ESRF X-rays reveal clues about life 100 million years ago trapped in opaque amber

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31-03-2008
Amber has always been a rich source of fossil evidence. The ESRF X-rays now make it possible for paleontologists to study opaque amber, previously inaccessible using classical microscopy techniques. Scientists from the University of Rennes (France) and the ESRF found 356 animal inclusions, dating from 100 million years ago, in two kilograms of opaque amber from mid-Cretaceous sites of Charentes (France). In a second study, synchrotron X-rays were used to determine the 3D structure of feathers found in translucent amber, to complement the information already known about the feathers. The feather fragments are unique because they may have belonged to a feathered dinosaur featuring feathers in an intermediate stage of evolution to those of modern birds.

 

Opaque amber accounts for up to 80% of the amber found in Cretaceous sites like those in Charentes. From the outside, it is impossible to tell whether something may be contained inside.  Malvina Lak and her colleagues from the University of Rennes and Paul Tafforeau of the ESRF, together with the National Museum of Natural History of Paris, have applied a synchrotron X-ray imaging technique known as propagation phase contrast microradiography to the investigation of opaque amber. This technique permits light to reach the interior of this dark amber, which resembles a stone to the human eye. “Researchers have tried to study this kind of amber for many years with little or no success. This is the first time that we can actually discover and study the fossils contained within”, says Paul Tafforeau.

The scientists imaged 640 pieces of amber from the Charentes region in south-western France. They discovered 356 fossil animals, ranging from wasps and flies, to ants and even spiders and acarians. The team was able to identify the family for 53% of the inclusions.

Pieces opaque amber. Credits: V. Girard/D. Néraudeau, UMR CNRS 6118.  

Figure 1: Pieces of opaque amber. Image credits: V. Girard/D. Néraudeau, UMR CNRS 6118.

Most of the organisms they discovered are tiny. For example, one of the acarians measures 0.8 mm and a fossil wasp only 4 mm. “The small size of the organisms in the amber would suggest that bigger animals were able to escape from the resin before becoming trapped; the smaller ones were captured more easily”, explains Malvina Lak.

ambre_tomo_med.jpg (a) radiography of a bloc with inclusions in absorption...

 

Figure 2: a) radiography of an amber block with inclusions viewed in absorption mode. b) the same radiograph in propagation phase contrast mode with 990 mm of propagation distance (pixel size: 5 µm). Credits: M. Lak, P. Tafforeau, D. Néraudeau (ESRF Grenoble and UMR CNRS 6118 Rennes).

Add water to see tiny fossils better
Surface features such as cracks in the pieces of amber stood out more prominently in the images than the fossil organisms in the interior when using synchrotron radiation. To get around this problem, scientists soaked the amber pieces in water before the experiment. Because water and amber have very similar densities, immersion made the outline of the amber pieces and the cracks almost invisible. Consequently, inclusion visibility was increased, leading to better detection and characterisation of the fossils.

Classification of species
Once discovered on the radiographs, some of the organisms were imaged in three dimensions using phase contrast microtomography and virtually extracted from the resin. The high quality of these 3D reconstructions allowed the paleontologists to study the organisms in detail and to describe them. The success of this experiment shows the high value of the ESRF for the study of fossils. “Opaque amber hosts many still unknown aspects of the past life on our planet, and the use of third generation synchrotron sources will continue to play an important role in unveiling them”, asserts Malvina Lak.

Organisms embedded in amber.

 

Figure 3: Examples of virtual 3D extraction of organisms embedded in opaque amber: a) Gastropod Ellobiidae; b) Myriapod Polyxenidae; c) Arachnid; d) Conifer branch (Glenrosa); e) Isopod crustacean Ligia; f) Insect hymenopteran Falciformicidae. Credits: M. Lak, P. Tafforeau, D. Néraudeau (ESRF Grenoble and UMR CNRS 6118 Rennes).

 

Missing link between bird and dinosaur feathers hidden in amber?
Using the same technique that allowed scientists to find 356 inclusions in amber, the team studied some translucent amber samples containing feathers. Knowledge of the structure of feathers of the earliest birds and of the few feathered dinosaurs is poor. In particular, the formation of the long fused shaft in which the feather barbs are inserted, typical of so-called “modern feathers”, was not well understood. Until recently, no material evidence supported the theory of the various developmental stages of feathers.

The morphology of modern feathers was already present in theropod dinosaurs, from which today’s birds have evolved. Also, Archaeopteryx, the oldest known bird who lived about 135-150 million years ago, presented feather imprints of the modern type. However, the preservation of fossil feathers in calcareous plates does not permit a detailed investigation of their three-dimensional structure.

The missing link to the modern feather might be hidden in a large piece of amber (4 x 3 x 2 cm) discovered in 2000 in Western France by Didier Néraudeau, from the University of Rennes (France) and Vincent Perrichot from the Museum für Naturkunde of Berlin (Germany). This piece of amber contains seven very particular feather fragments. In 2004, scientist Loïc Marion, from the University of Rennes, observed  that they have a structure unknown in bird feathers: their long shafts fuse progressively to compose the rachis or shaft (similar to a banana “trunk” which is formed by juxtaposition of leaves). This is contrary to the complete fusion observed in the rachises of all other fossils and almost all modern feathers.

3D reconstruction of the basis of one of the feathers. Credits: Paul Tafforeau/ESRF.  

Figure 4: 3D reconstruction of the basis of one of the feathers. Credits: Paul Tafforeau/ESRF.

Although the authors do not exclude that these fragments could have originated from an early bird featuring primitive feathers, they consider it more probable that they actually belonged to a feathered dinosaur.

By using X-ray synchrotron holotomography developed at the European Light Source, Paul Tafforeau succeeded in imaging these fragments in three dimensions at high resolution, revealing details smaller than one micrometre. He also detected numerous other interesting features within the block of amber. These investigations provided the final proof of the partial fusion of barbs with the shaft. They also revealed that this shaft is flattened, which indicates the formation of the planar form of feathers, a pre-requisite for using them to fly. 

This breakthrough is the first fossil evidence of an intermediate stage in the evolutionary diversification of feathers. Discovery and then investigations of many more fossils is necessary to fully understand how and when birds gained the feathers permitting them to fly.

 

References

M. Lak, D. Néraudeau, A. Nel, P. Cloetens, V. Perrichot and P. Tafforeau, Phase contrast X-ray synchrotron imaging: opening access to fossil inclusions in opaque amber, Microscopy and Microanalysis, Forthcoming article, doi: 10.1017/S1431927608080264.

V. Perrichot, L. Marion, D. Neraudeau, R. Vullo and P. Tafforeau, The early evolution of feathers: fossil evidence from Cretaceous amber of France. Proceedings of the Royal Society of London B 275, 1197-1202 ( 2008); Doi:10.1098/rspb.2008.0003.

 

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