Fossil Microbodies Are Melanosomes: Evaluating and Rejecting the “Fossilized Decay-Associated Microbes” Hypothesis

Melanosomes are membrane-bound organelles of varying geometry, commonly found within a range of vertebrate tissues, that contain the pigment melanin. Melanosomes have been identified in the fossil record in many exceptionally preserved fossils allowing reconstructions of the coloration of many extinct animals. However, these microstructures have also been interpreted as “microbial cells” or melanin-producing bacteria based on their geometric similarities to melanosomes. Here we test these two conflicting hypotheses experimentally. Our results demonstrate multiple lines of evidence that these fossil microbodies are indeed melanosomes: the geometry of decay-associated microbes differs significantly from fossil microbodies; fossil microbodies are very strongly localized to in vivo melanized tissues and are absent in tissues typically unmelanized in vivo, in all fossils examined regardless of lithology or age. On the basis of these results, as well as a thorough review of existing literature on melaninlike pigments, we are able to rule out a bacterial origin for fossil microbodies associated with exceptional vertebrate fossils and demonstrate that fossil microbodies are in fact preserved melanosomes. 1 Vertebrate Palaeontology Laboratory, Division of Earth and Planetary Science, the University of Hong Kong, Hong Kong. 2 School of Biological, Earth and Environmental Sciences and Environmental Research Institute, University College Cork, Cork, Ireland. 3 School of Geography, Earth and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, U.K. 4 School of Biological Sciences, the University of Hong Kong, Hong Kong. 5 Interface Analysis Centre, H.H. Wills Physics Laboratory, School of Physics, University of Bristol, Bristol, U.K. 6 School of Earth Sciences, University of Bristol, Bristol, U.K. INTRODUCTION Integumentary coloration and patterning are instrumental to comprehending an animal’s life history and evolution (Burley et al., 1982; Butcher and Rohwer, 1989). Coloration is commonly utilized by animals for aposematic signals, crypsis, sociosexual selection and optimizing physiological functions (Cuthill et al., 2017). Pigmentary systems (including melanins, carotenoids and porphyrins) produce coloration through selective absorbance and reflectance of wavelengths of light (Hill and McGraw, 2006a; 2006b). Additionally, structural integumentary coloration is produced by scattering of different wavelengths of light due to nanoscale variations in refractive indices of the tissue constituents making up the integumen252 BULLETIN AMERICAN MUSEUM OF NATURAL HISTORY NO. 440 somes” (Hill and McGraw, 2006b). It is important to appreciate, though, that natural melanosomes will vary in composition and the eumelanosome/phaeomelanosome dichotomy is only crudely instructive in describing this observed variation. The reconstruction of color patterns in ancient animals is a burgeoning field of paleontological research and is intricately linked with the geometry of melanin-bearing “melanosome-like microbodies” (Moyer et al., 2014; Lindgren et al., 2015; Schweitzer et al., 2015) preserved in fossils. Investigations into the distribution of melanosomes in fossils led to the identification of countershading in extinct animals allowing ecological and behavioral implications to be inferred (Vinther et al., 2016; Brown et al., 2017; Smithwick et al., 2017). However, the identity of such “microbodies” has been repeatedly called into question, with a counter view (Wuttke, 1983; Toporski et al., 2002; Moyer et al., 2014; Lindgren et al., 2015; Schweitzer et al., 2015) that interprets them as fossilized microbial cells, which they were traditionally identified as, prior to the work of Vinther et al. (2008). This traditional view has been challenged in several studies evaluating the evidence for these microbodies being melanosomes, based on four important characteristics: (1) geometric and spatial consistency with extant melanosomes (Vinther et al., 2008; Li et al., 2010; Zhang et al., 2010; Li et al., 2012); (2) concentration of microbodies in fossilized vertebrate tissues that originally contained melanosomes, i.e., feathers, eyes, and visceral organs (Vinther et al., 2008; Zhang et al., 2010; Gabbott et al., 2016); (3) preserved color patterns (Vinther et al., 2008); and (4) presence of chemical signatures of melaninderived compounds in the microbodies (Glass et al., 2012 , 2013; Colleary et al., 2015). Despite this body of direct and indirect evidence, a consensus regarding the identity of fossil microbodies has yet to be reached. Four main counterarguments have been posited in favor of the microbial identity of fossil microtary structures (Prum, 1999). Melanin pigments are widespread among vertebrates and have garnered much interest within the paleontological community in the last decade (Vinther et al., 2008, 2010, 2015, 2020; Li et al., 2010, 2012; Clements et al., 2016; McNamara et al., 2016; Roy et al., 2020) especially because the identification of fossilized pigments has allowed the color of extinct organisms to be interpreted, which has provoked significant scientific and cultural interest (Vinther, 2015, 2020; Roy et al., 2020). What makes melanin especially important to fossil color reconstruction is its intrinsic synthesis as a metabolic product (rather than the result of dietary uptake, e.g., carotenoids), and its potential for preservation in various taxa across deep time (Vinther et al., 2008; Glass et al., 2012). Melanin is most commonly found within vertebrates encased within lysosome-derived, membrane-bound and subcellular vesicles called melanosomes (Raposo et al., 2001). Melanosomes have been described in fossil fish (Lindgren et al., 2012; Tanaka et al., 2014; Clements et al., 2016; Gabbott et al., 2016), amphibians (McNamara et al., 2016, Rossi et al. 2019), marine reptiles (Lindgren et al., 2014), birds (Clarke et al., 2010; Pan et al., 2016; Gren et al., 2017; Zheng et al., 2017), nonavialan dinosaurs (Li et al., 2010, 2012; Zhang et al., 2010; Xu et al., 2015; Vinther et al., 2016; Hu et al., 2018) and mammals (Manning et al., 2019; Rossi et al., 2019). Animal melanins create a spectrum of colors within a tissue by varying the ratio of darkbrown/black, produced by eumelanin, and rufous to buff, produced by pheomelanin (Vinther et al., 2008). Observations of extant mammalian and avian melanosomes show a geometrical continuum from spherical to “elongate-oblong” shapes. The elongate-oblong integumentary melanosomes that contain high relative proportions of eumelanin and have been termed “eumelanosomes,” whereas spherical melanosomes are typically dominated by pheomelanin and are called “pheomelano2020 ROY ET AL.: FOSSIL MICROBODIES ARE MELANOSOMES 253 bodies (Moyer et al., 2014; Lindgren et al., 2015; Schweitzer et al., 2015): (1) frameworks seen in fossil microbodies are formed because microbes align themselves in biofilms (Toporski et al., 2002; Moyer et al., 2014); (2) decayrelated microbes readily colonize dead bodies and so would naturally be preserved and more readily visible than melanosomes, which would instead be encapsulated by fossilized keratin out of view (Toporski et al., 2002; Moyer et al., 2014; Schweitzer et al., 2015); (3) microbes are ubiquitous, fossilize easily, and are indistinguishable geometrically from melanosomes (Moyer et al., 2014; Schweitzer et al., 2015); and (4) certain microbes (bacteria and fungi) produce melanin pigments that may lead to erroneous chemical identification rather than the melanin in the tissues of the organism itself (Lindgren et al., 2015; Schweitzer et al., 2015). Herein, we test the validity of these arguments, by designing experiments to test the following hypotheses: (1) Melanosomes and microbial cells are geometrically indistinguishable (2) Microbes localize and align themselves in orientations akin to extant melanosomes (3) Microbes fossilize easily and are thus ubiquitous throughout fossils (4) Microbes are sources of melanin and there is a likelihood of extraneous preservation in fossils. TABLE 1 Melanosomes measured from feathers of modern birds No. Species sampled Feather color Sample size Melanosome shape 1 Tragopan sp. Black 162 Elongate rod