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Fig. 1. Melanosomes in extant frogs. a–v Histological sections stained with Fontana-Masson; melanosomes (and aggregates of melanosomes) appear black. Insets in f and v show isolated melanosomes. w–al Scanning electron micrographs. Scale bar in a, 20 µm, and same scale in a–v; scale bar in w, 2 µm, and same scale in w–al
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Fig. 2. Decayed tissues of extant frogs. a–c Scanning electron micrographs of rod-shaped decay bacteria within internal tissues of the torso in Kaloula (a), Xenopus (b) and Osteopilus (c). d–f Fluorescent micrographs of melanosomes (d) and rod-shaped bacteria (e) in the torso, and of rod-shaped bacteria in the thigh (f). Melanosomes appear black in fluorescent images. Bacteria appear green and red. Scale bars, 2 μm (a–c), 20 μm (d–f)
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Fig. 3. Fossil amphibian soft tissues. a–c Frogs from Libros (Miocene, Spain) (a MNCN 63776, Museu Nacional de Ciencias Naturales, Madrid), Bechlejovice (Oligocene, Czech Republic) (b NMP 39449, National Museum Prague), and Messel (Eocene, Germany) (c SMF-ME-00978, Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt). Torsos show dark patches of non-integumentary melanosomes. Inset in b shows melanosomes from the torso. d, e Tadpoles from Libros. Lungs are dark patches in the torso (d MNCN 63848); the notochord, two closely spaced parallel lines (d, e NHM 49999, Natural History Museum, London, UK). f Salamander from Daohuguo (Middle Jurassic; CNU-V-1264, Capital Normal University, Beijing, China). Torso shows conspicuous dark patches. g–j Scanning electron micrographs of melanosomes in frogs (g–i) and tadpoles (j) from Libros. g Non-integumentary melanosomes. h Integumentary melanosomes. i, j Size-specific layers of melanosomes. i Integumentary melanosomes (top right) overlying non-integumentary melanosomes (lower left). j Size-specific layers of non-integumentary melanosomes, reflecting vertical superposition of different internal tissues in vivo. Scale bars, 60 mm (a), 50 mm (b), 20 mm (c), 10 mm (d, f), 2 µm (g–j)
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Fig. 4. Layered soft tissues in the hindlimbs of fossil frogs from Libros. a–b MNCN 63775. Light micrograph of area indicated in inset, showing a plan view of the soft tissues in the thigh. b Interpretative drawing of soft tissue layers present, based on light microscopy. c Patterning in the integumentary melanosome layer. d Three-dimensional schematic illustration of the preserved 3D structure of the layered soft tissues in the Libros frogs, based on data presented in ref.12 and this figure. The terms ‘dorsal’ and ‘ventral’ are for illustrative purposes only. e–l MNCN 63798. e Scanning electron micrograph of soft tissues from the region indicated in the inset. f–k Details of regions indicated in e. f Sediment (s) underlying the ventral melanosome layer (vm). g Detail of ventral melanosomes. h Ventral layer of phosphatized skin (vs) overlain by non-integumentary melanosomes (im) and underlain by ventral melanosomes. i Detail of collagen fibres of phosphatized skin. j Detail of non-integumentary melanosomes. k Dorsal layer of phosphatized skin (ds) overlain by dorsal melanosome layer (dm). l Elemental maps of polished sections through the soft tissues from the thigh. Melanosome layers are defined by C, S and Zn. SE: secondary electron micrograph of region analysed, dm: dorsal melanosome layer, s: sediment, vm: ventral melanosome layer, vs: ventral skin layer. Scale bars, 300 μm (a), and same scale in b, 500 μm (c), 300 µm (e), 30 µm (f, h), 10 µm (g, i, j), 20 µm (k), 50 μm (l)
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Fig. 5. Distribution of soft tissues in part and counterpart of fossil amphibian specimens. In a–f the plane of splitting passes medially through the soft tissues, thus exposing non-integumentary melanosomes at the surface and producing near-identical distributions of soft tissues in part and counterpart. In g, the dissimilar distribution of soft tissues in part and counterpart demonstrates that the plane of splitting is not precisely medial within the soft tissues; non-integumentary melanosomes may still be exposed at the surface. a MNCN 63781. b MNCN 63848. c MNCN 63793. d MNCN 63864. e IPS 16465, Institut de Paleontología de Sabadell Miquel Crusafont, Sabadell, Barcelona, Spain. f MGB-33179a, Museu de Geologia de Barcelona, Barcelona, Spain. g MPV-1934-RMa, Museu de Paleontología de Valencia, Valencia, Spain. a–f are larval P. pueyoi from Libros; g is Chelotriton from the Middle Miocene of Rubielos de Mora, Spain. Scale bars, 10 mm
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Fig. 6. Melanosomes in extant reptiles. Scanning electron micrographs of extant black iguana (Ctenosaura similis (Squamata: Corytophanidae), a–c) and the common basilisk (Basiliscus basiliscus (Squamata: Iguanidae), d–f). a, d Connective tissue. b, e Liver. c Kidney. f Spleen. Scale bars, 5 μm
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