Murdoch University ancient DNA research laboratory

The extinct Equus quagga

The first aDNA study isolated and sequenced small pieces of DNA from this extinct equid. Somewhat unsurprisingly it showed its closest living relatives were the Horse and Zebra .

In 1984, the first published ancient DNA (aDNA) sequence was based on the extraction and sequencing of 229 base pairs of mitochondrial DNA (mtDNA) from the extinct quagga, Equus quagga (Higuchi et al. 1984). Two decades later, and following the invention of polymerase chain reaction (PCR), aDNA sequence data are applied in many fields, including; climate change, biodiversity, extinctions, taxonomy, conservation, anthropology, and molecular evolution. It is the goal of this lab's research to first assess the long-term survival DNA under Australian conditions and second, to use aDNA sequence data to answer a multitude of biological questions both in Australia and elsewhere.

DNA degrades post-mortem at a rate that depends on many environmental factors – eventually reaching the point where no authentic DNA can be recovered. Historically aDNA studies have focused on samples obtained from frozen or “cool” environments because temperature was thought to be the single most important factor in the survival of DNA. The prevalence of high and fluctuating temperatures, and extreme ranges of humidity, have meant that warm climates, such as Australia, have been largely dismissed as a place where aDNA techniques can be applied. In fact, some studies predict that such environments are so hostile to DNA preservation that it should not survive for more than a few years. However, these predictions are proving unreliable as authentic aDNA has been extracted from from middens, coprolites and sediments in arid desert environments in the South-Western USA (Hofreiter et al. 2000, 2003, Kuch et al. 2003 and Poinar et al. 2003). Only recently has the field started to explore the chemistry of preservation and begun to formulate techniques capable of extracting aDNA from samples/environments that were previously thought to be inimical to long-term DNA survival.

Ancient DNA studies showed the female moa (Dinornis) was much larger than its male counterpart. A pigeon skeleton (bottom right) is included for scale. 

The extinct Haast's eagle attacking a moa.      Image: John Megahan

80 million years ago the landmass that constitutes New Zealand separated from the southern “super-continent” called Gondwana.  Following the extinction of dinosaurs 65 million years ago New Zealand did not “follow” the evolutionary path of other landmasses, in that birds rather than mammals became the dominant herbivore. Prior to human settlement 700 years ago New Zealand had no terrestrial mammals  - apart from three species of bats - instead approximately 250 avian species dominated the ecosystem half of which are now extinct.

The lack of mammals is thought to of prompted the evolution of many flightless birds, the most striking of which is the herbivorous moa (pictured) - approximately 10 species of which lived in different habitats in New Zealand's North and South Island (pictured). Moa were ill-adapted to deal with the introduction of humans and rodents, and were driven to extinction about 500-800 years ago. 

Human males have an X and a Y chromosome, whereas females are XX, therefore the DNA on the Y chromosome can positively identify DNA as being male.  Researchers have employed a similar approach with ancient moa DNA to identify the sex of moa bones (Bunce et al. 2003 and Huynen et al. 2003) - this research was some of the first use of ancient nuclear DNA to determine characteristics of extinct fauna.  The data conclusively demonstrated that females were substantially larger than males (up to 3 times the mass), a phenomenon known as reversed sexual dimorphism. The image on this page compares an averaged sized male and female with a pigeon as a scale. Research using ancient DNA and isotopes to understand the taxonomy, evolutionary history and diet is ongoing. In 2006 we secured a 3 year Marsden grant (for $825,000) to AMS 14C date and profile (with stable isotopes and DNA) moa from two North Canterbury swamps. The goal of the research is to increase our understanding of moa ecology and the environment which they occupied.

"Dirt' DNA or Molecular caving

DNA obtained from sediments at this site in New Zealand have yielded DNA from extinct birds, plants and insects. DNA profiles offer insights into former (or extinct) ecosystems

In 2003 two key papers (from labs in Leipzig and Copenhagen) demonstrated that ancient plant and animal DNA could be retrieved directly from both frozen and non-frozen sediments (Hofreiter et al., 2003; Willerslev et al., 2003). These studies highlighted the potential of aDNA research to genetically reconstruct past ecosystems. Willerslev et al. (2003) used genetic profiles from permafrost cores discovered distinct shifts in floral composition over time and, for the first, time used genetics as a tool to study changes in climate and biodiversity over thousands of years. The extraction and amplification of ancient DNA from non-frozen sediments (or “molecular caving” - Hofreiter et al., 2000) is a novel approach to dissecting the components of past ecosystems. Plant, animal, and microbial DNA preserved in sediments appears to be widespread because it has been successfully recovered from geographically diverse sites such as permafrost in Siberia and caves in the south-western USA and New Zealand (Hofreiter et al., 2000; Willerslev et al., 2003). Other than knowing that aDNA is present, little else is known about how it is preserved in the sediments, or even how it got there. One possible source of plant aDNA in sediments is fine rootlets (Willerslev et al., 2003), whereas a variety of sources, including dung, urine, skin, hair, and keratin, have been suggested for animal aDNA (Lydolph et al., 2005). Although the initial reports of aDNA recovery from sediment have been promising, the possibility that DNA “leaches” between sediment layers (disturbing the original genetic chronology), is still a major concern (Paabo et al., 2004).

The techniques employed in ancient DNA labs (DNA isolation, PCR and sequencing) are common molecular biology techniques and are relatively straight forward. While this statement is true things get increasingly complex when dealing with cross-linked, nicked and fragmented DNA. If there is good DNA preservation then ancient DNA can be easily obtained. However, problems arise when there is only a few surviving copies where contamination and post-mortem DNA damage become a factor. The ease or difficulty of an ancient DNA project ultimately depends on the DNA preservation (it is a numbers game) however it is impossible to know how good (or bad) the preservation is before beginning work on a sample. 

Some researchers advocate ancient DNA analyses are something that should only ever be conducted by labs with an extensive track record of dealing with degraded DNA  (the so-called "ancient DNA: do it with me or not at all" school of thought). There are extensive sets of guidelines surrounding how ancient DNA studies should (and shouldn't) be conducted I would urge anyone thinking of embarking on aDNA projects should read these and take a common sense approach identifying if their facilities and controls are adequate. In the field of ancient DNA the onus is on the researcher to demonstrate that the result is reproducible and not a contaminant. If adequate controls/safeguards  are not employed it is unlikely the data will be publishable. A final, and salient point, is that aDNA studies involve destructively sampling specimens - some of which are rare or unique. The use of small scale pilot studies (and preferably qPCR) to determine preservation should be conducted before large scale "shotgun" sampling occurs.