 54 with those of extant turtles with the same life-style adaptations (Lawver & Jackson 2014). 55 The egg characteristics of extant (freshwater, terrestrial, and marine) and fossil turtles 56 57 58 59 1 60 Palaeontology Palaeontology Page 2 of 29 1 2 3 (freshwater and terrestrial) have been summarised by Hirsch (1983), Schleich & Kästle 4 (1988), Nuamsukon et al. (2009), and Lawver (2017). A potential third example of a 5 6 gravid fossil turtle comes from a Late Jurassic, eurysternid? (Cryptodira, Eucryptodira) 7 from Solnhofen, Bavaria, Germany. However, investigation of the microstructure 8 revealed complete diagenetic alteration of the “eggshell”. Therefore, the authors conclude 9 that these eggs must be referred to as pseudomorphs because no details of the eggshell 10 microstructure can be ascertained (Joyce & Zelenitsky 2002; Lawver & Jackson 2014), 11 leaving this finding highly controversial. 12 13 14 Here, we describe the first undisputable gravid marine fossil turtle ever found, with 15 eggs preserved inside its visceral cavity, found in a shallow-marine rock sequence 16 belonging to the Paja Formation in Villa de Leyva, a town in Colombia, South America. 17 We attribute these eggs as Testudoolithus oospp. Thus, we compare the egg development 18 strategies among the truly marine-adapted extant clades Dermochelyidae and 19 Cheloniidae, and the extinct Protostegidae and suggest possible explanations for the 20 21 differences among them. 22 23 GEOLOGICAL SETTINGS 24 25 The region of Villa de Leyva has yielded important remains of fossil vertebrates, 26 invertebrates, and plants from the early Cretaceous (upper Barremian–lower Aptian, > 27 120 Ma in age), dated based on ammonoid biostratigraphy (Reboulet & Hoedemaeker 28 29 2006; Reboulet et al. 2014). All these fossils belong to the Paja Formation, which is 30 characterised by a succession of mudstones interbedded with limestones, deposited in an 31 intertidal marine complex (Forero & Sarmiento 1985). Fossil vertebrates from Villa de 32 Leyva include the pliosaurs Kronosaurus boyacensis (Hampe 1992) and 33 Stenorhynchosaurus munozi (Páramo-Fonseca et al. 2016); the first dinosaur of Colombia 34 Padillasaurus leivaensis (Carballido et al. 2015); the turtle Desmatochelys padillai 35 (Cadena & Parham 2015), a basal marine protostegid; Leyvachelys cipadi (Cadena 2015), 36 37 the first sandownid turtle from South America; and several unpublished ichthyosaurs, 38 plesiosaurs, and fish. The gravid fossil turtle with its eggs reported herein was discovered 39 by Juan de Dios Parra-Ruge at Monsalve hill, located between the towns of Villa de 40 Leyva and Sutamarchan (Supporting Information Fig. S1A). 41 42 MATERIAL AND METHODS 43 44 45 Fossil material and preparation 46 The fossil turtle specimen was prepared at the Centro de Investigaciones Paleontológicas 47 (CIP). The bone surface of the turtle was originally preserved in a hard orange-reddish 48 layer of iron-rich mudstone. To expose the bone and properly identify the taxonomy, the 49 rock matrix was removed using airscribe tools (microjack) and dental picks. 50 51 52 Light and polarised transmitted microscopy 53 To establish the microstructural preservation and mineralogical composition of the eggs 54 and the rock matrix, we took one of the four isolated eggs collected together with the 55 specimen that separated during the collection and used mechanical preparation of the 56 57 58 59 2 60 Palaeontology Page 3 of 29 Palaeontology 1 2 3 shell to produce a thin section at the Paleontological Lab, North Carolina State 4 University, Raleigh, USA. The whole egg with the rock matrix was processed after it was 5 6 embedding in Silmar SIL66 resin. We extracted bubbles using a vacuum system, with 7 subsequent refrigeration for 24 hours to harden the Silmar resin. Then, we cut the sample 8 and mounted it on a glass slide. We used a precision saw and a grinding machine to reach 9 an average thickness of 70 µm. Finally, we observed the thin section and took 10 photographs using an Olympus BX53 polarised light microscope with 2x, 4x, and 10x 11 lenses at the Paleontological Lab of Yachay Tech University in San Miguel de Urcuquí, 12 Ecuador. 13 14 15 Computer tomography scanning of the specimen 16 To establish the total number of eggs preserved inside the shell, as well as their 17 characteristics, internal morphology, and preservation, we scanned the whole specimen 18 using computer tomography (CT scanning) at the Hospital San Ignacio X-ray facility, 19 Bogotá, Colombia, under the following settings: zoom factor 1.16x, 12,902 total images, 20 21 120 kV, 150 mA, and 2 mm interslice spacing (horizontal view (Data Archiving 22 Statement Video S1), coronal view (Data Archiving Statement Video S2), and sagittal 23 view (Data Archiving Statement Video S3)). We analysed the CT data using the iQ- 24 VIEW software. 25 26 Scanning electron microscopy and elemental analysis (SEM-EDS) 27 We performed elemental analysis combined with high-resolution imaging of the eggshell 28 29 structure and rock matrix of one of the isolated eggs using a scanning electron 30 microscope coupled with an energy-dispersive X-ray spectroscopy analyser, Phenom 31 ProX, at the Paleontological Lab of Yachay Tech University, San Miguel de Urcuquí, 32 Ecuador. SEM-EDS allowed us to explore the mode of preservation of the eggshell 33 ultrastructure and