Living the Scientific Life (Scientist, Interrupted)

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Elephant bird, Aepyornis maximus, egg
compared to a human hand with a hummingbird egg balanced on a fingertip.

To conduct my avian research, I’ve isolated and sequenced DNA from a variety of specimens, such as blood, muscle, skin and a variety of internal organs, dry toepads from long-dead birds in museum collections, feathers, the delicate membranes that line the inside of eggs, and even occasionally from bone. But I was surprised to learn that avian DNA can also be extracted directly from fossilized eggshells — eggshells that completely lack eggshell membranes.

An international team of scientists just published a paper demonstrating for the first time that fossil eggshells are a rich source of DNA.

“Researchers have tried unsuccessfully to isolate DNA from a fossil eggshell for years,” reports first author Charlotte Oskam, a doctoral student in biology at Murdoch University in Perth, Western Australia.

“It just turned out that they were using a method designed for bone that was not suitable for a fossil eggshell.”

According to Michael Bunce, a professor of biology at Murdoch University, extracting DNA from bone involves removing all the calcium from the bone. This calcium extract is then discarded — a mistake when dealing with eggshells.

“Turns out that the DNA is trapped within the calcium carbonate matrix,” said Dr Bunce.

Using their newly developed method, the team isolated ancient DNA (aDNA) from a variety of fossil eggshells, including a 19,000-year-old emu eggshell as well as eggshells from an extinct species of giant moa, Dinornis robustus, and the enigmatic elephant bird, Aepyornis maximus, from Madagascar. Eggshells from two other extinct species, the little bush moa, Anomalopteryx didiformis, and the heavy-footed moa, Pachyornis elephantopus, both from New Zealand’s north island, were estimated to be more than 3,000 years old. Attempts to isolate DNA from a 50,000-year-old flightless Australian Thunderbird, Genyornis, failed because the DNA was too fragmented.

In the eyes of the casual observer, avian eggshells are deceptively simple in appearance. However, a closer examination reveals that eggshells do not have a uniform structure (Figure 1):

Figure 1. Avian eggshells. (a) Stylized radial cross-section (upper) with corresponding pictorial view (lower) of a moa eggshell (Dinornis robustus). (b) Outer surface of a moa eggshell (D. robustus). Pores are visible and are aligned towards the poles of the egg. (c) Elephant bird (Aepyornis maximus). (d) Duck (Anas sp.). (e) Emu (Dromaius novaehollandiae). Scale bar, 2 mm. [larger view]
DOI: 10.1098/rspb.2009.2019

Using their new method, most eggshell samples from both ratites and from other Holocene birds yielded aDNA, indicating that avian eggshells can potentially preserve DNA for very long periods of time, even in hostile environments that have not traditionally been conducive to long-term DNA survival.

To further optimize their new method, the team stained fragments of fossil eggshells from a New Zealand giant moa and a Madagascar elephant bird with a fluorescent dye that binds only to double-stranded DNA to determine where aDNA is physically located and to determine its source. Using confocal microscopy, the team found that aDNA is distributed fairly uniformly throughout the eggshell matrix as well as on its inner surface (Figure 2):

Figure 2. Confocal images of ratite eggshells stained for DNA. (a) Confocal radial cross section of an Aepyornis eggshell, stained with SYBR Green, displaying the DNA distributed throughout the matrix: 5× objective lens, scale bar, 400 µm. Inset, orientation of confocal image. (b) Confocal inner surface of a D. robustus eggshell, stained with Hoechst dye, displaying mammillary cones (outlined) with peripherally located DNA: 40× objective lens, scale bar, 50 µm. Inset, orientation of confocal image. Red arrows, fluorescently labelled DNA. [larger view]
DOI: 10.1098/rspb.2009.2019

These data shows that the aDNA almost certainly comes from the mother rather than the developing embryo. As the developing egg moves through the hen’s oviduct, some the mother’s cells are mixed in the calcium carbonate shell as it thickens.

Eggshell is already used for a variety of scientific tests, such as radiocarbon dating and isotope analysis, which provides important information about diets, environments, past biodiversity and evolutionary processes. But obtaining genetic information from both mitochondrial and nuclear DNA isolated from the same samples promises even richer reconstructions of evolutionary history than is currently possible.

The aDNA has not yet been sequenced, but researchers are working on expanding their catalogue of aDNA samples from extinct birds: currently they are extracting genetic material from eggshells held in museums and excavated at archaeological and fossil sites throughout the world.

Combining this newly-devised aDNA extraction method with older, more established techniques will provide more clues into the lives of these ancient birds, and may shed some light on why they mysteriously died out.

The extinction of Aepyornis is especially mysterious. This giant bird lived on Madagascar for nearly 2 million years, but went extinct by the middle of the 17th century. Related to other ratites like the ostrich and emu, the herbivorous Aepyornis was approximately 10 feet tall and weighed roughly 1,000 pounds, making it the heaviest bird to ever live (at 11 feet tall, the now-extinct giant moa is the tallest bird to ever live). It is thought that Europeans saw (and were impressed by) this giant bird before it went extinct.

“This mysterious bird was probably the inspiration behind stories in the Thousand And One Nights as told by Marco Polo,” said study co-author Mike Parker-Pearson, an archaeologist at the University of Sheffield in the UK.

Even though it’s known that Aepyornis went extinct after the arrival of humans on Madagascar, there’s actually no convincing evidence that the bird was hunted by humans.

“There’s not even evidence that they ate the eggs — even though each one could make omelettes for 30 people,” remarked Dr Parker-Pearson.

It is possible that humans might have destroyed the birds’ habitat or perhaps their domestic animals, such as dogs and pigs, hunted and ate Aepyornis eggs and chicks, which then drove the species to extinction.

“It’s amazing that we now know so much about its genetic make-up, its diet and its habits,” said Dr Parker-Pearson. “Sadly, it seems to have been yet another casualty of human population growth.”

Will it be possible to use aDNA to resurrect these long-extinct birds? Unfortunately, this is unlikely: although aDNA can be sequenced, scientists must know how to correctly repackage the DNA into chromosomes, those giant molecules that contain millions of genes. This is the same challenge facing scientists who wish to bring woolly mammoths back to life, even though mammoth DNA was sequenced from well-preserved specimens recovered from the Siberian permafrost.

“As with all ancient DNA, the DNA we isolated from eggshell is very fragmented,” Ms Oskam pointed out. “It will be possible to sequence extinct genomes from fossil eggshell but it is a huge leap to imagine we can clone an extinct species.”

Source:

Oskam, C., Haile, J., McLay, E., Rigby, P., Allentoft, M., Olsen, M., Bengtsson, C., Miller, G., Schwenninger, J., Jacomb, C., Walter, R., Baynes, A., Dortch, J., Parker-Pearson, M., Gilbert, M., Holdaway, R., Willerslev, E., & Bunce, M. (2010). Fossil avian eggshell preserves ancient DNA. Proceedings of the Royal Society B: Biological Sciences DOI: 10.1098/rspb.2009.2019

Comments

  1. #1 arby
    March 10, 2010

    That’s really cool.

  2. #2 Greenpa
    March 10, 2010

    Sorry, all the “convincing evidence” needed, to assign this to human predation is- the utterly uniform behavior of hunter-gatherer peoples, and specifically the proto-polynesian peoples that wound up in Madagascar. Wherever they landed, they ate everything. Always. All the flightless birds wound up in the pot, and extinct (ok, except kiwis and kakapos, but don’t forget the many moas).

    This is a symptom of the distressingly common fallacy that “if I can’t find a peer-reviewed paper on it – then, it isn’t true, or demonstrated, and we don’t know it”. Such malarky- I’m quite sure the creators of “science” did not intend to just wipe out all human experience, and start from scratch- but somehow we’re training scientists who do not understand that.

    Most of these “fossils” incidentally, are only called that out of courtesy- they are not fossilized in the usual sense, of having minerals substituted for bone. Which doesn’t mean you can’t get DNA from true fossils.

  3. #3 "GrrlScientist"
    March 11, 2010

    greenpa: well, as they say; making assumptions can make an “ass out of you and me.”

    good point about the use of the word “fossil” in this paper. it appears that the use of this word is different for archaeologists than for paleontologists (dictionary implies it is a rather general word; specific definitions come from context). i think these eggshells might be more correctly known as “subfossils” by the paleontologists amongst us.

  4. #4 MadScientist
    March 11, 2010

    @Greenpa: Actually it would be silly to say that people must have eaten the bird and thus driven it to extinction. There are any number of other reasons the bird may have been hunted and any number of other reasons it could have been driven to extinction. So scientists suspend judgement as Bertrand Russel puts it, rather than make statements which they cannot demonstrate to be true because they want to imagine that they know something. Even if bird pieces were found in ancient garbage pits and indicated that humans ate the birds, scientists can still only say that hunting may have been a contributing factor. We know for example that the dodo did not become extinct because humans hunted it for food – so there goes your theory that they are always hunted to extinction for food. The Carrier Pigeon is another animal which was not driven to extinction due to its value as food. Otters were driven to extinction in many places due to their pelts, and the North American Buffalo was almost extinguished for its pelts; there are accounts of the animals being shot and skinned and the carcass left to rot, with only the occasional carcass taken for food.

  5. #5 John Hawks
    March 11, 2010

    The Carrier Pigeon is another animal which was not driven to extinction due to its value as food.

    Indeed, because it isn’t extinct! No doubt you meant the Passenger Pigeon…

  6. #6 llewelly
    March 11, 2010

    And the Passenger Pigeon was hunted for food, and quite aggressively.

  7. #7 Greenpa
    March 11, 2010

    Mad Scientist- you caught me writing too fast. :-) You’re absolutely correct, of course. I posted some very similar comments over on the post re ancient musk ox DNA.

    And, I did that terribly human thing- repeating something I’d heard repeatedly. Among many people studying the Pacific, the bit about “they ate everything!” is in vogue, though not intended as absolute truth.

    Good catch. :-)

  8. #8 Greenpa
    March 11, 2010

    llewelly – yes, indeed, but the picky ones will point out that it was not hunting, per se, that caused the extinction, but the interference with reproduction. Most ornithologists I think now accept the idea that Passenger Pigeons had a very large minimum flock size required for initiation of a nesting colony – like a half-million birds or so. Smaller flocks refused to nest. Our host may have the most recent information?

    Which points out of course the frustrating difficulties of scientists from different disciplines attempting to communicate. The ethologist might focus on the nesting disruption, and the anthropologist on the practice of feeding pigs on the knocked down nestlings.

    My own preference is to suggest that the introduction of any novel top predator into a naive population will render virtually the entire ecosystem metastable. Extinctions can easily result- and historically- its clear they do. One stellar example being the broad extinctions of the South American marsupial mega fauna – where Homo was not even involved.

  9. #9 Greenpa
    March 11, 2010

    And, actually, Mad scientist- Carrier Pigeon!!?? lol. We all do it. :-)

    And, re: American bison- the anthropologists and historians will claim that the near extinction was the direct result of a US Federal policy, established and pushed by General WT Sherman, of eradicating them to starve out the Native Americans. From Wikipedia (sorry):

    “In July 1865, only three months after Robert E. Lee’s surrender at Appomattox, General W. T. Sherman was put in charge of the Military Division of the Missouri, which included every territory west of the Mississippi. Sherman’s main concern as commanding general was to protect the construction and operation of the railroads from attack by hostile Indians. In his campaigns against the Indian tribes, Sherman repeated his Civil War strategy by seeking not only to defeat the enemy’s soldiers, but also to destroy the resources that allowed the enemy to sustain its warfare. The policies he implemented included the extensive killing of large numbers of buffalo, which were the primary source of food for the Plains Indians.[104]”

    Sherman, and his successors, I believe, greatly encouraged and facilitated the kind of slaughters you describe-where only the robe and tongue were taken; salted; and shipped east on the railroads the Army was protecting…

  10. #10 Bob O'H
    March 11, 2010

    but the picky ones will point out that it was not hunting, per se, that caused the extinction, but the interference with reproduction.

    Err, yes. And how did their population sizes get that low? Hunting, perhaps?

  11. #11 "GrrlScientist"
    March 11, 2010

    maybe not hunting, per se. didn’t the pigeon pope come to america and impose regulations on avian sexual behavior?

  12. #12 Greenpa
    March 11, 2010

    The pigeon pope !!?? lol!

    And Bob- yes, we’re talking about the “picky” ones; people who like to cherry pick which direction information leads. And hunting? Are you familiar with the primary harvest methods? Hard to call them hunting- if one is picky.

    Standard method- several hundred people would wait outside one of the miles-long nesting sites until well after dark. Then, with torches, they would move in, and club- everything. Some would be assigned to pick up unmangled adults and squabs big enough for eating. Afterward, they’d turn the pigs in, to clean up the ones missed or stepped on, or just superfluous.

    Appalling and shameful beyond words- and a far cry, many would say, from “hunting”.

    Now- where I live in SE Minnesota, unlimited market hunting DID eradicate whitetail deer and wild turkey, when they were hunted to feed the riverboats and loggers in the 1800s. The deer are pests now, and it’s hard to remember that until around 1950, seeing a deer around here would get your name in the paper; rare as Great Auks.

  13. #13 MadScientist
    March 12, 2010

    @John Hawks: Oops … silly me. Yes, the Passenger Pigeon.

  14. #14 MadScientist
    March 12, 2010

    @llewelly: The shooting of Passenger Pigeons was actually promoted, and not for food either. I don’t know if it’s that “shoot ‘em for fun” or shooting for food which eventually reduced their numbers too much; I’ve always heard it was shooting for fun. It’s easy to imagine shooting them for food though; pigeons are tasty and just large enough to be worthwhile hunting or trapping for food.

    @Greenpa: It’s easy to make those mistakes. Sometimes we see so much of one case that we forget about other cases. There are a number of migratory birds in Asia which will probably be driven to extinction due to (1) hunting for food and (2) extermination due to being (incorrectly) perceived as pests. The Asian Mynah is being driven towards extinction as well but I’m not aware of anyone using the bird as food (which doesn’t mean the bird isn’t used as food – but certainly not eaten in places I’ve been to).

  15. #15 MadScientist
    March 12, 2010

    @llewelly: Hmm … OK – according to the Wikipedia they were hunted for a variety of reasons including food. So hunting for food probably was a big part. The stories I remember (or think I remember) must be largely hearsay then.

  16. #16 Greenpa
    March 12, 2010

    Mad- I’m sure there was plenty of “fun” shooting, but Passenger Pigeons were also an abundant commodity at the time, and without refrigeration, the waste must have been phenomenal. They were typically just packed, un-plucked, ungutted, into wooden barrels, and tossed on the train. Here is a first-remove narrative; the story on pigeons is 3/4 of the way down the page-

    http://genealogytrails.com/ill/jefferson/articles/vol2hist4.html

    I’d never heard of this particular method- injuring a bird and then shooting the responding mobs- a rather un-pigeonlike behavior I think. A fascinating fragment about the behavior of the vanished.