- aDNA Research Overview
- Research Projects in the Murdoch aDNA lab
- Stick nest rat middens
- Ancient Sediment DNA
- Extinct New Zealand Megafauna
- I want to do some ancient DNA work - is it easy?
- Prospective PhD/Masters/Honours Students
What is ancient DNA (aDNA)
Ancient DNA can be loosely described as any "old" (usually degraded) DNA recovered from biological samples. Examples include the analysis of DNA recovered from archaeological material, mummified tissues, archival collections of museum/medical specimens, preserved plant remains, ice and soil cores, and so on. Unlike modern genetic analyses, ancient DNA studies have to deal with DNA that is highly degraded. Whereas most modern DNA exists as long intact stretches, ancient DNA is damaged and broken into small pieces making its recovery technically challenging. The extent of damage to the DNA places constraints on what analyses can achieved. Although DNA persists in the environment upper limits exist beyond which no DNA is deemed likely to survive. Current estimates suggest that in optimal environments, i.e environments which are very cold, such as permafrost or ice, an upper limit of between 400,000 and several million years exists. However, non frozen environments can also preserve DNA for extended periods (e.g. 40,000 year-old Neanderthal bones). Early studies (15 years ago) reported recovery of much older DNA from fossil dinosaur remains (as popularised by Jurassic park). However, these studies cannot be reproduced and are likely the result of contamination, as opposed to authentic ancient DNA.
aDNA Research Overview
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.
Research Projects in the Murdoch aDNA lab
Lab work first began in the Murdoch University ancient DNA lab in June 2006 - as a "new" laboratory we are in the process of developing research projects - some of which are listed below along with a description of how ancient DNA can be used as a tool to study extinct and endangered flora and fauna.
Extinct New Zealand Megafauna
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 rese
DNA from Fossil Eggshell
In 2010 our lab published the first ever report involving the isolation of ancient DNA from Fossil Eggshell. Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary (isotope) studies. The ability to characterise DNA from eggshell will enable definitive species identification and/or genotypes to be incorporated into these kind of studies. In our paper we describe the successful isolation and ampliﬁcation of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Ancient DNA from fossil and museum eggshell samples is a key focus of Charlotte Oskams' PhD program of study.
Ancient Sediment DNA
"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).
Stick nest rat middens
Stick-nest rats and its middens
The extinct lesser stick-nest rat; Leporillus apicalis (illustration by Peter Schouten). Fecal pellets (Left) and amberat (right) from 3000 year-old middens used in ancient DNA studies.
The middens of the Lesser and Greater Stick-nest Rats (Leporillus apicalis; L. conditor) are widespread across arid zones in Australia. Both species are now extinct on mainland Australia, but L. conditor survives on West and Eastern Franklin Islands off South Australia- and has been successfully introduced onto a few others. These rodents assemble remarkable (up to 1m high and 1.5m in diameter) nests of sticks, that become middens composed of sticks and plant and animal remains (Watts and Eves, 1976), cemented together by the viscous urine, which solidifies into a hard substance known as amberat. Dating shows that some nest/midden sites have been used continuously for centuries (Webeck and Pearson, 2005). The middens do not just include rat and plant remains because both species of Leporillus scavenged material from throughout their home ranges; nests contain bone, egg shell, hair, faecal matter of bats, echidnas, dingos, owls, insects and other species (Pearson and Dodson, 1993). The middens therefore represent a legacy of material from the environment at the time of deposition. Traditional macroscopic analysis (pollen profiling), and carbon dating have allowed researchers to investigate the history of Australia’s arid zones. DNA profiling of has been used to study the middens of American desert rodents (Kuch et al., 2002) and giant ground sloths (Poinar et al., 2003), and is an excellent way of investigating the former ranges of extinct and endangered species and how their diets may have changed through time. A major research program on fossil American pack-rat middens has, over 25 years, provided unparalleled spatial and temporal information on late Quaternary climates and ecological change in arid and semi-arid environments (Van Devender and Spaulding, 1979). Arguably, at the moment this is the richest archive of dated, identified, and well-preserved plant and animal remains in the world (Pearson and Betancourt, 2002).
I want to do some ancient DNA work - is it easy?
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.
Prospective PhD/Masters/Honours Students
From time to time the lab will have research projects (and possibly funding) for PhD, Masters and Honours students. These projects will be listed here. Unfortunately the lab is unable to take on all the students that are interested in working in the lab - there is simply not the space or resources. Prospective students are encouraged to do some background research into the kind of research (and methodologies) that are conducted in the field of ancient DNA labs before making contact with the lab. Information about Murdoch University scholarships can be found here: http://www.research.murdoch.edu.au/gradcentre/scholar.html
The lab is continually looking for new projects and samples that are suitable for ancient DNA analysis. If you are interested in establishing collaborations (on projects within Australia or Internationally) please contact the lab.