The Koala Genome

Led by Australian Museum Research Institute Director Dr Rebecca Johnson, a small group of Australian scientists with a passion for koalas gathered together in 2013 to share current knowledge and ideas about koala populations, genetics and diseases. Their collective aim was to steer their research towards ensuring the long-term survival of this important marsupial. They also recognised the importance of increasing Australia’s genome sequencing capability, since no de novo mammal genome had ever been sequenced and assembled by a solely Australian led group. It was from this small group that the Koala Genome Consortium was born.

In 2018 Consortium now comprises of 54 scientists from 29 different research institutions across seven countries. The group have strong links with international and domestic partners and end-users, including wildlife hospitals and governments responsible for managing koala conservation.

This is the first Australian-led big genome project. The Consortium sequenced the genomes of three animals, one using state of the art long-read sequencing technology from Pacific Biosciences (also known as ‘PacBio sequencing’). This technique delivers very long and uninterrupted sequences, and has produced the best quality marsupial genome to date, of comparable quality to the human genome (the human genome project took 13 years and cost US$3billion to complete).

The koala genome is slightly larger than the human genome (3.5Gb v 3.2Gb) and has a similar number of genes. We report 26,558 koala genes, an update on our original discovery of 15,500 Koala genes (Hobbs et al 2014). The long-read genome allowed the consortium to discover many genes that contribute towards the koala’s unique biology.

The Australian Museum’s collection of mammals was formed in the mid-1800s and has grown to become one of the most comprehensive collections in the world. The collection currently contains approximately 48,000 specimens from over 100 different countries.

The Australian Museum Koala collection currently comprises over 290 specimens dating from the late 1870s. There are over 100 skins, 130 skulls and approximately 50 spirit preserved specimens in the collection which are available to researchers both within Australia and overseas. Many of the early specimens are ex captives from Zoos. More recently, specimens are received after they have been found dead on the road or euthanised due to severe injuries or disease

Many specimens in the collection have great historical as well as scientific value having formed the basis of the original description of a particular species when it was first named. There are over 570 of these specimens known as “type” specimens” in the collection including the only known specimens of a number of species.

One of the most important specimens is the “type” specimen of a Koala subspecies from Victoria, Phascolarctos cinereus victor, described in 1935 by the museum’s mammal curator at the time, Ellis Troughton. Other significant specimens in the collection include those donated in the 1950s from the now endangered population on Barrenjoey Peninsula in Sydney’s north.

The Koala collection has formed the basis of a number of recent research projects. DNA extracted from historical specimens from the late 1800s and early 1900s was used to examine the origins and evolution of retroviruses which increase Koala’s susceptibility to infections such as Chlamydia. Specimens used in the Koala genome sequencing project are also lodged in the collection.

The koala is recognised worldwide as an iconic Australian species, and there is anxiety in Australia and internationally that this unique species is declining in the wild.

The koala is of interest to scientists, policy makers and the public because of their biological uniqueness, the high level of attention they receive for conservation, and their attraction to the visitor economy (koala-related tourism estimated at over AUD$1.5 billion per annum).

Koalas are evolutionarily distinct. They are the only living representative of the marsupial family Phascolarctidae, the last of up to 20 species recognised in the fossil record.

Koalas are biologically unique. They can eat highly toxic eucalyptus leaves that would kill most other mammals, but they are picky eaters, so very prone to habitat loss. With increasing threats of predation and habitat loss through urbanisation, koalas are particularly vulnerable to deleterious effects of fragmented habitat and population bottlenecks, and are further threatened by disease, low genetic diversity and climate change. Worryingly, some koala populations are expected to decline across Australia by up to 50% over the next three generations (approx. 20 years), so maintaining healthy populations will require significant conservation intervention measures.

New information from the Koala Genome Project is critical in understanding and communicating the threats that modern populations face.

We use a technique that estimates demographic history over thousands of generations from genome sequence. Looking back >100,000 years, koalas were at their peak population size of 50,000 – 86,000 animals. At around 30-40,000 years ago the population underwent a rapid decrease, then stabilised to about 10% of their previous numbers. This rapid decline coincided with the time that other Australian species, especially the megafauna, underwent widespread extinction, pointing to a significant climatic or habitat change.

Those stable population numbers were again dramatically reduced by European settlement around 200 years ago, when millions of koalas were hunted for their skins, resulting in extinctions and severe bottlenecking in southern Australia. This is the koala population we are now faced with conserving in the current day.

One of the biggest modern threats to koala survival is land clearing. This has led to loss of habitat, and loss of connectivity between habitats. One of the long-lasting outcomes has been reduced genetic diversity and possibly inbreeding. This is complicated by the wide geographic distribution of koalas throughout Australia, covering many bio-regions and four Australian states.

Good news has come from our populations studies using new genome markers. Using genome linked markers, we show, for the first time, that NSW and Queensland populations retain significant levels of genetic diversity and still show long term connectivity across regions. Ensuring this genetic diversity is conserved, is the key to the long term survival of this species, and genetic monitoring must accompany conservation measures to protect habitat, and reduce dog attacks, vehicle strikes and disease.

This work represents the new generation of science based conservation policy as it has been integrated as an important pillar into the NSW Koala Strategy 2018. Sequencing the koala genome represents a paradigm shift for Koala research and conservation efforts.

The genome sequence of the koala tells us more about their immunity and resilience to disease - one of the biggest threats to populations.

The long-term survival of the koala is significantly threatened by disease, so characterizing the immune system of the koala is important for management. Our long-read genome allowed characterisation of koala immune genes, many for the first time. These gene families are notoriously difficult to characterise because there are many copies with similar sequence, but they can be assembled, counted and identified by long read sequencing.

Once these genes were assembled we could compare the ones that were expressed in animals with symptoms of ocular chlamydia with those expressed in the healthy animals. Several of these animals were also involved in a chlamydial vaccine trial, with some showing ‘strong’ or ‘weak’ immune response to the vaccine. The genome has provided the first insights into the genetic basis for this response, and will provide essential information for future successful vaccine development.

New findings on koala reproduction enlighten us further about the uniques and specialised development that takes place in young.

Koalas are induced ovulators, which means they only ovulate in response to mating. Genes known to be involved in male induced ovulation were found in the koala genome (also highly conserved in other induced ovulators such as camels and llamas) suggesting a role for male koalas in inducing ovulation.

Like all marsupials, koalas do most of their development in the pouch. They are born after 34-36 days gestation and spend ~6 months developing before emerging from the pouch. As for other marsupials, the development of koala young depends on a supply of milk that changes in composition during pouch life. Thanks to the high-quality genome, we were able to analyse and discover koala-specific milk proteins that are critical for various stages of development. Excitingly, these proteins seem to have an antimicrobial role, showing activity against a range of bacteria and fungal species, including the strain of Chlamydia pecorum known to cause ocular and reproductive disease in koalas. This would be vital in the early stages of development, when the koala young lack a functional immune system.

What does the koala genome tell us about their interesting taste for low calorie, high toxin plants?

Koalas eat a diet largely of eucalyptus leaves (20 of the >600 species), which have a high level of toxins. The consortium found that koalas have two large expansions in a gene family known to be integral to detoxification, the Cytochrome P450 gene family of metabolic enzymes, which oxidise molecules that can then be excreted as water soluble metabolites. These detox genes are expressed in many koala tissues, especially the liver. The evolution of these extra detox genes is undoubtedly what allowed koalas to become dietary specialists, and thus evade competition from other species which could not detoxify as effectively.

There must have been strong selection for koalas to be able to metabolise this food source. The Consortium found that koala detox genes have experienced diversifying selection (creating more detox genes via expansion through tandem duplicates) and purifying selection (which keeps the genes functioning, presumably because they play a very important role).

Knowledge of the koalas’ detox genes is important for veterinary care of koalas because this gene family is known to metabolise non-steroidal anti-inflammatory drugs (typically used in pain relief) and possibly antibiotics used to treat koala diseases like chlamydia.

In addition to this, Eucalyptus leaves are very low in calories, so koalas typically need to eat 600-800g per day. They are often touted as the most expensive animal to feed in captivity outside of Australia. The koala genome offers us clues to the basis of koala ‘choosiness’.

The consortium found that koalas have an expanded ‘bitter taste’ repertoire of 24 genes, the most of any Australian marsupial. Bitter taste receptors recognise structural toxins (such as terpenes, phenols and glycosides which are found in high levels in Eucalyptus), so this would allow them to optimise their leaf choice by targeting nutrients and minimising toxins.

Koalas derive most of their water from their diet, eating only leaves with more than 55% water content. The genome points to the koala’s ability to ‘water taste’ as it has a duplication in the aquaporin 5 gene thought to be a central component to sense water concentration.

This project was made possible by access to infrastructure provided by Bioplatforms Australia through funding from the Australian Government National Collaborative Research Infrastructure Strategy; access to technology through NCI, Amazon Web Services and Pacific Biosciences. Stakeholders such as Featherdale Wildlife Park, The Koala Hospital in Port Macquarie, and Australia Zoo Wildlife Hospital provided access to live animals; and funding was obtained from various grants including the Australian Museum Foundation, the Australian Research Council, and the NSW Environmental Trust.

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