Koalas are one of Australia's greatest assets; not only a magnificent marsupial (not to mention really cute) they also draw in over three billion dollars to the Australian economy. Sadly, Koala numbers are rapidly diminishing - now deemed a vulnerable species, their habitats are being destroyed by urbanisation, and their populations challenged by chlamydia and Koala retrovirus (KoRV).
As part of a multidisciplinary team using molecular biology and population genetics to help protect the Koala, the Earlham Institute was instrumental in assembling and analysing the first complete genome sequence of the Koala. EI researchers used their bioinformatics expertise to uncover novel insights into Marsupial evolution and biology, including dietary and immune adaptations.
We speak to Dr Will Nash and Dr Graham Etherington to find out their story behind the paper - how the conservation study has impacted the new marsupial genome reference and its evolutionary findings.
How did the international project come to fruition – what was your motivation?
GE: We already had collaborations and many interests in common with researchers in Australia so when the opportunity arose to join the Koala Genome Consortium I was very keen to provide my expertise to the group. The aims of the project closely crossed with my interests in genome assembly and conservation genomics, so it was an easy decision to make.
WN: I was particularly interested in the work because of its potential for evolutionary insights. As the marsupials diverged from other mammals so long ago (160 million years ago) there is lots of time for genomic differences to occur, and lots of cool traits to link them to (eg, koala eucalyptus diet adaptation).
What role did EI play in exploring the species’ genome landscape?
WN: With Dr Wilfried Haerty at EI, we used the high quality reference sequence assembled by the consortium and we looked at how genes had duplicated through copying errors; these copied genes that share the same or very similar functions form gene families. Variations in gene families size are one major source for adaptive variation during evolution.
We compared the koala genome to the genomes of three other marsupials (Wallaby, Tasmanian Devil, Opossum), Eutherian mammals (human, mouse, dog), and ancient species (Platypus, Chicken). This enabled us to first annotate gene families by looking at similarity between the proteins created by all the genes in each genome. After identification of the gene families, we could then compare to see which were bigger in the Koala than in other species, and then look at the function of these families showing signatures of expansion. This is one way to see which genes and associated functions have potential adaptive significance in this enigmatic species.
We also did selection analysis on genes which had single copies in all the genomes we were studying. We first aligned these sequences, and then look at how the nucleotide and resulting protein sequence had changed at individual positions. From these alignments, we inferred the rate of change in nucleotides, and compared it to a theoretical model of change which is random (neutral model). Significant differences between the observed and expected random changes likely reflect that part of the sequence is important as evolutionary forces somehow linked to the Koala’s survival shape sequence evolution. We are currently finalising this work and will publish a companion paper on it soon.
What insights did the bioinformatic approaches we use at EI provide to the study?
GE: We were able to use both our advanced genomics facility and high-performance computer cluster at EI to aid in the assembly and annotation of the Koala genome. This allowed us different types of genomic data to improve the assembly and obtain more information about how Koalas have evolved and adapted to their environment over millions of years.
WN: Alongside the excellent work GE mentions above, our expertise in this field shaped one of the main findings in the paper. By using a specialised pipeline to group genes into families, we were able to identify one of the key adaptive changes discovered at this stage in studying the Koala’s genome: the expansion of a set of genes which make proteins to break down dietary toxins. Given that the Koala only eats the foliage of highly toxic eucalypts, this may represent a key change in the genome associated with it’s ecology. We also used this finding as a test case in the development of a new approach to this kind of analysis, one developed inhouse at EI. The GeneSeqToFamily workflow, developed by Anil Thanki, Nicola Soranzo and Robert Davey, aims to make similar findings accessible to the community with great ease.
Sadly, Koala numbers are rapidly diminishing - their habitats are being destroyed by urbanisation.
What’s the predicted impact our role will have for the koala’s survival in the wild?
GE: It is hard to assess the impact of research so soon but the paper addresses the expression of genes involved in disease response, Koala Retrovirus (KoRV) evolution and the evolutionary adaptation to a life in gum trees.
WN: Building from GE’s accurate summary, above, I’d say that the reference genome provides a basis for future conservation efforts and insights. In a worst case scenario, where Koala populations continue to decline, it could help in identifying population differences and inform breeding programmes aimed at strengthening the remaining populations. The genome paper acts as a milestone in the initiation of this process. The high quality data generated allowed the first genome wide population study of Koalas, showing evidence of population fragmentation and different levels of genetic diversity in different subpopulations. Also, many of our collaborators on the project have key importance in the efforts to conserve Australian marsupials. One key example is the work of Kathy Belov, using the genome of the Tasmanian Devil to understand it’s response to the really serious Devil Facial Tumor Disease which ravaged their population. Similar could be done for Koala, if needed.
Why do you think the evolutionary history of koalas is so poorly understood?
WN: I would say that the Marsupials as a whole are understudied, but this is also proportional to their abundance and commercial importance (both low, apart from the Koala’s visibility as a tourist icon). They aren’t crops though. In an era where it is now possible to assemble large and potentially complex non-model organism genomes (conveniently, EI’s speciality) we can start to address glaring omissions like this. For example, exciting projects like the Oz Mammal Genome initiative and the Koala Genome are an excellent foundation to future new findings. It also opens up a huge number of really interesting evolutionary questions, and will make further studies of all mammals more robust.
Did you learn new elements of genomics from this study that you didn’t know before?
GE: Although I currently work on vertebrates, this was my first exposure to marsupials. It has become apparent to me that marsupials are an extremely interesting group of animals and have huge potential for further research.
WN: As this was my first foray into vertebrate genomics, I have learnt a lot about the fascinating biology of Marsupials; about the bioinformatic approaches we used and how to link them together, and also about the dynamics of large scale international collaborations.
What challenges did you encounter with the research?
GE: We needed to transfer frozen koala tissue from Australia to EI, which was challenging! Also, teleconferences were at 11pm as collaborators were in Australia, the UK and the USA.
WN: Learning to use some of the software and/or computational approaches as I was also producing work to directly contribute to the drafts.
What did you enjoy the most about being involved in the paper?
GE: The Australians were great to collaborate with. Working on such an iconic wild species was quite a privilege and has lead to further collaborations on marsupials
WN: I totally agree with GE above, our collaborators are great, and it was always a pleasure to skype and meet them at various conferences (G10K hosted by EI, SMBE2017 in Austin TX). Also having the opportunity to delve deep into the biology of such a cool, distant relative of ours was amazing, and really fuelled my passion for the power of genomic approaches in understanding biological diversity.
How will the koala research help our other studies into endangered marsupials?
GE: It gives a really good genome assembly as a reference to use with further species.
WN: Our work is continuing, we have another manuscript focussed on the koala in the pipeline, but this will also encompass exploring genes which are under selection across all marsupials. As GE suggests, these could provide a starting points from projects in the future.
How would you like to progress our part in the research? What are the next steps for the study and how will EI be involved?
GE: We are extending our interest in marsupial genomics by our involvement in a new project looking to find novel antimicrobial peptides (AMPs) in the genomes of further marsupials.
WN: See above, we are also interested in non-coding evolution in the four genomes we have studied here.
In terms of conservation genomics, what would be the next marsupial that you would like to work on and why?
GE: We are planning on working with four endangered marsupials - Numbat, Bilby, Brush-tailed Bettong and Western Barred Bandicoot. We’ll be looking at the genetic diversity of their immune systems in order to identify genetically diverse groups that we can use to introduce into smaller, more inbred populations.
WN: Purely for the cool and weird factor, I would love to work on the Marsupial moles. Also, they would contribute a unique clade to comparative studies. From an evolutionary point of view, it would also be cool to look at any of the other South American marsupials (we currently only have the Opossum genome), particularly the ‘Monito del monte’ as it is a basal relative of all the marsupials that live in Australia but is still found in South America. It is super rare though, so tissue samples would prove difficult to get hold of.
Evolutionary Genomics expert Dr Wilfried Haerty at EI who worked on the study, said: “The completion of the genome of the koala is a milestone for the study of Mammals genomes evolution and towards the conservation of this emblematic but threatened species. It is the first high-quality genome for a marsupial species, a group of species that has long been overlooked. The analysis of this genome has led to the identification of candidate genes underlying unique traits in Koala (diet), but also in marsupials.”
Study lead Dr Rebecca Johnson, Australian Museum Research Institute, added: “The success of sequencing the Koala genome is already revolutionising the conservation of our koala, through data informed genetic management, while giving us insights into the koala’s highly specialised diet. The expert contributions from the teams at the Earlham Institute were a critical component of this study. I’m so proud of the work this great collaboration has produced and thrilled it will be assisting future koala conservation efforts.”