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Synchrotron X-ray absorption spectroscopy of melanosomes in vertebrates and cephalopods: Implications for the affinity of Tullimonstrum

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Abstract

Screening pigments are essential for vision in animals. Vertebrates use melanins bound in melanosomes as screening pigments, whereas cephalopods are assumed to use ommochromes. Preserved eye melanosomes in the controversial fossil Tullimonstrum (Mazon Creek, IL, USA) are partitioned by size and/or shape into distinct layers. These layers resemble tissue-specific melano-some populations considered unique to the vertebrate eye. Here, we show that extant cephalopod eyes also show tissue-specific size-and/or shape-specific partitioning of melanosomes; these differ from vertebrate melanosomes in the relative abundance of trace metals and in the binding environment of copper. Chemical signatures of melanosomes in the eyes of Tullimonstrum more closely resemble those of modern cephalopods than those of vertebrates, suggesting that an invertebrate affinity for Tullimonstrum is plausible. Melano-some chemistry may thus provide insights into the phylogenetic affinities of enigmatic fossils where melanosome size and/or shape are equivocal.

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... Melanin is an essential screening pigment in vertebrate eyes [38]. Dark-toned fossil eyespots preserve melanosomes in fossil fish [19,39,40], an unidentified bird [41], the mosasaur Platecarpus [42], and in the enigmatic fossil Tullimonstrum [43]. Size-specific layering of melanosomes in the eye of a fossil is diagnostic of (a) melanic tissue layer(s); for example, the vertebrate retinal pigmented epithelium (RPE) that protects the retina from photooxidative damage. ...
... Size-specific layering of melanosomes in the eye of a fossil is diagnostic of (a) melanic tissue layer(s); for example, the vertebrate retinal pigmented epithelium (RPE) that protects the retina from photooxidative damage. The chemistry of fossil eye melanosomes may carry taxonomic information and has been used to suggest an invertebrate affinity for Tullimonstrum [40]. ...
... This reflects, in part, an incomplete understanding of the phylogenetic distribution of pheomelanin. Our recent research shows that pheomelanin is not restricted to mammals, birds, and reptiles [6] but also occurs in amphibians and fish (and cephalopods) [7,40,80]. It is thus unlikely that pheomelanin has multiple independent origins in vertebrates [81]; a single deep origin (for vertebrates and potentially for Metazoa) is more parsimonious. ...
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Melanins are widespread pigments in vertebrates, with important roles in visual signaling, UV protection, and homeostasis. Fossil evidence of melanin and melanin-bearing organelles-melanosomes-in ancient vertebrates may illuminate the evolution of melanin and its functions, but macroevolutionary trends are poorly resolved. Here, we integrate fossil data with current understanding of melanin function, biochemistry, and genetics. Mapping key genes onto phenotypic attributes of fossil vertebrates identifies potential genomic controls on melanin evolution. Taxonomic trends in the anatomical location, geometry, and chemistry of vertebrate melanosomes are linked to the evolution of endo-thermy. These shifts in melanin biology suggest fundamental links between melanization and vertebrate ecology. Tissue-specific and taxonomic trends in melanin chemistry support evidence for evolutionary tradeoffs between function and cytotoxicity. Melanin in Vertebrates Melanins (see Glossary) are dark to rufous pigments that are widespread in vertebrates and underpin critical functions in physiology and behavior [1]. Fossil evidence of melanin extending to over 300 million years ago has triggered a paradigm shift in paleobiology, prompting remarkable reconstructions of the coloration and behavior of extinct vertebrates [2-6]. New discoveries of internal melanins in vertebrate fossils have broadened our understanding of the functional diversity of ancient melanins [7-9] and invite a re-evaluation of the macroevolu-tionary history of melanin and its functions. Here, we synthesize trends in the fossil record of melanin and explore fossil evidence for the evolution of melanin function and the genetic basis of melanization. This highlights the value of the fossil record as a resource for tracking melanin evolution through deep time.
... Fossil color is a major focus of paleobiological research (Roy et al., 2019). Melanosomes, micron-sized organelles rich in melanin in vivo, are preserved in diverse fossil taxa and tissues (Vinther et al., 2008;Lindgren et al., 2012;Manning et al., 2019;Rogers et al., 2019;Rossi et al., 2019) and provide information about the evolution of color (Li et al., 2014;Gabbott et al., 2016;Lindgren et al., 2018;Manning et al., 2019) and the internal anatomy (Rossi et al., 2019) and phylogenetic affinities (Clements et al., 2016;Rogers et al., 2019) of ancient animals. ...
... Fossil color is a major focus of paleobiological research (Roy et al., 2019). Melanosomes, micron-sized organelles rich in melanin in vivo, are preserved in diverse fossil taxa and tissues (Vinther et al., 2008;Lindgren et al., 2012;Manning et al., 2019;Rogers et al., 2019;Rossi et al., 2019) and provide information about the evolution of color (Li et al., 2014;Gabbott et al., 2016;Lindgren et al., 2018;Manning et al., 2019) and the internal anatomy (Rossi et al., 2019) and phylogenetic affinities (Clements et al., 2016;Rogers et al., 2019) of ancient animals. ...
... This renders anatomical interpretation difficult and questions whether melanosome-associated Cu in other fossils is original or reflects, at least in part, diagenetic binding of Cu to degraded melanin. Some fossil melanosomes are rich in metals absent from the melanosomal metal inventory in extant vertebrates (Rogers et al., 2019;Rossi et al., 2019). Collectively, these data suggest that the preserved metal complement of melanosomes in some vertebrate fossils has been altered by diagenetic overprinting. ...
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Fossil melanosomes are a major focus of paleobiological research because they can inform on the original coloration, phylogenetic affinities, and internal anatomy of ancient animals. Recent studies of vertebrate melanosomes revealed tissue-specific trends in melanosome-metal associations that can persist in fossils. In some fossil vertebrates, however, melanosomes from all body regions are enriched only in Cu, suggesting diagenetic overprinting of original chemistry. We tested this hypothesis using laboratory experiments on melanosomes from skin and liver of the African clawed frog Xenopus laevis. After maturation in Cu-rich media, the metal chemistry of melanosomes from these tissues converged toward a common composition, and original differences in Cu oxidation state were lost. Elevated Cu concentrations and a pervasive Cu(II) signal are likely indicators of diagenetically altered melanosomes. These results provide a robust experimental basis for interpretating the chemistry of fossil melanosomes.
... Since its discovery more than fifty years ago, Tullimonstrum has been compared to mollusks, chordates, annelids, and arthropods, although the two most favored hypotheses considered it a mollusk (Foster, 1979;Rogers et al., 2019;Sallan et al., 2017) or chordate (Beall, 1991;Clements et al., 2016;Denton & Goolsby, 2018;McCoy et al., 2016). The radulae of mollusks are chitinous, whereas the keratinous "teeth" of jawless vertebrates have a proteinaceous scaffold. ...
... The phylogenetic position of Tullimonstrum has been controversial since it was first described (Richardson, 1966). Recent anatomical results that strongly suggest a vertebrate identity (Clements et al., 2016;McCoy et al., 2016) have been considered equivocal (Rogers et al., 2019;Sallan et al., 2017). The molecular composition of Tullimonstrum soft tissues, analyzed here in a comparative framework, supports a vertebrate F I G U R E 2 In situ Raman microspectroscopy of Mazon Creek fossil soft tissues. ...
Article
The chemical composition of fossil soft tissues is a potentially powerful and yet underutilized tool for elucidating the affinity of problematic fossil organisms. In some cases, it has proven difficult to assign a problematic fossil even to the invertebrates or vertebrates (more generally chordates) based on often incompletely preserved morphology alone, and chemical composition may help to resolve such questions. Here, we use in situ Raman microspectroscopy to investigate the chemistry of a diverse array of invertebrate and vertebrate fossils from the Pennsylvanian Mazon Creek Lagerstätte of Illinois, and we generate a ChemoSpace through principal component analysis (PCA) of the in situ Raman spectra. Invertebrate soft tissues characterized by chitin (polysaccharide) fossilization products and vertebrate soft tissues characterized by protein fossilization products plot in completely separate, non‐overlapping regions of the ChemoSpace, demonstrating the utility of certain soft tissue molecular signatures as biomarkers for the original soft tissue composition of fossil organisms. The controversial problematicum Tullimonstrum, known as the Tully Monster, groups with the vertebrates, providing strong evidence of a vertebrate rather than invertebrate affinity.
... This is the case of Tullimonstrum gregarium from the Carboniferous of the Mazon Creek, USA (Kuratani and Hirasawa, 2016). To recognize its kinship, Rogers et al. (2019) analysed the melanosomes from the eyespots using XRF. Although they could not provide definitive evidence whether T. gregarium is a vertebrate or invertebrate, the chemical signature of the specimen suggested that it is rather similar to melanin from modern squids. ...
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... Evidence of melanin and melanosomes has been reported from fossils that vary in taxonomy, age and geological setting 14,15,22,24 ; this broad distribution in the fossil record provides an excellent opportunity to investigate the controls on melanin and melanosome taphonomy. Most studies have focussed on integumentary melanosomes, especially those from feathers, which can preserve as three-dimensionally preserved microbodies 6 or as external moulds 7,25 . ...
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Die Geiseltal-Sammlung des Zentralmagazins Naturwissenschaftlicher Sammlungen (ZNS) der Martin-Luther-Universität Halle-Wittenberg stellt eine wertvolle geowissenschaftliche Forschungsressource dar und ist seit 2011 als national wertvolles Kulturgut eingetragen. Die Fossilien bieten einen detaillierten Einblick in die eozäne Tier- und Pflanzenwelt vor 42,5 bis 47,5 Millionen Jahren, aber auch auf den geologischen Ablagerungsraum einer küstennahen Sumpflandschaft. In den kohledominierten Sedimenten des Geiseltals (Sachsen-Anhalt) wurde von Mitte der 1920er Jahre bis zum Ende des 20. Jahrhunderts kommerzieller Braunkohlebergbau mit wissenschaftlichen Ausgrabungen kombiniert. Die seit fast 100 Jahren in Halle (Saale) gelagerte Geiseltal-Sammlung verbindet wissenschaftliches Potential aus geowissenschaftlicher, biologischer, chemischer, museologischer und historischer Sicht. Im Rahmen des deutsch-irischen Dissertationsprojektes „Taphonomy of the Eocene Geiseltal Konservat-Lagerstätte, Germany“ werden die fossilen Wirbeltiere untersucht. In dem vorliegenden Beitrag präsentieren wir Forschungsansätze, um die außergewöhnliche Erhaltung der Fossilien besser zu verstehen. Es wurden hierfür exemplarisch 180 Anuren (Froschlurche) auf ihre Ausrichtung im Sediment sowie auf Skelettvollständigkeit und -artikulation untersucht. In weiteren Untersuchungen ist die chemische Analyse der beprobten, potentiellen Weichteilüberreste und der die Froschlurche einbettenden Sedimentschichten vorgesehen. Dieser Artikel gibt einen historischen Überblick zur Geiseltal-Sammlung und stellt die genannten Untersuchungsmethoden, vorläufige Resultate und die damit einhergehenden Vorzüge der Objektbearbeitung vor.
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The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 microns and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.
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Reductive hydrolysis of pheomelanin with hydriodic acid (HI) gives two aminohydroxyphenylalanine isomers, 4-amino-3-hydroxyphenylalanine ('specific AHP') and 3-amino-4-hydroxyphenylalanine (3-aminotyrosine, AT), which derive from the oxidative polymerization of 5-S-cysteinyldopa, and 2-S-cysteinyldopa, respectively. Since we first introduced this analytical method, the combined amount of AHP and AT ('total AHP') has been extensively used as a marker of pheomelanin. However, one problem with using total AHP as a marker is that background levels originate from precursors other than pheomelanin. Considerable and variable amounts of background AT are produced from other sources, most likely nitrotyrosine residues in proteins. In order to overcome this problem, we developed HPLC conditions which enable the direct injection of the HI reduction products into the HPLC system allowing good separation of AHP and AT. In this way we could study the importance of both degradation products separately and their specificity as markers for pheomelanin. The usefulness of the present method is validated using human hair samples of various colours which were divided into dark, fair or red colours. The combined amount of specific AHP and AT shows an excellent correlation with total AHP, and the amount of specific AHP also correlates with the amount of total AHP. We also examined total AHP and specific AHP values against pyrrole-2,3,5-tricarboxylic acid (PTCA) values in the human hair samples. These results show that specific AHP measurement gives a more prominent segregation for the ratio of specific AHP to PTCA among hairs of various colours than the ratio of total AHP to PTCA. Thus, we conclude that 'specific AHP' is a more specific marker of pheomelanin than is 'total AHP'.
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Among the various melanin-producing systems, the ink gland of the cuttlefish (Sepia officinalis) has traditionally been regarded as a most convenient model system for the studies of melanogenesis. The ink gland is a highly specialized organ with immature cells in the inner portion, from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes. When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand. Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses the rearrangement of dopachrome to 5,6-dihydroxyindole) and a peroxidase (presumably involved in the later stages of melanin biosynthesis). These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland. More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP (cGMP) signalling pathway. Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells. Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO-guanylyl cyclase interaction. This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.
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Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.
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Bovine iris and choroid melanosomes at two ages (<1 week and >2 years) were examined by inductively coupled plasma mass spectrometry (ICP-MS), elemental analysis, infrared spectrometry (IR) and X-ray photoelectron spectrometry (XPS). When iris and choroid melanosomes at the same age were compared, the quantification of metal elements by ICP-MS revealed that choroid melanosomes had a higher binding capacity for the carboxylate-binding metal ions (e.g. Na+ K+, Mg2+, Ca2+ and Zn2+). Elemental analysis showed a higher O:N ratio in choroid melanosomes. Both observations suggested that choroid melanosomes have a higher content of carboxylate-containing monomer than iris melanosomes. IR spectrometric analysis showed a red shift (approximately 8 cm(-1)) of the absorption peak of aromatic C=C, C=N and C=O at approximately 1630 cm(-1) in the IR spectrum of iris melanosomes relative to choroid melanosomes. Increased conjugation in the molecular structure of the pigment is proposed to contribute to this peak shift. It is also notable that although the elemental analysis showed different C, N and O contents in the two types of melanosomes, XPS showed almost the same elemental compositions on the surface of two types of iris and choroid melanosomes studied. When the melanosomes from the same tissues at different ages were compared, ICP-MS analysis suggested that the number of carboxylate groups in the melanosomes decreased with age. Both elemental analysis and XPS showed that C:N ratio decreased with age, which was proposed to be due to both a decrease in carboxylate groups in mature samples and to the fissure of phenol rings caused by age-associated oxidation. Such age-related oxidative damage diminishes conjugation and is manifested by blue shifts of absorption peaks for aromatic double bonds in the IR spectra of mature melanosomes. XPS analysis showed that the ratio of C-O:C=O decreased with age. These tissue-related and age-related chemical differences between samples affected the optic density and metal binding properties of melanosomes, which are believed to be closely associated with the biological functions of melanins.
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Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity, and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field.
The Tully Monster and a new approach to analysing problematica
  • B Beall
Beall B. 1991 The Tully Monster and a new approach to analysing problematica. In The early evolution of Metazoa and the significance of problematic taxa: international symposium (eds S Conway-Morris, AM Simonetta), pp. 271-286. Cambridge, UK: Cambridge University Press.