Digital species: Unravelling the mysteries of kākāpō through genetics

Saturday, 19 January 2019

It all starts with Jane.

A man and a dog found Jane in 1989, among the last of her kind, limping through thick Stewart Island bush like a lonely soldier still waging war long after the battle was lost.

Her discovery coincided with the arrival of an author who was about to take the plight of the kākāpō global. Douglas Adams, known for writing The Hitchhiker's Guide to the Galaxy, was researching a book on endangered species and planned a chapter on kākāpō.

In Last Chance to See, Adams's description of kākāpō – paraphrasing zoologist Mark Carwardine – has become forever associated with the species: 'The world's largest, fattest and least-able-to-fly parrot.'

Jane, the kākāpo whose genome has become the reference for her species.

* Only 151 kākāpō left in NZ after native bird Jimmy dies

* DNA study says there may be 11 kiwi species, ancestors driven apart by glaciers**

Adams donated $1000 to the recovery project, earning him the right to name the latest bird. He chose Jane.

A couple of decades later – in an appropriately futuristic turn, given who named her – Jane was thrust to the technological forefront of conservation science.

Jane's genome has been mapped in painstaking detail, to what geneticists call a 'platinum standard', allowing researchers to comb through the genetic matter that made Jane, Jane.

She became one of the first animals in the world to have her genome sequenced to that level of detail. It was the work of Jason Howard, a researcher at Duke University in the US, whose young daughter had come across kākāpō in a children's book.

After sequencing one kākāpō's genome, the Department of Conservation's (DOC) kākāpō scientist, Dr Andrew Digby, came up with the idea to sequence every kākāpō's genome.

Sirocco the kākāpō became one of the most famous members of his species.

After a long, complicated process - which included collecting blood from every kākāpō, and a crowdfunding campaign to raise money, which was coordinated by the Genetic Rescue Foundation, a non-profit that aims to preserve biodiversity through genetic techniques - it has now been completed.

In the time that lapsed, several kākāpō died, but many more were hatched; What began with a target of 125 genomes has become 172, including all 147 living birds (and several dead ones).

It marks the second time globally an entire species has had its genome mapped - The first, the Spix's macaw, is believed to be extinct in the wild - and has the potential to shake up what we thought we knew about kākāpō.

'We've basically got the set of all genes, of all the individuals, in the entire species,' Digby said. 'It's a massive resource… It's kind of limitless, in a way, what we can find out.'

Few bird species worldwide have attracted as much publicity and research interest as the kākāpō, but it remains frustratingly enigmatic.

Some of the outstanding questions are complicated. Why, for example, are some birds more prone to particular diseases, like the dreaded 'crusty bum', than others? But other questions are simple, and speak to how little we know about the ancient parrot.

Chief among those questions: How long does a kākāpō live for? The answer is likely a very long time, but until now, there has been no way to know for sure.

Around 32 living birds were discovered in the wild as adults - known as 'founders' - meaning their age is unknown.

'We know they're at least 35 or 40 years old, but are they 50? Or 100?' Digby said. 'Are they about to die next year? We've got no idea.'

Some of those kākāpo may well be 100 years old. Perhaps the most famous kākāpo, Richard Henry, was found near Milford Sound in 1975 and died in 2010. He was the only mainland kākāpo to breed, providing precious genetic diversity to a population otherwise entirely drawn from Stewart Island.

Because there had likely been no kākāpō breeding in Fiordland since the early 20th century, it meant Richard Henry would have been middle-aged when discovered. It would put his age between 80 and 100 at the time of his death, but he may have been even older.

Another question concerns the significance of kākāpō in the evolutionary history of birds. Research shows that kākāpō, along with kea and kākā, are in what is called the 'basal clade' of parrots, meaning they were the first to split from the common ancestor all living parrots share.

Of those three species, kākāpō are most distinct - the species has its own genus, of which it is the only member - meaning it is potentially the most ancient parrot species alive today. Its genes could hold the story of how parrots are so capable of mimicking human sounds, and why some (like kea) seem to have superior intelligence to other birds.

Richard Henry, who died in 2010, was the only mainland kākāpō in modern times to successfully breed. He may have been around 100 years old when he died.

The new genetic data has already started to shed light on some mysteries.

Swedish researchers last year compared modern kākāpō genes to older museum specimens, and found a significant decline in genetic diversity over the space of around 200 years.

The researchers said this meant the arrival of Polynesian settlers likely had a limited impact on kākāpō numbers in the South Island; Much of the population decline was associated with European settlers and the predators they brought with them.

It also partly answered another question: Where the Stewart Island kākāpō came from. It had long been thought they were brought there by humans in the 19th century, but the researchers concluded, based on genetic information, at least some kākāpō had likely been there for several thousand years.

They didn't rule out had some had been brought there from the mainland, however: If that was the case, some kākāpō may still hold precious scraps of genetic diversity.

While such questions were interesting, the primary intent of the genome project was to help make more kākāpo, Digby said - and for that purpose, the new data could be invaluable.

One of the cruellest barriers to increasing the kākāpo population is widespread infertility among the population. Only around half of eggs hatch, and only one third result in a chick that fledges.

During the 2016 breeding season, 122 eggs were laid, but only 34 chicks fledged. This level of infertility was likely a consequence of inbreeding, Digby said.

'We have strong reasons to suspect that some of the problems with infertility are genetic, but we don't have enough information.

'If we could do something about that, and make some of those eggs that don't hatch or those infertile eggs into chicks, that would really turn things around.'

Due to the low number of kākāpō, they are intensively managed, to an extent that is rare globally.

During breeding season, each nest has a person camped nearby monitoring what's happening; They weigh the chicks every night to ensure they're growing properly (the adult kākāpō are unbothered by the human presence).

'We manage kākāpō more as individuals, rather than as a species,' Digby said.

Having each bird's genome means researchers can construct a family tree - discover which birds are more closely related than others, and which pairings would lead to better genetic diversity.

Dr Andrew Digby, DOC kākāpō scientist, with kākāpō Blades.

They can't control which kākāpō mate, Digby said. Kākāpō have a lek mating system – more similar to a bird of paradise than a parrot – in which the female birds control the mate selection process.

Male kākāpō meet together in what is called an 'arena', keeping a short distance from each other and often out of sight. While defending their own patch, they try to impress the female kākāpō, which are free to shop around among the males. When mating is complete, male kākāpō play no part in raising chicks.

This system has resulted in genetically poor matches. In some cases, half-siblings have mated, and once, even a mother and son.

There have been attempts to 'override' these matches through artificial insemination, which was last successful in 2009, but will be attempted again this year. Knowing how each kākāpō is related means more genetically preferable matches can be made.

With 2019 gearing up to be a record breaking season - Digby said he'd be 'ecstatic' with around 50 chicks - the prospect of genetics aiding future years is tantalising.

'This is the kind of blue sky, looking ahead work we need to engage in, I think.'

Until now, interpreting the genetic information of kākāpō has been limited to a technique called 'microsatellite genotyping', which produces low-quality data.

If that low-resolution data was like a VHS tape played on a blurry, old television, Jane's platinum standard genome is like a blu-ray played on a 4k, LED screen.

By using Jane's genome as a reference point, it has become easier to assemble the genomes of remaining kākāpō, said Dr Bruce Robertson, a molecular ecologist at the University of Otago.

'This was a major step forward,' he said. 'We're able to take all of the genome fragments and assemble them like a puzzle, scaffolded against Jane's genome, which allows us to put it all together.

'Once we've put them together we can compare them across individuals. You can go to exactly the location where we know a gene is present in other birds and find it in kākāpō.'

Robertson has begun sorting through the reams of data, which has come through in batches. Some genomes have been fully assembled, while others still need to be put together, like a jigsaw puzzle.

Plans for research based on the data are already underway, however. Robertson received a three year grant from the Marsden Fund to study hatching failure - Specifically, he will be comparing the genes of dead embryos to live birds, to find if there's a genetic difference.

'We'll be able to look at a dead embryo in exactly the same position in the DNA sequence as we would in a live bird, and see if there's any genetic difference between them,' he said.

'If you can crack why that is, and put in place management strategies to resolve that, then you're increasing the probabilities that eggs will hatch so if you have an egg, you have a kākāpō, which in conservation is what you want.'

It's just the beginning for what can be discovered through genetic research. A long way down the line, techniques such as CRISPR could be used to recreate the genetic diversity of the museum specimens in modern kākāpō.

Associate Professor Bruce Robertson of Otago University has begun sorting through the reams of data from the kākāpō.

The genome data is available upon request for non-commercial use, allowing scientists across various disciplines to use it for their own research.

A few years ago, when Robertson was asked to provide kākāpō DNA to the US researchers for the genome project, he chose Jane.

It was partly due to practical considerations - they had recent samples of Jane's blood - but also because she was the only female founder unable to breed.

'I thought it was the perfect way for Jane to make a really important contribution to kākāpō - to her species, basically.'

When Jane was found dead on Anchor Island last year, her genome was just about to be completed.

A few months later, the Vertebrates Genomes Project - an international effort to assemble the genomes of all 66,000 vertebrate species - released its first 15 'platinum genomes', including Jane's.

The project was dedicated to her - the first immortal kākāpō, who will live forever, digitally.