Sweden-based researchers have unveiled optical microscopy, SEM and X-ray tomography images of a 180 million year old fossil fern with pristinely preserved subcellular structures.

Analysis reveals the royal fern - Osmundaceae - was preserved, almost instantaneously, before cellular decomposition took place, so much so that some cells were even preserved during different stages of cell division.

What's more, observations also indicate the fern's genetic material has remained the same over the last 180 million years; an incredibly long time to go without evolutionary change.

Transmitted light micrographs: 1) Cross section through the stem of the fossilized fern rhizome, showing complex vascular cylinder in the centre and roots and leaf traces in the surrounding cortex. 2) Longitudinal section through the stem of the fossilized fern rhizome, showing readily recognisable nuclei in the ground tissue of the pith. [Benjamin Bomfleur]

As Dr Stephen McLoughlin, from the Department of Palaeobiology, Swedish Museum of Natural History, explains, SEM studies on cut and polished surfaces of the fern stem, etched in dilute hydrochloric acid, revealed good evidence of nuclei and amyloplasts.

However, cell walls could not be clearly defined as these structures had either corroded or detached during the acid pretreatment.

"We also expected some [researchers] might challenge the presence of organelles, shown in SEM images, as an artefact of the actual acid treatment," he adds.

With this in mind, McLoughlin and colleagues turned to synchrotron X-ray tomographic microscopy to non-destructively image the tissues at sub-cellular resolution.

Studies on the basal portion of a frond, removed from the fossil, revealed nuclei within the delicate tissues, providing, says McLoughlin, 'incontrovertible evidence' of the presence of these structures.

As the researcher highlights, the sheer volume of tissue containing fossilized cell nuclei, as well as chromosomes, makes this Jurassic fern fossil truly unique.

"Individual X-ray slices and composite, 3D tomographic reconstructions of the cortical tissue clearly showed a single nucleus confined to each cell and in some cases resolved details down to the level of the nucleolus within individual nuclei," he says. "The ability to rotate the tomographic reconstruction through 360 degrees also provided a visually convincing means of clarifying the distribution of nuclei within cells."

Box-shaped portion of ground tissue of the fossilized fern rhizome showing preserved nuclei (in light blue), as revealed via synchrotron radiation X-ray tomographic microscopy at the Swiss Light Source of the Paul Scherrer Institute in Villigen, Switzerland. [Bomfleur, McLoughlin & Vajda]

According to the researcher, synchrotron observations also revealed 'exquisite' cellular structural details including intercellular pitting and cell-wall lamellae on water-conducting tracheids.

"The ability to optimise the colour balance and contrast in tomographic imagery also facilitated visualization of features down to the level of nucleoli," he adds.

Crucially, analysis revealed the subcellular structures of the fossil fern matched those of today's royal ferns, indicating genome size of these reputed 'living fossils' hasn't changed for at least 180 million years; a prime example of evolutionalry stasis.

Comparison showing a preserved nucleus in the fern fossil compared to one of its close living relative Osmundastrum cinnamomeum. [Bomfleur]

Forgotten fossils

As McLoughlin explains, the actual fossil site was originally discovered by a farmer, Gustav Andersson, in the 1960s. Samples were donated to the Museum of Natural History, but the fossils remained largely forgotten for more than forty years.

"Six months ago, during a gap between projects, we inspected thin sections of the fern using a light microscope," he says. "It was immediately apparent that this was an exceptional fossil... owing to such rarity of preservation, the fossil became our prime focus of research."

Analysis of fossil spores and pollen from the fossil's rock matrix dates the fern to the Pliensbachian age of the Early Jurassic epoch in northern Europe, some 180 million years ago.

"Sadly, Gustav Andersson died in 2002, so he did not live to see the full significance of his fossil brought to light," says McLoughlin. "Nevertheless, it is the hard work of avid amateur fossil hunters like Andersson that continue to make important contributions to the cutting edge of palaeontology around the world."

The researchers will now use the X-ray tomography to analyse features from other three-dimensionally preserved plant and animal fossils, including the reproductive structures of extinct plants.

"We suspect that intracellular structures in permineralized fossils are more common that previously reported," says McLoughlin. "Our discovery opens up a new field of palaeocytology, and synchrotron x-ray tomography will be a key tool in resolving the fine-scale details of subcellular architecture in ancient organisms."