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Introduction
Less than a thousand years ago New Zealand was an undiscovered paradise separated from the rest of the world by millions of years and lacking any mammalian predators. A unique island ecology had evolved with bird species filling many of the available niches. The role of main herbivore on the island had been filled by giant flightless birds of the order Dinornithiformes, known to western science by the Maori word Moa (Fig. 1 &2.).
Figure 1 A museum reproduction of the
moa Dinornis giganteus (with its neck
in a semi-relaxed position) beside a kiwi for
size comparison.
Figure 2 A reproduction
of Dinornis giganteus with its neck
and head in an extended position.
These creatures have fascinated people since their existence was first predicted but more recently it is their role in our understanding of continental drift and evolution that has caused most research. Study of the links between present-day species of flightless birds has led to very interesting ramifications for the field of paleogeography (how the present-day continents formed from older landmasses).
The Moa
At least 11 species of this intriguing creature co-existed on New Zealand exploiting different browsing tactics in the forest ecosystem. The moa ranged in size from the smallest, Euryapteryx curtus (15- 50 kg), to the largest Dinornis giganteus ("Giant terrible bird"). Dinornis giganteus probably weighed about 250kg and was able to extend its neck to a height of 3m, making it the tallest bird that ever lived2(Fig.3).
Figure 3 - size comparison
of Dinornis giganteus with
man and two of its living
ratite relatives
Moa belong to the family of birds known as Ratites. All Ratites are flightless and lack the keel in their breastbone that normally anchors the flight muscles3. Living Ratites include the ostrich, emu, cassowary, rhea and the kiwi.
Even though the moa is extinct a lot of information can be inferred from the available data. The beak shapes of the different species imply that the larger moa, family Dinornithidae, browsed on the branches of trees using their long legs, long necks and their powerful broad beaks to shear and clip at the higher branches. The shorter, stouter moa, family Emeidae, used their weak, rounded beaks to graze the soft fleshy leaves and berries of the forest4.
Due to their lack of teeth or any effective food processing adaptation, the moa are known to have swallowed stones to aid the breakdown of their fibrous diet5. These stones collected in the gizzard where they acted like a food processor to grind the leaves and twigs into tiny bits. With constant rubbing against one another the stones eventually became smooth like pebbles on a beach and have been found in situ where moa have died. Analysis of the geology of the rocks show they have been collected from a very limited locality which suggests moa were disinclined to travel very far from any one spot5.
All this changed in the 13th century when Polynesian explorers arrived in New Zealand6. These ancestors of the Maori found themselves in a land of giant, easily obtainable prey (Fig.4) and they began to hunt the moa as a major food item in their diet. Although seemingly defenceless, Maori legend tells of the tendency for moa to lash out with their huge taloned feet when cornered7 (Fig.5).
Figure 4 Polynesian/Maori
rock drawing of Moa
Figure 5 a moa foot compared
to a human hand
The early Polynesians also brought with them other mammals, which wreaked havoc on the island ecology. This coupled with the continuous hunting led to the moas demise and extinction in a short space of time8. The exact date of the moas extinction is unknown. It certainly happened before the arrival of the white man in the 18th century and may have occurred in as little as 60 years after the arrival of the Polynesians8.
It wasn't until 1839 that the moa was "rediscovered" by western science after the unearthing of some leg bones9. Since then complete skeletons and many other pieces of moa have been found including preserved flesh (Fig.6) and whole eggs (Fig.7).
Figure 6 The preserved
head of a moa
Figure 7 Moa egg and hen
egg comparison
The problem
About 200 million years ago the earth was separated into two distinct landmasses. Gondwanaland was the southern supercontinent whose movement by plate tectonics gave rise to the modern day continents of the Southern Hemisphere (Fig.8).
Since ratites are only found in the continents of the Southern Hemisphere it has been theorised that Gondwanaland was home to a flightless common ancestor that divergently evolved into present day ratite species as the smaller continents drifted apart10. Information on the divergence dates of these species could lead to new understanding of the lingering connections formed as the supercontinent broke up and provide further evidence for the existence of Gondwanaland.
Figure 8 Origin of the
southern continents
New Zealand was home to two ratite species in the recent past: the kiwi, and the giant moa. The appearance of these two very different flightless birds in close proximity has led to speculation as to whether they diverged from a single Gondwanan ancestor, or if they are the result of different colonisation events.
PCR
One way of resolving this problem involves an interesting application of the Polymerase Chain Reaction (PCR)1. This technique enables the creation of many millions of copies of DNA from as little as one copy of template DNA11. DNA consists of two strands in an antiparallel alignment bound together as a double helix. The strands are mirror images of one another with each strand being made up of nucleotides strung together like beads on a string. The nucleotide containing the base Adenine (A) always binds to the nucleotide containing the base Thymine (T) on the opposite strand and vice versa. The same rules apply to nucleotides containing Guanine (G) binding to those containing Cytosine (C). The order in which these nucleotides are joined together contains the genetic information to make the protein products the cell needs.
PCR involves separating the two strands so that each has the ability to form new bonds according to the rules of base pairing12. This means that if a G was bound to a C in the original double helix then after separation the original G can only bind a new C and the original C can only bind a new G.
Figure 9 Diagram of
the 3 stages of PCR
To make 2 copies of the original double helix does require more than just the separation of the 2 original strands and the addition of new nucleotides and a polymerising enzyme. To start the synthesis there must be short sequences complementary to the single strand to "kick-start" the polymerisation12. These "primer" sequences are made to flank both sides of the target sequence and bind to the DNA (hybridisation). Polymerisation (which can only proceed in one direction) now causes an overlap of the target sequence on both new strands formed (Fig.9). The end product from one round of PCR is therefore 2 new double helices of DNA, incorporating 2 copies of the target sequence of interest. Repeating the separation/hybridisation/synthesis cycle allows the amount of target sequence DNA to increase exponentially.
PCR applied to the problem
Cooper et al1 used PCR technology to amplify mitochondrial DNA from 3 Moa genera (Emeus, Megalapteryx and Dinornis), 2 Tinamou species (Eudromia and Crypturellus), Elephant bird (extinct), Cassowary, Kiwi and Emu1. The Dinornis giganteus DNA was so well preserved (Fig. 10) that the complete mitochondrial genome of the animal was sequenced; the first complete genome of any extinct taxa. Primers were created for the Moa mitochondrial DNA by using known sequences from chicken, ostrich and rhea mitochondrial genomes as an exact match is not needed with PCR primers to get authentic replication of the target sequence.
Figure 10 Extraction
of DNA from Dinornis
bone
Once the relevant DNA sequences for all the ratites had been compiled they were used to produce a phylogenetic tree (Fig. 11). The fact that mitochondrial DNA mutates at a steady rate allows its use as a "molecular clock" to time evolution with. This can be used for the creation of interspecies relationship maps. The basic tenet is that if the date of divergence of one species from another is known (e.g. by fossil evidence) then this can be used with the genetic data to calibrate the mitochondrial DNA "molecular clock". Using this data and the other DNA sequences together with statistical analysis and simulation allows a mathematical diagram of the relationships between all the species to be built13. The result of this analysis is a map of the interrelationships between different species of ratite.
Figure 11 Phylogenetic map
for the ratite birds
Conclusions of Cooper et al
The phylogenetic map corresponds quite well to that expected to arise from the breakup of Gondwanaland and dispersal of a flightless ratite ancestor. The most interesting inconsistency with the map is that kiwi and moa seem to represent two different colonisation events and are not two endproducts of a common ancestor stranded on New Zealand as it broke away from Gondwanaland. The Moa is the older species having diverged from a hypothetical Gondwanan ancestor about 82 million years ago. The Kiwi represents a newer divergence at only 68 million years ago, showing that there must have still been a fairly close link between New Zealand and some of the other continents formed by the breakup of Gondwanaland at this time to allow a flightless kiwi ancestor to arrive.
Problems with PCR and ancient DNA
The power of PCR as a technique also leads to its weaknesses. The ability to make millions of copies of a single sequence of DNA leads to the possibility of contamination from a foreign source. In working with prehistoric DNA the risk of corruption is even greater and the utmost care must be taken14.
The need to be in an isolated, dedicated lab is paramount and all manner of control steps must be observed to reduce the risk of foreign, especially human, DNA entering the PCR cycle. Duplicating the experiments in different labs (as done by Cooper et al1) to produce identical results is a necessary step in ancient DNA studies to try and rule out the possibility of contamination.
Further work
The Moa genes have caused quite a lot of excitement around the world and Yasuyuki Shirota of Hirosaki University in Japan is one of the interested parties15. After working with homeobox genes in chickens and quails he would like to try the same thing with moa. Homeobox genes are thought to control embryo formation by regulating a number of other genes, like the first domino in a development gene cascade. Shirota wants to try and insert some moa genes into a chicken embryo to see if he can find the right homeobox to trick the chicken into displaying some of the characteristics of a moa. Moa colour, moa behaviour or possibly moa size (which would be welcomed by chicken farmers) may result from the hybrid chicken embryo if everything works out.
The thylacine (Fig.12), or marsupial tiger, of Tasmania (Thylacinus cynocephalus) is another recently extinct and unique antipodean inhabitant.
Figure 12A thylacine
(Photograph by David Fleay)
The largest marsupial carnivore of recent times was hunted into extinction after the European sheep farmers put a price on its head at the beginning of the 20th century. Systematic extermination led to the last known specimen (Benjamin) being placed in Hobart Zoo, Tasmania, in 1924 and since his death in the first half of 1936 no conclusive evidence of any surviving thylacine has been found. In the autumn of 1936 the thylacine was put on the list of protected species16.
All may not be lost however, as around the world there are a number of thylacine pups (Fig. 13) preserved in alcohol (a preservative that does not extensively degrade DNA). The success observed with moa genes suggests that the thylacine would be an obvious choice to continue this line of work with16. Sequencing the genome of the thylacine would be a worthy project and with the advent of cloning this has led some to believe that there is still hope for resurrecting the thylacine (although not with present technology).
Figure 13 A thylacine pup
preserved in alcohol
Conclusion
The sequencing of a complete mitochondrial genome from an extinct species is a landmark in biological science. Not just for its value in understanding the breakup of Gondwanaland but also because it can help us learn about a whole order of birds that were unlike anything seen alive today. The onus is on Man to learn about these creatures whose extermination he is responsible for and maybe learn something to help prevent any future repetition.
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