Presented by Dr Matthew Lott

Research Assistant, Australian Centre for Wildlife Genomics, Australian Museum

I thought it might be useful to begin by just providing a little bit of context for this research. Now, an increasing number of experts are suggesting that we've entered a new geological epoch that they've dubbed the Anthropocene, which is basically a post Holocene Epoch, defined by increasingly significant human mediated impacts on geological biotic and climatic planetary processes. And unfortunately, some of the major hallmarks of the Anthropocene appear to be rapid climate change, widespread habitat loss and globally declining biodiversity. And among the species that are expected to be hardest hit by these accelerating human mediated environmental challenges in the near future are specialist folivores, like the koala.

Now, I often feel that as scientists were sort of forced to walk this really precarious tightrope where, on the one hand, you definitely don't want to give in to cynicism. But on the other hand, all the empirical data that we've collected to date does paint a pretty bleak picture of the future. And we can't really afford to shy away from that either. For example, how many of you knew that the destruction of core koala habitat actually increased after the species was listed as vulnerable in 2012? So, in New South Wales alone, koala habitat destruction increased by about 32% from an average annual loss of around 11,000 hectares between 2004 and 2012, jumping up to an annual loss of around 15,000 hectares between 2012 and 2017. So, if the recent decision to reclassify koalas as endangered doesn't exactly fill you with optimism, you are kind of right to be sceptical. On top of that, in 2017, the New South Wales coalition government scrapped three key pieces of legislation that were specifically aimed at protecting native vegetation and wildlife and replace them all with the Biodiversity Conservation Act. So, this new legislation essentially allows landholders to decide unilaterally whether or not tree clearing on their properties requires prior approval. And, spoiler alert, most of the time they don't think that it does. And what that means is that most rural and forestry lands, which by some estimates account for over 65% of remaining koala habitat, are now effectively beyond the reach of any enforceable conservation policies.

And I know some people feel that koalas kind of occupy too much of legislators time. But it's also worth remembering that a lot of our other endemic flora and fauna also kind of require this habitat. So, it's a bit of a problem across the board, not just for koalas. So, because there's unfortunately no reason to suspect that the loss of native habitat is really going to do anything but accelerate in the near future. There's growing consensus among stakeholders in government, academia and conservation that we really need to find a way to get out ahead of this. We can't always be on the backfoot reacting, we need to come up with a way of prioritising conservation targets ahead of time, so that we can allocate our often very limited resources in a way that they'll do the most good. And one way we can do that is by using genetics. Sorry about that.

Okay, so let's start really basic. What is DNA? Well, in a nutshell, DNA is basically just a large molecule, which is composed of two long chains of smaller molecules, which are known as nucleotides, that coil around each other to form that distinctive double helix shape that you're probably all familiar with. And as I'm sure you're all aware, DNA carries the genetic instructions for the development, functioning, growth and reproduction of all known organisms, with the possible exception of some viruses, depending on where you come down on them being living organisms or not. But DNA is actually a lot more than just a blueprint for making a plant or an animal. It's also a record of how that organism has changed over time. And by comparing the DNA of different species or even different populations within the same species, we can start to gain a bit of an understanding of how those groups have responded to environmental conditions in the past, and even make predictions about how they might respond to future challenges. So, by studying the DNA of koalas, we can identify, for example, the genetic boundaries between discrete populations, we can determine the approximate time periods at which genetically distinct populations diverged. We can estimate the level of migration between geographically proximate populations. And we might even be able to identify some of the environmental or anthropogenic selection pressures that may be driving the emergence of genetic variation in isolated populations. And those are some of the findings that I'm going to be walking you through today.

Before I do though, there's maybe one other part of this equation that we need to talk about which is the importance of natural history collections for wildlife conservation. Now, rapid advancements in high throughput next generation sequencing technologies are allowing researchers to retrieve genomic information from museum specimens, so things like old skins or residual tissue and skeletal elements, which can then not only be used to establish the historical boundaries between genetically distinct populations or lineages in areas where a species may no longer exist, but they can also be used to directly assess recent genetic responses to climate change and habitat loss over time. But historical specimens aren't actually the only way that museums can contribute to threatened species conservation. For example, the Australian Museum specifically is the custodian of one of the largest, if not the largest koala frozen tissue collections in the world. And we regularly receive tissue donations from veterinarians, wildlife carers and researchers from across the country, which are then stored here using our specialised infrastructure for use in future research projects.

So, you can sort of see by working at the Australian Museum, we were uniquely positioned to access a range of different sample types that then allowed us to address some really interesting questions about koala biology and ecology that are going to be foundational for developing more effective conservation strategies. So, in this study, we sequenced the protein coding gene regions, or exons of around 260 koala specimens that represented a mix of both contemporary and historical samples. So, these samples were sourced from around 92 locations that spanned the modern distribution of the koala across eastern and southern Australia, making it sort of the most comprehensive survey of koala genetics to date. And we included specimens from a range of areas where koalas are now recognised to be locally extinct, such as in Northern Sydney. So, I'm not going to spend too much time on our methods.

But just to give you a broad overview of what the process looked like, the first thing we did was compile a list of exons using the Tasmanian devil reference genome. And the reason we use the devil genome for this is that it was actually the best quality genome available for an Australian marsupial at the time, apologies if you can hear the trolleys in the background there. It was actually the best reference genome available at the time for an Australian marsupial because the koala genome hadn't actually been published yet. We then tried to find the same exons in seven other marsupial's, including the koala, using a combination of genome and transcriptome data depending on what was actually available at the time for each species. After that, we imposed a number of different selection criteria to decide whether an exon should be included in the study, and a total of about of 2167 exons met our selection criteria, and were subsequently kept as targets. We then designed and synthesised what's called a sequence capture library to separate out those 2100 odd exons that were identified as targets from the rest of the koalas genome. We prepare genomic libraries for each of the 260 quality samples. And then the individually index libraries were pooled and hybridised against the SeqCap EZ probes from the previous step. And then finally, DNA sequencing was performed and genetic markers called SNPs or single nucleotide polymorphisms were identified using various bioinformatics pipelines that I'll spare you the gory details off, and it was these SNPs that we used after several additional filtering steps for all of our downstream analysis. So hopefully, I didn't just overload you with too much information there.

Okay, so,

on a continental scale, both model based and multivariate clustering procedures supported the existence of five major genetic clusters or lineages of koalas. And what's more, most of these major genetic clusters appear to be separated by well-known Plio-Pleistocene biogeographic barriers, a geographic barrier here, meaning a feature of the landscape that acts as an impediment to the movement of animals. And when you examine them together, you do find several commonalities across these biogeographic barriers. For the most part, they’re lower elevation zones of kind of dry, warm, open woodland or grassland, that you would expect to provide suboptimal habitat for forest adapted species like the koala, and would therefore presumably be pretty difficult for them to move across. So, the most significant biogeographic barriers in koalas appear to be the Sydney basin, the Clarence river corridor and either the Brisbane Valley or the St. Lawrence gap in Queensland. It's a little hard to tell at present because there's a bit of a sampling gap through Central Queensland. So, in the past the Hunter Valley has been speculated to be a pretty significant biogeographic barrier for koalas, but with the inclusion of our historical samples from Sydney, we can now see that it really doesn't seem to be.

Okay, so there's one other thing that I wanted to quickly draw your attention to. And that's the…part of what makes koala conservation so tricky is that these animals are subject to wildly different legislation depending on what part of the country they're in. And as I alluded to earlier, koalas have been declared endangered under the EBPC act in Queensland, New South Wales and the ACT. But populations in Victoria and South Australia are currently excluded from that listing. Now in New South Wales, specifically, a number of different management frameworks have kind of come and gone over the years. And at one point, there were seven koala management areas, or KMAs, which were created to facilitate conservation work. And the idea was kind of that these KMAs were going to provide regional divisions across New South Wales, which would be partly based on the distribution of preferred koala tree species and partly on local council boundaries to make the management of resources easier. But recently, these KMAs have been replaced by 48 ARKS or areas of regional koala significance. And they were identified using, and I quote, analysis of koala observation densities followed by spatial filtering of non-habitat features incorporating barrier information where available, whatever that means.

Now, that's all well and good, but what you'll notice is that neither existing jurisdictional boundary, so the state borders or management units, so either the ARKS or the KMAs actually reflect contemporary koala population structure. Which means that, at the bare minimum, the movement of koalas between management units that represent the same major genetic clusters needs to be encouraged in order to ensure that established management frameworks don't artificially restrict gene flow between regions and populations that were previously interconnected. With the proviso, of course that appropriate precautions also need to be taken to minimise the risk of negative non genetic effects like disease transfer. Okay, so if this is how genetic diversity is currently distributed among koalas, I guess the next logical question would be what sequence of events may have led to this situation? And to investigate that question, I used something called approximate Bayesian computation or ABC analysis. So, in a nutshell, that little stick figure there is a graphical representation of a mathematical model which best describes the sequence of events, which led to the distribution of genetic diversity that we currently observe in koalas. And it seems like that what happened was around 300,000 years ago, if you assume an average koala generation time of about seven years, the southern Australia, Mid-Coast New South Wales and Queensland genetic clusters, all emerged more or less simultaneously from a hypothetical ancestral population. And while you do need to take precise divergence times with a bit of a grain of salt, these data strongly suggest that the demographic history of modern koalas has been heavily influenced by the mid-Brunhes transition, which was a major climatic reorganisation that occurred around 430 to 300,000 years ago, which is believed to have increased the severity of glacial interglacial cycles and drove progressive aridity across much of the Australian continent.

So, the more pronounced climatic fluctuations of the middle to late Pleistocene are in turn hypothesised to have caused cyclical range expansions and contractions in many taxa, which you would expect to present now as divergent, geographically discrete lineages as predicted by refugia hypothesis, and that is what we seem to see in koalas. So, what our data is suggesting is that at least three major refugia were exploited by koalas during the middle Pleistocene glacial cycles and that the foundation of both the southeast Queensland and South Coast New South Wales genetic clusters, were driven by secondary contact between these previously isolated genetic clusters, likely during periods of mesic, forest expansion that would have occurred during the warmer and wetter interglacial periods. So that interpretation is also fairly well supported by independent bioclimatic modelling which shows that at various points in the species recent evolutionary history, for example, during the last glacial maximum, suitable koala habitat retracted to geographically restricted areas of southeast Queensland, northeaster New South Wales and small coastal areas of Western Australia, South Australia and Victoria. What's interesting, though, is that the Last Glacial Maximum was only around 26,000 odd years ago, whereas these genetic clusters appear to be much older than that. So, what that might indicate is that these refugia were exploited koalas across multiple Pleistocene glaciations. And the reason that that's now potentially concerning is that climate modelling indicates suitable koala habitat is expected to undergo an eastward range contraction in response to anthropogenic climate change, similar to what is believed to have occurred during the Last Glacial Maximum. Only this time, a lot of their traditional refugia may no longer be available to them because of extensive human modification to the environment.

Alright, so now we've identified a number of different biogeographic barriers and koalas and we have some understanding of how those barriers developed. It's worth noting that we wouldn't necessarily expect any biogeographic barriers to be absolute, and that it might still be possible for koalas to move across them, however rarely, either now or at some point in their evolutionary past. So, what I did here was use a programme called STRUCTURE to look at genetic admixture or the level of mixing between previously isolated koala lineages. And what STRUCTURE basically does is identify populations from a genetic data set and then calculates the relative contribution of each population to the genome of each individual. So kind of think of it as but for koalas. And, using the New South Wales animals again, as an example, what we see is that there has actually been quite a lot of admixture in koalas, particularly in parapatric or neighbouring populations, which means that from a management standpoint, we probably don't need to be too precious about moving them around, provided that we're not you know, translocating them into different completely different climates or anything like that. One other potentially interesting thing that I'll quickly point out though, is that genotypes that were inconsistent with the Mid-Coast New South Wales genetic cluster were observed in two individuals from the now extinct Northern Sydney koala population. So, specimens from Palm Beach and Avalon were assigned to the Queensland and Southern Australian genetic clusters respectively. And that strongly suggests that koala colonies endemic to Northern Sydney were at some point, supplemented with captive-source individuals from other areas, possibly an attempt to reverse the rapid population declines of the mid to late 20th century.

Okay, so now we know that koalas can sometimes cross established biogeographic barriers, but what would be useful to know is how often that currently happens. And to answer that question, we investigated contemporary migration patterns across putative biogeographic barriers using a Bayesian statistical framework implemented in the very unfortunately named programme BayesAss. So, our analysis yielded consistently low estimates of contemporary gene flow across putative biogeographic barriers, but with a couple of notable exceptions. So, the largest proportion of migrants appeared to be the result of asymmetrical westward dispersal across the Great Dividing Range from coastal New South Wales. Now, that's potentially interesting for a couple of reasons. Firstly, it suggests that a source sink dynamic may exist across the Great Dividing Range in koalas, meaning that more densely populated eastern coastal regions are disproportionately contributing immigrants to more sparsely populated regions in the West. Secondly, it's well documented that a number of previously large and stable Western koala populations have declined dramatically in recent years with further habitat loss and associated local extinction events predicted under anthropogenic climate change. Which means that we potentially have a net movement of koalas into heavily modified sub-optimal habitat, which you have to imagine is not going to be great for the long term persistence of the species. And thirdly, this kind of seems to indicate that there is no real reproductive isolation or resistance to genetic introgression at locally adapted loci between Western and coastal koala populations.

Now the reason that that's potentially important is that populations to the west of the Great Dividing Range are often assumed to harbour unique adaptations to drought and heat waves, which has led in the past to suggestions that assist a gene flow between western and eastern koala populations might help mitigate maladaptation to anthropogenic climate change through processes such as adaptive introgression. Now personally, I've never really bought into that idea. Firstly, there's not even any real evidence that Western koala populations are actually adapted to tolerate droughts or heat waves. And there's actually quite a bit of evidence to the contrary. For example, when southeast Queensland experienced a drought in the early 2000s, the koala populations there declined by about 80% They just straight up all died, which doesn't suggest that they're terribly well adapted to dealing with drought or heatwaves. So that suggests to me that the ability of koalas to survive west of the Great Dividing Range may be more down to in situ behavioural adaptations rather than intrinsic genetic ones. For example, behavioural behavioural thermoregulation, which is adopting postures or seeking out microclimates which age the regulation of body temperature is a very well documented phenomenon in koalas and might have more to do with their ability to survive in arid or semi-arid Western environments. All right, so we've spoken now a little bit about genetic diversity in koalas, but I think it might be worth taking just a little bit more time to discuss how genetic diversity is actually distributed across contemporary populations. And what we sort of want to know is that we've shown that significant genetic structuring exists in koalas, but what would be good to know is how much diversity that structure actually represents.

So, to answer that question, I used an AMOVA or Analysis of Molecular Variance. And interestingly, what we find is that the amount of diversity represented by the major genetic clusters that we identified earlier is actually relatively minor. And that the vast majority of existing genetic diversity in koalas is found at the level of individual animals. Which means that from a management perspective, you really kind of need to think of the individual koalas as the primary reservoirs of genetic diversity for the species. And that's arguably what we need to focus on protecting rather than arbitrary populations or management units. Because it kind of appears that the loss of even a single koala represents a potentially significant loss of genetic diversity for the species. And that's going to require a pretty substantial shift in current management paradigms, which increasingly seem to be focused on identifying and protecting what people are calling high value koala populations, which a lot of us are kind of worried might just end up becoming code for koala populations in areas with low land values.

So, one other thing that I'll quickly point out is that coloured graph on the bottom right there, which highlights the different levels of genetic diversity that we observed in the five major clusters of koalas. And basically what we found was that contemporary levels of genome wide diversity were not significantly different between parapatric or neighbouring koala genetic clusters. But that on a continental scale, a clear geographical pattern of genetic structure emerged in which overall genetic diversity declined with increasing latitude. Now, one possible explanation for that, that pattern is that Southern koalas meaning those in Victoria and South Australia, are known to have experienced extensive and repeated population bottlenecks as a result of over hunting, of translocating small numbers of individuals and population crashes caused by population explosion and over browsing, which could now be reflected in the genetics of this cluster. In contrast, it's generally believed that the more northern koala populations in Queensland and New South Wales largely escaped similarly severe bottlenecks despite heavy hunting following European occupation.

So, our data broadly do seem to support this assumption as analyses incorporating historical museum specimens didn't really provide any evidence of significant declines in genetic diversity within these northern koala populations over the last 120 odd years. There is one other possibility though, which is that an older bottleneck event actually underlies the negative relationship between latitude and genetic diversity that we observed in our extant koala clusters. So as I mentioned earlier, there's a growing body of evidence which suggests that koala populations experienced severe continent wide demographic declines through the last glacial maximum. And climatic refugia are consequently believed to have played a pretty significant role in the species’ survival into the Holocene. So, if a greater amount of core koala habitat persisted in areas like Southeast Queensland and coastal New South Wales during the last glacial maximum, then that could have supported larger and more stable koala populations which may have survived in these areas and experienced less severe demographic bottlenecks than their conspecifics in the south.

So, it's just another theory we're working with, another hypothesis, I should say. Okay, so the last thing I wanted to talk about is what has been termed neutral and functional genetic diversity and what its implications are for koala conservation, or if there even are any implications for koala conservation. So the growing accessibility of genome scale data sets for non-model organisms has led to suggestions that greater emphasis needs to be placed on the maintenance of genetic variation that is thought to directly affect fitness related traits as opposed to more traditional approaches which prioritise conserving genome wide genetic diversity. So, one example of potentially functional genetic diversity would be what's called Bergmann's rule, which basically states that within broadly distributed taxa, populations and species of larger size are typically found in colder environments, while populations and species of smaller size are generally found in warmer environments. Basically, because a larger body retains heat better than a smaller one because of the relatively small surface area to volume ratio. And the genetic diversity that underlies those size differences could be termed functional genetic diversity because it directly affects a fitness related trait. Now that sounds great in theory, but in practice, the elements of genetic diversity which are considered adaptive often vary significantly across time and space. And selection pressures are subjected to continual fluctuations. And what that kind of means is that variation, which is putatively neutral at present may end up proving critical for fuelling adaptation to unpredictable future conditions and vice versa. Genetic diversity that is currently adaptive may not actually prepare animals to deal with unpredictable future conditions. So, I'm not really convinced that identifying putatively functional gene diversity really has any implications for koala conservation, per se, but it's still a potentially interesting research question.

So, to get at that question, I used a number of different gene environment association methods to identify putatively adaptive alleles in koalas. And not surprisingly on a continental scale genetic structure and koalas was primarily attributed to random genetic drift and geographically restricted gene flow for all the reasons that we've already discussed. But we also did detect signatures of polygenic climatic adaptation, so a total of 75 genes were putatively linked to local adaptation in koalas. So, temperature related environmental predictors appeared to be the most important drivers of putatively adaptive regional genetic variation, and they accounted for about 35% of loci that showed signatures of selection. So, this demonstrates that a suite of complex molecular mechanisms are likely to underlie thermal tolerance in koalas. Now, it's also well known that body size in the modern koala represents a latitudinal morphocline. So adult individuals within southern high latitude populations are significantly larger on average than their conspecifics from low latitudes in the north. Again in accordance with Bergman's rule.

As you can see from this picture here, the southern koala is the bigger one on the left. So we identified several exons, linked to adipogenesis, or the formation of fat cells. So that's the frizelled class receptor 4 protein and the RAB 11 family-interacting protein 1, in addition to a number of genes associated more generally with the regulation of cell growth, differentiation, and cell death or apoptosis. And because all of those processes are intimately linked to embryonic and postnatal development, it's possible that these loci represent components of the genetic pathways which drive climate-based size variation across the koala’s range. Now, interestingly, exon is associated with both innate immune function. So complement C complement C1q chain and hemostasis - coagulation factor x - so hemostasis, meaning blood clotting, that kind of thing, we're also found to be linked to temperature dependent selection. Now both hyperthermia and hyperthermia are associated with the impairment of immune function and coagulopathy. So basically, failure of the coagulation systems in your blood. And as marsupials typically display a much greater daily range of body temperature than eutherian mammals, it's highly likely that environmental temperatures could exert a proportionately greater influence on these physiological processes and could therefore create strong regional selection pressures.

Now again, it's worth stressing that linking polygenic selection to fitness traits is extremely difficult without observing associated changes in phenotype, which requires massive data sets that don't actually exist for most wild populations, and most likely never will. But at the very least, we've identified a bunch of genes of interest that could be the targets of future research that collects phenotypic and gene expression data. Just going to quickly finish up by pointing out that this research has spun off a number of ongoing collaborations with various researchers both across Australia and internationally. So it's kind of a good example of how at times koalas can bring the research community together. And with that, I'd like to thank you very much for the opportunity to present our research.

Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species’ capacity to adapt in the face of extreme selective pressures. Researchers at the Australian Museum have therefore used a high-throughput next-generation sequencing-based approach to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range.

In a recently published study, an examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. There was strong evidence for repeated, climate-associated range contractions and demographic bottleneck events - this suggests that geographically isolated locations may have played a more significant role in the survival of the koala through the Pleistocene period.

Consequently, genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of vulnerable populations to accelerating threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g., guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.