AUSTRALIAN FRONTIERS OF SCIENCE, 2003
Canberra, 31 July to 1 August 2003
Molecular ecology
meets conservation biology Australian mammal case studies
by Dr Andrea Taylor
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Andrea Taylor is a Logan Research Fellow in the School of Biological Sciences at Monash University, where she completed her undergraduate degree in 1986. Her PhD on molecular genetics in conservation management of a highly endangered wombat species, was from the University of NSW. This followed a research assistantship on koala conservation genetics at La Trobe University, and a 4-month research visit to the National Cancer Institute in Maryland, USA. As part of her PhD studies she spent an extremely valuable 18 months at the Zoological Society of London's Institute of Zoology. Her postdoctoral research at Macquarie University involved population genetics of marsupials introduced to New Zealand. Her research focuses on applying molecular genetic and analytical tools to issues in conservation management of Australia's faunal populations, and has resulted in almost 50 publications. She is a member of the editorial board of the UK journal, Animal Conservation. |
I am going to be talking about animals rather than plants, and I guess I don't really need to convince you all of how precious and wonderful and unique our native wildlife species are. I possibly also don't need to tell you how much in trouble they continue to be 20 or so of our species have become extinct since European settlement, and many are still in decline. We have quite a number of highly endangered species. And, because of the relatively small number of biologists in Australia studying these animals, most of them are going extinct before we even know very much about them, let alone how to save them.
We have limited time and resources. Most of the mammal data that we have today is based on long-term PhD studies, where people just disappeared into the bush for 10 years and came back and told us something about the ecology of an organism. We don't have that luxury any more, in terms either of money or of time, not the least because PhDs now have to be out in three years. So we need rapid methods for getting some basic information on a lot of these species and, hopefully, using that basic information to see if we can aid in their conservation.
Andrew Young has already touched on genetic markers; I just thought I would mention a couple of the very familiar ones, eye colour and blood group, which carry very little resolution. They don't tell us a lot about what is going on in populations because they don't carry a lot of information. So we use DNA, which is much more precise.
The strength of DNA markers is that they tell a story of the history of individuals and populations, which you can then tease out by using a DNA marker with the appropriate level of resolution. So you can tell something about the parents of an organism, you can tell perhaps what population it came from and also what species it belongs to.
These markers inform across a broad range of scales. Important recent advances mean that they are a rapid, practical and inexpensive method of collecting lots of data on organisms. And, importantly, one-off samples are highly informative. We can go to a population, gather up or trap 30 animals a few of each sex and we don't need to continually go back to that population and keep trying to keep track of individuals to see what they are doing. We can just get the one-off samples and work on those.
Finally, it is an important message that all individuals have DNA but few have eartags. So again we can always find out something about any individual without having trapped and tagged it before.
Click on image for a larger version of figure 1
Here are the case studies I am going to talk about today (figure 1). I am not going to go on too much more in the abstract; I think it is best to illustrate these points with case studies. I have used microsatellite markers, which again Andrew conveniently introduced. The sampling for all this work was done on a single timeslice make a single visit to a population, gather up 30 samples so most of the time we are clearly looking at partial samples of the population. We are not sampling every single individual in a population.
Most of the effort in obtaining the data I am going to show you is, therefore, in the lab; there is very little field effort involved. And it is often done by students some very hardworking students I have had in the past, who have contributed to the work I talk about today, are Matthew Sloane, Sam Banks and Faith Walker.
The first species I will talk about is the northern hairy-nosed wombat, which is perhaps Australia's most endangered mammal. It lives in a single population in Epping Forest National Park, in Central Queensland. Being a single population, it is highly vulnerable to extinction. One of the primary management goals we have is to create a second population, by translocating animals from the first. This is considered the only way to take the focus of possible extinction off this single population, so spreading the risk. But in order to do that we need a really good method, first of all, of deciding how many animals there are currently in Epping Forest, and also a good monitoring tool for monitoring the population trends in this population after we remove animals from it, and also at the new site. We need to be able to monitor numbers of animals quite closely to assess how well we are going with our translocation.
Click on image for a larger version of figure 2
Traditionally, this has been done with tracking. These traps are absolutely huge; you need two people to carry them. So it is an incredibly labour-intensive thing to do, to trap these animals. Therefore, it is expensive. It is highly invasive to both the wombats and their habitat, and it can be harmful to animals as well (figure 2).
It does also lead us to expect that there is a sex bias in the population. We trap many more males than females. But this sort of bias is often seen in trapping surveys, and often it does not reflect what is happening in the real population; it may reflect trap-shyness on the part of females, for example. So you are just trapping more males because females won't be trapped. So we do not know whether there really is a male-biased sex ratio in this population. Also, the trapping gives very inaccurate data, as shown on the next slide.
The last trapping survey was done in 1993, coming up with a most likely estimate of 65 animals but with very, very large confidence intervals. So there was a great deal of uncertainty in this estimate.
Click on image for a larger version of figure 3
What we have done is to develop a method of DNA profiling from remotely collected hairs from wombats, to identify individuals and therefore basically count the number of animals in the population. Hairs are very easily collected on tapes strung across burrow entrances. They contain loads of DNA; I am sure that you can see these very large follicles (figure 3). They contain lots and lots of DNA and a single hair provides enough to be able to determine which individual donated that hair sample.
You can then do a microsatellite assay and at the same time determine what sex the animal is, by using Y chromosome markers. So the lanes in which there is a band at the bottom are, obviously, from a male animal, and the ones with no bands are females. All lines have a microsatellite product though, acting as an internal amplification control.
Finally, the important point is that because we have got good maps of where every single active burrow is, we can simultaneously 'trap' the entire population, all 120 active burrows, just by going out and putting tapes over burrow entrances.
Click on image for a larger version of figure 4
So that is what we did in 2000, when we did the first full remote census of this species, by sampling every night for 7 nights. The first thing you notice from the hair-based estimate is that we have hugely increased the accuracy over what is possible with trapping (figure 4). The 95 per cent confidence intervals are much, much narrower. The most likely number that we came up with is 113 animals, so we think that we have detected a true increase in the population size, which is very heartening. We think it relates back to good rain years, the breaking of the drought, leading up to when we did the census. And we also confirmed male bias. There really is a male bias in the population; that wasn't a trap artefact.
At the same time as we were collecting hairs for the census, we made the very tragic discovery of seven carcasses of northern hairy-nosed wombats 5-10 per cent of the species. So what on earth had happened?
It was very obvious from the signs around the carcases that they had been predated, but we did not know who by. One important piece of missing information was who were the victims although all of the animals have ear tattoos, the tattoos were not readable on the carcases, so we needed to do a DNA profile on the animals to work out who they are. We need to be able to trace the fate of every animal in the population if possible, but also we wanted to know whether animals might be more vulnerable to predation by virtue of either their sex or their age, perhaps.
We also wanted to know who were the predators, because this is going to have a very big impact on the type of control measures that you use to remove the threat to the wombats. Clearly the predators were dogs, dingoes or hybrids, but we could not tell which, just by the signs that we found in the field.
So we compared DNA profiles of these carcases against those of 81 known wombats that had been trapped. Very sadly, Female 82 was amongst the victims. She was quite an old female, and she actually was one of the known breeders in the population, so that was very sad. Two known males [Male 10 and Male 102] as well were found amongst the dead. The other four carcasses were from unknown animals. They were all male, and based on their skull measurements they ranged in age from less than two years up to greater than three years old.
So we know that both adults and juveniles were killed. We had never considered dingoes or dogs to be a significant threat to wombats, because we had never thought that a hulking great wombat could be killed by a dingo. We needed some more information about the predation event, obviously.
There is a bit of a male bias here, in that most of the animals killed were males. But that is not significantly different from the sex ratio in the population, so it is not suggesting that dingoes or dogs are selectively predating males.
As far as the identify of the culprits go, we got the scats that we found round the murder site I think we had about four scats and we did DNA profiles on those. Using some background data from Alan Wilton, we were able to show that the test data or the profiles from the scats found at Epping Forest cluster here very nicely with the genotypes or the DNA profiles from pure-bred dingoes. So we are not looking at hybrids and we are not looking at dogs; we are talking about pure-bred dingoes being the culprits who were responsible for the predation.
So we know that the predators were pure dingoes. We know that they did actually eat the wombats, because one of the scats, as well as containing DNA that identified the culprit, also contained remains of Male 10. So clearly these animals were eating the wombat. We also were able to show that at least three individuals were involved, and likely got together and tracked down and killed these wombats. That actually goes some way to explaining why these large animals could be killed. It is known that dingoes will hunt as a pack if they want to bring down a kangaroo, for example, and I guess that's what they did: they got together and worked out the best way of killing wombats.
Fortunately, we have now constructed a dingo-proof fence around the entire National Park, so we are hoping that we have knocked that problem on the head. Of course, we still face many other problems in the conservation of the species.
Moving on now to a different biological issue: this is the issue of dispersal, a highly intractable issue in mammal populations. It is a very important one in terms of population dynamics, but we know very little about dispersal in the majority of mammal species, and particularly little in cryptic species like this. We are talking about a long-lived burrowing animal. We don't know when it disperses, we don't know how long we have to track individuals for to find out, to try to detect dispersal events by tracking the animals. So again we have looked at genetic methods of doing this.
We have gone into a big southern hairy-nosed wombat population and trapped a bunch of females and a bunch of males; we have done DNA profiling on those and used the DNA profiles to calculate relatedness between every pair of individuals. When you group together the relatedness values for all pairs of females, you get something around 0.15, and when you group together all the relatedness values for pairs of males you get around 0.3. So, clearly, groups of males living in a population are much more related to one another than are groups of females. This suggests very strongly that females are the dispersing sex. Females are moving away from where they were born and they are ending up in populations or areas where they are not related to the other females there. So this gives us a really nice indication, just from a very simple genetic assay, that in this species dispersal is by females. This is quite a find, because the great majority of the other mammal species that have been looked at are male dispersers, so there is something really interesting going on in wombats.
So we have got a rapid method of identifying sex bias in dispersal, but we also think we have got a very nice method for identifying isolated populations of animals, using this technology. When you look at a small, isolated population of the same species, on Yorke Peninsula, you find there is no difference between males and females. What that is telling us, we propose, is that there are no immigrants of either sex coming into that population. It is an isolated population. When you do the same thing at Epping Forest, you find exactly the same pattern. Isolated populations have a particular signature that is very easy to measure, and that tells us that there is no immigration by either sex. So, at least for species that have sex-biased dispersal, we have a really nice method of working out what constitutes landscape connectivity or fragmentation for that species given that we have the appropriate genetic markers for that species. That is a really nice recent outcome from my lab.
Keeping with that habitat disturbance theme, I now want to just briefly describe some work we have been doing at Tumut with David Lindenmayer. The Tumut pine plantation has been described as 'Islands of bush in a sea of pine'. Basically, what happened is that during the 1930s to 1980s the native vegetation was cleared to make way for pine plantation, leaving behind all these little islands, of various shapes and sizes, of eucalypt vegetation embedded within the plantation.
Given that there are quite a variety of native species marooned within this sea, on these islands, it gives us a really lovely experimental system to compare species in their response to this type of disturbance and fragmentation. This can give an indication of whether it is as we might have predicted from what we knew about each species' biology in advance.
The species I want to talk about today is the greater glider, which we expected beforehand to be particularly sensitive to fragmentation. They have a highly restricted diet they only eat eucalypt leaves which means that they are very habitat-specific. They are not found in the pine plantation because they need to eat eucalypt leaves. This diet also means that they have a very low energy budget. They don't move a lot because they don't get a lot of energy from this crummy food source.
That also would be largely responsible for their very conservative ranging behaviour. They have amongst the smallest home ranges known for an animal of this size. They also show a very high level of home range fidelity. So they die on site when the forest is cleared. When the forest was being cleared for the Tumut pine plantation, Hugh Tyndale-Biscoe showed that the animals would just die on their tree that had been felled, rather than moving into the adjacent bush only metres away.
We expected that they have a very limited dispersal capability. This is based on the fact that dispersal distances are highly correlated with home range size, so in this case we would then expect that to be small. And in fact it has been measured in continuous habitats as maximum dispersal distances of probably 8 km or so.
Without specifically modelling the situation, we expected that greater glider populations would be unlikely to survive in patches, because they would be small, isolated populations with no immigration.
But in fact when the plantation was surveyed, 40 per cent of patches were found to be occupied by greater gliders. So, perhaps populations are not as isolated as we expected. The question here is: are patch populations descended purely from original patch occupants the ones that just remained there when the forest was cleared around or do patches experience immigration? Is this a meta-population system where there is dispersal going on between patches? Maybe a patch population goes extinct but a couple of animals move in from elsewhere, creating this persistent meta-population over time.
Click on image for a larger version of figure 5
One way of looking at that is to look at the genetic distance between populations in the patch systems. If there is not much dispersal between two patches, you expect them to be quite genetically different, because genetic drift will change their allele frequencies over time.
So what we did here is this: the red squares in figure 5 represent the genetic distance values for pairs of patch populations at different distances within the pine plantation, and the black squares represent continuous forest sites at similar distances apart. As you can see, there is quite a difference in the genetic differentiation amongst patch populations versus continuous populations. That is a significant difference. So patch populations are genetically different from one another, which indicates that the pine matrix does inhibit movement, as we expected.
Click on image for a larger version of figure 6
Another way of looking at this is to look at the level of genetic diversity in patches. Again for a very small, isolated population we expected to not have a lot of genetic diversity. And in fact all these 'p' populations they are the patches down one end of figure 6 all of the patch populations do fall at the lower end of the range of allele richness or genetic diversity values. Some of them have quite low diversity indeed. So it does seem that these populations are small and partially isolated.
Another way of using genetics to look at this question is to actually infer dispersal events more directly from the genotypes or the DNA profiles of these animals. So you can look at the likely origin of a particular DNA profile of a given individual by seeing how it would fit in with other patches or other populations in the area. Alternatively, you can actually do parentage analysis and identify parent-offspring pairs, and if you find that a parent is in one patch and an offspring is in another patch, clearly one of those animals has dispersed.
Click on image for a larger version of figure 7
Figure 7 is a map of the pine plantation. The patches in red are the ones that we have sampled and genotyped from. What we find in this particular one I am just going to give you an example of the sort of data we got there was a patch that clearly consisted of animals that had come in from elsewhere. We don't know exactly where, but clearly there was a lot of dispersal into that patch. Just looking at this other little patch up here where the patches are all only less than a kilometre apart, we see reasonably frequent dispersal between them. So animals are moving through the pines.
Finally, we have got the really amazing result of one animal that moved very clearly, as the genetics are virtually 100 per cent clear on this point about 7 km through the pines. She was a sub-adult female, which is a class of animal that is known to disperse in the species. So we have demonstrated a really long-distance dispersal event.
So the effects of the pine plantation on the greater glider population, just to summarise, are clearly to somewhat restrict movement amongst patches we have been able to show that but there is nonetheless some degree of dispersal between patches. Some is long-distance dispersal, and it does seem to be by both sexes. So dispersal between patches may actually rescue these patch populations over time.
In this case I would say we would have to conclude that the known species biology was inadequate for predicting response.
I hope I have shown that molecular ecology is a very powerful tool in conservation biology. There are endless possibilities for the sorts of questions you can answer; I have only been able to show you a very, very small proportion of them here. It is not very clear with animals how fitness and viability are affected by various threatening processes. It is not as easy to measure it as with plants; we don't have seeds and compatibility that we can measure. So there is a missing body of information there. But what we can do is provide missing biological data needed for assessing the prospects of persistence using population viability analysis models, which really do need a lot of the data that genetic analysis can provide.
Finally, I want just to point out that of course, although scientists can provide recommendations, these are ineffective without the public and political will to make these changes happen.
