Chapter 9
THE EVOLUTIONARY RIDDLE

Genetic scientist Rod Peakall had watched the story of the Wollemi pines unfold and knew that something strange must have happened if those trees had indeed been locked for aeons in their canyon. Most of the plants that he and his team study are refugees: their homes have been destroyed, their families have been blown apart and no-one is exactly sure what to do with them. Many are among the last few dozen of their species. In some cases only males are left and the plant can only be reproduced by cloning; others will face certain extinction no matter what happens to them. But all of Peakall’s subjects have something in common—the bizarre and desperate circumstances in which they have found themselves have done weird and wonderful things to their DNA. Studying these organisms, whose lives are so abnormal, permits science insights into evolution.

The bearded, soft-spoken Peakall is a world leader in his field and it was only a matter of time before someone investigating the Wollemi pines would ask him to become involved. Wyn Jones called him first and about a month later John Benson spoke to him at a conference in Sydney in December 1995. Peakall was intrigued and leapt at Benson’s invitation to analyse the Wollemia DNA. By the time Jones and Allen received their 1997 letters from the National Parks and Wildlife Service banning them from the site and telling them that Peakall was to be the beneficiary of the money saved, his genetic work on the tree had been underway for nearly a year. In early 1996, he had received an offer from the Royal Botanic Gardens to fund genetic work on the Wollemi pine.

Peakall had warned that the project would take time and be expensive. He needed numerous Wollemia samples of around five grams of fresh young leaf material. They had to be hauled out of the canyon quickly, and airfreighted overnight in ziplock bags to his lab at the Australian National University in Canberra. On some of the samples DNA extraction could be commenced immediately; others would be placed into freezer storage of minus eighty degrees Celsius. So crucial a factor was speed that Peakall requested Wollemi samples only be delivered at the beginning of the week, that way there would be no delays over weekends. It was finally agreed that for speed and security the best approach was for DNA to be extracted at Sydney’s Royal Botanic Gardens and then sent to Peakall.

Genetic scientist Rod Peakall: he was astounded by his discoveries once he began analysing the DNA of Wollemia nobilis.

‘This ancient lineage has presumably survived as an extremely small and isolated population(s) for many generations,’ Peakall wrote in his outline of the project. ‘Under these conditions, theoretical models predict a significant loss of genetic diversity over time as a result of genetic drift and inbreeding. Genetic drift is a technical term which means that if a population stays small for long enough it will lose variability until individuals almost become the same. This species may thus provide a rare opportunity to test the theory in natural populations,’ he continued. ‘Furthermore, it is widely assumed that genetic diversity is essential for evolutionary success, hence the goal of conservation to maximise the preservation of genetic diversity.’

On 2 April 1996 the first DNA samples from site one arrived in Peakall’s laboratory. The Royal Botanic Gardens obtained the DNA by grinding down fragments of foliage. Nearly two months later, on 29 May, the first batch of Wollemi pine DNA from site two arrived. A second sample bag from site two arrived on 17 June. In all, some 100 samples were provided from the two sites and from Mount Annan seedlings. A buzz went through the university’s zoology and botany departments. Peakall began to study the deepest secrets of the Wollemi pine and to this day is staggered and bewildered by what he found.

Soon after the April-June Wollemi pine DNA material had been sent, I visited Peakall to see the preliminary results of the study. He took me to a sunlit, sterile lab in one of the mazes of buildings on the ANU campus and opened the door of a freezer in which was kept a polystyrene container holding dozens of tiny tubes. At the bottom of each was DNA from one of the wild Wollemi pines. This DNA, a white glob at the bottom of each tube, contains the secrets that have kept this tree alive for over 100 million years.

The first sample that I saw was labelled ‘Tree One’. This is the Bill Tree or King Billy, the biggest Wollemia in the stand, and it was hard to reconcile that spectacular life form with the bland little blob in the tube. Slowly, using equipment accurate to atomic levels, Peakall isolated the tree’s genes. He then dissolved the DNA and, to obtain a map of the tree’s genes, painstakingly ran it through the same machines used by detectives for DNA fingerprinting.

All but identical twins and clones have differences in their DNA that stand out starkly when subjected to sensitive analysis. The offspring of sexual reproduction between a pair of clones or identical twins, however, will be genetically different from the two identical parents. Genetic studies are revolutionising our understanding of life. One of the greatest scientific endeavours presently underway around the world is the mapping of the human genome, promising revolutionary changes in medical care. Every living thing has a genome, which is the total set of its genetic information—a code that governs every aspect of an organism’s growth and life. In the last two decades DNA fingerprinting has transformed criminal investigation. In fact Peakall has been commissioned by the Australian Federal Police to study the DNA of cannabis. The goal of the project is to determine where seized marijuana has originated—cannabis has subtle differences in its DNA depending on where it is grown.

Genetic engineering is a technological advance that can propel life in completely new directions. DNA studies of plants have led to a total reordering of sections of the botanical kingdom and are becoming an important tool in the understanding of plant evolution. Ken Hill, for example, has used DNA analysis to work out Wollemia’s relationship to other members of the Araucariaceae. Hill’s DNA results backed up what his experience as a botanist had already told him—Wollemia nobilis is genetically distinct from the two other genera in the family. It seems that at some point in the distant past Araucaria and Agathis split from a common ancestor. And some time after this Wollemia branched off from Agathis.

I followed Peakall to another room off his lab, which is kept cool to protect delicate machinery, including a row of computers and the DNA fingerprinting equipment. The machine is the size of a chest of drawers and looks like something ‘Star Trek’ crew members would use to warp themselves to another time and place. He booted up a computer and after a few minutes a graph flashed up on a screen: the DNA analysis of a Wollemi pine. The graph displayed the results of a detailed search on part of the genome from one of the wild trees in stand one. It was generated by plotting the location of different DNA fragments. All DNA fingerprints are made up of a series of bands, which represent minute DNA fragments of varying size and intensity. Often these fingerprints resemble a barcode. On a barcode it is the position and width of the bars that permit it to be read and identified as a specific product. This is very similar to how DNA is read but when Peakall booted up the computer I didn’t see a barcode, I saw a jagged line not unlike the printout from a seismograph, with the occasional major spike. A few minutes later the graph of a second tree—this time from stand two—was on the screen. When the first graph was placed on the other, the two jagged lines become one. The map of the DNA of the first Wollemi pine was indistinguishable from the second.

‘When the DNA graphs from the two different trees are overlaid they are absolutely identical,’ Rod told me, one hand clutching at his beard, the other resting on the keyboard as I watched over his shoulder. ‘It indicates that the tree is either highly inbred or else an impossibly perfect clone. We may still find some variation but we have not been able to yet.’

So far Peakall’s team has searched 1000 points on the Wollemi’s genome. Although the number of points they could search is almost infinite, geneticists would normally expect to find variation after searching a handful of them. This was an especially odd result because the ANU team was targeting parts of the DNA that in other plant species typically have the most variability. Peakall has compared genetic material from the crown of King Billy to the foliage from fifty seedlings produced by five different mothers. Throughout site one no trace of genetic variability was found. This is something that Peakall has never before witnessed and in fact it is unknown in the scientific literature.

Each graph above represents the DNA profile of the same region of the genome for different members of the family Araucariaceae. The peaks and troughs represent the intensity of genetic variation. The Wollemi pine profiles are identical while the others, even among individuals in the same species, are different.

Peakall’s first thought was that Wollemia must have become a single organism. He also hypothesised that genetic drift could explain the lack of variability. ‘The puzzling thing about that is that while a population of plants may become identical in theory and on computer, in the wild you can lose variability but you never have none.’ Maybe, he said to me, genetic drift was being combined with clonality, that is, all of the individuals in the canyon were one big organism. To understand how both these forces can be at work at the same time, imagine a pine sending out a root that comes to the surface and then grows into a tree. When it matures the tree could conceivably reproduce with its parent. This means that both cloning and inbreeding could be at work in the canyon.

But this still did not explain why Wollemia had no variation. Normally a population of trees that finds itself facing extinction begins to clone itself before it loses all of its variability. This is a defence mechanism that ensures that if good times return the members of a species are not all identical. The problem became greater as soon as Peakall began to run DNA samples from site two through his equipment. ‘We expected variation between the two sites,’ Peakall said. A greater array of genetic material was then analysed from site two and compared to site one. Incomprehensibly, the same result was obtained. When the points on the genome were compared there was no genetic variability between site one and site two. They were totally identical.

As the results had flashed up on the computer during the months that Peakall was studying Wollemia DNA, a sickening feeling began to hit. In the wild and left undisturbed such low genetic diversity is not necessarily such a disaster because it appears that the tree has become perfectly adapted to its environment. But now that the wild pines are being visited diseases could be introduced. His intuition and then his machines told him that perhaps all of the trees being grown by Offord were also identical. With hundreds of genetically identical pines in cultivation a time bomb is ticking. If the population of plants in cultivation at Mount Annan and Mount Tomah Botanic Gardens—then their only locations out of the wild—were hit by disease then the entire collection could be lost. Identical genes means identical susceptibility to disease. Peakall urgently recommended that all pines immediately be protected by even more stringent hygiene during visits and that the cultivated populations be separated.

A fundamental tenet of evolutionary theory derives from Charles Darwin’s remark in The Origin of Species: ‘Amongst organic beings in a state of nature there is some individual variability: indeed I am not sure that this has been disputed.’ Variability is the fuel of evolution, the mechanism which allows the mutations that benefit an organism to happen. If no variability can be found in a Wollemi pine then only two explanations can be put forward—evolution in the trees has ceased or it has slowed to the point where it is no longer detectable. Until the entire genome of Wollemia is searched this riddle remains unsolved, yet Peakall remains hopeful that variation lurks somewhere in the tree’s DNA.

The easiest explanation of why the two sites are identical is that Wollemia nobilis is a giant clone, spread downstream from the second site and the trees are one enormous organism that has been divided in two by some catastrophe. In this theory, stems would have emerged from roots underground and become new trees. The existence of seedlings added to the confusion; Peakall was under the impression that both sites consisted only of teenage or adult trees. There was no evidence given to him that any of the seedlings were reaching a reproductive age. According to theory, if this were happening he ought not to have got the results that he did: when two genetically identical clones sexually reproduce with each other their offspring should have DNA different from its clonal parents. Even if two identical seedlings had reproduced then Peakall would expect to find variability in the population. The only other possibility was that Wollemia was growing its seed by apomixis—this means that the female cone is able to produce seeds without pollen. But this has never before been observed in any conifer in the world.

Even trees in far direr straits than Wollemia have genetic diversity—the world’s entire population of Allocasuarina portuensis is made up of five sick-looking adult males. This tree is so little known that it does not even have a common name. It grows on the edge of Sydney Harbour and has a multimillion-dollar view straight up the embayment towards the bridge and the Opera House. The five males are all past their prime and will never reproduce sexually again. Efforts are now being made to grow the tree from seeds collected from the females before they died. This may buy some more time for the diminutive scraggly plant that also has a history stretching back to Gondwana, when flowering plants were first evolving.

Peakall’s results were so odd that he requested permission to travel to the canyon in order to determine if there was something that he was missing or not understanding about the population. On 12 June 1998 Peakall arrived on site and was forced to reconsider all of his previous thoughts about the trees. The mystery of the genus deepened further. After he abseiled down the cliff, Peakall expected to see an organism similar to Allocasuarina—struggling to survive. But in the Wollemi wilderness he stood staring at trees that seemed to be flourishing. The vision splendid matched neither his laboratory findings nor his intuition. ‘When I went into the site I thought, “This is not like other rare species which are on the brink of extinction.” I think the Wollemi pine is functioning normally. It’s just highly localised. There is no obvious evidence to suggest that the tree has a serious problem and, if it’s been in that canyon for a long time, the opposite is the case.’

The first individual Wollemia he saw battered his theories even more. ‘It was a strapping young teenager-juvenile probably only a hundred years old and it has come up in the wake of a tree fall,’ Peakall told me. ‘It made me realise that there was at least one seedling that had made it through to adulthood.’ Here was the evidence that young new trees were being recruited to the adult population and that these should produce genetically distinct offspring. Clearly they were not. ‘In every other respect these seem like normal rainforest trees.’

The other big shock of the day was when Peakall flew upstream to the second stand of Wollemi pines. As he hovered above the site that had been found by Haydn Washington he realised that something extraordinary was happening in the astonishing terrain below him and that his original ideas about the Wollemi’s genes needed a radical rethink. The two stands are a mere two kilometres away from each other and in the same canyon system, but for all intents and purposes they might as well be on different continents. Clearly the landscape unfolding under the chopper was so rugged it made any genetic interchange between the two colonies unlikely. Even the possibility of genetic material in the form of stems or roots floating downstream to establish the first site seemed remote.

A month later Peakall wrote to the head of the Wollemi pine conservation team, Bob Conroy. ‘While the distance between the sites is not that great “as the crow flies”, it now seems to me that genetic exchange among the populations via pollen flow is very unlikely given the convoluted pathway required and the density of the intervening vegetation,’ Peakall told Conroy. ‘For similar reasons, while it can never be ruled out, the likelihood that site one has been derived from site two also seems unlikely.’

A new theory was needed to explain Peakall’s results. Wollemi pines, he told Conroy, may not have become genetically identical in the canyon. They must have been extraordinarily genetically similar before climate change forced them in there and this low variability, over thousands of years, has been further exaggerated by cloning in one big stand of pines that had somehow been split in two.

Cathy Offord’s explanation on how the population had been divided was found out the hard way. Her first trip to see Wollemia in December 1995 was led by Jan Allen. It began to rain as the party arrived at the camp-cave Dave Noble had found on his first visit to the canyon. The Wollemi gods decided to put on a show for the scientists—a storm of the likes that maybe strikes once every few years. Wind and rain blasts pummelled the canopy and all hope of seeing the trees on the afternoon her party arrived had to be abandoned. The wilderness had made up its mind to keep these intruders where they were for the evening. The tiny creek outside the camp-cave, normally just a metre wide, surged up 1.5 metres and increased its width five times. As the stream rose the team moved further and further inside the cave. Offord’s colleague, Graeme Errington, watched in awe as in the darkness tree ferns were spotlit by gashing sheets of lightning as if they were made of solid silver. Even bush rats and invertebrates sought shelter under the overhang. The cracking of log jams could be heard through the night and the party’s biggest fear was that a dam would break and water would wash away their camp.

A photograph taken the next morning (below) shows Offord in her sleeping-bag, wedged into the cave, looking as if she had been in a washing machine for a month. The creek subsided as quickly as it rose. The storm, however, had answered two of her most puzzling problems—why had the trees been obliterated elsewhere, and why were the two stands now separated? ‘To ask why it is not in other canyons is to discount the power of nature,’ Offord told me. ‘During that flood it was truly scary. We were trapped. You could get events in those canyons that wash away everything.’

In other words where the pines weren’t burnt away or hammered by rock falls, they were probably scoured out by weather conditions that are exaggerated by funnelling canyons—as if they were growing in a pot being scrubbed clean by steel wool.

Peakall sent off his report to Bob Conroy on 23 July 1998 yet he still had more questions than answers to the riddle of Wollemia. Soon after his visit to the pines I joined him again in his lab to discuss his thoughts. We talked about how, in the case of Wollemi pines, evolutionary theory did not sit comfortably.

‘Maybe we need to think about evolutionary potential in different ways,’ he said in his office, again having a good tug on his beard. He told me about some preliminary data he had obtained showing that other members of the family Araucariaceae had unusually low variability—though all except Wollemia had some. The monkey puzzle trees appear to be the shipwrecked sailors of the plant kingdom, finding themselves in circumstances of extreme hardship but miraculously immune to the genetic demands placed on the rest of life. Norfolk Island pines have lived for millions of years on a shard of Gondwana in the Pacific Ocean and have proven to be one of the toughest coastal conifers on earth. Other Araucariaceae, like those on Fraser Island off the coast of Queensland, have also survived biological isolation that would have destroyed many species. ‘Maybe what is happening here,’ Peakall reflected, ‘is that over a long evolutionary history and despite low diversity these plants have developed an allpurpose genotype.’

Perhaps, he speculated, relics like this are proof that there are other ways of surviving than by gambling on genetic diversity to ensure that certain individuals within a species do not succumb to an unexpected force that cripples other members. ‘Wollemia is likely an exception that disproves a rule,’ Peakall said. ‘The assumption has always been that genetic diversity is good because it is the basis of natural selection. The Wollemi pine might actually prove that in some systems it is possible to have exceptionally low variability and stay reasonably happy.’

Perhaps genetic variability is an asset to some, in particular to life’s newcomers and those expanding into new ecological niches. The variability is like a high-risk investment portfolio—it increases the chance of mutations producing an ecological windfall. The downside is that it also increases the chance of a freak wipeout. Perhaps the old-timers—ancient relic tree families, which have been around since before the advent of flowers and which have experienced just about everything a plant could encounter—are able to take a different approach. To put all their assets into one account but make sure it is a safe investment. This may be why Wollemi pines are thriving and healthy but a mere two score in number.

Whatever crash-tackled the tree—one of the most conservative organisms that life has ever thrown up—must have been bordering on apocalyptic. ‘So serious,’ Peakall told me, ‘the best genetic constitution hasn’t been able to get it out of its canyon. But the flipside is, once it settled down in there, its all-purpose genome has allowed it to do as well as it can. I think there’s a lot of luck in this story. Good luck in that Wollemi pines have had the constitution to hang in there in that canyon and bad luck in that whatever catastrophe drove them down there has left them stuck.’

For 100,000 millennia the atom-sized particles in a Wollemi pine’s DNA have been slowly learning a little bit more about surviving. Every particle in that little white glob in the bottom of Peakall’s test tubes is like a reference library containing all the knowledge and wisdom the tree has drawn on to survive.