Prehistoric Animals, in Living Color Paleontologists Are Looking Beyond Bones to Reveal the Hues of Prehistoric Animals That Vanished Millions of Years Ago

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Prehistoric animals, in living color Paleontologists are looking beyond bones to reveal the hues of prehistoric animals that vanished millions of years ago. But the young field has its share of disagreements.

Amber Dance, Science Writer

Michael Benton used to tell his paleontology students that Most of these colorful revelations have emerged from they would never know the true color of a dinosaur. After fossils that contain evidence of melanin pigments— all, even fossils that sport light or dark patches may not responsible for earth tones, such as red, black, brown, ’ indicate the creature s original hue. But in recent years, the and buff—or the tiny cellular bags, called melano- vertebrate paleontologist at the University of Bristol in the somes, which produce and store melanins. But some United Kingdom has had to revise those lectures. scientists are already identifying the brighter hues of In 2010, Benton and colleagues found evidence ancient snakes and insects. “Probably all of the colors that the feathered dinosaur Sinosauropteryx prima can eventually be identified,” predicts Benton. had reddish-brown stripes on its tail (1). The same year, The work is not only helping to repaint the colorful another group claimed that birdlike Anchiornis huxleyi pictures of dinosaurs that charm schoolchildren and bore a red crest on its head (2). Since then, others have discovered that a marine ichthyosaur was dark-colored, museum-goers. Color can provide clues about how and that the early bird Confuciusornis sanctus possessed animals lived, mated, or died. Bright feathers suggest dark feathers with light wing tips (3, 4) (Table 1). Sud- that animals used those hues to select a desirable denly, the color of prehistoric animals has become an mate, for example, whereas green coloration would active topic for research rather than speculation, turning offer camouflage amid foliage, indicating there must Benton into an optimist: “If you’re a scientist, never say have been predators about. “Color has roles in lots of anything is impossible,” he now tells students. different parts of animal behavior and social interactions,”

Researchers reconstructed the plumage color of a Jurassic birdlike dinosaur called A. huxleyi using color-imparting melanosomes, the morphology of which had been preserved in fossil feathers. Image courtesy of Michael DiGiorgio (artist).

8552–8556 | PNAS | August 2, 2016 | vol. 113 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1610795113 Downloaded by guest on September 27, 2021 Illustrations courtesy of Lucy Reading (graphic artist).

says Nick Edwards, a paleontologist at the University be responsible for helping to preserve fossil feathers, of Manchester in the United Kingdom. says Benton. But the fledgling field of paleo-color has also gen- Nineteenth century fossil hunters certainly knew that erated controversy. Some scientists dispute whether melanins could stand the test of time. Some even wrote melanosomes could be preserved in fossils, and argue letters with the melanin pigment ink that they found that researchers are being fooled by what are in fact preserved in the ink sacs of fossilized squids. And it was fossilized bacteria. Others question the use of certain during his own studies of squid fossils in 2006 that Jakob metals as proxies for pigments. “I think what’s happening Vinther, then a graduate student at Yale University in is what often happens when people come up with new New Haven, Connecticut, began to wonder whether ideas,” says Luis Chiappe, Vice President for Research and melanins could persist in other fossils. A handful of sci- Collections at the Natural History Museum of Los Angeles. entists, including his supervisor Derek Briggs, had pre- “We got super excited because we had figured out ways viously seen minuscule, sausage-shaped formations in a of potentially devising the colors of these extinct ani- variety of fossils, and labeled them fossil bacteria (5, 6). mals... now we’re starting to realize it’snotassimple.” Vinther suggested these formations could be melanosomes instead. Melanosome shape, he reasoned, The Race to Color a Dinosaur could even give clues to color: sausage-shaped mela- In retrospect, it’s not so surprising that pigment traces nosomes contain the black pigment eumelanin, whereas can survive for so long. Melanins, derived from the amino round melanosomes hold reddish-brown pheomelanin. acid tyrosine, are remarkably tough polymers, and they Briggs, a paleontologist, was skeptical, but willing to tightly cross-link with proteins, such as the keratin in test the idea. He suggested that Vinther take a closer look feathers. This strengthens tissues, and could even at a fossil feather that had dark and light stripes. If the blobs

Dance PNAS | August 2, 2016 | vol. 113 | no. 31 | 8553 Downloaded by guest on September 27, 2021 were melanosomes, they should be found only in the dark A. huxleyi specimen (8, 9). Lindgren concludes that sections. If they were bacteria, they ought to be all over the at least some of the microbodies in fossils are indeed feather. When Vinther examined the light sections with a melanosomes. scanning electron microscope, he saw nothing more than Vinther, now a paleontologist at the University of the imprint of the feather itself. But in the dark sections he Bristol, also uses ToF-SIMS; he and other scientists say found the oblong bodies, along with their impressions in this direct evidence of the chemical signature of the rock (7). His hypothesis appeared to be correct. melanin pigments clinches their argument. “As far as Once Vinther’s idea went public in 2008, the race I’m concerned, case closed,” says Briggs. was on to color in a dinosaur. It ended in a dead heat: However, some critics point out that these analysis Benton’s and Vinther’s teams unveiled the colors of methods may offer a misleading picture of pigmentation. stripe-tailed S. prima and red-headed A. huxleyi within Essentially, they can only look at a limited number of days of each other in early 2010 (1, 2). spots from a given specimen. Scientists have to chip off a Other discoveries followed fast, and Benton says small bit of the fossil to subject it to mass spectrometry; that the majority of paleontologists have come to accept to minimize damage they typically only look at a handful that the preserved blobs are melanosomes. Even Michael of chips. And Vinther and colleagues studied only Wuttke, the paleontologist recently retired from the one isolated feather to decide that Archaeopteryx lith- General Department of Cultural Heritage Rhineland- ographica was probably black (10). This kind of extrapo- Palatinate in Germany, who originally thought they were lation could be wrong, say Schweitzer and others. Imagine bacteria (5), says he is now “absolutely convinced” that trying to determine the coloration of a modern-day pea- melanosomes are preserved in hairs and feathers. cock from pigments at just a few dozen spots, she cau- However, evolutionary biologist Mary Higby Schweit- tions: you might not come up with the right pattern. zer, of North Carolina State University in Raleigh, calls To avoid this problem, Edwards and colleagues use the conclusions “overstated,” a position she admits has X-ray analysis (which causes no damage) to probe pig- made her unpopular among most paleo-color aficio- ments across an entire fossil. Along with collaborators nados. “I’m not against melanosomes preserving,” says Schweitzer. But, she adds, it’s “more parsimonious to at the Stanford Synchrotron Radiation Lightsource (SSRL) assume a microbial origin until disproven with data in Menlo Park, California, these researchers use a tech- other than pictures.” nique called synchrotron rapid-scanning X-ray fluo- rescence that causes different elements to emit a Rainbow of Possibilities characteristic burst of light. In fossil organisms such as Researchers have started to collect such data. Johan C. sanctus, the scientists see copper, which is one of Lindgren, a paleontologist at Lund University in Swe- several metal ions that can bind to melanins, and is also den, uses time-of-flight secondary ion mass spec- found in tyrosinase, an enzyme that controls the produc- trometry (ToF-SIMS) and other methods to analyze tion of melanins (4). Another technique, X-ray absorption the chemical composition of fossils. By exciting the spectroscopy, revealed that the copper was bound to or- fossil surface with an ion beam, Lindgren can mea- ganic molecules, which were “most probably derived from sure the masses of the atoms and molecules that precursor pigment molecules,” says Roy Wogelius, a Uni- fly off, and he has discovered black eumelanin pig- versity of Manchester geologist on the team. Based on ment itself in fossils, such as a fish eye and an these techniques, the researchers concluded that the tips

Elucidating Structural Color Pigments are not the only source of animal colors. The bright hues of a butterfly’s wing and the metallic sheen of a beetle are structural colors, produced when light bounces off multilayer, nanoscale structures that scatter the rays. Maria McNamara, of the University College Cork in Ireland, has shown that these nanostructures can occasionally survive the fossilization process (15), allowing researchers to decipher the color of insects that lived long ago. McNamara and her team examined the greenish-blue fossils of 47-million-year-old forester moths by using electron microscopy and spectrophotometry to analyze how they scatter light (16). The researchers concluded that the moths’ wings contained multilayer structures that would have produced color in life, but the wings probably weren’t originally greenish-blue. That’s because the fossilization process can change the architecture of the nanostructures, and therefore the color they produce. To simulate this process, McNa- mara exposed modern-day beetles to high temperatures and pressure in an autoclave. She found that the highest temperatures turned their wings black, whereas lower temperatures shifted the hue toward the blue end of the spectrum (17). Based on their measurements, McNamara and colleagues decided that the forester moth would have actually been a matte yellowish-green, likely matching leaves in its environment. This implies that these prehistoric moths had already evolved camouflage mechanisms to deter predators. Meanwhile, other researchers have identified structural color in a 50-million-year-old beetle, and McNamara and others have found it in fossil feathers (18–21). “It is an important area,” says Nick Edwards, a paleontologist at the University of Manchester in the United Kingdom. “The structural aspects to coloration ... can have a profound effect on how coloration can be perceived by another organism.”

8554 | www.pnas.org/cgi/doi/10.1073/pnas.1610795113 Dance Downloaded by guest on September 27, 2021 Based on samples taken from a leatherback turtle fossil (A) (scale bar, 10 cm), this ion image (B) (scale bar, 3 μm) shows the spatial distribution of peaks characteristic of eumelanin (green), silicon oxide (blue), and sulfate (red) superimposed onto a scanning electron microscopy image of the “skin.” An enlargement (C) (scale bar, 300 nm) of the demarcated area in B (white box) shows a melanosome-like microbody. Reproduced from ref. 3, with permission from Macmillan Publishers Ltd.: Nature, copyright 2014.

and one side of A. lithographica feathers were darkly (12). Back at the surface, the dark pigment might also pigmented, whereas the other side was lighter (11). have helped the reptiles absorb heat while basking in Vinther and Ryan Carney, a paleontologist at the the sun, as it does for modern leatherbacks. University of South Florida in Tampa who worked with Recently, Vinther and colleagues used melanosomes him on the A. lithographica feather, are not convinced. to discern the identity of the mysterious Tullimonstrum The researchers point out that copper binds to all kinds gregarium fossils, first found in Illinois in the 1950s by of organic compounds, including the humic acids in amateur paleontologist Francis Tully. The creature’s decomposing biomaterials. Briggs and Schweitzer add squishy finned body, elephantine trunk ending in eight that copper compounds could have moved around be- sharp teeth, and bizarre, barbell-like midbody organ tween fossilizing organisms and the surrounding rock. earned it the nickname “Tully monster,” and paleon- Nevertheless, Wogelius and Edwards argue that be- tologists have long debated whether it might have cause they map the entire fossil, they can correlate or- been a mollusk, a worm, or a vertebrate. Using electron ganic copper complexes with the places they microscopy and ToF-SIMS, the team identified mela- expect to see pigmentation, such as in feathers and nosomes in the creature’s eyes, arranged in layers typ- fossil melanosomes (4, 11). “That’s why the whole ical of vertebrates. The team concluded that the Tully image is so important,” says Edwards. “It’s another monster was a vertebrate, albeit an unusual one (13). way to check whether we’re being fooled by some These kinds of studies demonstrate the impact that sort of geochemical process.” paleo-color is having on the wider field, says Carney: “It allows us to gain more information than just what From Color to Behavior they looked like; we can infer things about their func- Armed with these discoveries, paleontologists are tion and behavior.” now considering how color fits in with their suspicions about prehistoric animal behavior. For example, Vinther Beyond Melanins suggests that the findings of colorful fossil feathers could Melanins are only part of the natural color palette. help scientists understand why feathers evolved. The Other pigments, such as the plant-derived orange very first proto-feather filaments were probably no good carotenoids, are commonly found in modern species, for flight, and paleontologists think they helped keep from flamingoes to goldfish. (And pigments aren’t dinosaurs warm. But it’s a big leap from single filaments always the color source; see Elucidating Structural to the branched, flat-feathers needed to soar into the Color.) But these molecules are much less stable than prehistoric skies. Pigments seem to appear around the melanin pigments, and lack the heavy elements needed same time as flat feathers, and Vinther suggests they for X-ray analysis at SSRL, making it challenging to find served as a “billboard” for displays—to attract a mate or them in fossils. indicate one’sspecies—before they ever took creatures Still, it might be possible to deduce their presence. airborne. Scientists already knew that birds and dino- Maria McNamara, a paleobiologist at University College saurs had ornamental feathers or even bony headgear, Cork in Ireland, has found a way to infer these other notes Chiappe, but the color findings certainly support colors in a 10-million-year-old, exquisitely-preserved the idea that those structures were for show. snake skin from Spain (14). The snakeskin is mineral- Pigmentation isn’t always about flash, though. In ized, its biological structures replaced by calcium phos- one study, Lindgren found dark eumelanin pigment in phate. Nonetheless, the snakeskin preserves the three fossil reptiles: a leatherback turtle, a lizard-like characteristic microscopic structures of individual aquatic mosasaur, and an ichthyosaur (3). Lindgren cells. In reptiles, three distinct cell types are largely re- suspects the ichthyosaur was uniformly dark, like mod- sponsible for producing color: melanophores make ern deep-diving sperm whales, which are camouflaged black; xanthophores hold red, yellow, and orange ca- by their coloration in the ocean’s depths. This theory rotenoids; and iridophores contain crystals that scatter is supported by other evidence that the icthyosaur light. A combination of melanophores with iridophores might have been a deep diver, such as its large eyes or xanthophores produces green coloration.

Dance PNAS | August 2, 2016 | vol. 113 | no. 31 | 8555 Downloaded by guest on September 27, 2021 Each pigment cell type has a different shape, size, Although some scientists remain to be convinced and location, so McNamara and her team used electron about paleo-color studies, the public has embraced the microscopy to identify them in the fossil snakeskin, and discoveries, a side benefit of the work that’s not lost on the concluded that the creature once sported blotches of researchers. Both Edwards’ and McNamara’steamhave green, black, and yellow-green, with a pale underbelly. taken part in the United Kingdom’s Royal Society Summer “These types of blotches are really good at breaking up Science Exhibition in London, a prestigious outreach the body outline for camouflage,” McNamara explains. event, and McNamara also showcased her research at She believes that the snake probably evaded predators the World Science and Technology Fair in Bangkok in “ during the day by hiding among leaves, as many 2012. Science can often be intimidating to students ” “ modern snakes do. and kids, says Carney. Dinosaurs, especially colored ” “It’s an amazing specimen, and it really opens the flying dinosaurs, are a great way to get them excited. The discoveries are certainly enticing, agrees Philip door to expanding our palette of color reconstruction,” Donoghue, a paleobiologist at the University of Bristol. says Carney. McNamara suspects other fossils might But then, it’s not too surprising to find that ichthyosaurs yield to her technique; fish and amphibians also have were dark or that early birds flashed ornamental feathers. diverse types of pigment cells. He wants more. To achieve truly novel insights, scientists But Matthew Shawkey, an evolutionary biologist at may need to identify colors in many more fossils. That the University of Ghent in Belgium, is skeptical of the could allow them to investigate the geographical distri- findings because, he says, the fossil cells that McNamara bution of different color patterns, how they vary within a identified do not look much like the modern versions, species, or how colors evolved over time. meaning they may not be pigment cells at all. Briggs, Following the initial thrill of the first paleo-color stud- who advised McNamara during her postdoctorate ies, Donoghue says that researchers now need to pose studies, suspects that the cells changed shape as the some key questions: “Whatworkcanweputthatdatato?” skin mineralized (as, for example, muscle cells have he asks. “What hypotheses can we now test?” With any been shown to do in some fossils), and so he believes luck, as the paleo-color field matures, such inquiries will the results are valid. Briggs doubts that the method will offer new insights into the lives of prehistoric animals, find wide use, though, because it requires fossils with even as it brightens museum exhibits and children’s books finely preserved details. with true-to-life colors from the distant past.

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