By Matt Kaplan

Extremely well-preserved DNA discovered in the fossilized eggshells of extinct bird species suggests that they could be a source of ancient genetic material for sequencing efforts.

Eggshells are commonly found at fossil sites worldwide. But, to date, no one has described recovering DNA from them successfully.

Now, evolutionary biologist Michael Bunce and his graduate student Charlotte Oskam at Murdoch University in Perth, Western Australia, together with an international team of scientists, have recovered DNA from fossil eggshells by dramatically improving on the process used to extract it. The results were published online this week in the Proceedings of the Royal Society B.

Conventionally, scientists remove calcium salts from fossil eggshells using decalcifying solutions before extracting DNA. Unless they are removed, the salts prevent DNA from sticking to the silica column used during the purification process. But eggshells are predominantly (97 percent) made up of calcium carbonate crystals that are embedded in a protein matrix that forms the shell. This led Bunce and Oskam to wonder if the decalcification process itself might be washing away DNA preserved within the shell.

To find out, the team separately measured the amount of DNA in both decalcified eggshell powder and the waste solution used to decalcify them. They found DNA in the decalcification solution but not in the powder--suggesting that researchers have been throwing away precious ancient DNA.

"This is a quite exciting piece of work. It is rather embarrassing for the ancient-DNA community that we have not tried this before," says biologist Michi Hofreiter of the University of York, UK.

But just discovering that ancient DNA was housed within the eggshell's calcium matrix was not enough -- the team wanted to recover more of it for further studies. "Collecting ancient DNA is a numbers game and our job gets a whole lot easier if we can maximize DNA yields," says Bunce.

In a bid to recover every shred of DNA from fossil eggshell, the team next tried raising the temperature to relax the eggshell's crystalline matrix and release the DNA. When they heated up the DNA extraction solutions to 95 degrees Celsius for 10 minutes, the team found that eggshells yielded twice as much DNA as they did without heating.

Unscrambling egg DNA

The researchers tested their method on 18 fossilized eggshell specimens, from species including moa (Dinornithiformes), emu (Dromaius) and elephant birds (Aepyornis and Mullerornis) from New Zealand, Australia and Madagascar. The eggs were between 400 and 19,000 years old, and the team collected good-quality DNA from all specimens--a feat that no-one has reported before.

"To date, the 19,000-year-old emu egg DNA is the oldest DNA that has ever been recovered from Australian fossils," says Bunce.

"This may bring birds a bit more into the spotlight of ancient DNA research--they have been neglected so far," notes Hofreiter.

Ancient DNA has been recovered from the fossilized bones of mammals, such as polar bears that are around 130,000 years old, but these fossils are usually found in cold, dry environments that protect the DNA.

"I find it intriguing that it was possible to recover ancient DNA from warm environments like those of Australia and Madagascar, which are thought to not be favorable for long-term preservation of DNA," says molecular biologist Charlotte Lindqvist of the University at Buffalo in New York.

The team speculates that DNA finds its way into eggshells because when the egg forms inside the female, some of her cells slough off and get mixed into the calcium carbonate and protein matrix. They think that the DNA stays well preserved because eggshells form a barrier against water and oxygen that would otherwise damage the DNA.

Pure and simple

The fossilized eggshell DNA is not only well preserved, but also very pure compared with DNA from fossilized animal bones, which is often badly contaminated with the DNA of bacteria that fed on the animal after its death.

When the team compared the level of bacterial DNA contamination in moa bone and moa eggshell from the same period, the level of microbial DNA contamination was 125 times lower in the DNA extracted from eggshells than from bone.

The researchers do not know if this difference between bone and eggshell contamination is because bacteria cannot get into the eggshell matrix or if it is because discarded shells provide little food on which bacteria can grow.

"These findings show great promise for the recovery of DNA from ancient birds, and perhaps reptiles," says Lindqvist. "It would be exciting to see if using this technique on eggshells from climates that are more favorable for DNA preservation, like permafrost, push back the date for ancient DNA recovery even further."