03/10/2019 in RZSS
Rhinos being difficult: the trials and tribulations of identifying rhino parents
This month we have a guest blog from Tarid Purisotayo, a PhD student who has been working in the RZSS WildGenes lab on a project that aims to inform the conservation of Botswanan Rhinos.
Since 2010, the World Wide Fund for Nature (WWF) announced that September 22nd of every year will be World Rhino Day, for the celebration of all five living rhino species: black, white, greater-one-horn, Sumatran and Javan rhinos. We, a collaboration between the WildGenes Lab at the Royal Zoological Society of Scotland (based in Edinburgh Zoo) and the institute of Biodiversity Animal Health and Comparative Medicine at the University of Glasgow, would like to take this opportunity to share our ongoing white rhino research project. Initially, our primary aim was to construct pedigrees for three southern white rhino (SWR) populations in the Republic of Botswana. However, things were more challenging than planned and we thought it would be good to share our trials as we sought solutions.
To understand what we have been working on, it will help to understand a brief history of rhino conservation. The conservation story of the SWR has been a positive example of successful wildlife conservation but there is still much to do. During the mid and late nineteenth century, southern white rhinos were indiscriminately hunted until the 1890s, when they were cornered at Hluhluwe–Umfolozi, Kwazulu-Natal, South Africa. This became the single remnant population, with less than 100 SWRs. Hluhluwe–Umfolozi park became the oldest proclaimed African national park in 1895, just in time to save the last population of SWRs and with the intensive protection in the park, the population size of the species rapidly increased.
In the 1960s, advances in capturing and anesthetic techniques arrived at a time when the number of rhinos had started to exceed the resources available in the park. Some of the rhinos were initially transferred to the Kruger National Park, hundreds of kilometers north. After this initial success, other former range states continued the translocation story. At this point, the remnant population in the Hluhluwe–Umfolozi park became the founder of most (if not all) re-introduced populations. Since then modern populations of SWRs have been kept in separated and intensively protected areas, often fenced. Apart from the state-protected areas, the emergence of private game ranches across South Africa have contributed most to the increasing populations. This has proved to be an efficient strategy as the population sizes of most re-introduced populations have rapidly increased. This success has led to the overall continental population size of SWR reaching a milestone of 20,000 in 2012, with more translocations now required to continue this rise.
Continued transfers of rhinos between populations have also been used to introduce unrelated rhinos into the populations. This is very important, as the historical bottleneck of the species means that all individuals are going to be closely related and so it is extremely important to avoid inbreeding. The challenge is keeping track of which rhinos are closely related and which rhinos are unrelated (as far as possible). Currently, such information is based on observational pedigrees. Rangers patrol a protected area to prevent poaching, but they also record the behavioural relationships between rhinos e.g. which rhino is the mother of an offspring and which rhino is most likely to be the father. Often, these observations can yield only limited data and even mother-offspring relationships can be incorrectly identified. Many factors are responsible for the inaccuracy of observational pedigree. For example, a female may have an overlapping territory with multiple males, making it difficult to identify the father or difficulty in remote identification of an individual rhino may compromise the accuracy of maternal assignment.
Fieldwork on Southern White Rhinos in Botswana (credit: Nicholas Jonsson)
Scientists have other tools to resolve the pedigrees. DNA analysis is commonly used in parentage assignment and this is where our research project steps in. Often, tens of DNA markers are enough to resolve parentage. However, things are complicated for the SWR because of the historical population bottleneck when only 100 rhinos were left in the single population in South Africa. This means that all the descendants are very closely related and so this number of markers will not be able to provide unique genetic fingerprints for the individuals in the current population. Our research project has confirmed low variation in the twenty-three DNA markers currently available, and showed that the current level of marker variation was not enough to construct a complete pedigree, even when combined with observational pedigrees (find out more about this here).
To further resolve the pedigrees using more sensitive markers, we were lucky to obtain DNA samples from almost all SWRs within private-managed populations in the Republic of Botswana, thanks to Erik Verreynne our research partner from Agric Vet Consultants in Botswana. As the currently available DNA markers did not provide enough power for complete parentage assignment, we have needed to locate more.
However, developing new DNA markers is not simple. We needed these new DNA markers to be variable and reproducible across different populations to facilitate future studies. We explored two different ways of finding new markers: 1) reduced representation sequencing techniques; and 2) targeted capture sequencing. Don’t worry, as this is not a genetic textbook, we won’t go into the details here. All we need to know is that the former group of techniques does not require prior genetic knowledge but does require good quality DNA. The latter techniques, on the contrary, perform well with low quality DNA but requires prior genetic knowledge of the species. Unfortunately for the SWRs, we could not use either of them alone.
Reduced representation sequencing has been widely used in population genetic analyses for conservation because it provides cost effective screening of genetic variation across large numbers of individuals. The SWR samples were collected from populations living in natural habitats where the conditions available to preserve DNA samples are difficult to meet. Quality of DNA samples has been one major issue for our project as more than half of the samples were degraded, and so we could not use reduced representation sequencing for all samples.
Our research project would have to pave a new path. Fortunately, European zoological parks under the European Endangered Species Programme and the Science and Advice for Scottish Agriculture (SASA) generously provided us with additional samples containing good quality DNA that we could use to develop an alternative approach. After we had obtained some successful sequencing from reduced representation techniques, we were able to identify new DNA markers. From 23 traditional DNA markers, we now had ~8,000 candidate markers. No typo, it was indeed 8,000.
But our job isn’t finished.
We used information about these markers to develop a panel of probes to be used for targeted sequence capture. We have now tested the targeted capture process and have just obtained usable DNA sequences even from some of our lowest-quality DNA samples.
We now aim to use the newly developed DNA markers to determine parents of rhino offspring. However, these DNA markers could benefit other conservation studies on SWRs. In the past, DNA markers have been used to aid wildlife criminal prosecution. DNA can be extracted from mysterious powders detained in the wildlife black market and used to identify whether the powder is made of rhino horn or other substances (or other animal species). With more genetic markers, theoretically, we could identify the specific population where the horn comes from or even identify the individual.
(Seized rhino horn being analysed in the laboratory)
With the information of thousands of DNA markers, we should be able to identify parents for most offspring and we hope this set of new DNA markers will benefit many research studies and the conservation of rhinos into the future.
Until next time,
This work has been made possible by a partnership between the University of Glasgow and the WildGenes Lab, this work has been funded by a PhD grant from the Royal Thai government. We thank the Department of Wildlife and National Parks of Botswana for providing research permits, and the Vet and Agric Consultants in Botswana and the Science and Advice for Scottish Agriculture for providing samples.