Molecular, micro- and ultrastructural investigations of labile tissues in deep time Gren, Johan 2017 Document Version: Publisher's PDF, also known as Version of record Link to publication Citation for published version (APA): Gren, J. (2017). Molecular, micro- and ultrastructural investigations of labile tissues in deep time. Lund University, Faculty of Science, Department of Geology, Lithosphere and Biosphere Science. 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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Download date: 06. Oct. 2021 LITHOLUND THESES 31 Molecular, micro- and ultrastructural investigations of labile tissues in deep time JOHAN A. GREN LITHOSPHERE AND BIOSPHERE SCIENCE | DEPARTMENT OF GEOLOGY | LUND UNIVERSITY 2018 Molecular, micro- and ultrastructural investigations of labile tissues in deep time Lund 2017 NORDIC ECOLABEL, 3041 0903 Printed by Media-Tryck, This dissertation encompasses six scientific papers presenting investigations of microstructures and molecular analyses of various fossil tissues. Using an array of analytical tools, combining established methods with state-of-the art techniques, a series of case studies are presented. These cases take on fossils belonging to a variety of taxa of different geological ages and localities. I provide compelling evidence for the presence of the pigment melanin in the fossil record, often occurring in close association with microstructures that resemble melan- osomes. The microstructures do, however, overlap in size and shape with bacteria, making chemical analyses necessary to discern their origin and, subsequently, basing interpretations of biology, ecology or colour reconstructions on their presence. The author, Johan Gren, is a geologist trained in the profession at Lund University. His interest for palaeontology has been present since childhood. In high school, Johan volunteered in the collections of the Royal Tyrell Museum of Paleontology in Drumheller, Canada, and since then the path has been clear. His interest in the life of long extinct organisms is still as vivid as it can be, and the ever growing collection of miniature dino- saurs serves to remind everyone of his true passion. Lithosphere and Biosphere Science Department of Geology Lund University Sölvegatan 12 SE-223 62 Lund, Sweden Telephone +46 46 222 78 80 ISSN 1651-6648 ISBN 978-91-87847-34-9 LITHOLUND THESIS 31 Molecular, micro- and ultrastructural investigations of labile tissues in deep time Johan A. Gren Lithosphere and Biosphere Science Department of Geology DOCTORAL DISSERTATION by due permission of the Faculty of Science, Lund University, Sweden. To be defended in Pangea, Department of Geology, Sölvegatan 12, Lund on the 19th of January 2018 at 13:15. Faculty opponent Dr. Maria E. McNamara University College Cork © Johan Gren Front cover: FEG-SEM image of elongate microstructures in the fossil feather FUM-1980 Back cover: The author. In a bow tie. Photo credit: Stefan Johansson Lithosphere and Biosphere Science Department of Geology Faculty of Science ISBN 978-91-87847-34-9 (print) ISBN 978-91-87847-35-6 (pdf) ISSN 1651-6648 Printed in Sweden by Media-Tryck, Lund University, Lund 2018 Organization Document name LUND UNIVERSITY DOCTORAL DISSERTATION Department of Geology Sölvegatan 12 SE-223 62 Lund Sweden Date of issue: 19 January 2018 Author: Johan A. Gren Sponsoring organization: – Title and subtitle: Molecular, micro- and ultrastructural investigations of labile tissues in deep time Abstract This thesis comprises investigations of microstructures and molecular remains, preserved in a variety of fossil speci- mens. The results are presented in six papers, collectively aiming to thoroughly examine fossil traces of such remains by employing a combination of both established and relatively new analytical methods. The main questions asked are: What can we learn about the biology, ecology and behaviour of ancient organisms by studying these fossilised remnants, and what methods are best suited to accomplish this? The work presented herein has been conducted through a series of case studies performed on fossils representing an array of taxa, collected from different geological ages and settings. Methods applied include light and electron microscopy, computed tomography, and molecular analyses, such as time-of-flight secondary ion mass spectrometry, IR microspectroscopy and X-ray absorption spectroscopy. Microscopic analysis and histology of teeth from Mesozoic marine reptiles allowed calculations of dentine formation and tooth replacement rates. My findings include evidence that while teeth of larger mosasaur taxa took longer time to develop, their dentine formation rates were more rapid, relative to that of smaller species. In other fossils, microscopic investigations of eye, skin and feather remnants revealed aggregations of micro- metre-sized, sub-rounded to elongate structures. Because similar microbodies have previously been described al- ternatively as relict melanosomes (i.e. pigment-containing, eukaryotic cellular organelles) and lithified bacteria, my co-authors and I performed chemical and molecular analyses in order to explore the affinity of these structures. To as- certain the chemical identity of our fossil samples, corresponding analyses were carried out also on molecularly sim- ilar compounds, including modern eumelanin, as controls. My studies show chemical evidence of animal eumelanin in close association with the microbodies, advocating the melanosome interpretation for the analysed specimens. Beyond the results reported in the included papers, this thesis provides a short review of melanin formation in ver- tebrate eyes and integument, as well as in fungi and bacteria which also produce this type of pigment. An overview of the various methods applied is presented and followed by a discussion about possible outcomes and pitfalls when studying fossil microstructures and molecular palaeontology. Whereas remnant melanosomes could potentially pro- vide new insight into a multitude of biological and ecological aspects of ancient life, there is still no straight-forward approach to determine the affinity of ‘melanosome-like’ microstructures found in fossils. Therefore, a thorough in- vestigation of such remains, including chemical analyses, should be applied in each study – at least until a consensus has been reached regarding the minimum amount of criteria to be used for a confident recognition. Key words: eumelanin, fossils, histology, integument, IR microspectroscopy, melanin, melanosomes, molecular palaeontology, ToF-SIMS, XAS Classification system and/or index terms (if any): – Supplementary bibliographical information: – Language: English ISSN and key title: 1651-6648 LITHOLUND THESES ISBN: 978-91-87847-34-9 Recipient’s notes: – Number of pages 105 Price: – Security classification: – I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sourcespermission to publish and disseminate the abstract of the above-mentioned dissertation. Signature: Date: 4 December 2017 Preface To begin this thesis, I would like to take a moment for reflection. I believe that science is completely driven by curiosity, and that new discoveries always start with a question. And there is one question to rule them all: “Why?” One word, simple as that, can sprout ideas, inspire explorations and investiga- tions and lead to great scientific discoveries. But it can also be immensely frightening. Why? Because there always comes a point when there is no answer to the question. Whatever the context, if the question “Why?” is repeatedly posed, the answers will become successively vaguer, until the one inevitable answer left is “I don’t know”. I guess that the question itself is not what horrifies us. It is the fear of not having an answer. And that is the foundation of science: To find answers to new ques- tions. Therefore, in a way, you can say that science is built on fear. I would argue that it is healthy kind of fear, though, a fear that always keeps us moving. We are chasing the “Why?”, while running as fast as we can from ignorance. If we hesitate, or even slow down, “I don’t know” will eventually catch up. So I will keep running. Keep chasing the question in pursuit of a better answer, whatever my questions may be. Never stop being curious. Always learn. I will do it because I am afraid of not having an answer. Why it scares me? I don’t know. Contents 1. SCOPE OF THE THESIS 9 2. INTRODUCTION 9 3. METHODS 10 4. MELANIN 10 Melanin chemistry 10 Melanosomes 10 Melanin in different tissues